UNIVERSITI PUTRA MALAYSIA
OPTIMIZATION OF INVITRO CULTURES AND EFFECTS OF ELICITATION THE FLAVONOID CONTENTS OF PEGAGA (CENTELLA
ASIATICA L. URBAN)
ONG MEI KYING
FBSB 2008 11
OPTIMIZATIO� OF I� VITRO CULTURES A�D
EFFECTS OF ELICITATIO� O� THE
FLAVO�OID CO�TE�TS OF PEGAGA
(CE�TELLA ASIATICA L. URBA�)
O�G MEI KYI�G
MASTER OF SCIE�CE
U�IVERSITI PUTRA MALAYSIA
2008
OPTIMIZATIO� OF I� VITRO CULTURES A�D
EFFECTS OF ELICITATIO� O� THE FLAVO�OID CO�TE�TS OF
PEGAGA (CE�TELLA ASIATICA L. URBA�)
By
O�G MEI KYI�G
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfilment of the Requirement for the Degree of Master of Science
February 2008
Dedicated with loveDedicated with loveDedicated with loveDedicated with love and gratitudeand gratitudeand gratitudeand gratitude to:to:to:to:
MMMMy fy fy fy father, Ong Siow Kinather, Ong Siow Kinather, Ong Siow Kinather, Ong Siow Kin
aaaand nd nd nd
My mMy mMy mMy mother, Ng Siew Chengother, Ng Siew Chengother, Ng Siew Chengother, Ng Siew Cheng
ii
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of
the requirement for the degree of Master of Science
OPTIMIZATIO� OF I� VITRO CULTURES A�D
EFFECTS OF ELICITATIO� O� THE FLAVO�OID CO�TE�TS OF
PEGAGA (CE�TELLA ASIATICA L. URBA�)
By
O�G MEI KYI�G
February 2008
Chairman : Associate Professor �orihan Mohd Saleh, PhD
Faculty : Biotechnology and Biomolecular Sciences
Secondary metabolites in Centella asiatica, especially flavonoids is known to possess
strong antioxidative activity and widely investigated as a new source of active
compound for health benefits. The flavonoids content in field planted Centella
asiatica is very low, therefore high volume of Centella asiatica plant supply is needed
to fulfill the increase demand for the active compounds as an important nutraceutical
resources. The main aim of this research done was to obtain the most applicable,
simplest and effective in vitro approaches that can increase the flavonoids content in
Centella asiatica (CA05). Many research studies done have proved that flavonoids
content can be successfully enhanced through elicitation. In this study, elicitation of
important flavonoids such as catechin was investigated and their presence in various
tissues of Centella asiatica (CA05) planted using various cultivation methods were
assessed. The initial work involved the optimization of plantlet regeneration and root
iii
culture of Centella asiatica (CA05). Regeneration of Centella asiatica (CA05) from
various explants is necessary in order to produce continuous supply of plant materials
for further manipulation. In addition to in vitro plantlets and root culture,
hydroponically grown plantlets were also used for flavonoids elicitation using biotic
elicitors, namely chitosan and yeast extract. Various techniques of sterilization and
media formulation using different plant growth regulators such as auxin and cytokinin
were applied to regenerate the plant. The explants were sterilized using 30% Clorox
(1.58% sodium hypochlorite) for 5 minutes (leaf), 30% Clorox (1.58% sodium
hypochlorite) for 15 minutes (stem), 0.05% mercury chloride for 5 minutes and
followed by 30% Clorox (1.58% sodium hypochlorite) for 20 minutes (seed) for
56.7% of viable leaf explant, 75.0% of viable stem explant and 48.3% of sterile
seedlings, respectively. For plant regeneration, MS supplemented with 2.26µM
Indole-3-acetic acid (IAA) and 2.26µM 6-Benzylaminopurine (BAP) exhibited 38.0%
of shoot regeneration frequency from leaf explants. MS supplemented with 2.26µM
Indole-3-butyric acid (IBA) showed rapid induction of root from stem (63.0%) after 7
days. In this study, the seed of Centella asiatica were treated with various techniques
to break the seed dormancy. The hard seed were pretreated by scarification and
soaking for various time regime. Effect of presoaking in MSO liquid medium for 1
day before culture gave the highest percentage means of germination (81.0%). High
pressure liquid chromatography (HPLC) was used to determine the flavonoids in
various Centella asiatica plant parts (root, stem, leaf) grown in vitro and hydroponics.
