universiti putra malaysia optimization of invitro...

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


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


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



(Internal Examiner)

Mohd Puad Abdullah, PhD Faculty of Biotechnology and Biomolecular Sciences


(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


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



(Chairman)

Azizah Abdul Hamid, PhD

Associate Professor

Faculty of Food Science and Technology


(Member)

AI�I IDERIS, PhD

Professor and Dean

School of Graduate Studies


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



4.7.4 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



4.8.3 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


seeds pretreated with different concentration of sucrose and

scarification treatments

65


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


growth regulators, which were incubated for 4 weeks in the dark

73

11 Assessment of the responses of Centella asiatica stem explant in


growth regulators, which were incubated for 4 weeks in the light

75

12 Assessment of the responses of Centella asiatica stem explant in


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


101

24 Flavonoids level in elicited in vitro Centella asiatica with 10mg/l


101

25 Flavonoids level in elicited in vitro Centella asiatica with 0.5mg/l

yeast extract for a period of 4 weeks

104



104



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


112


mg/l yeast extract for a period of 4 weeks

112



113



115

34 Flavonoids level in elicited root culture Centella asiatica with 0.5


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


hesperidin and rutin at concentration of 500 ppm

161


myricetin and quercetin at concentration of 500 ppm

162


kaempferol and apigenin at concentration of 500 ppm

163

C6 Representative HPLC Chromatogram of control and

elicited leaf in in vitro Centella asiatica

164


elicited leaf in hidroponics-grown Centella asiatica

165


elicited root culture Centella asiatica

166

C9 Representative HPLC Chromatogram of leaf in

field-grown Centella asiatica

167

xxiii

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