Isolation, purification and characterization of...

Isolation of bioactive compounds from banana rhizomeIsolation of bioactive compounds from banana rhizomeIsolation of bioactive compounds from banana rhizomeIsolation of bioactive compounds from banana rhizome

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Chapter 3: Chapter 3: Chapter 3: Chapter 3:

Isolation, purification and characterization Isolation, purification and characterization Isolation, purification and characterization Isolation, purification and characterization

of bioactive compounds from banana of bioactive compounds from banana of bioactive compounds from banana of bioactive compounds from banana

rhizome rhizome rhizome rhizome var. var. var. var. Nanjanagudu RasbaleNanjanagudu RasbaleNanjanagudu RasbaleNanjanagudu Rasbale


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INTRODUCTION

Extensive use of plant extracts in alleviating various human health diseases and

disorders are in practice since, time immemorial in Ayurveda, Unani and other traditional

medicinal systems throughout the world. The search for bioactive components with

multiple bioactivities from plant source has gained increasing importance in recent time,

due to growing worldwide concern about alarming increase in the rate of infection by

antibiotic-resistant microorganisms and carcinogenicity of synthetic compounds. Natural

products continue to provide greater structural diversity than standard combinatorial

chemistry and they offer great opportunities for finding novel bioactive compounds with

potential bioactivities. Thus plant extracts offer a significant role in the discovering of

bioactive compounds of our interest. However, it is important to establish the scientific

rationale to defend their use in nutraceutical and functional foods and as potential source

of drug. In this regard, potentiality of banana pseudostem and rhizome has been explored

as a source of bioactive compound. The earlier study indicated that pseudostem of banana

var. Nanjanagudu Rasbale as a highest source of polyphenols among all the eight

commercial varieties screened. Interestingly, highest source of polyphenolic content was

observed in banana rhizome in comparison with pseudostem. Consequently, the banana

rhizome of var. Nanjanagudu Rasbale has been focused for isolation and characterization

of bioactive compounds. The detail work carried out is presented in this chapter.


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Materials and Methods

Preparation of extracts

The extraction of banana rhizome powder using serial extraction process with

increasing polarity of solvents was followed as discussed in the chapter 1 (materials and

methods).

Isolation and purification of bioactive compounds from banana rhizome var.

Nanjanagudu Rasbale extracts

The isolation and purification of bioactive compounds were carried out using

repeated silica gel column chromatography and thin layer chromatography (TLC). The

purified bioactive compounds were characterized by subjected to UV, IR, LC-MS and

NMR spectroscopy studies.

Isolation and purification of antioxidant compound from banana rhizome var.

Nanjanagudu Rasbale

The acetone extract of banana rhizome showed high total phenolic and total

flavonoid content and multiple antioxidant activity in all the in vitro tested models.

Hence, antioxidant activity (DPPH radical scavenging method) guided fractionation was

employed to isolate and purify antioxidant compound from acetone extract of banana

rhizome.

Fractionation of the crude acetone extract

To isolate antioxidant compound from acetone extract of banana rhizome,

activated silica gel (60-120 mesh) was packed onto a glass column (450 mm x 40 mm)

using n-hexane solvent and 20 g of crude acetone extract was loaded on the top of silica

gel. The column was eluted stepwise at a flow rate of 1 mL min-1

with 500 mL of hexane

and linear gradient of 2000 mL of hexane : chloroform (75 : 25 to 0 : 100 v/v), 2600 mL


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of chloroform : ethyl acetate (75 : 25 to 0 : 100 v/v), 8000 mL of ethyl acetate : acetone

(75 : 25 to 0 : 100 v/v) and 3500 mL of acetone : methanol (75 : 25 to 0 : 100 v/v). In this

column chromatographic separation, about 83 fractions measuring 200 mL each were

collected and concentrated by using the rotary evaporator. Weight of each fraction was

measured.

Thin-layer chromatography (TLC)

An aliquot of all the concentrated fractions were loaded on the activated silica gel

TLC plates (20 cm x 20 cm). The plates were developed in ascending direction from 18

to 19 cm of height with different proportions of chloroform and methanol as mobile

phase. After air-drying, the spots on the plate were located by exposure to iodine.

Fractions were pooled based on the spotting pattern and Rf values on the TLC plate. The

pooled fractions were numbered (Fr.1'-Fr.18'). All the eighteen pooled fractions were

tested for antioxidant activity using DPPH radical scavenging method as discussed in

chapter 1 (materials and methods).

Purification of bio-active fraction

Since, fraction nine (Fr.9') obtained from first step of column chromatography

(Fig. 3.1) showed high antioxidant activity and yield, it was selected for further

purification. About 2 g of bioactive Fr.9' were further purified using silica gel (60-120

mesh) column (450 mm x 40 mm). The column with Fr.9' was eluted stepwise at a flow

rate of 1 mL min-1

with 100 mL of hexane, followed by linear gradient of 200 mL of

hexane : chloroform (90 : 10 to 0 : 100 v/v), 1100 mL of chloroform : ethyl acetate (90 :

10 to 0 : 100 v/v), 900 mL of ethyl acetate : acetone (90 : 10 to 0 : 100 v/v) and 400 mL

of acetone : methanol (90 : 10 to 0 : 100 v/v). In this second column chromatographic

separation, about 27 fractions from Fr.9' measuring 100 mL each were collected and

concentrated by using the rotary evaporator. Weight of each fraction was measured. An

aliquot of all the fractions were loaded on the TLC plate, fractions having similar Rf

values and spotting pattern were pooled and numbered into sub-fractions (Fr.9.1''-


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Fr.9.5''). These sub-fractions were tested for antioxidant activity using DPPH radical

scavenging method.

Figure 3.1: Schematic representation of isolation and purification of antioxidant

compound from acetone extract of banana rhizome var. Nanjanagudu Rasbale

Sub-fraction three (Fr.9.3'') obtained from second step column chromatography

(Fig. 3.1) showed high antioxidant activity, hence selected for further purification. About

800 mg of bioactive sub-fraction three Fr.9.3'' was further purified on a silica gel (100-

200 mesh) column (600 mm x 15 mm). The column was eluted stepwise at a flow rate of

1 mL min-1

with linear gradient of 100 mL of hexane: chloroform (90: 10 to 0: 100 v/v),

400 mL of chloroform: ethyl acetate (95: 05 to 0: 100 v/v), 400 mL of ethyl acetate:


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acetone (95: 05 to 0: 100 v/v) and 200 mL of acetone: methanol (90: 10 to 0: 100 v/v). In

this third column chromatographic separation, about 22 fractions measuring 50 mL each

were collected and concentrated. Weight of each fraction was measured. Fractions with

similar Rf values and spotting patterns on the TLC plate were pooled and numbered

(Fr.9.3.1'''-Fr.9.3.3'''). Among these, sub-fraction two (Fr.9.3.2''') obtained from the third

step column chromatography showed purity in the TLC profile. This pure compound was

subjected to various spectroscopic techniques for elucidation of the structure. The

chromatographic separation of sub-fraction two (Fr.9.3.2''') afforded one pure compound.

This pure compound was subjected to various spectroscopic techniques to elucidate the

structure and studied for their antioxidant activity using different in vitro models in detail.

The entire process of purification is shown in figure 3.1.

High performance liquid chromatography (HPLC)

These purified compound was tested for their purity by using HPLC, on C-18

column (model LC-10A, Shimadzu Corporation, Japan), with ultraviolet (UV) detection

using a diode array detector (DAD) operating at 220, 280 and 320 nm. An isocratic

solvent system, consisting of methanol: water: trifluoro acetic acid (89.5:10:0.5), was

used as a mobile phase at a flow rate of 1 mL min-1

. Ultraviolet (UV) detection was

carried out with a diode array detector (Shimadzu).

Isolation and purification of antimicrobial compounds from banana rhizome var.

Nanjanagudu Rasbale

Chloroform extract showed high antimicrobial activity against wide spectrum of

bacterial (Gram +ve and Gram -ve bacteria) and fungal strains. Hence, antimicrobial

compounds were aimed to isolate from chloroform extract by antimicrobial activity

guided fractionation method.


