UNIVERSITI PUTRA MALAYSIA

PRODUCTION AND ANTIBACTERIAL PROPERTIES OF FLAVOUR AND FLAVONOID COMPOUNDS FROM CULTURED TISSUES OF

CITRUS HYSTRIX D.C. (‘LIMAU PURUT').

SURI BIN ROOWI

FSAS 2001 34

PRODUCTION AND ANTIBACTERIAL PROPERTtES OF FLAVOUR AND FLAVONOID COMPOUNDS FROM CULTURED TISSUES OF CITRUS

HYSTRIX D.C. r LIMAU PURUT').

By

SURI BIN ROOWI

Thesis Submitted In Fulfilment of the Requirement for the Degree of Master of SCience in the Faculty of Science and Environmental Studies

Universiti Putra Malaysia

May 2001

DEDICATION

"Dedicated especially to mom (Jainab), my late father (Hj Roowi) , my wife

(Zaharinah), my sons (Abu Hanifa and Zaid) , my brothers and sisters (Hj .

Sumali, Kalsom, Basirah , Oayah, Ahmad and family, Habsah, Jumeyah ,

Amnah), my father and mother-in-law and those whose sacrifice and support

me to complete my study successful ly."

ii

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

PRODUCTION AND ANTIBACTERIAL PROPERTIES OF FLAVOUR AND FLAVONOID COMPOUNDS FROM CULTURED TISSUES OF CITRUS

HYSTRIX D.C. (,LIMAU PURUT')

By

SURI BIN ROOWI

May 2001

Chairman : Associate Professor Radzali Muse, Ph.D.

Faculty : Science and Environmental Studies

Analyses of the flavour compounds from the selected parts of flower,

seedlings grown in-vitro and callus cultures of Citrus hystrix D.C. were

performed using the gas chromatography (GC) technique. The results

showed that the major flavour compound obtained from the flower was

citronella/. Young seedlings grown in-vitro on the Murashige and Skoog

(MS) basal medium without plant growth regulators did not produce any

citronella! . Nevertheless, the quantity of l imonene was remarkably higher

( 1 01 .00 ± 5.24 119/g fwt. tissue) than in the petal and ovary and pollen plus

anther. Callus derived from stem treated with 2.0 mgll (w/v) NAA plus 1 .0

mgll (w/v) kinetin reached a maximum growth (0.94 ± 0.08 g fwt.lculture)

after six weeks of culture . No callus was found from the leaf and peel.

Maximum production of flavour compounds Le. cyclohexanol , l imonene and

p-pinene were obtained after three and four weeks of culture.

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Treatment of the C. hystrix stem-derived cal lus with 1 .0 mg/I (w/v) of

kinetin showed higher growth (0.42 .:!: 0.04 g fwt.lculture) than al l treatment

with NAA. Most of the flavour compounds in the callus were found highest

after being treated with 5.0 mgll (w/v) of NAA. Addition of various

concentrations of salicylic acid (0 to 20.0 mM), yeast extract (0 to 0.5%) and

alginate (0 to 0.5%) into the medium, decreased the callus growth. However,

treatment of stem-derived cal lus with 0.3% of (w/v) of alginate resulted in

higher growth (0.88 .:!: 0.05 g fwt.lculture) than the other treatments. On the

other hand, callus treated with yeast extract and salicylic acid were able to

syntheses two and six additional compounds respectively compared to the

control. Treatment of C. hystrix stem-derived callus with proline and

phenylalanine decreased the callus growth but Significantly increased the

production of flavour compounds i .e. p-cymene, terpineol , citronellal ,

citronellol , y-terpene and �-pinene.

Analyses of flavonoid compounds from young seedlings, callus and

d ifferent parts of C. hystrix intact plant such as leaf, flower, stem and fruit

was performed by high performance liquid chromatography technique

(HPLC). The highest production of flavonoids i.e. naringin ( 1 1 .66 .:!: 0.76

mg/g dwt. tissue), rutin (34.63 .:!: 1 .69 mg/g dwt. tissue) and kaempferol

(3.01 .:!: 0.02 mg/g dwt. tissue) was found in the peel; hesperidin (3.21 ±

0.23 mg/g dwt. tissue) in the leaf and quercetin (0.68 ± 0.04 mg/g dwt.

tissue) in the whole flower. In the young seedlings, naringin (5.26.:!: 0.25

iv

mg/g dwt. tissue) , and rutin (0.91 1: 0.03 mg/g dwt. tissue) were found in

high concentration in the stem compared to the leaf and root. The naringin

and rutin content of stem-derived callus showed the maximum values at 2 .29

±. 0.09 and 0 .90 ±. 0 .03 mg/g dwt. tissue respectively after six weeks of

culture.

