Nowsheen Shameem Nowsheri

152 Pages·2012·4.86 MB·English

Antimicrobial and Antioxidant activities of
Arnebia benthamii (Wall ex. G. Don Johnston) - A
Critically Endangered Medicinal plant of Kashmir

Dissertation Submitted in Partial Fulfillment of the
Requirements for the Award of Degree of

MASTER OF PHILOSOPHY (M.PHIL.)
IN
ENVIRONMENTAL SCIENCE

By
Nowsheen Shameem Nowsheri
M.Sc.

Under the Joint Supervision of

Dr. Rabia Hamid Prof. Azra N. Kamili
Sr. Assistant Professor Head/ Director
Department of Biochemistry Department of Environmental Science/CORD
(Co-Supervisor) (Supervisor)

POST GRADUATE DEPARTMENT OF ENVIRONMENTAL SCIENCE
Faculty of Physical and Material Sciences
University of Kashmir
NAAC Accreditation ‘A’
Srinagar-190006, J&K
2012
Phone: Office: 0194-2420078, 2420405, Ext. 2155
email: [email protected];
[email protected]

Centre of Research for Development
University of Kashmir, Srinagar -190006

No.
Dated:

Certificate

Certified that the M.Phil Dissertation entitled “Antimicrobial and
Antioxidant activities of Arnebia benthamii (Wall ex. G. Don Johnston) - A
Critically Endangered Medicinal plant of Kashmir” substantiates the original
work carried out by Ms. Nowsheen Shameem Nowsheri for submission as partial
fulfilment of the requirements for the award of Master of Philosophy (M. Phil)
Degree in Environmental Science. This study has been carried out under our
supervision for the period required under statutes and the same has not been
submitted elsewhere for any degree before.
We deem it fit for submission.

Dr. Rabia Hamid Prof. Azra N. Kamili
Sr. Assistant Professor Head/ Director
Department of Biochemistry Department of Environmental Science/CORD
University of Kashmir Srinagar. University of Kashmir, Srinagar.
(Co-Supervisor) (Supervisor)

Prof. Azra N. Kamili
Head/ Director
Department of Environmental Science/CORD
University of Kashmir, Srinagar.
i
ACKNOWLEDGMENTS

BISMILLAH-IR-RAHMAN-IR-RAHEEM
Allah is the Light of the heavens and the earth. The parable of His
Light is as if there were a Niche And within it a Lamp: The Lamp
enclosed in glass; The glass as it were a brilliant star: Lit from a blessed
tree, an Olive, Neither of the East nor of the West, Whose oil is well-nigh
luminous, Though fire scarce touched it: Light upon Light! Allah do
guide whom He will to His light: Allah do set forth parables for men: And
Allah do know all things. (Lit is such a light) in houses, which Allah have
permitted to be raised to honour; For the celebration, in them, of his
name: In them is He glorified in the mornings And in the evenings, (again
and again). (Surah ; Al Nur Verse 35 &36)
All praises and thanks to Almighty Allah (most merciful and
beneficent) whose grace and special succour enabled me to successfully
complete M. Phil programme with great zeal and zest.
I take this opportunity to express my enthusiastic gratitude and
indebtedness to acknowledge my guide Prof. Azra N. Kamili, H.O.D.
Department of Environmental Science / Director CORD University of
Kashmir, for believing in me and suggesting me the work of vital interest.
This study was conducted at her initiative and under her supervision. I
am thankful to her for her guidance, invaluable suggestions, enthusiastic
encouragement and necessary research facilities during the course of my
studies. I must confess that without her constant endeavour in guiding me
in completion of this work, it could not have been completed successfully.
I wish to pay due and respectful obeisance and indebtedness to Dr.
Rabia Hamid, Sr. Assistant Professor Department of Biochemistry
ii
University of Kashmir for her guidance, kind supervision, continued
encouragement, generous assistance, timely cooperation valuable
suggestions and constructive Criticism during the course of present study.
I also express my profound gratitude Prof. A. R. Yousuf (former
Dean Academic Affairs), Prof. A. K. Pandit (CORD/Environmental
Science) and Prof. G. A. Bhat (CORD/Environmental Science), Dr.
Ruqeya (Asst. Prof. CORD) for their moral support, useful suggestions
and continuous encouragement all through the course of study.
My sincere thanks also go to other technical and non-technical staff
members of CORD especially Mrs. Bilquis Qadri (Scientific Officer), Ms.
Rubiya Dar (Sr. Technical Assistant), Ms. Sumaira Tyub (Sr. Technical
Assistant) and Mr. Javeed A. Javeed (Jr. Technical Assistant) for their
cooperation timely help and whole hearted support.
The successful completion of the research programme was possible
only because of the active cooperation of my friends and colleagues. I
would like to put on record my thanks to Scholars of CORD in general
and Microbiology in particular namely Suhaib A. Bandh, Javid A.
Parray, Sana shafi, Humera Nissa, Samira Saleem and Bashir A. Lone
who rendered full cooperation and support which helped me to sustain
through long hours of creation and completion of this research
programme. I am also thankful to my friends Uzma, Navish, Sana,
Afeefa and Habiba for their help and fruitful guidance.
I fail in my duty if I don’t make a special mention here of my parents
and my sisters (Shumail and Zufishan) and other family members who
encouraged and cooperated with me in several capacities throughout the
research work.

