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INVESTIGATION OF ANTIOXIDANT AND ANTIMICROBIAL EFFECTS OF

PLANTAGO MAJOR LEAVES

A THESIS SUBMITTED TO

THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES

OF

MIDDLE EAST TECHNICAL UNIVERSITY

BY

ZGE KAYA

IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS

FOR

THE DEGREE OF MASTER OF SCIENCE

IN

BIOCHEMISTRY

SEPTEMBER 2011

Approval of the thesis:

INVESTIGATION OF ANTIOXIDANT AND ANTIMICROBIAL EFFECTS

OF PLANTAGO MAJOR LEAVES

submitted by ZGE KAYA in partial fulfillment of the requirements for the degree

of Master of Science in Department of Biochemistry, Middle East Technical

University by,

Prof. Dr. Canan zgen ________________

Dean, Graduate School of Natural and Applied Sciences

Prof. Dr. G.Candan Grakan ________________

Head of Department, Biochemistry,METU

Assoc. Prof. Dr. Nursen oruh ________________

Supervisor, Chemistry Dept., METU

Assist. Prof. Dr. A. Glin Sadolu Celep ________________

Co-Supervisor, Family and Consumer Sci. Dept., Gazi Unv.

Examining Committee Members:

Prof. Dr. Musa Doan ________________

Biology Dept., METU

Assoc. Prof. Dr. Nursen oruh ________________

Chemistry Dept., METU

Prof. Dr. Orhan Adal ________________

Biology Dept., METU

Prof. Dr. Ceyhan Kayran ________________

Chemistry Dept., METU

Assist. Prof. Dr. A. Glin Sadolu Celep ________________

Family and Consumer Sci. Dept., Gazi University

Date: 09.09.2011

iii

I hereby declare that all information in this document has been obtained and

presented in accordance with academic rules and ethical conduct. I also declare

that, as required by these rules and conduct, I have fully cited and referenced

all material and results that are not original to this work.

Name, Last name: zge KAYA

Signature:

iv

ABSTRACT

INVESTIGATION OF ANTIOXIDANT AND ANTIMICROBIAL EFFECTS OF

PLANTAGO MAJOR LEAVES

KAYA, zge

M.Sc., Department of Biochemistry

Supervisor: Assoc. Prof. Dr. Nursen ORUH

Co-Supervisor: Assist. Prof. Dr. A. Glin Sadolu CELEP

September 2011, 96 pages

This study was designed to investigate Plantago major leaves, which is widely used

in ethnobotanical uses in Turkey as is the case in all over the world, for their

antioxidant capacities and antimicrobial effect on two gram-positive and two gram-

negative bacteria.

Extracts of P. major leaves prepared in the methanol and fraction samples were

collected in solvents in increasing polarity, from non-polar to polar, to separate

bioactive compound in crude extract. Antioxidant capacities of the crude extracts and

fractions were examined by radical scavenging methods, namely 2,2-azinobis(3-

ethylbenzothiazoline-6-sulfonic acid) ABTS and 1,1-diphenyl-2-picryl-hydrazyl

(DPPH); as well, total phenolic and total flavonoid contents were determined.

Furthermore, antimicrobial activities of crude extract and fractions were calculated in

terms of minimum inhibitory concentration (MIC) and minimum bactericidal

concentration (MBC) methods together with disc diffusion method.

v

Among the investigated samples, ethyl acetate phase had the highest radical

scavenging capacity with the ABTS value of 2130.49.845 mol equivalents of

trolox/ g of ethyl acetate fraction DW, and with EC50 value of 0.0110.001 mg/mL;

further, the highest phenolic content was found in ethyl acetate phase with values of

0.452 GAE mg/mg of ethyl acetate fraction DW and 0.434 0.0017 mg catechin

equivalent in per mg of ethyl acetate fraction DW.

In general, it was found that the crude extract and aqueous phase of Plantago major

had a weak antimicrobial effect on S. aureus, P.mirabilis, S.pyogenes and E. coli,

along with the highest bacteriostatic and bactericidal upshot on selected bacterial

populations in ethyl acetate fraction.