Catechin is detected to be major flavonoids in Centella asiatica in addition to naringin,
hesperidin, rutin and myricetin. Result from the study showed that in vitro plantlets
iv
contain high total flavonoids (4456.9 + 287.5 µg/g) compared to that of hydroponics-
grown plantlets (2401.0 + 148.4 µg/g) and that of field plant (2323.5 + 376.8 µg/g).
In general, leaf of Centella asiatica was found to contain the highest flavonoids
content compared to either root or stem. Effects of both elicitors, namely chitosan
and yeast extract on flavonoids production were evaluated. Result of the study
showed that root culture treated with 0.5 mg/l chitosan for one week showed the
highest flavonoids (2968.2 + 66.1 µg/g) with 2.28 fold increase of catechin (128.1%
higher than control). While, for the elicitation in in vitro plantlet with 5 mg/l chitosan
in 1 week, the highest total flavonoids amount was recorded at 5530.6 + 385.5 µg/g
with increase of 17.9% or 1.2 fold compared to control. Prolong exposure to elicitors
up to four weeks did not show any increase of flavonoids content in all cultivated
plant tissues. Therefore, it can be concluded that, flavonoids production was
improved with chitosan elicitation, and it can be helpful in increasing the productivity
of flavonoids.
v
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk Ijazah Master Sains
PE�I�GKATA� KULTUR I� VITRO DA� KESA� PE�GARUHA�
KA�DU�GA� FLAVO�OID DALAM PEGAGA
(CE�TELLA ASIATICA L. URBA�)
Oleh
O�G MEI KYI�G
Februari 2008
Pengerusi : Profesor Madya �orihan Mohd Saleh, PhD
Fakulti : Bioteknologi dan Sains Biomolekul
Metabolit sekunder yang dikandungi oleh Centella asiatica, terutamanya flavonoids,
diketahui mempunyai aktiviti antioksidatif dan telah dikaji dengan meluas sebagai
sumber baru sebatian aktif yang baik untuk kesihatan. Disebabkan kandungan
flavonoids yang rendah dalam pokok Centella asiatica tanaman ladang, bekalan
pokok Centella asiatica yang tinggi diperlukan untuk memenuhi permintaan yang
meningkat terhadap bahan aktif ini sebagai sumber nutraceutika yang penting. Tujuan
utama kajian ini dijalankan adalah untuk memperolehi pendekatan secara in vitro
yang paling mudah, berkesan dan berkeupayaan untuk meningkatkan kandungan
flavonoids dalam pokok Centella asiatica (CA05). Kebanyakan kajian penyelidikan
telah berjaya membuktikan kandungan flavonoids boleh ditingkatkan melalui
pengaruhan. Dalam kajian ini, oleh kerana kandungan flavonoids boleh ditingkatkan
melalui pengaruhan secara in vitro, pengaruhan flavonoids penting seperti catechin
telah dikaji dan kewujudannya di dalam pelbagai tisu Centella asiatica (CA05) yang
vi
dibiakkan menggunakan pelbagai kaedah penanaman telah dibuat penilaian. Kajian
awal melibatkan regenerasi anak pokok dan kultur akar Centella asiatica (CA05).
Regenerasi anak pokok Centella asiatica (CA05) daripada pelbagai eksplan adalah
perlu untuk menghasilkan sumber pokok yang berterusan bagi manipulasi seterusnya.
Selain kultur akar dan pokok in vitro, pokok yang ditanam melalui teknik hidroponik
juga digunakan untuk pengaruhan flavonoids dengan menggunakan agen perangsang
biotik, seperti chitosan dan ekstrak yis. Pelbagai teknik pensterilan dan formulasi
media dengan penggunaan pengawalatur tumbuhan seperti auksin dan sitokinin
digunakan untuk meregenerasikan pokok ini. Eksplan daun disteril dengan 30%
Klorox (1.58% sodium hipoklorit) selama 5 minit menghasilkan 56.7% eksplan hidup,
75% eksplan batang hidup dihasilkan dengan 30% Klorox (1.58% sodium hipoklorit)
selama 15 minit, manakala pensterilan biji benih dengan menggunakan 0.05%
merkuri klorida selama 5 minit, diikuti dengan 30% Klorox (1.58% sodium
hipoklorit) selama 20 minit adalah perlu untuk menghasilkan 48.3% pokok in vitro.