Nowsheen Shameem
iii
CONTENTS
DESCRIPTION
Page No.
CERTIFICATE
i
AKNOWLEGMENTS
ii-iii
CONTENTS
iv-v
LIST OF TABLES
vi
LIST OF FIGURES
vii-viii
LIST OF PHOTOPLATES
ix
ABBREVATIONS
x
ABSTRACT
xi-xiv
1. INTRODUCTION 1-15

1.1. Phyto chemicals
1.2. Mechanism of action
1.3. Major groups of Antimicrobial and Antioxidant
substances
1.4. Plant Profile
16-37
2. REVIEW OF LITERATURE
2.1. Phytochemistry
2.2. Antimicrobial activity
2.3. Antioxidant activity

iv
3. MATERIAL AND METHODS 38-48
3.1. Instrumentation
3.2. Chemicals and Reagents
3.3. Sampling

3.4. Preliminary Phyto-chemical Qualitative screening
3.5. Antimicrobial Susceptibility Testing
3.6. Anti-oxidant activity
3.7. Statistical Analysis
4. RESULTS 49-75

4.1. Phytochemical screening
4.2. Antimicrobial activity
4.3. Antibacterial Activity
4.4. Antifungal activity

4.5. MIC and MBC determination
4.6. Antioxidant activity
5. DIS CUSSIONS 76-92
5.1. Phytochemical screening
5.2. Antibacterial activity