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Fractionation of the crude chloroform extract

Figure 3.2: Schematic representation of isolation and purification of antimicrobial

compounds from chloroform extract of banana rhizome var. Nanjanagudu Rasbale

The crude chloroform extract from serial extraction of banana rhizome powder of

about 10 g (Fig. 3.2) was subjected to column (450 mm x 40 mm) chromatography using

silica gel (60-120 mesh) and eluted stepwise with linear gradient of 1000 mL of hexane :

Ethyl acetate (95:5 to 75:25 v/v), 2000 mL of chloroform : ethyl acetate (90:10 to 0:100

v/v), 800 mL of ethyl acetate : acetone (90:10 to 0:100 v/v) and 400 mL of acetone :

methanol (90:10 to 0:100 v/v). About 42 fractions, measuring 100 mL, were collected,

concentrated by using the rotary evaporator. Weight of each fraction was measured.


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Thin layer chromatography (TLC)

An aliquot of column fraction was spotted on to a silica gel TLC plate (20 cm x

20 cm). The plates were developed in ascending direction from 18 to 19 cm of height

with different proportions of hexane and ethyl acetate as mobile phase. After air-drying,

the spots on the plate were located by exposure to iodine. Fractions were pooled based on

the spotting pattern and Rf values on the TLC plate. The pooled fractions were numbered

(Fr.1'-Fr.8'). All the eight pooled fractions were tested for antimicrobial activity as

discussed in chapter 1 (materials and methods)

Purification of bio-active fraction

Since fraction three (Fr.3’), obtained from first step column chromatography

showed high antimicrobial activity and yield, it was selected for further purification (Fig.

3.2). About 1.7 g of bioactive Fr.3' was further purified using silica gel column (450 mm

x 40 mm) chromatography and eluted with linear gradient of 1700 mL of hexane and

ethyl acetate (95:5 to 5:95 v/v) and with 500 mL of ethyl acetate and methanol (90:10 to

50:50 v/v). About 22 fractions measuring 100 mL were collected, concentrated by

distillation and after TLC analysis, based on Rf value and spotting pattern, fractions were

pooled into five sub-fractions (Fr.3.1''-3.5''). Weight of each fraction was measured and

assayed for their antimicrobial activity.

Sub-fraction (Fr.3.2'') obtained from the second step column chromatography

(Fig. 3.2) showed high antimicrobial activity and yield, hence selected for further

purification. About 900 mg of bioactive sub-fraction two (Fr. 3.2'') was further purified

on a silica gel (100-200 mesh) column (600 mm x 15 mm). The column was eluted

stepwise at a flow rate of 1 mL min-1

with linear gradient of 600 mL of hexane:

chloroform (90: 10 to 0: 100 v/v), 400 mL of hexane: ethyl acetate (95: 05 to 0: 100 v/v)

and 200 mL of ethyl acetate: acetone (95:05 to 0:100 v/v). In this third column


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chromatographic separation, about 24 fractions measuring 50 mL each were collected and

concentrated. Weight of each fraction was measured. Fractions having similar Rf value

and spotting pattern on the TLC plate were pooled and numbered (Fr. 3.2.1'''- Fr. 3.2.3''').

Among these, sub-fraction one and two (Fr.3.2.1''' and 3.2.3''') obtained from the third

step column chromatography showed purity in the TLC profile. These pure compounds

were subjected to various spectroscopic techniques to elucidate the structure and studied

for their antimicrobial activity in detail. The entire process of purification is shown in fig

3.2.

High performance liquid chromatography (HPLC)

These purified compounds were tested for their purity by using HPLC, on C-18

column (model LC-10A, Shimadzu Corporation, Japan), with ultraviolet (UV) detection

using a diode array detector (DAD) operating at 220, 280 and 320 nm. An isocratic

solvent system, consisting of acetonitrile: water: trifluoro acetic acid (89.5:10:0.5), was

used as a mobile phase at a flow rate of 1 mL min-1

. Ultraviolet (UV) detection was

carried out with a diode array detector (Shimadzu).

Acetylation of purified compounds

For acetylation of purified compounds, about 20 mg of compound, dissolved in

0.5 mL anhydrous pyridine, to which 0.5 mL anhydrous acetic anhydride was added and

stirred in a stoppered conical flask overnight at room temperature. Reaction mixture was

then poured into ice-cold water (≈50 mL) with constant stirring and left for 1 h. The

insoluble acetylated compound was separated by filtration, using a Whatman filter paper

No. 1.


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NaBH4 reaction

Reduction of acetylated pure fraction was carried out using NaBH4 at ice cold

condition in methanol. In a typical reaction, 100 mg of fraction was taken in 5 mL

methanol and 0.5 equivalent of NaBH4 was added. The reaction mixture was stirred at ice

cold condition for 1 hour.

Characterization of bioactive compounds from banana rhizome var. Nanjanagudu

Rasbale extracts

UV-Visible Spectroscopy

UV-Visible spectrum of the isolated compound was recorded on a UV-Visible

spectrometer (Shimadzu UV-160A, Singapore) at room temperature. About 1 mg of

compound dissolved in 20 mL of acetone/chloroform and was used to record the

spectrum in the wavelength range of (λ) 200 to 800 nm.

Infra red (IR) Spectroscopy

IR spectrum of isolated compound was recorded on a Perkin-Elmer FT-IR

Spectrometer (Spectrum 2000) at room temperature. About 1 mg of isolated compound

was mixed with spectroscopic grade KBr and well ground before preparing the pellet.

The IR spectrum was taken in the frequency range (ν) of 4000 cm-1

- 400 cm-1

.

Liquid chromatography-Mass spectrometry (LC-MS)

Mass spectrum of the compound was recorded on instrument HP 1100 MSD

series (Palo Alto, CA) by electro spray ionization (ESI) technique with a flow rate of 0.2

mL min-1

on C-18 column and total run time of 40 min. The sample used for recording


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the mass spectrum was prepared by dissolving 0.1 mg of compound in 10 mL of

methanol/acetonitrile.

NMR Spectroscopy

The 1H NMR was recorded for the isolated compound (5mg in 600µL DMSO-D6)

at 500MHz on BRUKER AQS NMR spectrometer, Bruker biospin AG, Switzerland. The

J-modulated spin-echo for 13

C -nuclei coupled to proton to determine number of attached

protons (SEFT) was recorded at 125 MHz. The spectral width for 1H NMR was 0-12 ppm

and 0-220 ppm for 13

C NMR.

Bioactive properties of isolated compounds

The isolated pure compounds from acetone (chlorogenic acid) and chloroform (4-

epicyclomusalenone and cycloeucalenol acetate) extracts were used to study their bio-

active properties viz., antioxidant activity, antimicrobial activity, platelet aggregation

inhibition activity and cytotoxicity in detail.

Antioxidant activity

The antioxidant activities of the isolated compounds (10-300 µl from 1 mg/ mL of

stock) were investigated by using eight different in vitro assays Viz., DPPH radical

scavenging activity (DPPH RSA), superoxide radical scavenging activity (SRSA), β-

carotene bleaching inhibition (βCBI) assay, anti-lipid peroxidation (ALPO) activity,

metal chelating activity (MCA), hydrogen peroxide scavenging activity (HPSA), nitric

oxide scavenging activity (NOSA) and total reducing power (TRP) assay. BHT, EDTA,

ascorbic acid and curcumin were used as standard antioxidants. Results were expressed

as EC50 value, which represents the sample concentration require to show 50%

antioxidant activity. The methodology for all the antioxidant assays followed as described

in chapter 1 (materials and methods section), except for total reducing power (TRP)

assay, which is followed as described in chapter 2 (materials and methods section).


