Determination and manipulation of biologically active ...

Determination and manipulation of biologically active triterpenoid secondary metabolites in

Centella asiatica

by

JACINDA TERRY JAMES

THESIS

submitted in fulfilment

of the requirements for the degree

PHILOSOPHIAE DOCTOR

in

BIOCHEMISTRY in the

FACULTY OF SCIENCE at the

UNIVERSITY OF JOHANNESBUG Auckland Park

Study Promoter: Prof IA Dubery NOVEMBER 2012

Angels are watching over you when times are good or stressed.

Their wings wrap gently around you, whispering you are loved and blessed…

For gran who was always watching

Jean Ou Kow

(31/10/1925 – 7/3/2005)

To accomplish great things, we must not only act, but also dream; not

only plan, but also believe

- Anatole France

Summary

Plants are able to recognise and respond to signals from the environment through a complex

array of biochemical pathways, which enables them to deter pathogenic micro-organisms and

herbivores. Thousands of different structures of low-molecular weight organic compounds /

natural products can be produced through an inducible chemical defence system; that can be

manipulated for biotechnological purposes. The importance of natural products in medicine,

agriculture and industry has led to numerous studies such as this, to understand the

biosynthesis and biological activity of these substances.

This study provided a comprehensive overview of the pentacyclic triterpenoids present in

Centella asiatica cultivated in South Africa. Literature has reported triterpenoids with duplicate

names, synonyms and contradictory findings for the triterpenoids of C. asiatica, all dependent

on either the environmental conditions and / or location of the plant. In addition to the

morphological features, the triterpenoid composition in C. asiatica can greatly differ, and an

array of centelloids of the oleanane or ursane subtypes can occur. Hence for this study, a

method to quantify specific triterpenoids, namely asiaticoside, madecassoside, asiatic acid

and madecassic acid (which have not previously been investigated concurrently) was

developed based on quantitative thin-layer chromatography (QTLC). The applicability of this

method for the analysis of leaf extracts was shown to quantify the four principal centellosides

under study. The concentrations, expressed as 1-3 g/g tissue, were confirmed by reverse-

phase high pressure liquid chromatography (HPLC), thus supporting the accuracy of this

TLC-densitometric method. A comparative, quantitative analysis of the active triterpenoids in

C. asiatica samples from two different phenotypes occurring in South Africa was performed

by means of HPLC to evaluate the natural variability in triterpenoid content. It was seen that

the four metabolites were present in leaf tissue, with the free acids to glycosides occurring in

a ratio of approximately 1:2.5. All four centelloids were also found to be present in C. asiatica

callus and cell suspensions.

Since the four pentacyclic triterpenoids were present in C. asiatica cell suspensions,

metabolic changes due to a chemical induction by methyl jasmonate (MeJa) was

investigated. After treatment with 0.2 mM MeJa for 4 days, an increase was observed in the

number of metabolites after anisaldehyde detection on TLC. The results showed that MeJa is

able to trigger differential changes in the metabolome of C. asiatica cells, leading to changes

in the biosynthesis of secondary metabolites. These triterpenoids were then analysed

(qualitatively and quantitatively) with the aid of chromatographic and mass spectrometric

techniques and multivariate statistical models, since the manipulation of biochemical

pathways resulted in altered metabolic profiles, and presumably enhanced the level of

secondary metabolite production. In order to fully understand secondary metabolites and their

functioning, metabolomics tools were employed, which allowed for all metabolic changes

occurring due to the treatment to be captured. Inspection of the base peak intensity (BPI)

chromatograms indicated the changes as increases or decreases in peak intensities, new

peaks and peak suppression. The targeted pentacyclic triterpenoids (in ethanolic extracts

from C. asiatica cell suspensions), were analysed by ultra-performance liquid

chromatography coupled to mass spectrometry (UPLC-MS) and quantified by QuanLynx™

software. Prior to treatment with MeJa for 4 d, the concentrations of the targeted triterpenoids

were found to be in the region of 0.07 – 0.3 g/g fresh weight. There was a 5- and 9-fold

increase in the concentration of asiaticoside and asiatic acid respectively after elicitation, with

a 2-fold increase for madecassoside and madecassic acid, although from the BPI

chromatograms, only asiaticoside and asiatic acid were visually detected. Their identities

were confirmed by MS in ESI negative mode.

In addition, MeJa was also shown to have an effect on the production of other metabolites, as

seen from the PCA loading plot and investigation of the discriminatory ions resulted in the

identification of two metabolites, putatively identified as jasmonic acid and rishitin.

Many medicinal claims for C. asiatica have been reported and studies suggest the biologically

active ingredients are mainly attributed to the presence of several triterpenes. These

centelloid molecules have raised great interest due to the numerous reports describing their

medicinal properties and antimicrobial activity, and thus the potency against fungal and

bacterial pathogens by C. asiatica was investigated. Bioautographic screening showed that

centelloids present in crude extracts of C. asiatica cell suspensions occurred in very low

concentrations. Leaf extracts were able to inhibit the growth of micro-organisms due to the

presence of asiatic- and madecassic acid, with minimum inhibitory concentrations (MICs) in

the 1.56-6.25 mg/ml range. These C. asiatica leaf extracts also exhibited a greater activity

against Gram-positive bacteria than Gram-negative bacteria.

Preface

Parts of the material in this thesis have been presented in the following:- Publications

James, J.T.; Tugizimana, F.; Steenkamp, P.A. and Dubery, I.A. (2013) Metabolic analysis of methyl jasmonate-induced triterpenoid production in the medicinal herb, Centella asiatica (L.) Urban. Molecules. 18: 4267-4281

Presented in Chapter 6 James J.T. and Dubery I.A. (2011) Identification and quantification of triterpenoid centelloids in Centella asiatica (L.) Urban by densitometric TLC. Journal of Planar Chromatography. 24: 82-87

Presented in Chapter 4 James J.T. and Dubery I.A. (2009) Pentacyclic triterpenoids from the medicinal herb, Centella asiatica (L.) Urban. Molecules.14: 3922-3941.

Presented in Chapter 3 James J.T.; Meyer R. and Dubery I.A. (2008) Characterisation of two phenotypes of Centella asiatica in Southern Africa through the composition of four triterpenoids in callus, cell suspensions and leaves. Plant, Cell and Tissue Organ Culture. 94: 91–99

Presented in Chapter 5

Posters James J.T.; Meyer R., Dubery I.A. (2005). Secondary metabolite production in Centella asiatica and Centella cordifolium. Poster at: SASBMB XIX

th conference, Stellenbosch (South Africa). 16-20 January. James J.T.; Meyer R., Dubery I.A. (2008). Improved HPLC method for the analysis of centellosides

from Centella asiatica. Poster at: 6th ChromSAAMS conference, Bela Bela (South Africa). 12-15 October.

Each chapter has its own list of references which is combined in Chapter 9

Acknowledgements and thanks

I would like to extend my sincere thanks and gratitude to all who made this possible, especially to.... My study promoter, Prof Ian Dubery, for his invaluable assistance, guidance and advice with this

project.

Dr Marianne Cronjé, Prof Liza Bornman and Mrs Lynette de Kooker in the Department of Biochemistry at the University of Johannesburg all their support, help and encouragement. Lynette, thank you for your willingness to do all the little things that helped in such a big way.

Prof Paul Steenkamp at the CSIR, for his assistance with the UPLC-MS, method development and providing me with an extensive amount of data to work with for the metabolomics chapter. Your expertise and contribution were invaluable.

Mr Fidele Tugizimana for his assistance with the UPLC-MS data analysis; for all the advice, suggestions and most importantly, your willingness to help at all hours. Courage as you always said. Ms Caryn Beets and Mr Edwin Madala for their help with the structures and input with the metabolomics. Edwin, you have been a fantastic metabolomics advocate!

Mr Riaan Meyer for his expertise on the HPLC and chromatography.

Dr Safi Traore and Dr Aurelia Williams for assisting me with the cytotoxicity and viability studies.

Dr Ju-Chi (Carol) Huang for helping to reproduce the figures and diagrams and helping me with all the formatting.

The many student assistants and Honours students who somehow contributed to this study.

Dr Lizelle Piater, thank you for sharing this journey with me - first as a laboratory partner and then a colleague in the Biochemistry department. A huge thank you for giving your time so unselfishly to review everything, for your constructive criticism, and suggestions to improving this thesis.

All my friends, who have been supportive and encouraging especially Rabia, Sherrie-Ann, Lindy, Nicola and Carol. For those friends and former students (there are too many to name), that have come and gone but always provided some inspiration, company and good laughs – Denise, Melissa, Ashleigh, Christina, Aurelia, Safi, Karolina and Prof Debra Meyer – thank you.

My dearest friend Heather-Anne for the conversations in the car and during many, many tea breaks. Thank you for being constantly at my side and helping me to see the lighter and funnier side of things.

Melo, Chloe and Bruce-Lee, for your companionship and willingness to sit through the many long hours, late into the evenings and over weekends with me.

Gareth, my sincere gratitude for going through each step and each day with me, this has been a long journey, with many bumps and curves. Thank you for being a part of all the highs and lows, all the disappointments and the break-throughs.

My parents, Johnny and Joyce for always believing in me, for their love and patience, and to my brother Bradley and sister-in-law Cindy who took an interest, even though they did not really understand the science.

The Lord above who granted me with the ability and talents to do this, and made all things possible.

Contents

List of Abbreviations i

List of Figures iv

List of Tables vii

Chapter 1: Introduction and Objectives of the study 1

Chapter 2: Literature Overview The Biosynthesis of Natural Products and Triterpenes 7

1. Natural products 9

2. Secondary metabolism and metabolites 9

3. Terpenoids as natural products and secondary metabolites 14

3.1. Terpenes and triterpene glycosides in plants 14

3.1.1. The structures and classification of terpenes, terpenoids

and isoprenoids 15

3.1.2. The biosynthetic pathway of terpenoids 16

3.2. Biological activities and the distribution of plant saponins 23

Chapter 3: Pentacyclic Triterpenoids (Centelloids) from the Medicinal Herb, Centella

asiatica (L.) Urban 27 1. Abstract 29

2. Introduction 29


4. Chemical diversity of terpenoids 30

5. The biosynthesis of Centella triterpenes and triterpenoids 32

6. Biological activities of Centella triterpenoid saponins and sapogenins 40

7. Variation in triterpene production in C. asiatica chemotypes 45

8. Manipulation of centelloside production in cell and tissue culture 47

9. Conclusions 50

Chapter 4: Identification and Quantification of Triterpenoid Centelloids in Centella

asiatica (L.) urban by Densitometric TLC 51 1. Abstract 53

2. Introduction 53

3. Experimental 55

3.1. Preparation of triterpenoid standards 55

3.2. Plant material 55

3.3 Sample preparation 56

3.4. Chromatography 56

3.5. Visualisation of the spots for densitometry 56

3.6. Densitometry analysis of triterpenoid spots 57

3.7. Method validation 57

3.7.1. System suitability 57

3.7.2. Statistical analysis 57

3.7.3. Linear working range for known Centella triterpenes 58

3.7.4. Limits of detection and quantification 58

3.7.5. Specificity 58

4. Results and Discussion 58

5. Conclusion 66

Chapter 5: Characterisation of two phenotypes of Centella asiatica in Southern Africa through the composition of four triterpenoids in callus, cell suspensions and leaves 67

1. Abstract 69

2. Introduction 69

3. Materials and Methods 71


3.2. Preparation of callus and cell cultures 72

3.3. Determination of dry / wet weight 72

3.4. Ethanolic extracts of C. asiatica tissues 72

3.5. TLC analysis of triterpene saponins 73

3.6. Quantitative analysis using HPLC 73

3.7. Metabolite profiling using densitometric analysis 74


Chapter 6: Enhanced secondary metabolite production by means of chemical induction 82

1. Methyl jasmonate (MeJA) and jasmonic acid (JA) 84

2. Production of centellosides in C. asiatica 87

3. Enhanced biosynthesis of triterpenoids through biotechnology – elicitation

of C. asiatica by MeJa 88

4. Metabolomics 90

4.1. Analytical platforms 94

4.2. Multivariate data analysis (MVDA) 97

5. Significance of this study 98


6.1. Preparation and elicitation of the cell cultures 99

6.2. Cell viability assessment using the Alamar blue® assay 100

6.3. Extraction of the secondary metabolites - triterpenoids 100

6.4. Partial characterization and fractionation of the extracted

triterpenoids 100

6.5. Ultra-performance liquid chromatography – high definition mass

spectrometry (UPLC-HDMS) analysis 101

6.5.1. Experimental parameters 101

6.5.2. Data analysis 103

7. Results 106


7.2. TLC analysis 107



8. Discussion 123

9. Conclusion 130

Chapter 7: Antimicrobial effects of Centella asiatica 131

1. Introduction 133

2. The development of antibiotics and the emergence of antibiotic

resistance 133

3. Alternative therapies: Plants as a source of anti-microbial agents 136

4. The plant genus Centella: the medicinal value of Centella asiatica and

variations in triterpene production 137

5. C. asiatica as a source of ethnopharmacological agents: Biological

activities of C. asiatica triterpenoid saponins and sapogenins 139


6.1. Cytotoxicity evaluation 142

6.2. Isolation of PBMCs 143

6.3. Experimental design for the bioassays: the preparation and

extraction of C. asiatica centelloids 144

6.4. Investigation of potential cytotoxicity of ethanolic C. asiatica

extracts To PBMCs 145

6.4.1. Cell counting and trypan blue staining 145

6.4.2. Determining the cell viability after C. asiatica treatments

by means of colourimetric assays 146

6.4.2.1. XTT assay 147

6.4.2.2. MTT assay 148

6.5. Culturing the micro-organisms for the inhibition studies 149

6.6 Bioautographic screening for anti-microbial activity 150

6.6.1. Antibacterial and anti-mycobacterial screening 150

6.6.2. Antifungal screening 151

6.7. Quantification of the anti-microbial activity 151


7.1. Investigation into the potential cytotoxicity of ethanolic C. asiatica

extracts to PBMCs by means of the tetrazolium salts XTT and

MTT 154

7.2. Anti-microbial screening 158

7.3. MIC Determinations 163

8. Conclusion 167

Chapter 8: Concluding discussion 169

Chapter 9: Reference list 173

Addenda 212

- i -

List of Abbreviations

-AS alpha-amyrin synthase -AS beta-amyrin synthase

13-HPOT 13(S)-hydroxy linolenic acid 2,4-D 2,4-Ddchlorophenoxyacetic acid A600 Absorbance at 600 nanometers ACN Acetonitrile amu Atomic mass unit AOC Allene oxide cyclase AOS Allene oxide synthase APCI Atmospheric pressure chemical ionization AS Anisaldehyde suphuric acid BAP 6-Benzylaminopurine BPI Base pair intensity CabAS Centella asiatica -amyrin synthase CaCYS Centella asiatica cycloartenol synthase CAS Cycloartenol synthase CaSQS Centella asiatica squalene synthase CE-MS Capillary electrophoresis-mass spectrometry CID Collisional induced dissociation CPI Cyclopropyl sterol isomerase CV Coefficient of variation DBE Double bond equivalent DIMS Direct-injection mass spectrometry DIOS Direct ionization on silicon DMAPP Dimethylallyl diphosphate DMSO Dimethlsulphoxide DRE Dynamic range extended EDTA Ethylenediamine tetra acetic acid EED Ethanolic extracts solubilised in DMSO EI Electron impact EIC Extracted ion chromatogram ESI Electrospray ionization F254 Fluorescent at 254 nm FCS Fetal calf serum FDA Food and Drug Administration FPP Farnesyl diphosphate FPS Farnesyl diphosphate FTIR Fourier transform-infrared spectroscopy

GAP Glyceraldehyde-3-phosphate. GC-MS Gas chromatography mass spectrometry GHP German Homeopathic Pharmacopeia GS Gentamycin sulphate

- ii -

HCA Hierarchical cluster analysis HCA Hydroxycinnamates HDMS High definition mass spectroscopy HIV Human immunodeficiency virus HMG-CoA (S)-3-hydroxy-3-methylglutaryl co-enzyme A HPLC High pressure / performance liquid chromatography INT 3-[4-iodo-phenyl]-2-[4-nitrophenyl]-5-phenyl-2H-tetrazolium chloride IPP Isopentenyl diphosphate JA Jasmonic acid JMT Jasmonic acid carboxyl methytransferase

kMN k-mean cluster analysis KNN K-nearest neighbour

LA Linolenic acid LC-MS Liquid chromatography mass spectrometry LC-NMR-MS Liquid chromatography nuclear magnetic resonance mass

spectrometry LDI Laser desorption ionization LOD Limit of Detection LOQ Limit of Quantification LOX Lipoxygenase LS Lupeol synthase LUP Lupeol synthase

m/z Mass to charge ratio MALDI Matrix-assisted laser desorption ionization MeJa Methyl jasmonate MIC Minimum inhibition concentration MS Mass spectrometry MS/MS Tandem mass spectrometry MS media Murashige and Skoog media MTT 3-(4,5-dimethylethiazol-2yl)-2,5-diphenyltetrazolium bromide MVDA Multivariate data analysis n number of repeats Na2HPO4 2H2O Sodium phosphate dibasic dihydrate NAA Naphthalene acetic acid NADPH Nicotinamide adenine dinucleotide (reduced form) NADPH2 Nicotinamide adenine dinucleotide NMR Nuclear magnetic resonance

OPDA 12-oxo-phytodienoic acid OPLS Orthogonal projection to latent structures OPLS-DA Orthogonal projection to latent structures-discriminant

analysis OPLS-DA Orthogonal partial least squares discriminant analysis OSC(s) Oxidosqualene cyclase(s)

- iii -

PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline PC Principal component PCA Principal component analysis PDA Photodiode array PH-A Phytohemagglutinin from Phaseolus vulgaris PMS Phenazine methosulphate

QTLC Quantitative thin layer chromatography r Correlation coefficient RDA Recommended daily allowance RDBE Rings plus double bond equivalent Rf Retardation factor RF Radio frequency RI Retention index rpm Rotations per minute RSD Relative standard deviation Rt Retention time

S/N Signal to noise ratio SA Salicylic acid SCoA S-coenzyme A SD Standard Deviation SIMCA / SIMCA-P Soft independent modelling of class analogy SOM Self-organizing mapping SQE Squalene epoxidase SQS Squalene synthase TECA Titrated extract of C. asiatica TGF-β Transforming growth factor beta TIC Total ion chromatograms TOF Time of flight TTF Total triterpenoid fraction. TTFCA Total triterpenoid fraction of C. asiatica UPLC Ultra performance liquid chromatography UPLC-HDMS Ultra performance liquid chromatography high-definition mass

spectroscopy UPLC-PDA Ultra performance liquid chromatography photodiode array UV Ultra violet WHO World Health Organization XTT 2,2-bis-(methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-

carboxanilide

YE Yeast extract

http://en.wikipedia.org/wiki/Transforming_growth_factor_beta

- iv -

List of Figures

Chapter 2

Figure 2.1: Pathway events in the synthesis of secondary metabolites 14

Figure 2.2: The relationship of the terpenes 16

Figure 2.3: Overview of terpenoid biosynthesis in plants showing the basic stages

of their process and the major groups of end products 17

Figure 2.4: Outline of the mevalonate pathway for the formation of C5 isoprenoid

units 18

Figure 2.5: The formation of C10, C15 and C20 prenyl diphosphates from the fusion

of isoprenoid units 19

Figure 2.6: The structures of humulene, menthene and cembrene 20

Figure 2.7: Schematic representation of triterpenoid biosynthesis in Arabadopsis 21

Figure 2.8: Phylogenetic tree of plant oxidosqualene cyclase (OCSs) 22

Figure 2.9: Triterpenes structures can be classified according to the oleanane

and ursane-types 23

Figure 2.10: Aglycone skeletons of steroidal spirostane, steroidal furostane and

triterpenoid saponins 24

Chapter 3

Figure 3.1: Aglycone skeletons of pentacyclic steroidal spirostane,

steroidal furostane and triterpenoid saponins 32

Figure 3.2: A simplified scheme of triterpenoid biosynthesis in C. asiatica 33

Figure 3.3: The model triterpenoid compound from C. asiatica 36

Chapter 4

Figure 4.1: Structures of the main identified active components in C. asiatica

which are claimed to have medicinal properties 54

Figure 4.2: Separation of ethanolic leaf extracts containing the sapogenins and

saponins of C. asiatica and equimolar concentrations of the four

commercially available standards 60

- v -

Figure 4.3: Standard curves for densitometric analysis of each of the

investigated triterpenoids from C. asiatica 61

Figure 4.4: Reverse phase-HPLC chromatograms representing characteristic

profiles from ethanolic extracts of C. asiatica leaves 65

Chapter 5

Figure 5.1: Two phenotypes of South African C. asiatica 71

Figure 5.2: The growth curves (wet and dry mass) of C. asiatica Type-1

and Type-2 75

Figure 5.3: Reverse phase-HPLC chromatograms representing characteristic

profiles from ethanolic extracts of Centella asiatica 79

Chapter 6

Figure 6.1: Biosynthesis of jasmonic acid from linolenic acid 86

Figure 6.2: Schematic representation of the -omic hierarchy: genomics,

transcriptomics, proteomics, and metabolomics 93

Figure 6.3: Viability assessment of C. asiatica cell suspensions after MeJa

treatment using the Alamar Blue® assay 106

Figure 6.4: TLC plates of crude C. asiatica extracts induced with different

concentrations of MeJa for 2 and 4 d 107

Figure 6.5: UPLC-MS BPI chromatograms of the four authentic

standards (Extrasynthase) each with a concentration of 200 ng/ml 109

Figure 6.6: MS spectra of asiatic acid standard 110

Figure 6.7: MS spectra of madecassic acid standard 111

Figure 6.8: MS spectra of asiaticoside standard 111

Figure 6.9: MS spectra of madecassoside standard 111

Figure 6.10: UPLC-MS BPI chromatogram of the four authentic standards

(Extrasynthase) mixed together in equimolar concentrations 112

Figure 6.11: UPLC-MS BPI chromatograms obtained for ethanolic extracts of C.

asiatica cell suspensions under control and treated conditions 113

Figure 6.12: Representative extracted ion chromatogram of the full mass

chromatogram of the extracts after 2 d treatments with 0.2 mM MeJa 114

Figure 6.13: MS spectrum for two ion peaks corresponding to asiaticoside and

asiatic acid 115

- vi -

Figure 6.14: ESI- mass spectra for m/z 252 and 957 and MS/MS mass spectra for

co-eluting ions at m/z 252 and m/z 957 116

Figure 6.15: UPLC-PDA chromatograms 117

Figure 6.16: UPLC-MS (BPI) chromatograms of MeJa-treated ethanolic extracts

from C. asiatica cell suspensions at day 2, 4 and 7 118

Figure 6.17: PCA scores plot of a representative experiment 119

Figure 6.18: Representative PCA loading plot for all time points (2, 4 and 7 d) 120

Figure 6.19: Standard curves for asiaticoside, madecassoside, asiatic acid

and madecassic acid obtained by UPLC-MS analysis 122

Chapter 7

Figure 7.1: Global distribution map of C. asiatica 138

Figure 7.2: C. asiatica is a small creeping herb with shovel shaped leaves

emerging alternately in clusters at the stem nodes 139

Figure 7.3: Metabolism / reduction of XTT to a water soluble formazan salt by

viable cells 147

Figure 7.4: PBMCs allowed to proliferate for 3 d with treatments 153

Figure 7.5: MTT results showing the effect on viability of PBMCs exposed to EED

extracts from C. asiatica cells induced for 4 d with 0.2 mM MeJa 155

Figure 7.6: MTT results showing the effect on the viability of PBMCs under

different test conditions 157

Figure 7.7: A silica gel-60 plate was chromatographed with ethyl acetate,

methanol, H2O (80:10:10 (v/v/v)) as mobile phase for

C. cucumerinum inhibition studies 159

Figure 7.8: TLC plate depicting bacterial inhibition studies by C. asiatica leaves 161

Figure 7.9: TLC plate depicting bacterial inhibition studies by a commercially

available C. asiatica extract from Linnea 162

Figure 7.10: Quantification of microbacterial activity by means of INT 164

- vii -

List of Tables

Chapter 2

Table 2.1: The number of known secondary metabolites from higher plants 10

Chapter 3

Table 3.1: Structures of the pentacyclic triterpenes reported to occur in C. asiatica

to date 37

Table 3.2: Product range of extracts from C. asiatica indicating the specific

chemical composition and treatment 41

Table 3.3: Summary of the medicinal claims for C. asiatica 43

Table 3.4: Various saponins occur in C. asiatica due to the location and

diverse environmental conditions 46

Chapter 4

Table 4.1: Densitometric analysis of triterpene saponin and sapogenin standards 63

Table 4.2: Investigation of Centella asiatica leaf tissue by densitometric analysis of

TLC plates after AS detection 64

Chapter 5

Table 5.1: Quantitative determination by HPLC of asiatic acid, madecassic acid and

their glycosides, asiaticoside and madecassoside 77

Chapter 6

Table 6.1: Terminology and definitions related to metabolomics 91

Table 6.2: UPLC conditions - gradient composition 102

Table 6.3: MS parameters 103

Table 6.4: Elemental composition method for metabolite identification using

MassLynxTM 104

Table 6.5: Parameters for the MarkerLynx PCA method 105

- viii -

Table 6.6: Rt values obtained for the four targeted triterpenoids analyzed separately 112 Table 6.7: Table for discriminating ions 120

Table 6.8: Estimated concentration of the targeted triterpenoids 121

Chapter 7

Table 7.1: Some of the product range of C. asiatica extracts indicating the

specific chemical composition and treatment 140

Table 7.2: The minimum inhibitory concentrations 166

Introduction and Objectives of the Study

Chapter 1

Chapter 1 - Introduction

- 2 -

The genus Centella compromises some 50 species, including the medicinally important

Centella asiatica. This small perennial herbaceous plant belongs to the plant family Apiaceae

(Umbelliferae) and has been used as a traditional herbal medicine in Malaysia, India and

other parts of Asia for centuries (Brinkhaus, 2000). Both the leaves and the entire plant can

be used therapeutically (EMEA, 1998), and it can also be eaten fresh as salad, cooked as a

vegetable or blended as a drink (Hashim et al., 2011). Due to its utilization in nutraceutical

food preparations, C. asiatica has become an important commercial plant (James and

Dubery, 2009).

C. asiatica is found in south-east Asia, Sri Lanka, in parts of China, the western South Sea

Islands, Madagascar, South Africa, the south-east of the USA, Mexico, Venezuela, Columbia

and in the eastern regions of South America. This tropical plant has been reported to be used

for various medicinal purposes such as wound healing (Tenni et al., 1988; Marquart et al.,

1999), improved circulation, memory enhancement, cancer, vitality, respiratory ailments, as a

general tonic detoxifying the body, for the treatment of skin disorders (such as eczema and

psoriasis (Sampson et al., 2001), revitalizing connective tissue (Montecchio et al., 1991), burn

and scar treatment, skin infections, slimming and edema, arthritis, rheumatism, periodontal

disease, sedative, anti-stress, anti-anxiety, an aphrodisiac, an immune booster and for the

treatment of liver and kidney diseases. In fact, the latter has been used for centuries in both

traditional Chinese and Indian systems of medicine and has become a popular alternative

treatment for people suffering from hepatitis and alcoholic liver disease. It has been claimed

that extracts from C. asiatica are able to assist in destroying toxin accumulation in the brain

as well as in the nerves, while it helps to clear the body from heavy metals and drugs –

including recreational drugs. In alternative health, this herb has been used to treat tumours

and cancerous growths (Babu et al., 1995), without suppressing the autoimmune system or

creating toxic wastes within the body. This tropical plant has been used in Ayurvedic and

traditional medicines not only for wound healing but general well being as well as an anti-

bacterial and anti-viral agent (Ullah et al., 2009). However, since none of these above claims

have been evaluated by the FDA, research has been done by various institutes and

universities and it was concluded that more research is called for on this ancient herb.

C. asiatica accumulates large quantities of pentacyclic triterpenoid saponins. These valuable

secondary compounds include centellasaponins, asiaticoside, madecassoside, centelloside,

brahmoside, brahminoside, thankuniside, sceffoleoside, centellose, asiatic-, brahmic-,


- 3 -

centellic- and madecassic acids amongst others (Matsuda et al., 2001; James and Dubery,

2009). Asiaticoside is one of the principal triterpene saponins present in the leaves and it is

used commercially as a wound-healing agent because of its potent anti-inflammatory effects

(Pointel et al., 1987).

The triterpenoid saponins are a large class of isoprenoidal natural products present in higher

plants. More than eighty different skeletal types of triterpenes are known as natural products,

which are elaborated at the cyclization step of a common substrate, 2,3-oxidosqualene (Wink,

1999). Following structural modifications on cyclization, such as oxygenation, esterification

and glycosylation, further structural diversity is obtained (Abe et al., 1993). A hydrophobic

triterpenoid structure (aglycone) containing a hydrophilic sugar chain (glycone) can thus be

produced which has characteristics that attribute to the biological activity of saponins.

To deter pathogenic micro-organisms and herbivores, plants have developed an inducible

chemical defence system. It is known that the induced synthesis of low molecular weight

compounds called phytoalexins can be provoked by exposing cultured cell cultures to various

elicitors (Gunlach et al., 1992). The elicitor interacts with a plant membrane receptor (Cosio et

al., 1990) leading to the activation of specific genes (Chappell and Hahbrock, 1984), resulting

in the synthesis of secondary metabolites (Gunlach et al., 1992). Elicitors or defense-related

signal molecules (such as methyl jasmonate (MeJa) and salicylic acid (SA)) have been found

to induce secondary metabolite accumulation not only in the intact plants but also in plant

tissue cultures. Several studies have indicated that many plant tissue cultures are stimulated

by elicitors and that secondary metabolites accumulate rapidly in response to treatment with

elicitors (Discomo and Misawa, 1985; Eilert et al., 1987; Mukundan and Hjortso, 1990; Ning

et al., 1994).

Although living organisms produce thousands of different structures of low-molecular weight

organic compounds; many of these have no apparent function in the basic process of growth

and development, and have been referred to as natural products or secondary metabolites.

The importance of natural products in medicine, agriculture and industry has led to numerous

studies on the synthesis, biosynthesis and biological activity of these substances, yet little is

known about their role in nature.


- 4 -

In order to understand secondary metabolites and their functioning, metabolomics may be

employed. Plant metabolomics can be defined as the study of metabolic pathways and

processes through the use of analytical methods in a model species. This technique allows all

metabolic changes occurring due to a treatment to be captured and the information gained

from this research is used to understand how plants grow and carry out functions, as well as

improve the quality of food or medicines. Its use in the cell culture area is still under-

developed (Khoo and Al-Rubeai, 2007).

As previously described, the active constituents in C. asiatica described to date include

pentacyclic triterpene derivatives, which consist of the tripene saponins (madecassoside and

asiaticoside) and their sapogenins (madecassic and asiatic acid). The biochemical pathways

and genetic machinery required for the elaboration of this important family of plant secondary

metabolites are still largely uncharacterized, despite the considerable commercial interest in

this important group of natural products. This is likely to be due in part to the complexity of the

molecules and the lack of pathway intermediates for biochemical studies.

In this thesis, the hypotheses investigated are as follows:- The basal levels of four triterpenes,

namely asiaticoside, madecassoside, asiatic acid and madecassic acid can be enhanced by

MeJa treatment and that other induced, defence-related secondary metabolites in C. asiatica

cell suspensions may be synthesized. These new or altered metabolites can be isolated,

characterized and identified by various chromatography techniques.

The objectives of this study were to:

[1] Provide a comprehensive overview of the pentacyclic triterpenoids present in C.

asiatica since many of the triterpenoids have duplicate names and synonyms.

[2] Asses the basal levels of specific triterpenes in C. asiatica with the aim of developing

a HPLC method to yield the optimum conditions for separation and quantification.

[3] To analyse (qualitatively and quantitatively) the changes in defence-related secondary

metabolites in C. asiatica cell suspensions treated with MeJa, with the aid of

chromatographic techniques and multivariate statistical models since the biochemical


- 5 -

pathway(s) were manipulated to result in altered metabolic profiles, and enhance the

level of secondary metabolite production.

[4] To identify the induced metabolites with a focus on the four (4) active triterpenoids,

namely madecassoside, asiatic acid and madecassic acid and to compile a profile of

the defence-related secondary metabolites found in C. asiatica cell suspensions.

[5] Lastly, it has been demonstrated that C. asiatica exhibits potency against fungal and

bacterial pathogens (Hanawa et al., 1992; Cos et al., 2002; Oyedeji and Afolayan,

2005). Although herbal medicines are perceived as being natural and therefore

harmless, the use of various medications without proper knowledge of the plant

constituents and possible toxicity can lead to accidents and fatalities. Thus, there is a

need for potential medicinal plants to be evaluated. The biological activities of extracts

containing said secondary metabolites were tested for cytotoxicity and anti-microbial

capabilities.

This study will provide a comprehensive overview of the pentacyclic triterpenoids present in

C. asiatica and describe metabolic changes due to MeJa treatment on secondary metabolism

in cell suspensions, with the focus on four active triterpenoids, which has not previously been

investigated concurrently.

There are two possible outcomes: if the result confirms the hypothesis, then

you’ve made a discovery. If the result is contrary to the hypothesis, then you’ve

made a discovery.

Enricco Fermi –

20th Century physicist

Literature Overview: The Biosynthesis of

Natural Products and Triterpenes

Chapter 2

Chapter 2- Literature overview Triterpenoids

- 8 -

1. Natural products 9

2. Secondary metabolism and metabolites 9


3.1. Terpenes and triterpene glycosides in plants 14

3.1.1 The structures and classification of terpenes, terpenoids and

isoprenoids 15

3.1.2. The biosynthetic pathway of terpenoids 16

3.2. Biological activities and the distribution of plant saponins 23


- 9 -

1. Natural products

Natural products are compounds produced by living organisms. They have provided

challenging synthetic targets, and their biological activity has lead to the development of

valuable medicines. These natural occurring compounds may be divided into three

categories.

1. The general metabolism that is common to all cells involves natural products, the

primary metabolites. These compounds occur in all cells and play a central role in

metabolism and the reproduction of cells and are involved in the storage and release

of energy in the synthesis of essential constituents such as nucleic acids, the common

amino acids and sugars. These are known as primary metabolites.

2. The high-molecular-weight polymeric materials such as cellulose, the lignins and

proteins which form the cellular structures.

3. Other natural products that have been found in a limited range of species and give

species their distinctive characteristics. These are the secondary metabolites. Most

primary metabolites exert their biological effect within the cell or organism that is

responsible for their production. Secondary metabolites have often attracted interest

because of their biological effect on another organism.

The biologically active constituents of medicinal, commercial and poisonous plants have been

studied; many of these compounds are secondary metabolites. It has been estimated that

over 40% of medicines have their origins in these active natural products. Natural products

often have an ecological role in regulating the interactions between plants, micro-organisms,

insects and animals. They can be defensive substances, such as phytoalexins and

phytoanticipins, anti-feedants, attractants and pheromones (Hanson, 2003).

2. Secondary metabolism and metabolites

Aside from the primary metabolic pathways common to all life forms, some reactions lead to

the formation of compounds unique to a few species or even to a single cultivar. These

reactions are classified under the term “secondary metabolism” and their products known as


- 10 -

“secondary metabolites” (Luckner and Nover, 1977). These substances include, inter alia,

anthocyanins flavanoids, phenylpropanoids, coumarins, alkaloids, antibiotics, volatile oils,

resins, tannins, sterols and saponins. It has become evident that secondary metabolites are

not just waste products or otherwise functionless molecules. They may also function as signal

molecules within the plant or between the plant producing them and other plants, microbes,

herbivores, pollinating or seed-dispersing animals. More often, they serve as chemical

defence compounds against herbivorous animals, microbes or competing plants (Wink,

1999).

Table 2.1: The number of known secondary metabolites from higher plants (Wink, 1999).

* Approximate number of known structures ** Total number exceeds 22 000 at present (Dictionary of Natural Products, 1996)

An increase in knowledge related to secondary metabolism and known secondary

metabolites in all living organisms have been accompanied by technical advances in

chromatography and tracer techniques. However, the boundaries of secondary metabolism

are not very distinct and the term “secondary products” has been used to indicate products

Type of secondary metabolite Number *

Nitrogen containing

Alkaloids 12 000

Non protein amino acids (NPAAs) 600

Amines 100

Cyanogenic glycosides 100

Glucosinolates 100

Without Nitrogen

Sesquiterpenes ** 3 000

Monoterpenes ** 1 000

Diterpenes ** 1 000

Triterpenes, steroids, saponins ** 4 000

Tetraterpenes ** 350

Flavonoids 2 000

Polyacetylenes 1 000

Polyketides 750

Phenylpropanoids 500


- 11 -

derived from primary metabolites. This is true for most alkaloids whose N- and C-atoms are

usually derived from protein amino acids with the basic ring system being modified by methyl,

acetyl, isoprene or other groups. This concept however also applies to cyanogenic

compounds and tannins. Nevertheless, many secondary metabolites (including some

alkaloids) have their own unique biosynthetic pathways (Figure 2.1) which are not directly

related to primary metabolism (Bell and Charlwood, 1980).