Untuk regenerasi pokok, media Murashige and Skoog (MS) yang ditambahkan
dengan 2.26 µM IAA dan 2.26 µM BAP telah menunjukkan 38.0% frekuensi
regenerasi pucuk daripada eksplan daun. Media Murashige and Skoog (MS) yang
ditambahkan dengan 2.26 µM IBA menunjukkan kadar penghasilan akar daripada
eksplan batang (63.0%) selepas 1 minggu. Dalam kajian ini juga, biji benih Centella
asiatica telah dirawat dengan pelbagai teknik memecahkan kedormanan benih. Benih
yang berkulit keras ini dipra-rawat dengan sagatan dan pra-rendaman dalam pelbagai
masa rendaman. Kesan pra-rendaman benih di dalam MSO medium cecair selama
sehari sebelum dikultur memberi peratusan percambahan yang tertinggi iaitu 81.0%.
vii
Kromatografi cecair bertekanan tinggi (HPLC) digunakan untuk menentukan
flavonoids yang terkandung dalam pelbagai bahagian tisu Centella asiatica (akar,
batang, daun) yang dikultur secara in vitro mahupun hidroponik. Catechin dalam
Centella asiatica dikesan paling tinggi selain naringin, hesperidin, rutin dan
myricetin. Keputusan kajian ini menunjukkan pokok in vitro mengandungi kandungan
flavonoids yang tertinggi iaitu 4456.9 + 287.5 µg/g berbanding dengan pokok
hidroponik (2401.0 + 148.4 µg/g) dan pokok tanaman ladang (2323.5 + 376.8 µg/g).
Secara amnya, daun Centella asiatica ditemui mengandungi kandungan flavonoids
yang tertinggi berbanding akar mahupun batang. Kesan peningkatan kandungan
flavonoids melalui penggunaan agen perangsang biotik seperti chitosan dan ekstrak
yis telah dinilai. Keputusan kajian menunjukkan kultur akar yang dirawat dengan 0.5
mg/l chitosan selama seminggu menunjukkan kandungan flavonoids tertinggi iaitu
2968.2 + 66.1 µg/g, dengan peningkatan catechin sebanyak 2.28 ganda (128.1 % lebih
tinggi berbanding kultur asal tanpa rawatan). Sementara, pengaruhan pokok in vitro
dengan 5 mg/l chitosan selama seminggu, mencatat kandungan jumlah flavonoids
tertinggi sebanyak 5530.6 + 385.5 µg/g dengan peningkatan 17.9 % atau 1.2 ganda
berbanding kultur asal tanpa rawatan. Pendedahan tumbuhan yang berlanjutan kepada
agen pengaruh biotik melebihi empat minggu tidak menunjukkan peningkatan
kandungan flavonoids dalam semua tisu Centella asiatica. Oleh itu, adalah dapat
dirumuskan bahawa producktiviti flavonoids boleh dimajukan dengan pengaruhan
chitosan dan ini dapat membantu meningkatkan produktiviti flavonoids.
viii
ACK�OWLEDGEME�TS
My special and utmost appreciation and gratitude to Associate Profesor Dr. Norihan
Mohd. Saleh for her compassionate and valuable guidance and mentor. Her ceaseless
patient and encouragement has moulded me into a researcher full of high spirit and
inner strength.
My heartfelt gratitude to Associate Profesor Dr. Azizah Abdul Hamid for her
kindness, patience and continuous motivation to excellence. A special thanks is also
recorded to Dr. Mohd Azmuddin Abdullah who was involved in supervisory
committee at the early part of this study.
My appreciation is also extended to the tissue culture lab members especially Kak
Fadhilah, Kak Liza, Kak Roszaimah and Mr. Ong. Besides, all the lab comrades and
in time of woe and joy companions especially Kak Norrizah, Kak Umi, Nor Aliza,
Tzer Miin, Chiang Lim, Christina, Mai Anh, Thuc, Yohannes, Kak Watima, Elham
and Siong Thong for their ever ready help and support. I would also like to thank
nutraceutical lab members especially Abang Khairi, Abang Reza, Rosnizam, Kak
Aya, Kak Lah, Kak Mahanom and Syikin. Not to forget, I am also grateful to the
staffs at the HPLC laboratory (Encik Halim) and Food Chemistry laboratory (Encik
Azman) of the Food Science Department (UPM), and Hydroponics Unit (Encik
Yunus) of Agriculture Faculty (UPM).