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Antimicrobial activity

The minimum inhibitory concentrations (MIC) of the isolated compounds against

bacterial and fungal strains were determined as the procedure discussed in chapter 2

(materials and methods)

Determination of minimum bactericidal concentration (MBC)

Minimum bactericidal concentration was determined according to the method of

Smith-Palmer et al., (1998). Test tubes containing nutrient broth with different

concentrations (10-1000 ppm) of isolated compounds were inoculated with 100 µ l of the

bacterial suspension (105 CFU mL

-1) Inoculated tubes were incubated for 24 h at 37ºC

and growth was observed both visually and by measuring OD at 600 nm. About 100 µ l

from the tubes not showing growth were plated on nutrient agar as described earlier.

MBC is the concentration at which bacteria failed to grow in nutrient broth and nutrient

agar inoculated with 100 µ l of suspension. Triplicate sets of tubes were maintained for

each concentration of the test sample.

Determination of minimum fungicidal concentration (MFC)

The minimum fungicidal concentration (MFC) was determined using the method

of Rotimi et al. (1988). The mixtures of the fungus and isolated compounds in MIC

studies which showed no visible growth after 7 days of incubation were subcultured onto

a potato dextrose agar (PDA) plate using an inoculum of 10 µl. The plates were incubated

at 27°C for 72 h. The MFC was regarded as the lowest concentration that prevented the

growth of any fungal colony on the solid medium.

Platelet-aggregation inhibitory activity

The platelet aggregation inhibition activity of the purified compounds was studied

by the methodology followed as described in chapter 1 (materials and methods).


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Cytotoxicity

Cytotoxicity of the purified compounds was studied by using HepG-2 cell lines.

The methodology followed as described in chapter 1 (materials and methods).

Results and discussion

Characterization of Bioactive Compounds

Elucidation of structure of antioxidant compound from acetone extract of banana

rhizome var. Nanjanagudu Rasbale

The pure compound was subjected to various spectroscopic analysis viz. UV,

FTIR, LC-MS and NMR to elucidate the structure. The compound exhibited UV λ

maxima at 340 nm corresponding to π- π* transition of C=C double bonds. FTIR spectral

data showed characteristic stretching frequencies corresponding to carbon skeleton

present in the molecule as follows; 3452 cm-1

OH stretching, 2963 cm-1

Broad, carboxylic

acid OH stretching, 2726 cm

-1 Sharp, CH2, 1771 cm

-1 ester carbonyl C=O stretching,

Table 3.1: UV, FTIR and LC-MS spectral data of chlorogenic acid

Spectra Chlorogenic acid

UV 340 nm

FTIR

810 cm-1 (alkane = C-H stretching)

1107 cm-1 (ester C-O stretching)

1449 cm-1 (aromatic C=C stretching)

1771 cm-1

(ester carbonyl C=O stretching)

2726 cm-1

(alkane C-H stretching )

2963 cm-1

(OH-carboxylic stretching)

3452 cm-1

(O-H stretching)

Mass 355.2 (M+1

)


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1449 cm-1

aromatic C=C stretching, 1107 cm-1

ester CO stretching and 810 cm-1

Sharp,

alkane CH stretching. LC-MS data showed major parent molecular ion [M+1

] peak at m/z

355.2 (100%). The elemental analysis of this compound reveals the presence of C-

54.24%, H-5.12% and O-40.64%. The elemental analysis and mass spectrum indicated

the chemical formula as C16H18O9.

The characteristic signals corresponding to cyclohexanyl moiety integrating for four

protons appear at 1.70-2.10 ppm. The related carbon signals (36.51, 37.37) appear with

negative intensity in 13

C-SEFT experiment confirming the presence of two –CH2 groups.

Two vicinal protons attached to hydroxyl group at position 4 and 5 of cyclohexane ring

appeared at 3.57 and 3.93 ppm, the related 13

C signals appeared as positive signals

indicating the presence of two –CH’s. Proton signal for ester attached cyclohexane ring

was appeared at 5.07 ppm and related carbon signal appeared at 71.01ppm confirming the

ester linkage to the cyclohexane ring. α,β-unsaturated double bond attached protons

respectively appeared at 6.15 and 7.42 as doublet with J value of 15.85 Hz , related

olefinic methine carbons appeared at 114.5 & 145.02 ppm with positive intensity. The

two negative quaternary carbon signals appearing at 175.04 & 165.88 ppm indicated

presence of two carbonyl groups. The presence of broad signal integrating to single

proton in the 1H experiment at 12.41 ppm leads to the conclusion of presence of carboxyl

group. The aromatic region of the spectrum presented signals integrating for three

protons as doublet (J = 8 Hz), double doublet (J1= 8 Hz, J2= 1.9 Hz) and doublet (J = 1.9

Hz) appeared at 6.77, 6.98 & 7.04 ppm respectively. Two broad singlets corresponding to

phenolic hydroxyl appear at 9.16 & 9.59 ppm. All these spectral features indicated the

molecule has close resemblance with the standard chlorogenic acid spectral

characteristics. The spectral assignments were again confirmed by the two dimensional

NMR experiments (HSQC, HMBC & COSY) comparison with that of standard

chlorogenic acid.


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Table 3.2: The NMR Spectral data of chlorogenic acid

C 1H J (Hz) 13

C SEFT

1H-

1H 2D

COSY Correlated H

13C-

1H 2D

HMBC Correlated C

1 - 73.69 C - -

2 2.03 1.94

m (dd, 12.8,

7.80) 37.37 CH2

H2 (1.94), H2' (2.03), H5 (3.93)

C1 (73.69), C3 (71.01) C4 (70.64), C5 (68.33)

C7 (175.04)

3 5.07 m 71.01 CH H2' (2.03), H4 (3.57)

C1 (73.69), C3 (71.01) C4 (70.64)

4 3.57 m 70.64 CH H3 (5.07), H5 (3.93)

C3 (71.01)

5 3.93 m 68.33 CH H2 (1.79), H4 (3.57)

-

6 2.01 1.79

m (dd, 13.83,

3.42) 36.51 CH2

H6 (1.79), H3 (5.07) H6' (2.01)

C1 (73.69), C3 (71.01) C4 (70.64), C5 (68.33)

C7 (175.04)

7 - 175.04 CO - -

8 - 165.88 CO - -

9 6.15 (d, 15.85) 114.50 CH H10 (7.42) C8 (165.88), C11 (125.78)

10 7.42 (d, 15.85) 145.06 CH H9 (6.15) C8 (165.88), C12 (114.93),

C16 (121.46)

11 - 125.78 C - -

12 7.04 (d, 1.90) 114.93 CH H16 (6.98) C16 (121.46), C10(145.06), C13 (148.48), C14 (145.72)

13 - 148.48 C - -

14 - 145.72 C - -

15 6.77 (d, 8.08

Hz) 115.92 CH H16 (6.98)

C11 (125.78), C13 (148.48) C14 (145.72)

16 6.98 (dd, 8.08,

1.92) 121.46 CH H15 (6.77)

C12 (114.93), C13 (148.48) C10 (145.06), C14 (145.72)


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Based on all these spectral data and comparison with standard chlorogenic acid,

the structure was elucidated to be a (1S, 3R, 4R, 5R)-3-((E)-3-(3, 4-Dihydroxyphenyl)

acryloyloxy)-1, 4, 5-trihydroxycyclohexanecarboxylic acid and designated it as a

“Chorogenic acid” (Fig 3.3). This is the first report of isolation and characterization of

chlorogenic acid from acetone extract of banana rhizome.

Figure 3.3 Structure of chlorogenic acid

Yield of purified chlorogenic acid

The yield of compound chlorogenic acid is 350 mg/10 g of crude extract, 35

mg/kg of dried banana rhizome powder and 6.3 mg/kg of fresh banana rhizome of var.

Nanjanagudu Rasbale.

