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