Keywords: Plantago major, Antioxidant, Antimicrobial, Free Radicals.

vi

Z

PLANTAGO MAJOR YAPRAKLARININ ANTOKSDAN VE

ANTMKROBYAL ETKLERNN ARATIRILMASI

KAYA,zge,

Yksek Lisans, Biyokimya Blm

Tez Yneticisi: Do.Dr. Nursen ORUH

Ortak Tez Yneticisi: Yrd. Do. Dr. A. Glin Sadolu CELEP

Eyll 2011, 96 sayfa

Bu alma, Dnyada olduu gibi Trkiyede de geni bir alanda etnobotanik olarak

kullanlan Plantago major bitkisinin antioksidan kapasitesini belirlemek ve iki gram-

pozitif iki gram negatif bakteri zerindeki antimikrobial etkilerinin tesbiti iin

tasarlanmtr.

Plantago major yapraklarnn total zt methanolde hazrlandktan sonra total zt

iindeki biyoaktif maddelerin znrlk farklarndan yararlanlarak total zte artan

polaritelerde apolardan polara doru organik solventler uygulanarak fraksiyonlama

ilemi yaplmtr. Total zt ve fraksiyonlarn antioksidan kapasiteleri radical

yakalama kapasitesini lme methodlar olan olan 2,2'-azino-bis(3-

ethylbenzthiazoline-6-sulphonic acid) ABTS ve 1,1-diphenyl-2-picryl-hydr`azyl

DPPH methodlar ile belirlenmitir. Bunun yansra total fenolik ve total flavonoid

madde miktarlarda hesaplanmtr. Antimikrobiyal aktivite tayininin belirlenemesi

vii

iin minimum inhibe edici konsantrasyon, minimum bakterisidal konsantrasyon ve

disk difzyon teknikleri kullanlmtr.

Total zt ve fraksiyonlar iinde en yksek radikal snmleme kapasitesini etil

asetat faz ABTS 2130.49.845 mol trolox elenii/g, 0.0110.001 mg/mL DPPH

EC50 deerleri ile gstermitir.Ayn zamanda toplam fenolik madde miktar 0.452

GAEmg/mg ve total flavonoid miktar 0.434 mg catechine edeer madde/mg olarak

hesaplanmtr.

Genel olarak Plantago major bitkisinin S. aureus, P.mirabilis, S.pyogenes ve E. coli

bakterileri zerinde zayf bir etkisi olduu saptanm. Etil asetat fraksiyonunun en

etkili antimikrobiyal faz olmann yan sra antioksidan olarak da en etkili olduu

gzlenmitir.

Anahtar Kelimeler: Plantago major, Antioksidan, Serbest Radikal, Antimikrobiyal.

viii

To my sister and my mother

ix

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation and thanks to my supervisor Assoc. Prof.

Dr. Nursen oruh for her guidance, valuable advices, moral support and for

enlightening my professional and academic vision throughout my study.

I would like to thank to my co-supervisor Assist. Prof. Dr. A. Glin Sadolu

Celep for her guidance and valuable advices.

I would like to also thanks to Prof. Dr. Mesude can to give me opportunity to use

her laboratory equipment.

I appreciate for patience, criticism and advice of my thesis examining committee

members Prof. Dr. Musa Doan, Prof. Dr. Orhan Adal, Prof. Dr. Ceyhan Kayran,

and Assist. Prof. Dr. A. Glin Sadolu Celep, while reading and commenting on

my thesis study.

I would like to express my great thanks to all the members of Nursen oruh

Research Group Yeim Kmbet, Nizamettin zdoan , Can Nebigil, and my friends

Sule Sahin, Asligl Aksoy, Ayten Eroglu , Elif Asik, Emrah Sagr for their helps and

friendship. I express my special thanks to research assistant Can Ylmaz helping me

in the usage of ELISA

I would like to express my sincere thanks to Gktu Seymen, Neslihan alc

Akdoan, Serap Altnay, Mnire Ekmekigil, Tevfik Akdoan, Buket arba, Serdar

arba, Yeim Kmbet for their endless support, patience and motivation throughout

this work.

Finally, I would like to express my deepest thanks to my family my mother, my

sister, my cousin rem Gzbyk for helping, encouraging and loving me all

through my life. I cannot ask for more from them as they are simply perfect.

x

TABLE OF CONTENTS

ABSTRACT .............................................................................................................. iv

Z .............................................................................................................................. vi

ACKNOWLEDGEMENTS ..................................................................................... viii

TABLE OF CONTENTS .............................................................................................x

LIST OF TABLES .................................................................................................... xii

LIST OF FIGURES ................................................................................................. xiii

LIST OF ABBREVIATIONS .................................................................................. xvi

CHAPTERS

1.INTRODUCTION ..................................................................................................1

1.1 PLANTAGO MAJOR L...........................................................................................2

1.1.1 Botany of the Plantago maj

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