Although the structures of secondary metabolites are very diverse, the majority of these

compounds belong to one of a number of families, each with particular structural

characteristics arising from their biosynthesis (Hanson, 2003). The classes of secondary

metabolites are:

Alkaloids

These are a structurally diverse group of natural occurring chemical compounds that

contain nitrogen, with the exception of proteins and peptides. The nitrogenous parts of

the alkaloids are derived from amino acids like ornithine, lysine, tyrosine or

tryptophan. Alkaloids are classified according to the heterocyclic ring system present

in their structure e.g. indole alkaloid, pyridine alkaloids and benzylisoqunoline. These

compounds function as poisons or repellents towards predators, parasites and

competitors (Robinson, 1974).

Flavanoids These polyphenolic compounds are found naturally in plants and possess 15 carbon

atoms; two benzene rings joined by a linear three carbon chain. Anthocyanins are

flavonoids that are widely distributed throughout plants and are the cause of the

colours of flowers and fruits. They also play a role in protecting the plants from

microbe and insect attacks. Flavonoids also have apparent roles in plant stress

defence, such as in protection against damage caused by pathogen attack, in

wounding or in excess of UV-light. The low availability of nitrogen or phosphorus, and

low temperatures affect flavonoid levels in plants (Dixon and Paiva, 1995; Winkel-

Shirley, 2002).


- 12 -

Phenylpropanoids

The phenolic compounds with a C6-C3 skeleton that are products of the

phenylpropanoid pathway (phenyalanine / hydroxycinnamate) pathway which includes

reactions from L-phenylalanine to the hydroxycinnamates (HCAs, Wink, 1999). The

structural diversity of phenylpropanoids is due to the action of enzymes and enzyme

complexes which cause amongst others, cyclization, aromatization, acylation and

glycosylation (Noel et al., 2005).

Polyketides and fatty acid synthase products

Polyketides are formed by the linear combination of acetate (ethanoate) units derived

from the building block acetyl-CoA that is very similar to fatty acid biosynthesis. The

difference between polyketide and fatty acid biosynthesis are in the number and type

of acyl precursors used the extent and position of keto-group reductions and the

cyclisation pattern of the products (Revill et al., 1996).

Terpenoids and steroids. These are assembled in nature from isoprenoid C5 units linked in a head-to-tail

manner. These isoprenoid C5 units are derived from isopentenyl (3-methylbut-3-en-1-

yl) diphosphate and have a characteristic branched chain structure. Many sterols

occur as glycosides typified by the steroidal saponins. A number of plant steroids

possess a useful pharmacological activity while other steroids such as ecdysteroids

are insect hormone and brassinosteroids are plant hormones. The carotenoids are red

or yellow pigments that are found in many plants, these carotenoids are good anti-

oxidants and contribute beneficial effects to many foods.

Benzenoids

Benzenoid compounds (C6-C1) are generally assumed to be derived from

phenylpropanoids (C6-C3) but a route for benzenoid biosynthesis via isochorismate

has been proposed (D’Auria and Gershenzon, 2005). Most floral derivatives are

classified as either terpenoids or benzenoids and plants can use these volatile

molecules together with floral form and colour to attract pollinators that allow or

facilitate reproduction (Verdonk et al., 2005).


- 13 -

Specialized amino acids and peptides. Proteins are biological polymers that are structural, in that they maintain the cell’s

shape and carry out cell movements while others function enzymatically, which

generates energy for the cell to carry out biochemical reactions e.g. the synthesis of

nucleotides and the assembly of nucleic acids. Although amino acids are normally

considered primary metabolites, there are some unusual amino acids that are of

restricted occurrence.

Specialized carbohydrates.

Sugars (carbohydrates) such as glucose are typical primary metabolites, which are

essentially synthesized from a series of complex reactions during photosynthesis.

These carbohydrates store energy in the form of starch and provide carbon for the

synthesis of other compounds or are the structural components in cells and tissues.

However, there are other sugars that are of a much more limited occurrence, some of

these less common sugars are attached to natural products as part of a glycoside, the

non-sugar portion, known as the aglycone may be a flavonoid, terpenoid, alkaloid or

polyketide (Hanson, 2003).

Despite the enormous variety of secondary metabolites, the number of corresponding

basic biosynthetic pathways is restricted and distinct. Precursors usually derive from

basic metabolic pathways (Figure 2.1) such as glycolysis, Krebs cycle or the shikimate

pathway (Wink, 1999).


- 14 -

Figure 2.1: Pathway events in the synthesis of secondary metabolites. Several thousand different types of molecules from very different plant groups have been isolated and characterized but despite their varied structures, all of them are synthesized by only a few pathways (adapted from Edwards and Gatehouse, 1999). The complex alkaloids include several families of polycyclic pyrrolidine, piperidine, and guanidine alkaloids which exhibit a wide array of biological activities including, antitumor, antiviral, antibacterial, and antinociceptive properties.

3. Terpenoids as Natural Products and Secondary Metabolites

3.1. Terpenes and triterpene glycosides in plants

The terpenes form one of the largest and most diverse groups of natural plant products. This

group of compounds include sterols and triterpenes, which can accumulate as glycosides

(saponins) in extensive amounts in plants (Sparg et al., 2004).


- 15 -

3.1.1. The structures and classification of terpenes, terpenoids and

isoprenoids

The terpenes (also known as terpenoids or isoprenoids) are a diverse class of secondary

metabolites that may have been formally considered to be constructed from isoprene (2-

methylbutadiene, C-C-C-C) units (Ruzicka, 1953; Gershenzon and Kreis, 1999) linked in a

C

head-to-tail manner.

Most of the terpenes have cyclic structures. The majority of the terpenoid cyclizations which

take place in living systems are of an acid-catalyzed type. The branched chain nature of the

isoprenoid backbone coupled with readily protonated functional groups (e.g. alkenes) allows

them to be in an acid-catalyzed arrangement during the course of these biosynthetic

reactions.

Several thousand terpenes occur in many genera of higher plants and organisms (Devon and

Scott, 1972; Darnley-Gibbs, 1974) and although often the structures of the various classes

seem unrelated, detailed biochemical studies have revealed the pattern of biosynthesis (Bell

and Charlwood, 1980). The steroids are derived from the tetracyclic triterpenoids and

isoprene units and have sometimes been found to be components of other natural products.

The terpenes are classified by the number of these C5 isoprene units that they contain (Figure

2.2). The classes are:

Monoterpenoids (C10) e.g. geraniol, limonene and terpineol

Sesquiterpenoids (C15) e.g. farnesol

Diterpenoids (C20) e.g. cafestol, kahweol, cembrene and taxadience.

Sesterterpenoids (C25)

Triterpenoids (C30)

Carotenoids (C40)

Hemiterpenes e.g. isoprene which consist of a single isoprene unit

Polyterpenes consisting of long chains of many isoprene units.


- 16 -

Figure 2.2: The relationship of the terpenes (Banthorpe and Charlwood, 1980). The starting molecule for all the different groups is mevalonic acid that is phosphorylated and converted to a phosphorylated isoprene. The isoprenes polymerize and subsequently fix the number and position of the double bonds. In this way, all green plants are able to generate linear isoprenoids.

Given the many ways the basic C5 units can be combined, it is not surprising to observe the

numerous amount and diversity of the structures (Gershenzon and Dudareva, 2007).

3.1.2. The biosynthetic pathway of terpenoids

Although the “isoprene rule” for structure elucidation was established in the 1920’s by

Ruzicka, the biosynthetic origin of the C5 unit was unknown. There are now known to be two

major pathways, one based on mevalonic acid and the other one 1-deoxyxylulose

(Gershenzon and Kreis, 1999).


- 17 -

Terpenoid biosynthesis can be divided into four stages (Figure 2.3). Firstly there is the

formation of the isoprene unit isopentenyl diphosphate, secondly there is the association of

these units to form the (C5)n isoprenoid backbone of the terpenoid families, thirdly there is the

cyclization of these to generate the carbon skeletons; and finally there are the

interrelationships, hydroxylations and oxidations that lead to the individual terpenoids.

Figure 2.3: Overview of terpenoid biosynthesis in plants showing the basic stages of their process and the major groups of end products (Wink, 1999) (abbreviations: Co-A, Coenzyme A; GAP, glyceraldehyde-3-phosphate).

The first irreversible step in terpenoid biosynthesis in the mevalonate pathway (Figure 2.4),

involves the enzymatic reduction of (S)-3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)

with hydrogen from nicotinamide adenine dinucleotide (NADPH2) to produce (R)-mevalonic

acid. The HMG-CoA arises mainly by the condensation of acetyl-CoA with acetoacetyl-CoA.

Two successive phosphorylations of mevalonic acid produce the 5-diphosphate. This


- 18 -

undergoes trans elimination of the tertiary hydroxyl group and the carboxyl group to form 3-

methylbut-3-enyl diphosphate (isopentenyl diphosphate, IPP).

Figure 2.4: Outline of the mevalonate pathway for the formation of C5 isoprenoid units (Wink, 1999). (abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; NADPH, nicotinamide adenine dinucleotide phosphate (reduced from); SCoA, S-coenzyme A (to which acetate is attached; CoASH, free coenzyme A).


- 19 -

A steriospecific but reversible isomerization of the double bond of IPP (Figure 2.5) produces

3-methylbut-2-enyl diphosphate (dimethylallyl diphosphate, DMAPP). The significance of this

isomerization is the creation of a reactive allylic diphosphate which plays an important role in

the association of the two isoprene units to form the C10 geranyl diphosphate. The allylic

diphosphate is used in an enzyme catalysed (prenyl transferase) alkylation reaction of IPP.

The loss of a diphosphate anion to form the alkylating unit together with a proton from the IPP

generates a further reactive allylic diphosphate. The formation of the new carbon-carbon

bond between the isoprene units accompanied by inversion of the configuration of the allylic

carbon atom was demonstrated by labelling experiments.

Figure 2.5: The formation of C10, C15 and C20 prenyl diphosphates from the fusion of isoprenoid units

(Wink, 1999).

The stereochemistry of the loss of the proton from IPP suggests that this alkylation reaction

may take place in two steps. When an intermediate is bound to a chiral enzyme surface, two

apparently identical hydrogens lose their identity and may be distinguished by the enzyme.

Chirally labeled mevalonates have established the steriochemistry of these reactions. The

addition of further units gives farnesyl diphosphate (C15) and geranylgeranyl diphosphate


- 20 -

(C20), which are parents of the sesqui- and diterpenoids. Squalene (C30), the parent of

triterpenes and steroids, is formed by two molecules of farnesyl diphosphate, which are

joined together in a “tail-to-tail” manner.

There are two general modes of cyclization of the acylic terpenoid precursors. In the first, the

cyclization is initiated by protonation of an alkene or, as in the case of the triterpenoids, an

epoxide. In the second mode, the cyclization is initiated by electrophillic attack of the allylic

diphosphate or the isomeric diphosphate of a 3-hydroxy-1-ene on a double bond in another

isoprene unit. In the monoterpenoids, this can generate the menthene skeleton (Figure 2.6),

and in the sesquiterpenoids it can lead to the medium sized rings of the humulene series. In

the diterpenoids it leads to the formation of the monocyclic ring of the cembrenes (or

sometimes neocembranes, Figure 2.6).

Figure 2.6: The structures of humulene, menthene and cembrene respectively (Tholl et al., 2005).

Cembrene itself has little importance as a chemical entity, being a trail pheromone for

termites, but the chemical structure of cembrene is central to a wide variety of other natural

products found in plants and animals (Tholl et al., 2005). Further cyclizations of these lead to

a wide variety of different skeletons.

Plants synthesize diverse triterpenoids and their genomes encode multiple oxidosqualene

cyclases (OSCs) enzymes to form these skeletons. Synthesis of triterpenes involves

cyclizations of 2,3-oxidosqualene into various triterpene skeletons (Figure 2.7), including -

and -amyrin and lupeol (Jenner et al. 2005). These conversions are catalysed by OSCs,

known collectively as triterpene synthases (Haralampidis et al., 2002).

Figure 2.7: Schematic representation of triterpenoid biosynthesis in Arabadopsis. Solid arrows indicate the enzymes involved which include lupeol synthase (LUP), cycloartenol synthase (CAS) and cyclopropyl sterol isomerise (CPI) (Aadapted from Matsuda and Bartel webpage (Rice University, www-bioc.rice.edu/~bartel/ projects/2010Fig1.gif; Phillips et al., 2006).


- 22 -

A phylogenetic tree analysis (Figure 2.8) shows that OSCs have the same enzyme function

from respective branches in the tree even though they were derived from different plants

species. All triterpene synthases appear to have diverged from an ancestral cycloartenol

synthase (CAS) gene (Zhang et al., 2003) but an independent origin for - amyrin synthase

( -AS) in eudicots and monocots has been proposed (Phillips et al., 2006). The triterpenoid

cyclases are distinct from lanosterol synthase (LS) and CAS, and form discrete subgroups

within the OSC superfamily (Haralampidis et al., 2002).

Figure 2.8: Phylogenetic tree of plant oxidosqualene cyclases (OCSs). The distances between each clone and groups were calculated with the indicated scale representing 0.1 amino acid substitution per site (Zhang et al., 2003).

Cyclization of 2,3-oxidosqualene through a protosteryl cation intermediate generates

lanosterol and cycloartenol, the structural precursors to all the steroids, while cyclization

through a dammarenyl, baccharenyl and lupenyl cation intermediates generates lupeol and α/

-amyrin (Jenner et al., 2005), the precursors of the pentacyclic triterpenoid saponins (Figure

http://en.wikipedia.org/wiki/Cycloartenol

http://en.wikipedia.org/wiki/Steroid


- 23 -

2.7). The α / -AS enzymes cyclize oxidosqualene via the dammarenyl cation and allows for

further ring expansion and some rearrangement before deprotonation to α-amyrin and -

amyrin respectively (Haralampidis et al., 2002).

α-Amyrin contains the ursane (C19, C20 dimethyl) and -amyrin the oleanane (C20 dimethyl)

substitution patterns respectively (Matsuda et al., 2001; Figure 2.9)

Figure 2.9: Triterpenes structures can be classified according to the oleanane (left) and ursane-types.

Following cyclization, further diversity is conferred by modification of the products by

oxidation, hydroxylation, glycosylation and other substitutions mediated by cytochrome P450-

dependent monooxygenases, glycosyl transferases and other enzymes. Little is known about

the enzymes required for these chemical elaborations. One common feature shared by all

saponins is the presence of a sugar chain attached to the aglycone. Glycosylation is

particularly important as the sugar chain is critical for the biological activity of many saponins

(Morrissey and Osbourn, 1999; Achnine et al., 2005). The oligosaccharide chains are likely to

be synthesized by sequential addition of single sugar residues to the aglycone, but little is

known about triterpenoid glycosylation (Haralampidis et al., 2002).

3.2. Biological activities and the distribution of plant saponins

Saponins are glycosylated (aglycone = sapogenin) plant triterpenoids or steroids found in a

variety of species including major food crops (Papadopoulou et al., 1999). Their surface-

active properties are what distinguish these compounds apart from other glycosides (Sparg et

al., 2004). Due to some of these saponins forming the starting points for the semi-synthesis of

steroidal drugs, these metabolites are highly sought after by the pharmaceutical industry (Liu

and Henkel, 2002).


- 24 -

Saponins are classified according to their aglycone skeleton. The first group consists of non-

steroidal saponins, which are the most common and occur mainly in the dicotyledonous

angiosperms. The second group consists of the steroidal saponins which are almost

exclusively present in the monocotyledonous angiosperms. Some claim a third class called

steroidal amines, which are also referred to as steroidal alkaloids (Bruneton, 1995).

In higher plants, 2,3-oxidosqualene is a common precursor in the synthesis of both sterols

and triterpenes (Jenner et al., 2005). Steroidal saponins consist of a steroidal aglycone, a C27

spirostane skeleton which generally consists of a six-ring structure (Figure 2.10A). The

hydroxyl-group in the 26-position may be engaged in a glycosidic linkage so that the aglycone

structure remains pentacyclic (Figure 2.10B). This is referred to as a furostane skeleton.

Triterpenoid saponins consist of a triterpenoid aglycone, which consists of a C30 skeleton,

compromising a pentacyclic structure (Figure 2.10C).

Figure 2.10: Aglycone skeletons of (A) steroidal spirostane, (B) steroidal furostane and (C) triterpenoid saponins. The R-group is a sugar moiety and indicates a methyl (-CH3) group with unknown steriochemistry (Sparg et al., 2004).

Pentacyclic triterpenoids are usually concentrated in the outermost layers such as the plant

cuticle, fruit peel and bark. For example, an apple peel contains about 0.1 grams of ursolic

acid per fruit, and the outer bark of white birch species contains up to 40% w/w of betulin. The

amount of betulin obtainable from the birch bark waste in the wood-working industry in

Finland is estimated at about 150,000 tons per annum (Kuznetsov, et al., 2005).


- 25 -

The biological and pharmalogical activities of saponins include (Sparg et al., 2004):

Haemolytic activity Saponins have the ability to disrupt erythrocytes. This has led to the development of

haemolytic assays to detect the presence of saponins in drugs or plant extracts.

Molluscicidal activity Saponins are highly toxic to molluscs and have been investigated as molluscicides.

Anti-inflammatory activity There are numerous reports of saponins with anti-inflammatory activity (Just et al.,

1998).

Antimicrobial activity In view of the fact that many saponins have potent antifungal and antibacterial

properties and are usually present in healthy plants in high concentrations, these

molecules may act as preformed chemical agents against pathogen attack. The

isolation of plant mutants defective in saponin biosynthesis represents a strategy to

evaluate the importance of these compounds in plant defence (Crombie and Crombie.

1986). This group of secondary metabolites may have a significant role as

antimicrobial phytoprotectants as these saponins are widespread throughout the plant

kingdom. In plant tissue cultures, stress induced by inactivated fungi or fungal

enzymes, has been used to enhance production of biologically active secondary

metabolites. It has been reported that this fungal elicitation can lead to an

overproduction of pentacyclic triterpenes instead of some other expected metabolites

(Van der Heijden et al., 1988). These compounds can therefore be regarded as

constitutive phytoanticipins, but also as inducible phytoalexins.

Antiparasitic activity

Cytotoxicity and antitumor activity

Antiviral activity

Literature supplies numerous examples of enzymes that can be inhibited by pentacyclic

terpenoids, indicating the ability of pentacyclic triterpenoids to act broadly in a non-specific

mode on multiple targets. Such examples include the inhibition of rat renal 11-β-

hydroxysteroid dehydrogenase and the in vitro inhibition of adenosine deaminase.

Pentacyclic triterpenes derived from Maprounea africana are potent inhibitors of HIV-1

reverse transcriptase (Pengsuparp et al., 1994). Ursolic acid showed toxicity and feeding


- 26 -

deterrent effects towards insects and their survival (Varanda et al., 1992). The mode of

inhibition of enzymes seems to be non-specific and based primarily on hydrophobic

interaction with an enzyme's hydrophobic domain (Patent US 6303589, Pentacyclic

triterpenes, Free patents online).

In Chapter 3 the focus is specifically on the triterpenoids of C. asiatica. A review of the

literature has revealed duplicate names, synonyms and contradictory findings for the

triterpenoid compounds of C. asiatica which will be addressed.

Pentacyclic Triterpenoids (Centelloids) from

the Medicinal Herb, Centella asiatica (L.)

Urban

Chapter 3

- 28 -

Published as: Molecules (2009) 14, 3922-3941; doi: 10.3390/molecules14103922

1. Abstract 29

2. Introduction 29


4. Chemical diversity of terpenoids 30

5. The biosynthesis of Centella triterpenes and triterpenoids 32

6. Biological activities of Centella triterpenoid saponins and sapogenins 40

7. Variation in triterpene production in C. asiatica chemotypes 45

8. Manipulation of centelloside production in cell and tissue culture 47

9. Conclusions 50

Chapter 3- Centelloids of C. asiatica

- 29 -

1. Abstract

Centella asiatica accumulates large quantities of pentacyclic triterpenoid saponins,

collectively known as centelloids. These terpenoids include asiaticoside, centelloside,

madecassoside, brahmoside, brahminoside, thankuniside, sceffoleoside, centellose, asiatic-,

brahmic-, centellic- and madecassic acids. The triterpene saponins are common secondary

plant metabolites and are synthesized via the isoprenoid pathway to produce a hydrophobic

triterpenoid structure (aglycone) containing a hydrophilic sugar chain (glycone). The biological

activity of saponins has been attributed to these characteristics. In planta, the Centella

triterpenoids can be regarded as phytoanticipins due to their antimicrobial activities and

protective role against attempted pathogen infections. Preparations of C. asiatica are used in

traditional and alternative medicine due to the wide spectrum of pharmacological activities

associated with these secondary metabolites. Here, the biosynthesis of the centelloid

triterpenoids is reviewed; the range of metabolites found in C. asiatica, together with their

known biological activities and the chemotype variation in the production of these metabolites

due to growth conditions are summarized. These plant-derived pharmacologically active

compounds have complex structures, making chemical synthesis an economically

uncompetitive option. Production of secondary metabolites by cultured cells provides a

particularly important benefit to manipulate and improve the production of desired

compounds; thus biotechnological approaches to increase the concentrations of the

metabolites are discussed.

2. Introduction

Centella comprises some 50 species, inhabiting tropical and sub-tropical regions. This genus

belongs to the plant family Apiaceae (Umbelliferae) and includes the most ubiquitous species

Centella asiatica. This perennial creeper flourishes abundantly in moist areas and is a small,

herbaceous annual plant of the subfamily Mackinlaya (Liu et al., 2003), previously included in

Hydrocotyle (Brinkhaus et al., 2000), occurring in swampy areas of India, Sri Lanka,

Madagascar, Africa, Australia (Schaneberg et al., 2003), China, Indonesia, Malaysia,

Australia and Southern and Central Africa (Verma et al., 1999). The plant is clonally

propagated by producing stolons that are characterized by long nodes and internodes which

http://en.wikipedia.org/wiki/Herbaceous

http://en.wikipedia.org/wiki/Annual_plant


- 30 -

bear crowded cordate, obicular or reniform leaves and sessile flowers in simple umbels

(Zheng and Qin, 1999). Depending on environmental conditions, the form and shape of the C.

asiatica plant can differ greatly (Adamson, 1950). C. asiatica, also known as Gotu kola or

Indian pennywort (Bruneton, 1995), is a medicinal plant that has probably been used since

prehistoric times and has been reported to have been used for various medicinal and

cosmetic purposes, thus becoming an important commercial product. This plant is listed as a

drug in the Indian Herbal Pharmacopoeia, the German Homeopathic Pharmacopoeia (GHP),

the European Pharmacopoeia, and the Pharmacopoeia of the People’s Republic of China

(Schaneberg et al., 2003). According to World Health Organisation (WHO) monographs,

Herbae Centellae should not contain less than 2% of the triterpene ester glycosides

asiaticoside and madecassoside (WHO, 1999).

3. Terpenoids as natural products and secondary metabolites

Secondary metabolites are natural products that often have an ecological role in regulating

the interactions between plants and their environment. They can be defensive substances,

such as phytoalexins and phytoanticipins, anti-feedants, attractants and pheromones

(Hanson, 2003). The importance of plant triterpenes or steroids in medicine, agriculture and

industry has led to numerous studies on the synthesis, biosynthesis and biological activity of

these substances. It has been estimated that over 40% of medicines have their origins in

these active natural products (Gershenzon and Kreis, 1999). A prominent group of natural

products are the terpenes and derivitized terpenoids.

4. Chemical diversity of terpenoids

Several thousand terpenes and terpenoids occur in many genera of higher plants and

organisms (Devon and Scott, 1972; Darnley-Gibbs, 1974) and although often the structures

of the various classes seem unrelated, detailed biochemical studies have revealed their

biosynthesis patterns (Bell and Charlwood, 1980). The terpenes are biosynthetically

constructed from isoprene (2-methylbutadiene) units (Ruzicka, 1953; Gershenzon and Kreis,

1999). The C5H8 isoprenes polymerise and subsequently fix the number and position of the


- 31 -

double bonds. The basic molecular formulae of terpenes are thus multiples of C5H8. Most

terpenes have cyclic structures and are classified by the number of C5 isoprene units that

they contain. Given the many ways the basic C5 units can be combined, it is not surprising to

observe the amount and diversity of the structures (Gershenzon and Dudareva, 2007). The

classes are: hemiterpenes consisting of a single C5 isoprene unit, monoterpenes (C10),

sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), carotenoids

(C40) and polyterpenes consisting of long chains of many isoprene units.

The triterpene group of compounds include sterols and triterpenes, which can accumulate as

glycosides (saponins) in extensive amounts in plants (Sparg et al., 2004). Saponins are

glycosylated (aglycone = sapogenin) secondary metabolites found in a variety of plant

species (Papadopoulou et al., 1999). Their surface-active properties are what distinguish

these compounds from other glycosides (Sparg et al., 2004). Due to the fact that some of

these saponins are the starting points for the semi-synthesis of steroidal drugs, these

metabolites are highly sought after by the pharmaceutical industry (Liu and Henkel, 2002).

Saponins are classified according to their aglycone skeleton. The first group consists of non-

steroidal saponins, which are the most common and occur mainly in the dicotyledonous

angiosperms. The second group consists of the steroidal saponins which are derived from the

tetracyclic triterpenoids and isoprene units and are almost exclusively present in

monocotyledonous angiosperms. Some claim a third class called steroidal amines, which are

also referred to as steroidal alkaloids (Bruneton, 1995). Steroidal saponins consist of a

steroidal aglycone, a C27 spirostane skeleton which generally consists of a six-ring structure

(Figure 3.1A). The hydroxyl-group in the 26-position may be engaged in a glycosidic linkage

so that the aglycone structure remains pentacyclic (Figure 3.1B). This is referred to as a

furostane skeleton. Triterpenoid saponins consist of a triterpene aglycone, which consists of a

C30 skeleton, compromising a pentacyclic structure (Figure 3.1C).


- 32 -

Figure 3.1: Aglycone skeletons of pentacyclic (A) steroidal spirostane, (B) steroidal furostane and (C) triterpenoid saponins. The R-group is a sugar moity (Sparg et al., 2004). Tetracyclic terpenes such as lanosterol, sitosterol and cycloartenol can also be derived from oxidosqualene through a different pathway utilizing cycloartenol synthase (CAS1) (Phillips et al., 2006).

5. The biosynthesis of Centella triterpenes and triterpenoids

Terpene biosynthesis can be divided into four stages (as generally described in Chapter 2).

Firstly, there is the formation of the isoprene unit isopentenyl diphosphate. There are two

known major pathways for the biosynthesis of the isoprene unit, one based on mevalonic acid

and the other one on 1-deoxyxylulose (Gershenzon and Kreis, 1999). Secondly, there is the

association of these units to form the (C5)n isoprenoid backbone of the terpene families;

thirdly there is the cyclization of these to generate the carbon skeletons. Finally, there are the

interrelationships, hydroxylations and oxidations that lead to the individual terpenoids. The

general biosynthesis of terpenes leading to sterols has been reviewed extensively by

Benveniste and others (Gershenzon and Kreis, 1999; Collins, 2001; Haralampidis et al.,

2002; Benveniste, 2004; Jenner et al., 2005; Kalinowska et al., 2005). Triterpenes consist of

six isoprene units and have the molecular formula C30H48. The linear triterpene squalene is

derived from the reductive coupling of two molecules of farnesyl pyrophosphate by squalene

synthase (SQS). Squalene is then oxidized biosynthetically by squalene epoxidase (SQE) to

generate 2,3-oxidosqualene. Oxidosqualene cyclases (OSCs) cyclize 2,3-oxidosqualene

through cationic intermediates to triterpene alcohols or aldehydes including - and -amyrin

and lupeol (Figure 3.2, Jenner et al., 2005 and Phillips et al., 2006).

http://en.wikipedia.org/wiki/Squalene


- 33 -

Figure 3.2: A simplified scheme of triterpenoid biosynthesis in C. asiatica. Farnesyl diphosphate synthase (FPS) isomerizes isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to farnesyl diphosphate (FPP), which squalene synthase (SQS) converts to squalene. Squalene epoxidase (SQE) oxidises squalene to 2,3-oxidosqualene. Oxidosqualene cyclase (OSC) enzymes cyclize 2,3-oxidosqualene through cationic intermediates (e.g. dammarenyl cation) to one or more cyclic triterpene skeletons. Other enzymes involved include α / -amyrin synthases (α / -AS) which can also form the lupenyl cation but further ring expansion and rearrangements are required before the deprotonation to α / -amyrin, the precursors of the sapogenins, to generate the products listed in Table 3.1. (Adapted from Haralampidis et al., 2002; Phillips et al., 2006). These conversions are catalysed by OSCs (Figure 2.7. in Chapter 2), known collectively as

triterpene synthases (Haralampidis et al., 2002). Plants biosynthesize diverse triterpenoids

and their genomes encode multiple OSC enzymes to form these skeletons. The level at which

the structural diversity of triterpenes is generated depends on the cyclization of 2,3-


- 34 -

oxidosqualene by different OSCs such as cycloartenol synthase (CAS), lupeol synthase (LS)

and α / -amyrin synthase (AS) (Mangas et al., 2006). A phylogenetic tree analysis shows

that OSCs have the same enzyme function from respective branches in the tree even though

they were derived from different plants species. All triterpene synthases appear to have

diverged from an ancestral CAS gene (Zhang et al., 2003), but an independent origin for -AS

in eudicots and monocots has been proposed (Phillips et al., 2006). The triterpenoid cyclases

are distinct from LS and CAS, and form discrete subgroups within the OSC superfamily

(Haralampidis et al., 2002).

Cyclization of 2,3-oxidosqualene through a protosteryl cation intermediate generates

lanosterol and cycloartenol, the structural precursors to all the steroids, while cyclization

through a dammarenyl, baccharenyl and lupenyl cation intermediates generates lupeol and α/

-amyrin (Jenner et al., 2005), the precursors of the Centella pentacyclic triterpenoid

saponins. The α / AS enzymes cyclize oxidosqualene via the dammarenyl cation and allow

further ring expansion and some rearrangement before deprotonation to α-amyrin and -

amyrin respectively (Haralampidis et al., 2002). α-Amyrin contains the ursane (C19, C20

dimethyl) and -amyrin the oleanane (C20 dimethyl) substitution patterns respectively.

Following cyclization, further diversity is conferred by modification of the products by

oxidation, hydroxylation, glycosylation and other substitutions mediated by cytochrome P450-

dependent monooxygenases, glycosyl transferases and other enzymes. Little is known about

the enzymes required for these chemical elaborations. One common feature shared by all

saponins is the presence of a sugar chain attached to the aglycone. Glycosylation is

particularly important as the sugar chain is critical for the biological activity of many saponins

(Morrissey and Osbourn, 1999; Achnine et al., 2005). The oligosaccharide chains are likely to

be synthesized by sequential addition of single sugar residues to the aglycone, but little is

known about triterpenoid glycosylation (Haralampidis et al., 2002).

In the case of C. asiatica, the biochemical pathways involved in the synthesis of terpenes are

active, as can be seen from the presence of monoterpenes and sesquiterpenes (Oyedeji and

Afolayan, 2005), and the well described pentacyclic triterpenes (Brinkhaus et al., 2000; Kim et

al., 2002a; Aziz et al., 2007; James et al., 2008). The level at which the structural diversity of

triterpenes is generated depends on the cyclization of 2,3-oxidosqualene by different OSCs

such as cycloartenol synthase, lupeol synthase and -amyrin synthase (Mangas et al.,

http://en.wikipedia.org/wiki/Cycloartenol

http://en.wikipedia.org/wiki/Steroid


- 35 -

2006). Further diversity is conferred by modification of the cyclization products by

hydroxylation, glycosylation and other substitutions. Glycosylation is particularly important as

the C-3 chain is critical for the biological activity of many saponins (Morrissey and Osbourn,

1999).

An AS, putatively involved in the synthesis of asiaticoside, has recently been described

(Ca AS, GenBank accession AAS01523; Kim et al., 2005).

The basic structures of the Centella pentacyclic triterpenoid metabolites are represented in

Figure 3.3. These can be divided according to the methyl substitution pattern on the C19 and

C20 into the oleanane and ursane subtypes (Matsuda et al., 2001a). The most prominent of

the Centella saponins are madecassoside and asiaticoside and their sapogenins

(madecassic and asiatic acid). Other pentacyclic triterpenic acids and their respective

glycosides which occur in C. asiatica, and reported in the older literature, include names like

bramic-, madasiatic- (Matsuda et al., 2001b), centic-, centoic-, centellinic-, centellic- (Labadie

and De Silva, 2001), isodencentic acid, brahmoside, brahmioside (Rastogi and Dhar, 1963),

thankuniside, isothankuniside (Dutta and Basu, 1962), and centelloside, amongst others.

Structures of the pentacyclic triterpenes reported are compatible with the model scheme

(Figure 3.3), with the exception of isothankunic -, centic - and centoic acids, that have an

additional hydroxyl group attached to C5 (Bhattacharyya, 1956). A review of the literature has

revealed duplicate names, synonyms and contradictory findings for the triterpenoid

compounds of C. asiatica. Lack of structural data also hinders the assignment of names to

structures. Isobrahmic acid, for example, was reported as identical to madecassic acid (Dutta

and Basu, 1962), with the latter being an isomer of terminolic acid (Jian et al., 2007). Another

report states that brahmic acid has been demonstrated to be identical to madecassic acid,

while isobrahmic acid has been reported to be a mixture of asiatic - and madecassic acids

(Rao and Seshadri, 1970). The compounds brahmoside and brahminoside are recognised as

sugar esters, similar to asiaticoside and madecassoside (Rao and Seshadri, 1970) but also

containing arabinose. According to Dutta and Basu (1968), thankunic and isothankunic acid

are isomers of madecassic acid and the sugar-containing derivatives would be the glycosides

thankuniside and isothankuniside respectively. C. asiatica plants from Sri Lanka were

reported to contain centic, centoic, centellinic (the agylcone of centelloside) acids as well as

indocentoic acid (the aglycone of indocentelloside) (Hegnauer, 1966; Castellani et al., 1981),

but whether these names represent unique structures could not be ascertained.


- 36 -

Table 3.1 summarizes a list of the ursane and oleanane centelloids where the compound

name could be verified with a reported structure. Minor components such as the lupaene

pentacyclic triterpene betulinic acid (3ß-hydroxy-20(29)-lupaene-28-oic acid), though

structurally similar to the centelloids, are not included.

Figure 3.3: The model triterpenoid compound from C. asiatica. These triterpenes can occur in the ursane (R6, R7 = methyl) or oleanane (R7, R8 = methyl) types with double bonds occurring at C12-C13, C13-C18 or C20-C21.

Table 3.1: Structures of the pentacyclic triterpenes reported to occur in C. asiatica to date.