Treatment of stem-derived callus with 0.3% and 0.5% (w/v) of

agarose gave the highest production of naringin and rutin at 12.13 ± 0.07

and 3.09 ± 0.05 mg/g dwt. tissue, respectively. Addition of 2 .0 mM

phenylalanine into the culture medium also increased the production of

naringin (24.05 ±. 1 .02 mg/g dwt. tissue) and rutin (3.52 ± 0.12 mg/g dwt.

tissue).

The essential oils (contains flavour compounds) and the methanolic

extracts (contains flavonoid compounds) were obtained from peel, leaf, juice

and stem-derived callus. Results showed that most extracts were able to

inhibit g ram positive and gram negative bacterial g rowth.

v

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

PENGHASILAN DAN SIFAT ANTIBAKTERIA PADA BAHAN PERISA DAN FLAVONOID DARI KULTUR TISU LlMAU PURUT (CITRUS

HYSTRIX D.C.)

Oleh

SURI BIN ROOWI

Mei 2001

Pengerusi : Profesor Madya Radzal i Muse, Ph.D.

Fakulti : Sains dan Pengajian Alam Sekitar

Analisis kandungan bahan perisa pada beberapa bahagian bunga

limau purut (Citrus hystrix) , anak pokok yang ditanam secara in-vitro dan

kal us telah dilakukan menggunakan teknik kromatografi gas (GC) . Hasi l

ujikaji menunjukkan komponen bahan perisa yang tertinggi adalah sitronelal .

Anak pokok yang d itumbuhkan d i atas media asas pertumbuhan Murash ige

dan Skoog (MS) tanpa pengawal atur pertumbuhan tidak berkemampuan

mensintesis bahan sitronelal . Walau bagaimanapun, kandungan bahan

l imonen d idapati lebih tinggi (101.00 ± 5.24 J.1g/g berat basah tisu)

berbanding dengan kelopak, ovari dan debunga dan cepu debunga (anter) .

Kalus yang diaruh dari batang menggunakan 2.0 mgl/ (beratlisipadu) asid 0.-

naftalena asetik (NAA) dan 1 .0 mgl/ (beratlisipadu) kinetin menunjukkan

pertumbuhan yang tertinggi (0.94 ± 0.1 0 9 berat basah/kultur) selepas 3 dan

4 minggu pengeraman. Sebaliknya, tiada pembentukan kalus pada

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bahagian lain seperti daun dan kulit. Penghasilan bahan peri sa maksima

contohnya sikloheksanol , l imonena dan �-pinena seterusnya didapati

selepas tiga dan empat minggu pengkulturan.

Kalus batang yang telah dirawat menggunakan 1 .0 mg/l

(beratlisipadu) kinetin menunjukkan pertumbuhan yang lebih tinggi (0.42 ±

0.04 9 berat basah/kultur) berbanding dengan rawatan menggunakan NAA.

Kebanyakkan kuantiti bahan perisa tertinggi didapati pada kalus yang

dirawat dengan 5.0 mgll (beratlisipadu) NAA. Rawatan menggunakan asid

salisilik, ekstrak Vis dan alginat pada kepekatan yang berbeza, telah

merencatkan pertumbuhan kalus. Sebaliknya, rawatan kultur kalus dengan

menggunakan 0.3% (beratlisipadu) alginat pula telah mencatatkan

pertumbuhan kalus yang lebih tinggi (0.88 ± 0.05 9 berat basah/kultur) . Hasil

analisis kandungan perisa menunjukkan bahawa rawatan dengan

menggunakan ekstrak Vis dan asid salisi l ik berkeupayaan mensintesis dua

dan enam komponen perisa berbanding dengan kawalan. Penambahan

bahan prolina dan fenilalanina ke daJam media pertumbuhan kaJus C. hystrix

juga telah merencatkan pertumbuhan kalus tetapi meningkatkan

penghasilan bahan peri sa contohnya p-simena, terpineol , sitronellal ,

sitronellol , y-terpinena dan �-pinena.

Analisis sebatian flavonoid daripada anak pokok, kaJus dan bahagian

pokok yang berbeza contohnya daun, bunga, batang dan buah pada

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C. hystrix telah di lakukan dengan menggunakan teknik kromatografi turus

berprestasi tinggi (HPLC) . Tahap penghasilan bahan flavonoid tertinggi

contohnya naringin ( 1 1 .66.:!: 0 .76 mg/g berat kering tisu), rutin (34.63.:!:

1 .69 mg/g berat kering tisu) dan kaempferol (3.0 1 .:!: 0.02 mg/g berat kering

tisu) d i dapati d i dalam kulit buah; hesperidin (3.2 1 .:!: 0.23 mg/g berat kering

tisu) daJam daun dan kuercetin (0.68 .:!: 0.04 mg/g berat kering tisu) dalam

bunga. Naringin (5.26.:!: 0 .25 mg/g berat kering tisu) dan rutin (0.91 .:!: 0 .03

mg/g berat kering tisu) juga d idapati tinggi pada batang anak pokok. Kalus

C. hystrix juga mencatatkan penghasilan naring in (2.29 .:!: 0.09 mg/g berat

kering tisu) dan rutin (0.90 .:!: 0.03 mg/g berat kering tisu) maksimum

selepas enam minggu pengkulturan.

