UNIVERSITI PUTRA MALAYSIA - psasir.upm.edu.mypsasir.upm.edu.my/31914/1/FP 2012 7R.pdfuniversiti putra malaysia in-vitro antibacterial activity and phytochemical screening of bioactive

UNIVERSITI PUTRA MALAYSIA

IN-VITRO ANTIBACTERIAL ACTIVITY AND PHYTOCHEMICAL SCREENING OF BIOACTIVE COMPOUNDS FROM GUAVA

(Psidium guajava L.) CRUDE LEAF EXTRACTS

SITI NUR SARAH BINTI SHAFIEI

FP 2012 7

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IN-VITRO ANTIBACTERIAL ACTIVITY AND

PHYTOCHEMICAL SCREENING OF

BIOACTIVE COMPOUNDS FROM GUAVA

(Psidium guajava L.) CRUDE LEAF EXTRACTS


MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2012

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IN-VITRO ANTIBACTERIAL ACTIVITY AND PHYTOCHEMICAL

SCREENING OF BIOACTIVE COMPOUNDS FROM GUAVA (Psidium

guajava L.) CRUDE LEAF EXTRACTS

By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Master of Science

August 2012

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Master of Science

IN-VITRO ANTIBACTERIAL ACTIVITY AND PHYTOCHEMICAL

SCREENING OF BIOACTIVE COMPOUNDS FROM GUAVA (Psidium

guajava L.) CRUDE LEAF EXTRACTS

By


August 2012

Chair: Associate Professor Kamaruzaman bin Sijam, PhD

Faculty: Agriculture

Plant pathogenic bacteria are known to be detrimental microbes able to reduce the

quality and quantity of crop production around the world. Psidium guajava leaf was

screened for its potential use as biological control agent against plant pathogenic

bacteria. P. guajava leaf was successfully extracted using n-hexane, ethyl acetate and

methanol by maceration. The yield percentage of crude extracts obtained for each

extract was 0.93% for hexane, 1.28% for ethyl acetate and 1.70% for methanol leaf

crude extracts. The higher yield obtained by methanol showed that methanol

extracted more compounds that are readily soluble to methanol than hexane and ethyl

acetate. For in vitro antibacterial activity, seven different species of plant pathogenic

bacteria were used namely Pectobacterium carotovorum subsp. carotovorum,

Pectobacterium chrysanthemi, Ralstonia solanacearum, Xanthomonas axonopodis

pv. citri, Xanthomonas euvesicatoria, Xanthomonas oryzae pv. oryzae and

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Xanthomonas oryzae pv. oryzicola. For all crude extracts, four different

concentrations 25, 50, 100 and 200 mg/ml were used in cup-plate agar diffusion

method. Streptomycin sulfate at concentration of 30 µg/ml was used as positive

control, while each respective solvent used for leaf extraction was used as negative

control. The results obtained from in vitro studies showed only methanol extract

possessed antibacterial activity tested on the plant pathogenic bacteria. Hexane and

ethyl acetate did not show any antibacterial activity against plant pathogenic bacteria

where no inhibition zones were recorded. X. oryzae pv. oryzae recorded the highest

diameter of inhibition zones for all range of concentrations introduced followed by X.

oryzae pv. oryzicola, X. euvesicatoria, X. axonopodis pv. citri, P. chrysanthemi and

P. carotovorum subsp. carotovorum. For minimum inhibitory concentration (MIC)

and minimum bactericidal concentration (MBC) determination, only methanol

extract was subjected for the assay as only methanol extract exhibited antibacterial

activity. The lowest concentration of methanol extract used in MIC assay was at 0.39

mg/ml which inhibited X. oryzae pv. oryzae, followed by X. euvesicatoria (0.78

mg/ml), and X. oryzae pv. oryzicola, X. axonopodis pv. citri, P. chrysanthemi and P.

carotovorum subsp. carotovorum at concentration 1.56 mg/ml. This assay showed

that methanol extract had bacteriostatic effects at concentration 0.39, 0.78 and 1.56

mg/ml for different species of plant pathogenic bacteria. The lowest MBC

concentration recorded was at 0.78 mg/ml against X. oryzae pv. oryzae, 1.56 mg/ml

against X. euvesicatoria, 3.13 mg/ml against X. oryzae pv. oryzicola and X.

