UNIVERSITI PUTRA MALAYSIA

CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF FLAVONOIDS FROM HYDROPONICALLY GROWN PEGAGA

(CENTELLA ASIATICA, LINN.,URBAN) EXTRACTS

FEZAH BT. OTHMAN

FSAS 2003 16

CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF FLA VONOIDS FROM HYDROPONICALLY GROWN PEGAGA (CENTELLA

ASIATICA, LINN., URBAN) EXTRACTS

By

FEZAH BT. OTHMAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of Requirements for the Degree of Master of Science

March 2003

11

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science

CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF FLAVONOIDS FROM HYDROPONICALLY GROWN PEGAGA (CENTELLA

ASIATICA, LINN., URBAN) EXTRACTS

By

FEZAH OTHMAN

February 2003

Chairman: Associate Professor Dr. Radzali B. Muse, Ph.D.

Faculty: Science and Environmental Studies

This study was carried out mainly to determine the major flavonoid

compounds present in Centella asiaticae extracts and their allelopathic, anti-microbial

and anti-inflammatory activities. The yield of extracts of C. asiatica was also

determined and it was found that methanol was the most effective solvent to obtain

crude extract from C. asiatica. Analysis on the nutrient composition of C. asiatica

showed that the mean values of the nutrients analysed were about the same among the

four groups (leaf, petiole including stolon, root and whole plant). The mineral profiles

showed that C. asiatica leaf contains significantly (p<O.05) higher levels of P, Fe, Na

and Mg compared to other parts; Ca and K contents were significantly (p<O.05) higher

in the root part compared to other parts of C. asiatica. The level of vitamin C also was

found significantly (p<O.05) higher in the leaf part compared to petiole including

stolon, root and whole plant of C. asiatica.

ill

Determination of total polyphenol content showed that fresh samples of C.

asiatica exhibited significantly (p<O.05) higher content of total polyphenol

compared to air-dried samples. Similarly, the content of salicylic acid was found

significantly (p<O.05) higher in fresh samples (especially from the root part)

compared to air-dried samples of C. asiatica. In the antioxidant studies using the

ferric thiocyanate (FTC) and thiobarbituric acid (TBA) methods, it was found that

the root extract of C. asiatica exhibited significantly (p<O.05) higher percentage of

total antioxidant activity compared to other C. asiatica extracts but lower when

compared to the reference antioxidants (a-tocopherol and butylated

hydroxytoulene, BHT).

Analysis of flavonoid compounds usmg HPLC method revealed that

catechin, luteolin, quercetin and kaempferol were present as the major flavonoid

compounds in the leaf extracts of C. aSiatica, while catechin, rutin, quercetin and

naringin were present as the major flavonoid compounds in C. asiatica root

extracts. Results from both seed germination and anti-microbial assays indicated

that the extracts of C. asiatica, especially the methanol extract might contain some

bioactive compounds that are able to affect the biological activity of the seeds and

microbes used in the experiments. In the anti-inflammatory studies, both C.

asiatica methanol and water extracts exhibited significant (p<O.05) levels of anti­

inflammatory activities. Thus, with the presence of flavonoids and other

compounds with possible cytotoxicity and antioxidative action in C. aSiatica,

coupled with favourable amounts of minerals and vitamins, it is worth promoting

this plant (c. asiatica) as a potential anti-inflammatory agent in the future.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

lV

UNSUR-UNSUR KIMIA DAN AKTIVITI-AKTIVITI BIOLOGI FLAVONOID EKSTRAK PEGAGA (CENTELLA ASIATICA, LINN., URBAN) YANG

DITANAM SECARA HIDROPONIK

Oleh

FEZAH OTHMAN

Febuari 2003

Pengerusi: Profesor Madya Dr. Radzali B. Muse, Ph.D.

Fakulti: Sains dan Pengajian Alam Sekitar

Kajian ini telah dijalankan terutamanya untuk mengenalpasti sebatian

flavonoid utama dan kesan ekstrak-ekstraknya ke atas aktiviti-aktiviti allelopatik, anti-

mikrobial dan anti-intlamatori. Hasil-hasil ekstrak C. asiatica juga telah dikenalpasti

dan didapati metanol merupakan pelarut yang paling berkesan untuk mendapatkan

ekstrak kasar dari C. asiatica. Analisis terhadap komposisi nutrien C. asiatica

menunjukkan nilai nutrien purata dalam C. asiatica adalah hampir sarna di antara

empat kumpulan (daun, petiol terrnasuk stolon, akar dan tumbuhan keseluruhan).