Rawatan kalus batang dengan menggunakan 0 .3 dan 0.5%

(beratlisipadu) agarosa telah menghasilkan naringin ( 1 2. 1 3 .:!: 0.07 mg/g

berat kering tisu) dan rutin (3.09 .:!: 0.05 mg/g berat kering tisu) tertinggi .

Penambahan 2 .0 mM fen ilalanina ke dalam kultur med ia juga boleh

men ingkatkan penghasilan naringin (24.05 .:!: 1 .02 mg/g berat kering tisu)

dan rutin (3.52 .:!: 0 . 1 2 mg/g berat kering tisu) .

Kajian ke atas perencatan pertumbuhan bakteria dengan

menggunakan minyak pati yang mengandungi bahan perisa dan ekstrak

metanol yang mengandungi bahan flavonoid , juga telah di lakukan. Hasil

kajian telah menunjukkan bahawa hasil ekstrak berkemampuan

merencatkan pertumbuhan bakteria gram positif dan negatif.

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ACKNOWLEDGEMENTS

I would l ike to express my sincere gratitude to my supervisors ,

Associate Professor Dr. Radzali Muse, Professor Dr Marziah Mahmood and

Associate Professor Dr Mohd. Aspollah Hj Sukari for their guidance and

encouragement throughout this study.

Gratitude and thanks are also due to Ministry of Science, Technology

and Environment, Malaysian Agricultural Research and Development

Institute (MARD I) for providing me a postgraduate scholarship, research

grant, facil ities and study leave.

I wish to thank Associate Professor Datin Dr Khatijah Mohd. Yusoff,

the Head of B iochemistry and Microbiology Department, U niversiti Putra

Malaysia, Serdang, Selangor for allowing me to use all the research

facilities. Sincere thanks are also due to Pn Normah Omar (MARD I), En

Mohd. Shaib Jaafar (MARDI) , En Arif Zaidi Jusoh (UPM) , En Ratna Agung

Samsumaharto (UPM) and Hj Sumal i Alwi for helping me in technical

analysis.

I also wish to thank my fellow lab-mates for their encouragement and

support and last but not least, my special thank goes to my parent for their

endless love.

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I certify that an Examination Committee met on 9th May 2001 to conduct the final examination of Suri B in Roowi on his Master of Science thesis entitled "Production and Antibacterial Properties of Flavour and Flavonoid Compounds from Cultured Tissues of Citrus hystrix D.C. CLimau Purut')" i n accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1 980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1 98 1 . The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as fol lows:

Johari Ramli , Ph . D Faculty of Science and Envi ronmental Studies , Universiti Putra Malaysia. (Chairman)

Radzal i Muse, Ph .D Faculty of Science and Environmental Studies, Universiti Putra Malaysia. (Member)

Marziah Mahmood, Ph .D Faculty of Science and Environmental Studies, Universiti Putra Malaysia. (Member)

Mohd . Aspollah Hj Sukari , Ph.D Faculty of Science and Environmental Studies, Universiti Putra Malaysia . (Member)

AIN I IDERIS, Ph.D , Professor/Dean of Graduate School, Universiti Putra Malaysia

Date: 2 3 MAY 2001

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This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Master of Science.

xi

AIN I I DERIS, Ph.D, Professor, Dean of Graduate School , U niversiti Putra Malaysia .

Date: 1 4 JUN 2001

DECLARATION

I hereby declare that the thesis is based on my original work except for quotation 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.

Date: '23 MAY 2001

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TABLE OF CONTENTS Page

DEDiCATION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i ABSTRACT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i i ABSTRAK. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix APPROVAL SHEETS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x DECLARATION FORM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi i LIST OF F IGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi i i LIST OF PLATES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx LIST OF ABBREViATIONS . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi i

CHAPTER

INTRODUCTION . . . . . . . , .......... ... . . . '" . .. ... . . . ... ... ... ... ... . .. 1 Objective of the Studies . . . . . . . . . . . . .. . .. . ... ... . . . .. . .. . .. . ... .. . . . . 8

" LITERATURE REVIEW. . . . . . ... . . . .. . . . . . .. . .. . . . ... ... . .. ... .. . . . . 9 Production of Flavour Compounds . . . . . . ............ ............. 9