Elucidation of structure of antimicrobial compounds from chloroform extract of

banana rhizome var. Nanjanagudu Rasbale

HO

OH

HO

O

OH

O

O

OH

OH


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Elucidation of structure of compound 1

Table 3.4: UV, FTIR and LC-MS spectral data of cycloeucalenol acetate

Spectra 4-Epicyclomusalenone

UV 243 nm

FTIR

763, 887 cm-1

(alkane =C-H stretching)

1376 cm-1

(alkane –C-H stretching)

1453 cm-1

(aromatic C=C stretching)

1711 cm-1

(aliphatic carbonyl C=O stretching)

2868 cm-1

(alkane C-H stretching)

2935.37 cm-1


Mass 425.2 [M+1

]

The structure of the isolated bioactive compound was characterized by analyzing

UV, FTIR, LC-MS and NMR spectral data. UV spectra of the compound displayed

characteristic absorption maxima at 243 nm corresponds to π- π* transition indicating the

presence of C=C double bonds (Table 3.3). IR spectral data showed alkane C-H

stretching at 2935 and 2868 cm-1

, aliphatic carbonyl C=O stretching at 1711 cm-1

, C=C

stretching at 1453 cm-1

, alkane –C-H at 1375 cm-1

, alkane =C-H stretchings at 959 and

887 cm-1

indicating the presence of aliphatic carbonyl and olefinic double bond groups.

LC-MS spectrum showed major parent molecular ion [M+1

] peak at m/z 425.2 (100%).

The elemental analysis of this compound reveals the presence of C-84.84%, H-11.39%

and O-3.77%. The elemental analysis and mass spectrum indicated the chemical formula

as C30H48O.

This compound also showed positive reaction for liebermann-Burchard reaction

indicating it to be a triterpeniod. Proton NMR spectrum (Table ….) showed the presence

of six methyl groups at δ (ppm) 0.89 (3H), 0.92 (3H), 1.01 (3H), 1.02 (3H), 1.03 (3H)

and 1.64 (3H) attached at C-20, 14, 13, 24, 4 and 25 respectively. It also indicated the

presence of protons of olefinic methylene group at δ (ppm) 4.69 (bs, 2H) on C-27

attached to C-24. Another methylene group in a trimembered (C-9, 10 and 19) ring at C-


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19 is showed the signals at δ (ppm) 0.41 (d, 4.0 Hz, 1H) and 0.64 (d, 4.0 Hz, 1H).

Signals at δ (ppm) 2.44 (m, 2H) and 2.24 (m, 1H) indicated the presence of methylene

and methyne groups on the adjacent carbons of carbonyl at C-3. The downfield shift of

these signals is due to anisotropic effect of carbonyl group. All the remaining protons on

methylene and methine carbons are resonated between δ (ppm) 1.13-2.11.

Carbon NMR spectrum (Table…..) showed the signals at δ (ppm) 213, 149.9 and

109.4 for C-3, C-25 and C-27 respectively, confirmed the presence of carbonyl group at

C-3 and double bond between C-25and C-27. All other methyl, methylene, methyne and

quaternary carbons resonated between 10.8-52.2 ppm. Assignments are confirmed with

the help of SEFT, HSQC and HMBC spectra (table ……). All these signals are

comparable with that of the reported values for 4-Epicyclomusalenone (Akihisa et al.,

1997). The structure of the isolated compound is elucidated as 4-Epicyclomusalenone.

Based on all these spectral data the structure was elucidated to be a (3aS, 3bS,

5aS, 6R, 91R, 10

1S, 12aR) - 1 - ((2R, 5R) – 5, 6 - dimethylhept - 6 - en - 2- yl) - 3a, 6, 12a

- trimethyltetradecahydrocyclopenta (a) cyclopropa (e) phenanthren - 7 (3bH) – one, and

designated it as a “4-Epicyclomusalenone” (Fig 2.2). This is the first report of tritepene

compound 4-Epicyclomusalenone from chloroform extract of banana rhizome.

Figure 3.4 Structure of 4-Epicyclomusalenone

OH

H


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Table 3.3: NMR spectral data of 4-Epicyclomusalenone

H/C 1H

δδδδ (ppm) Multiplicity

JH-H

(Hz)

13C

δδδδ (ppm)

HMBC

1-CH2 1.88 1.60

m m

- 32.9 C-5,19

2- CH2 2.44 m - 41.0 C-1,3 3-C=O - - - 213.0 - 4-CH 2.24 m - 50.0 C-29 5-CH 2.11 m - 41.6 C-1,6,7,10

6-CH2 1.13 1.37

m m

- 25.2 -

7-CH2 0.75 1.71

m m

- 25.9 -

8-CH 1.66 m - 47.1 - 9-C - - - 21.5 - 10-C - - - 25.2 -

11-CH2 2.06 1.25

m m

- 27.2 -

12-CH2 1.67 m - 32.8 - 13-C - - - 45.3 - 14-C - - - 48.5 -

15-CH2 1.32 m - 35.4 -

16-CH2 1.32 1.91

m m

- 28.1 C-17

17-CH 1.60 m - 52.20 - 18-CH3 1.01 s - 18.3 -

19-CH2 0.41 0.64

d d

4.0 4.0

27.0 -

20-CH 1.39 m - 36.0 - 21-CH3 0.89 d 6.5 18.4 -

22-CH2 0.94 1.37

m m

- 33.9 -

23-CH2 1.19 1.46

m m

- 31.5 -

24-CH 2.11 m - 41.6 C-23 25-C - - - 149.9 -

26-CH3 1.64 s - 18.6 -

27-CH2 4.69 bs - 109.4 C-24, 25,26

28-CH3 1.02 d 7.0 20.2 - 29-CH3 1.03 d 6.5 10.8 - 30-CH3 0.92 s - 19.2 -


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Yield of purified compound

The yield of compound 4-epicyclomusalenone is 420 mg/10 g of crude extract, 42

mg/kg of dried banana rhizome powder and 7.6 mg/kg of fresh banana rhizome.

Elucidation of structure of compound 2

Table 3.5: UV, FTIR and LC-MS spectral data of cycloeucalenol acetate

Spectra Cycloeucalenol acetate

UV 244 nm

FTIR

886 cm-1 (alkane =C-H stretching)

959 cm-1 (alkane =C-H stretching)

1166 cm-1 (ester C-O stretching)

1248 cm-1

(ether C-O stretching)

1458 cm-1

(aromatic C=C stretching)

1735 cm-1

(ester carbonyl C=O stretching)

2859 cm-1


2925 cm-1


3448 cm-1

(O-H stretching)

Mass 469.1 (M+1

) and 409

The pure compound was subjected to various spectroscopic analysis viz. UV,

FTIR, LC-MS and NMR to elucidate the structure (Table 3.5). The compound exhibited

UV λ maxima at 244 nm corresponding to π- π* transition of C=C double bonds. FTIR

spectral data showed O-H stretching at 3448 cm-1

, alkane C-H stretching at 2925 and

2859 cm-1

, ester carbonyl C=O stretching at 1736 cm-1

, C=C stretching at 1458 cm-1

,

alkane –C-H at 1375 cm-1

, ester C-O stretchings at 1249 and 1166 cm-1

, alkane =C-H

stretchings at 959 and 887 cm-1

indicating the presence of ester carbonyl and olefinic


79

double bond groups. LC-MS data showed major parent molecular ion [M+1

] peak at m/z

469.1 (100%) and along with other fragment m/z 409 (M+ - CH3COO, 20%). Fragment

m/z 409 indicated the presence of acetoxyl group in the molecule. The elemental analysis

of this compound reveals the presence of C-81.99%, H-11.18% and O-6.83%. The

elemental analysis and mass spectrum indicated the chemical formula as C32H52O2.

Compound showed positive reaction for liebermann-Burchard reaction indicating

it to be a triterpeniod. Proton NMR spectrum (Table 3.5) showed the presence of six

methyl groups at δ (ppm) 0.86 (3H), 0.90(3H), 0.91(6H), 1.01(3H) and 1.02(3H) attached

at C-14, 13, 4, 20, 25 and 26 respectively. It also indicated the presence of protons of

olefinic methylene group at δ (ppm) 4.69 (bs, 1H) and 4.74(bs, 1H) on C-28 attached to

C-24. Protons on adjacent carbons C-23 and 25 showed the signals at δ (ppm) 2.04 (m,

2H) and 2.24 (m, 1H) respectively due to the anisotropic effect as well as electron

withdrawing effect of double bond. Another methylene group in a trimembered (C-9, 10

and 19) ring at C-19 is showed the signals at δ (ppm) 0.17 (bs, 1H) and 0.41(bs, 1H).