R1 R2 R3 R4 R5 R6 R7 R8 R9

C=C Name Molecular

Formula

MW Ref

Ursane (C19,C20 – dimethyl) -OH -OH -CH3 -CH2OH -H -CH3 -CH3 -OH -COOH - 2α,3ß,20,23-tetrahydroxy-urs-28-oic acid C30H52O6 506 Bhattacharyya, 1956

-OH -OH -CH3 -CH2OH -H -CH3 -CH3 -H -COOH 20-21 2α,3ß,23-trihydroxy-urs-20-en-28-oic acid C30H50O5 488 Jian et al., 2007

-OH -OH -CH3 -CH2OH -H -CH3 -CH3 -H -COO-glc(1-6)glc(1-

4)rha

20-21 Scheffuroside B

2α,3ß,23-trihydroxy-urs-20-en-28-oic acid O-α-L-

rhamnopyranosyl- (1-4)-O- ß-D-glucopyranosyl-(1-6)-O- ß-

D-glucopyranosyl ester

C48H78O19 959 Jian et al., 2007

Rao and Seshadri,

1970

-OH -OH -CH3 -CH2OH -H -CH3 -CH3 -H -COOH 12-13 Asiatic acid

2α,3ß,23-trihydroxy-urs-12-en-28-oic acid

C30H48O 488 James et al., 2008;

Dutta and Basu, 1968

-OH -OH -CH3 -CH2OH -H -CH3 -CH3 -H -COOCH3 12-13 Methyl asiatate C31H50O5 502 Dutta and Basu, 1968

-OH -OH -CH3 -CH2OH -H -CH3 -CH3 -H -COO-glc(1-6)glc(1-

4)rha

12-13 Asiaticoside

2α,3ß,23-trihydroxy-urs-12-en-28-oic acid O-α-L-

rhamnopyranosyl- (1-4)-O- ß-D-glucopyranosyl-(1-6)-O- ß-

D-glucopyranosyl ester

C48H78O19 959 Kim et al., 2002a;

Aziz et al., 2007;

Hegnauer, 1966

-OH -OH -CH3 -CH2OAc -H -CH3 -CH3 -H -COO-glc(1-6)glc(1-

4)rha

12-13 Asiaticoside C C50H80O20 1001 Rao and Seshadri,

1970

-OH -OH -CH3 -CH3 -H -CH3 -CH3 -H -COO-glc(1-6)glc(1-

4)rha

12-13 Asiaticoside D C48H78O18 943 Rao and Seshadri,

1970

-OH -OH -CH3 -CH2OH -H -CH3 -CH3 -H -COO-glc(1-6)glc 12-13 Asiaticoside E C42H68O15 812 Rao and Seshadri,

1970

-H -OH -CH3 -CH2OH -H -CH3 -CH3 -H -COO-glc(1-6)glc(1-

4)rha

12-13 Asiaticoside F C48H78O18 943 Rao and Seshadri,

1970

-OH -OH -CH3 -CH2OH -OH -CH3 -CH3 -H -COOH 12-13 Brahmic acid, Madecassic acid

(6ß-hydroxy-asiatic acid)

C30H48O6 504 Kim et al., 2002a;

Aziz et al., 2007;

Dutta and Basu, 1968,

Castellani et al., 1981

-OH -OCH3 -CH3 -CH2OH -OH -CH3 -CH3 -H -COOCH3 12-13 Methyl brahmate C32H50O6 532 Dutta and Basu, 1968

-OH -OH -CH3 -CH2OH -OH -CH3 -CH3 -H -CH2OH 12-13 Brahmol C30H50O5 490 Dutta and Basu, 1968

-OH -OH -CH3 -CH2OH -OH -CH3 -CH3 -H -COO-glc(1-6)glc 12-13 Centellasaponin B C42H68O16 828 Kim et al., 2002a

-OH -OH -CH3 -CH2OH -OH -CH3 -CH3 -H -COO-glc(1-6)glc(1-

4)rha

12-13 Brahminoside

Madecassoside

C48H78O20 975 Kim et al., 2002a;

Aziz et al., 2007; Yu

et al., 2006

-OH -OH -CH3 -CH3 -OH -CH3 -CH3 -H -COO-glc(1-6)glc(1-

4)rha

12-13 Centellasaponin C C48H78O19 959 Kim et al., 2002a

-OH -O-L-Ara -CH3 -CH2OH -OH -CH3 -CH3 -H -COOH 12-13 Arabinoside

3-O-[α-L-ara]-2α,3ß,6ß,23-tetrahydroxy-urs-12-en-28-oic

acid

C35H56O11 652 Castellani et al., 1981

-H -OH -H -CH2OH -OH -CH3 -CH3 -H -COOH 12-13 Isothankunic acid

3α,5α,6ß,24-tetrahydroxy-urs-12-en-28-oic acid

C29H46O5 474 Labadie and De Silva,

2001

-H -OH -H -CH2OH -OH -CH3 -CH3 -H -COO-glc(1-6)glc(1-

4)rha

12-13 Isothankuniside C47H76O19 945 Matsuda et al., 2001a

-OH -OH -CH3 -CH3 -OH -CH3 -CH3 -H -COOH 12-13 Madasiatic acid C30H48O5 488 Kim et al., 2002a

-H -OH -CH3 -CH3 -H -OH -CH3 -CH3 -COOH 12-13 Pomolic acid C30H48O4 472 Yoshida et al., 2005

-H -OH -CH3 -CH3 -H -CH3 -CH3 -CH3 -COOH 12-13 Pomolic acid C31H50O3 470 Yoshida et al., 2005

-OH -OH -CH3 -CH3 -H -CH3 -CH3 -H -COOH 12-13 Corosolic acid C30H48O4 472 Yoshida et al., 2005

-H -OH -CH3 -CH3 -H -CH3 -CH3 -H -COOH 12-13 Ursolic acid C30H48O3 456 Yoshida et al., 2005

-OH -OH -CH3 -CH3 -H -H -CH3 -CH3 -COOH 12-13 Maslinic acid C30H48O4 472 Yoshida et al., 2005

-OH -OH -CH3 -CH2OH -H -CH3 -CH3 -H -COO-glu(1-6) 12-13 Quadranoside (IV) C36H58O10 650 Nhiem et al., 2011

-OH -OH -CH3 -CH3 -OH -H -CH3 -CH3 -COO-glc(1-6)glc(1-

4)rha

12-13 2α, 3ß,6ß,trihydroxyolean-12-en-28-oic acid 28-O-[α-L-

rhamnopyranosyl -(1-4)- ß-D-glucopyranosyl-(1-6)-ß-D-

glucopyranosyl] ester

C48H78O19 950 Nhiem et al., 2011

Oleanane (C20, C20-dimethyl) -OH -OH -CH3 -CH2OH -H -H -CH3 -CH3 -COOH 12-13 2α,3ß,23-trihydroxy-olean-12-en-28-oic acid C30H48O5 488 Aziz et al., 2007

-OH -OH -CH3 -CH2OH -OH -H -CH3 -CH3 -COOH 12-13 Terminolic acid C30H48O6 504 Schaneberg et al.,

2003

-OH -OH -CH3 -CH2OH -OH -H -CH3 -CH3 -COO-glc(1-6)glc(1-

4)rha

12-13 Asiaticoside B C49H80O20 989 Castellani et al., 1981;

Kim et al., 2002a;

Schaneberg et al.,

2003

-OH -OH -CH3 -CH2OH -H -H -CH3 -CH3 -COOH 13-18 Centellasapogenol A

2α,3ß,23-trihydroxy-olean-13-en-28-oic acid)

C30H48O5 488 Aziz et al., 2007;

-OH -OH -CH3 -CH2OH -H -H -CH3 -CH3 -COO-glc(1-6)glc(1-

4)rha

13-18 Centellasaponin A

Scheffoleoside A

C48H78O19 959 Kim et al., 2002a,

Aziz et al., 2007

-H -OH -CH3 -CH2OH -OH -H -CH3 -CH3 -COOH 12-13 3ß,6ß,23-trihydroxy-olean-12-en-28-oic acid C30H48O5 488 Kim et al., 2002a

-H -OH -CH3 -CH2OH -OH -H -CH3 -CH3 -COO-glc(1-6)glc(1-

4)rha

13-18 Centellasaponin D C48H78O19 959 Kim et al., 2002a

* New triterpenoids discovered since the publication of James and Dubery (2009) are indicated in red. ** Centelloids which are isomers of each other are highlighted with the same the colour.


- 40 -

The biochemical pathways and genetic machinery required for the elaboration of this

important family of plant secondary metabolites are still largely uncharacterized, despite the

considerable commercial interest in this important group of natural products. This is likely to

be due in part to the complexity of the molecules and the lack of pathway intermediates for

biochemical studies.

6. Biological activities of Centella triterpenoid saponins and

sapogenins

C. asiatica synthesizes triterpenoid saponins as secondary metabolites as part of normal

growth and development. Other chemical constituents that may contribute to the biological

activities of C. asiatica may involve essential oils from this plant. Analyses of these oils have

revealed monoterpenoids, oxygenated monoterpenoids, sesquiterpenoids, and oxygenated

sesquiterpenoids with -humulene, -caryophyllene, bicyclogermacrene, germacrene B / D,

myrcene, trans -farnesene and p-cymol as the predominant constituents (Yoshinori et al.,

1982; Oyedeji and Afolayan, 2005; Rajkumar and Jebanesan, 2007).

Interest in the centelloid molecules stems from their medicinal properties, antimicrobial

activity, and their likely role as determinants of plant disease resistance (Haralampidis et al.,

2002). Although classified as saponins, the saponin-like sugar esters of the triterpenoid acids

exhibit low hemolytic activity (Kartnig, 1988).

The active constituents are well known for their clinical effects in the treatment of chronic

venous diseases and wound healing disorders (Pointel et al., 1987; Montecchio et al., 1991).

Many commercial formulations available contain madecassoside and asiaticoside in different

ratios, depending on the source of the plant used to manufacture the final formulation (Table

3.2). Pharmacological studies performed have investigated the effects of undefined alcohol or

aqueous extracts of Centella, as well as defined extracts. The following extracts are reported

in the literature: TECA = titrated extract of C. asiatica, TTFCA = total triterpenoid fraction of C.

asiatica and TTF = total triterpenoid fraction. TECA and TTFCA are combinations comprising

asiatic acid (30%), madecassic acid (30%) and asiaticosides (40%) while TTF comprises

asiatic acid and madecassic acid (60%) in combination with asiaticosides (40%) (Brinkhaus et


- 41 -

al., 2000). Some commercial products used in West Germany and France include

Centasinum, Centelase, Emdecassol and Madecassol (Kartnig, 1988; Brinkhaus et al., 2000).

Table 3.2: Product range of extracts from C. asiatica indicating the specific chemical composition and treatment (WHO, 1999; Advanced Cosmeceutical Technology, 2006).

Extract Chemical composition

Applications

Asiatic acid >95% Asiatic acid Anti-ageing cosmetics, application after laser therapy, cosmeceutics

Titrated Extract of Centella Asiatica (TECA)

55-66% Genins 34-44% Asiaticoside

Anti-cellulite, slimming products, breast creams, stretch marks, scarred skin, anti-ageing cosmetics, moisturizing care

TECA cosmetics >40% Genins > 36% Asiaticoside

Anti-cellulite, slimming products, breast creams, stretch marks, scarred skin, anti-ageing cosmetics, moisturizing care

Heteroside >55% Madecassoside >14% Asiaticoside

Slow release effect, anti-ageing cosmetics, for moisturizing night-creams

Asiaticoside >95% Asiaticoside Anti-inflammatory, against irritated and reddened skin, anti-allergic

Genins >25% Asiatic acid >60% Madecassic acid

Natural antibiotic, antibacterial properties, for anti-acne products, intimate hygiene

In addition to the applications mentioned in Table 3.2, Centella extracts have been used for

many ailments which have led to successful treatments (Table 3.3). Although none of the

claims listed have been evaluated by the Food and Drug Administration (FDA), positive

investigations have been done by various institutes and universities, which concluded that

more research on the pharmacological and bio-medical activities of C. asiatica is called for.

No recommended daily allowance (RDA) or dosage has been determined, but fresh leaves

have been used in salads, or dried leaves used to make tea. Supplements are usually

available in varying strengths and levels of purity. Crude preparations can cause allergic

responses and nausea has been reported in cases of high levels of intake. A toxic dose of


- 42 -

asiaticoside by intramuscular application to mice and rabbits was reported as 40-50 mg per

kg body weight (Abou-Chaar, 1963). In oral applications, 1 g of asiaticoside per kg body

weight has not proven toxic, and nearly all chemical trials have shown good tolerance by

patients to extracts from C. asiatica or asiaticoside (Abou-Chaar, 1963; Boely, 1975). No

cases of intolerance were observed following injections of Madecassol preparations (Wolfram

von St., 1965) which is a C. asiatica extract comprising 40% asiaticoside, 29-30% asiatic acid

and 1% madasiatic acid (Brinkhaus et al., 2000).

Although great progress has been made over the past decade in the study of biologically

active components and the bioactivities of C. asiatica, the underlying mechanisms involving

the physiological effects are poorly understood (Zheng and Qin, 2007). Most triterpenoid

compounds in adaptogenic and medicinal plants are found as saponin glycosides. These

sugars can be cleaved off in the gut by bacterial enzymes, allowing the aglycone triterpenoids

to be absorbed. Uptake can be followed by insertion into cell membranes and modification of

the composition. Membrane fluidity can be influenced to potentially affect signalling by many

ligands and cofactors. In addition, the centelloids can potentially inhibit enzymes specifically

or non-specifically. Literature supplies numerous examples of enzymes that can be inhibited

by pentacyclic terpenoids, indicating the ability of these compounds to act broadly in a non-

specific mode on multiple targets. The mode of inhibition of enzymes seems to be non-

specific and based primarily on hydrophobic interaction with an enzyme's hydrophobic

domain (Glinski and Branly, 2002).

http://en.wikipedia.org/wiki/Aglycone

Table 3.3: Summary of the medicinal claims for C. asiatica. This table contains information on how Centella is used in alternative herbal treatments to treat various ailments and problems.

Medical claim Description of treatment Reference

Skin

ailm

ents

Wound healing

Treatment of skin

disorders (such as

eczema and psoriasis)

Revitalising

connective tissue

Burn and scar treatment

Cleaning up skin

infections

Leprosy

This tropical plant has been used in the Ayurvedic and traditional medicine in China,

Malaysia and Madagascar, not only for wound healing but general well being as well

as an anti-bacterial and anti-viral agent. Both the leaves and the entire plant can be

used therapeutically.

In traditional African medicine, it has been used for the treatment of leprosy. The

asiaticoside content dissolves the waxy coat of the leprosy bacteria, thus allowing the

bacteria to be destroyed by the immune system.

Centella extracts are reported to be used topically in the adjunct treatment of surgical

wounds and minor burns.

Montecchio et al., 1991

Oyedeji and Afolayan, 2005

Bonfill et al., 2005

Mathur et al., 2000

Circulation Acts as a complementary treatment of ulcers of venous origin. Montecchio et al., 1991

Arthritis and rheumatism Extracts are taken orally to relieve symptoms of venous and lymphatic vessel insufficiency. Bruneton, 1995


Memory enhancement, vitality and

longitivity.

In India, for the past 3 000 years of Ayurvedic medicine, it has been used from wound

healing, a mild diuretic, increasing concentration and alertness, and well as for the treatment

of anxiety and stress.

Mathur et al., 2000


Cancer In alternative health, this herb has been used to treat tumours and cancerous

growths without suppressing the auto immune system or creating toxic wastes

within the body.

Cytotoxic and anti-tumour properties of the crude extract and in particularly purified

fractions.

Babu et al., 1995



Medical claim Description of treatment Reference

Res

pira

tory

ailm

ents

Bronchitis

Asthma

Mathur et al., 2000


A general health tonic, an aphrodisiac

and immune booster

Centella assists in destroying toxic accumulation in the brain as well as in the nerves,

while it helps to clear the body from heavy metals as well as drugs – including

recreational drugs.


Detoxifying the body Stimulates lipolysis and blood microcirculation and are thus used in the management of

local adiposity or cellulite.

Cristoni and Pierro (1998)



Slimming

Diuretic

Treatment of liver and kidneys It has been used for centuries in the treatment of liver and kidney problems and has

become a popular alternative treatment for people suffering from hepatitis and

alcoholic liver disease.

Managing diabetes.

Sarma et al., 1996


Oyedeji and Afolayan, 2005

Sedative,

Anti-stress, anti-anxiety and the treatment

of depression

Ethanol extracts of roots had significant anti-stress activity.

Activity against stress-induced gastric ulcer formation.

Anxiolytic and sedative effects of the hydroalcoholic extracts of the leaves.

Sarma et al., 1996

Lucia et al., 1997


Mathur et al., 2000

Antifungal properties Jatisanienr and Tragoolpua (1996)

Insect anti-feedant

Mosquito repellent Properties of isolated compounds of the extracts of rhizomes.

Volatiles of Centella.

Srivastava et al., 1997b Rajkumar and Jebanesan, 2007

Ant

ibac

teria

l

activ

ity

Periodontal disease

Syphillis

Hepatitis

Newell an Linda, 1996

Srivastava et al., 1997a


Mathur et al., 2000


- 45 -

7. Variation in triterpene production in C. asiatica chemotypes

Natural products are an unsurpassed source of bioactive compounds and constitute a

relevant economic resource for the pharmaceutical, cosmetic and food industry. Differences

between varieties in medicinal plants of the same species (chemotypes) are common and

variation in secondary metabolites has been observed with identical phenotypes and growth

conditions, depending on plant origin (Aziz et al., 2007). Not surprisingly, significant

differences in active constituents have therefore also been observed between samples of C.

asiatica originating from different countries (Das and Mallick, 1991). Moreover, the

biosynthesis of major secondary metabolites is often either tissue or organ specific (Aziz et

al., 2007). This also seems to be the case in C. asiatica where triterpenoid saponins,

especially asiaticoside, could not be detected in undifferentiated cells of a Korean chemotype

(Kim et al., 2002a). In contrast, detectable levels of the triterpenoids in cultured cells (callus

and cell suspensions) were reported in South African chemotypes (James et al., 2008).

Asiaticoside biosynthesis seems to be concentrated in the leaves (0.4-1.4% dry weight) and

the level of asiaticoside content is quite low in the roots of whole plants (Kim et al., 2002a;

2004; 2005; 2007).

In addition to the asiatic – and madecassic acids and their glycosides, other chemically

diverse centelloid compounds as summarized in Table 3.1, have been isolated from C.

asiatica and studied (Das and Mallick, 1991; Inamdar et al., 1996; Matsuda et al., 2001a;

Monti et al., 2005). The reported composition of saponin mixtures of different sources of C.

asiatica varies considerably (Table 3.4) as does the concentration of these compounds. The

occurrence of these related triterpene ester glycosides and triterpene acids show that there

are different varieties of C. asiatica, which can be summarized in Table 3.4 (Labadie and De

Silva, 2001).


- 46 -

Table 3.4: Various saponins occur in C. asiatica due to the location and diverse environmental conditions (Kartnig, 1988; Labadie and De Silva, 2001; James et al., 2008).

Location /

Source Glycosides

Saponin Sapogenin Sugar

Associated triterpene

acids Madagascar South Africa

Asiaticoside Madecassoside

Asiatic acid Madecassic

acid

Glucose and rhamnose

Ceylon Sri Lanka

Centelloside Centellic acid Glucose and rhammose

Centic acid Centoic acid

India

Asiaticoside and Madecassoside

Asiatic acid Madecassic

acid


Brahmic acid

Indocentoic acid

Asiaticoside Brahmoside Brahminoside

Asiatic acid Brahmic acid Brahmic acid

Glucose and rhamnose Glucose, rhamnose and arabinose

Isobrahmic acid Betulinic acid

Thankuniside Thankunic acid Glucose and rhamnose

Asiatic acid

Isothankuniside Isotankunic acid


Asiatic acid

Gupta et al. (1999) reported variable asiaticoside content in five lines of C. asiatica from

India. Similarly, Rouillard-Guellec et al. (1997) investigated the secondary metabolites in India

and Madagascar, and reported that plants from the latter contained the highest level of

asiaticoside. The distribution of asiaticoside and madecassoside throughout the plant was

organ specific with leaves of both lines containing the higher content of these compounds. In

a study of C. asiatica from Madagascar, asiaticoside content of between 2.6 and 6.42% dry

weight was reported (Randriamampionona et al., 2007). The authors achieved in vitro

propagation of C. asiatica in a hormone free medium but these in vitro plants displayed lower

asiaticoside content. Aziz et al. (2007) reported two phenotypes of C. asiatica exhibiting

differences in terpenoid content that were tissue specific and varied between glasshouse

grown and tissue derived material. Triterpenoid saponin content was highest in leaves


- 47 -

(asiaticoside and madecassoside concentrations of 0.7-0.9 and 1.1-1.6% dry weight were,

respectively, reported), and roots contained the lowest content of asiaticoside. In their study,

asiaticoside and madecassoside were undetectable in transformed roots and undifferentiated

callus. Two morphologically distinct phenotypes of C. asiatica in South Africa were analysed

in relation to the levels of triterpenoid saponins (madecassoside and asiaticoside) and their

sapogenins (madecassic and asiatic acid), produced in undifferentiated cultured cells and

leaves (James et al., 2008). In both cases the triterpenoids present in undifferentiated cells

(callus and cell suspensions) were lower compared to the levels in leaf tissues. The total

content of the triterpenoids were generally comparable to that reported from India, Korea and

Madagascar, but differences in the ratios of free acids to glycosides were observed. The

reasons for this variability in the ratio between glycoside and aglycone can be due to climate,

seasonal and geographical conditions, harvesting times and storage conditions (Pannizi et

al., 1993).

Furthermore, the differences in the composition and type of triterpenoid molecules

synthesized (Table 3.1) by various C. asiatica chemotypes can perhaps be attributed to

genomic diversity and variation in the OSC and other genes involved in their biosynthesis

(Das and Mallick, 1991; Haralampidis et al., 2002; Kim et al., 2007), as well as the presence

and activity of enzymes involved in the attachment of the sugar residues to the aglycones.

Metabolic pathways for these triterpenoids should therefore be further investigated and the

flux through these pathways elucidated to obtain a better understanding of the biochemical

conversions that will allow the manipulation and exploitation of secondary product synthesis

in C. asiatica.

8. Manipulation of centelloside production in cell and tissue culture

As in the case of most plant-derived pharmacologically active compounds, pentacyclic

triterpenoids have complex structures, making chemical synthesis an economically

uncompetitive option. Plant cell culture has been used in attempts to increase the production

of bio-active secondary metabolites of pharmaceutical interest (Giri and Naraseu, 2000;

Gaines, 2004). A particular important benefit is the potential ability to manipulate and improve


- 48 -

the production of desired compounds within the plant cell through experimentation with cell

culture.

However, the relationship between cell differentiation and tissue organisation and the

biosynthesis of secondary compounds is somewhat obscure. Secondary metabolite

production may require interaction between roots and leaves with metabolic precursors

generated in roots and passing to aerial parts of plants for bioconversion in leaves (Giri and

Naraseu, 2000). The biosynthesis of major secondary metabolites is often either tissue or

organ specific (Aziz et al., 2007), as found also in the case of C. asiatica triterpenoid

saponins (Kim et al., 2002b). Plant secondary metabolites are normally synthesised by

specialised cells, often at distinct stages of plant development and certain compounds are not

synthesised, or synthesized at a low level, if cells remain undifferentiated as in cell

suspensions (Kim et al., 2002b). The distribution of mRNA transcripts, enzymes and

biosynthetic precursors within and between cells is an important component of regulation for

secondary plant metabolic processes. In addition, many metabolic pathways are

compartmentalised, enabling the separation of incompatible or competing reactions, and

concentrating enzymes and metabolites (Samanani and Facchini, 2006).

One approach used to regulate metabolic pathways favouring the production of specific

secondary metabolites has been to add precursors to the culture medium (Bouhouche et al.,

1998), though it is not known if this option has been investigated for enhanced production of

triterpenoids in C. asiatica cells. The instability of cell cultures for the continued production of

secondary products poses another problem; some cell lines lose the ability to synthesize the

desired compound after prolonged culture.

In plant tissue cultures, stress induced by biotic and abiotic elicitors has been used to

enhance production of biologically active secondary metabolites. It has been reported that

fungal elicitation can lead to an overproduction of pentacyclic triterpenes in

Tabernaemontana species instead of some other expected metabolites (Van de Heijden et

al., 1988). Another approach is to use plant-specific signal molecules such as methyl

jasmonate (MeJa) to up-regulate key enzyme levels. It is known that exogenously applied

MeJa can induce the biosynthesis of many secondary metabolites, including triterpenoid

saponins (Hayashi et al., 2003). The enzymes SQS and OCS were reported to be


- 49 -

upregulated by MeJa treatment in cultured Glycyrrhiza glabra cells (Hayashi et al., 2003).

This upregulation was accompanied by enhanced concentrations of triterpenoid saponins.

OCSs catalyze regulating steps in the isoprenoid pathway (Figure 3.2) and is responsible for

the cyclization of 2,3-oxidosqualene, the common intermediate of both triterpene and

phytosterol biosynthesis (Haralampidis et al., 2002). A significant increase in the asiaticoside

levels of MeJa treated plantlets that were accompanied by a decrease in the content of free

sterols were reported (Mangas et al., 2006). Previously, Kim et al. (2005) reported an

activation of ß-AS (an OSC) and a corresponding inhibition of expression of CS, responsible

for the first step in sterol biosynthesis, in C. asiatica roots treated with MeJa. Thus, the

inhibition of the branch point enzyme CS seems to result in increased flux through the

triterpenoid pathway.

Biotechnological attempts to overproduce the quantities of asiaticoside through cell or tissue

culture derived from a Korean chemotype have thus far encountered limited success (Kim et

al., 2002a; 2004; 2007). Future studies to manipulate asiaticoside production, should be

broadened to include all the triterpenoids obtainable from a specific chemotype in cultured

plants and cells.

The biosynthesis of the Centella triterpenoids can also be engineered by means of

recombinant DNA technology along different steps of the pathways, once a particular rate-

determining factor in a pathway has been identified. One approach to enhance terpenoid

synthesis is to increase the flux of IPP and DMAPP by over expression of their respective

genes (Roberts, 2007). This potentially allows for the increased synthesis of all the

triterpenes, but also for the manipulation of specific centellosides. Also, specific terpene

synthases and OCSs may be modified or over expressed to either regulate or enhance

particular terpenoids (Degenhardt et al., 2003). In this regard, C. asiatica calli were cultivated

in different media and the expression levels of the genes belonging to the biosynthetic

pathway determined using RT-PCR (Mayano et al., 2007).


- 50 -

9. Conclusions

Due to its medicinal properties, interest in C. asiatica has increased over the years and there

have been studies on the enhanced production of these centellosides as well as cloning of

genes in their biosynthetic pathway. The production of these compounds and expression in

differentiated (leaves and roots) and non-differentiated (calli) cells have been investigated.

Metabolic pathways for these triterpenoids should be elucidated to obtain a better

understanding of the biochemical conversions that will allow the manipulation and exploitation

of secondary product synthesis in C. asiatica. There is a need for additional studies to be

done to evaluate the genetic resources of the plant for variation in growth, morphology, and

yield related characteristics which can, in turn, be utilized to identify high yielding populations

suitable for agronomical and plant breeding programs.

Identification and Quantification of

Triterpenoid Centelloids in Centella asiatica

(L.) Urban by Densitometric TLC

Chapter 4

Chapter 4- Densitometry

- 52 -

Published as: Journal of Planar Chromatography (2011), 24(1), 82–87

1. Abstract 53

2. Introduction 53

3. Experimental 55

3.1. Preparation of triterpenoid standards 55


3.3 Sample preparation 56

3.4. Chromatography 56

3.5. Visualisation of the spots for densitometry 56

3.6. Densitometry analysis of triterpenoid spots 57

3.7. Method validation 57

3.7.1. System suitability 57

3.7.2. Statistical analysis 57

3.7.3. Linear working range for known Centella triterpenes 58

3.7.4. Limits of detection and quantification 58

3.7.5. Specificity 58


5. Conclusion 66


- 53 -

1. Abstract

A simple and rapid method for the determination of four major triterpenoids from Centella

asiatica preparations is described. Thin layer chromatography is widely used for saponin

analysis and here it was utilised for identification and quantification of pure compounds and

for compounds in complex mixtures when densitometric analysis is used subsequent to TLC.

Ethanolic leaf preparations from C. asiatica, along with authentic standards of asiatic acid,

asiaticoside, madecassic acid, and madecassoside, were separated by normal phase TLC

with chloroform, methanol, acetic acid and water 60:32:12:8 (v/v/v/v) as the mobile phase.

This solvent mixture was found to be successful in separating the triterpenoid acids and their

respective glycosides from other components in crude extracts. The separated compounds

were detected with anisaldehyde-sulphuric acid solution. The resulting violet spots displayed

intensity proportional to the amount of saponin or sapogenin present. The standard curves

had correlation coefficients between 0.9904 and 0.9982 and were linear over the range of

1.25 - 10 nmoles of applied standard, corresponding to approximately 0.6 - 5 g for the acids

and 1.2 - 10 g for the glycosides. The applicability of this method was to use TLC to

determine the specific saponins present of C. asiatica leaves and the results found were

consistent with previous reports from the literature.

2. Introduction

Centella comprises some 50 species belonging to the plant family Apiaceae which includes

the medicinally important C. asiatica Urban (James and Dubery, 1999). This herbaceous

plant is an umbellifer which has many common names including Gotu Kota and Indian

Pennywort (Bruneton et al., 1995; Matsuda et al., 2001). Centella terpenoids, known as

centelloids, include asiaticoside, centelloside, madecassoside, brahmoside, brahminoside,

thankuniside (Dutta and Basu, 1962), sceffoleoside, centellose, asiatic, brahmic, centellic

(Bhattacharyya, 1956; Labadie and De Silva, 2001) and madecassic acids (Aziz et al., 2007).

Depending on the origin of the Centella plant material, these saponins can account for

between 1 and 8% of the constituents (Schaneberg et al., 2003).


- 54 -

Different therapeutic uses for the plant are claimed, which are attributed to active pentacyclic

triterpenoids, namely asiatic acid and madecassic acid, and their glycosides asiaticoside and

madecassoside (Figure 4.1). This plant is listed as a drug in the Indian Herbal

Pharmacopoeia, the German Homeopathetic Pharmacopoeia (GHP), the European

Pharmacopoeia and the Pharmacopoeia of the People’s Republic of China (Brinkhaus et al.,

2000). The most abundant triterpenoid saponin, asiaticoside, has antibacterial, fungicidal and

cell proliferative activities which have been shown to aid in the treatment of wounds (Shukla

et al., 1999), ulcers, various skin diseases, vein insufficiency, tuberculosis and in the

treatment of mental disorders (Mathur et al., 2001). Recent studies have led to the isolation of

other centellosides with healing abilities, namely terminolic acid, madecassoside and

asiaticoside-B (Schaneberg et al., 2003). The biological activities of the centelloids were

recently reviewed by James and Dubery (2009).

Figure 4.1: Structures of the main identified active components in C. asiatica which are claimed to have medicinal properties. (A) madecassic acid (MW 504.17), (B) asiatic acid (MW 488.70), (C) madecassoside (MW 975.1) and (D) asiaticoside (MW 959.12).

TLC has been used for many years as the primary screening method for plant-based drugs

and preparations (Wagner and Bladt, 2001). Methods used and results acquired for

compounds being identified as promising would need to be reproducible and reliable. In

addition, a large number of samples would be required for analysis, which is allowed for by

TLC. For this, TLC still has the advantages of achieving high reproducibility at lower costs.

Various TLC methods for the identification of the Centella saponins in plant extracts and

pharmaceutical powders have been described (European Pharmacopoeia, 2005; Bonfill et al.,

2006).


- 55 -

The combination of TLC and densitometry, known as quantitative thin-layer chromatography

(QTLC) to determine soy saponins has been reported (Curl et al., 1985; Gurfinkel and Rao,

2002), and this procedure is adapted and evaluated in this communication for the

quantification of the major saponins and sapogenins of C. asiatica. The two saponins,

asiaticoside and madecassoside and their aglycones, asiatic acid and madecassic acid, for

which commercially available standards are available, were used to demonstrate this

application. Here we describe a simple and relatively inexpensive method for quantification of

the four principal centellosides and show the applicability of the method for analysis of

extracts obtained from leaves.

3. Experimental

3.1. Preparation of triterpenoid standards

Authentic, purified triterpenoids (asiatic acid, madecassic acid and their glycosides

asiaticoside and madecassoside were purchased (Extrasynthase, France; purity was

assessed by HPLC as >99%). Each standard was reconstituted in absolute ethanol to a

concentration of 1 mM. Standards were diluted to cover a range of 0.04 – 0.8 mM and applied

in volumes of 25 µl, using an automatic micro-pipette, in rows to pre-coated silica gel 60

plates (Merck, Darmstadt, Germany).

3.2. Plant material

Commercially cultivated Centella asiatica was obtained from a local nursery and identified by

Dr. N. A. Moteetee, University of Johannesburg. A voucher specimen (J. James 1-JRAU)

was deposited in the herbarium of the Botany and Plant Biotechnology department, University

of Johannesburg, South Africa.


- 56 -

3.3. Sample preparation

Leaves of C. asiatica were extracted in absolute ethanol as previously reported (James et al.,

2008). The leaves (fresh material) were weighed, cut into strips and placed in absolute

ethanol (1:12 (w/v)) to extract secondary metabolites. This was placed on a magnetic stirrer

for 24 h and then centrifuged at 2200 xg for 20 min. The supernatant was decanted and

concentrated 10 fold with a rotary evaporator at 45 °C under vacuum. The extracts were then

used for analysis. Extraction efficiency was studied by adding known concentrations of pure

standards to plant extracts to be studied. The extractions efficiency (n=6) for madecassoside

was 98.3% (CV of 0.35%), asiaticoside 98.7% (CV of 0.45%), madecassic acid 96.4% (CV of

0.67%) and asiatic acid 95.8% (CV of 0.52%).

3.4. Chromatography

Various dilutions of crude preparations of C. asiatica leaf extracts were applied as described

above to silica gel 60G 20 x 20 cm plates, thickness, 250 µm, with a pre-concentration zone

which allow for band formation (Merck, Darmstadt, Germany). Plates were developed in a

chloroform, glacial acetic acid, methanol and dH2O (60:32:12:8 (v/v/v/v)) mobile phase and

allowed to run until the flow front was 1 cm from the edge of the plate.

3.5. Visualisation of the spots for densitometry

Detection of triterpenoids was achieved by spraying with anisaldehyde-sulphuric acid (AS)

reagent (Kraemer et al., 2002) which resulted in the development of violet spots with a

density proportional to the total saponin present. The mobile phase was allowed to evaporate

by placing the plates in an oven for 10 mins at 100 C. The AS reagent was prepared with 0.5

ml anisaldehyde (Sigma-Aldrich, St. Louis, USA) mixed with 10 ml glacial acetic acid,

followed by 85 ml methanol and 5 ml concentrated sulphuric acid, in that order. The TLC

plates were sprayed with 10 ml of the spray reagent and then heated at 95 ºC for 10 min or

until the coloured bands appeared. Triterpenoids corresponding to the Rf values of the


- 57 -

authentic standards were identified in the leaf extracts subsequent to generating standard

curves for the four known saponins.

3.6. Densitometry analysis of triterpenoid spots

Purple zones obtained on the TLC plate were analysed using Quantity-One software (Bio-

Rad). All plates were set up in monochromatic mode and then converted to grey scale for

band intensity analysis. The intensity per mm2 for each spot was plotted against the amount

of triterpenoid present to produce a standard curve from which the saponin content of the

unknown samples can be calculated.

3.7. Method validation

3.7.1. System suitability

TLC is regarded as a suitable analytical technique for the triterpenoids from Centella

preparations (Wagner and Bladt, 2001; European Pharmacopoeia, 2005). A system

suitability test was conducted to determine whether the combination with densitometric

detection gave accurate results. Standard solutions of 0.4 – 0.8 mM of asiatic acid,

madecassic acid, asiaticoside and madecassoside were applied nine times to TLC plates and

analysed under optimized conditions for the calculation of standard deviation (SD) and

coefficient of variation (CV) for each standard.

3.7.2. Statistical analysis

The values obtained for band intensities were statistically analysed using Microsoft™ Excel.

Results are expressed with SD and CV.


- 58 -

3.7.3. Linear working range for known Centella triterpenes

For QTLC, sample volumes of 25 µl were used; final concentrations of 0.1 to 0.8 mM (1 - 20

nmoles / spot) were prepared and applied.

3.7.4. Limits of detection and quantification

The limit of detection (LOD) and quantification (LOQ) were determined at signal to noise

ratios of 3:1 and 10:1 respectively (Hiai et al., 1976; Xing et al., 2009).

3.7.5. Specificity

The four authentic compounds; asiatic acid, madecassic acid and their glycosides

asiaticoside and madecassoside, were used as external standards for identification (Rf and

colour reaction) purposes.

4. Results and Discussion

Well described protocols exist for the analysis of extracts and preparations of C. asiatica by

TLC (Wagner and Bladt, 2001). These protocols focus on the major metabolites, as TLC is

not able to resolve isomers which might occur as trace compounds (Oleszek, 2002), due to

the small number of theoretical plates inherently associated with the technique. In this study,

a mobile phase consisting of chloroform, glacial acetic acid, methanol and dH2O (60:32:12:8

(v/v/v/v)) was used as it was found to resolve the compounds effectively as depicted in Figure

4.2. Depending on the complexity of the extracts, decreasing the pH of the mobile phase may

improve the band broadening of the glycosides, which might be due to ionisation of the

carboxyl groups not being completely suppressed, and lowering the polarity might improve

the separation of the acids close to the front.


- 59 -

Due to weak absorbance properties of the pentacyclic triterpenoid saponins, colorimetric

determination methods are used for the evaluation of Centella extracts and preparations

(Wagner and Bladt, 2001). There are many color reactions for steroidal saponins but few for

triterpenoid sapogenins and saponins in general, where vanillin and anisaldehyde based

spray reagents can be used (Nix and Wilson, 1990). The purple color formed by the

anisaldehyde spray exhibited a slow decline in spot intensity over time; thus after

development, the same amount of time should pass when processing in order to obtain a

lower level of variance between results. The calculated concentrations of the results

corresponded with the observations of the TLC plates. When equal molar concentrations of

the four commercial standards are compared to each other by densitometry (Figure 4.2), it

can be seen that there is a variation in colour intensity and thus a density difference, hence

showing that these four triterpenes react differently with the AS reagent, depending on their

molecular configuration. The detection reagent is sensitive to most functional groups,

especially those which are strongly and weakly nucleophilic such as phenols and sugars. It

tends to be insensitive to alkenes, alkynes and aromatic compounds unless other functional

groups are present in the molecules being analysed. Vanillin spray is an alternative to the

detection of these saponins, and although the colour of the compounds relative to the

background improves over time (results not shown), the anisaldehyde detection method was

more discernable even though less stable. Since detection of saponins can be sensitive to

structural variations of individual saponins, they should ideally be standardised to saponin

mixtures isolated from the plant species in which the concentration is to be measured

(Oleszek, 2002).


- 60 -

Figure 4.2: Separation of (A): ethanolic leaf extracts containing the sapogenins and saponins of C. asiatica and (B): equimolar concentrations of the four commercially available standards on QTLC with chloroform, glacial acetic acid, methanol and dH2O (60:32:12:8 (v/v/v/v)) as mobile phase and AS reagent for detection. The densitometric trace is of the standards: madecassoside, Rf = 0.45; asiaticoside, Rf = 0.55; madecassic acid, Rf = 0.94 and asiatic acid, Rf = 0.97. The front was excluded from the densitometric analysis.

The standard curves obtained following densitometric analysis of the four authentic standards

are shown in Figure 4.3. The graphs show the analytical range, LOD and precision profiles.