Prospects in the Production of Secondary Metabolites Using Plant Cell Cultures. . . . .. . .. . . . . . . . .. ... . . . . . . .. . . . . . . . . 9 Brief DeSCription About Citrus hystrix or 'Limau purut' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. .. . .. . ... . . . 12 Techniques in Sample Preparation and Identification of Flavour Compounds . . . . . . . . . . . . . . . ' " .. . . 14 Isolalion and Concentration of Headspace Volatiles. . . . 1 4 Extraction . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . .. 1 6 Liquid Carbon Dioxide Extraction . . . . . . . . . . . . . . . . . . . " . " '" 17 Simultaneous Distil lation Adsorption . . . . . . . . . . . . . . . . . . . . . . . 17 Simultaneous Distil lation Extraction . . . . . . . . . . . . . . . . . . . . . . . . 18 Location of Flavour Compounds in Plants . . . . . . . . . ... . .. . 1 9 Flavour Compounds i n C. hystrix. . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Natural Food Flavours in Malaysia . . . . . . . . . . . . . . . . . . . . . . . . . 25 Uses of Natural Flavours . . . . .. ... .. . .. . ...... ... ... . ...... ... 27 Tissue Culture of Citrus . . . .. . ... ... ... ... ... . .. ... ... . . . ... ... 29 Problems in Producing Flavour Compounds in Plant Cell Cultures. . . . . . ... . . . . . . ... . . . . . . . . . . . . . . . . . . . .. . .... . . . . . .. . . 30 Use of High Product-Yield Plants . . . . . . . . . '" ... .. . '" ... . .. 32 Effects of Plant G rowth Regulators . . . . . . . . . . . . . . . . . . . . . . . 33 Effects of I l luminations . . . . . . . . . . . . . . . . . . . . . . . . . . . . " .. . ... . .. . 35 Effects of Elicitors . . . . . . . .. .. . ... . . . . .. ... . .. ... . . . . .. . . . ... . . . .. 37 Effect of E licitors on the Production of F lavour Compounds. . . . . . . .. . . . .. . . . . . . . . . . . .. . . . . . . . .. .. . .. . ... .. . . . . ... 38 Effects of Ami no Acids . .. ... . .. ... ........ . . .... . ... .. ... . .. . . . 39

xii i

Effect of Amino Acids on the Production of Flavour Compounds . . . . . . '" .. . .... . , . . . . . . . , ... . . . , . .... . . , . .. , . .. ... .. . . 40

Production of Flavonoids . . . . . , . . . '" . . . . . , . . . . . . . . . . . . . . . . . . , . . . . . . 41 Medicinal Plants in Malaysia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Role of F lavonoids in Plant. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Flavonoids as Antimicrobial Compounds. . . . . .. . . . ........ 44 Flavonoids as Medicinal Compounds . . . . . . . . . . . . . . . . . . . . . . 45 Location of Flavonoids in Plants . . . . ' " . . . . . . . . . . . . . . . . . . . . . 46 Flavonoids i n Citrus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Flavonoids in Callus . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Effect of Plant Growth Regulators . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Effects of Lights . . . . . . '" . . .. .. .. . .. , .. . . . . . . . . .. . . , .. . . .. '" .,. . 52 Effects of Amino Acids . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . .. . . . .. . . 52 Effect of Elicitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

I I I MATERIALS AND M ETHODS . . . . . . . . . . . . . . . . . . . . . . . . . . , . .. . . . . . . 56 Plant Materials . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 56 Induction of Callus.. . . . . . . . . . . . . . . . . . .. . . . ... ... . .. . . . . . . . . . . . . . . . ... . 56 Determination of Callus Growth for Optimum Production Flavour and F lavonoids Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 I l lumination Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Elicitors Treatment. . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . " . . . . . . . . . . . . . . . . . . . 58 Phytohormones Treatment. . . . " .. . . . . '" '" .. . .. . . . . . . . . . . . . . . . . . .. 59 Flavour Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Preparation of Standard Flavour Compounds. . . . . . . . . . . . 6 1 Apparatus for Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . 6 1 Gas Chromatography Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Flavonoids Extraction . . . . . . . . . . . . . " .. . . . . . . . . . , ..... . . . . ... .. , . . . . . . 62 Preparation of Standard Flavonoids . . . . . . . . . . . . . . '" . . . . ,. . 62

Apparatus for H igh Performance Liquid Chromatography . . . . . , ...... . ... . . . . . '" '" . . . . , . . .. '" . . . . . . . . . . 63 HPLC Cond itions . . . . . . . . . . . . . . . . , . . .. .. , . . . . . . . , . . . . . . . . . . . . . . . . . 63

Amino Acids Analysis . . . . " . .. . . . '" . . . . . . . . . . . . .. . ........ , . . . '" ... 64 Preparation of Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 HPLC Cond itions. . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . .... . . . . . . . . . 65 Preparation of Standard Amino Acids... . . . ... .... ......... 65 Preparation of Reagents . . . . . . . . . . , . . . , . ... .. . . . . , . . . , . . . . . . . . . 65