Analisis profil mineral bahagian daun menunjukkan kandungan P, Fe, Na dan Mg

signifikan (p<O.05) paling tinggi berbanding dengan bahagian lain; Ca dan K

signifJkan (p<O.05) paling tinggi di bahagian akar. Paras vitamin C juga telah didapati

signifikan (p<O.05) paling tinggi pada bahagian daun berbanding dengan bahagian

petiol terrnasuk stolon, akar serta keseluruhan pokok C. asiatica.

Penentuan polifenol total menunjukkan sampel segar ada mengandungi

polifenol total yang signifikan (p<O.05) paling tinggi jika dibanding dengan sampel

kering-udara. Kandungan asid salisilik juga didapati signifikan (p<O.05) paling tinggi

pada sampel segar (terutama pada bahagian akar) berbanding dengan sampel C.

asiatica yang kering-udara. Dalam kajilidikan antioksidan, menggunakan kaedah

ferrik tiosianat (FTC) dan asid tiobarbiturik (TBA), didapati bahagian akar C. asiatica

menunjukkan aktiviti antioksidan total yang signifikan tertinggi (p<O.05) berbanding

dengan kawalan dan bahagian lain tetapi lebih rendah daripada kawalan rujukan

(tokoferol ex dan hidroksitoulena butilat, BHT). Analisis sebatian-sebatian flavonoid

dengan menggunakan kaedah HPLC telah menunjukkan katekin, luteolin, kuersetin

dan kaempferol adalah komponen flavonoid utama dalam ekstrak daun, sementara

katekin, rutin, kuersetin dan naringin adalah komponen flavonoid utama dalam ekstrak

akar. Keputusan yang didapati dari asai-asai percambahan biji benih dan anti­

mikrobial telah menunjukkan ekstrak C. asiatica, terutamanya ekstrak metanol

berkemungkinan mengandungi sebatian-sebatian bioaktif yang mampu mempengaruhi

aktiviti-aktiviti biologi biji benih dan mikrob yang digunakan di dalam ujikaji-ujikaji.

Dalam kajilidikan aktiviti biologi anti-inflamatori, kedua-dua ekstrak metanol

dan air C. asiatica menunjukkan paras aktiviti anti-inflamatori yang signifikan

(p<O.05) berbanding dengan kawalan. Oleh itu, dengan kehadiran flavonoid dan lain­

lain sebatian yang bersifat sitotoksik dan antioksidatif pada C asiatica, tumbuhan ini

(C asiatica) berpotensi untuk dicadangkan sebagai agen anti-inflamatori pada masa

akan datang.

VI

ACKNOWLEDGEMENTS

First and foremost, I would like to express my deepest appreciation to my

supervisors, Associate Professor Dr. Radzali Muse, Prof Dr. Maziah Mahmood,

Associate Professor Dr. Johari Ramli and Associate Professor Dr. Mohd. Aspollah

Sukari for their guidance, help, knowledge and encouragement and critical review

towards the completion of my thesis. I would like to thank Associate Professor Dr.

Nor Aripin Shamaan, En. Ismail Omar, Associate Professor Dr. Mohd Arif Syed,

Associate Professor Dr. Juzuhayati Arshad, Dr. Nazamid Shaari, Dr. Azizah Abd.

Hamid, Dr. Mohd. Yunus Shukor, Dr. Azlan Jualang and Dr Aziz Ahmad for their

useful comments and suggestions throughout the run of the research. I also would

like to extend my gratitude to En. Onn, En. Ibrahim, En. Osman, En. KhaIid, Pn.

Siti Rodiah, En. Shamsuddin, En. Zamros and En. Hussin (from Biochemistry and

Microbiology Department, FSAS), En. Bakari and En. Ibrahim (Nutrition

Laboratory, FKVSP), En. HaIim and En. Mohd. Khairi (Biochemistry Laboratory,

FSMB), En. Sugeng and Pn. Ratna Asmah (Chemistry Laboratory, FSAS), En.

Hajaraih (Microbiology Laboratory, FKVSP), Pn. Sapiah (Histology Laboratory,

FKVSP) and Pn. Siti Muskinah (Nutrition Laboratory, FPSK) for their technical

assistance and support in the field of trial experiment. Not forgetting the

Government of Malaysia and Universiti Putra Malaysia, I am truly grateful for the

financial support from which I was able to complete my research work

successfully. Last but not least, I want to thank my family and friends for their

love, support and encouragement throughout my study.