axonopodis pv. citri, and 6.25 mg/ml against P. chrysanthemi and P. carotovorum

subsp. carotovorum. The phytochemical screening of P. guajava methanol leaf

extracts showed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids

and phenols. The thin layer chromatography (TLC) profiling of methanol extract

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using hexane and ethyl acetate with ratio of 7:3 (v/v) gave 13 maximum colorful

bands when visualized with 10% ethanolic sulfuric acid solution, with different

retention factor, Rf values which proved the presence of various active secondary

metabolites within methanol extract. The antibacterial activity of methanol extract

was also screened through direct bioautography technique in order to detect the

location of active bands on chromatograms developed in the same matter for TLC

profiling. The most active Rf values which inhibited all tested plant pathogenic

bacteria at the same Rf values location were at 0.63, 0.85, and 0.98. The Rf values

obtained were compared to the previous studies performed using the same solvent

system indicated that the Rf values recorded at >0.90 belongs to hydrocarbons, and

0.60-0.80 belongs to epoxides and esters which were found to fall within oxygenated

terpenes group.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains

AKTIVITI ANTIBAKTERIA IN-VITRO DAN PENYARINGAN FITOKIMIA

SEBATIAN AKTIF DARIPADA EKSTRAK MENTAH DAUN JAMBU BATU

(Psidium guajava L.)

Oleh


Ogos 2012

Pengerusi: Profesor Madya Kamaruzaman bin Sijam, PhD

Fakulti: Pertanian

Bakteria penyakit tanaman dikenali sebagai mikrob pemusnah yang berupaya

mengurangkan kualiti dan kuantiti penghasilan tanaman di seluruh dunia. Daun

Psidium guajava telah disaring untuk mengenalpasti potensinya sebagai agen

kawalan biologi dalam mengawal bakteria patogen tumbuhan. Daun P. guajava telah

berjaya diekstrak menggunakan n-heksana, etil asetat dan metanol melalui kaedah

rendaman. Peratusan hasil ekstrak mentah yang diperolehi bagi setiap ekstrak adalah

0.93% untuk heksana, 1.28% untuk etil asetat dan 1.70% untuk ekstrak metanol daun

mentah. Hasil yang lebih tinggi yang diperolehi oleh metanol menunjukkan bahawa

metanol mengekstrak lebih banyak sebatian yang mudah larut metanol daripada

heksana dan etil asetat. Bagi aktiviti anti-bakteria secara in vitro, tujuh spesies

bakteria patogen tumbuhan yang berbeza telah digunakan iaitu Pectobacterium

carotovorum subsp. carotovorum, Pectobacterium chrysanthemi, Ralstonia

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solanacearum, Xanthomonas axonopodis pv. citri, Xanthomonas euvesicatoria,

Xanthomonas oryzae pv. oryzae dan Xanthomonas oryzae pv. oryzicola. Bagi

kesemua ekstrak mentah, empat kepekatan yang berbeza iaitu 25, 50, 100 dan 200

mg/ml digunakan dalam kaedah peresapan agar cawan-plat. Streptomisin sulfat pada

30 kepekatan μg / ml telah digunakan sebagai kawalan positif, manakala setiap

pelarut yang digunakan untuk pengekstrakan daun digunakan sebagai kawalan

negatif. Keputusan yang diperolehi dari kajian secara in vitro menunjukkan hanya

ekstrak metanol memiliki potensi anti-bakteria apabila diuji terhadap bakteria

patogen tumbuhan. Heksana dan etil asetat tidak menunjukkan sebarang potensi

sebagai anti bakteria terhadap bakteria patogen tumbuhan di mana tiada zon

perencatan direkodkan dan diperhatikan untuk ekstrak ini. X. oryzae pv. oryzae

mencatatkan diameter tertinggi zon perencatan bagi semua julat kepekatan yang

diperkenalkan diikuti oleh X. oryzae pv. oryzicola, X. euvesicatoria, X. axonopodis

pv. citri, P. chrysanthemi dan P. carotovorum subsps carotovorum. Bagi penentuan

kepekatan perencatan minimum (MIC) dan kepekatan minimum bakterisida (MBC),

ekstrak metanol digunakan sebagai cerakin kerana hanya ekstrak metanol

mempamerkan aktiviti anti bakteria. Kepekatan terendah metanol ekstrak yang

digunakan dalam MIC adalah pada 0.39 mg/ml yang menghalang pertumbuhan X.

oryzae pv. oryzae, diikuti oleh X. euvesicatoria (0.78 mg/ml), dan X. oryzae pv.

oryzicola, X. axonopodis pv. citri, P. chrysanthemi dan P. carotovorum subsp.

carotovorum pada kepekatan 1.56 mg/ml. Cerakin ini menunjukkan bahawa ekstrak

metanol mempunyai kesan bakteriostatik pada kepekatan 0.39, 0.78 dan 1.56 mg/ml

untuk spesies yang berbeza bagi bakteria patogen tumbuhan. Kepekatan MBC

terendah yang dicatatkan adalah pada 0.78 mg/ml terhadap X. oryzae pv. oryzae, 1.56

mg/ml terhadap X. euvesicatoria, 3.13 mg/ml terhadap X. oryzae pv. oryzicola dan X.