Preparation of Eluent A. . . . . . . . . . . . . . . . . . . . , . .. '" .. . ... . 66 Preparation of Eluent B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Precursor Stud ies . . . . " ... '" .. . .. . . . . . . . . . . . .. . . . . " .. . . . . . . . .. . 66 Antibacterial Assay . . . . . . . . . . . . . . . . . . . . . ... . .. . . . . . . .. . . . . . . . .. . . .. . . . 66

Preparation of Nutrient Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Preparation of N utrient Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '" 67 Preparation of Paper Disc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . 67 Antibacterial Screening . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . , . . . , . . . . . 68

xiv

IV RESULTS AND DISCUSSION . . . . . . . . , . . . . . . . , . . . . . . . . . . . . , . . . . . . 69 Production of Flavour Compounds . . . '" . . . . . , . . . . . . . . . . . . . . . . . . . 69

Flavour Compounds in C. hystrix Flower. . . . . . . . . . . . . . . . . . . 69 Effect of Explant Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Flavour Compounds in Different Parts of C. hystrix Young Seedl ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Flavour Compounds i n Callus Derived from

Different Parts of Young Seedl ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Effects of I l luminations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Effect of Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Effect of Phytohormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Effect of a-NAA and Kinetin on Stem-Derived Callus Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Effect of Kinetin on the Production of Flavour Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Effect of a-NAA on the Production of Flavour Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Effect of Elicitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Effect of Elicitors on Stem-Derived Callus Growth . . . . . . 86 Effect of Alginate and Agarose on the Production of F lavour Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Effect of Salicylic Acid on the Production of Flavour Compounds... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Effect of Yeast Extract on the Production of Flavour Compounds . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Amino Acids in C. hystrix Parent Plant and Stem-Derived Callus . . . '" ., . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . , . . . . 94 Effect of Amino Acids on Stem-Derived Cal/us Growth 97 Effect of Phenylalanine on the Production of Flavour Compounds . . . . . . . . . . , . . . , . . . . . . . .. . . . . . . . , . . . . . . . . , . . . , 99 Effect of Proline on the Production of Flavour Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 01

Production of Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . 1 03 Flavonoids in C. hystrix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 03 Effect of Selected Explants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 06 Effect of Illuminations............................................ 107 Effect of Growth . . . '" . , . . . . . . . . . . . . , . . . . . . . . . . . . . . . . , . . . . . . . . . . . . , 1 09 Effect of Plant Growth Pegulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1 Effect of a-NAA and Kinetin on Stem-Derived Callus Growth . . . . " . . . . . . '" . . . . . . . . . . . . . , . . . , . . . . . . . . . . . . . . . 1 1 1

Effect of a-NAA on the Production of Naringin and Ruti n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2

Effect of Kinetin on the Production of Naringin and 1 14 Rutin .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Elicitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 6

xv

Effect of Elicitors on Stem-Derived Callus Growth . . . . . . . 1 1 6 Effect of Salicylic Acid on the Production of Naringin and Rutin . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 1 1 8 Effect of Yeast Extract on the Production of Naringin and Rutin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 9 Effect of Alginate on the Production of Naringin and Rutin . . . . . . . . , '" . . . . . . . . . . . . . . . . . . . , . . . . . . , '" . . . . , . . . . . . . '" . . . . , . . , . 1 2 1 Effect of Agarose on the Production of Naringin and Rutin . . . . . . . . . . . . . . . . . . . '" . . , . . , . . . . . . . , . . . . . . , . . . . . . . . . . . . . . , . . . . . . . . 1 22 Effect of Selected Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . 1 23 Effect of Proline and Phenylalan ine on Stem-Derived Callus Growth . . . . , . . . . . . . . . . . . . . , . . . . . . . . . . . . . . , . . . , . . . . . . . . . . . . . . . 1 23 Effect of Phenylalanine on the Production of Naringin and Rutin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . , 1 25 Effect of Proline on the Production of Naringin and Rutin . . . . . , '" . . . '" . . . . . . . . . . . . '" ., . . . , '" . . . . . . . , . . . . . . . . . . . . . . . . . . , 1 27

Antibacterial Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 28 Antibacterial Assay of Standard Flavour Compounds. 1 28 Antibacterial Activity of Standard Flavonoids. . . . . . . . . . . . 1 30 Antibacterial Assay of Essential Oils Obtained from C. hystrix Plant and Stem-Derived Callus . . . . . . . . . . . ,. . . . . . . . . . 1 32 Antibacterial Activity of Methanolic Extracts. . . . . . . . . . . . . . . 1 36