ix
Lastly but not the least, to my loving and supportive husband, Andrew Chong, to all
my dedicated teachers especially Bro. Tan, my friends, my family members, my
relatives, all Sri Serdang house residents, and to those unnamed, I would like to
present this thesis as testimony of each and everyone’s unique loving kindness,
endless support and contribution directly and indirectly. Financial supports from
research grant Vot. Pegaga 63537 and the sponsorship of PASCA graduates studies
from The Ministry of Science, Technology and Environment of Malaysia are
gratefully acknowledged.
x
I certify that an Examination Committee has met on 5th February 2008 to conduct the
final examination of Ong Mei Kying on her Master of Science thesis entitled
“Optimization of In Vitro Cultures and Effects of Elicitation on the Flavonoids
Contents of Pegaga (Centella asiatica L. Urban)” in accordance with Universiti
Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia
(Higher Degree) Regulations 1981. The Committee recommends that the student be
awarded the degree of Master of Science.
Members of the Examination Committee were as follows:
Janna Ong Abdullah, PhD
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Suhaimi �apis, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Mohd Puad Abdullah, PhD Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Muhammad �or Omar, PhD Product Development and Advisory Services
Malaysian Palm Oil Board (MPOB)
Malaysia
(External Examiner)
________________________________
HASA�AH MOHD. GHAZALI, PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 26 May 2008
xi
This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Master of Science.
The members of the Supervisory Committee were as follows:
�orihan Mohd. Saleh, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Chairman)
Azizah Abdul Hamid, PhD
Associate Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Member)
AI�I IDERIS, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 12 June 2008
xii
DECLARATIO�
I declare that the thesis is my original work except for quotations and citations, which
have been duly acknowledged. I also declare that it has not been previously, and is not
concurrently, submitted for any other degree at Universiti Putra Malaysia or at any
other institution.
O�G MEI KYI�G
Date: 24 April 2008
xiii
TABLE OF CO�TE�TS
Page
DEDICATIO� ii
ABSTRACT iii
ABSTRAK vi
ACK�OWLEDGEME�TS ix
APPROVAL xi
DECLARATIO� xiii
LIST OF TABLES xvii
LIST OF FIGURES xix
LIST OF APPE�DICES xxii
LIST OF ABBREVIATIO�S xxiv
GLOSSARY xxvi
CHAPTER
1 I�TRODUCTIO�
1.1 Introduction 1
1.2 Objectives 3
2 LITERATURE REVIEW
2.1 Centella asiatica 4
2.1.1 Basic morphology 4
2.1.2 Benefits and uses 5
2.2 Secondary Metabolite 7
2.2.1 Importance of secondary metabolite 9
2.2.2 Roles of plant cell culture in secondary metabolite
enhancement
10
2.3 Flavonoids 12
2.3.1 Flavonoids in nature 12
2.3.2 Functional properties of flavonoids 17
2.3.3 Flavonoids isolation and determination 19
2.4 Establishment of tissue culture system for Centella asiatica 21
2.4.1 Callus induction and maintenance 21
2.4.2 Plant regeneration 21
xiv
2.5 Establishment of root culture 22
2.5.1 Effect of plant growth regulator on root formation 24
2.5.2 Effect of elicitor on secondary metabolite production
in root culture
25
2.6 Elicitation 25
2.6.1 Types of elicitors 26
2.6.2 Action mechanism of elicitors 28
2.6.3 Advantages of elicitor on production of secondary
metabolite
28
3.0 MATERIALS A�D METHODS
3.1 Plant material 31
3.1.1 Seed, leaf and stem 31
3.2 Sterilization 31
3.2.1 Seed 31
3.2.2 Leaf and stem 33
3.3 Pretreatment of seeds 35
3.3.1 Pre-soaking treatment 35
3.3.2 Scarification 38
3.4 Medium formulations 39
3.4.1 MS basal medium supplemented with different plant
growth regulators for seed germination
39
3.4.2 Regeneration medium for Centella asiatica plantlet 39
3.4.3 Regeneration of plantlet from explants 40
3.4.4 Induction of organogenenesis 40
3.5 Preparation of hydroponics system 43
3.6 Preparation of elicitors 43
3.7 Elicitation 44
3.7.1 In vitro root culture 44
3.7.2 In vitro plantlet 45
3.7.3 Hydroponics plantlet 47
3.8 Determination of flavonoids 49
3.8.1 Preparation of flavonoid standards 49
3.8.2 Extraction of flavonoids 49
3.8.3 Reversed-phase High Performance Liquid
Chromatography