5.3. Antifungal activity

5.4. MIC and MBC determination
5.5. Antioxidant activity
6. CONCLUSIONS 93-94
7. BIBLIOGRAPHY 95-121
APPENDIX 122

v
List of Tables
Table Page
Description
No No.
1. Extraction yield and macroscopic characteristics of the crude 58
extracts of aerial parts (AP) and root parts (RP) of A.
benthamii L.
2. Qualitative analysis for various secondary metabolites in 59
aerial part (AP) extracts of A. benthamii L.
3. Qualitative analysis for various secondary metabolites in root 59
part (RP) extracts of A. benthamii L.
4. Antibacterial activity of different solvent extracts of aerial 60
parts (250µg/ml) of A. benthamii L.
5. Antibacterial activity of different solvents of root part (RP) 61
extracts (250µg/ml) of A. benthamii L.
6. Antifungal activity of different solvent extracts (400µg/ml) of 62
Aerial parts (AP) of A. benthamii L.
7. Antifungal activity of different solvent extracts (400µg/ml) of 63
root (RP) extracts of A. benthamii L.
8. MIC & MBC of the extracts of aerial/root part extracts of A. 64
benthamii against tested bacterial strains.
9. Radical Scavenging activities of extracts of AP & RP extracts 65
of A. benthamii as measured by DPPH method at different
concentrations.
10. Superoxide dismutase (SOD) activity of extracts of aerial & 66
root parts of A. benthamii measured by riboflavin photo-
oxidation method at different concentrations presented as
%age of inhibition rate.
11. DNA Damage assay of extracts of aerial/root parts of A. 67
benthamii measured by Hydroxyl scavenging method at
different concentrations presented as %age of inhibition rate.
12. Lipid peroxidation assay of extracts of aerial/root parts of A. 68
benthamii measured by TBAS method at different
concentrations presented as %age inhibition rate.
vi
LIST OF FIGURES
Fig. Description Page
No No.
1 Standard Gallic acid curve for estimating (TP) Total phenolic 69
content Gallic Acid EQ (µg/mg)
2 Total Phenolic (TP) Content of the extracts of Aerial parts (AP) 69
and Root parts (RP) of A. benthamii L.
3 Comparative analysis of antibacterial activity of aerial and root 70
part extracts of A. benthamii L. against Shigella flexnerii.
4 Comparative analysis of antibacterial activity of aerial and root 70
part extracts of A. benthamii L. against Staphylococus aureus.
5 Comparative analysis of antibacterial activity of aerial and root 70
part extracts of A. benthamii L. against Psuedomonas
aeruoginosa.
6 Comparative analysis of antibacterial activity of aerial and root 71
part extracts of A. benthamii L. against Escherchia coli.
7 Comparative analysis of antibacterial activity of aerial and root 71
part extracts of A. benthamii L. against Klebseilla pneumonia.
8 Comparative analysis of antibacterial activity of aerial and root 71
part extracts of A. benthamii L. against Salmonella
typhimurium.
9 Comparative analysis of antibacterial activity of aerial and root 72
part extracts of A. benthamii L. against Asperigillus flavus.
10 Comparative analysis of antifungal activity of aerial and root 72
part extracts of A. benthamii L. against Acremonium spp.
11 Comparative analysis of antifungal activity of aerial and root 73
part extracts of A. benthamii L. against Candida albicans.
12 Comparative analysis of antifungal activity of aerial and root 73
part extracts of A. benthamii L. against Candida krusie.
vii
13 Comparative analysis of antifungal activity of aerial and root 73
part extracts of A. benthamii L. Against Aspergilus versicolor.
14 Comparative analysis of radical Scavenging activities of 74
extracts of aerial and rootparts of A. benthamii L. w.r.t. control
as measured by DPPH method at higher concentrations
(300µg) presented as %age of inhibition rate.
15 Comparative analysis of antioxidant activity of extracts of aerial 74
and root parts of A. benthamii L. w .r. t. control measured by
superoxide dismutase (SOD) assay method at higher
concentrations (300µg) are presented as %age of inhibition
rate.
16 Comparative analysis of DNA Damage assay of extracts of 75
aerial and root parts of A. benthamii L. w.r.t. control measured
by Hydroxyl scavenging method at higher concentrations
(300µg) are presented as %age inhibition rate.
17 Comparative analysis of Lipid per-oxidation assay of extracts of 75
aerial and root parts of A. benthamii L. w. r. t. Control measured
by TBARS method at higher concentrations (300µg) are
presented as %age inhibition rate.

viii
List of plates
Plate
Description
No
Phytochemical screening Tests of extracts of Arnebia benthamii L.
1.
A =Tests for Alkaloids; B= Tests for Phenols
C= Tests for Terpenoids; D= Tests for Flavonoids
Antibacterial activity of aerial part extracts of Arnebia benthamii L.
against
2.
A =Shigella flexnerii; B= Psuedomonas aeruoginosa
C= Klebseilla pneumonia; D= Escherchia coli
Antibacterial activity of root extracts of Arnebia benthamii L.
against
3.
A = Salmonella typhimurium; B= Shigella flexnerii
C= Staphylococcus aureus; D= Escherchia coli
Antifungal activity of aerial part extracts of Arnebia benthamii L.
against
4.
A = Acremonium spp; B= Aspergillus flavus
C= Candida parapsilosis; D= Candida kruesie
Antifungal activity of root extracts of Arnebia benthamii L. against
5.
A = Candida albicans; B= Acremonium spp
C= Candida parapsilosis; D= Aspergillus versicolor

ix