V CONCLUSION . . . . , . . . , . . . . . . . . . . , . . . , . . . . . . . . . . . . . . , . . . '" . . . . . . . . , '" 1 4 1

REFERENCES . . . . . . . . , . . . . . . '" '" . . , . . . '" . . . . . . . . . . . . . . . . . . . . . . . , '" 1 43

APPENDiCES. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 67 A. Composition of Murashige and Skoog Medium. . . . . . . . 1 67 B . Add itional figures . . . . . . . . . . . . . , . . . . . . . , . . . , . . . . . . . . . . . . . . , . . . . . . 1 68

ViTA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 79

xvi

LIST OF TABLES

Table

1 List of Companies in Japan i nvolved in the Production of

Page

Secondary Metabolites Using plant Cell Cultures... . . ... . . . . . 1 0

2 Percentage Compositions of Extracts i n Solvent Free Basis . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. . . . . . . . . . . . . . . . . . . . . . . . 1 6

3 Flavour Compounds of Selected Parts of C. hystrix Flower 70

4 Flavour Compounds of Selected Parts of C. hystrix Young Seedling . . . . , . . . . . . . . . . . . . . . , . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . 74

5 Flavour Compounds in Callus Derived From Different Parts of C. hystrix. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 77

6 Quantity of Flavonoids in Different Parts of C. hystrix. . . . . . . . . 1 04

7 Antibacterial Activity of Standard Flavour and Flavonoid Compounds . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . , . . . . . , . . . . . . . . . . . . . 1 29

8 Yield of Volatile Oils in Selected Parts of C. hystrix and Stem-Derived Callus . . . . . . . . . . . . . . . . , . . . . , . . . . . . . . . . . . . . . . . . . . . , . . . . . .. 1 33

9 Antibacterial Activity of C. hystrix Flavour Extracts. . . . . . . . . . 1 33

1 0 Antibacterial Activity of C. hystrix Methanol ic Extracts . . . . . . 1 37

xvii

Figure 1 2

3 4 5 6 7

8

9

1 0

1 1

1 2

1 3

14

1 5

1 6

1 7

1 8

1 9

LIST OF FIGURES

DLGG and LPGG in Citrus hystrix Leaf Extract. . . . . . . , .

Arrangement of Traps for Headspace Collection of Volatiles from Liquids (a) Light-weight Materials b) Dense Solids . . . . . . . . . . . . . . . . . . . . . . , . ' " . . . . . . . . , . . , . . . . . . . . . . . . .

Simultaneous Distillation Adsorption . . . . , . . , . . . . . . , . . , . . . . .

Modified Lickens and Nickerson Apparatus . .. . . . . . . . . . . . .

Flavour Compounds found in C. hystrix Extracts . . . . . . . . Pathway of Flavonoids Biosynthesis . . . . . . . . , . . . . . . . . . . . . . .

Effect of Different Types of I l lumination on C. hystrix Callus Growth . . . . . , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . , . . . . Effect of Different Types of I l lumination on the Production of Flavour Compounds in C. hystrix Stem-Derived Callus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '" . . . . " . . . . . . . . . . . .

Growth of C. hystrix Callus Derived From Stem Explant and Incubated at 27 ± 2°C Under White Cool Fluorescent Light . .. . . . . " . . . . . . . . . . . . . . , . . , . . . . . . . . . . . . . . . . . . . . Production of Major Flavour Compounds From C. hystrix Stem-Derived Callus Culture During Incubation at 27 ± 2°C Under Cool Fluorescent Light. . . . . , . . , '" . . . . . . ' " . . . . . . . . . . . . . . . . ' " . , . . , . . , . . . . . . , . . . . . . Effect of Supplied NAA and Kinetin on C. hystrix Stem-Derived Callus Growth after Three Weeks of I ncubation . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . .

Effect of Supplied Kinetin on the Production of Flavour Compounds in C. hystrix Stem-Derived Callus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of Supplied NAA on the Production of Flavour Compounds in C. hystrix Stem-Derived Callus . . . . . . . . Effect of Supplied Salicylic Acid o n C. hystrix Stem­Derived Callus Growth After 3 Weeks of Incubation . . . . . . . . . . . , . . . . . . . . . ' " . . . . . . . . . . , . . . . . . . . . , . . , . . . ' " . . .

Effect of Supplied Alginate, Agarose and Yeast Extract on C. hystrix Stem-Derived Callus Growth after 3 Weeks of I ncubation . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .

Effect of Supplied Salicylic Acid o n the Production of Flavour Compounds in C. hystrix Stem-Derived Callus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of Supplied Yeast Extract on the Production of Flavour Compounds i n C. hystrix Stem-Derived Callus . . . . . . . . ... . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .

Percentage of Amino Acids i n C. hystrix Stem-Derived Callus, Leaf and Fruit Extracts .. . .. . . . . . . . . . . . . . .