The lines were linear from 0.08 - 0.4 mM with correlation coefficients (r) of 0.9972, 0.9982,

0.9904 and 0.9977 for asiatic acid, asiaticoside, madecassic acid and madecassoside

respectively. LOD is generally defined as the lowest analyte content which can be detected,

with a reasonable statistical certainty and can be identified according to the identification

criteria of the method if present (Hiai et al., 1976; Xing et al., 2009), this is usually determined

at a signal: noise (S/N) ratio of 3:1. The LODs were visually determined by the analysis of

samples with known concentrations of analyte and by establishing the minimum level at

which the analytes can be reliably detected, thus at 0.03 mM, corresponding to 0.3 and 0.72

g of the sapogenins and saponins respectively. The limit of quantification (LOQ) (generally a

S/N ratio of 10:1) was determined to be 0.5 g (1.25 nmole or 0.05 mM) for the sapogenins

and 1.2 g (1.25 nmoles or 0.05 mM) for the saponin standards. Ranges outside the linear

regions of the curve cannot be used to determine sample concentrations and dilutions of

samples need to be prepared. In addition, the accuracy of the method is compromised if the

linearity across the range is not taken into account.


- 61 -

Figure 4.3: Standard curves for densitometric analysis of each of the investigated triterpenoids from C. asiatica.

It is generally assumed that densitometric analyses provide an accurate determination if there

is a linear relationship between the increasing amounts of a particular analyte and the

densitometric measurements of these bands. Pitre et al. (2007) provided evidence that this

assumption is invalid because even in a linear relation, densitometric ratios differ substantially

from known actual ratios of sample analysed, unless specific standards are available for

analysis. The gradients of the standard curves show that the intensity / response do not

increase linearly with concentration for each of the selected terpenoids. Curve-fit analysis

[Madecassoside], mM [Asiaticoside], mM

[Asiatic acid], mM [Madecassic acid], mM

y = 9353.1x +1449.7 y = 2.667.1x +1429.8

y = 314.51x +1489.7 y = 1199.31x +1827.7


- 62 -

reveals the curve obtained is well described by an indirect linear relation (R2>0.99), and that a

relation of y = ax + b with b 0 is obtained (Figure 4.3). The indirect linear relation existing

between the triterpenoid concentrations and their densitometry measurements implies that a

theoretical zero mM concentration of saponin sample would provide a non-zero optical

density value and this in spite of background correction. The reasoning behind this

inconsistency is that the physical relation, the Beer-Lambert law, between the incident and

emergent energy of light transversing a solid substance (in this case a spot or band on the

TLC plate) is logarithmic and not linear (Das and Mallick, 1991). Hence, linear approximations

of the curve do not include the origin to intercept the y-axis at a non-zero value.

Table 4.1 indicates the precision of the method for the four compounds at various

concentrations with the calculated SD and CV based on at least 9 determinations. The

average CV was less than 4% for the standards and between 4-8% for the experimental

samples. The variation in the calculated average CV for the standards and samples may be

explained by the TLC method which depends on the exact amount of sample that is applied

and, it needs to ensure that all the spots are of equal width. A constant sample application

technique is required in order to achieve good precision and accuracy. Equal volumes of

sample with different concentrations were applied to minimise this variation. In addition, it

should be considered that data analysis is done in a grey-scale mode which generates a

discrepancy in monochromatic intensity as conversions from a coloured plate allows for

variations of grey, for this reason, the background colour must be subtracted.


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Table 4.1: Densitometric analysis of triterpene saponin and sapogenin standards (asiaticoside, asiatic acid, madecassoside and madecassic acid) with known concentrations. Saponin / sapogenin (mM) Mean (na) SD CV (%)b

Asiatic acid 0.04 1562.46 74.93 4.79 0.08 1613.84 78.06 4.83 0.20 2115.87 77.65 3.67 0.40 2399.08 55.78 2.32 0.60 2722.70 157.42 5.78 0.80 2900.62 116.92 4.03 Asiaticoside 0.04 1687.51 79.59 4.72 0.08 2614.43 154.91 5.93 0.20 4470.09 395.51 8.84 0.40 6782.46 203.60 3.00 0.60 7106.63 372.23 5.24 0.80 7195.86 167.42 2.33 Madecassic acid 0.04 1996.33 32.92 1.65 0.08 2311.53 69.58 3.00 0.20 3365.97 138.66 4.11 0.40 3974.96 157.06 3.95 0.60 4181.66 251.19 6.00 0.80 4654.71 127.65 2.74 Madecassoside 0.04 1841.66 116.95 6.35 0.08 1986.52 43.59 2.19 0.20 3542.55 179.69 5.07 0.40 5100.11 79.37 1.56 0.60 4614.19 86.25 1.86 0.80 5769.73 143.99 2.53 a Each band intensity determination is the average of at least 5 replicate spots, SD = standard deviation b CV = SD x 100% / mean

The concentration of the selected centellosides was determined in crude extracts prepared

from leaf tissue by means of the constructed standard curves (Figure 4.3; Table 4.2).


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Table 4.2: Investigation of Centella asiatica leaf tissue by densitometric analysis of TLC plates after AS detection. The estimated concentrations of triterpenoid centellosides were determined from the standard curves. HPLC quantification and identification were as previously described (James et al., 2008).

Compound Densitometric intensity (na)

CV b (%) Estimated concentration HPLC data

(% dw) (mM ) (mg/g fw) (% dw)**

Asiatic acid* 1930.12 6.65 0.22 1.29 1.29 1.44 Asiaticoside 4030.58 6.94 0.18 2.07 2.07 2.38

Madecassic acid 3973.74 4.60 0.49 2.96 2.96 3.29 Madecassoside 3719.75 0.25 0.24 2.92 2.92 3.22

a Each band intensity determination is the average of at least 5 replicate spots. b CV = SD x 100% / mean. * Samples were diluted so the concentrations could be determined on the linear range of the standard curve. ** For comparative purposes, the dry weight (dw) is calculated as 10% of the fresh weight (fw).

The concentrations, expressed as g/g tissue were confirmed by reverse-phase HPLC

(Figure 4.4) analysis (James et al., 2008), thus supporting the accuracy of the TLC

densitometric method. However, in all cases, the concentrations determined by TLC

densitometery were underestimated by an average of about 14%. Although not observed at

the dilutions used, the density values obtained can potentially be influenced by the presence

of non-saponin contaminants which contribute to the total density measured. Sample cleanup

may allow for contaminants to be eliminated or a reduction in their concentrations to below

detectable values, but introduces human error and additional costs.


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Figure 4.4: Reverse phase-HPLC chromatograms representing characteristic profiles from ethanolic extracts of C. asiatica leaves. HPLC conditions have been previously described (James et al., 2008). A chromatogram of the standards, madecassoside (1), asiaticoside (2), madecassic acid (3) and asiatic acid (4) at 25 ng each, is displayed for comparative purposes. Leaf samples from 2 different geographical locations in South Africa, labeled Type 1 and 2 were analyzed. The y-axes were offset with 250 mAbs units and the x-axes for Type I (upper trace) offset by 2 min, in order to facilitate visual comparison. The peaks indicative of the target triterpenoids are designated by an asterisk. Differences between varieties in medicinal plants of the same species (chemotypes) are

common and variation in secondary metabolites has been observed with identical phenotypes

and growth conditions, depending on plant origin (Aziz et al, 2007; James and Dubery, 2009).

Not surprisingly, significant differences in active constituents have therefore also been

observed between samples of C. asiatica originating from different countries (Das and

Mallick, 1991; James and Dubery, 2009). The determination of saponins by means of

biological activity tests is often non-selective (Oleszek, 2002), and cannot detect the

presence of precursor molecules. Most studies on the quantification of terpenoid saponins

from C. asiatica only report on the concentration of asiaticoside alone. The combination of

densitometric analysis following TLC as herein described generates a simple, rapid and

robust procedure for the determination of all four major triterpene saponins / sapogenins in C.

asiatica. The individual identification and quantification of the two main acids, asiatic acid and

madecassic acid, as well as their glycosides, asiaticoside and madecassoside in C. asiatica

extracts can now be individually quantified in order to determine the free acids to glycosides

ratios in plant extracts and preparations.


- 66 -

5. Conclusion

The combination of TLC and densitometry can be utilised to assess the purity of C. asiatica

extracts, and to estimate the concentration of the four major pentacyclic triterpenoids in fresh

plant material. As such, QTLC can be utilised for the analysis and quantification of the

centelloids in laboratories that lack more advanced equipment like HPLC and mass

spectrometers.

Characterisation of two phenotypes of

Centella asiatica in Southern Africa through

the composition of four triterpenoids in callus,

cell suspensions and leaves

Chapter 5

Chapter 5- Phenotypes and HPLC

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Published as: Plant Cell Tissue Organ Culture (2008) 94, 91-99

1. Abstract 69

2. Introduction 69



3.2. Preparation of callus and cell cultures 72

3.3. Determination of dry / wet weight 72

3.4. Ethanolic extracts of C. asiatica tissues 72

3.5. TLC analysis of triterpene saponins 73

3.6. Quantitative analysis using HPLC 73

3.7. Metabolite profiling using densitometric analysis 74



- 69 -

1. Abstract

Two morphologically distinct phenotypes of Centella asiatica (Type-1 and Type-2) in South

Africa were compared in relation to the levels of triterpenoid saponins with the aim of

assessing their potential for biotechnological manipulation of triterpenoid synthesis. The

metabolites investigated included madecassoside and asiaticoside and their sapogenins

madecassic- and asiatic acid; produced in cultured undifferentiated cells (cell suspensions

and calli) and leaves. Weight determination in plant cell suspensions and the accumulation of

secondary metabolites after 16 days for Type-1 and 20 days for Type-2 were investigated

since these secondary metabolites accumulate during the period that follows the active

growth phase. The four triterpenoids of interest were analyzed and quantified by HPLC in

crude ethanolic extracts. A difference in bioactive triterpenoids was exhibited that was tissue

specific and varied between the two phenotypes. The triterpenoids from leaf tissue were more

easily quantifiable in each phenotype than in the case of the undifferentiated cells (callus and

cell suspensions), which had lower, but still quantifiable, levels of these targeted secondary

metabolites. Leaves contained the highest triterpenoid levels (ranging from 1.8 to 5% dry

weight for the triterpenoid acids and their glycosides, respectively), with the free acids

occurring in a ratio of approximately 1:2.5 in relation to the glycoside content.

2. Introduction

Saponins are a vast group of glycosides, widely distributed in higher plants. A number of

different plant species synthesize triterpenoid saponins as part of normal growth and

development with the most predominant group being pentacyclic triterpene derivatives and

their sapogenins (Haralampidis et al., 2002). Their surface active properties are what

distinguish these amphiphilic compounds from other glycosides (Sparg et al., 2004). The

triterpenoid structure (aglycone) is hydrophobic and contains a hydrophilic sugar chain

(glycone) and these characteristics are responsible for the biological activity of saponins

(Singelton et al., 2000). The sugar(s) are attached to the aglycone and varies both in type

and number. The substances of therapeutic interest are the saponins containing triterpene

acids and their sugar esters (Sparg et al., 2004).


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Triterpenoid saponins are synthesized via the isoprenoid pathway by cyclization of 2,3-

oxidosqualene to give primarily oleanane (beta-amyrin) or dammarane triterpenoid skeletons.

The triterpenoid backbone then undergoes various modifications (oxidation, substitution and

glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyl

transferases and other enzymes. In general very little is known about the enzymes and

biochemical pathways involved in saponin biosynthesis. The genetic machinery required for

the elaboration of this important family of plant secondary metabolites is still largely

uncharacterized, despite the considerable commercial interest in this important group of

natural products. This is likely to be due in part to the complexity of the molecules and the

lack of pathway intermediates for biochemical studies (Haralampidis et al. 2002).

Centella compromises some 45 species belongings to the plant family Apiaceae which

includes the medicinally important C. asiatica. This slender, weakly aromatic, small creeping

perennial herbaceous plant is an umbellifer which has many common names including Gotu

Kota and Indian Pennywort (Matsuda et al., 2001). It has been utilized for centuries in

Ayurvedic medicine to alleviate symptoms of anxiety (Wijeweera et al., 2006) and to promote

fibroblast proliferation and collagen synthesis (Maquart et al., 1999). Centella terpenoids

include asiaticoside, centelloside, madecassoside, brahmoside, brahminoside, thankuniside,

sceffoleoside, centellose, and asiatic, brahmic, centellic and madecasic acids (Aziz et al.,

2007). Depending on the origin of the Centella plant material, these saponins can account for

between 1 and 8% of the constituents (Brinkhaus et al., 2000). It was reported that C. asiatica

extracts contained three bioactive triterpenoids, namely asiatic acid, madecassic acid and

asiaticoside, which have healing properties. The most abundant triterpenoid saponin,

asiaticoside, has antibacterial, fungicidal and cell proliferative activities which have been

shown to aid in the treatment of wounds (Shukla et al., 1999), ulcers, various skin diseases,

vein insufficiency, tuberculosis and in the treatment of mental disorders (Mathur et al., 2000).

Recent studies have led to the isolation of other triterpenoids with healing abilities, namely

terminolic acid, madecassoside and asiaticoside-B (Schaneberg et al., 2003).

Most plant derived pharmacologically active compounds have complex structures, making

chemical synthesis an economically uncompetitive option. Plant cell culture has been used in

attempts to increase the production of bioactive secondary metabolites of pharmaceutical

interest (Gaines, 2004). A particular important potential benefit is the ability to manipulate and


- 71 -

improve the production of desired compounds within the plant cell through manipulation of

cultured cells by elicitors and plant hormones.

Two phenotypes of C. asiatica which will be referred to as Type-1 (a reniform (broad and

round) leaf shape with crenate leaf margins) and Type-2 (a cardate leaf with sinuated leaf

margins) (Figure 5.1), are found in South Africa. A comparison and evaluation of the

triterpenoid content (asiatic acid, madecassic acid and their glycosides) in callus, cell

suspensions and leaves of these phenotypes are reported. The reported data will contribute

to the establishment of knowledge about the triterpenoid saponin composition of C. asiatica

found in Southern Africa in comparison to other geographical areas, and lays a foundation for

future studies on the manipulation of the phytochemical composition of C. asiatica.

Figure 5.1: Two phenotypes of South African C. asiatica (left): a broad and round leaf shape with crenate leaf margins (Type-1) and (right) a cardate leaf with sinuated leaf margins (Type-2)

3. Materials and methods

3.1. Plant material

Commercially cultivated C. asiatica was obtained from a local nursery and designated Type-

1. The morphologically wild relative, Type-2, (previously known as swamp Centella or C.

cordifolia) was collected from a marshy area in the Gauteng province, South Africa. Voucher

specimens of both Type-1 and Type-2 were deposited in the herbarium of the Botany and

Plant Biotechnology Department, University of Johannesburg, South Africa.


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3.2. Preparation of callus and cell cultures

Explants from stems of Type-1 and Type-2 was cultivated and maintained in agar solidified

Murashige and Skoog (MS) medium with vitamins (50 mg nicotinic acid, 50 mg thiamine HCl,

10 mg pyridoxine HCl and 10 g myoinositol per 100 ml, Highveld Biologicals, South Africa)

supplemented with 1 µM 2,4-D, 1 µM BAP, 30 g/L sucrose and 1g/L casein hydrolysate

(Bouhouche et al., 1998). To obtain callus proliferation, 1 g of callus was transferred

aseptically to solid MS salt solution as described above. The cultures were kept in a culture

room with 18 / 6 h light / dark cycle and the temperature was regulated at 23 °C for 21 days.

In addition, callus was transferred to sterile liquid medium, to initiate cell suspensions. These

were incubated on an orbital shaker in the dark at 23°C. Every 7 days the homogenous cell

suspension was subcultured in a 1:1 (v/v) ratio with fresh MS medium.

3.3. Determination of dry / wet weight

A series of Erlenmeyer flasks, each containing 40 ml of culture medium were all individually

inoculated with 10 ml of an established cell suspension and allowed to grow in the dark at

23°C. The growth was terminated on alternate days by filtering the cell suspension through a

0.45 µm nylon membrane. The wet weight of the cells were determined and these cells were

either dried in pre-weighed polypropylene tubes (Merck, Darmstadt, Germany) at 65 °C for 48

h to determine the dry weight, or added to the required amount of ethanol for metabolite

extraction as described below.

3.4. Ethanolic extracts of C. asiatica tissues

After the required growth period was achieved, the cell suspensions of Type-1 and Type-2

were filtered through a 0.45 µm nylon membrane and cells were added to absolute ethanol

(Saarchem, South Africa) in a 1:3 (w/v) ratio. Extracts of callus were prepared similarly by

transfer to absolute ethanol. The suspension were homogenized for 10 min and centrifuged

at 2200 xg for 20 min. The supernatants were collected and vacuum dried to remove the

excess solvent.


- 73 -

Leaves of C. asiatica (Type-1 and Type-2) were weighed, cut into strips and placed in

absolute ethanol (1:12 (w/v)) to extract secondary metabolites. This was placed on a

magnetic stirrer for 24 h and then centrifuged at 2200 xg for 20 min. The supernatant was

decanted and concentrated in 10 times with a rotary evaporator at 45 °C under vacuum. The

extracts were then used for analysis.

3.5. TLC analysis of triterpene saponins

Concentrated crude extracts of callus, cell suspensions and / or leaves were reconstituted in

a minimal amount of ethanol and applied to Silica gel 60 F254 TLC plates (20 x 20 cm, Merck,

Darmstadt, Germany). These were developed in a chloroform, glacial acetic acid, methanol

and dH2O (60:32:12:8 (v/v/v/v)) developing solution. Detection of triterpenoids was achieved

by spraying with anisaldehyde-sulphuric acid (AS) reagent prepared according to Kraemer et

al. (2002). The TLC plates were sprayed with 10 ml of the spray reagent and then heated at

95 °C for 10 min. Authentic standards of asiatic acid, madecassic acid and their respective

glycosides (Extrasynthase, France) were also chromatographed on the plates to calculate

corresponding Rf values. Violet spots develop with a density proportional to the total saponin

content present. This approach, described by Gurfinkel and Rao (2002) was referred to as

direct densitometry, which allows for the rapid analysis of many samples.

3.6. Quantitative analysis using HPLC

Chromatography was carried out on a Shimadzu 10AVP system consisting of a dual set of

10AT solvent delivery modules, a 10 AD Shimadzu auto-sampler and a Shimadzu SPD-M VP

diode array detector. Data were collected and analyzed using the Shimadzu CLASS VP

software supplied. The eluate was monitored continuously from 200 to 600 nm. Column

temperature was maintained at 25°C. A reverse-phase C18 Zorbax (250 x 2.6 mm) column

coupled to a Phenomenex Security Guard column was used. Acetonitrile and water (Sigma-

Aldrich, St. Louis, USA) were used as the mobile phase; the column was eluted with a

gradient from 20% acetonitrile in water to 100% acetonitrile over 35 min, maintaining

acetonitrile for a further 10 min. Flow rates of 1 ml/min were used. Quantification was carried


- 74 -

out by injection of the four standards, the peak areas of the standards were determined at the

wavelength providing maximum absorbance using the Shimadzu CLASS VP software.

Concentrations from 1 to 250 g/ml were injected in order to construct response versus

concentration standard curves for the linearity range and regression equation. All the samples

and standards were filtered through a 0.2 m Millex-HV-filter and then put into a 2 ml SRI vial.

Samples of 25 µl were injected for analysis. Under these conditions, the obtained retention

times (Rt) for madecassoside, asiaticoside, madecassic acid and asiatic acid were 6.54, 8.94,

14.34 and 16.27 min, respectively. Identification of the metabolites in extracts was based on

Rt values and UV absorbance spectra. Further verification of the four compounds was done

by spiking with authentic standards to confirm retention time and spectral properties.

3.7. Metabolite profiling using densitometric analysis Zones (by calculated Rf -values) on the TLC plate which, correlating to authentic standards

commercially available, were analyzed using Quantity-One software (BioRad).

4. Results and discussion

Primary metabolism is associated with the log or exponential phase of a culture during where

the sole products of metabolism are either essential for growth or are the byproducts of

energy yielding metabolism (Prescott et al., 1999). Ideophase refers to the period in a batch

culture in which secondary metabolites are synthesized in preference to primary metabolites;

it generally corresponds to the stationary phase and the end of the log phase. Growth curves

of C. asiatica cells were constructed in order to determine the active and stationary phases of

growth as is illustrated in Figure 5.1. In some cases the production of secondary metabolites

does not show a positive correlation with the maximal growth rate of the culture. It was

ascertained that Type-1 reaches the stationary phase at day 13 of growth in suspension

under the conditions described, whereas Type-2 was able to sustain a longer latent and

growth phase up to day 19. Under the described experimental culture conditions, cell

suspensions of C. asiatica Type-1 exhibited a lag phase from day 1-3, followed by an active

exponential growth phase to day 12. The stationary phase was achieved after 13-14 days.


- 75 -

Similar findings were reported by Bouhouche et al. (1998) who reported a latent phase

between 0 and 3 days and an exponential growth phase from 3 to 12 days and a stationary

phase from 12 to 14 days under the same conditions. Nath and Buragohain (2005) found an

initial lag phase up to day 10 of incubation for C. asiatica cell suspensions followed by a

steep rise in growth rate until the third week. In contrast, C. asiatica Type-2 was able to

sustain a longer growth cycle. There is a latent phase from 1 to 11 days in culture before an

exponential growth phase is encountered for 7 days; the stationary phase is only achieved

after day 19.

Figure 5.2: The growth curves (— wet and dry mass) of C. asiatica Type-1 (a) and Type-2 (b) in liquid medium, indicative of when the stationary phase is achieved ( ) and the production of secondary metabolites is predominant.

The highest concentrations of the targeted triterpenoids in cell suspensions detected by TLC

with the AS reagent were seen on day 16 for Type-1 and day 20 for Type-2 once the

stationary phase has been attained. Media filtered off from the cell cultures were analyzed

under the same conditions as for extracts from callus and cell suspensions. None of the

investigated triterpenes could be detected (results not shown), indicating that the metabolites

are not secreted to a significant extent and therefore that harvesting of the triterpenoid

saponins from the culture medium is not an option.

These observations may reflect a competition for metabolites utilized in primary metabolism

with those pathways leading to the formation of secondary products. One approach used to


- 76 -

regulate metabolic pathways favouring the production of specific secondary metabolites has

been to add precursors to the culture medium, though it is not known if this option has been

investigated for C. asiatica cells. Another is the manipulation of metabolic flux through specific

pathways in response to elicitors and signal molecules of plant defense responses. The

instability of cell cultures for the continued production of secondary products poses another

problem; some cell lines loose the ability to synthesize the desired compound after prolonged

culture. The relationship between cell differentiation and tissue organization and the

biosynthesis of secondary compounds is also obscure. Plant secondary metabolites are

normally synthesized by specialized cells, often at distinct stages of plant development and

certain compounds are not synthesized if cells remain undifferentiated as in cell suspensions

(Kim et al., 2002b). The distribution between mRNA transcripts, enzymes and biosynthetic

products within and between cells is an important component of regulation for secondary

plant metabolic processes. Many metabolic pathways are compartmentalized, enabling the

separation of incompatible or competing reactions, and concentrating enzymes and

metabolites (Samanani and Facchini, 2006). Biotechnological attempts to overproduce the

quantities of asiaticoside through cell or tissue culture have encountered limited success (Kim

et al., 2002a).

Callus is undifferentiated tissue which has the ability to develop into any plant organ whether

it is a root, shoot or leaf, under the correct growth hormone concentrations. This might be the

reason behind the presence of all the four triterpenes in the callus samples, albeit in low

concentrations (Table 5.1). The detection of asiaticoside and madecassoside in callus

contrasts with the findings of Kim et al. (2004) who failed to detect asiaticoside in

undifferentiated cells of Korean C. asiatica, but is supported by Nath and Buragohain (2005)

who stated that callus and cell suspensions of Indian origin did, in fact, synthesize

asiaticoside.

All four of the triterpenes under consideration were found in leaves of both Type-1 and Type-

2 C. asiatica and this finding thus supports the claimed medicinal value of this herb. This point

is indirectly confirmed by the fact that it is the leaves of C. asiatica that are used as material

for various medicinal products and health foods (Brinkhaus et al., 2000).

Analysis of callus, cell suspensions and leaf tissue by means of TLC showed differences in

the asiatic acid, madecassic acid and their glycoside content for both phenotypes. These


- 77 -

findings were verified by HPLC analysis which allowed the quantification of the four targeted

metabolites.

Table 5.1: Quantitative determination by HPLC of asiatic acid, madecassic acid and their glycosides, asiaticoside and madecassoside, in ethanolic extracts from C. asiatica Type-1 and Type-2 from cultured cells (suspensions and callus) and leaves. Sample Asiatic acid Madecassic acid Asiaticoside Madecassoside

C. asiatica Type-1 Cell suspensions 0.16 ± 0.032a 0.28 ± 0.036a 1.38 ± 0.020a 1.67 ± 0.012a

Callus 0.19 ± 0.016a 0.24 ± 0.013a 2.46 ± 0.092a 2.35 ± 0.098a

Leaves 1.89 ± 0.080a 1.97 ± 0.007a 5.23 ± 0.025a 4.76 ± 1.342a

C. asiatica Type-2 Cell suspensions 0.14 ± 0.024a 0.15 ± 0.012a 1.23 ± 0.015a 1.31 ± 0.018a

Callus 0.24 ± 0.021a 0.19 ± 0.010a 2.84 ± 0.012a 1.98 ± 0.056a

Leaves 1.79 ± 0.102a 1.88 ± 0.070a 4.52 ± 0.138a 4.28 ± 0.124a

Authentic

standards

Rf-value (TLC) 0.95 0.86 0.39 0.32

Rt-value (HPLC) (min) 16.27 14.34 8.94 6.54

Cells suspensions at day 16 (Type-1) and 20 (Type-2), respectively, were used for analyses. Quantification and identification were as described under Materials and methods (values presented are expressed as % dry weight and standard deviationa n = 5, calculated to be in the magnitude of 0.100-0.032). The values presented in this table are an average of at least 5 individual experiments carried out. Authentic standards were used to set up standard curves for quantification purposes. R2 values of 0.97, 0.98, 0.99 and 0.99 were obtained for the regression lines for asiatic acid, madecassic acid, asiaticoside and madecassoside, respectively.

HPLC offers the advantages of speed, sensitivity and the capability to analyze without

derivatization. However, two major difficulties of saponin characterization is the lack of a

chromophore and available standards. The fact that the compounds do not contain

chromophores limits the detection and types of solvents which can be utilized in the HPLC

method as these may absorb in the UV region. The use of acetonitrile / water on reverse-

phase columns at a detection wavelength of 205 nm made the qualitative and quantitative

determination of the triterpenes of C. asiatica and its pharmaceutical preparations possible

(Gunther and Wagner 1996; Inamdar et al.,1996; Schaneberg et al., 2003).

By means of these parameters and our own modifications, our solvent of choice was ethanol

for extraction and an eluent consisting of a linear gradient of acetonitrile and water for HPLC

in order to effectively separate all four compounds in a single run. Chromatograms of plant

extracts, which demonstrated the optimum separation and ideal evaluation of individual

constituents in the plant extract is shown in Figure 5.3. The detection limit by HPLC for the

four standards was determined to be 1.5 g/ml for madecassoside and asiaticoside with a


- 78 -

slightly higher value of 1.8 g/ml for madecassic acid and asiatic acid (the limit of detection

(LOD) was calculated using peak height to three times the baseline noise). This is somewhat

higher to the detection limit of 1 g/ml of each standard obtained by TLC. Standard curves

exhibited linearity of the above mentioned concentration ranges with R2-values of 0.97-0.99.

Extraction efficiency was studied by adding known concentrations of pure standards to plant

extracts to be studied. The extraction efficiency (n = 6) for madecassoside was 98.3%

(relative standard deviation (R.S.D) of 0.35%), asiaticoside 98.7% (R.S.D. of 0.45%),

madecassic acid 96.4% (R.S.D. of 0.67%) and asiatic acid 95.8% (R.S.D. of 0.52%). The

retention times of the four metabolites are given in Table 5.1.

HPLC quantification indicated that leaves contained higher levels of triterpenoids than the

undifferentiated cultured cells for both types, with Type-1 (the commercial species) having a

higher triterpenoid content generally. In addition, all four the targeted metabolites were found

in the undifferentiated cultures cells, with calli exhibiting the higher concentrations compared

to cell suspensions (Figure 5.3, Table 5.1). Secondary metabolite production may require

interaction between roots and leaves with metabolic precursors generated in roots and

passing to aerial parts of plants for bioconversion in leaves (Giri and Naraseu, 2000). The

biosynthesis of major secondary metabolites is often either tissue or organ specific (Aziz et

al., 2007), as also indicated by Kim and colleagues in the case of C. asiatica terpenoid

saponins. In contrast to our data, they could not detect asiaticoside in undifferentiated cells.

Their results have shown that asiaticoside biosynthesis is concentrated in the leaves (0.4-

1.4% dry weight) and that the level of asiaticoside content is quite low in the roots of whole

plants (Kim et al., 2002a, 2004, 2005, 2007).


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Figure 5.3: Reverse phase-HPLC chromatograms representing characteristic profiles from ethanolic extracts of Centella asiatica leaves (a), callus (b) and cell suspensions (c); (Type-1 (upper trace) and Type-2 (lower trace). HPLC conditions are as described in Materials and methods. A chromatogram of the four standards (25 ng of each): madecassoside (1), asiaticoside (2), madecassic acid (3) and asiatic acid (4) are displayed for comparative purposes. All chromatograms were normalized to the second largest peak. The y-axes were offset with 250 mAbs units and the x-axes for Type-1 (upper trace) offset by 2 min, in order to facilitate visual comparison. The peaks indicative of the target triterpenoid are designated by an asterisk.

Differences between varieties in medicinal plants of the same species (chemotypes) are

common and variation in secondary metabolites has been observed with identical phenotypes

and growth conditions, depending on plant origin (Aziz et al., 2007). Significant differences in

active constituents have also been observed between samples of C. asiatica originating from

different countries, possibly as a result of genomic diversity (Das and Mallick, 1991). Gupta et


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al. (1999) reported variable asiaticoside content in five lines of C. asiatica from India.

Similarly, Rouillard-Guellec et al. (1997) investigated the secondary metabolites in India and

Madagascar, and reported that plants from the latter contained the highest level of

asiaticoside. The distribution of asiaticoside and madecassoside throughout the plant was

organ specific with leaves of both lines containing the higher content of these compounds. In

a study of C. asiatica from Madagascar, Randriamampionona et al. (2007) reported

asiaticoside content of between 2.6 and 6.42% dry weight. The authors achieved in vitro

propagation of C. asiatica in a hormone free media but these in vitro plants displayed lower

asiaticoside content. Aziz et al. (2007) reported two phenotypes of C. asiatica exhibiting

differences in terpenoid content that were tissue specific and varied between glasshouse

grown and tissue derived material. Triterpenoid saponin content was highest in leaves

(asiaticoside and madecassoside concentrations of 0.7-0.9 and 1.1-1.6% dry weight were,

respectively, reported), and roots contained the lowest content of asiaticoside. In their study,

asiaticoside and madecassoside were undetectable in transformed roots and undifferentiated

callus.

Most studies on the quantification of terpenoid saponins from C. asiatica only report on the

concentration of asiaticoside. The individual identification and quantification of the two main

acids, asiatic acid and madecassic acid, as well as their glycosides, asiaticoside and

madecassoside in C. asiatica extracts can be accomplished by our adapted HPLC protocol.

Our data indicates that (i) the total triterpenoid saponin content of the two South African

phenotypes is generally comparable to that reported from India, Korea and Madagascar, (ii)

all four targeted metabolites were found to occur together (as end products of a shared, initial

biosynthetic pathway), even in cell suspensions and calli, (iii) the two phenotypes we studied

were found to vary in triterpenoid composition and concentration and that (iv) the four

metabolites occur in leaf tissue with the free acids to glycosides in a ratio of approximately

1:2.5 (Table 5.1). The fourth point differs from the findings of Randriamampionona et al.

(2007) who reported free acid: glycoside ratios ranging from 1:5 to 1:30 for leaf tissue of C.

asiatica from Madagascar.

The differences in the triterpenoid saponin composition and content of various C. asiatica

chemotypes can perhaps be attributed to genetic variation in the oxidosqualene cyclase and

other genes involved in the biosynthesis (Haralampidis et al., 2002; Kim et al., 2007), as well

as the presence and activity of enzymes involved in the attachment of the sugar residues to


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the aglycones. Metabolic pathways for these triterpenoids should therefore be further

investigated and the flux through these pathways elucidated to obtain a better understanding

of the biochemical conversions that will allow the manipulation and exploitation of secondary

product synthesis in C. asiatica.

Enhanced secondary metabolite production in

C. asiatica cell suspensions by means of MeJa

induction

Chapter 6

Chapter 6 – Elicitor Induction and Metabolomics

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1. Methyl jasmonate (MeJA) and jasmonic acid (JA) 84

2. Production of centellosides in C. asiatica 87

3. Enhanced biosynthesis of triterpenoids through biotechnology – elicitation

Of C. asiatica by MeJa 88

4. Metabolomics 90

4.1. Analytical platforms 94

4.2. Multivariate data analysis (MVDA) 97

5. Significance of this study 98


6.1. Preparation and elicitation of the cell cultures 99


6.3. Extraction of the secondary metabolites – triterpenoids 100

6.4. Partial characterization and fractionation of the extracted triterpenoids 100



6.5.1. Experimental parameters 101

6.5.2. Data analysis 103

7. Results 106


7.2. TLC analysis 107



8. Discussion 123

9. Conclusion 130


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Many higher plants produce economically important organic compounds e.g. resins, oils,

tannins, flavors and fragrances and are steady sources of commercially valuable materials

(Belandrin, 1985). Advances in biotechnology, particularly methods for culturing plant cells

and tissues are approaches to provide new means for the commercial processing of even

rare plants and the chemicals that they produce.

Plant chemicals are often classified as either primary or secondary metabolites. Primary

metabolites are substances widely distributed in nature, occurring in one form or another in

virtually all organisms, whereas secondary metabolites are compounds biosynthetically

derived from primary metabolites, and have no apparent function in a plant's primary

metabolism. These often play an ecological role, in that they are pollinator attractants,

represent chemical adaptations to environmental stresses, or serve as chemical defenses

against microorganisms, insects and higher predators, and even other plants (allelochemics,

Belandrin, 1985). In plants, secondary metabolites accumulate in smaller quantities than

primary metabolites and tend to be synthesized in specialized cell types, at distinct

developmental stages, thus making their extraction and purification difficult. Since natural

products offer a diversity of chemical structures which can serve as precursor molecules, the

activities of these can be enhanced by manipulation, through combinations with chemicals

and synthetic chemistry (Koppula et al., 2010).

1. Methyl jasmonate (MeJa) and jasmonic acid (JA)

Jasmonates, namely JA and its ester, MeJa, are naturally occurring plant growth regulators

(Ketabchi and Shahrtash, 2011). These plant lipid derivatives are similar to mammalian

eicosanoids and play a critical role(s) in plant defenses against herbivores and pathogens

through up-regulating the expression of defense-related genes (Kim et al., 2004). Plants that

come under attack by insects, or are damaged mechanically, produce higher levels of JA and

MeJa, which accumulate in the damaged parts of the plant.

The biosynthesis of JA starts with linolenic acid (LA) and proceeds through a number of

stages involving lipoxidation, cyclization and β-oxidation (Figure 6.1). JA is further catabolized

by JA carboxyl methytransferase (JMT) to form MeJa, a volatile fatty acid-derived compound

that occurs widely in plants. Another form of signaling in herbivore-damaged plants can


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occur when oral secretions of the herbivore contact damaged plant tissue; this prompts the

release of volatile organic molecules that serve as attractants for predators of the herbivores,

and it seems that MeJa may be involved in this too (Sembdner and Parthier, 1993).

Metabolic pathways that originate from squalene, a common precursor for the synthesis of

triterpenes (C30) and sterols (C18-29), forms an extensive range of end products (Chapter 2).

The triterpenoids from C. asiatica, namely madecassoside and asiaticoside with their ursane

type sapogenins madecassic- and asiatic acid, proceed from the cyclization of 2,3,-

oxidosqualene by a specific oxidosqualene cyclase (OCS), namely -amyrin synthase (Figure

2.7). Studies suggest that elicitor action (by 100 µM MeJa) affects some steps of ursane

biosynthesis which leads to the increase in triterpenoid saponins levels in aerial parts of the

plant (Mangas et al., 2006). A few jasmonate-responsive genes in C. asiatica involved in the

biosynthetic pathways have been cloned (Hernandez-Vazquez et al., 2010); these include the

genes coding for the enzymes farnesyl diphosphate synthase (Kim et al., 2005a), squalene

synthase (Kim et al., 2005c), and OCS (Kim et al., 2005b). The jasmonate signaling pathway

is connected to other signaling pathways, thus constituting a complex regulatory network. The

genes up-regulated by MeJa treatment include those involved in jasmonate biosynthesis,

secondary metabolism, cell-wall formation, and those encoding stress-protective and defense

proteins (Cheong and Choi, 2003).

http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313X.2002.01328.x/full#b1


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Figure 6.1: Biosynthesis of jasmonic acid from linolenic acid (LA). Jasmonates are cyclopentanone derivatives that originate biosynthetically from LA via an inducible octadecanoid pathway consisting of several enzymatic steps. A phospholipase A1 releases a linolenic acid from membrane lipids. The -linolenic acid is oxygenated by lipoxygenase (LOX) to form 13(S)-hydroxy linolenic acid (13-HPOT), which is then converted to 12-oxo-phytodienoic acid (OPDA) by allene oxide synthase (AOS) and allene oxide cyclase (AOC). Jasmonic acid (JA) is synthesized from OPDA through reduction and three steps of -oxidation, and is further converted by JA carboxyl methyltransferase (JMT) to the end product methyl jasmonate (MeJA). This volatile methyl ester is used as a signaling molecule in biotic and abiotic stresses (Farmer and Ryan, 1990; Cheong and Choi, 2003).