Signals at δ (ppm) 2.08 (S, 3H) and 4.52 (dt, 10.5, 4.5 Hz, 1H) indicated the presence of

acetoxyl group and it attachment at C-3. The signals at δ (ppm) 2.02 (m, 1H) and 1.45

(m, 1H) indicated the two protons on C-2. One proton is shifted to downfield due the

anisotropic effect of carbonyl group on C-3. All the remaining protons on methylene and

methine carbons are resonated between δ (ppm) 1.50-2.00.

Carbon NMR spectrum (Table 3.6) showed the signals at δ (ppm) 106.1 and 156.5

for C-28 and C-24 double bond carbon respectively. The signals at δ (ppm) 78.5, 170.6

and 21.2 for C-3, acetoxyl carbonyl and methyl carbons confirmed the presence of

acetoxyl group on C-3. All other methyl, methylene, methyne and quaternary carbons

resonated between 14.5-52.1 ppm. Assignments are confirmed with the help of SEFT,

HSQC and HMBC spectra (table 3.6). All these signals are comparable with that of the

reported values for cycloeucaloenol acetate (Kikuchi et al. 1986). The structure of the

isolated compound is elucidated as “cycloeucaloenol acetate”.


80

Table 3.5: NMR spectral data of cycloeucalenol acetate

H/C 1H

δδδδ (ppm) Multiplicity

JH-H

(Hz)

13C

δδδδ (ppm)

HMBC

1-CH2 1.59 m

- 30.9 - 1.45 m

2-CH2 2.02 m

- 31.3 - 1.45 m

3-CH 4.52 dt 10.5

4.5 78.5 -

4-CH 1.41 m - 41.5 -

5-CH 1.28 m - 43.4 -

6-CH2 1.68

0.61

m

m - 24.6 -

7-CH2 1.08 m

- 25.0 - 1.32 m

8-CH 1.60 m - 46.8 -

9-C - - - 23.6 -

10-C - - - 29.7 -

11-CH2 1.96 m

- 26.9 C-9,10,12,19 1.22 m

12-CH2 1.63 m - 32.8 C-10,11,13,14,18

13-C - - - 45.3 -

14-C - - - 48.8 -

15-CH2 1.39 m - 35.3 -

16-CH2 1.95 m - 28.1 C-13

17-CH 1.59 m - 52.1 -

18-CH3 0.90 s - 17.8 C-13,14

19-CH2 0.17 bs

- 27.1 - 0.41 bs

20-CH 1.39 m - 36.0 -

21-CH3 0.91 d 6.5 18.4 -

22-CH2 1.57 m - 34.9 -

23-CH2 2.04 m - 31.4 -

24-C - - - 156.5 -

25-CH 2.24 m - 33.9 C-26,27

26-CH3 1.01 d 6.0 22.0 -

27-CH3 1.02 d 6.5 21.8 -

28-CH2 4.69 bs

- 106.1 C-25

4.74 bs C-23

OCOCH3 2.08 s - 21.2 OCOCH3

30-CH3 0.86 s - 14.5 -

OCOCH3 - - - 170.6 -

32-CH3 0.91 s - 19.2 -


81

Based on all these spectral data and comparison with literature, the structure was

elucidated to be a ((3aS, 6S, 7S, 91R, 10

1S, 12aR)-3a, 6, 12a-trimethyl-1-((R)-6-methyl-5-

methyleneheptan-2-yl) hexa decahydrocyclopenta [a] cyclopropa [e] phenanthren-7-yl

acetate), and designated it as a “Cycloeucalenol acetate” (Fig 3.5). This is the first report

of tritepene compound cycloeucalenol acetate is isolated and characterized from

chloroform extract of banana rhizome.

Figure 3.5 Structure of cycloeucalenol acetate

Yield of purified compound

The yield of compound cycloeucalenol acetate is 400 mg/10 g of crude extract, 40

mg/kg of dried banana rhizome powder and 7.2 mg/kg of fresh banana rhizome of var.

Nanjanagudu Rasbale.

OO


82

Bioactive properties of isolated compounds

In vitro antioxidant activities of isolated compounds

Table 3.6: Antioxidant activity (EC 50 value in µg/mL) of isolated compounds and

standard antioxidants using different in vitro assays

Sl.

No

Antioxidant

assays

Chlorogenic

acid 4-Epicyclomusalenone

Cycloeucalenol

acetate Standards

1 DPPH RSA 12±0.3a 260±5.0b 296±5.3c BHT <10

2 SRSA 22±1.2a - - BHT <10

3 βCBI 62±2.2a 170±3.0c 152±3.2b BHT <10

4 ALPO 26±1.2a 60±1.8b 80±2.4c BHT <10

5 MCA 90±2.5a 110±2.0c 102±2.2b EDTA <10

6 HPSA 44±1.6a 180±2.5b 196±3.8c Asc. acid <10

7 NOSA 28±1.5a 206±3.6b 242±4.6c Curcumin <10

8 TRP♣ 4.5±0.2a 44±1.2c 35±1.0b Asc. acid 24±2.0

Mean values in a column with different superscripts differ significantly at p<0.05. EC50: Effective concentration of the sample to

show 50% of antioxidant activity; DPPH RSA-1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, SRSA-superoxide radical

scavenging activity, βCBI-β-carotene bleaching inhibition assay, ALPO-Anti-lipid peroxidation activity, MCA-metal chelating

activity, HPSA-hydrogen peroxide scavenging activity, NOSA- Nitric oxide scavenging activity and TRP-total reducing power

assay; Asc.acid-ascorbic acid. ♣

sample concentration to get 0.5 of absorbance at 700 nm

DPPH radical scavenging activity (DPPH RSA)

Any molecules, which donate an electron or hydrogen to a reaction mixture, can

react with and bleach DPPH. Further, DPPH is reduced from a purple compound to a

light yellow compound by electrons from oxidant compounds. Substances which are able

to perform this reaction can be considered as antioxidants and therefore radical

scavengers (Brand-Williams et al., 1995). The reaction of DPPH with hydroxyl groups

involves a hemolytic substitution of one the DPPH phenyl rings, which yields 2-(4-

hydroxyphenyl)-2-phenyl-1-picryl hydrazine as a major product, and 2-(4-nitrophenyl)-2-

phenyl-1-picrylhydrazine via a series of secondary processes. Table 3.6 explains the

effect of purified compounds on DPPH radical scavenging activity. The compound

chlorogenic acid showed highest activity (EC50 of 12±0.3 µg/ mL) followed by 4-

epicyclomusalenone (EC50 of 260±5.0 µg/mL) and cycloeucalenol acetate (EC50 of


83

296±5.3 µg/mL). The DPPH radical scavenging activity of chlorogenic acid was

comparable to the standard antioxidant BHT (EC50 of <10 µg/mL).

Superoxide radical scavenging activity (SRSA)

Superoxide radical is known to be very harmful to cellular components as a

precursor of more reactive oxygen species (Soares et al., 1997). This radical is a powerful

oxidizing agent that can react with biological membranes and induce tissue damage

(Castelluccio et al., 1996). It may also decompose to singlet oxygen, hydroxyl radical, or

hydrogen peroxide (Halliwell and Gutteridge, 1985). Effect of purified compounds on

superoxide radical is shown in table 3.6. Compound chlorogenic acid showed high

superoxide radical scavenging activity (EC50 value of 22 µg/mL). However, triterpenoid

compounds both cycloeucalenol acetate and 4-epicyclomusalenone did not demonstrated

EC50 value upto the concentration of 300 µg/mL.