Effect of Supplied Amino Acids on C. hystrix Stem-

xvii i

Page 1 3

1 6 1 8 20 25 43

78

78

80

8 1

83

84

84

88

89

9 1

93

95

Derived Callus Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 20 Effect of Supplied Phenylalanine on the Production of

Flavour Compounds in C. hystrix Stem-Derived Callus . . . . . . . . . . . . . . . . . . . . . . . . . . . '" ., . . . . '" . . . . . . . . . . . . . . . . . . . . . . . . 1 01

2 1 Effect of Supplied Proline o n the Production of F lavour Compounds in C. hystrix Stem-Derived Callus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '" . . , .. . . . . . . . . . . 1 03

22 Production of Naringin, Rutin and Kaempferol in Different Parts of C. hystrix Plantlet Grown In-vitro. . . . . 1 07

23 Effect of Light on the Production of Naringin and Rutin in C. hystrix Stem-Derived Cal lus . . . . . . . . . . . . . . . . . . . 1 08

24 Effect of Growth on the Production of Naringin and Rutin in C. hystrix Stem-Derived Callus . . . . . . . . . . . . . . . . . . 1 1 0

25 Effect of Supplied NAA and Kinetin on C. hystrix Stem-Derived Callus Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2

26 Effect of Supplied NAA on the Production of Naringin and Rutin in C.hystrix Stem-Derived Callus . . . . . . . . . . . . . . 1 1 3

27 Effect of Supplied Kinetin on the Production of Naringin and Rutin i n C. hystrix Stem-Derived Callus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 1 1 5

28 Effect of Supplied Salicylic Acid on C. hystrix Stem­Derived Call us Growth after 6 Weeks of Incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7

29 Effect of Supplied Yeast Extract, Alginate and Agarose on C. hystrix Callus Growth... . .. . . . . . . . . . . . . . . . 1 1 7

30 Production of Naringin and Rutin from C. hystrix Stem-Derived Callus after Treatment with Salicylic Acid . . . . . . . , . . . . . . . . , . . . . . . .. . . . . . . . . . . , . . . . . . . . , . . . . , . . . . . . . . ,. . . . . . 1 1 9

3 1 Production o f Naringin and Rutin from C. hystrix Stem-Derived Callus after Treatment with Yeast Extract. . . . . . . . . . . . . , . . . . . . . . . . '" . . . . . . . . . . . . . . . '" . . . . . , . . . . ,. . . . . 1 20

32 Production of Naringin and Rutin from C. hystrix Stem-Derived Callus after Treatment with Alginate. . . . 1 22

33 Production of Naring in and Rutin from C. hystrix Stem-Derived Callus after Treatment with Agarose. . . . 1 23

34 Effect of Supplied Proline and Phenylalanine on C. hystrix Stem-Derived Callus Growth . . . . . . . . . . . . . . . . . . . . . . 125

35 Effect of Supplied Phenylalan ine on the Production of Naringin and Rutin i n C. hystrix Stem-Derived Callus . . 1 26

36 Biosynthesis of Flavonoids and Other Secondary Metabolites from Phenylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 26

37 Effect of Suppl ied Proline on the Production of Naringin and Rutin from C. hystrix Stem-Derived Callus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 1 27

xix

LIST OF PLATES

Plate Page 1 C. hystrix Fruits .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

2 Modified Lickens and Nickerson Apparatus for Simultameous Distil lation Extraction . . . . . . . . . . . . . . . . . . . . . . . . . 60

3 C. hystrix Young Seedlings Cultured on the basal MS Medium without Phytohormone after Four Weeks of I ncubation in Light Condition . . . . . . . . . . . . . . . . . . . . . . . . . ' " . . . . . . . . 73

4 Callus Derived from Stem of C. hystrix Young Seedling Cultured on the Basal MS Medium Supplemented with 2.0 mgl/ (w/v) NAA and 1.0 mgl/ (w/v) Kinetin Under Light Condition after 3 Weeks of I ncubation . . . ' " . . , . . . . , . . . . . . , . . . . . . . . . . . . . . . ' " . . . ' " . . . . . , . . . . . . . . . 75

5 Treatment of C. hystrix Stem-Derived Callus with Salicylic Acid after Three Weeks of I ncubation . . . . . . . . . 87

6 Treatment of C. hystrix Stem-Derived Callus with Yeast Extract after Three Weeks I ncubation . . . , . . . ' " . . . . 87

7 Treatment of C. hystrix Stem-Derived Callus with Proline after Three Weeks of I ncubation . . . . . . . . . . . . . . . . . . . 98

8 Treatment of C. hystrix Stem-Derived Callus with Phenylalanine after Three Weeks of I ncubation . . . ' " . . . 99

9 Treatment of C. hystrix Stem-Derived Callus with Phenylalanine after Six Weeks Incubation i n Light Condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 25