There are two chiral carbon atoms in the MeJa molecule; each can have either the R- or S-

absolute configuration, so that there are four potential isomers. Methyl (+)-epijasmonate, (3R,

7S)-(+)-methyl 3-oxo-2-(2-(Z)-pentenyl) cyclopentane-1-acetate, has the strongest odour of

the isomers, demonstrating the importance of molecular shape in fitting receptors and


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activating the sensory response (Wang, 2008). There are many applications listed for MeJa.

Since MeJa is more volatile than JA, it can act as a messenger to neighboring undamaged

plants, signaling that an attack is under way and prompting them to produce defensive

chemicals before they are attacked. Another promising application for methyl jasmonate lies

in prolonging the shelf-life of fresh fruit (Corbo, 2010). The role of methyl jasmonate is again

believed to involve the production of defense proteins, which encourage formation of

fungicides and anti-bacterial agents.

2. Production of centellosides in C. asiatica

The content of four principal triterpenoid bioactive compounds, namely asiaticoside,

madecassoside, asiatic acid and madecassic acid has been extensively evaluated and there

are many different reports on the content and concentration of these triterpenoids in C.

asiatica, in different regions (Chapter 4, Section 7), as well as in different parts of the plant

itself (Chapter 4, Section 8).

Mangas et al. (2008) described the relationship between gene expression and centelloid

production in non-differentiated tissues of C. asiatica from East Asia. Their data showed there

was a significantly lower expression of the gene encoding -amyrin synthase in calli, in

comparison to in vitro plant cultures, which was consistent with the lower production of

centellosides. In contrast to our results (Chapter 5), in callus tissue, the main triterpenoid was

madecassoside > madecassic acid > asiaticoside > asiatic acid. The concentration of

madecassoside was 6-8 times greater than asiaticoside, and madecassic acid was 2-3 times

higher than asiatic acid. The detection of asiaticoside and madecassoside in callus contrasts

with the findings of Kim et al. (2004) who failed to detect asiaticoside in undifferentiated cells

of Korean C. asiatica, but is supported by Nath and Buragohain (2005) who stated that callus

and cell suspensions of Indian origin did, in fact, synthesize asiaticoside. In vitro, the saponin

content varied depending on the part of the plant; the main terpenoid found in the aerial parts

was asiaticoside but in the roots it was madecassoside (Mangas et al., 2008). There are also

differences in triterpenoid production between calli and cell suspensions. Hernandez-

Vazquez et al. (2010) found more asiaticoside produced in cell suspensions than

madecassoside, asiatic- and madecassic acid, which is similar to our findings for both calli

and cell suspensions of Southern African C. asiatica (Chapter 5, Table 5.1).


- 88 -

The accumulation of secondary metabolites can also be enhanced or reduced by changing

the composition of the nutrient medium. Reports of callus induction have been carried out

with the focus on manipulating the triterpenoid production, in particular asiaticoside by

treatment with different growth regulators. Loc and An (2010) found MS medium with 1.0

mg/ml benzylaminopurine (BAP) and 1.0 mg/L naphthalene acetic acid (NAA) resulted in the

most growth after 21 d, and Nath and Buragohain (2005) found that leaf explants of C.

asiatica plantlets profusely formed calli on MS medium supplemented with a lower

concentration of BAP (1.0 mg/L) but the same amount NAA (1.0 mg/L) after 35 d of culture in

the dark. Callus formation from stem and leaf explants of C. asiatica on semi-solid modified

MS medium has been reported; however the medium was supplemented with a different ratio

of the growth regulators, namely 2.0 mg/L kinetin and 4.0 mg/L NAA (Patra and Rai., 1998) or

0.5 mg/L kinetin and 2.0 mg/L NAA (Rao et al., 1998) to allow for maximum callus induction

from C. asiatica stem explants. Martin (2004) developed calli on MS medium supplemented

with 2.32 μM kinetin in combination with either 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-

D) or 5.37 μM NAA. In order to increase the accumulation of secondary metabolites, studies

have suggested that plant growth regulators be combined with chemicals such as MeJa into

the culture medium as an elicitor (Sharma et al., 2011).

3. Enhanced biosynthesis of triterpenoids through biotechnology –

elicitation of C. asiatica by MeJa

The induced synthesis of low molecular weight compounds called phytoalexins can be

provoked by exposing cultured cell cultures to various elicitors (Gunlach et al. 1992). Elicitors

have been found to induce secondary metabolite accumulation not only in intact plants (to

deter pathogenic micro-organisms and herbivore attack), but also in plant tissue cultures.

Several studies have indicated that many plant tissue cultures are stimulated by elicitors and

that secondary metabolites accumulate rapidly in response to treatment with elicitors (Eilert et

al., 1987; Mukundan and Hjortso, 1990; Ning et al., 1994; Dicosmo and Misawa, 1995). In

general, elicitors act with plant membrane receptors (Cosio et al., 1990) resulting in the

activation of specific genes (Chappell and Hahbrock, 1984), leading to the synthesis of

almost all chemical classes of secondary metabolites (Gunlach et al., 1992).


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Due to its medicinal properties, there has been an interest to overproduce the centellosides

through in vitro cultures of C. asiatica (Matsuda et al., 2001; Nath and Buragohain, 2005).

The effects of a number of different elicitors, including yeast extract (YE), cadmium chloride

(CdCl2), copper chloride (CuCl2) and MeJa have been used to enhance asiaticoside

production in cultured whole plants (Kim et al., 2002b) with only MeJa and YE being able to

significantly stimulate asiaticoside production. Different approaches to modify the biosynthetic

pathway have also been carried out, given that the last steps of the biosynthetic pathway for

triterpenoid saponins are still unknown (Bonfill et al., 2005; Bonfill et al., 2011), and are likely

to involve enzymes that catalyze reactions such as oxidation and glycosylation (Faria et al.,

2009). The most common strategy is feeding plant cell cultures with commercially available or

easily extractable metabolic precursors (Kiong et al., 2005). Approaches which have led to

the enhancement of natural product accumulation include the addition of exogenous -amyrin

(see Figure 2.7, showing the biosynthetic pathway) as a substrate to cell suspensions at a

concentration of 0.01 mg/ml for 7 d. This produced a four-fold increase in centelloside content

(Hernandez-Vazquez et al., 2010; Kim et al., 2005a, 2005b, 2005c) have cloned some of the

genes involved in the biosynthetic pathway of triterpenoid saponins, such as -amyrin

synthase (CabAS), cycloartenol synthase (CaCYS) and squalene synthase (CaSQS), and

Kiong et al. (2005) used squalene, farnesyl pyrophosphate (FPP), isopentenyl pyrophosphate

(IPP) and leucine as precursors to increase the terpenoid production in callus cultures.

Exogenously applied MeJa is frequently used in attempts to enhance secondary metabolite

production in a variety of plants species, with 2,3-oxidosqualene being a common precursor

for both sterols and triterpenoids (Mangas et al., 2006). Centellosides are derived from the

cyclisation of 2,3-oxidosqualene but by a specific OSC, -amyrin synthase (see Figure 2.7).

The effect of MeJa or any elicitor may be attributed to many aspects such as the elicitor’s

specificity and concentration, the duration of the treatment and the growth stage of the culture

(Holden et al., 1988).

Many researchers have used concentrations of 100 µM of MeJa to increase secondary

metabolism in in vitro cultures (Cusidó et al., 2002; Palazón et al., 2003; Kim et al., 2004) with

plant cells or organs being in direct contact with the elicitor. In the case of C. asiatica,

asiaticoside has gained much attention due to its specific medicinal properties (Babu et al.,

1995, Table 3.3). Kim et al. (2004) reported an enhancement of asiaticoside production in

whole plant cultures of C. asiatica due to MeJa treatment. Bonfill et al. (2011) increased the


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production of the triterpenoids in C. asiatica from East Asia using 100 and 200 µM MeJa in

cell suspensions and found that the centelloside pattern was not altered by MeJa treatment,

and that madecassoside was the main triterpenoid followed by asiaticoside. However, the

effect of MeJa elicitation is dose-dependent and 100 µM MeJa for 3 weeks was more

effective. Elicitation with MeJa after genetic transformation of C. asiatica using Agrobacterium

rhizogenes was found to enhance the bioaccumulation of asiaticoside in hairy roots (Kim et

al., 2007).

4. Metabolomics

Metabolomics has been developed into an important field of plant sciences and natural

product chemistry in the last decade due to improved analytical capabilities, together with

newly designed, dedicated statistical, bioinformatics and mining strategies (Shulaev et al.,

2008). The primary purpose of metabolomics is to measure all the metabolites in an organism

both qualitatively and quantitatively, in order to provide a complete metabolic picture of a

living organism under specific conditions (Kim et al., 2010), and to broaden our horizons of

understanding how plants are organized and how metabolism can be both controlled and

highly flexible (Hall, 2005). The metabolome consists of two types of compounds, the primary

and secondary metabolites. All organisms share the same type of primary metabolites for

basic functions of the living cells, whereas secondary metabolites are species specific and

play a role in the interaction of a cell with its environment, e.g. the ability of a plant to defend

itself against pests (Verpoorte et al., 2007).

Previously, the analyses of metabolites have been focused on small metabolites, but since

information about novel compound classes and new metabolic pathways has increased, it

has been realized that metabolic pathways do not operate in isolation but are part of an

extensive network. The term metabolomics is also used in conjunction with genomics,

transcriptomics and proteomics, the terminology employed is diverse and often

interchangeable (Table 6.1).


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Table 6.1: Terminology and definitions related to metabolomics (Goodacre et aI., 2004; Hall, 2005; Ellis et al., 2007; Shulaev et al., 2008).

Terms

Definitions

Metabolite

Metabolome

Metabolomics

Metabonome

Metabonomics

Metabolite (or metabolic) profiling / targeted analysis

Metabolic

fingerprinting

Metabolic footprinting

Small molecules (<1500 Da) that are required for the maintenance, growth and normal function of a cell; these participate in general metabolic reactions e.g. amino acid, fatty acids, carbohydrates, vitamins and lipids The entire set of metabolites in an organism under physiological conditions. The metabolome is divided into exometabolome (metabolites outside the cell) and endometabolome (intracellular metabolites) Identification and quantification of all metabolites in a biological system, or a complete set of metabolites in a cell, body fluid or tissue type The overall metabolites, products and interactions of all individual compartments and / or metabolomes distributed in a complex organism The quantitative measurement of time related multi-parametric metabolic responses of living systems to pathophysiological stimuli or genetic modification (Nicholson et al., 1999). It assess the tissue and biological fluids for changes in endogenous metabolite levels due to disease or toxicology Quantitative analysis of a set of metabolites or derived products (either to identify or unknown) in a selected biochemical pathway or a specific class of compounds. This includes targeted analysis, the targeted analysis of a limited number of often structurally closely related analytes High-throughput, rapid global analysis; unbiased global screening approach to classify samples based on metabolite patterns or “fingerprints” that change in response to disease, environmental, or genetic perturbations, analyte identification and quantification are not necessarily involved Fingerprinting analysis of extracellular metabolites in cell culture medium as a reflection of metabolite excretion or uptake by cells. Also known as exometabolome (Kell et al., 2005)

Plants have the ability to synthesize an enormous variety of secondary metabolites since

there is a necessity for them to respond in order to survive, in a continuously changing and


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often hostile environment, and also to attract pollinators for seed distribution for their

reproduction (Hall, 2005). Stress in plants can be characterized by a change in growth

conditions (such as environmental change, physical stress, abiotic stress and nutritional

stress) that disrupts homeostasis, and brings about a change in metabolic pathways in a

process known as acclimation. Metabolomics could contribute significantly towards the

investigation of stress biology in plants by identifying different compounds such as by-

products of stress metabolism, stress signal transduction molecules or molecules that are

involved in the acclimation responses. In the preliminary stages, when plants sense a change

in environmental conditions, a network of signaling pathways is activated which triggers the

production of different proteins and compounds that restore or achieve a new state of

homeostasis. Generally, the plant defense response is associated with the production of

phytoalexins, the activation of the phenylpropanoid pathways and the induction of lignin

synthesis. Signal molecules which are produced as a result of stresses, such as MeJa and JA

are able to activate such systemic defense and acclimation responses (Shulaev et al., 2008).

The ultimate potential of the -omics approaches is the ability to look at the studied response

on a number of different levels, namely gene expression (transcriptomics), protein translation

(proteomics), including post-translational modifications, and the metabolic network with an

approach of defining the phenotype and bridging the genotype-to-phenotype gap (Goodacre

et al., 2004, Figure 6.3).


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Figure 6.2: Schematic representation of the -omic hierarchy: genomics, transcriptomics, proteomics, and metabolomics (adapted from Lucio, 2009).

There are major advantages of analyzing the metabolome in comparison to the transcriptome

or proteome (Dunn and Ellis, 2005). These include:

1) The number of metabolites in a cell should be lower than the number of genes and

proteins. However, this differs for prokaryotes and eukaryotes; for example,

Saccharomyces cerevisiae contains more than 6 000 genes but only 600 metabolites

which reduces the sample complexity. In the case of Arabidopsis thaliana, there are 27

000 genes encoding 35 000 proteins, and an estimated > 2000 metabolites.

2) The concentrations of metabolites can be altered considerably even though the enzyme

concentrations and metabolic flux does not change significantly,

3) The metabolome is a downstream product of gene expression so the anticipated

functional level of the cell is more suitably reflected and changes in the metabolome are

expected to be more amplified relative to the genome or transcriptome,

4) Metabolic fluxes are regulated not only by gene expression but also by environmental

stresses (phenotype) and measurement of the metabolites is appropriate here.

http://en.wikipedia.org/wiki/Gene


- 94 -

However, a crucial challenge for plant metabolomics is the lack of a comprehensive

metabolome for any plant species; of the estimated 90 000 - 200 000 primary and secondary

metabolites produced, the actual number present in any individual plant species is unknown

(Fiehn et al., 2000). These metabolites all differ in polarity, chemical behavior, stability and

concentration, which makes analysis in a single experiment difficult (Kim et al., 2010). In

addition, a standard method has not been established and metabolomics is only applicable

for on-demand restricted research subjects, nonetheless it possesses the advantage of not

requiring genomic information like transcriptomics and proteomics (Fukusaki and Kobayashi,

2005).

4.1. Analytical platforms

To investigate total metabolic changes, several technology platforms are utilized which are

based on chromatography (gas chromatography-mass spectroscopy (GC-MS)) or liquid

chromatography-mass spectroscopy (LC-MS) and other MS spectroscopy or NMR

spectroscopy (Verpoorte et al., 2010), but none are able of analyzing all the metabolites in an

organism both qualitatively or quantitatively (Choi et al., 2005).

MS (including combined chromatography-MS) is the most widely employed technology in

metabolomics. It provides a combination of rapid, sensitive and selective qualitative and

quantitative analyses with the ability to identify metabolites. MS spectrometers operate by ion

formations, separation of ions according to the mass to charge (m/z) ratio and detection of

separate ions (Dunn and Ellis, 2005).

GC-MS is a combination system where volatile and thermally stable compounds are first

separated by GC, and then the eluting compounds are further detected by electron-impact

(EI) mass spectrometers. This method is ideal for volatile, low molecular weight metabolites,

thus biased against metabolites which are non-volatile with high molecular weights. High

chromatographic resolution of compounds and high sensitivity (typical limits of detection are

pmol or nmol concentrations) is achieved, although the chromatograms are complex

(containing hundreds of metabolite peaks) and may be further complicated by multiple

derivatization products. Quantification is provided by either external calibration (through the

preparation of a commercially available metabolite) or response ratio (the peak area of


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metabolite / peak area of internal standard, Dunn and Ellis, 2005). The metabolome coverage

is largely determined by the volatility of the sample components, and metabolite identification

is provided by matching the retention time (Rt) or retention index (RI) and mass spectrum of

the sample peak with those of a pure compound previously analyzed on the same instrument

and under identical instrumental conditions (Wagner et al., 2003).

Another combined system is LC-MS. This method provides metabolite separation by LC

followed by electrospray ionization (ESI) or, less typically, atmospheric pressure chemical

ionization (APCI, Bakhtiar et al., 2002). It differs from GC-MS in that samples do not need to

be volatile; analysis occurs at lower temperatures and sample derivatization is not always

required, although it can be of use to improve chromatographic resolution and sensitivity or to

provide ionizable groups on metabolites that would be undetected by ESI-MS.

ESI-MS instrumentation operates in positive and negative ion modes (either as separate

experiments or by polarity switching during analyses) and only those metabolites that can be

ionized by the addition or removal of a proton or by the addition of another ionic species are

detected. Thus, wider metabolome coverage can be obtained by analysis in both modes,

since metabolites are detected in either positive or negative ion mode (never both);

quantification is performed by external calibration or peak areas. ESI does not result in

fragmentation of molecular ions as observed in EI mass spectrometers, thus it does not allow

for direct metabolite identification by comparison of ESI mass spectra (Dunn and Ellis, 2005).

Metabolite identification can be performed by the use of accurate mass measurements and /

or tandem MS (MS/MS) to provide collisional induced dissociation (CID) and related mass

spectra (MS/MS) (Lenz et al., 2004a, Lenz et al., 2004b). Applications of LC-MS

metabolomics have been mainly focused on clinical applications with NMR as a

complementary tool. This screening technique allows some metabolite identification validation

by means of targeted analysis or with exact mass and / or MS/MS. LC-NMR-MS techniques

are currently being explored to aid in metabolite identification (NMR) together with sensitive

analyses (MS).

Direct-injection mass spectrometry (DIMS) is a high-throughput screening tool and involves

the injection or infusion of crude sample extracts into an ESI-MS resulting in a mass

spectrum, which is representative of the composition of the sample. As for LC-MS, the

metabolome coverage depends on the ability of the metabolite to be ionized and the mass


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spectrum or mass list (m/z vs. response) is used for sample classification. Due to the

occurrence of ionization suppression and the lack of sensitivity (limits of detection in the µM

range), this is more of a screening tool and not ideal for quantification. Metabolite

identification needs to be performed with high-resolution instruments and accurate mass

determination e.g. time-of-flight (TOF) instruments, however, the absence of chromatographic

separation can allow for peaks with differences in monoisotopic masses to overlap and hence

a reduction in the mass accuracy of accurate mass determinations. Since structural isomers

have the same monoisotopic mass, and cannot be detected separately, chromatographic

separation would be required.

Other MS-based techniques such as capillary electrophoresis-mass spectrometry (CE–MS)

have been employed to a lesser extent in analyzing the metabolome. Another field of interest

is the application of matrix-assisted laser desorption ionization (MALDI), laser desorption

ionization (LDI) or direct ionization on silicon (DIOS) to provide ionization of metabolite

solutions spotted directly onto a target plate, thus allowing minimal sample preparation and

high-throughput analysis.

Fourier transform-infrared spectroscopy (FTIR) is an analytical technique that enables the

rapid, nondestructive and high-throughput of a diverse range of samples. The principle of FT-

IR involves a sample being interrogated with light (or electromagnetic radiation). Chemical

bonds at specific wavelengths absorb this light and vibrate in one of a number of ways such

as stretching or bending vibrations, which can be correlated to single bonds or functional

groups of a molecule for the identification of an unknown compound.

NMR spectroscopy is a rapid, non-destructive, high-throughput method that requires minimal

sample preparation (Lindon et al., 2003) and is widely used in metabolomics (or

metabonomics). NMR spectroscopy occurs through the application of strong magnetic fields

and radio frequency (RF) pulses to the nuclei of atoms. For atoms with either an odd atomic

number (e.g. 1H) or odd mass number (e.g. 13C), the presence of a magnetic field will cause

the nucleus to possess spin, termed nuclear spin. Absorption of RF energy will then allow the

nuclei to be promoted from low-energy to high-energy spin states, and the subsequent

emission of radiation during the relaxation process is detected. Since the majority of

applications employ 1H (proton) NMR for clinical studies and, and because the majority of

known metabolites contain hydrogen atoms, the system is unbiased to particular metabolites.


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The NMR spectra (specifically the chemical shift) are complex and are dependent on the

effect of electron shielding. The chemical shift for 1H NMR is determined as the difference (in

ppm) between the resonance frequency of the observed proton and that of a reference proton

present in a reference compound. Thus, complex samples do not interfere with measured

intensity as ionization suppression does with ESI and quantification can be performed as the

signal intensity depends on the number of identical nuclei. In metabolomics, NMR

spectroscopy is mainly used as a high-throughput fingerprinting technique (Dunn and Ellis,

2005). In NMR spectroscopy, the signal intensity for all compounds is dependent on the

molar concentration and reproducibility is high even though the sensitivity is lower and more

sample is required (Choi et al., 2005). Although a smaller number of compounds are

measured in a single analysis for NMR than with the other methods, every compound that

has a proton will be detected. By using 2D-NMR the identification of compounds can be

facilitated and minor compounds can be better observed, even allowing for structural

elucidation of unknowns in crude extracts (Verpoorte et al., 2010). The 2D-J-resolved spectra

have been shown to be a very useful tool, which provides two types of information, namely a

plot of chemical shifts versus coupling constants (Choi et al., 2006; Kim et al., 2010).

4.2. Multivariate data analysis (MVDA)

Changes in metabolite levels may be dramatic, which are easily recognizable, or subtle,

which requires statistical processing to determine if these changes are significant or not

(Miller and Miller, 2000). The purpose of this analysis is for the characterization of data

structure and preliminary mining of significant tendencies included in the data. Multivariate

analysis includes multiple regression, discriminate analysis, principal component analysis

(PCA), hierarchical cluster analysis (HCA), factor analysis, and canonical analysis (Fukusaki

and Kobayashi, 2005). PCA, HCA and self-organizing mapping (SOM, also known as

Kohonen neural networks) are the most commonly used multivariate analysis methods.

Other multivariate methods available for plant metabolomics are soft independent modeling of

class analogy (SIMCA-P), K-nearest neighbor (KNN) and k-mean cluster analysis (kMN).

SIMCA is a useful method for large sample numbers and for the classification and prediction

of unknown samples by means of the principal component models for each group of known


- 98 -

samples, whereas KNN and kMN allow for sample classification (Fukusaki and Kobayashi,

2005).

However, even though these tools are available, the biggest challenge of metabolomics for

bioinformatics is the lack of appropriate databases and data exchange formats (Sumner et

al., 2003). Metabolomics generates large volumes of data that require specialized

bioinformatics and data mining tools to gain knowledge. It requires automated raw data

processing software that can manage data from various instruments (which each have their

expected limitations), extensive mass spectral libraries and powerful database management

systems that can store both raw and metadata (Shulaev et al., 2008). Reproducibility is the

most important criteria for developing a metabolomics technology platform; other factors

include the ease of quantification and identification, the number of metabolites that can be

evaluated and time, including sample preparation (Choi et al., 2005).

5. Significance of this study

The purpose of this study was to qualitatively and quantitatively analyze the changes in

secondary metabolites in C. asiatica cell suspensions, subsequent to exogenous MeJa

treatment by means of chromatographic techniques and multivariate statistical models, given

that metabolic pathways are manipulated through the application of chemical elicitors. This

chapter focuses on the changes in the occurrence of four targeted triterpenoids, namely

asiatic acid, madecassic acid, asiaticoside and madecassoside after MeJa treatments. It is

hypothesized that a new / altered metabolic profile (with an enhanced secondary metabolite

profile) will be attained for C. asiatica cell suspensions since the potential exists for new and

novel defense-related metabolites to be synthesized. To date, a metabolic approach to MeJa

induced defense-related secondary metabolism in C. asiatica cell suspensions, using

metabolic tools, has previously not yet been described.

6. Materials and Methods

Homogeneous cell suspension cultures (for the large-scale culturing of plant cells) possess

the advantage of providing a continuous and reliable source of natural products from which


- 99 -

secondary metabolites are extracted (Bonfill et al., 2011). Plant cell suspensions have been

widely used in many areas of research, including gene expression (Axelos et al., 1992),

hormone action (Brault et al., 2004) and stress physiology (Duval et al., 2005; Hirai et al.,

2007). Cell suspensions offer the advantages of providing homogenous and rapidly growing

cells, together with the ease of controlling the environment, culture conditions and treatment.

6.1. Preparation and elicitation of the cell cultures

Cell suspensions were maintained in MS medium with vitamins supplemented with 1 µM 2,4-

D, 1 µM BAP, 30 g/L sucrose and 1 g/L casein hydrolysate (Bouhouche et al., 1998) on an

orbital shaker in the dark at 23 °C (Chapter 3, Section 3.2). For the experiments, 10 ml of the

homogenous cell suspension was subcultured with 40 ml fresh MS medium in 100 ml

Erlenmeyer flasks and grown for 10 d. This was to ensure that sufficient nutrients remain for

the culture during the extended stationary phase when secondary metabolite production

occurs.

Since a detailed time-course metabolic profiling analysis of plants subject to stress could lead

to the identification of many compounds (Shulaev et al., 2008) a concentration and time study

for MeJa exposure was investigated. For concentration studies, cell suspensions were

treated by the application of exogenous MeJa (4.33 M, Sigma-Aldrich, Munich, Germany) to

obtain final concentrations of 0.05 - 0.3 mM. In addition, a time study was done for 2, 4 and 7

d of treatments with the different concentrations of MeJa. Treatments with MeJa commenced

at day 10 of culture growth, when the cells had attained the stationary phase of the growth

curve (see Figure 5.2). After 17 days of culture, the cells enter the death phase of the growth

curve due to the accumulation of waste products and the deficit of nutrients (see viability

results). All results are based on 3 biological repeats, each with 3 analytical replicates (n= 9).

In addition, each analytical replicate was analyzed in triplicate to include technical repeats.

Non-treated C. asiatica cell suspensions were used as negative controls for the respective

time points. Since the cells were continuously grown in batch culture, even cell cultures not

treated with MeJa have altered metabolite profiles due to their changing environments.

https://mail.uj.ac.za/owa/#_ENREF_2






- 100 -

6.2. Cell viability assessment using the Alamar blue® assay

To verify that the cells were viable after treatment with MeJa, the Alamar blue® assay was

performed according to Byth et al. (2001). Whole cells were assessed by removing 200 µl of

the treated cell suspension and filtering off the medium. These cells were incubated with 180

µl 0.05 M sodium phosphate buffer, pH 7.4 and 20 µl Alamar blue® (AbD Serotec, United

Kingdom) for 1 h with gentle agitation in the dark. The cells were then sonified (50% power

for 30 sec) and centrifuged at 15 871 xg (13 000 rpm in an Eppendof 345-R centrifuge) for 10

min. The supernatant (180 µl) was transferred into a 24 (6 x 4) well microtiter plate and

readings were taken at an excitation wavelength of 540 nm and emission wavelength of 620

nm on a Fluoroscan (Ascent fluorimeter (AEC-Amersham)). A blank sample was prepared

with 20 µl Alamar blue® and 180 µl 0.05 M sodium phosphate buffer, pH 7.4.

6.3. Extraction of the secondary metabolites – triterpenoids

The crude ethanolic extracts for TLC analysis were prepared as described in Section 3.4.

Briefly, after the required growth period was achieved, the cell suspensions were filtered

through a 0.45 µm nylon membrane to remove the medium, and the cells were added to

absolute ethanol (Saarchem, South Africa) in a 1:3 (w/v) ratio. These were left overnight on

an orbital shaker and then the suspensions were centrifuged at 2200 xg for 20 min. The

supernatants were collected and vacuum dried to remove the excess solvent. The remaining

residue was reconstituted into 2 ml ultra-pure UPLC-grade methanol (Waters, Manchester,

UK) and filtered through a 0.22 µm filter into pre-labeled UPLC vials fitted with slitted caps

(Waters).

6.4. Partial characterization and fractionation of the extracted

triterpenoids

TLC analysis of the crude ethanolic extracts allowed for the partial characterization and

fractionation of the extracted C. asiatica metabolites. This was done as for Section 3.5. The


- 101 -

concentrated crude cell suspensions extracts were reconstituted in a minimal amount of

ethanol and applied to Silica gel 60 F254 TLC plates (20 x 20 cm, Merck, Darmstadt,

Germany). These were developed in a chloroform, glacial acetic acid, methanol and dH2O

(60:32:12:8 (v/v/v/v)) developing solution. The plates were visualized under UV light to

assess for any fluorescent compounds followed by detection of the triterpenoids by spraying

with anisaldehyde-sulphuric acid (AS) reagent as previously described (Chapter 4, Section

3.5).


spectrometry (UPLC-HDMS) analysis

UPLC and UPLC-HDMS analysis was performed on a Waters Acquity Ultra-Performance

Liquid Chromatography (UPLC) system coupled in tandem to a Waters photodiode array

(PDA) detector and a SYNAPT G1HDMS mass spectrometer (Waters, Milford, USA).

The UPLC-HDMS system is ideal for the analysis of small particles. Properties of a particular

compound, such as the accurate mass, fragmentation pattern, elemental composition, and

shape and isomer analysis can be obtained. UPLC makes use of sub 2 m particles (1.7 m)

presents many advantages such as enhanced efficiency, higher resolution and / or faster

analysis. Efficiency can also be significantly enhanced by coupling columns at elevated

temperature and using conventional HPLC system (Shaaban and Go´recki, 2011).

6.5.1. Experimental parameters

Chromatographic separation was achieved on a Waters Acquity UPLC column (BEH C18

2.1mm x 150 mm, 1.7 m). The volume of samples injected was 10 µl and gradient elution

was performed as described below. Under these conditions, madecassoside eluted at 6.58

min, asiaticoside at 7.1 min, madecassic acid at 9.94 min and asiatic acid at 11.4 min.

A binary solvent was utilized, consisting of H2O with 0.1% formic acid (Eluent A) and

acetonitrile with 0.1% formic acid (Eluent B). The initial conditions were 95% A at a flow rate


- 102 -

of 0.4 ml/min and kept constant for 1 min. A gradient elution was introduced to change the

chromatographic conditions (Table 6.2) and then the conditions were kept constant for 2 min

to flush the analytical column, followed by restoring the column to the initial conditions at 18

mins followed by equilibrium for 2 min. The total run time was 20 min and the injection volume

was 10 l. All measurements were done in triplicate to account for any analytic variability.

The PDA scan was from 200 to 500 nm (1.2 nm resolution) and with a sampling rate of 20

spectra per sec.

Table 6.2: UPLC conditions - gradient composition.

%A %B Curve Step

Initial 95.00 5.00 0 1.00 95.00 5.00 6 13.00 30.00 70.00 6 14.00 30.00 70.00 6 15.00 10.00 90.00 2 17.00 10.00 90.00 2 18.00 95.00 5.00 2 Wash 20.00 95.00 5.00 6 Equilibration

To detect compounds of interest for quantification, the SYNAPT G1 high definition mass

spectrometer was used. The MS parameters are given in Table 6.3. Both ESI positive and

negative mode for MS analysis was initially investigated, but following comparative

evaluation, all subsequent analyses were performed in ESI- mode.


- 103 -

Table 6.3: MS parameters.

6.5.2 Data analysis

Mass accuracy of all m/z values in all the acquired spectra were automatically corrected

based on calibration curves, lockmass and dynamic range extended (DRE), and the

molecular weight assignments were obtained using the MassLynxTM software. For

multivariate data analysis (MVDA), the raw data was analyzed with the following parameters

(Table 6.4).

MS VALUE Polarity Capillary voltage (kV) Sample cone voltage MCP detector voltage (V) Source temperature (ºC) Desolvation temperature (ºC) Cone gas flow (L/hr) Desolvation gas flow (L/hr) Analyzer m/z range Scan time (sec) Interscan time (sec) Data format Lockmass Lockmass flow rate (ml/min) Mass accuracy window (Da)

ES – / ES + 3 60 1700 120 450 50 800 V mode 100-1100 1 0.02 Centroid Leucine enkephalin (556.2614 µg/ml) 0.04 0.5

Molecular formula assignments software

MassLynx™ 4.1


- 104 -

Table 6.4: Elemental composition method for metabolite identification using MassLynxTM.

Property VALUE Number of results (maximum)

10

Mass tolerance (Da) Mass mode Electron state Elements C H O

1.5 Monoisotopic Even electron ion 1-50 0-100 0-20

For multivariate data analysis (MVDA), ESI negative data was extracted from MassLynxTM

and then analyzed using MarkerLynxTM. The processed dataset was then exported to the

SIMCA-P software version 12.0 (Umetrics, Umea, Sweden) to obtain PCA models (Table

6.5). The data was Pareto-scaled.


- 105 -

Table 6.5: Parameters for the MarkerLynx™ PCA method.

Property VALUE Initial retention time Final retention time Low mass High mass Mass tolerance (Da) Apex track parameters Peak width at 5% height (seconds) Peak to peak baseline noise Apply smoothing Collection parameters Intensity threshold (counts) Mass window Retention time window Noise elimination level Deisotope data

2.00 18.00 250.00 1000.00 0.5 1 0.00 Yes 10 0.05 0.20 4.00 Yes

PCA scores plots were used to depict the clustering of biological groups, either as controls or

MeJa treated samples. All results are based on 3 biological repeats, each with 3 analytical

replicates (n= 9). In addition, each analytical replicate was analyzed in triplicate to include

technical repeats. Non-treated C. asiatica cell suspensions were used as negative controls

for the respective time points.


- 106 -

7. Results

7.1. Cell viability assessment using the Alamar blue® assay

In order for the cell suspensions to be able to produce secondary metabolites by reacting to

the exogenous MeJa and triggering a response, the cells need to be viable. The viability of

whole cells was determined by means of the Alamar Blue® assay. In this assay, a redox

indicator that changes colour or fluorescence in response to metabolic activity is integrated in

order to assess viability quantitatively.

The Alamar blue® assay (Figure 6.3) after 7 d of treatment (essentially amounting to 17 d in

batch culture) showed a decrease in viability for all cells (control and MeJa treated), indicating

the death phase of the growth cycle. There is need for new nutrients to be introduced into the

suspension culture and the removal of the accumulated waste products. Thus, this time point

is disregarded as the untreated (control) cells only had a viability of 50%.

Figure 6.3: Viability assessment of C. asiatica cell suspensions after MeJa treatment using the Alamar Blue® assay. This graph shows the viability for 4 different concentrations of exogenously applied MeJa (0.05 – 0.3 mM) over 3 different time periods (2, 4 and 7 days, n= 8). The fluorescence values were converted to %viability by normalizing all the arbitrary fluorescence values to the value obtained for the control sample on day 2 of treatments after 10 d of growth. Error bars indicate standard deviations.

0

20

40

60

80

100

120

Control 0.05 0.1 0.2 0.3

Viab

ility

(%)

MeJa (mM)

Day 2

Day 4

Day 7


- 107 -

Although there is a decrease by approximately 20% in viability for the MeJa treated cell

suspensions, there is no significant difference between cells that have been exposed to

treatments for 2 or 4 d, nor the concentrations of MeJa added.

7.2. TLC analysis

Ethanolic C. asiatica extracts analyzed with TLC using chloroform, glacial acetic acid,

methanol and dH2O (60:32:12:8 (v/v/v/v)) as the mobile phase and AS reagent for detection

Figure 6.4). There was a increase in the amount of spots for the extracts after 4 d of

treatment with MeJa, especially those having Rf values correlating to asiatic- and madecassic

acid (near the migration front). Although fluorescing compounds were detected under UV

light, these did not correspond to any of the four targeted triterpenoids, nor did they react to

the AS spray which is sensitive to most functional groups, especially those which are strongly

and weakly nucleophilic such as phenols and sugars. The AS detection solution tends to be

insensitive to alkenes, alkynes and aromatic compounds unless other functional groups are

present in the molecules being analyzed (see Chapter 4).

Figure 6.4: TLC plates of crude C. asiatica extracts induced with different concentrations of MeJa for 2 (left) and 4 d (centre). Chloroform, glacial acetic acid, methanol and dH2O (60:32:12:8 (v/v/v/v)) was used as the mobile phase and AS reagent for detection. Fluorescing compounds are present in the C. asiatica extracts (right), that do not react to the AS reagent. More compounds are present after 4 d of treatment with MeJa than at 2 d. Treatments of the cell suspensions with MeJa were initiated at 10 d of growth.


- 108 -

Since a viability of 80% was maintained and 4 d of treatment showed a greater variety of

different metabolites, an exogenous treatment of 0.2 mM MeJa for 4 d was selected for

further experimentation. This concentration coincides with studies by Cusidó et al. (2002),

Palazón et al. (2003), Fritz et al. (2010) and Bonfill et al. (2011).


spectrometry (UPLC-HDMS) analysis

A targeted-metabolomic approach was adopted since the four main triterpenoids, asiatic acid,

madecassic acid, asiaticoside and madecassoside were the focus and a UPLC-MS platform

was used. Analysis of the standards (Extrasynthase, France) provided two parameters

(retention time, Rt and mass-to-charge ratio, m/z) that would enable the identification of these

compounds in the samples. Representative chromatograms of the standards are shown in

Figure 6.5.