β -carotene bleaching inhibition (βCBI) assay

In this model, β-carotene undergoes rapid discoloration in the absence of an

antioxidant. The presence of an antioxidant can hinder the extent of β-carotene

destruction by ‘‘neutralizing” the linoleate free radical and any other free radicals formed

within the system (Kamath and Rajini, 2007). In term of β-carotene bleaching effect, the

purified compounds exhibited in the following order: BHT > chlorogenic acid >

cycloeucalenol acetate > 4-epicyclomusalenone. Chlorogenic acid exhibited a marked

antioxidant activity (EC50 of 62±2.2 µg/mL) followed by triterpenoid compounds

cycloeucalenol acetate (EC50 of 152±3.2 µg/mL) and 4-epicyclomusalenone (EC50 of

170±3.0 µg/mL). While, standard BHT showed EC50 <10 µg/mL (table 3.6). It was clear

that hydroperoxides formed in this system may be decomposed by the purified

compounds of banana rhizome. Hence, the degradation rate of β-carotene depends on the

antioxidant activity of the purified compounds. This suggests that the purified compounds

may have potential use as antioxidative preservatives in emulsion-type systems.


84

Anti-lipid peroxidation (ALPO) activity

Lipid peroxidation can inactivate cellular components and plays an important role

in oxidative stress of biological systems. Furthermore, several toxic byproducts from the

peroxidation can damage other bio-molecules (Box and Maccubbin, 1997). Lipid

peroxidation causes destabilization and disintegration of the cell membrane, leading to

liver injury, atherosclerosis, kidney damage, aging, and susceptibility to cancer (Rice-

Evans and Burdon, 1993). It is well established that transition of metal ions, such as iron

and copper, can stimulate lipid peroxidation through various mechanisms. They may

either promote the generation of hydroxyl radicals to initiate the lipid peroxidation

process or propagate the chain process via decomposition of lipid hydroperoxides

(Braughler et al., 1987). Lipid peroxidation is a free radical mediated propagation of

oxidative insult to polyunsaturated fatty acids involving several types of free radicals. Its

termination occurs in biological system through enzymatic means or by radical

scavenging activity by antioxidants (Heim et al., 2002). In this study, high ALPO activity

with EC50 value of 26±1.2 µg/mL was shown by chlorogenic acid followed by 4-

epicyclomusalenone (60±1.8 µg/mL) and cycloeucalenol acetate (80±2.4 µg/mL) (table

3.6).

Metal chelating activity (MCA)

Iron is known to generate free radicals through the Fenton and Haber-Weiss

reaction (Halliwell and Gutteridge, 1990). Ferrous ions can stimulate lipid peroxidation

by Fenton reaction, and also accelerates peroxidation by decomposing lipid hydro

peroxides into peroxyl and alkoxyl (Halliwell, 1991; Gulcin et al., 2003). Metal ion

chelating activity of an antioxidant molecule prevents oxyradical generation and the

consequent oxidative damage. Metal ion chelating capacity plays a significant role in

antioxidant mechanism since it reduces the concentration of the catalyzing transition

metal in lipid peroxidation (Duh et al., 1999). The compound chlorogenic acid was found

to be more potential antioxidant activity with an EC50 value of 90±2.5 µg/mL followed

by cycloeucalenol acetate and 4-epicyclomusalenone with an EC50 value of 102±2.2 and


85

110±2.2 µg/mL respectively (Table 3.6). The action of these compounds as antioxidant

may be related to its iron-binding capacity. It was reported that chelating agents, which

form σ-bonds with a metal, are effective as secondary antioxidants because they reduce

the redox potential thereby stabilizing the oxidised form of the metal ion (Gordon, 1990).

High chelating ability of these compounds may be beneficial as a most effective

proxidants in the food system.

Hydrogen Peroxide Scavenging Activity (HPSA)

The compound chlorogenic acid was found to be more potent in scavenging

hydrogen peroxide (44±1.6 µg/mL), followed by 4-epicyclomusalenone (180±2.5 µg/mL)

and cycloeucalenol acetate (196±3.8 µg/mL) (Table 3.6). The H2O2, formed by the two-

electron reduction of O2 is not a free radical, but is an oxidizing agent. In the presence of

O2 and transition metal ions especially iron and copper, H2O2 can generate OH. via the

Fenton reaction (Shahidi, 1992). H2O2 can cross membranes and may slowly oxidize a

number of biomolecules and compounds. H2O2 is formed in vivo when superoxide

dismutates and also by many oxidase enzymes. H2O2 at micromolar levels is poorly

reactive. However, higher levels of H2O2 can attack some energy-producing systems.

H2O2 inactivates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase.

Hence, metal chelating and H2O2 scavenging processes are important for living organisms

(Halliwell and Gutteridge, 1985; Aruoma, 1998; Marcocci et al., 1994).

Nitric oxide scavenging activity (NOSA)

In the present study, nitric oxide radical generated from sodium nitroprusside at

physiological pH was found to be inhibited by purified compounds chlorogenic acid,

cycloeucalenol acetate and 4-epicyclomusalenone (Table 3.6). Incubation of sodium

nitroprusside with purified compounds from banana rhizome revealed that the inhibition

of nitrite production was highest in compound chlorogenic acid (EC50 of 28±1.5 µg/ mL)

whereas, 4-epicyclomusalenone and cycloeucalenol acetate showed moderate inhibition

of nitrite production and exhibited EC50 of 206±3.6 and 242±4.6 µg/ mL respectively. In

addition to reactive oxygen species, nitric oxide is also implicated in inflammation,


86

cancer and other pathological conditions (Moncada et al., 1991). The plant/plant products

may have the property to counteract the effect of NO formation and in turn may be of

considerable interest in preventing the ill effects of excessive NO generation in the

human body. Further, the scavenging activity may also help to arrest the chain of

reactions initiated by excess generation of NO that are detrimental to human health.

Total reducing power (TRP) assay

The reducing capacity of a compound from Fe3+

/ferricyanide complex to the

ferrous form may serve as a significant indicator of its antioxidant capacity (Meir et al.,

1995). The compounds viz., chlorogenic acid, cycloeucalenol acetate and 4-

epicyclomusalenone were showed high reducing power with EC50 value of 4.5±0.2,

35±1.0 and 44±1.2 µg/mL respectively (Table 3.6). The activity of chlorogenic acid was

found to be higher than the standard ascorbic acid (24±2.0). The result revealed that these

compounds are the potential electron donor and also could react with free radicals,

converting them to more stable products and terminating the radical chain reaction.

Multiple antioxidant activities of the isolated compounds and their structure activity

relationship

Chlorogenic acid, 4-epicyclomusalenone and cycloeucalenol acetate were

exhibited multiple antioxidant activities in all the in vitro models tested. The activity

shown by chlorogenic acid in some of the in vitro antioxidant assays viz., total reducing

power (4.5±0.2 µg/mL), DPPH radical scavenging activity (12±0.3 µg/mL), superoxide

radical scavenging activity (22±1.2 µg/mL), anti-lipid peroxidation activity (26±1.2

µg/mL) and nitric oxide scavenging activity (28±1.5 µg/mL) were comparable to the

standard antioxidants viz., BHT, curcumin and ascorbic acid. Whereas, cycloeucalenol

acetate and 4-epicyclomusalenone were showed high antioxidant activity with low EC50

value in total reducing power assay (EC50 of 35±1.0 and 44±1.2 µg/ mL, respectively),

anti-lipid peroxidation activity (EC50 of 80±2.4 and 60±1.8 µg/ mL, respectively) and

metal chelating activity (EC50 of 102±2.2 and 110±2.0 µg/mL, respectively).