Vll

I certify that an Examination Committee met on 27th February 2003 to conduct the final examination of Fezah Bt. Othman on her Master of Science thesis entitled "Chemical Constituents and Biological Activities of Flavonoids from Hydroponically Grown Centella asiatica, L., Urban (Pegaga)" in accordance with Universiti Putra Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) regulation 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

MOHD. ARIF SYED, Ph.D. Associate Professor Department of Biochemistry and Microbiology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia. (Chairman)

RADZALI MUSE, Ph.D. Associate Professor Department of Biochemistry and Microbiology, Faculty of Science and Environmental Studies, U niversiti Putra Malaysia. (Member)

MAZIAH MAHMOOD, Ph.D. Professor Department of Biochemistry and Microbiology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia. (Member)

JOHARI RAMLI, Ph.D. Associate Professor Department of Biochemistry and Microbiology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia. (Member)

MOHD. ASPOLLAH HJ. SUKARI, Ph.D. Associate Professor Department of Chemistry, Faculty of Science and Environmental Studies, Uni·versiti Putra Malaysia. (Member)

Professor / Deputy ean, School of Graduate Studies, Universiti Putra Malaysia.

Date: 2 6 JUN 2003

V1ll

This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirements for the degree of Master of Science. Members of the Supervisory Committee are as follows:

RADZALI MUSE, Ph.D. Associate Professor Department of Biochemistry and Microbiology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia. (Chairman)

MAZIAH MAHMOOD, Ph.D. Professor Department of Biochemistry and Microbiology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia. (Member)

JOHARI RAMLI, Ph.D. Associate Professor Department of Biochemistry and Microbiology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia. (Member)

MOHD. ASPOLLAH HJ. SUKARI, Ph.D. Associate Professor Department of Chemistry, Faculty of Science and Environmental Studies, Universiti Putra Malaysia. (Member)

AINI IDERIS, Ph.D. Professor / Dean School of Graduate Studies, Universiti Putra Malaysia. Date: 12 JUN 2UlJ3

1X

DECLARATION

I hereby declare that thesis is based on my original work except for equations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

FEZAH OTHMAN Date:: 2 4 MAR 2003

x

TABLE OF CONTENTS

Page

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CHAPTER

I

II

INTRODUCTION .. . . , . .. , . . . . . . . . . . . . ... . .. .. . . . . ... .. . . .. .. . . .. . . . 1 Background ..... , '" '" ... . , . .. , ... ... ... . , ... , ...... '" ., . .. , ... .... 1 Objectives of the Study . . . . . . . , . . .. . . . .. . . . . . . . . . . . . , .. . .. . ... ... . . . 3

�I����� . . . . .. . . , . . . .. . . . . . , . .. , ... . .. . . . . ,. .. . . . . . 4 Phytochemicals in Funtional Medicine . . . . . . . . . . . . '" . , . . , . .. , '" 4

How are Phytochemicals Produced in Plants? .. ... ... ... ... . 5 Flavonoids ... . . . . , . . .. . . . . . . .. . . . . . . . .. . . . . '" '" . . . . . , . . . . .. . ,. . .. 6

Detennination of Flavonoids .. . . . , . . . . .. . . . . , . . . , . " ... . . . . 8 �hat is Hydroponics? . . . . . . . . .. . . .. . , . . . , '" . . . .. . . , . . . , . . . . . . . . . . 9

Hydroponics: A Promising Technique for Plant Secondary Metabolite Production .. . . . . . . . . . . .. . '" . . , . . . '" . . . . , . . . , . . . . . . . 10

Biological Activities ofPhytochemicals . . . . . . '" . . . . . . ... ... . . . . 12 Phytochemicals as Antioxidants .. . . . . '" . . . .. . . .. .. . ... ... '" . 12 Phytochemicals as Anti-microbial Agents .. . .. , ... ... ... ... ... 17

Anti-microbial Activity ofFlavonoids. . . . . . . . . . .. . . . ... . . . 18 Mechanisms of Anti-microbial Actions . .. . , . . . , . . . '" . . . . . 20

Phytochemicals in Inflammation . . . . . , . . . . . . . . . . , . . . , .. . . . . . . . 23 Flavonoids Modulation of Inflammatory Cells . . . . . . .. . .. . 26 Non-steroidal Anti-inflammatory Drugs (NSAIDs). . . . . 29 How NSAIDs Relieve Inflammation .. . . ,. . . . . .. ... .. . ... . 30 Phenylbutazone ... . .. .. . . .. ... . .. . . .... ... . . . . . , . . . . . . . . . . . . ... 30

Medicinal Plants in Malaysia .. . . .. . . . .. . . .. . .. . . . . . . . . . . . . .. . . . , . . 32 Centella asiatica (�inn. , Urban) . .. . . , '" . . . . . . . . . . . . . . . . .. . ... 33

Morphology of C. asiatica . . . . . . . . , ... . . . . . . . ,. .. . . . . . . . ... 34 Traditional Uses of C. asiatica . . . . . .. ,. .. . . . . . . . . . . . . . . .. . . 36 Chemical Constituents of C. asiatica . . . . . . . , . .. , . . . . . . . ... 37 Scientific Evidence for the Effectiveness of C. asiatica 40