50
3.9 Statistical analysis 52
xv
4.0 RESULTS A�D DISCUSSIO� 53
4.1 Sterilization technique 53
4.1.1 Seed 54
4.1.2 Leaf and stem 56
4.2 Pretreatment prior to seed germination 60
4.2.1 Pre-soaking of explants in MSO and distilled water 60
4.2.2 Effect of scarification on seed germination 63
4.3 Effect of different types of plant growth regulators on
germination of Centella asiatica seed
66
4.4 Induction of calli 70
4.5 Organogenesis of Centella asiatica 77
4.6 Establishment of root culture 83
4.7 Determination of flavonoids content in Centella asiatica 87
4.7.1 Fresh field plant 87
4.7.2 Hydroponics plantlet 89
4.7.3 In vitro plantlet 89
4.7.4 Root culture 90
4.8 Assessment of different types and concentrations of elicitors
on flavonoids content in tissues of Centella asiatica from
various cultivation method
92
4.8.1 Hydroponics plantlet 93
4.8.2 In vitro plantlet 97
4.8.3 Root culture 108
5.0 CO�CLUSIO� A�D RECOMMME�DATIO�S FOR FUTURE
RESEARCH
118
BIBLIOGRAPHY 122
APPE�DICES 138
BIODATA OF STUDE�T 168
xvi
LIST OF TABLES
Table Page
1 Classification of elicitors 27
2 Examples of elicitation for enhancement of secondary metabolites
used as food ingredients
30
3 Mean percentages of sterile viable seeds according to various
sterilization treatments
55
4 Sterilization of Centella asiatica leaf explants using different
concentration and duration of Clorox
57
5 Sterilization of Centella asiatica stem explants using different
concentration and duration of Clorox
59
6 Efficiency and duration of initial germination of Centella asiatica
seeds pretreated with presoaking treatments
62
7 Efficiency and duration of initial germination of Centella asiatica
seeds pretreated with different concentration of sucrose and
scarification treatments
65
8 Efficiency and duration of initial germination of Centella asiatica
seeds cultured onto MS medium supplemented with different
concentration of plant growth regulators
69
9 Assessment of the responses of Centella asiatica leaf explant in
MS media (1962) supplemented with different types of plant
growth regulators, which were incubated for 4 weeks in the light
72
10 Assessment of the responses of Centella asiatica leaf explant in
MS media (1962) supplemented with different types of plant
growth regulators, which were incubated for 4 weeks in the dark
73
11 Assessment of the responses of Centella asiatica stem explant in
MS media (1962) supplemented with different types of plant
growth regulators, which were incubated for 4 weeks in the light
75
12 Assessment of the responses of Centella asiatica stem explant in
MS media (1962) supplemented with different types of plant
growth regulators, which were incubated for 4 weeks in the dark
76
xvii
13 Percentage of fresh weight increase of root cultures in MS media
containing different combination of plant growth regulators in 4
weeks
155
14 Root morphological characteristic in media contained different
plant growth regulators
86
15 Different flavonoids content found in fresh field, in vitro and
hydroponics cultivated Centella asiatica
88
16 Different flavonoids content found in fresh root culture of Centella
asiatica in week 1-week 7
156
17 Different flavonoids content found in fresh hidroponics cultivated
Centella asiatica after 1 week of elicitation
157
18 Different flavonoids content found in elicited in vitro Centella
asiatica with 5 mg/l chitosan for a period of 4 weeks
99
19 Different flavonoids content found in elicited root culture of
Centella asiatica with 0.5 mg/l chitosan for a period of 4 weeks
109
xviii
LIST OF FIGURES
Figure Page
1 Basic morphology of Centella asiatica
6
2 Synthesis of major classes of secondary metabolites from primary
metabolites
8
3 Biosynthesis of flavonoids
13
4 Flavonoids structure
14
5 The structures of flavanol, flavone, flavanones and flavonols
16
6 The parts in Centella asiatica plant
32
7 Sterilization process for Centella asiatica explants
34
8 Flow chart of the designed experiment for presoaking treatment 36
9 In vitro Centella asiatica (CA05) plantlets in liquid medium of
elicitation
46
10 Stock solution for hydroponics cultivation
48
11 Whole hydroponics-grown plantlets
48
12 Summary flow chart on sample preparation for flavonoids
determination via reverse-phase HPLC method
51
13 Germinated seed of Centella asiatica 67