ESI positive mode of MS analysis resulted in decreased sensitivity and produced an

extensive fragmentation pattern (results not shown). The same was seen by other

researchers (Li et al. 2005, Hanisa et al., 2012). Hence ESI negative was used as the

targeted metabolites were able to ionize and be detected in this mode.

Figure 6.5: UPLC-MS (BPI) chromatograms of the four authentic standards (Extrasynthase) each with a concentration of 200 ng/ml. A gradient elution was done with H2O with 0.1% formic acid as eluent A and acetonitrile with 0.1% formic acid as eluent B. The retention times (Rt) and m/z values are indicated for (A) asiatic acid (13.55min, 487.3428), (B) madecassic acid (12.47 min, 503.3404), (C) asiaticoside (10.14 min, 957.5087) and (D) madecassoside (9.63 min, 973.4989). The peak present at Rt = 12.95 mins (m/z 221.15) is not related to the standards but was present in the solvent blank as well.


- 110 -

MS spectra and the elemental composition of each standard were done to verify the

identification the metabolite.

The embedded methodologies of the MassLynxTM software (used on UPLC-HDMS) such as

isotopic fit (i-FIT) and rings-plus-double-bond equivalent (RDBE or simply DBE) provided a

level of certainty in calculating and selecting elemental compositions from MS data. The i-FIT

algorithm is a measure of the likelihood of a collection of peaks (cluster of mass ions) in the

spectrum matching a theoretical isotope mode and is calculated as the log-likelihood based

on a χ2 distribution and it scores zero for a perfect isotope match. DBE, on the other hand, is

an estimative value of the number of rings and double bonds or degree of unsaturation in a

chemical structure. The DBE is calculated based on the lowest valence state for each

element following the formula below (Kind and Fiehn, 2007; Moco et al., 2007; Castillo et al.,

2011).

RDBE = C + Si + ½ (H+ F+ Cl+ Br + I) + ½ (N+ P) +1

Representative MS spectra for these authentic standards are shown in Figures 6.6, 6.7, 6.8

and 6.9.

Figure 6.6: MS spectra of asiatic acid standard. Ion peak [M-H]- eluted at 13.55 min with m/z of 487.3428. Based on the spectrum, the formula calculated is C30H47O5, i-FIT of 0.4; DBE of 7.5; negative mode (one H+ less).


- 111 -

Figure 6.7: MS spectra of madecassic acid standard. Ion peak [M-H]- eluted at 12.47 min with m/z of 503.3389. Based on the spectrum, the formula from calculated elemental composition is C30H47O6, i-FIT of 0.5; DBE of 7.5; negative mode (one H+ less).

Figure 6.9: MS spectra of madecassoside standard. Ion peak [M-H]- eluted at 9.63 min with m/z of 973.5045. Based on the spectrum, the formula calculated is C48H77O20, i-FIT of 0.9 and a DBE value of 10.5. The following Rt values were obtained which correlates with the more polar glycosides eluting

first, followed by the more non-polar aglycones (Table 6.6).

Figure 6.8: MS spectra of asiaticoside standard. Ion peak [M-H]- eluted at 10.14 min with m/z of 957.5092. Based on the spectrum, the formula calculated is C48H77O19, i-FIT of 0.9 and a DBE value of 10.5.


- 112 -

Table 6.6: Rt values obtained for the four targeted triterpenoids analyzed separately.

A mixture of the four standards was also analyzed and showed consistent Rt and m/z values

(Figure 6.10). Thus the method that was developed for analysis could be used for the

ethanolic C. asiatica plant extracts.

Figure 6.10: UPLC-MS (BPI) chromatogram of the four authentic standards (Extrasynthase) mixed together in equimolar concentrations (each with a final concentration of 1mM final). The same protocol was applied as described in Materials and Methods with a gradient elution. The 4 targeted triterpenoids were identified based on the values obtained for Rt and m/z.

Equimolar concentrations (final concentration of 1 mM) for each of the 4 authentic standards

were analysed together (Figure 6.10) and it can be seen that asiatic acid and then

asiaticoside have the highest intensities under these elution conditions. The same was found

for the TLC densitometry analysis of these 2 metabolites (Figure 4.2).

The effect of MeJA on secondary metabolism of C. asiatica cell suspensions was investigated

by visual inspections of chromatograms of the treated and non-treated samples. Differences

were observed between the chromatograms, BPI and PDA (Figures 6.11 and 6.15) for the

control and treated samples for all the time points (2, 4 and 7 d) with 0.2 mM MeJa.

Triterpenoid Molecular formula

[M-H]- Rt (min)

m/z (MS ESI-)

Madecassoside Asiaticoside Madecassic acid Asiatic acid

C48H78O20

C48H78O19 C30H48O6 C30H48O5

C48H77O20

C48H77O19

C30H47O6

C30H47O5

9.63

10.14

12.47

13.55

973.4989 957.5088 503.3404 487.3428


- 113 -

Treatments of the cell suspensions with MeJa were initiated at 10 d of growth in batch

culture.

Figure 6.11: UPLC-MS BPI chromatograms obtained for ethanolic extracts of C. asiatica cell suspensions under control (bottom, red chromatogram) and treated (top, green chromatogram) conditions. The treated cell suspensions were exposed to 0.2 mM MeJa for 2, 4 and 7 d (A, B and C respectively). Ion peaks corresponding to asiaticoside (Rt = 10.14 mins, m/z = 957.52) and asiatic acid (Rt = 13.52 mins, m/z = 487.34) are indicated as 1 and 2 respectively. Treatments of the cell suspensions with MeJa were initiated at 10 d of growth in batch culture. Since the cells are continuously adapting to the changing environment during growth, untreated samples (controls) at each time point were included.

2 d

4 d

7 d


- 114 -

A closer inspection of the BPI chromatograms of 0.2 mM MeJa treated and non-treated

samples (Figure 6.11) showed two treatment-related ion peaks (1 and 2) with Rt and m/z (ion

peak 1: Rt = 10.14 min, m/z = 957.52, ion peak 2: Rt = 13.52 min, m/z = 487.34) similar to

two of the targeted metabolites, namely asiaticoside and asiatic acid (Figure 6.5 and Table

6.6). The extracted ion chromatogram (EIC) confirms this by displaying the extracted m/z of

asiatic acid and asiaticoside respectively, from the full mass chromatogram of treated

samples (Figure 6.12).

Figure 6.12: Representative (A) extracted ion chromatogram (EIC) and (B) the full mass chromatogram of the extracts after 2 d treatments with 0.2 mM MeJa. The presence of asiaticoside (peak 1) and asiatic acid (peak 2) is indicated.

Furthermore, the MS-spectra (and elemental composition calculation) of these ion peaks 1

and 2 (Figure 6.13) matched those of asiaticoside and asiatic acid respectively (Figures 6.6

and 6.8). Visual analysis of the BPI chromatograms also revealed that these ion peaks (1 and

2) appear to be related to the MeJA treatment: the intensity of the ion peak 1 changes in

treated samples compared to the non-treated samples, and the ion peak 2 appears only in

the treated samples.


- 115 -

Figure 6.13: MS spectrum for (A) ion peak 1 and (B) ion peak 2 from Figure 6.11. The elemental composition is determined to confirm the identity of the metabolite. Based on the spectrum, the formula obtained is C48H17O19, i-FIT of 0.8; DBE of 10.5, this is indicative of asiaticoside (m/z 957.5067). Peak B has an elemental composition of C30H47O5, i-FIT of 0.6; DBE of 7.5 which is indicative of asiatic acid (m/z 487.3428).

In all the experiments for 4 d of treatment, the ion peak m/z 957.5067 was not visually

present in the BPI chromatograms; however an ion of m/z 252.0816 was prominently

displayed at Rt 10.13 min (Figure 6.11). The MS spectrum of the latter contains the peak m/z

957.5067 (Figure 6.14A). Further analysis (MS/MS experiment) was then performed and

indicated that the two ions of m/z 252.0816 and 957.5067, were actually two different ions /

compounds co-eluting at Rt 10.13 min (Figure 14 B and C) with the ion m/z 252.0816 being a

more intense peak (compared to m/z 957.5067). Thus, MS/MS confirmed that at day 4 the ion

peak of m/z 957.5067 (putatively identified as asiaticoside) is actually present but is not

visually displayed on the BPI chromatogram because it co-elutes with the ion of m/z 252.0816

which is of a high intensity.


- 116 -

Figure 6.14: ESI- mass spectra for (A) m/z 252 and 957 and MS/MS mass spectra for co-eluting ions at (B) m/z 252 and (C) m/z 957 to confirm the presence of asiaticoside in 0.2 mM MeJa treatments for 4 d.

Visual inspection of both UPLC-PDA and UPLC-MS-BPI chromatograms revealed time-

dependent variations for the ethanolic extracts of C. asiatica cell suspensions after treatments

with 0.2 mM MeJa for 2, 4 and 7 d. In Figure 6.15, the PDA chromatogram window from 9-11

mins showed prominent differences between the extracts from day 2-7. More visible changes

are seen after 4 d of exposure to 0.2 mM MeJa indicating that metabolic changes due to the

treatment are occurring. The overall decrease in peak intensity / absorbance observed for the


- 117 -

7 d extracts is probably due to the cells entering the death phase. As mentioned before

(Chapter 5, Section 3.6), the centellosides do not exhibit a significant absorbance in the UV

range and thus does not make a significant contribution to the absorbance spectra. Figure

6.16 of representative BPI chromatograms shows changes in the intensity and the presence /

absence of ion peaks. These changes / variations in chromatograms (in both PDA- and BPI

chromatograms, Figures 6.15 and 6.16) revealed time-dependent intracellular metabolic

reprogramming, as a response to MeJa treatment of the C. asiatica cells. However, it has to

be re-emphasized that the approach used in this study was a targeted metabolomic analysis,

with a focus on the four triterpenoids, namely asiaticoside, madecassoside and their

respective aglycones.

Figure 6.15: UPLC-PDA chromatograms (photodiode array range: 200 - 500 nm) showing time-dependent variations for ethanolic extracts of C. asiatica cells after treatments for 2, 4 and 7 d treated with 0.2 mM MeJa.


- 118 -

Figure 6.16: UPLC-MS (BPI) chromatograms of MeJa-treated ethanolic extracts from C. asiatica cell suspensions at day 2, 4 and 7. The targeted triterpenoids (asiatic acid and asiaticoside, encircled in blue) and representative new peaks due to the treatment are encircled in orange.

The UPLC-MS data were further analysed by PCA modelling to highlight the MeJA-induced

metabolic changes. PCA is an unsupervised multivariate linear method used to identify

patterns in data and express the data in such a way to highlight the similarities and

differences. The quality of the PCA models are evaluated based on model diagnostic tools

such as (i) the cumulative modelled variation in matrix X, R2X(cum) (known also as the

goodness-of-fit parameter) and (ii) the predictive ability parameter, Q2(cum): the fraction of

the total variation of matrix X that can be predicted by the extracted components. For a robust

mathematical model with a reliable predictive accuracy, the values of these diagnostic

parameters must be close to 1.0 (or above 0.5) and the difference between them must be

less than 0.2. The PCA scores plot gives visual information about sample variations, and the

PCA loadings scatter plot explains the variation in scores and illustrates the putative

discriminating variables responsible for sample clustering. Since PCA is a non-parametric

analysis, the generated model is independent of the user, hence unsupervised (Goodacre et

al., 2004; Trygg et al., 2007; Boccard et al., 2007).


- 119 -

Thus, the data matrix obtained from MarkerLynx XS™ processing was exported into the

SIMCA-P12 software for PCA modelling. A five-component model was computed and

explained 84.1% of the total variation in the X matrix [R2X(cum) of 0.841] with the goodness

of prediction [Q2(cum)] of 0.657. A scores plot was constructed using the first two

components (PC1 and PC2, explaining 58.2% of the variance), showing samples differentially

clustered into different groups with minimal variation within groups (Figure 6.17): all the non-

treated samples (controls) are grouped together and significantly separated from treated

samples; the 0.2 mM MeJA-treated samples are differentially grouped into three clusters

corresponding to the three time points (2, 4, and 7 d). Since PCA clustering is based on a

large number of analytes / data points, the clustering of the controls cannot be based solely

on the absence of MeJa in comparison to the treated samples. The PCA scores plot

illustrates the variation within the groups along the y-axis and the variation between the

groups on the x- axis.

Figure 6.17: PCA scores plot of a representative experiment. A 5-component model explains 84.1% variation. The above scores plot is computed using the first two components (PC1 and PC2) which explain 58.2% of the variation. All control samples are clustered together, with R2X(cum) = 0.841 and Q2(cum) = 0.657.

The PCA loading plot (Figure 6.18) shows that ions of m/z 958.5088 (Rt 10.13 min) and

487.3418 (Rt 13.52 min) are discriminating ions contributing to the sample clustering in the

scores plot. These ions have Rt and m/z similar to those of ion peaks 1 and 2 (respectively)

from Figure 6.11, and they were putatively identified as asiaticoside and asiatic acid,

respectively (Figure 6.12). A list of selected discriminatory ions (from the PCA loadings plot)


- 120 -

is given in Table 6.7. For most of these ions, only the calculated empirical formula is

presented here, since this study focussed on the 4 targeted metabolites, as mentioned

previously.

Figure 6.18: Representative PCA loading plot for all time points (2, 4 and 7 d). The m/z 487.3418 (Rt =13.52 min) associated with asiatic acid (blue circle) and m/z 957.5088 (Rt = 10.13 min) relating to asiaticoside (red circle), contributes to the clustering of the samples.

Table 6.7: Table for discriminating ions (from Figure 6.18)

Rt (min) m/z

Empirical formula Compound name

9.79 9.89 10.13 11.00 12.91 13.52

212.1384 209.1172 957.5058 573.1284 221.1530 487.3418

C12H20O3 C12H18O3 C48H78O19 C27H26O14 C14H22O2 C30H48O5

Jasmonic acid Asiaticoside Rishitin Asiatic acid

The quantitative analysis of the MeJA effect on secondary metabolism of C. asiatica cell

suspensions was carried out by quantifying the targeted triterpenoids (asiaticoside,


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madecassoside, asiatic acid and madecassic acid) in the ethanolic extracts from the control

and treated cell suspensions. The standard curves (Figure 6.19) were constructed from the

UPLC-MS analyses of the four standards. Peak area was estimated using mean-based peak

integration, and QuanLynx™ 4.1 of the MassLynxTM software was used for automated

quantification. The concentration values for triterpenoids of interest were expected to be in

the low ng/ml region (Table 6.8).

Table 6.8: Estimated concentration of the targeted triterpenoids ( g/g fw).

Triterpenoid Rt (min) M z Time

point (day)

Concentration ( g/g fw)

Control samples Treated samples

Madecassoside 9.63 973.51 2 0.186 0.036 0.524 0.296

4 0.216 0.042 0.276 0.078 7 0.156 0.044 0.835 0.058

Asiaticoside 10.14 957.51 2 0.708 0.112 3.305 2.804 4 ** 0.381 0.427 2.176 1.873

7 0.396 0.265 7.463 0.233

Madecassic acid 12.47 503.33 2 0.151 0.011 0.824 0.143 4 0.193 0.017 0.498 0.015 7 0.194 0.017 0.186 0.036

Asiatic acid 13.55 487.34 2 0.023 0.005 1.213 0.042 4 0.071 0.003 0.675 0.018 7 0.017 0.003 1.302 0.016

** not an absolute value due to co-elution of two ions at m/z 252.0784 and 95.5011 at Rt 10.13 min

Figure 6.19: Standard curves for asiaticoside (A), madecassoside (B), asiatic acid (C) and madecassic acid (D) obtained by UPLC-MS analysis. A concentration range of 0 – 1 000 ng/ml was used and each point on the curve is indicative of triplicate values. Peak areas representing asiaticoside, madecassoside, asiatic- and madecassic acid respectively, was quantified automatically by means of QuanLynx™ and the procedure of mean smoothing / filtering was applied.

C

A B

C D

[Asiaticoside], ng/ml [Madecassoside], ng/ml

[Asiatic acid], ng/ml [Madecassic acid], ng/ml

R2 = 0.999

R2 = 0.999

R2 = 0.998

Are

a A

rea

Are

a A

rea

R2 = 0.973


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8. Discussion

Extracts from C. asiatica are widely used in the food, health and cosmetic industries and due

to their commercial importance, there is great interest to enhance the production of these

plant secondary metabolites through biotechnology (Bonfill et al., 2011). Studies have

indicated that many plant tissue cultures are stimulated by elicitors and that secondary

metabolite accumulation occurs rapidly in response to elicitors (Eilert et al., 1987; Mukundan

and Hjortso, 1990; Ning et al., 1994; Collin, 2001). Cell suspension cultures provide a

continuous and reliable source of natural products, from which secondary metabolites can be

extracted, and the production of these secondary metabolites (in particular the centelloids),

can be further enhanced by the application of exogenous MeJa (Kim et al,. 2004; Mangas et

al., 2006; Bonfill et al., 2011).

The active metabolites under investigation were the centelloids (asiaticoside, madecassoside,

asiatic- and madecassic acid) and the aim was to increase the production of these

pentacyclic triterpenoids in C. asiatica cell suspensions. This was done by establishing viable

cell suspensions and after 10 d into the growth cycle, when the production of secondary

metabolites occurred as the stationary phase of the growth cycle was achieved, treating these

cell suspensions with 3 different concentrations of MeJa namely, 0.1, 0.2 and 0.3 mM for 3

different time points (2, 4 and 7 d).

By means of the Alamar blue® assay, the cells were shown to be 80% viable after treatments

for 2 and 4 d but longer incubation periods exhibited >50% decline in viability (Figure 6.3) due

to nutrient depletion and, possibly, the stress of elicitation. Similar results for viability were

reported by Bonfill et al. (2011). In addition to its role in defense responses, MeJa is also an

important signalling molecule that is reported to be able to promote plant senescence, a

process consisting of deterioration events that ultimately leads to death (Noodén, 1988),

usually in leaves and flowers.

An increase in the number of metabolites detected by AS spray (which is specific for

saponins) was observed for TLC after 4 d of treatment with 0.2 mM MeJa (Figure 6.4). Zones

corresponding to asiatic- and madecassic acid were more visible after 4 d of treatment. This

can be ascribed to the fact that MeJa is a key signal molecule that is utilized as an elicitor and


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it induces the accumulation of triterpenoid accumulation in plants (Yoo et al., 2011).

Quantification of these triterpenoids by means of densitometry (Chapter 4) was unsuccessful

because of the low concentrations of triterpenoids present in the ethanolic extracts of C.

asiatica cell suspensions. Kiong et al. (2005) found that C. asiatica cell suspensions

contained the lowest amounts of triterpenes and suggested that contributing factors for low

triterpene yield in cell suspensions of C. asiatica were due to possible secretion of the

triterpenes into the surrounding medium, as well as the degradation of the products. The

medium from the cell suspensions was also analysed on TLC plates (results not shown) and

it was found that it did not contain any of the investigated triterpenoids. Kim et al. (2004) also

did not find any release of asiaticoside from tissues of C. asiatica into the medium due to any

treatments. Fluorescent compounds were also detected on TLC plates (Figure 6.4), which

may have been enhanced by the chemical induction of MeJa, and be involved in plant

defence responses. These however, did not correspond to the targeted triterpenoids as they

do not possess significant absorbance properties which make them easily detectable in the

UV range.

Phytochemical techniques are rapidly developing, and tools are now available which allow for

the analysis of complex mixtures in novel ways. Enormous developments have occurred in

separation methods that have led to the development of hyphenated techniques such LC-MS,

which have become essential for the early detection and identification of new compounds in

crude plant extracts (Hostettmann and Marston, 2002). However, due to the chemical

complexity, the biological variance inherent in most living organisms and the range of

limitations for most instrumental approaches, no single technique offers the ability to gain a

complete overview of the metabolic complement of a plant (Sumner et al., 2003; Hall, 2005).

Previous work has been reported to increase the production of centelloids by means of MeJa,

in particular asiaticoside, in cell cultures (cell suspensions and calli) and whole plants and the

levels of asiaticoside, madecassoside, asiatic- and madecassic acid, have usually been

analysed by means of HPLC. The introduction of UPLC, which utilises columns with smaller

particle sizes and therefore higher pressures, has the benefit of improved chromatographic

separation and resolution. UPLC also allows for a more rapid analysis without the loss of

resolution (Croixmarie et al., 2009).

After centelloid screening by means of TLC, quantitative and qualitative analysis was

performed, through the use of UPLC-PDA and UPLC-MS. A method was developed for the


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separation of the targeted triterpenoids as per Table 6.2, to obtain distinctive Rt values for

targeted analysis. Under these conditions, madecassoside eluted at 6.58 min, asiaticoside at

7.1 min, madecassic acid at 9.94 min and asiatic acid at 11.4 min (Figure 6.5), and the

identities of these triterpenoids were confirmed by MS (Figures 6.6, 6.7, 6.8 and 6.9). These

authentic standards were then mixed in equal concentrations (final concentration of 1 mM for

each) to verify the reliability of the method and to confirm the characteristic Rt values for the

targeted triterpenoids (Figure 6.10) under these conditions.

UPLC-PDA chromatograms (Figure 6.15) confirm that the targeted triterpenoids does not

display significant absorbance in the UV range (which was seen on the TLC plates (Figure

6.4)) and thus does not make a significant contribution to the absorbance spectra. Visible

changes are seen after 4 d of exposure to 0.2 mM MeJa, indicating that the cells are reacting

to the treatment but the overall decrease in peak intensity observed for the 7 d extracts is

probably due to the cells entering the death phase.

UPLC-MS in the ESI- mode was used for further analyses, since ESI in the positive mode was

less sensitive and produced extensive fragmentation (Hanisa et al., 2012). Inspection of the

BPI chromatograms for 2 and 4 d clearly indicated that MeJa treatment induced differential

metabolic changes, and were exhibited as increases or decreases in peak intensities, new

peaks and peak suppression. This corresponds with the findings of Bonfill et al. (2011) who

established maximum centelloside production in the stationary phase at 15 d with 0.1 mM

MeJa-elicited cultures, showing a time lag between gene activation and centelloside

biosynthesis. Under the experimental conditions followed in this study, two of the targeted

metabolites were identified in the MeJa treated cell suspension extracts based on the

characteristic Rts, and the identity of asiaticoside and asiatic acid (Figure 6.12 and 6.13) was

confirmed by means of MS. The other two targeted triterpenoids (madecassoside and

madecassic acid) exhibited borderline detectability, possibly due to poor ionisation in the

samples or co-elution with other metabolites that might be present in the extracts. In general,

mass spectrometers are able to detect large numbers of compounds simultaneously, even if

they are co-eluting, selectivity is based on the identification and quantification of the

compound’s specific molecular masses and fragmentation patterns. Since mass

spectrometers can only detect ions, it is essential that there is perfect ionization of all

compounds throughout the chromatogram for quantification. Unfortunately this is not always

the case since one compound can hamper the ionization efficiency of another, even if these


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are chemically related and co-elute at the same time. Thus, ultimate separation is needed to

ensure robust quantification, especially for very complex mixtures consisting of hundreds of

components to be analysed and quantified (Tolstikov and Fiehn, 2002). This was true for an

ion of m/z 252.0816 which was prominently displayed at Rt 10.13 min (Figure 6.11). Further

analysis by MS/MS indicated that two ions were present (m/z 252.0816 and 957.5067) which

were two different compounds co-eluting at the same Rt (Figure 6.14). Thus, MS/MS

confirmed that at day 4, asiaticoside is present (ion peak of m/z 957.5067), but is not visually

displayed on the BPI chromatogram because it co-elutes with the ion of m/z 252.0816 which

is present in a higher concentration, and thus generates ions of a higher intensity. Due to the

high polarity, thermal labiality and low content in plants, losses in active constituents such as

such as flavonoids, triterpenoids, terpenes, and caffeic acid derivatives may also occur during

the processing steps (Li et al., 2005).

Chromatographic methods have been shown to be very useful for quantitative analysis;

however, there is a need for calibration curves for the compounds that are being quantified

since each compound gives a different detector response. Thus, the retention behaviour and

physical characteristics are tools for identification in qualitative analysis. Authentic standards

were separated using UPLC and detected using MS (as described in Materials and Methods)

in concentrations of 0 – 1 000 ng/ml, with the objective of quantifying the four centelloids in

the C. asiatica extracts, and investigating the changes in triterpenoid levels due to MeJa

treatment. Chromatograms for each of the standards are shown in Figure 6.4 and standard

curves were constructed using the peak area (Figure 6.19). A table of Rts for the authentic

triterpenoids and MW values are given in Table 6.6 when separated with the conditions

described. For quantitative purposes, metabolites with the characteristic Rt as the targeted

triterpenoid were isolated and automatic quantification was done by means of QuanLynx™

software. The estimated concentration of the four targeted triterpenoids is provided in Table

6.8 with asiaticoside being the main targeted metabolite present after treatment with 0.2 mM

MeJa. Prior to treatment with MeJa for 4 d, the concentrations of the targeted triterpenoids

were found to be in the region of 0.07 – 0.3 g/g fresh weight. There was a 5- and 9-fold

increase in the concentration of asiaticoside and asiatic acid respectively after elicitation, with

a 2-fold increase for madecassoside and madecassic acid. The exact concentration of

asiaticoside (m/z 957.50) on day 4 is subjective due to the co-elution of an ion with m/z of

252.08 at the same Rt, with a higher intensity on the UPLC-MS BPI chromatograms (Figure

6.11). The apparent absence of madecassoside and madecassic acid in the UPLC-MS BPI


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chromatograms is substantiated by their estimated low levels (<300 ng/ml). Findings by

Hanisa et al. (2012) reported the presence of only madecassic acid in extracts of leaf tissue

even though all four active triterpenoids were screened for. These authors suggested that

discriminatory results are due to different ions being reported for the different active

constituents. The undetectable madecassoside in the cell suspensions of C. asiatica

observed in this study corresponds to results obtained from other researchers (Kim et al.,

2004; Aziz et al., 2007). Kim et al. (2004) showed that the levels in whole plants are generally

low, even though whole plants derived from nodes were richer in asiaticoside than those

obtained from other plant material (Kim et al., 2002a). They suggested that asiaticoside

production is tissue specific and mainly occurs in the leaves, and this is indirectly confirmed

by the fact that the leaves of C. asiatica are used as material for various medical products

and health foods (Brinkhaus et al., 2000). Long et al. (2012) also described the co-occurrence

of madecassoside with asiaticoside in dried leaf samples, with asiaticoside as a main

compound together with other lesser prominent saponins. Nevertheless, the presence of

asiaticoside in undifferentiated cell suspensions of C. asiatica was described by Nath and

Bouragohain (2005). These researchers detected asiaticoside in callus and cell suspension

cultures of C. asiatica of Indian origin, but the quantity was less than that found in naturally

growing plants (Sholapur and Dasankoppa, 2011). The low secondary metabolite production

in culture cells could be due to the lack of single specialized cells, cell compartments and

tissues or specialized parts of organs that serve as the synthesis and storage sites for

secondary metabolites (Kiong et al. 2005), although tissue cultures are a more convenient

and reliable source of secondary products than intact plants and offer independence from

fluctuations in supply of the raw material (Collin, 2001).

The results showed that MeJa is able to trigger differential changes in the metabolome of C.

asiatica cells, leading to a change in the biosynthesis of secondary metabolites (Figure 6.11).

The method of analysis and the choice of a sample preparation method are extremely

important in metabolomic studies, as it affects both the observed metabolite content and

biological interpretation of the data (Vuckovic, 2012). It was established that not all the

targeted centelloids were detectable in the extracts from MeJa treated cells, and if they did

occur, they existed in changing / differing concentrations. To allow for minimal variations, not

only the growth stage, but the exact time of sampling should be controlled. The levels of

metabolites present in plants are dependent on the developmental stages in an individual

plant, and metabolites in leaves of different age are not the same (Abdel-Farid et al., 2009).


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Analytical methods and instruments should be decided on depending on the characteristics of

the target metabolites (including the number and amounts of these metabolites,

Fukusaki and Kobayashi, 2005). It has been generally observed that biological variability is

greater than analytical variability (Kim et al., 2010), even when controlled sampling and

sample preparation are employed (Roessner et al., 2000). In addition, chromatography in

combination with MS, suffers with problems of reproducibility, and calibration curves for all the

individual compounds are required for absolute quantification (Choi et al., 2006). Technical

variations can occur due to sampling, metabolite extraction and instrument platform variability

whereas platform variability can arise from variations in sample injection, LC conditions (such

as contaminant build up, column degradation and / or pressure / temperature changes), MS

conditions (ionization efficiency fluctuations, matrix effect, a decrease in the detector

sensitivity or source contamination) and detector electronic noise (Masson et al., 2011).

By means of UPLC-MS, highly complex data matrices are produced and through MVDA

models, the metabolite distribution patterns due to biological variations can be deciphered

(Madala et al., 2012). The use of chemometric tools e.g. PCA and OPLS-DA is of great

importance as these include efficient, validated and robust methods for modelling information-

rich chemical and biological data. PCA is a useful technique to reduce the dimensionality of

large data sets and it is an effective method to identify significant signals in noisy data.

Patterns in data can be identified and expressed in such a way as to highlight their

differences and similarities (T’Kindt et al., 2009). Since PCA is a non-parametric analysis, the

generated model is independent of the user, hence unsupervised (Guedes et al., 1980; Kuc

and Rush, 1985). In PCA, the targeted metabolites are used as an independent variable and

the amount of corresponding metabolite is used as a dependent variable. The use of PCA to

a metabolome data set provides two quantities namely, the score and the loading. The PCA

score is defined as the co-ordinate of data vectors in the base of the PCA

(Fukusaki and Kobayashi, 2005) and provides a visual image of the differences between

samples from a global view (Figure 6.17). The loading plot (Figure 6.18) is useful to evaluate

the contribution that each ion / individual metabolites makes to the total information of the

metabolome to be evaluated (Tugizimana et al., 2012). Metabolomic analysis by means of

PCA scores plots (Figure 6.17) showed clusters of sample replicates for 0.2 mM MeJa

treatments and time-dependent variation at 0, 2, 4 and 7 d. The samples were found to be

clustered into distinct groups corresponding to control and treated, with no significant intra-

group variation. A six-component model was computed for the concentration study data (ESI-)


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and explained 68.67% of the variance. The first two principal components (PC1 and PC2)

explain 39.39% of the variance. PC1 and PC2 were then used to graphically show the

variation in the data.

As mentioned, variations indicated by examination of UPLC BPI chromatograms (Figure

6.11and Figure 6.16) showed that the C. asiatica cells did respond to the application of

exogenous MeJa and that these responses caused alterations in cellular metabolite profiles.

These changes include variation in the concentration levels of the constitutively expressed /

synthesized metabolites, and also lead to the production of new metabolites. The compound-

identification approach involves comparing control and treated conditions and then extracting

the ion peaks that show differences (either in intensities or presence / absence). Mass

spectra of the extracted ion peaks can then be used to deduce the putative empirical

formulae of the compounds and databases such as the Dictionary of Natural Products

(www.dnp.chemnetbase.com) and ChemSpider (www.chemspider.com) can be consulted for

compound identification. Although MS allows for the determination of a molecular weight and

in the case of high resolution, the elemental composition, this is not always sufficient to

determine the structure (Verpoorte et al., 2007). A list of several discriminatory ions is

presented in Table 6.7 with asiatic acid (m/z 487.3418) and asiaticoside (m/z 958.5088)

contributing to the sample clustering (Figure 6.18). It is worth noting that at Rt 12.91 min and

m/z 221.1530, a molecule putatively identified as rishitin was found. This sesquiterpene

phytoalexin is known to be produced in cell cultures due to elicitation (Namdeo, 2007).

Sesquiterpenoids are known to share some of the metabolic pathways / steps with

triterpenoids (refer to the literature overview, Sections 3.1.1 and 3.1.2).

Lastly, the recent development of TOF-MS has increased the resolution of MS that has

become a standard when hyphenated with chromatographic techniques such as LC and GC.

Another technique frequently used in metabolomics analyses is NMR. Using NMR-based

metabolomics, Liang et al. (2006) also showed clear separation between MeJa treated and

non-treated Brassica rapa leaves from the PCA score plot. The potential use of metabolite

fingerprinting using NMR- based in combination with MVDA was reported by Maulidiani et al.

(2011) to discriminate between three Centella varieties, which could be distinguished based

on the presence of different classes of metabolites, that include the triterpenoids asiaticoside

and madecassoside and chlorogenic acids. Although NMR has the advantage of being able

to measure intact biomaterial non-destructively, the sensitivity of NMR spectroscopy is

http://www.dnp.chemnetbase.com/

http://www.chemspider.com/


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relatively poor, compared to MS methods, and the detection of potential targeted metabolites

may also be below detection limits.

9. Conclusion

This chapter provides a new and novel approach to investigate the effects of exogenous

MeJa as a signalling molecule and elicitation agent on C. asiatica cell suspensions, by means

of metabolic profiling and metabolomic techniques. The manipulation of the centelloid

metabolic profiles of C. asiatica cell suspensions by external addition of MeJa was found to

be feasible, with both quantitative and qualitative changes occurring. However, the use of cell

suspensions was associated with low concentrations of madecassic acid and

madecassoside. The combination of PCA with metabolic profiling clearly demonstrated the

metabolic changes occurred in the cells as a function of time through clustering of data values

obtained at 2, 4 and 7 d. Asiaticoside and asiatic acid were discriminatory biomarkers in the

treated extracts, confirming the increase in their concentration due to MeJa treatment. In

addition, preliminary data also indicated the presence of rishitin, a sesquiterpenoid

phytoalexin, in treated extracts. MeJa therefore seems to target not only the pentacyclic

triterpenoid pathway, but also other branches of the terpenoid metabolic tree in C. asiatica.

Anti-microbial effects of Centella asiatica

Chapter 7

Chapter 7-Anti-microbial effects

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1. Introduction 133

2. The development of antibiotics and the emergence of antibiotic resistance 134

3. Alternative therapies: Plants as a source of anti-microbial agents 136

4. The plant genus Centella: the medicinal value of Centella asiatica and

variations in triterpene production 137

5. C. asiatica as a source of ethnopharmacological agents: Biological activities

of C. asiatica triterpenoid saponins and sapogenins 139


6.1. Cytotoxicity evaluation 142

6.2. Isolation of PBMCs 143


extraction of C. asiatica centelloids 144

6.4. Investigation of potential cytotoxicity of ethanolic C. asiatica

extracts to PBMCs 145

6.4.1. Cell counting and trypan blue staining 145

6.4.2. Determining the cell viability after C. asiatica treatments

by means of colourimetric assays 146

6.4.2.1. XTT assay 147

6.4.2.2. MTT assay 148

6.5. Culturing the micro-organisms for the inhibition studies 149

6.7 Bioautographic screening for anti-microbial activity 150

6.6.1. Antibacterial and anti-mycobacterial screening 150

6.6.2. Antifungal screening 151

6.7. Quantification of the anti-microbial activity 151


7.1. Investigation into the potential cytotoxicity of ethanolic C. asiatica

extracts to PBMCs by means of the tetrazolium salts XTT and MTT 154

7.2. Anti-microbial screening 158

7.3. MIC Determinations 163

8. Conclusion 167


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C. asiatica is a medicinal plant that has been used for various medicinal and cosmetic

purposes, thus becoming an important commercial product (Bruneton, 1995). This tropical

plant has been used in Ayurvedic and traditional medicine in China, Malaysia and

Madagascar, not only for wound healing but general well being, as well, in addition to an anti-

bacterial and anti-viral agent. It has been demonstrated that C. asiatica exhibits potency

against fungal and bacterial pathogens (Hanawa et al., 1992; Cos et al., 2002; Oyedeji and

Afolayan, 2005). Although herbal medicines are perceived as being natural and therefore

harmless, the use of various medications without proper knowledge of the plant constituents

and possible toxicity can lead to accidents and fatalities. Thus, there is a need for potential

medicinal plants to be evaluated for safety and effectiveness. The potential cytotoxic effect in

mammalian cell cultures by extracts from C. asiatica was evaluated and abiity of these crude

plant extracts to inhibit micro-organisms was evaluated.

1. Introduction

The use of herbal products is on the increase due to the vast therapeutic potential thereof.

The use of plants as traditional medicines has been occurring for thousands of years and

continues to bring about new remedies. These plant-based medicines are available as crude

mixtures such as teas, powders and other herbal formulations, which now serve as a basis

for novel drug discovery (Jachak and Saklani, 2007). To utilize the full potential, methods of

standardization are required to validate authenticity, quality and purity. The usefulness of the

medicines is substantiated where the clinical effect is more effective than toxic (Shafiqul-

Islam et al., 2003).

Plant–based indigenous knowledge has been passed down from generation to generation,

and has contributed to the development of different traditional systems of medicine. It is

estimated that of the 250 000 flowering plant species that occur globally, half of these are

found in tropical forests and these plants have the potential to prove invaluable compounds

for the development of new drugs. To date, only about 1% of tropical species have been

studied for their pharmaceutical potential (Jachak and Saklani, 2007). The isolation and

characterization of pharmalogical active compounds from medicinal plants has been

designed for drug discovery techniques in an attempt to obtain some system of herbal

medicine standardization and to elucidate analytical marker compounds. The development of


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products with new pharmacological modes of action is required for new drug discoveries from

plants but this is associated with several obstacles. The dosage forms are not successful due

to absorption and therapeutical efficacy; drug discovery from plants has been time consuming

and faster and better methods are required for plant collection, bioassay screening and

compound isolation (Jachak and Saklani, 2007; Gertsch, 2009).