Xl

III MATERIALS AND METHODS .... , . ........ , ... '" ... ... ... .... 43 Plant Materials ...... '" .. , '" '" ... ... ... ... ... ... ... '" ... ... ... .... 43 Yield of Extracts of C. asiatica ... ...... ... ...... ... ... '" ... ...... 44

Reagents... ... ... ... ... ... ... .. . ... ... ... ... ... ... ... ... .. . ... ..... 44 Determination of Yield of Extract of C. asiatica... . . . ... ... 44

Nutrient Composition of C. asiatica (Linn., Urban)... ... ... ... 44 Preparation of Plant Materials ... ... ...... .. , .,. '" .. , .... ,. '" 45 Moisture Analysis... ...... ... ... ...... ... ... ... ... ...... ... ..... 45 Crude Protein Analysis ... '" ... ... ... ... ... ... ... ... ... ... ... .. 46

Fat Analysis... ...... ...... ...... ... ... ...... ...... ......... ... .... 47 Crude Fibre Analysis... ...... .................................. 47 Ash Analysis... ... ... ... ... ... ... ... ... ... .. . ... ... .. . ... ... .... 48 Mineral Analysis: Calcium, Iron, Sodium, Magnesium, Phosphorus and Potassium ... ........ , ... ........ , .. , ... ..... .. . 48 Ascorbic Acid: Indophenol Dye Method ... ... . ,. '" ... .. , . .. 51

Reagents... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. . ... ... . 51 Standardization of the Indophenol Reagents... ... ... .... 52 Preliminary Test for Appreciable Amount of Basic Substances... ... ... ... ... ... ...... ... ...... ...... ... ...... ..... 53 Sample Preparation ...... ..... , ... '" " . .. . .. . . .. ... ... ... .. . 54

Total Polyphenol Assay... ... ... ... ...... ... ............... ... ...... 55 Reagents ... . , . ........ , ., . ........ , ......... ... '" ... ... ... . . . ... . 55 Preparation of Standard Polyphenol Solutions and Extracts ofe. asiatica ... ......... ...... ........................... ....... 55 Determination of Total Polyphenol. .. ... ... ... ... ... '" '" .. , 56

Salicylic Acid Assay... ... ............ ... ...... ... ... ... ... ... ... ... 57 Reagents... ...... ... ............ ... ... ......... ... ......... 57 Preparation of Standard Salicylic Acid Solutions and Extracts of C. asiatica ... ... ... ... ......... ... ... ... ... ... ... ..... 58 Determination of Salicylic Acid in C. asiatica ... ... ... ... . ,. 58

Total Antioxidant Assays... ... ... ... ... ... ... ... ... ... ... ... ... .... 59 Reagents... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .. 60 Preparation of C. asiatica Extracts. " ... ..... , '" '" .. , .. , .,. . 60

Ferric Thiocyanate (FTC) Method ...... . " .. ... . . " .. . . . . .. , . 60 Thiobarbituric Acid (TBA) Method ...... ... ... ... .. , .,. ... . .. 61

Analysis ofFlavonoids in C. asiatica Extracts ...... .. , ... . ,. .... 62 Thin Layer Chromatography (TLC) Method... ... ... ... ... .. 62

Reagents ..... . ........ , ......... .. , ... ... ..... , ........... , .... 62 Preparation of C. asiatica Extracts ... ... ... ... ... .. , ... ..... 63 Standard Flavonoids...... ... ... ... ...... ...... ... ... ...... ... 63 Adsorbent. .. ... ... ... ... ... ... ... ... '" .. . ... ... ... ... ... ... ... 63 Chromatography Solvents... ............ ... ...... ...... ...... 64 Detection ... ... '" ... ... . , . ... ... ... '" ... .. , .. , ... ... ..... , .... 64

Spectrophotometry Method ... '" ... ...... ... .. , ... . " '" ... ..... 65 Preparation of Extracts... ......... ... ...... ... ...... ... ... ... 65 Determination of Flavonoid Contents... ... ... ... ... ... .... 65

High Performance Liquid Chromatography Method... ... ... 66 Extraction and Hydrolysis Conditions ... ... . , . ...... '" ... 66

Determination ofFlavonoids Content. .. ............ ...... . Biological Activities of C. asiatica . . . . . . . . . . ,. ' " ... ... ' " ....... .

Seed Germination Assay ............ ........ , ., . ......... ... ' " . An

' . b

'IA tl-rrucro Ia ssay .. , ...... ... .... ,. '" ... ... ... ... ... ... .... .