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axonopodis pv. citri, dan akhirnya pada kepekatan 6.25 mg/ml terhadap P.

chrysanthemi dan P. carotovorum subsp. carotovorum. Pemeriksaan fitokimia

ekstrak metanol daun P. guajava menunjukkan kehadiran alkaloid, flavonoid, tanin,

saponin, terpenoid dan fenol. Pemprofilan lapisan kromatografi nipis (TLC) ekstrak

metanol menggunakan sistem pelarut heksana dan etil asetat dengan nisbah 7:3 (v/v)

memberi 13 lapisan maksimum yang berwarna-warni apabila disembur dengan 10%

larutan asid sulfurik etanol, dengan faktor tahanan berbeza, nilai Rf menunjukkan

kehadiran pelbagai metabolit sekunder aktif dalam ekstrak metanol. Aktiviti anti

bakteria ekstrak metanol juga telah dijalankan melalui teknik bioautografi langsung

untuk mengesan lokasi lapisan aktif pada kromatogram yang dibangunkan dengan

sistem pelarut yang sama didalam pemprofilan TLC. Nilai-nilai yang Rf paling aktif

yang menghalang semua bakteria patogenik yang diuji terdapat pada lokasi nilai R f

yang sama iaitu pada 0.63, 0.85, dan 0.98. Nilai Rf yang diperolehi berbanding

dengan kajian yang dilakukan sebelum ini dengan menggunakan sistem pelarut yang

sama menunjukkan bahawa nilai Rf yang direkodkan pada >0.90 adalah milik

hidrokarbon, dan 0.60-0.80 milik epoksida dan ester yang didapati tergolong dalam

kumpulan terpenes teroksigen.

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ACKNOWLEDGEMENTS

I wish to express my gratitude to all who had helped me to achieve my research

objectives during my studies in Universiti Putra Malaysia. My gratitude will first be

pointed to my mother, Rohiham who had supported my ideas of pursuing my studies

for Master of Science in UPM, and my supervisor Assoc. Prof. Dr. Kamaruzaman

Sijam and my co-supervisor Prof. Dr. Dzolkhifli Omar. Thank you for both of you

for being wonderful advisor and knowledge that both of you shared during my

studies. Not to forget all the labmates and lab assistant who had helped me a lot

during my research in the laboratory, thank you for all of your guidance and warmth.

Not to forget, my gratitude to School of Graduate Studies for supporting my studies

by giving me scholarship through Graduate Research Fellowship and Special

Graduate Research Assistantship for my two and half years of studies.

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I certify that a Thesis Examination Committee has met on 7th

August 2012 to conduct

the final examination of Siti Nur Sarah binti Shafiei on her thesis entitled “In-vitro

Antibacterial Activity and Phytochemical Screening of Bioactive Compounds from

Guava (Psidium guajava L.) Crude Leaf Extracts” in accordance with the

Universities and University Colleges Act 1971 and the Constitution of the Universiti

Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the

student be awarded the Master of Science.

Members of the Thesis Examination Committee were as follows:

Halimi bin Mohd Saud, PhD

Associate Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

Zainal Abidin bin Mior Ahmad, PhD

Associate Professor

Faculty of Agirculture


(Internal Examiner)

Mohd Aspollah bin Hj Md Sukari, PhD

Professor

Faculty of Science


(Internal Examiner)

Siti Zaiton Mat So’ad, PhD

Department of Pharmaceutical Chemistry

International Islamic University

Malaysia

(External Examiner)

_____________________________

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies


Date: 22 October 2012

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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:

Kamaruzaman bin Sijam, PhD

Associate Professor



(Chairman)

Dzolkhifli Omar, PhD

Professor



(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

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.


Date: 7th

August 2012

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TABLE OF CONTENTS

Page

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS ix

APPROVAL x

DECLARATION xii

LIST OF TABLES xvi

LIST OF FIGURES xviii

LIST OF ABBREVIATIONS xx

CHAPTER

1 INTRODUCTION 1

1.1 Overview 1

1.2 Objectives 4

1.3 Problem statements 4

2 LITERATURE REVIEW 5

2.1 Plant pathogenic bacteria 5

2.2 Genus Pectobacterium 6

2.2.1 Pectobacterium carotovorum subsp. carotovorum 7

2.2.2 Pectobacterium chrysanthemi 8

2.3 Genus Ralstonia 8

2.4 Genus Xanthomonas 9

2.4.1 Xanthomonas axonopodis pv. citri 10

2.4.2 Xanthomonas euvesicatoria 11

2.4.3 Xanthomonas oryzae pv. oryzae 12

2.4.4 Xanthomonas oyrzae pv. oryzicola 13

2.5 Psidium guajava Linn. 14

2.5.1 Phytochemistry and biological activity of Psidium

guajava L.