14 Induction of calli 71
15 Induction of calli and shoot organogenesis on MS supplemented
with 2.26µM IAA and 2.26µM BAP under light condition
78
16 Induction of calli and shoot organogenesis onto MS media
supplemented with 2.26 µM IBA and 2.26 µM BAP under light
condition
80
17 Root organogenesis of different explants cultured on MS
supplemented with 2.26 µM IBA in the dark after 1 week
82
18 Growth rate of Centella asiatica root cultured in MS liquid media
supplemented with various plant growth regulators
84
xix
19 Flavonoids conent in Centella asiatica root culture grown in MS
medium supplemented with 2.26 µM IBA from week 1 to week 7
91
20 Different flavonoids level in elicitors treated hydroponics
cultivated Centella asiatica (1 week)
95
21 Flavonoids level in untreated in vitro Centella asiatica as control
for a period of 4 weeks
100
22 Flavonoids level in elicited in vitro Centella asiatica with 0.5 mg/l
chitosan for a period of 4 weeks
100
23 Flavonoids level in elicited in vitro Centella asiatica with 5 mg/l
chitosan for a period of 4 weeks
101
24 Flavonoids level in elicited in vitro Centella asiatica with 10mg/l
chitosan for a period of 4 weeks
101
25 Flavonoids level in elicited in vitro Centella asiatica with 0.5mg/l
yeast extract for a period of 4 weeks
104
26 Flavonoids level in elicited in vitro Centella asiatica with 5mg/l
yeast extract for a period of 4 weeks
104
27 Flavonoids level in elicited in vitro Centella asiatica with 10mg/l
yeast extract for a period of 4 weeks
105
28 Flavonoids level in elicited root culture Centella asiatica with 5
mg/l chitosan for a period of 4 weeks
110
29 Flavonoids level in untreated root culture Centella asiatica as
control for a period of 4 weeks
110
30 Flavonoids level in elicited root culture Centella asiatica with 0.5
mg/l chitosan for a period of 4 weeks
112
31 Flavonoids level in elicited root culture Centella asiatica with 10
mg/l yeast extract for a period of 4 weeks
112
32 Flavonoids level in elicited root culture Centella asiatica with 5
mg/l yeast extract for a period of 4 weeks
113
33 Flavonoids level in elicited root culture Centella asiatica with 10
mg/l chitosan for a period of 4 weeks
115
34 Flavonoids level in elicited root culture Centella asiatica with 0.5
mg/l yeast extract for a period of 4 weeks
115
xx
35 Root culture after 1 week of incubation in control (MS+IBA).
147
36 Root culture after 1 week of incubation in chitosan of 0.5 mg/l
147
37 Root culture after 1 week of incubation in yeast extract of 0.5 mg/l
147
xxi
LIST OF APPE�DICES
Appendix Page
A General 139
A1 Summary of experimental approach 140
A2 Basic morphology of Centella asiatica 141
A3 Composition of MS (Murashige and Skoog) basal
medium (1962)
142
A4 Notes on solutions and list of compounds and
molecular weights
143
A5 Preparation of stock hormone 144
A6 Combination of plant growth regulators to regenerate
plantlet from explant.
145
A7 Copper formulation
146
A8 Morphology different between control root culture with
elicited root culture
147
A9 The eight flavonoids and its individual functions and
benefits
148
A10 Retention time of 8 types of flavonoids standards
153
B Result tables
154
B1 Percentage of fresh weight increase of root cultures
155
B2 Flavonoids content in Centella asiatica root cultures
from week 1 to week 7
156
B3 Flavonoids content in hydroponically grown Centella
asiatica
157
xxii
C HPLC Chromatograms
158
C1 HPLC Chromatogram of mix solution of 8 standards
monitored at 280 nm
159
C2 Representative HPLC Chromatogram of standard
catechin and naringin at concentration of 500 ppm
160
C3 Representative HPLC Chromatogram of standard
hesperidin and rutin at concentration of 500 ppm
161
C4 Representative HPLC Chromatogram of standard
myricetin and quercetin at concentration of 500 ppm
162
C5 Representative HPLC Chromatogram of standard
kaempferol and apigenin at concentration of 500 ppm
163
C6 Representative HPLC Chromatogram of control and
elicited leaf in in vitro Centella asiatica
164
C7 Representative HPLC Chromatogram of control and
elicited leaf in hidroponics-grown Centella asiatica
165
C8 Representative HPLC Chromatogram of control and
elicited root culture Centella asiatica
166
C9 Representative HPLC Chromatogram of leaf in
field-grown Centella asiatica
167
xxiii