Antimicrobial activity and cytotoxicity of active compounds of Withania somnifera extracts

112 Pages·2015·2.7 MB·English

Republic of Iraq
Ministry of Higher Education
and Scientific Research
Al-Nahrain University
College of Science
Department of Biotechnology

Antimicrobial activity and cytotoxicity of
active compounds of Withania somnifera
extracts

A Thesis
Submitted tothe council of College of Science / Al-Nahrain University as a

partial fulfillment of the requirements for the Degree of Master of Science in
Biotechnology

By
Waham Saad Atta
B.Sc. Biotechnology/ College of Science/ Al-Nahrain University, 2012

Supervised by
Dr. Nabeel K. Al-Ani
(Professor in Plant Biotechnology)

January 2015 Rabeea- Alawwal 1435

Supervisors Certification 
we, certify that this thesis entitled (Antimicrobial activity and
Cytotoxicity of Active Compounds of Withania somnifera extracts) was
prepared by (Waham Saad Atta) under my supervision at the College of
Science / Al-Nahrain University as a partial fulfillment of the requirements
for the Degree of Master of Science in Biotechnology.

Signature :
Name: Dr. Nabeel K. Al-Ani
Scientific Degree: Professor
Date:

In review of available recommendation, I forward this thesis for debate by
examining committee

Signature :
Name: Dr. Hameed M. Jasim
Scientific Degree: Professor
Title: Head of Biotechnology Department
Date:
Committee Certification 
We, the examining committee certify that we have read this thesis
entitled (Antimicrobial activity and Cytotoxicity of Active
Compounds of Withania somnifera extracts )and examined the student
(Waham Saad Atta) in its contents and that in our opinion; it is
accepted for the degree of Master of Science in Biotechnology.

Signature :
Name: Dr. khulood W. Abood
Scientific Degree: Professor
Date:
(Chairman)

Signature : Signature :
Name: Dr. Kamil M. Malih Name: Dr. Nedhaal S. Zbar
Scientific Degree: Professor Scientific Degree: Instructor
Date: Date:
(Member) (Member)

Signature :
Name: Dr. Nabeel K. Al-Ani
Scientific Degree: Professor
Date:
(Member and Supervisor)

I, hereby certify upon the decision of the examining committee
Signature :
Name: Dr. Hadi M. A. Abood
Scientific Degree: Assistant Professor
Title: Dean of College of Science
Dedication

To those whom always give
me support and

encouragement to insist

moving toward my glory
future
My Father and My Mother

My uncle

Mustafa Ahmed Reshan

Waham….....
Acknowledgments

First of all praise to Allah the lord of the universe, peace be upon
Mohammed the messenger of Allah and upon his relatives and
companions.
Great thanks to my supervise Professor Dr. Nabeel Al-Ani for his support,
patience and important guidance throughout my study.
I would like to express my gratitude and feelings of love to my mother, My
Father and my brothers and my sister for their encouragement, patience
and many sacrifices they presented during all stages of study.
I would like to thankful My uncles (Mustafa Ahmed Reshan and Jamal
Ahmed Reshan) for their love, support and encouragement.
I would like to extend my thankful to Biotechnology Research center/ Al-
Nahrain University.
I am very thankful to my Friends Zainab, Islah, Yasamin, Mina, Albab and
Marwa for their appreciable help in this study.

For all of them, I said “Thank you very much”………..
Summary
In this study Withania somnifera leaves were collected from
gardens of Al-Nahrain University. Then the leaves powder was
used to get three type of extracts, each type of Withania extracts
(water, ethanol and acetone extract) were subjected to chemical
analysis to identify the active compounds in each extract which
indicated that alkaloids, glycoside, saponins, flavonoids, fixed oil
were present in ethanol, acetone and water extracts, while absent
of terpene in water extract and protein in acetone extract, Tannins
were not found in all extract types. Numbers of active group in
Withania extracts were detected by FTIR method. HPLC analysis
was done to detect the concentration of important active
compounds (alkaloid, flavonoid and saponin) present in the water,
acetone and ethanol extracts of Withania. All three types of
Withania extracts contained two types of Alkaloids (Withanolide-
A and Withaferin-A), seven types of Flavonoids (Naringenin,
Catechin, Luteolin, Hesperetin, Kaempfero, Apigenin and
Naringin) and two types of Saponins (Sitoindosides VII and
Sitoindosides VIII) appeared as different peaks.
All three types of Withania extracts (acetone, water and ethanol
extracts) showed antibacterial activity on the following bacteria
(Escherichia coli, Enterobacter sakazakii, Klebsiella pneumonia,
Staphylocuss aureus, Staphylocuss epidermis, Streptococcus pyogenes,
Proteus mirabilis, Pseudomonas aeruginosa) with probability (P<0.05)
and only Acetone extract showed activity on fungal isolate (Trichophyton
violaceum). The cytotoxic activity of the plant extracts was determined
by evaluating the effects on the growth of HepG2 cell line after
incubation for 72 hour with different concentrations (80, 100 and 120
mg/ml) of plant extracts (water, ethanol and acetone extracts).
  I
W. somnifera extracts have cytotoxic effect on HepG2 cell line with
probability (P< 0.01), higher cytotoxic effect was belonged to ethanol
extract at 120 mg/ml concentration followed by the water extract and the
acetone extract was the lowest one.

  II
List of Contents

Number Title Page No.
Chapter One
Introduction and Literature review
1.1 Introduction 1
1.2 Aim of study 2
1.3 Literature Review 3
1.3.1 Medicinal Plants 3
1.3.2 The Withania genus 3
1.3.2.1 Common name and Taxonomy 4
1.3.2.2 Plant morphology 5
1.3.2.3 Plant Distribution 6
1.3.2.4 Chemical Composition 6
1.3.3 \ Biological activity and pharmaceutical 7
application
1.3.3.1 Anti-oxidant Activity of Withania 7
1.3.3.2 Anti-inflammatory Activity of Withania 8
1.3.3.3 ImunomodulImatory activity 9
1.3.3.4 Other Activities of Withania 10
1.3.4 Metabolite profiling 10
1.3.5 Antimicrobial Activity 11
1.3.5.1 Bacterial skin infection 12
1.3.5.2 Fungal skin infection 13
1.3.6 Pathogenicity of other studies 14
Microorganism
1.3.6.1 Enterobacter sakazakii 14
III
1.3.6.2 Escherichia coli 14
1.3.6.3 Proteus mirabilis 15
1.3.6.4 Klebsiella pneumonia 15
1.3.7 Effect of active compounds on Tumor cell 16
line
1.3.7.1 Cytotoxicity activity of Withania Somnifera 17
1.3.7.2 Human liver carcinoma cell line HepG2 18
Chapter Two
Materials and Methods
2.1 Materials 20
2.1.1 Equipment’s and Instruments 20
2.1.2. Chemicals 21
2.1.3 Ready to use media 21
2.1.3.1 Bacterial and fungi Media 21
2.1.3.2 RPMI=1640 Medium 22
2.1.4 Standards 22
2.1.4.1 Alkaloid Standards 22
2.1.4.2 Flavonoid Standards 22
2.1.4.3 Saponin Standards 23
2.1.5 Solutions and Dyes Preparation 24
2.1.5.1 Lead Acetate Solution 1 %( w/v) 24
2.1.5.2 Ferric Chloride Solution 1% (w/v) 24
2.1.5.3. Potassium Hydroxide Solution 50% (w/v) 24
2.1.5.4 Fetal Calf Serum (FCS) 24
IV