10 Antibacterial Activity of Standard Flavour Compounds Against Bacillus subtilis . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 130

11 Antibacterial Assay of Standard Flavonoids Against Bacillus subtilis . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . .. . . . . . . .. . . . . . . 1 31

1 2 Antibacterial Activity of the Essential Oils from Different parts of C. hystrix Against Bacillus cereus . . . . 134

13 Antibacterial Assay of the Essential O ils from Stem-Derived Callus of C. hystrix Against Bacillus Subtilis and Staphylococcus aureus . . . . " . . , . . . . . . . . . . . . . . . . . . . . . . . . . . 134

xx

14 Antibacterial Assay of the Methanolic Extract from Different Parts of C. hystrix Against Bacillus cereus. . . 1 38

1 5 Antibacterial Assay o f the Methanolic Extract From Different Parts of Plants Against Bacillus subtilis. . . . . . . . . 1 39

1 6 Antibacterial Assay of the Methanolic Extract from C. hystrix Stem-Derived Callus Against Bacillus subtilis . . . 1 39

xxi

Abs CH cm dwt. F lO fwt. g GC HP HPLC i .d . kg m M M �g �I �M mg min ml MS N NAA NaOEOTC PAL PEG PGRs PITC R I SA SOA SOE TEA TFA Tiss. U YE (w/v) (w/w)

LIST OF ABBREVIATIONS

- absorbance - casein hydrolysate - centimeter - dry weight basis - flame ionized detector - fresh weight - gram - gas chromatography - Hewlett Packard - high performance liquid chromatography - inner d iameter - kilogram - meter - molar - magnificent - microgram - microliter - micromolar - milligram - minute - millil iter - Murashige and Skoog - normality - a-naphthalene acetic acid - sodium d iethyldithiocarbamate - phenylalanine ammonium lyase - polyethylene glycol - plant growth regulators - phenylisothiocynate - retention index - salicylic acid - simultaneous distillation adsorption - simultaneous distillation extraction - triethylamine - trifluoroacetic acid - tissue - unit - yeast extract - weight/volume - weight/weight

xxii

CHAPTER I

INTRODUCTION

Secondary metabolites can be defined as compounds which are

biosynthetically derived from primary metabol ites (Blandrin and Klocke,

1 985). These include alkaloids, phenolic compounds, flavonoids, steroids,

carotenoids and terpenoids. Among the most valuable plant secondary

metabol ites are vinblastine (anticancer) , vincristine (antileukemia) and taxol

(anticancer) , scopolamine (sedative drug) , atropine (as anticholergen) and

digoxin (heart stimulants) (Coll in and Edwards, 1 998). In the higher plants,

these compounds are frequently produced in small quantities compared to

the primary metabolites and need special cel l types at distinct developmental

stages (Blandrin and Klocke, 1 985).

Secondary metabolites are compounds which have no apparent

function in primary metabolism but often have ecological roles, e.g . as a

pollinator, represent chemical adaptations to environmental stress, or serve

as chemical defenses against microorganism, insects and higher predators

(Harborne, 1 982). Secondary metabolites were also used to detoxify

substances accumulated during primary metabolism, act as physiological

effectors, provisioned of chemical signals to coordinate the metabolism of

cells and used to develop the ecological relationship (Luckner, 1 986) .

However, due to the difference in structure and their sites of location in

2

plants , it is difficult to identify a common function of all secondary

metabol ites (Col l in and Edwards, 1 998). Some secondary metabolites may

have detrimental effects on man and domesticated animals. For example,

an excessive i ntake of certain secondary metabolites can inhibit digestive

enzymes, i nduce Parkinson and Alzheimer disease, anemia, toxic and liver

damage (Endress, 1 994) .

Most spices, condiments , tea , citrus and other plants produce their

secondary metabol ites for specific properties, for example flavour and

fragrance. Although such compounds can be produced synthetically, high

prices are stil l being paid in some cases for extracted compounds from the

natural sources, especially to be used as food additives, natural colour or

flavouring agents (Slandrin and Klocke, 1 985) . For example, synthetic

benzaldehyde sells at US$1 .50 to US$1 .75/kg whereas natural

benzaldehyde sells for over US$200/kg . Similarly , synthetic methyl

anthranilate sells for US$5.75/kg compared to natural methyl anthranilate

which sells at more than US$400/kg to US$500/kg (Sahai , 1 994) . The market

for natural flavours in the US was estimated at US$200 mil l ion per year and

there have been an i ncreasing demand on the use of natural flavours rather

than synthetic flavours which were obtained throug h organic synthesis

(McCormick and Wild, 1 989).

C itrus flavour is widely used to flavour or aromatize food, non­

carbonated or carbonated beverages and also in household products as it