2. The development of antibiotics and the emergence of antibiotic

resistance

Since the early 1940s, antibiotics have been used to treat infectious diseases caused by

bacteria and other microbes. Anti-microbial chemotherapy has been a leading cause for the

dramatic rise of average life expectancy (Todar, 2008). For an antibacterial agent to be

successful in systematic use, it must be effectively absorbed and distributed in the host

system and have the ability to penetrate or concentrate in the micro-organism. It should have

a selective action on the target site within the cell, causing no damage to the host and kill the

pathogen before it can mutate and become resistant to it.

An antibiotic is defined as any substance of biological, semi-synthetic or synthetic origin that

shows selective activity against bacteria and is thus a part of a potential treatment for

infections. Antibiotic agents generally cure diseases by killing the causative agent through the

inhibition of a unique and vital function in the pathogen (Baqueo and Blazque, 1997).

Antibiotics are classified as bacteriocidal if they kill the susceptible bacteria or bacteriostatic if

it is able to reversibly inhibit the growth of bacteria. Antibiotics usually work in one of five

ways:

a) Inhibition of nucleic acid synthesis

b) Inhibition of protein synthesis

c) Interference with enzyme systems

d) A specific action on a bacterial cell membrane

e) A specific action on a bacterial cell wall (Burton and Engelkirk, 2000).


- 135 -

The first antibiotic, penicillin, discovered by Alexander Fleming came about after the inhibition

of staphylococci on an agar plate contaminated by Penicillium mould was observed. While

Flemming was working on penicillin, a German doctor, Gerard Domagk announced the

discovery of a synthetic molecule with antibacterial properties called sulfonamides or sulfa

drugs. Even though these sulfa drugs are not as effective as natural antibiotics, they are

widespread for the use in the treatment of many conditions such as urinary tract infections

and pneumonia (Todar, 2008).

In the late 1940s and early 1950s, new antibiotics were introduced, including

chloramphenicol, streptomycin and tetracycline. These were effective against a full arrange of

bacterial pathogens, including Gram-positive and Gram-negative bacteria, intracellular

parasites and tuberculosis bacilli.

Several mechanisms have evolved in bacteria which give them antibiotic resistance. These

mechanisms can either:

a) chemically modify the antibiotic though inactivation by a cellular enzyme in such a way

it no longer affects the micro-organism

b) render it inactive through physical removal (export) from the cell or

c) modify or alter the antibiotic target site so that it is not recognized by the

antibiotic.

Bacterial infections which contribute to most human diseases are also those that display the

most microbial resistance to antibiotics, which make them more difficult and expensive to

treat (Coleman, 1994). Gram-negative pathogens, such as Pseudomonas aeruginosa,

Enterobacteria cloaceae, Klebsiella pneumoniae and Stenotrophomonas maltophilia and

several of the most important Gram-positive pathogens such as Enterococci, Staphylococci

and Streptococci species are resistant to multiple antibiotic agents. They are responsible for

diseases such as puerperal sepsis, toxic shock syndrome, scarlet fever, pneumococcal

pneumonia, rheumatic fever, surgical wound infections and food poisoning (Barrett et al.,

1993).

The use of antibiotics has even been extended to the farming industry, but with the over- and

misuse of these antibiotics, the problem of antibiotic-resistant micro-organisms has occurred

(Viksveen, 2003). Between 20 and 50% of antibiotic prescriptions in community settings are


- 136 -

believed to be unnecessary and, in turn, bacteria and microbes that cause infections have

developed ways to resist antibiotics and other anti-microbial drugs.

Significant increase in the prevalence of resistance to antibiotics has been observed in

common human pathogens worldwide, with the consequences of increased morbidity,

mortality and cost in health care (Hellinger and Fleisman, 2000). In hospitals, about 70% of

the bacteria that cause infections are resistant to at least one of the drugs most commonly

used for treatment. The introduction of new antibiotics has not solved the problem. Previously

treatable infectious diseases such a tuberculosis, malaria and acute respiratory diseases

have become more difficult to treat and the drug resistant pathogens have further

complicated the treatment of such diseases in immunocompromised AIDS and cancer

patients (Chattopadhyay and Naik, 2007). It would seem appropriate that the potential for

other therapies should be investigated which claim to be effective in treating infectious

diseases and as a part of the struggle to reduce antibiotic resistance (Viksveen, 2003).

3. Alternative therapies: Plants as a source of anti-microbial

agents

An infection, caused by either bacteria or another micro-organism, occurs when the body’s

immune system is unable to protect itself effectively against the pathogenic influence. Many

symptoms that occur during infection are produced by the body itself, in an attempt to fight

the micro-organism, with the best known example being fever. Patients who are treated with

antibiotics for infections may become more susceptible to these and other micro-organisms

as a result of not launching an active / effective immune response and thus, those patients

who have been repeatedly treated with antibiotics may end up with a vicious cycle of

recurrent infections.

The basic principles of herbal medicines are to assist the body’s own healing mechanisms to

rid itself of disease (WHO, 2001). The use of natural medicines has been used for more than

200 years and the belief is that the correctly chosen herbal remedy will assist the body in its

own fight against a micro-organism, so that the body overcomes the infection and develops

own natural immune resistance to it.


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Medicinal plants have become useful and economically essential since they contain active

constituents that are utilized for the treatment of many human diseases (Stary and Hans,

1998). Such plant extracts have been further developed to be used as anti-microbial

substances (Del Campo et al., 2000). Various chemicals and secondary metabolites such as

tannins, alkaloids, cyanoglycosides, saponins, terpenoids, oleic and stearic acids which are

naturally present in plants have been implicated in the conferment of anti-microbial activities

on the plants containing them (Ingham, 1973; Osbourn, 1996).

The demand for more drugs from plant sources is continuously increasing and it is essential

to evaluate traditional medicinal plants for their therapeutic value (Sumanthi and Parathi,

2010).

4. The plant genus Centella: the medicinal value of Centella

asiatica and variations in triterpene production

The genus Centella compromises some 50 species, inhabiting tropical and sub-tropical

regions. The generic name has been derived from the Latin word “centum” which denotes

“hundred”, referring to abundantly branched prostrate herbs. This genus belonging to the

plant family Apiaceae (Umbelliferae) includes the most ubiquitous species Centella asiatica.

This perennial creeper flourishes abundantly in moist areas and is a small, herbaceous

annual plant of the subfamily Mackinlaya (Liu et al., 2003), previously included in Hydrocotyle

(Brinkhaus et al., 2000), occurring in swampy areas of India, Sri Lanka, Madagascar, Africa,

Australia (Schaneberg et al., 2003), China, Indonesia, Malaysia, Australia and Southern and

Central Africa (Verma et al., 1999). With the exception of four species, all are prevalent in the

Cape Floristic Region of South Africa (Karnitig and Hoffmann-Bohm, 1992; Schubert, 2000;

Figure 7.1).

http://en.wikipedia.org/wiki/Herbaceous

http://en.wikipedia.org/wiki/Annual_plant


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Figure 7.1: Global distribution map of Centella asiatica (adapted from Global biodiversity information faculty

information sheet).

The plant is clonally propagated by producing stolons that are characterized by long nodes

and internodes which bear crowded cordate, obicular or reniform leaves (Figure 7.2) and

sessile flowers in simple umbels (Zheng and Qin, 2007). Depending on environmental

conditions, the form and shape of the C. asiatica plant can differ greatly (Adamson, 1950). C.

asiatica, also known as Gotu kola or Indian pennywort (Bruneton, 1995), is a medicinal plant

that has probably been used since prehistoric times and has been reported to have been

used for various medicinal and cosmetic purposes, thus becoming an important commercial

product. This plant is listed as a drug in the Indian Herbal Pharmacopoeia, the German

Homeopathic Pharmacopoeia (GHP), the European Pharmacopoeia, and the Pharmacopoeia

of the People’s Republic of China (Schaneberg et al., 2003). According to World Health

Organisation (WHO) monographs, the main components of Herbae Centellae are the

triterpenes asiatic acid and madecassic acid and their derived triterpenes glycosides. The

asiaticoside and madecassoside content was no less than 2% as determined by means of

TLC and spectroscopic analysis (Kartnig, 1988).


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Figure 7.2: C. asiatica is a small creeping herb with shovel shaped leaves emerging alternately in clusters at the stem nodes. The runners lie along the ground and the inch long leaves with their scalloped edges rise above on long reddish petioles. The only other species of Centella, that have been reported to be of medicinal interest, are C.

glabrata and C. montana. Both have been used in the early days in the Cape as a remedy for

diarrhoea and dysenteries (Watt and Breyer-Brandwijk, 1962).

5. C. asiatica as a source of ethnopharmacological agents: Biological

activities of C. asiatica triterpenoid saponins and sapogenins

C. asiatica extracts have been used for many ailments which have led to successful

treatments (see Table 3.3). C. asiatica synthesizes triterpenoid saponins as secondary

metabolites as a part of normal growth and development. Interest in these molecules stems

from their medicinal properties, anti-microbial activity, and their likely role as determinants of

plant disease resistance (Haralampidis, 2002). Notable bioactive compounds are the

pentacyclic triterpene saponins madecassoside and asiaticoside and their sapogenins

(madecassic and asiatic acid, Table 3.1; Figure 4.1) These compounds are referred to as

centelloids / centellosides and proceed from the cyclisation of 2,3-oxidosqualene (Figure 2.7)

by a specific oxidosqualene cyclase (OSC), -amyrin synthase (Mangas et al., 2006).

Due to their medicinal properties, anti-microbial activity, and their likely role as determinants

of plant disease resistance, interest in these centelloid molecules has developed

(Haralampidis, 2002). Although classified as saponins, the saponin-like sugar esters of the

triterpenoid acids exhibit low hemolytic activity (Kartnig, 1988).

http://images.google.co.za/imgres?imgurl=http://www.greenchem.biz/Images/ProductImages/centellaAsiatica.jpg&imgrefurl=http://www.greenchem.biz/Images/ProductImages/pageproducts.php?no=25&usg=__e1C8vcPUd73CSt_Mm06INtXgT1g=&h=321&w=406&sz=19&hl=en&start=8&tbnid=DSQFtbVGNxWLwM:&tbnh=98&tbnw=124&prev=/images?q=centella&gbv=2&hl=en


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The bioactive constituents are known for their clinical effects in the treatment of chronic

venous diseases and wound healing disorders (Pointel et al., 1987, Montecchio et al., 1991).

Depending on the source of the plant used to manufacture the final formulation, many

accessible commercial formulations consist of madecassoside and asiaticoside in different

ratios (the ratio of madecassic acid to asiaticoside varies between 1.5 and 2.3 to 1, Table

7.1). The effects of undefined alcohol or aqueous extracts of C. asiatica, as well as defined

extracts have been used in pharmacological studies (Kartnig, 1988; Brinkhaus et al., 2000).

These include titrated extracts of C. asiatica (TECA), total triterpenoid fractions of C. asiatica

(TTFCA) and total triterpenoid fractions (TTF). TECA and TTFCA are combinations consisting

of 30% asiatic acid, 30% madecassic acid and 40% asiaticosides (40%) while TTF contains

60% asiatic- and madecassic acid in combination with 40% asiaticosides (Brinkhaus et al.,

2000). Table 7.1: Some of the product range of C. asiatica extracts indicating the specific chemical composition and treatment (WHO, 1999; Advanced Cosmeceutical Technology, 2006).

Extract Chemical composition Usage

Asiatic acid >95% Asiatic acid Anti-ageing cosmetics, application after laser

therapy, cosmeceutics

Titrated Extract of Centella

Asiatica (TECA)

55-66% Genins

34-44% Asiaticoside

Anti-cellulite, slimming products, breast creams,

stretch marks, scarred skin, anti-ageing cosmetics,

moisturizing care

TECA cosmetics >40% Genins

> 36% Asiaticoside

Anti-cellulite, slimming products, breast creams,

stretch marks, scarred skin, anti-ageing cosmetics,

moisturizing care

Heteroside >55% Madecassoside

>14% Asiaticoside

Slow release effect, anti-ageing cosmetics, for

moisturizing night-creams

Asiaticoside >95% Asiaticoside Anti-inflammatory, against irritated and reddened

skin, anti-allergic

Genins >25% Asiatic acid

>60% Madecassic acid

Natural antibiotic, antibacterial properties, for anti-

acne products, intimate hygiene

In addition to the applications mentioned in Table 7.1, C. asiatica extracts have been used for

many ailments which have led to successful treatments (Table 3.3). Although none of the

claims listed have been evaluated by the Food and Drug Administration (FDA), positive


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investigations have been done by various institutes and universities, which concluded that

more research on the pharmacological and bio-medical activities of C. asiatica is called for.

No recommended daily allowance (RDA) or dosage has been determined, but fresh leaves

have been used in salads, or dried leaves used to make tea. Supplements are usually

available in varying strengths and levels of purity. Crude preparations can cause allergic

responses and nausea has been reported in cases of high levels of intake. A toxic dose of

asiaticoside by intramuscular application to mice and rabbits was reported as 40-50 mg per

kg body weight (Abou-Chaar, 1963). In oral applications, 1 g of asiaticoside per kg body

weight has not proven toxic, and nearly all chemical trials have shown good tolerance by

patients to extracts from C. asiatica or asiaticoside (Abou-Chaar, 1963; Boely, 1975). Some

commercial products used in West Germany and France include Centasinum, Centelase,

Emdecassol and Madecassol (Kartnig, 1988; Brinkhaus et al., 2000). No cases of intolerance

were observed following injections of Madecassol preparations (Wolfram, 1965) which is a C.

asiatica extract comprising 40% asiaticoside, 29-30% asiatic acid and 1% madasiatic acid

(Brinkhaus et al., 2000).

The underlying mechanisms relating to the physiological effects of biologically active

components and the bioactivities of C. asiatica, are poorly understood (Zheng and Qin,

2007). Most triterpenoid compounds in adaptogenic and medicinal plants are found as

saponin glycosides. These sugars can be cleaved off in the gut by bacterial enzymes,

allowing the aglycone triterpenoids to be absorbed. Uptake can be followed by incorporation

into cell membranes and alterations in the composition thereby affecting membrane fluidity

and potentially affect signalling by many ligands and cofactors. In addition, the centelloids can

potentially inhibit enzymes specifically or non-specifically. Literature supplies numerous

examples of enzymes that can be inhibited by pentacyclic terpenoids, indicating the ability of

these compounds to act broadly in a non-specific mode on multiple targets. The mode of

enzyme inhibition seems to be non-specific and is based primarily on the hydrophobic

interaction with the hydrophobic domain of the target enzymes (Glinski and Branly, 2002).

In addition to the ethanol / water soluble centelloids, other factions of the plant can also

contain bioactive molecules. Analysis of essential oils from C. asiatica by Oyedeji and

Afolayan (2005), showed 11 monoterpenoid hydrocarbons, 9 oxygenated monoterpenoids, 14

sesquiterpenoid hydrocarbons, 5 oxygenated sesquiterpenoids and 1 sulfide sesquiterpenoid.

-Humulene (Figure 1.6), -caryophyllene, bicyclogermacrene, germacrene B and myrcene

http://en.wikipedia.org/wiki/Aglycone


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were the predominant constituents. This essential oil exhibited antibacterial activities against

Gram-positive and Gram-negative organisms. The essential oil composition reported by

Yoshinori et al. (1982), consisted of undetermined terpenic acetate while other prominent

constituents were -caryophyllene, trans -farnesene and germacrene D. In another study,

the volatile components in Centella were found to contain p-cymol, b-caryophyllene and

farnesene which exhibit mosquito repellent activity (Rajkumar and Jebanesan, 2007). Tests

for inhibition of rat lens aldose reductase indicate that flavanols such as kaempferol 3-O- -D-

glucuronide and petuletin may be additional active principles of this natural medicine

(Matsuda et al., 2001).

Due to the emergence of antibiotic resistance, it would seem appropriate to investigate the

potential of other therapies which are able to treat infectious diseases and contribute to the

effort to reduce antibiotic resistance. Research carried out on herbal / natural product

treatments of infectious diseases is encouraged due to it also being an environmentally

friendly alternative to antibiotics (Viksveen, 2003). In this chapter the anti-microbial claims of

C. asiatica will be evaluated as related to its ability to inhibit the growth of Gram-positive and

Gram-negative bacteria, a fungal and yeast culture.

6. Materials and Methods

6.1. Cytotoxicity evaluation

To test for potential C. asiatica cytotoxicity, peripheral blood mononuclear cells (PBMCs)

were obtained from blood donated by healthy volunteers from the University of

Johannesburg. All individuals were recruited after ethical approval by the WITS Ethics

Committee (University of the Witwatersrand, Johannesburg) and informed consent was

obtained.

6.2. Isolation of PBMCs

The cytotoxicity and viability of peripheral blood mononuclear cells (PMBCs) with and without

exposure to C. asiatica extracts at different concentrations for 4 and 7 d were investigated.


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RPMI media (Highveld Biologicals, South Africa) containing 2 mM L-glutamine, 1%

gentamycin sulphate (GS; Highveld Biologicals, Modderfontein, South Africa), other

antibiotics (from Adcock Ingram, Midrand, South Africa) such as penicillin C (10 mg/ml),

streptomycin sulphate (10 mg/ml) and fungizone (25 mg/ml) and 1 ng/ml purified human

interleukin-2 (IL-2, Sigma-Aldrich, Munich, Germany) was filter sterilised using 0.22 µm filters

and stored at 4 C. PMBCs were isolated from the blood of healthy adult female volunteers

that were seronegative. Blood was collected in EDTA vaccutainer tubes and the anti-

coagulant blood was diluted 50% with incomplete RPMI 1640 media and the PBMCs were

isolated by density gradient centrifugation using Ficoll-histopque (ICN Biomedicals Inc, Ohio,

USA). Briefly, the diluted blood was slowly layered on top of the Ficoll-histopaque with a ratio

of 2:1 blood-Ficoll. This was followed by centrifugation at 1912 xg (3000 rpm on a Beckman-

Coulter Allegra R25 swing bucket centrifuge) for 30 min at room temp. The cloudy layer of

cells was carefully removed and washed by the addition of RPMI 1640 media to a final

volume of 50 ml and centrifugation at 1028 xg (2200 rpm) for 10 mins. The supernatant was

decanted and the pellet was incubated for 5 min with 10 ml ammonium chloride potassium

(ACK) buffer (0.15 M NH4Cl, 0.001 M KHCO3 and 0.0001 M EDTA, pH 7.2) to lyse any

contaminating red blood cells that may be present. The cells were washed once again with 25

ml RPMI 1640 media and centrifuged at 212 xg (1000 rpm) for 10 mins. The pellet was

resuspended in 10 ml of complete media (RPMI 1640 media supplemented with 10% heat

inactivated (56 C for 30 min) fetal calf serum (FCS; Highveld Biological) with the addition of

60 l of a 1 mg/ml purified phytohemagglutinin from Phaseolus vulgaris (PH-A; Sigma-

Aldrich, St Louis, USA) stock solution. PH-A assists in cell proliferation.


extraction of C. asiatica centelloids

Callus originally initiated and maintained in agar solidified MS medium with vitamins

(Highveld Biologicals, South Africa; for more detail see Chapter 5, Section 3.2).

supplemented with 10 M 2,4-D, 10 M BAP, 30 g/L sucrose (Merck, Modderfontein, South

Africa) and 1 g/L casein hydrolysate (Merck, Modderfontein, South Africa) according to

Bouhouche et al. (1998), was transferred to sterile medium, made up as described before

without agar to initiate cell suspensions. All experimental work was done under strict aseptic


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conditions in a laminar flow cabinet. The cell suspensions were maintained on an orbital

rotary shaker at 23 C in the dark.

C. asiatica cells suspensions were induced with various concentrations of MeJa (Chapter 6).

A ratio of 1 g filtered cells to every 3 ml 100% ethanol was used. The extracting solvent was

added to the filtered cells, homogenized for 1 min and then centrifuged at 2200 xg in a

Beckman Allgera™25R swing bucket centrifuge for 20 mins. The supernatant was decanted

and the extracting solvent was evaporated off using a Buchi rotavapor apparatus at 45 C

under vacuum until all the ethanol (EtOH) was removed prior to exposure to the PBMCs.

These extracts were then used for analysis.

The concentrated ethanolic extracts were dissolved in minimal amount of dimethyl sulfoxide

(DMSO, Merck, Darmstadt, Germany). DMSO is the solvent of choice as it is miscible with

water and at concentrations lower than 3% (v/v) it is non-toxic (Cos et al., 2006). The final

concentration of this extract was 1.25 g/ml, ensuring that the DMSO concentrations were less

than the toxic amounts. These ethanolic extracts solubilised in DMSO will now be referred to

as EED.

A serial dilution was done with complete RPMI media to determine which EED plant extract

concentration(s) were non-toxic to the cells. The final concentrations of the extract tested

were at a concentration range of 4.875 - 625 g/ml.

Extracts obtained from cell suspensions treated with 0.2 mM methyl jasmonate (MeJA)

induced the most extensive variety of secondary compounds after 4 d (as determined by TLC

analysis – Chapter 6). These extracts were used for the cytotoxicity assays as it was

assumed the highest concentration and variety of compounds would be present for the

screening. These ethanol extracted metabolites were concentrated to dryness and then

redissolved and diluted in the appropriate media as described.


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6.4. Investigation of potential cytotoxicity of C. asiatica ethanolic

extracts to PBMCs

After the PMBCs were stimulated for 3 d (72 h) in humidified air at 37 C and 5% CO2 with 2

g/ml purified PH-A, the cell suspension was centrifuged at 212 xg (1000 rpm) for 10 mins.

The resulting pellet was resuspended in 10 ml fresh complete RPMI medium.

6.4.1. Cell counting and trypan blue staining

The cell viability and cell concentration was determined by means of the trypan blue

exclusion method and a haemocytometer (cell counting chamber). Trypan blue is a stain

used to selectively colour dead cells blue while live cells with intact cell membranes are not

coloured. An aliquot of cells (20 µl) was added to 180 µl trypan blue solution (0.4% solution in

0.85% saline, Sigma-Aldrich, St Louis, USA), mixed and 10 l of the cell-stain mixture was

loaded onto the haemocytometer. The cells were counted within 5 mins.

% Viability = number of live cells x 100

Total number of cells counted

[Cell] = number of live cells x dilution factor x 10 000

Number of quadrants counted

Once the cells were determined to be viable and the cell concentration was calculated, the

original stock of cells was diluted in complete RPMI medium to give a concentration of 1 x 106

cells/ml.

The bioassays were done in microtiter plates with 100 µl of the EED C. asiatica extract added

to 100 µl PBMCs (concentration of 1 x 106 cells/ml in complete RPMI media). The control well

contained 100 µl RPMI medium and 100 µl cells. The plates were incubated at 37 C in a 5%

CO2 incubator for 4 and 7 d.

http://en.wikipedia.org/wiki/Cell_(biology)

http://en.wikipedia.org/wiki/Cell_membrane


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6.4.2. Determining the cell viability after C. asiatica treatments by

means of colourimetric assays

Various assays have been developed to investigate cellular viability. Some of these assays

rely on the integrity of the plasmalemma of the cell and others reflect not only the cell

membrane integrity but also the cellular metabolic state. Such include the reduction of

tetrazolium salts into formazan pigments that is caused by the enzymes in the mitochondria

(Kairo et al., 1999). The most frequently used of these dyes are 3-(4,5-dimethylethiazol-2yl)-

2,5-diphenyltetrazolium bromide (MTT) and 2,2-bis-(methoxy-4-nitro-5-sulphophenyl)-2H-

tetrazolium-5-carboxanilide (XTT).

MTT is a non-soluble single reagent system based on the in situ metabolic reduction or

cleavage of the yellow tetrazolium salt by mitochondrial dehydrogenase (succinate-

tetrazolium reductase) in living cells to produce an insoluble purple formazan crystal (Mshana

et al., 1998; Kairo et al., 1999). The produced crystals are solubilised with DMSO or acidified

alcohol which destroys the cells and allows only a single measurement. In the XTT assay the

metabolically active cells reduce XTT to a red formazan product (Figure 7.3).


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Figure 7.3: Metabolism / reduction of XTT to a water soluble formazan salt by viable cells. The assay is based on the cleavage of the yellow tetrazolium salt XTT to form a red formazan dye by metabolic active cells (adapted from Dojindo Laboratories catalogue, Japan).

The absorbance of the formazan products can be determined spectrophotometrically and

both assays measure the metabolic activity of viable cells, are non-radioactive and suitable

for measuring cell proliferation, cell viability or cytotoxity.

6.4.2.1. XTT assay

A colourimetric XTT-based cell proliferation assay was used to determine the cytotoxic effect

of the extracts on the cells after 4 d. This assay is designed for the spectrophotometric

quantification of cell growth and viability. It is used for the measurement of cell proliferation in

response to growth factors, cytokines and nutrients. It can also be used for the quantitive

measurement of cytotoxicity (Roche, cell proliferation kit II (XTT) pamphlet).

After the plates were prepared and incubated as described earlier in Section 6.4.1, 150 µl of

the supernatant from each well was carefully removed and discarded. To each well, 50 µl


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XTT reagent (1 mg/ml XTT (Roche) with 0.383 mg/ml PMS in sterile RPMI 1640 media) was

added. The plates were left overnight at 37 C in a CO2 incubator. The absorbance of the

plates was determined at 450 nm (reference at 690 nm) and the amount of XTT reduction

was calculated.

% Viability = Sample containing extract A540nm-A690nm x 100

(XTT) control sample A540nm-A690nm

6.4.2.2. MTT assay

The 96-well microtitre plate was prepared as for Section 6.4.1, where 100 µl of EED C.

asiatica extract was added to 100 µl PBMCs (1 x 106 cells/ml in complete RPMI medium) and

left for 4 d. The control well contained 100 µl RPMI medium and 100 µl cells. Complete

medium (sterile RPMI 1640 media with 10% FCS) was plated in the outer wells of the plate

and served as the blank. To each well of the microtiter plate, 20 l of MTT solution was

added. MTT was prepared at a concentration of 5 mg/ml in phosphate buffered saline (PBS)

solution, filtered through a 0.2 M pore membrane and stored at 4 C. The plate was

incubated at 37 C in a CO2 incubator. After 4 h of incubation, solubilization of the formazan

crystals was achieved by adding 100 l of 0.1 N HCl in isopropanol. Complete dissolution of

crystals was achieved by placing the plate on a shaker for 10 min. The optical density was

measured on a 96-well plate reader (Labsystems Multiscan MS, Helsinki, Finland) with a filter

setting at 540 nm (the reference filter setting was 690 nm). The absorbance at 690 nm was

subtracted from the absorbance at 450 nm to eliminate the influence of non-specific

absorbance (Liu and Schubert, 1997). Control wells were included from the test absorbance

in each assay.

% MTT reduction = Sample containing extract A540nm-A690nm x 100

Viability (MTT) control sample A540nm-A690nm


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6.5. Culturing the micro-organisms for the inhibition studies

Representative stains of Gram-negative bacteria (Pseudomonas aeruginosa (ATTC 9027)

and Escherichia coli (ATTC 1175)), Gram-positive bacteria (Bacillus subtilis (ATTC 6051),

Enterococcus faecalis (ATTC 29212) and Mycobacterium smegmatis, yeast (Candida

albicans (ATTC 10231)) and fungi (Cladosporum cucumerninum) was used as test

organisms.

E. coli was stored in LB media (10 g/L Bacto-tryptone, 5 g/L Bacto-yeast extract, 5 g/L NaCl,

pH 7.0), and M. smegmatis was stored in Bacto Middelbrook 7H9 broth (Difco, Nevada, USA)

with 50% glycerol (Unilab, South Africa). All the other bacterial stains (Gram-positive and

Gram-negative) and C. albicans were stored as frozen stocks in tryptone soy broth (TSB,

Biolab, Merck, Darmstadt, Germany). All stocks were supplemented with 50% glycerol and

kept at -20 C.

C. cucumerinum was grown and maintained on V8 media plates (150 ml V8 Vegetable juice

(Campbell’s, New Jersey, USA), 3 g CaCO3, 15 g agar (Sigma-Aldrich, Munich, Germany),

850 ml H2O).

Before the antibacterial / yeast screening, 40 ml of the respective media was inoculated with

1 ml of the stock organism and sustained on a shaking incubator at 37 C overnight. The A600

readings were determined and all cultures were diluted in media prior to carrying out the

experiment to a working solution which provided A600 readings of approximately 0.2

absorbance units.

A glucose mineral salt medium was prepared with 7 g KH2PO3, 3 g Na2HPO4 2H2O, 4 g

KNO3, 1 g MgSO4 7H2O and 1 g NaCl per litre dH2O. This solution was autoclaved for 20 min

at 120 C. Under sterile conditions, 10 ml of 30% glucose (D+ glucose monohydrate,

Saarchem) was added. A fungal spore suspension of C. cucumerinum was prepared by

adding 100 ml of the glucose mineral salt to a Petri dish containing an established,

sporulating C. cucumerinum culture. Due to the hydrophobicity of the fungal spores, a few

drops of a wetting agent (Insure, Effekto, South Africa) were added to increase the “solubility”

of the spores. This suspension was filtered through miracloth prior to application.


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6.6. Bioautographic screening for anti-microbial activity

One hundred microlitres (100 l) of the ethanolic C. asiatica extracts from cell suspensions

(control and MeJA induced) were applied to silica gel 60 F254 TLC plates (20 x 20 cm, Merck,

Darmstadt, Germany). In addition, 100 l of the C. asiatica ethanolic leaf extracts (prepared

as described in Chapter 4, Section 3.3) and 100 l of a commercially available C. asiatica (10

mg/ml, Linnea, Locarno, Switzerland) was also assayed. This commercial extract is available

as a powder which contains between 36-44% asiaticoside and between 56-64% of other

genins (by HPLC analysis). These TLC plates were developed in an ethyl acetate / methanol

/ H2O (80:10:10 (v/v/v)) developing solution. The developed TLC plates were allowed to dry

for complete removal of the remaining solvents.

6.6.1. Antibacterial and anti-mycobacterial screening

A simple and direct assay on TLC plates was used to screen for compounds with activity

against the micro-organisms. The procedure as described by Hamburger and Cordell (1987)

was used with slight modifications to obtain optimal conditions. The principle of the assay

involves a suspension of a micro-organism in suitable growth medium being applied to a

developed TLC plate. Incubation occurs in a humid atmosphere and zones of inhibition are

visualized by the conversion of a tetrazolium salt by metabolically active bacteria into a

corresponding intensely coloured formazan product; thus antibacterial compounds appear as

clear spots against a coloured background.

The prepared bacterial suspensions (and C. albicans) was applied to the sides of the

developed TLC plate and spread with a glass rod until just wet. This method ensured an even

distribution of the organisms. Plates were incubated in a clean container lined with wet filter

paper for 8 h at 37 C. The plates were then sprayed with a 2 mg/ml solution of -

iodonitrotetrazolium violet (INT, 3-[4-iodo-phenyl]-2-[4-nitrophenyl]-5-phenyl-2H-tetrazolium

chloride, Sigma-Aldrich, Munich, Germany) and incubated in a sealed container overnight at

37 C for colour development. Bacterial growth was indicative of a reddish background

against which antibacterial compounds were identified as clear zones. Comparison with a

duplicate TLC plate developed under the same conditions but visualized with anisaldehyde-


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sulphuric acid (AS) reagent allowed for the detection of the separated compounds

(centelloids) with antibacterial properties present in the extracts.

6.6.2. Antifungal screening

Homans and Fushs (1970) descried a biography method based on the direct spraying and

growing of a fungal spore suspension on a developed TLC plate. This method was used to

screen for compounds in the C. asiatica ethanol plant extracts for antifungal activity. The TLC

plate was sprayed with a spore suspension of C. cucumerinum in a glucose-mineral salt

medium. Plates were incubated in a clean container lined with wet filter paper for 37 C and

examined at regular intervals for fungal growth – this was presented by a green lawn of

mycelial growth. Inhibition was observed as a clear zone indicative of reduced growth or lack

of growth.

6.6. Quantification of the anti-microbial activity

The minimum inhibition concentration (MIC) value was determined for extracts and each

organism. The MIC value was taken as the lowest concentration of the extract that inhibited

any bacterial growth after 24 h of incubation. The serial micro-dilution method described by

Eloff (1999) was done.

Two hundred microlitres (200 l) of each test solution was added to all the wells in the 2nd

column of a 96 (12x8) well microtitre plate. These were then serially diluted with media in a

1:1 ratio in the subsequent columns so that all wells contained a final volume of 200 l. Serial

dilutions of the following ethanolic extracts (which were concentrated to dryness and

dissolved in media) were tested in TSB / LB or Bacto-Middlebrook 7H9 medium (depending

on the organism studied):

a) A non-MeJa control from C. asiatica cell suspension (25 mg/ml)

b) MeJA induced C. asiatica cell suspension extract (25 mg/ml)

c) C. asiatica leaf extract (33 mg/ml) and


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d) commercially available C. asiatica extract (10 mg/ml, Linnea, Switzerland)

A higher concentration of the ethanolic cell suspension extracts was used as this was a very

crude extract in comparison to the crude commercial leaf extract from Linnea.

Control tests included were

a) 200 l of the diluted organism (A600 0.2) was added into each well of the first column

and

b) 200 l media was pipetted into the last test column of the 96-well microtiter plate.

c) Absolute ethanol was used to supply a non-growth control (the extracts were

concentrated to dryness and reconstituted in the respective media, so no trace of

ethanol should be present, thus ensuring that the test extracts were accountable for

the inhibitory effect).

All the wells received 100 l of the bacterial suspensions (A600 0.2), so that the final volume

was 300 µl in each well. To ensure no / minimal evaporation at 37ºC occurred; the plates

were covered with optical adhesive film.

The antibiotics neomycin sulphate (10 mg/ml, Sigma-Aldrich, Munich, Germany), gentamicin

sulfate (40 mg/ml, Micro Healthcare, South Africa) and kanamycin sulfate (500 mg/ml, Sigma-

Aldrich, St Louis, USA) for Gram-positive and Gram-negative bacteria and fluconazole (1H-1,

2, 4-trizole-1-ethanol, - (2,4-diflurophenyl)- (1-1,2-riazol-1-ylmethyl, 2 mg/ml, Pfizer,

Sandton, South Africa) for C. albicans were tested. Gentamycin sulphate and flucanazole

were purchased from a local pharmacy in the mentioned concentrations; neomycin sulphate

was purchase as a solution from Sigma-Aldrich and the concentration of kanamycin sulphate

was chosen as this exceeds the stock concentration of 50 mg/ml used in bacteriology.

The microplates were incubated at 37 C overnight. As an indication of growth, 40 l of INT (2

mg/ml) was added to the microplate and incubated at 37 C for 30 min to ensure adequate

colour development. Biologically active organisms are able to reduce the colourless

tetrazolium salt to a red-coloured product whereas the inhibition or a decrease in growth is

indicative by a clear solution or reduced colour intensity.


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7. Results and Discussion

In each of the in vitro tests, untreated PMBCs were used as a control. All results obtained are

compared to this control in order to understand what the effects of the plant extracts are. The

viability of these untreated cells was set to 100% and all subsequent values for the tests are

reflected as a percentage for comparison.

The morphology and amount of PBMCs shown in the control are very alike to cells that were

exposed to EED extract (Figure 7.4 (A) and (B)). However, there is a major decrease in the

amount of cells in the population that was treated with absolute ethanol (Figure 7.4 (C)).

Ethanol can affect the viability tests due to the disruption of various metabolic pathways

functions and cytosolic and mitochondrial redox states being altered by ethanol metabolism

(Bailey and Cunningham, 1998; Costa et al., 2004; Nurmi, et al., 2009).

The cells were determined to be viable by means of trypan blue and then diluted to a

concentration of 1 x 106 cells/ml for further experiments.

Figure 7.4: PBMCs allowed to proliferate for 3 d. (A) 1 ml RPMI media untreated which was used as a control, (B) 1 ml EED extract of C. asiatica and (C) 1 ml absolute ethanol added to PBMCs These PBMCs were incubated for a further 24 h at 37 C in a 5% CO2 incubator. Certain natural compounds have the ability to stimulate lymphocytes to divide. The

compounds of most importance are called lectins. They all act as T cell mitogens (a

substance that stimulates cells to divide) and these lectins bind sugars and carbohydrates

specifically. PH-A binds N-acetyl-D-galactosamine while Con A specifically binds -D-

mannose and -D-glucose residues. The binding of these lectins to lymphocyte membranes

stimulates nucleoside incorporation, phospholipid synthesis, DNA synthesis and cell division.

Not all lymphocytes respond equally well to all lectins. PH-A primarily stimulates Y-cell

A B C


- 154 -

division although it has a slight effect on B cells. It had been shown that extracts of C.

asiatica has an immunostimulating effect on mitogen-stimulated proliferation of human

PBMCs. Although the mechanism is still unclear, it may be mediated by interactions between

active components of the extracts and growth factors or cell surface molecules involved in

mitogen activation (Punturee et al., 2005b). The other possible action of C. asiatica extracts

may be interference with cell signalling and cytokine production but further investigation is

warranted (Punturee et al., 2005a).