Preparation of Extracts ... ... ... ............................ . Preparation of Media ... ... ... ... ... ........ , ' " .. , '" ...... . T

. 14 An

' . bial A

' " estmg or u-rrucro ctlVltles ..... , ........... . . . .

Anti-inflammatory Assay ... ' " ... . .. .. . ... .. . . , . ' " . , . ' " .. . . . Experimental Animals ... ' " .. , '" .. , ........ . .... ......... . . Preparation of Extracts ... ...... ... ..................... '" .. . Carrageenan-induced Paw Oedema Assay ...... ... ...... . TPA-induced Ear Oedema Assay ......... ................. .

Histopathological Studies of the Oedematous Ear ... . Staining and Coverslipping ..... , ..... , ..... , ...... ... ' "

xu

67 68 69 70 70 71 72 73 73 73 74 77 78 81

IV RESULTSAND DISCUSSIONS................................. 82 Yield of Extracts of C. asiatica ... ........ , ... ... . , . .. , '" ... . ,. ... 82 Nutrient Composition of C. asiatica... ... ... ... ... ... ... ... ... ... 84 Total Polyphenol Content in e. asiatica ... . ,. ... ... ... ... ... ... 87 Salicylic Acid Content in e. asiatica ........ , ... .. , .,. " . . .. ... ... 89 Total Antioxidant Activity of e. asiatica ..... , ., . ... . ,. '" ... ... . 92 Analysis of Flavonoid Compounds in C. asiatica Using TLC, Spectrophotometry and HPLC Methods ... . ,. ... ... ... ... ... .... 96 Biological Activities of C. asiatica Extracts ... '" ............ .. , 1 08

Allelopathic Activity of C. asiatica Extracts. .. ... ... . .. ... .. 1 09 Anti-microbial Activity of C. asiatica Extracts ..... , .,. ..... 1 1 7 Anti-inflammatory Activity ofe. asiatica Extracts... ...... 1 21

V CONCLUSIONS... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..... 1 32

REFERENCES ... ... ... .. , ... ... ... ... ... ... ................. , ... ... 1 34

APPENDICES ... ........ . ... ... ...... ........ . .................. ... 155 Appendix A 1 . .. .. .. ... ... ... .. . ... ... ... ... ... ... ... ... ... ... ... ... 1 55 Appendix A2 ..... , . .. , ... ... ...... ... " ....... ...... '" . .. ... ... ... 1 56 Appendix A3 ......... , ., . ........ , '" ... ...... ... ... ... ... .. , ... ... 1 57 Appendix A4 ............... , ... ... ... ..... , ... ...... .. , ........... , 1 58 Appendix A5....... ... ... ... ......... ... ............... ... ... ...... 159 Appendix A6 ......................... '" .............. , ., . ........ , 160 Appendix A7 ....... ...... ... ... ... .. , ..... , ., . ... ...... '" .. , .,. ... 1 61

BIODATA OF THE AUTIIOR ............... ....... " .. , ... ... ... 162

xiii

LIST OF TABLES

Table Page

1 Sources of antioxidants from higher plants. . . . . . . . . . . . . . . . . 14

2 Plants with anti-microbial activity............ . ..... ... . . . . . . . . . . . . 19

3 Major classes of anti-microbial compounds from plants . . . . " . 2 1

4 Terpenoids of Cent ella asiatica... . . . . . . . . . . . . . . . . . . . . . . . . ... . . . ... 39

5 Composition of mobile phase for RP-HPLC analysis of flavonoid compounds... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . .. . . . 68

6 Yield of extracts from different parts (leaf, petiole and stolon, and root) of C. asiatica using solvents of different polarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . , .. . . . . . . . . . .. . . . . 83

7 Nutrient composition of different parts (whole plant, leaf, petiole and stolon, and root) ofe. asiatica., . . . . , . . . . . , .. .. , .. . . 85

8 Qualitative and Quantitative Analyses of Flavonoid Compounds in e. asiatica by (i) Thin Layer Chromatography, (ii) Spectrophotometry and (iii) High Performance Liquid Chromatography Methods... . . . . . . . . . . . . .. 97

9 Maximal UV absorption by standard flavonoids in analar grade methanol. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 98

10 Anti-microbial activities of methanol, petroleum ether and water extracts of e. asiatica . . . .. . . . . . . , .. . . . . . . . . . . . . . . . . . . . . . . . " 1 1 8

1 1 Time course of anti-inflammatory activity of C. asiatica extracts in carrageenan-induced hind paw oedema in ICR mice...... .. . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . 1 22

12 Effect of topical application of C. asiatica extracts on the TP A-induced ear oedema in ICR mice . . . .. . . . . . . . . . . '" . . . ... . . . . 124