87

DPPH radical scavenging by antioxidants has been attributed to their hydrogen-

donating ability of -OH and -CH3 groups. The antioxidant activity of the compound may

be attributed to the presence of -OH and C=O groups (Chen and Ho, 1995; Nikolaos, et

al., 2003). The compound chlorogenic acid possess -OH and C=O groups, whereas,

compound 4-epicyclomusalenone and cycloeucalenol acetate possess -CH3 and C=O

groups. Lipid peroxidation inhibitory activity was mainly attributed to the number of

hydroxyl groups of the compounds (Sopheak and Betty, 2002). Presence of six hydroxyl

and two methylene groups in chlorogenic acid and methyl group in teriterpenoid

compounds (4-epicyclomusalenone and cycloeucalenol acetate) may be responsible for

high lipid peroxidation inhibitory activity. It was reported that the structures containing

two or more of the following functional groups: -OH, -SH, -COOH, -PO3H2, C=O, -NR2,

-S- and -O- in a favorable structure-function configuration is responsible for metal

chelating activity (Lindsay, 1996; Yuan et al., 2005). Chlorogenic acid, which is having -

OH, -COOH, C=O and -O- functional groups might have contributed for the metal

chelating activity. Whereas, 4-epicyclomusalenone and cycloeucalenol acetate have only

C=O may explain the less activity, when compare to the compound chlorogenic acid. The

solubility and hydrophobicity of these compounds plays important role in their

antioxidant activity (Sopheak and Betty, 2002).

High antioxidant activities of chlorogenic acid than triterpenoid compounds (4-

epicyclomusalenone and cycloeucalenol acetate), due to their possessing an O-dihydroxy

B-ring structure, which conferred higher stability in the radical form and participated in

electron delocalisation. This conclusion was consistent with those reported in the

literature (Pietta, 2000). This also depends on the hydrogen-donating ability of

antioxidants. Studies on oxidation potentials and redox reactions between and transition

metal ions have shown that the o-dihydroxyl feature is a crucial factor for the reducing

efficiency (Makris and Kefalas, 2005). Antioxidant activity of phenolics increased when

there were more hydroxyl groups in the molecule (Kumaran and Karunakran, 2006).

Presence of two phenolic hydroxyl groups apart from four other hydroxyl groups in the

compound chlorogenic acid explains the antioxidant potential. Moreover, presence of


88

trihydroxy cyclohexane and α hydroxy carboxylic acid might have contributed the

antioxidant activity of the compound chlorogenic acid. In vitro antioxidant properties of

chlorogenic acid with multiple mechanisms involving free radical scavenging, metal ion

chelation, and inhibitory effect on specific enzymes responsible for free radical and

hydroperoxide formation were reported earlier (Chen and Ho, 1997; Zhou and Zheng,

1991; Kono et al., 1997). Triterpenes, which are known to have diverse physiological and

pharmacological activities (Consolacion et al., 2007 Fernandes et al., 2003; Harmand et

al., 2003). The antioxidant activity of terpenoids was well documented (Chae et al., 2009;

D’Abrosca et al., 2006).

Antibacterial activity of isolated compounds

Minimum inhibitory concentration (MIC)

The triterpenoid compounds 4-epicyclomusalenone and cycloeucalenol acetate

isolated from chloroform extract were exhibited higher antimicrobial activity when

compared to chlorogenic acid isolated from acetone extract of banana rhizome (table 3.7).

Further, these compounds showed antibacterial and antifungal activities against wide

spectrum of bacterial and fungal species and were more effective against Gram +ve

bacteria (high activity with low MIC value) than Gram -ve bacteria. Highest activity with

lowest MIC value were observed against Gram +ve bacteria of 60 ppm was observed in

4-epicyclomusalenone against M. luteus followed by cycloeucalenol acetate against M.

luteus (90 ppm). While, chlorogenic acid (90 ppm), and 4-epicyclomusalenone (90 ppm)

against B. subtilis. Among Gram -ve bacteria high activity with low MIC value of 160

ppm was shown by cycloeucalenol acetate against K. pneumoniae, followed by 180 ppm

and 220 ppm were shown by 4-epicyclomusalenone and cycloeucalenol acetate

respectively against S. typhi. Interestingly, chlorogenic acid showed MIC value only for

two Gram -ve bacteria viz., P. aeruginosa (320 ppm) and K. pneumoniae (770 ppm).


89

Table 3.7: Antimicrobial activity (MIC and bactericidal and fungicidal) in ppm

concentration of isolated compounds

Microbial

strains

Chlorogenic acid 4-Epicyclomusalenone Cycloeucalenol acetate

MIC Bactericidal

activity MIC

Bactericidal

activity MIC

Bactericidal

activity

Bacterial strains

Gram +ve bacteria

M. luteus 100 Yes 60 Yes 90 Yes

S. aureus 260 Yes 270 No 550 No

E. fecalis 380 No 100 Yes 190 Yes

B. cereus 150 Yes 120 Yes 150 Yes

B. subtilis 90 Yes 90 Yes 170 Yes

L. monocytogenes 110 Yes 180 Yes 110 Yes

Gram -ve bacteria

P. aeruginosa 320 Yes - - 280 Yes

E. coli - - - - - -

S. typhi - - 180 Yes 220 Yes

K. pneumoniae 770 No 240 Yes 160 Yes

E. aerogenes - - 820 No 790 No

P. mirabilis - - - - - -

Y. enterocolitica - - - - - -

Fungal strains

Fungal strains MIC Fungicidal

activity MIC

Fungicidal

activity MIC

Fungicidal

activity

A. niger 260 Yes 280 Yes 210 Yes

A. flavus 340 No 410 No 560 No

A. fumigatus 180 Yes 320 Yes 450 Yes

A. parasiticus 220 Yes 400 No 650 No

P. rubrum - - 750 No 850 No

F. moniliforme 300 No 780 No 900 No

*Each value represents mean of three different observations

Determination of bactericidal effect

The isolated compounds viz., chlorogenic acid, 4-epicyclomusalenone and

cycloeucalenol acetate were found to be bactericidal against wide range of bacterial

species tested (table 3.7). All the three compounds showed bactericidal effect against all


90

the Gram +ve bacteria tested, except against S. aureus (by 4-epicyclomusalenone and

cycloeucalenol acetate) and E. fecalis (by chlorogenic acid). Among Gram -ve bacteria,

4-epicyclomusalenone showed bactericidal effect against S. typhi and K. pneumoniae,

and cycloeucalenol acetate showed bactericidal effect against P. aeruginosa, S. typhi and

K. pneumoniae. Interestingly, chlorogenic acid showed bactericidal effect against only

one Gram -ve bacteria (P. aeruginosa). It appeared that effective MIC also represents the

effective bactericidal concentration for the bacteria tested.

The sensitivity difference between Gram +ve and Gram –ve bacteria towards the

tested pure compounds could be ascribed to the morphological differences between these

microorganisms. The structures of cell envelope, including cytoplasmic membrane and

cell wall component are different between Gram +ve and gram -ve bacteria. Gram -ve

bacteria possess an outer membrane surrounding the cell wall, which restricts diffusion of

hydrophobic compounds through its lipopolysaccharide covering. Without outer

membrane, the cell wall of Gram +ve bacteria can be permeated more easily and

polyphenols can disturb the cytoplasmic membrane, disrupt the proton motive force

(PMF), electron flow, active transport and coagulation of cell contents (Burt, 2004).