14

2.6 Natural products from plants 15

2.7 Plant extracts as bio-control agents 17

2.8 Plant allelopathy 18

2.9 Plant defense and secondary metabolites 19

2.10 Major groups of plant secondary metabolites 21

2.10.1 Terpenes 22

2.10.2 Phenolics 24

2.10.2.1 Flavonoids 25

2.10.2.2 Tannins 26

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2.10.3 Nitrogen (N) containing compounds 27

2.10.3.1 Alkaloids 27

2.10.3.2 Saponins 28

2.11 Experimental approaches for plant extraction methods 28

2.11.1 Plant material and extracts preparation 29

2.11.2 Solvents used for plant extraction 30

2.11.3 Plant extraction methods 32

2.12 Antimicrobial susceptibility testing 33

2.12.1 Agar cup plate method 34

2.12.2 Broth macro dilution method 35

2.13 Bio autography 37

2.14 Tetrazolium salts as colorimetric indicator for living cells 37

3 ANTIBACTERIAL ACTIVITY OF PSIDIUM GUAJAVA L.

LEAF EXTRACTS AGAINST PLANT PATHOGENIC

BACTERIA

39

3.1 Introduction 39

3.2 Materials and methods 40

3.2.1 Plant pathogenic bacteria sources and

confirmation by polymerase chain reaction (PCR)

40

3.2.2 Psidium guajava L. Leaf collection and pre-

preparation for extraction

41

3.2.3 Preparation of Psidium guajava leaf extracts 41

3.2.4 In-vitro antibacterial activity of Psidium guajava

leaf extracts, cup plate agar diffusion method

42

3.2.5 Statistical analysis 43

3.2.6 Minimum inhibitory concentration (MIC) and

minimum bactericidal concentration (MBC) of

Psidium guajava leaf extracts

43

3.3 Results and discussion 44

3.3.1 Plant pathogenic bacteria confirmation by PCR

assays

44

3.3.2 Psidium guajava leaf extracts physical

characteristics and percent yield

45

3.3.3 In-vitro antibacterial activity of Psidium guajava

L. leaf extracts against plant pathogenic bacteria

46

3.3.4 Minimum inhibitory concentration (MIC) and

minimum bactericidal concentration (MBC) of

Psidium guajava methanol leaf extracts

53

3.4 Conclusion 57

4 PHYTOCHEMICAL SCREENING AND THIN LAYER

CHROMATOGRAPHY (TLC) PROFILING OF PSIDIUM

GUAJAVA L. METHANOL LEAF EXTRACT

58

4.1 Introduction 58


4.2.1 Protocol for phytochemical screening 59

4.2.1.1 Alkaloids 59

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4.2.1.2 Flavonoids 60

4.2.1.3 Terpenoids 60

4.2.1.4 Tannins 60

4.2.1.5 Saponins 60

4.2.1.6 Phenols 61

4.2.2 Thin layer chromatography (TLC) analysis of the

fractions

61

4.2.3 TLC spot visualization by daylight, UV light and

10% ethanolic sulfuric acid (H2SO4) solution

62


4.3.1 Phytochemical screening analysis 63

4.3.2 TLC analysis of the fractions and screening with

different visualization methods

66

4.4 Conclusion 69

5 LOCALIZATION OF ANTIBACTERIAL COMPOUNDS

FROM PSIDIUM GUAJAVA L. METHANOL LEAF

EXTRACT ON CHROMATOGRAMS BY DIRECT

BIOAUTOGRAPHY TECHNIQUE

70

5.1 Introduction 70


5.2.1 Direct TLC bioautography assay 71


5.3.1 Direct TLC bioautography assay 72

5.4 Conclusion 79

6 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATIONS FOR FUTURE RESEARCH

80

6.1 Summary 80

6.2 General conclusion 84

6.3 Recommendations for future research 86

REFERENCES 87

APPENDIX A 109

APPENDIX B 110

APPENDIX C 111

APPENDIX D 112

APPENDIX E 113

BIODATA OF STUDENT 114

LIST OF PUBLICATIONS 115

Documents

UNIVERSITI PUTRA MALAYSIA - psasir.upm.edu.mypsasir.upm.edu.my/31914/1/FP 2012 7R.pdfuniversiti putra malaysia in-vitro antibacterial activity and phytochemical screening of bioactive