XlV

LIST OF FIGURES

Figure Page

1 Chemical structures of some flavonoid compounds.. . . . . . . . . . . 7

2 Modulation of key enzymes in the arachidonic acid cascade by phytochemicals during inflammation. . . . . . . . . . . . . . . . . . . . . . . . . 27

3 Chemical structures of Phenylbutazone . . . . . , . . . '" . . . . . . . . . . . . . . . 3 1

4 Cente/la asiatica (Linn., Urban), "Pegaga Kampung" variety . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . '" . . . . . . . . , . , . . .. . . , . . . . 35

5 Chemical structures of some triterpenoids... . . . .. . . . . . . . . . . . . . . . 37

6 Injection of carrageenan into the sub plantar region of the mouse hind paw . .. . . . . . . . . , .. . . . . . . . . . . .. . .. . . , . . . . . . .. . '" . . . . . . . . . . 75

7 Measurement of oedematous hind mouse paw using a Vernier caliper .. .. , . .. . . . . '" .. , ., . . . .... .. .. . .. .. .. . . . . . , . .. . . . , . . . . 76

8 A general procedure for making tissue slides. . . . . . . .. ... . . . . . . . 79

9 Total polyphenol content (mg pyrogallol eq.lg dry weight tissue) in different parts (leaf, petiole and stolon, and root) of fresh and air-dried samples ofe. asiatica . . . . ,. ...... ... ......... 88

10 Salicylic acid content (mg salicylic acid eq.lg dry weight tissue) in different parts (leaf, petiole and stolon, and root) of fresh and air-dried samples ofe. asiatica. . . . .. . . . . . . . . . .. . . . . . . . 91

11 (i) Absorbance values of extracts from different parts (leaf, petiole including stolon and root) of e. asiatica using FTC method. (ii) Total antioxidant activity of extracts from different parts of e. asiatica using FTC method... . . . . . . . . . . . . . . 93

12 Total antioxidant activity of extracts from different parts (leaf, petiole and stolon, and root) of e. asiatica using TBA method...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 95

13 Thin layer chromatogram ofe. asiatica using n-buthanol: acetic acid: water, BA W (4: 1 :5) as the development chromatography solvent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

14 Thin layer chromatogram of e. asiatica using benzene: ethyl acetate: methanol, BEM (5:1 :4) as the development chromatography solvent.. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 1 02

Figure

15

16

17

18

1 9

20

21

22

23

24

25

26

27

28

Effect of different concentrations (0, 10, 100, 250, 500 and 1000 mglml) of methanol extract of C. asiatica on the germination of S. oleracea and C. frutescens seeds ...... ...... .

Effect of different concentrations (0, 10, 100, 250, 500 and 1000 mglml) of petroleum ether extract of C. asiatica on the germination of S. oleracea and C. frutescens seeds ......... ' " .

Effect of different concentrations (0, 1 0, 100, 250, 500 and 1000 mglml) of water extract of C. asiatica on the germination of S. oleracea and C. frutescens ......... ... ' " ... .

Effect of methanol extract of C. asiatica at a concentration of 250 mglml on the growth of S. oleracea seeds ...... ' " ... .... .

Effect of methanol extract of C. asiatica at a concentration of 250 mglml on the growth of C. frutescens seeds ... ... . , . ' " ... .

Cidal effect (clear inhIbition zone) of C. asiatica extracts on B. subtilis ......... ' " ' " . . . ...... .. , ' " ' " . . . . . . . . , '" '" ... . . , . . . . . . . , . . . .

HE-stained vertical sections of mouse ear ... . , . ' " ... . .. . . . ..... .

A standard curve for estimation of total polyphenol concentration (mg pyrogallol eq. / ml) in C. asiatica extracts.

A standard curve for estimation of salicylic acid concentration (mg salicylic acid eq. / ml) in C. asiatica extracts ... ' " ...... ... ... . .. .. . ...... . .. .. , ... . , . ... ' " .. , .. . .. . . . , ... .

A standard curve for estimation of apigenin concentration (mg apigenin / ml) in C. asiatica extracts ...... ......... ..... , ' "

A standard curve for estimation of kaempferol concentration (mg kaempferol / ml) in C. asiatica extracts ...... ' " ... . .. ... .. .

A standard curve for estimation of quercetin concentration (mg quercetin / ml) in C. asiatica extracts ... ... ' " ... . , . ... .... .

A standard curve for estimation of rutin concentration (mg rutin / ml) in C. asiatica extracts ... ........... , .. . ... ..... . ... .. .