Antifungal activity

All the three isolated compounds were found to be effective against tested fungal

strains (table 3.7). Unlike bacterial strains, fungal strains were found to be more

susceptible to chlorogenic acid, followed by 4-epicyclomusalenone and cycloeucalenol

acetate. Triterpene compounds showed antifungal activity (MIC value) against all the

fungal strains tested. Whereas, chlorogenic acid failed to inhibit P. rubrum. High

antifungal activity with low MIC values were observed in chlorogenic acid against A.

fumigatus (180 ppm), cycloeucalenol acetate against A. niger (210 ppm) and chlorogenic

acid against A. parasiticus (220 ppm). The isolated compounds viz., chlorogenic acid, 4-

epicyclomusalenone and cycloeucalenol acetate were found to be fungicidal against wide

range of fungal species tested. Compound chlorogenic acid showed fungicidal effect

against A. niger, A. fumigatus and A. parasiticus. Whereas, compounds 4-


91

epicyclomusalenone and cycloeucalenol acetate were exhibited fungicidal effect against

A. niger and A. fumigatus.

Multiple antimicrobial activities of the isolated compounds and their structure

activity relationship

Polyphenols participate mainly in plant protection, structural, photosynthesis and

nutrient uptake, among other functions in vascular plants. Moreover, it has been

demonstrated that induction of resistance in several plant species is followed by

production of phenolics with antimicrobial activity (Puupponen-Pimiä et al., 2001). Many

plant polyphenols are known to possess antimicrobial properties, so they might change

the composition of microflora in any environment in which these compounds are applied

and/or induced in a proper kind and concentration (Puupponen-Pimiä et al., 2001;

Heinäaho et al., 2006). In fact, the antimicrobial activity of phenolics is well known and

it is related to their ability to denature proteins, being generally classified as surface-

active agents (Sousa, 2006). High antimicrobial activity of chlorogenic acid against wide

range of bacterial and fungal strains may be attributed to its structural components. It

possesses six-hydroxyl, two-methylene and two-carbonyl groups along with ester bond

linkage which are proposed to be responsible for increased antibacterial activity (Ultee et

al., 2002). Hydroxyl group of the compound is responsible for depletion of ATP

dependent metabolic functions, ultimately leading to cell death (Ultee et al., 2002).

Further, presence of oxygen function in the framework of the compound chlorogenic acid

increases the antimicrobial properties (Naigre et al., 1996).

The antibacterial and antifungal activities of triterpenes were well established

(Ragasa et al., 2007). Most of the terpenoids tested were found to inhibit oxygen uptake

and oxidative phosphorylation (Knobloch et al., 1985). Terpenoid compounds were

shown to permeabilize the membranes, making them swell. This inhibits respiratory

enzymes, which led to a partial dissipation of the pH gradient and electrical potential,

which are crucial to the energy system in a cell (Sikkema et al., 1992 and 1994). The

structural components six-methyl and one-carbonyl groups may account for the enhanced


92

antimicrobial activity of compounds 4-epicyclomusalenone and cycloeucalenol acetate.

Presence of carboxyl and acetoxyl groups along with oxygen function of the triterpenoid

compounds may also increases the antibacterial properties (Naigre et al., 1996). Further

investigations are required to test the mode and site of action of the isolated compounds.

Platelet-aggregation inhibitory activity

Platelet aggregation is a complex phenomenon that involves several intracellular

events involving many biochemical pathways (Son et al., 2004) and causes thrombosis,

cardiovascular diseases, thromboembolic complications of atherosclerosis, heart attacks,

strokes and peripheral vascular diseases (Ross, 1986). Therefore, the inhibition of platelet

function represents a promising approach for the prevention of thrombosis (Ross, 1986).

Platelet activate by agonists viz., collagen, arachidonic acid, thrombin, epinephrine,

calcium ionophore, restocitin through binding to seven transmembrane receptors coupled

with G proteins (GP IIb - IIIa). Activation of G proteins is followed by an increase in the

free calcium levels in the platelets. Second messengers such as Ca2+

, IP3 and protein

tyrosine kinases increase platelet aggregation, whereas cAMP inhibits (Bloekmans et al,

1995). In this study, platelet aggregation was induced by collagen. It is a triple stranded

coil composed of one α and two β-chains, the latter being intramolecularly cross linked

(Siess, 1989). Collagen can interact with very diverse proteins, such as fibronectin,

fibrinogen, vWF, factor VIII (Dessall et al., 1978; Girma et al., 1986). The expression of

endothelium collagen following vascular injury plays an important dual role in the

contribution of platelets to both the haemostatic by atherosclerotic process, in vitro.

Firstly, platelets adhere to expressed collagen via the α2β1 integrin (glycoprotein (GP)

Ia/Iia) forming the initial haemostatic layer, and secondly, collagen activates platelets,

thereby recruiting additional platelets to and consolidating the thrombosis (Lockhart et

al., 2001).


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Table 3.8: Platelet aggregation inhibitory activity

(EC50 value in µg mL−1

) of isolated compounds from

banana var. Nanjanagudu Rasbale

Isolated compounds EC50 value µg mL−1

Chlorogenic acid 102±1.6a

4-Epicyclomusalenone 126±3.3c

Cycloeucalenol acetate 115±1.9b

Each value represents means ± SD (n = 3) and values with different superscripts

differ significantly at p<0.05. *EC50: Effective concentration of the sample to show

50% of platelet aggregation inhibitory activity

The isolated compounds chlorogenic acid, 4-epicyclomusalenone and

cycloeucalenol acetate were exhibited platelet aggregation inhibitory activity with EC50

value of 102±1.6, 126±3.3 and 115±1.9 µg/mL, respectively (table 3.8). Isolated

compounds inhibit the platelet aggregation induced by collagen indicating that it is likely

to not only competing with GP IIb–IIIa receptors along with these agonists, but may also

reduce their interaction with these receptors to cause inhibition (Bloekmans et al., 1995).

The platelet-aggregation inhibitory properties of the isolated compounds may be

attributed to the inhibition of TxA2 formation (You et al., 1999), thromboxane receptor

antagonism (Hubbart et al., 2003), protein kinase C activation (Ganet-Payrastre et al.,

1999) and phosphoinositide synthesis. Further experiments are required to elucidate the

exact biochemical alterations taking place in platelets as a result of their interaction

within the purified compounds, which may be causing the inhibition of platelet

aggregation.

Cytotoxicity

All the three compounds showed toxicity against HepG-2 cells. Chlorogenic acid

exhibited high cytotoxicity against HepG-2 cell line with CTC50 of 82±1.1 µg/mL,


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followed by cycloeucalenol acetate (93±1.5 µg/mL) and 4-Epicyclomusalenone (108±1.8

µg/mL), in MTT assay (table 3.9).

Table 3.9: Cytotoxicity ( CTC50 value in µg mL−1

) of isolated

compounds from banana var. Nanjanagudu Rasbale

Isolated compounds CTC50 value µg mL−1

Chlorogenic acid 82±1.1a

4-Epicyclomusalenone 108±1.8c

Cycloeucalenol acetate 93±1.5b

Each value represents means ± SD (n = 3) and values with different superscripts differ

significantly at p<0.05. * CTC50: Effective concentration of the sample to show 50% of

cytotoxicity

The cytotoxicity results of isolated compounds indicate that the compounds are

toxic towards the HepG-2 cells. Human epidemiology and animal studies have indicated

that cancer risk may be modified by dietary components. The naturally occurring,

phytochemicals or active compounds from fruits, vegetables, grains, nuts, tea and seeds,

may prevent or reduce the risk of cancer (Patriäcia et al., 2003). Terpenoids (Kim et al.,

2000; Fernandes et al., 2003; Rumjanek et al., 2001; Harmand et al., 2003) and

polyphenols (Jing et al., 2010) were known to be cytotoxic against cancer causing cell

lines. The mode of action on cytotoxicity is not well established, but there are reports that

protein binding ability, in particular membrane protein of cell lines may affects the cell

growth and its viability (Damianaki et al., 2000). Cytotoxic properties exerted by the

isolated compounds merit further investigations to identify the activity in animal models.


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Conclusion

Three bioactive compounds were successfully isolated and characterized from

banana rhizome extracts. This is the first report of the bioactive compounds viz.,

chlorogenic acid, 4-epicyclomusalenone and cycloeucalenol acetate from banana (var.

Najanagudu Rasbale) rhizome. All the three bioactive compounds exhibited multiple

antioxidant activity, antimicrobial activity against broad spectrum bacterial and fungal

species, platelet aggregation inhibition activity and cytotoxicty.

The acetone extract of banana rhizome showed high polyphenolic content,

antioxidant activity, antimicrobial activity, platelet aggregation inhibitory activity,

cytotoxicity properties and also yielded multifunctional compound chlorogenic acid.

Hence, acetone extract of banana rhizome var. Nanjanagudu Rasbale was selected for

alleviation of antioxidant mediated diabetes in streptozotocin induced diabetic rats.

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