The most frequently used solvents to make up the test compound solution include DMSO,

methanol and ethanol. The latter two have the disadvantages of rapidly evaporating so that

the stated concentration of stock solutions cannot be maintained. The use of DMSO offers

the advantage of eliminating microbial contamination and reducing the need for sterilization

by autoclaving or other strenuous methods which affect the quality of the samples. In

addition, DMSO has good compatibility with test automation and integrated screening

platforms, assuring for example solubility during the serial dilution procedures. For natural

products or compound mixtures where the exact molecular weight is not known,

concentrations are usually expressed as µg/ml (Cos et al., 2006).

7.1. Investigation into the potential C. asiatica cytotoxicity to

PBMCs by means of the tetrazolium salts XTT and MTT

The mechanisms of cell activation and cell damage are widely investigated by means of

tetrazolium assays. The advantages of MTT over XTT have been reported, and it is generally

found that XTT requires the addition of an intermediate electron acceptor, such as phenazine

methosulphate (PMS) to accelerate their bioreduction and the production of formazans.

Goodwin et al. (1995) demonstrated that the tetrazolium salt XTT forms an unstable reagent

mixture with PMS which leads to the subsequent decrease in formazan production by

mitogen activated Nb2 lymphoma cells. In the XTT assay, some fluctuations can be observed

which can be explained by the decline in XTT-formazan production and manifestation in the

microculture tetrazolium assay as poor within assay precision and serious assay drift

(Goodwin et al., 1995). For these reasons it was decided that the MTT assay for cell viability

would be employed.


- 155 -

Results show that the viability of the PBMCs was maintained with exposure to EED extracts

for 4 d and the same trend is evident after 7 d (Figure 7.5 shows data for 7 d). At lower

concentrations, the viability is similar to untreated PBMCs, but at higher concentrations the

viability and proliferation is evident suggesting that the effects are dose dependent. Further

incubation times could not be assessed due to the limited life span of the primary cell line.

Figure 7.5: MTT results showing the effect on viability of PBMCs exposed to EED extracts from C. asiatica cells induced for 4 d with 0.2 mM MeJa. This graph represents measurements done after 7 d of EED exposure. A serial dilution of the plant extract was done to test for any potential toxic effects. Error bars indicate the standard deviation (a measure of how widely values are dispersed from the mean value). C. asiatica cells suspensions were induced with 0.2 mM MeJa for 4 d. The levels of toxicity

and / or any effects due to enhanced metabolite concentrations within the extracts were also

tested. Secondary metabolite production is enhanced by exposure of C. asiatica cell

suspensions to 0.2 mM MeJa (Chapter 6). PBMCs were exposed to EED extracts for 4 and 7

d and these results, using XTT also showed no decrease in cell viability encountered, but

rather encouraged proliferation at high(er) concentrations (results not shown). The MTT

assay showed that at higher concentrations of EED extract, the viability is increased to above

100% (more than 3-fold with 625 g/ml). Even at the higher end of EED concentrations

tested, these quantities do not appear to be toxic to the cells. This increase in metabolites,

particularly the triterpenoids, can potentially be the cause for the enhanced viability.

Reports regarding cell stimulatory / proliferation effects are found in the literature. Hashim et

al. (2011) reported that stimulatory effects on collagen synthesis by C. asiatica extracts was

dose dependent and only at higher concentrations that 50 mg/ml did the cell viability of

0

50

100

150

200

250

300

350

400

Control 4.875 9.75 19.5 39 78 156 312.5 625

% V

iabi

lity

C. asiatica extracts ( g/ml)


- 156 -

human dermal fibroblasts decrease. Since this plant has long been used for its medicinal

qualities, it would be expected that there is limited toxicity and side effects for those taking in

the herb in low concentrations. C. asiatica has been used for wound healing, for the treatment

of skin disorders (such as eczema and psoriasis, Awang 1998), revitalizing connective tissue

(Montecchio et al., 1991), burn and scar treatment, and cleaning up skin infections, amongst

other health impairments (Kakkar, 1988; Bevege, 2004). The triterpenes are thought to

stimulate the production of human collagen I, a protein associated with wound healing (Wei et

al., 2008), however, the mechanism, especially at a molecular level, remains only partially

understood. Asiaticoside was determined by Lee et al. (2006) to induce type I collagen

synthesis via the activation of the TbetaRI kinase-independent Smad pathway. Smads are

intracellular proteins that transduce extracellular signals from transforming growth factor beta

(TGF-β) ligands to the nucleus where they activate downstream TGF-β gene transcription.

Asiatioside is able to induce the phosphorylation of both Smad 2 and Smad 3 and bind to

Smad 3 and Smad 4. Since the nuclear translocation of the Smad 3 and Smad 4 complex

was induced via treatment with asiaticoside, this indicates the involvement of asiaticoside in

Smad signaling.

According to Marquart et al. (1999), in cell culture studies, asiatic acid is the only triterpene

component responsible for stimulating collagen synthesis of human fibroblasts. In contrast,

some studies associate asiatic acid, madecassic acid and asiaticoside and their mixtures

responsible for the stimulation of collagen synthesis in skin fibroblast cultures (Bonté et al.,

1994) while, asiaticoside from C. asiatica is able to stimulate type-I collagen and

madecassoside type-III (Bonté et al., 1995; Lu et al., 2004). C. asiatica extracts have also

been reported to exhibit anti-oxidant properties (Hamid et al, 2002; Chen et al., 2003; Pittella

et al., 2009) and asiaticoside and flavanoids are reported to be the responsible factors for the

induction of antioxidant levels in wound healing (Mustafa et al., 2010). Nhiem et al. (2011)

reported concentrations of 100 M having no cytotoxic effects on LPS-stimulated RAW 254.7

cells after 24 h.

The claim of beneficial rather than detrimental effects can be substantiated from Figure 7.5.

The active components of this plant have been isolated in adequate amounts to enhance the

proliferation and viability of PBMCs. The results for the MTT assay were confirmed using a

commercially available C. asiatica extract obtained from Linnea containing no less than 36-

44% asiaticoside, asiatic- and madecassic acid. Here, no decrease in the viability of PBMCs

http://en.wikipedia.org/wiki/Protein

http://en.wikipedia.org/wiki/Transforming_growth_factor_beta

http://en.wikipedia.org/wiki/Ligands


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was seen (Figure 7.6), instead, the commercial extract (1 mg/ml) is able to maintain the

viability whereas the EED extracts (non- and induced) are able to stimulate almost a 2-fold

increase.

Figure 7.6: MTT results showing the effect on the viability of PBMCs under different test conditions. Similar results were obtained for 4 and 7 d of exposure. The PBMCs were exposed to non-induced EED extracts (25 mg/ml) and EED extracts (25 mg/ml) induced with 0.2 mM MeJa for 4 d to enhance secondary metabolite production. The commercial C. asiatica extract available from Linnea (10 mg/ml) was used as a positive experimental control and absolute ethanol for a negative experimental control.

Although ethanol was used to generate the C. asiatica extracts and then dried off to ensure

minimal (if any) interference with residual ethanol, as stated by Cos et al. (2006), such

solvents are likely to evaporate in the bioassay. To assess this, the solvent of choice, namely

ethanol was included in the assay - PBMCs were treated in the same manner as the plant

extracts. Ethanol (in DMSO and RPMI medium) was added to the cells as an additional

control to investigate the effect that traces of ethanol might have on the assay as mentioned

before. For those cells that were exposed to the ethanol solution; the viability decreased by

more than 50% (Figure 7.6). Therefore, metabolite enhanced C. asiatica extracts appear to

be non-toxic to PBMCs.

0

50

100

150

200

250

PBMCs Non-Induced MeJa Induced

Linnea EtOH

% V

iabi

lity

Test condition


- 158 -

7.2. Anti-microbial screening

A commonly used method to screen for anti-microbial compounds in crude plant extracts is

the agar diffusion assay. However, this has the disadvantage in that agar has been found to

hamper the diffusion of certain test compounds and is thus unsuitable for certain compounds

(Eloff, 1999). The direct (agar diffusion) bioassay has the advantage of being quick, easy to

perform and relatively cheap, requiring a small amount of sample and having a high

throughput. It is suitable for testing of all compounds that can be separated by TLC, against

any organism that can grow directly on the surface of the TLC plate; however, this is not a

quantitive method and cannot necessarily determine if the extracts are active, it only shows

how many compounds could inhibit the pathogen if they were separated.

TLC plates were initially developed as for Chapter 4 (Section 3.4) in chloroform, glacial acetic

acid, methanol and dH2O (60:21:12:8 (v/v/v/v)). However, even though these plates were left

in a fume hood to evaporate off the mobile phase, there were trace amounts of the glacial

acetic acid which affected the growth of the micro-organisms. A compromise in separation

was opted for and the developing solution was changed to ethyl acetate, methanol, H2O

(80:10:10 (v/v/v)). The following results were obtained for TLC plates after development with

C. cucumerinum for 4 d (Figure 7.7) or INT following incubation with the micro-organisms for

8 h at 37 C or AS spray (Figure 7.8, Figure 7.9).

Volumes of 50 µl and 100 µl aliquots of the leaf extract (stock concentration of 33 mg/ml) and

the commercial extract available from Linnea (stock concentration of 10 mg/ml) were used for

the C. cucumerinum plates to obtain inhibition zones, but there is a compromise in the

resolution of the active aglycones; the more concentrated aliquots produced larger zones as

can be seen by the AS detection (Figure 7.7). Unfortunately, smaller volumes applied (and

thus a lower concentration) did not show any significant inhibition of growth of C.

cucumerinum. The bacteria and C. albicans had a better response to lower concentrations of

the leaf and Linnea extract (Figure 7.8 and Figure 7.9). Lower concentrations of the C.

asiatica extract provide improved resolution of the triterpenoids and defined zones of

inhibition was attained.


- 159 -

Figure 7.7: Chromatogram and bioautogram of ethanol extracts of C. asiatica leaves and a commercial extract from Linnea against the fungus C. cucumerinum. A silica gel-60 plate was chromatographed with ethyl acetate, methanol, H2O (80:10:10 (v/v/v)) as mobile phase. An aliquot of 50 µl and 100 µl of the leaf extract (stock concentration of 33 mg/ml) and the commercial extract available from Linnea (stock concentration of 10 mg/ml) were applied. These plates were sprayed with a sporulating C. cucumerinum culture after development and incubated at 37 C for 4 d to allow the fungi to grow. White inhibition zones can be seen on a lawn of green fungi. Inhibition zones corresponding to asiatic acid (red blocks) can be seen for leaf extracts (A) and two zones correlating to asiatic- (red blocks) and madecassic acid (black blocks) were seen with the commercial Linnea extract of C. asiatica (C). The TLC plate developed under the same conditions but developed with AS can be seen for C. asiatica leaf extracts (B) and the commercial Linnea sample (D) with volumes of 50 and 100 l respectively.

Growth inhibition was seen for all the organisms tested (both Gram-positive and Gram-

negative bacteria, yeast (C. albicans) and fungi. Two prominent inhibition zones

corresponding to asiatic acid and madecassic acid can be seen (Figure 7.7, Figure 7.8 and

Figure 7.9). It was observed that not all organisms grow well in unfavourable conditions on a

TLC plate; M. smegmatis does not produce the same characteristic red background after

contact with INT (Figure 7.8, Figure 7.9). In addition, B. subtillis has the ability to form highly

resistant resting stages called endospores that decrease its sensitivity to anti-microbials

(Moat et al., 2002). Extracts from cell suspensions of C. asiatica induced with MeJa did not

show any inhibition zones (results not shown). The concentration of the triterpenoids in these

extracts, even though enhanced through chemical induction, could be too low to effectively

inhibit the growth of the micro-organisms. Typically, bioactive compounds in herbal plants are


- 160 -

present in low concentrations (Kim et al., 2009a) and even more so in undifferentiatial cell

suspensions. By comparison, the Linnea commerical extract contained 36-44% asiaticoside,

asiatic- and madecassic acid. In addition, there are many examples in literature of active

extracts that do not show inhibition of pathogen growth by means of the bioautography

method.

It has previously been reported that the aglycones were more active than their glycosides

(Yun et al., 2008; Won et al., 2010) due to the presence of sugar moieties at C-28 (Figure

3.3, -R9 position). Nhiem et al. (2011) reported a decrease in anti-inflammatory activity of

nitric oxide and tumor necrosis factor- production in LPS-stimulated macrophage-derived

RAW 254.7 cells for this reason. Furthermore, the absence of a hydroxyl group at C-2 caused

inactivity (Figure 3.3, -R1 position). Asiatic acid alone has been shown to have cytotoxic

activiy on fibroblast cells (Coldren et al., 2003) and induces apoptosis in different cancers

(Babu et al., 1995, Park et al., 2005; Gurfinkel et al., 2006) in a dose-dependent manner.

However, it was shown to be non-cytotoxic against lung carcinoma and normal hamster

kidney (BHK-21) cell lines. The selectivity of action could be attributed to the differences in

morphology and physiology between tested cell lines, which would give reasons for why no

cytotoxic effects were seen with the PBMCs, yet, inhibition was displayed against microbial

organisms. Although madecassocide and madecassic acid are associated with asiaticoside

and asiatic acid in wound healing and other medicinal claims, there is very little known about

their therapeutic properties in isolation.

Preliminary in vivo studies have been described in the literature. Pittella et al. (2009) found

asiaticoside to be the most abundant triterpene glycoside in C. asiatica leaves from plants in

Malasyia and found that in water, it is tranformed into asiatic acid, in vivo by hydrolysis

(Pittella et al., 2009). Rattanachaikunsopon and Phumkhachorn (2010) have reported the

strong anti-microbial activity and bactericidal action of aqueous C. asiatica extracts displayed

against Flavobacterium columnare, a bacterium which causes columnaris disease in tilapia

aquaculture. High concentrations of 100 mg/L were used and showed no adverse effect on

affected fish.

Figure 7.8: Chromatogram and bioautogram of ethanol extracts of C. asiatica leaves separated into different fractions. Ten microlitres (10 µl) of a 33 mg/ml reconstituted ethanolic C. asiatica leaf extract was applied onto silica gel-60 plates and chromatographed with ethyl acetate, methanol, H2O (80:10:10 (v/v/v)) as mobile phase. These plates were treated as described in Materials and Methods and exposed to the following test organisms: B. subtilis (ATTC 6051), E. faecalis (ATTC 29212), E. coli (ATTC 1175), P. aeruginosa (ATTC 9027), M. smegmatis and C. albicans (ATTC 10231). After development with 2 mg/ml ρ-iodonitrotetrazolium violet (INT), white (inhibition) zones appeared on a red / pink background. Another plate developed under identical conditions with 10 l of the leaf extract and 5 l of a 1 mM mixture of C. asiatica triterpenoids standards (Std, Extrasynthase, France) was sprayed with anisaldehyde-sulphuric acid (AS) spray. This mixture of C. asiatica triterpenoids consisted of asiatic acid, asiaticoside, madecassic acid and madecassoside, final concentration 1 mM of each). Triterpenoids in the leaves corresponding to the 4 triterpenoids could be identified and the inhibition zones could be correlated to asiatic- and madecassic acid.

Figure 7.9: Assessment of anti-microbial activity by a commercially available C. asiatica extract from Linnea (Switzerland). TLC plates were chromatographed as described in Materials and Methods, where 10 l of a 10 mg/ml C. asiatica commercial extract was used and exposed to the various micro-organisms for 8 h and then developed with INT. Once again 5 l of a 1 mM mixture of C. asiatica authentic triterpenoids standards (Std, Extrasynthase, France) was included. The zones of inhibition corresponding to asiatic acid and madecassic acid displayed inhibition to the micro-organisms. The Linnea commercial extract contained 36-44% asiaticoside, asiatic- and madecassic acid.


- 163 -

7.3. MIC Determinations

Many reports use agar diffusion assays to determine antimicrobial activity for plant

extracts. This technique works well with defined inhibitors, however, when extracts

containing unknown components is examined, there are problems that arise such as false

positive and negative results. The antimicrobial effect may be inhibited or increased by

intrinsic factors or contaminants, such as the agar type, salt concentration, temperature

for incubation and the molecular size of the anti-microbial component. In addition, this

technique can also not distinguish between bactericidal and bacteristatic effects and the

MIC cannot be determined (Eloff, 1998). As a result, quantification of the anti-

microbacterial activity of the C. asiatica extracts was carried out using the serial plate

micro-dilution assay.

Figure 5.10 is a representative plate for P. aeruginosa (ATTC 9027). The highest

concentrations of extract are present in the 2nd column and 200 l of untreated bacteria

(column 1) and 200 l of culture medium (column 11) were added as a positive and

negative control respectively.

The results showed all the C. asiatica extracts (from cell suspensions and leaves) are able

to inhibit the growth of the micro-organisms, and the MIC values were in the low mg/ml

range, which is indicative of a high inhibitory activity against the pathogen (Table 7.2).

Plant extracts are generally more active against Gram-positive bacteria than Gram-

negative bacteria (McCutcheon et al., 1992; Abu-Shanab et al., 2004; Basari and Fan,

2005) because of the permeability barrier provided by the cell wall or membrane

accumulation mechanism. The concentrations of the C. asiatica cell suspensions (non-

and induced with 0.2 mM MeJa) extracts needed to hinder growth of the Gram-negative

micro-organisms are 2-fold more than for the Gram-positive bacteria (Table 7.2). Lower

MIC values are obtained for leaf extracts than extracts from cell suspensions suggesting

the composition of metabolites in the leaf extracts are more potent than in cell

suspensions substantiating the use of the aerial parts of the plant.

When treated with the C. asiatica commercial extract from Linnea, Gram-positive bacteria

(E. faecalis and B. subtilis), showed lower MIC values of 0.16 mg/ml (Table 7.2) in

comparison to Gram-negative bacteria (E. coli and P. aeruginosa), which have higher MIC

values for the commercial extract (0.63 mg/ml). This again verifies that plant extract are

more active against Gram-positive bacteria. Jagtap et al. (2009) also reported a C.


- 164 -

asiatica extract showing a high extent of inhibition against B. subtilis compared to other

strains of bacteria tested. Thus, extracts from C. asiatica are shown to displayed strong

anti-microbial activity, since MIC values lower than 100 g/ml are considered to possess

significant any-microbial activity (Gibbons, 2004; Rios and Recio, 2005).

Figure 7.10: Quantification of anti-microbacterial activity by means of INT. The colourless tetrazolium salt is reduced to a red product by biologically active organisms; the inhibition of growth was detected when the solution remained clear after incubation with INT. Representation of such results is given for (top) P. aeruginosa (ATTC 9027) and (bottom) the antibiotics tested. TSB / LB / Middlebrooks media was used as a sterility control and absolute ethanol was included to exclude any reaction with the solvents. One hundred microlitres (100 µl) of bacteria (A600 0.2) was added to react with either MeJa induced C. asiatica extracts, the commercial extract from Linnea or C. asiatica leaf extracts. Extracts were serially diluted (1:1) and the plates were incubated overnight at 37 C. As an indicator of bacterial growth, 40 µl of 2 mg/ml INT was added and left for at least 30 min to ensure adequate colour development. Although no zones of growth inhibition were detected on the bioautograms for C. asiatica

cell suspension extracts (induced and non-induced), the micro-dilution assay showed

inhibition of growth of the selected micro-organisms occurred, when the whole extract was

used. It is possible these separated triterpenoids could be in concentrations too low to

cause growth inhibition on the TLC plates or there is a need for the triterpenes to be


- 165 -

associated with other metabolites in the crude extracts to cause an inhibition of microbial

growth, i.e. some mode of synergism between two or more agents present in the extract.

It should be emphasized the higher MIC values obtained for the cell suspensions as

compared to the leaf extracts and commercial preparation is also due to the sample being

a crude extract and is comprised of many metabolites, not only the isolated triterpenoids,

which seem to be present in low concentrations.

Inhibition by whole plant extracts of C. asiatica has been described against Xanthomonas

sp., Bacillus megaterium, E. coli and Proteus vulgaris (Koppula et al., 2010, Koppula et

al., 2011). These extracts had more effect when compared to standard antibiotics. This

was also found in is study when the commercial antibiotics, namely kanamycin sulphate

(500 mg/ml), gentamycin sulphate (40 mg/ml), neomycin sulphate (10 mg/ml) and

flucoanazole (2 mg/ml) were tested. No distinct positive control was obtained for this

experiment under these experimental conditions. Very little, if any, inhibition of growth was

found when micro-diluted concentrations of the antibiotics were investigated except

against M. smegmatis with kanamycin sulphate (250 mg/ml), gentamycin sulphate (5

mg/ml), and neomycin suphate (1.15 mg/ml). This could be due to the slower growth rate

that occurs with M. smegmatis thus exposing the cells to a longer period of contact with

the antibiotics. The growth of E. coli was inhibited by gentamycin at a MIC of 5 mg/ml.

There are no specific lethal dose (LD50) values reported in the literature for the antibiotics

used. Parameters for these values would depend on the growth medium that is used for

the organism and the animal model used for the study. MIC values of gentamycin against

E. faecalis and P. aeruginosa have been reported as 10 µg/ml and 1 µg/ml against E. coli

in Mueller-Hindoton (HM) broth in agar diffusion assays (Kianbakht and Jahaniani, 2003)

but higher doses of gentamycin required to be effective have been reported in animal

models. In addition, there is also the need for multiple doses of antibiotics to hinder

microbial growth or to eliminate micro-organisms.

Factors that influenced the assay was the inoculum size (the number of bacteria in

suspension calculated with respect to the final volume) and the presence of chlorophyll in

the leaf extract. Adequate dilutions of the overnight cultures had to be prepared to allow

sufficient growth over 24 h but still provide acceptable MIC values since an excessive

amount of bacteria would provide higher MIC values. Some of the test organisms clumped

to the bottom and the presence of chlorophyll caused some difficulty in determining the

end point but after reduction of the colourless tetrazolium salt to a coloured formazan

product, it was easier to differentiate the coloured product from the green colour of the


- 166 -

plant extracts, especially when growth occurred at the lower concentrations when the

green colour was diluted out.

Table 7.2: The minimum inhibitory concentrations (MIC) of C. asiatica cell suspensions (non-treated and induced with 0.2 mM MeJA), leaf extracts and commercially available preparations from Linnea showing inhibition of microbial activity (n=5)

Micro-organism

MIC values (mg/ml)

Non-induced cell

suspension extracts (25 mg/ml)*

0.2 mM MeJa induced

cell suspension extract (25 mg/ml)*

C. asiatica leaf extract (33 mg/ml)*

Commercial C. asiatica

extract from Linnea (10 mg/ml)*

Bacillus subtilis

(ATTC 6051)

Gram-positive

3.13

3.13

4.13

0.16

Enterococcus faecalis (ATTC 29212)

Gram-positive

3.13

3.13

2.06

0.16

Escherichia coli

(ATTC 1175)

Gram-negative

6.25

6.25

2.06

0.63

Pseudomonas aeruginosa

(ATTC 9027)

Gram-negative

6.25

6.25

4.13

0.63

Mycobacterium smegmatis

Mycobacterium Gram- positive

1.56

1.56

4.13

0.63

Candida albicans (ATTC 10231)

Yeast

3.13

3.13

2.06

No inhibition

* Concentrations as used in the microtitre plate assay


- 168 -

The MIC experiments were done to evaluate the claims of C. asiatica as a medicinal plant. It

was seen that this method did not provide optimal results as the commercial antibiotics (used

as a positive controls) did not inhibit any of the investigated pathogens. As mentioned

previously, the MIC of gentamycin against E. coli was reported to be 1 g/ml and the

concentration used for this study was 40 mg/ml, thus 40 000-fold higher, but no inhibition was

observed, thus leading to some uncertainty for the method used here. In addition the extracts

were prepared using absolute ethanol. For MIC determinations, ethanol was used as a no

growth control and also to indicate if there was any reaction with the reagents in the

experiment.

8. Conclusion

Medicinal plants have the potential to be sources of new, bioactive (including anti-microbial)

compounds (Runyoro et al., 2006a, 2006b; Cowan, 1999). Should they prove to be non-toxic

and more tolerable than patent drugs, they offer an affordable and valuable source of

pharmacologically active substances, which can be made in sufficient quantities though

cultivation. In this chapter, we investigated the bioactive, anti-microbial potential of

triterpenoids from C. asiatica cell suspension and leaf extracts.

Many medical claims have been reported for C. asiatica, therefore cytotoxicity levels were

assessed. This study showed that ethanolic C. asiatica extracts from cell suspensions and

leaves did not display any cytotoxic effects on PBMCs for 4 and 7 d. Moreover, enhanced cell

proliferation was observed at the concentrations tested which would substantiate the use of

C. asiatica to treat wounds, burns and ulcers and to accelerate healing thus preventing the

formation of scar tissue following surgery (Wei et al., 2008).

The active ingredients or the main compounds in C. asiatica are the triterpenes asiatic acid

and madecassic acid, together with triterpenoid ester glycosides, known as asiaticoside and

madecassoside. Also present are volatile compounds such as p-cymol, -caryophyllene,

farnesene and polyphenolic antioxidants such as quercetin, myricetin, and kaempferol

(Mustafa et al., 2010).

Metabolites in the crude extracts of C. asiatica cell suspensions and leaves were separated

with ethyl acetate, methanol and H2O (80:10:10 (v/v/v)) as mobile phase and in combination


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with the direct bioautography method, anti-microbial activity in C. asiatica extracts were

tested. C. asiatica leaf extracts provided two main zones of inhibition, corresponding to

asiatic- and madecassic acid against the test micro-organisms, namely, Gram-positive (B.

subtilis and E. faecalis) and Gram-negative bacteria (E. coli and P. aeruginosa),

mycobacterium (M. smegmatis), yeast (C. albicans) and fungi (C. cucumerinum). The

ethanolic extracts from cell suspensions did not exhibit any inhibition of microbial growth,

possibility due to the low concentration of active triterpenoids present in the extracts. The

glycosides (asiaticoside and madecassoside) also did not show any anti-microbial effects,

most likely due to the presence of sugar moieties, indicating that the aglycones (asiatic- and

madecassic acid) are more active than their glycosides against the tested organisms. A

commercial C. asiatica extract obtained from Linnea (Switzerland), used in various

dermatological applications, functioned as a positive control. This extract also exhibited anti-

microbial inhibition against all micro-organisms tested.

Since the bioautography method is not a quantitive technique, to obtain MIC values, the serial

plate micro-dilution assay was carried out. Extracts were diluted in a 1:1 ratio, with the

appropriate media, and the results displayed an inhibition in the growth of the micro-

organisms, with MIC values in the low mg/ml range (indicative of a high anti-microbial activity)

by the C. asiatica leaf and cell suspension extracts. Furthermore, the plant extracts were

more active against Gram-positive than Gram-negative bacteria. Unfortunately, in this study,

a comparison with standard antibiotics could not be done. Various LD50 values have been

reported for commercial these antibiotics, which are affected by factors such as the animal

model and the concentration of bacteria.

The shortcomings for clinical studies are the lack of standardization of the C. asiatica

preparations used. Differences in extraction procedures, sources of plant material and

geographical origin may contribute to the differences in results. Traditional organic solvent-

based extractions can be subject to low extraction yields, long extraction times and residual

toxic organic solvents in the final products, which could also be problematic and deteriorate

the quality of the extracts (Kim et al., 2009b). Nevertheless, in view of the low toxicity and

wide range of bioactivities, it seems plausible to evaluate the potential pharmacological

properties and applications of C. asiatica further.

Conclusion

Chapter 8

Chapter 8 – Conclusion

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This study provided a comprehensive overview of the pentacyclic triterpenoids present in C.

asiatica, and described the presence of four specific, active triterpenoids. These four

centelloids have not previously been investigated concurrently. Metabolic changes due to

MeJa treatment on secondary metabolism in cell suspensions were also explored, and to our

knowledge, no metabolic profiling study has previously been conducted to investigate the

effects of MeJa treatment on C. asiatica cell suspensions.

Interest in C. asiatica has increased over the years due to its medicinal properties. The active

constituents in C. asiatica described to date include pentacyclic triterpenes derivatives;

however, the exact number of these centelloids remains unknown since many of these

pentacyclic triterpenoids have synonyms or duplicate names. An overview of the triterpenoids

in C. asiatica disclosed an extensive number of centelloids that occurred as either the ursane-

or oleanane type. This study followed a targeted approach, where four triterpene saponins,

namely madecassoside and asiaticoside, and their sapogenins- madecassic and asiatic acid

were investigated.

A method combining TLC and densitometry demonstrated that it was possible to assess the

purity of C. asiatica extracts, and to estimate the concentration of the four major pentacyclic

triterpenoids in fresh leaf material. These triterpenoids were however present in

concentrations below the limits of detection of QTLC in callus and cell suspensions. Thus, by

means of more advanced techniques such as reverse-phase HPLC analysis, the

concentrations of the targeted centelloids, could be quantified in leaf tissue, callus and cell

suspensions. By means of a modified method, a linear gradient elution of acetonitrile and

water on a reverse-phase C18 column could separate all four compounds in a single run, and

two phenotypes of C. asiatica found in Southern Africa could be differentiated on the basis of

their triterpenoid content. Differences between varieties in medicinal plants of the same

species have been reported, and variations in secondary metabolites have also been

observed in plants with identical phenotypes and growth conditions. In addition, significant

differences in the active constituents and their relative ratios have been reported for C.

asiatica originating from different countries. These differences can be attributed to genetic

variation in the oxidosqualene cyclase and other genes involved in the biosynthesis.

In order to understand secondary metabolites and their functioning, metabolomics tools were

employed. The use of analytical methods in a model species allows for all metabolic changes


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occurring due to a treatment to be captured and the information gained from this research

can be used to understand how plants grow and carry out specific functions. MeJa has been

reported to induce secondary metabolite accumulation not only in intact plants, but also in

plant tissue cultures, thus the effect of 0.2 mM MeJa as a signal / elicitation molecule was

investigated. Viability assays confirmed the cell suspensions were affected by the treatment

and by means of UPLC-MS, alterations in the metabolic profile of C. asiatica cells were

investigated and confirmed.

By means of non-linear regression curves and specialized software such as QuanLynx™, the

four targeted centelloids were quantified and found to be in the low ng/ml range

(corresponding to 0.07 – 0.3 g/g fresh weight). Of the targeted centelloids, asiaticoside was

found to be present in the highest concentration and could be visually detected in UPLC-MS

BPI chromatograms, sometimes co-eluting with a molecule of m/z 252, as confirmed by

MS/MS. There was a 5-fold increase in asiaticoside due to treatment after 4 d. The other

centelloid which could be seen was asiatic acid (9-fold increase in the concentration for the

treated cells). These contributed to the clustering of the samples in a representative PCA

loading plot for all the time points. By means of discriminating ions identification (from the

PCA loading plot) and obtaining information such as the elemental composition, Rt and m/z

values, other metabolites such as JA and rishitin, which are synthesized in the presence of

MeJa could be putatively identified.

Many medical claims have been reported for C. asiatica, therefore cytotoxicity levels were

assessed. This study showed that ethanolic C. asiatica extracts from cell suspensions and

leaves did not display any cytotoxic effects on PBMCs for 4 and 7 d. Moreover, enhanced cell

proliferation was observed at the concentrations tested, which would substantiate the use of

C. asiatica to accelerate wound healing, thus preventing the formation of scar tissue. The

anti-microbial activities in C. asiatica extracts were tested by separating metabolites in the

crude extracts of C. asiatica cell suspensions and leaves on TLC, and then utilizing the direct

bioautography method. C. asiatica leaf extracts provided two main zones of inhibition,

corresponding to asiatic- and madecassic acid against the test micro-organisms. The

ethanolic extracts from cell suspensions however did not exhibit any detectable inhibition of

microbial growth, possibility due to the low concentration of active triterpenoids present in the

extracts. Since the bioautography method is not a quantitative technique, to obtain MIC

values, the serial plate micro-dilution method was carried out. These results showed that all


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the C. asiatica extracts (from cell suspensions and leaves) were able to inhibit the growth of

the micro-organisms, although the MIC values were in the low 1.56 – 6.25 mg/ml range,

showing a high activity of anti-microbial activity. Furthermore, the plant extracts were more

active against Gram-positive than Gram-negative bacteria. Thus, it seems plausible to

evaluate the potential pharmacological properties and applications of C. asiatica further.

It is important to take into consideration that differences in extraction procedures, the sources

of plant material and geographical origin may contribute to the differences in results. Thus,

there is a need for additional studies to be done to evaluate the genetic resources of the plant

for variation in growth, morphology, and yield-related characteristics. The biochemical

pathways and genetic machinery required for the elaboration of this important family of plant

secondary metabolites are still largely uncharacterized, despite the considerable commercial

interest in this important group of natural products. This is likely to be due in part to the

complexity of the molecules and the lack of pathway intermediates for biochemical studies.

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Chapter 4

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characterisation and response against ethanol. Plant Cell, Tissue and Organ Culture, 68, 257-263.

Labadie, R.P. and De Silva, K.T.D. (2001) Centella asiatica (L.) Urban in perspective: an evaluative

account. In Studies in Indian Medicinal History; Wujastyk, D., Zysk, K.G., (eds), Vol. V, Motilal

Banarsidass, New Delhi, India, p.191-205.

Mathur, S., Verma, R.K. and Gupta, M.M., Ram, M., Sharma, S. and Kumar, S. (2001) Screening of

genetic resources for the medicinal-vegetable plant Centella asiatica for herb and asiaticoside

yields under shaded and full light conditions. Journal of Horticultural Science and Biotechnology,

75, 551-554.


constituents of Gotu Kola (2): Structures of new ursane- and oleanane-type triterpenene

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Shukla, A., Rasik, A.M., Jain, G.K., Shankar, R., Kulshrestha, D.K. and Dhawan, B.N. (1999) In vitro

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Ethnopharmacology, 65, 1-11.

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Chapter 5

Aziz Z.A., Davey, M.R., Power, J.B., Anthony, P., Smith, R.M. and Lowe K.C. (2007) Production of

asiaticoside and madecassoside in Centella asiatica in vitro and in vivo. Plant Biology, 51, 34-42.

Bouhouche, N., Solet, J.M., Simon-Ramiasa, A., Bonaly, J. and Cosson, L. (1998) Conversion of 3-





Das, A. and Mallick, R. (1991) Correlation between genomic diversity and asiaticoside content in

Centella asiatica (L.) Urban. Botanical Bulletin of Academia Sinica, 32, 1-8.

Gaines, J.L. (2004) Increasing alkaloid production from Catharanthus roseus suspensions through

methyl jasmonate elicitation. Pharmaceutical Engineering, 24, 1-6.

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Gupta, A.P., Gupta, M.M. and Kumar, S. (1999) High performance thin layer chromatography of

asiaticoside in Centella asiatica. Journal of the Indian Chemical Society, 76, 321-322.

Gurfinkel, D.M. and Rao, A.V. (2002) Determination of saponins in legumes by direct densitometry.

Journal of Agricultural and Food Chemistry, 50, 426-430.


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Kim, O.T., Kim, M.Y., Huh, S.M., Bai, D.G., Ahn, J.C. and Hwang, B. (2005) Cloning of a cDNA

probably encoding oxidosqualene cyclise associated with asiaticoside biosynthesis from Centella






bioreactors. In vitro Cellular and Development Biology - Plant, 38, 1-10.

Kraemer, K.H., Schenkel, E.P. and Verpoorte, R. (2002) Ilex paraguariensis cell suspension culture

characterisation and response against ethanol. Plant Cell, Tissue and Organ Culture, 68, 257-263.





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Mathur, S., Verma, R.K., Gupta, M.M., Ram, M., Sharma, S. and Kumar, S. (2000) Screening of

genetic resources for the medicinal vegetable plant Centella asiatica for herb and asiaticoside

yields under shaded and full light conditions. Journal of Horticultural Science and Biotechnology,

75, 551-554.





Nath, S. and Buragohain, A.K. (2005) Establishment of callus and cell suspension cultures of Centella

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Mahillion, J., Ratsimamanga, S. and Jaziri, M.E.J. (2007) Comparative analysis of active

constituents in Centella asiatica samples from Madagascar: application for ex situ conservation

and clonal propagation. Fitoterapia. 78, 482-489.

Rouillard-Guellec, F., Robin, J.R., Ratsimamanga, A.R., Ratsimamanga, S. and Rasaoanaivo, R.

(1997) Comparative study of Centella asiatica of Madagascar origin and Indian origin. Acta

botanica gallica. , 144, 489-493.

Samanani, N. and Facchini, P.J. (2006) Compartmentalization of plant secondary metabolism. In:

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Pharmazie, 58, 381-384.

Shukla, A., Rasik, A.M., Jain, G.K., Shankar, R., Kulshrestha, D.K. and Dhawan, B.N. (1999) In vitro

and in vivo wound healing activity of asiaticoside isolated from Centella asiatica. Journal of

Ethnopharmacology, 65, 1-11.


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Singleton, J.A., Stikeleather, L.F. and Haney, C.A. (2000) Microextraction and characterization of

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Sparg, S.G., Light, M.E. and van Staden, J. (2004) Biological activities and distribution of pant

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Gotu Kola (Centella asiatica) extracts and asiaticoside in rat behavioral models. Phytomedicine

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asiaticoside and madecassoside in Centella asiatica in vitro and in vivo. Plant Biology, 51, 34-42.


48, 53-57.

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