HPLC profiles of mixed standard flavonoids (A), leaf (B), root (C) and petiole (including stolon) (D) extracts of Centella asiatica and. A: apigenin, C: catechin, K:

kaempferol, L: luteolin, N: naringin, Q: quercetin and R: rutin ... ... ' " ' " ., . . . . ... ... ' " ... ...... ... ... . .. . . . ... .. , ' " ., . ....... .

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1 60

LIST OF ABBREVIATIONS

5, 12 -HPETE -5, 12 - hydroperoxyeicosatetraenoic acid

A - absorbance

AAS - atomic absorption spectrophotometer

AMP - adenosine monophosphate

AOA - activity of antioxidant

BA W - n-buthanol: acetic acid: water

BEM -benzene: ethyl acetate: methanol

BHA - butylated hydroxyanisole

BRT - butylated hydroxytoulene

l3C NMR -

l3e nuclear magnetic resonance

COX - cyclooxygenase

CV - coefficient of variation

Da-dalton

DCPIP - 2, 6 - dichloro-phenol-indophenol

DNA - deoxyribonucleic acid

FTC - ferric thiocyanate

RCI - hydrochloric acid

HPLC - high perfonnance liquid chromatography

L TB4 - leukotriene B4

M - Molar

mg - milligram

min-minute

ml - milliliter

XVI

mm - millimeter

mM - milliMolar

MPO - myeloperoxidase

NA -nutrient agar

Na2C03 - sodium carbonate

NaOH - sodium hydroxide

NB - nutrient broth

nm - nanometer

NSAIDs - non-steroidal anti-infl ammatory drugs

OH - hydroxyl group

PDA -potato dextrose agar

PGE2 - prostaglandin E2

PGH2 - prostaglandin H2

PVP - polyvinyl pyrrolidone

RNA - ribonucleic acid

ROS - reactive oxygen species

RP-HPLC - reversed-phase high-performance liquid chromatography

tR - retention time

SEM - standard error of the mean

SD - standard deviation

SOD - superoxide dismutase

TBA - thiobarbituric acid

TBHQ -tert-butylhydroquinone

TECA - titrated extract of Cente/la asiatica

TF A - trifluoroacetic acid

XVll

TLC - thin layer chromatography

TPA - 12- 0- tetradecanoyl-phorbol-acetate

UV - ultra violet

v/v - volume per volume

wt -weight

w/v - weight per volume

XVlll

CHAPTER!

INTRODUCTION

Background

Over the years, plants play important roles both as staple foods and sources

of natural products for food supplements. The dependence of early human

civilisation on herbs and medicinal plants for the purpose of healing the sick was

well documented in the history of Egyptians, Chinese and Romans (Esnon, 2000).

Inflammatory-related diseases such as arthritis, asthma and rheumatic fever

are always considered serious health problems in many countries. Although many

drugs and therapeutic techniques have been developed, treatment of these diseases

remains unsatisfactory (Samuelsson, 1993). With the increasing number of global

elderly population and rapid changing lifestyle in many countries, the demand for

drugs to treat inflammatory-related diseases especially arthritis and asthma is

expected to increase.

Many drugs from aspirin to cortisone control inflammation. At present,

aspirin-like non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids and

methotrexate are commonly used for treating arthritis and other inflammatory

diseases. Unfortunately, many of these drugs are claimed to have less side effects

but usually they also reduce anti-inflammatory activity (Frishman, 2002; Buttgereit

et ai., 2001; Takeuchi et ai., 2001; Fosslien, 1998; Simon, 1997). Therefore, there

2

is an urgent need in finding new anti- inflammatory agents that are more potent but

with minimal side effects.

In Malaysia, the indigenous and naturalised plants are recognised by most

rural communities as important sources of medicines. It is estimated that there are

about 12,000 species of flowering plants and out of these about 1,300 species have

been claimed to be medicinal (Teo et al., 1990). One of these plants that naturally

have some potential sources of fine phytochemicals and some interesting

biological activities is Centella asiatica. C. asiatica is usually consumed fresh or

as a fresh juice and the Malays sometimes used it as a tonic to treat mental fatigue,

anxiety and post partum wounds. A range of biological activities has been found

for C. asiatica extracts including wound healing activity, anti-inflammatory, anti­

microbial and toxicity effects (Teik, 1997). Some of these effects may be attributed

to the use of C. asiatica in folk medicine.

Today, products of Centella such as Centella juice, tea and tablets can be

easily found at pharmacies and departmental stores. Many researchers and ethno­

botanists from developed countries have exploited C. asiatica in order to

investigate the biological activities of the fine chemicals presents in this plant. As a

result, numerous data have been reported on the therapeutic effects of Centella

especially the triterpenoid compounds from Centella (Kartnig, 1988). The

triterpenes of Centella are extensively studied for their potential as improving

factor in wound healing (Shukla et ai, 1999a; Morriset et aI, 1987), anti-ageing

(Kartnig, 1988) and memory enhancing (Veerendra and Gupta, 2002).

3

Objectives of The Study

Even though recent studies conducted internationally have confinned the

efficacy of Centella asiatica triterpenoids, primarily in the treatment of skin

injuries and diseases, data on the content of flavonoids and anti-inflammat01y

activities of C. asiatica are still lacking. In view of this reason, studies were carried

out based on the following objectives:

1) To detennine and to quantify the major flavonoid compounds present in C.

asiatica using thin layer chromatography (TLC), spectrophotometry and reversed­

phase high-performance liquid chromatography (RP-HPLC) methods. In addition,

total antioxidants as well as total polyphenol and salicylic acid content in C.

asiatica extracts were also determined.

2) To examine the biological activities of C. asiatica extracts using seed

germination, anti-microbial and anti-inflammatory assays.

CHAPTER 2

LITERATURE REVIEW

Phytochemicals in Functional Medicine

Since the beginning of life, plants have played a major role in influencing

human being and similarly, men have influenced the forms and characteristics of

plants in helping them to adapt to man's progress. Historically, plants have been

beneficial to the medical community and humans have looked to plants as their

primary medicinal source (Carper, 1988).

According to Lin (1994), the term phytochemical refers to any compound

found in plants and the word is commonly used to define the biologically active

molecules in plants that are not classified as vitamins or nutrients by more

traditional definitions. While phytochemicals have key functions in plants, they

may also play a significant role in human health. Phytochemicals have recently

gained much attention from the research community since studies have

consistently shown several benefits from consuming these natural phytochemical

compounds.

For nearly thirty years, synthetic drug research dominated the

pharmaceutical industry until natural drug research began to make a come back

around 1990 when it was realised that even with the advanced computerized

technology that exists today, scientist cannot abandon nature as a source for drugs

(Pizzomo & Murray, 1 987). This is because, plants contain plenty of powerful

phytochemical defences, many of which are too complex to be accurately

replicated in the laboratory. This chemical complexity is probably due to the

building of defence mechanisms to avoid enemies in nature and thus made the

plants as a rich source of "medicines" (Das, 1 989).

Many important drugs today are plant-derived and the most widely used

phannaceutical in the world is aspirin, which was originally derived from the plant

Filipendula ulmaria (Middleton & Drzewieki, 1985). Ephedrine, which is derived

from the species Ephedra sinica, is presently used as a bronchodilator (White et

al. , 1 997). Several drugs come from the opium poppy (Papaver somniferum),

including morphine, an analgesic and papaverine, an antispasmodic (Calixto et al.,

2000). The rosy periwinkle (Catharanthus roseus) has given the medical

community two very important drugs: vinblastine, a treatment for Hodgkin's

disease and vincristine, a treatment for paediatric leukaemia (Noble, 1 990). Despite

this, less than 1,325 of the world's 265,000 flowering plant species have been

thoroughly tested for their medicinal potential (Soepadmo, 1999).

How Are Phytochemicals Produced in Plants?

Plants have developed extensive systems for managing reproduction,

response to injury, protection from ultraviolet light, and resistance to disease.

Attending to these important functions requires communication between plant cells

and between the plant and its environment. Plant use phytochemicals both as a

mean of "communication" and as a response to environmental stress.

6

"Communication" also occurs between phytochemica1s and the human

organism when we eat plant-based foods (Borris, 1996). The process by which the

plant produces the chemicals needed for this communication, i.e., signalling

molecules, is referred to as secondary metabolism (Cuppett, 1998). The secondary

metabolites produced from this process influence diverse communication activities.

This is much agreed by Alaluf et af., (2002) who mentioned that secondary

metabolites such as carotenoids, not only provide colour to signal pollinators such

as birds and insects but they also protect DNA from damage in dry and/or light

intensive environments.

There may be well over 10,000 different phytochemical secondary

metabolites that have biological activity in humans. Based on chemical structure

and synthesis pathways, these phytochemicals are catergorized into at least 14

general classes: carotenoids, coumarins, flavonoids, glucurates, indoles,

isothiocyanates, lignans, monoterpenes, diterpenes and triterpenes, phenolic acids,

phtalides, phytates, polyacetylenes, and sulfides. Of these, the flavonoids are the

largest class, with over 4,000 different representatives (Harborne and William,

2000).

Flavonoids

The flavonoids are polyphenolic compounds possessing 15 carbon atoms;

two benzene rings joined by a linear three-carbon chain. Figure 1 shows chemical

structures of some flavonoid compounds.