identification of bioactive metabolites from endophytic actinomycetes isolated from medicinal plants

302 Pages·2016·8.78 MB·English

IDENTIFICATION OF BIOACTIVE METABOLITES
FROM ENDOPHYTIC ACTINOMYCETES ISOLATED
FROM MEDICINAL PLANTS

Dissertation

Submitted to the Punjab Agricultural University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
in
MICROBIOLOGY
(Minor Subject: Biochemistry)

By

Preeti Saini
(L-2012-BS-76-D)

Department of Microbiology
College of Basic Sciences and Humanities
© PUNJAB AGRICULTURAL UNIVERSITY
LUDHIANA-141 004

2016
CERTIFICATE I
This is to certify that the thesis entitled, “Identification of bioactive metabolites
from endophytic actinomycetes isolated from medicinal plants” submitted for the degree
of Ph.D. in the subject of Microbiology (Minor subject: Biochemistry) of the Punjab
Agricultural University, Ludhiana, is a bonafide research work carried out by Preeti Saini
(L-2012-BS-76-D) under my supervision and that no part of this thesis has been submitted for
any other degree.
The assistance and help received during the course of investigation have been fully
acknowledged.

_______________________
Major Advisor
Dr. (Ms) Madhurama Gangwar
Senior Microbiologist
Punjab Agricultural University
Ludhiana-141004
CERTIFICATE II
This is to certify that the thesis entitled, “Identification of bioactive metabolites
from endophytic actinomycetes isolated from medicinal plants” submitted by Preeti Saini
(L-2012-BS-76-D) to Punjab Agricultural University, Ludhiana, in the partial fulfillment of
the requirements for the degree of the Ph.D. in the subject of Microbiology (Minor subject:
Biochemistry) has been approved by the Student’s Advisory Committee along with the Head
of the Department after an oral examination on the same.

_________________________ ______________________
(Dr. (Ms) Madhurama Gangwar) (Dr. K. Annapurna)
Major Advisor External Examiner
Principal Scientist-cum-Head
Division of Microbiology,
Indian Agricultural Research Institute (IARI)
New Delhi

___________________________
(Dr. (Mrs.) Param Pal Sahota)
Head of the Department

_______________________
(Dr. (Mrs.) Neelam Grewal)
Dean Postgraduate Studies
ACKNOWLEDGEMENTS
This project is one of the most significant accomplishments in my life and it would
not have been possible without people who supported me and believed in my caliber. At the
very onset with folded hands, I bow my head with reverence and dedicatedly accord my
gratitude to the Almighty Lord, the merciful and compassionate, whose grace, glory and
blessings gave me the courage in odd critical times for the successful completion of the
degree.
I would like to extend my gratitude and sincere thank to my honorable supervisor
Dr. Madhurama Gangwar, Senior Microbiologist, Department of Microbiology. She is not
only a great lecturer with deep vision but also most importantly a kind person. I sincerely
thank for her exemplary guidance and encouragement. Her trust and support inspired me in
the most important moments of making right decisions and I am glad to work under her
supervision.
Acknowledgement perhaps is too narrow in scope to express my reverence to Dr.
(Mrs.) Param Pal Sahota, Head, Department of Microbiology, CoBS&H, PAU, Ludhiana
for her indelible help to elucidate my problems, unreserved enthusiasm and providing me
the necessary research facilities for this study.
I owe my sincere gratitude & thankfulness to Dr. (Mrs.) Veena Khanna, Senior
Microbiologist, Department of Plant Breeding and Genetics and Dr. Neerja Sharma,
Senior Biochemist, Department of Plant Breeding and Genetics, for showing sustained
interest and providing help throughout the period of my work. Words fail to express my
thanks to Dr. Narinder Singh, Senior Plant Pathologist, Department of Plant Pathology
and Dr. Shammi Kapoor, Senior Mycologist, Department of Microbiology, for their apt
suggestions and help throughout the study and going through this manuscript.
Special thanks to Dr. (Mrs.) Anu Kalia for her assistance with Scanning Electron
Microscopy. My sincerest thanks are due to Mr. Anoop Patyal for his generous help in LC-
MS/MS work. I am genuinely appreciative of my labmates Sukhjinder and Khushboo for
their suggestions and moral support during my work. The tireless help received from Mr.
Daljeet Singh, Mrs. Leelawati and Mr. Raj Kumar is greatly acknowledged.
The positive and loving feedback received from my abiding friends Ameek, Kusum
and my beloved brother Devender, is something inexpressible. Words at my command are
inadequate in form and spirit to convey the depth of feelings to my revered parents and
grandparents- whose love, affection, moral support, guidance, sacrifices, blessings and
encouragement enabled me to put this tireless venture to a fruitful result.
All may not have been mentioned but none is forgotten.

Place:
Date: (Preeti Saini)
Title of the Dissertation : “Identification of bioactive metabolites from
endophytic actinomycetes isolated from medicinal
plants”
Name of the Student : Preeti Saini
and Admission No. (L-2012-BS-76-D)
Major Subject : Microbiology
Minor Subject : Biochemistry
Name and Designation : Dr. (Ms.) Madhurama Gangwar
of Major Advisor Sr. Microbiologist
Degree to be Awarded : Ph. D.
Year of award of Degree : 2016
Total Pages in Thesis : 228+ Appendices+Vita
Name of University : Punjab Agricultural University, Ludhiana- 141004,
Punjab (India)
ABSTRACT
A total of 144 endophytic actinomycetes were isolated from roots, stems and leaves
of Aegle marmelos (n=51), Murraya koenigii (n=35) and Syzygium cumini (n=58). Another 71
isolates (35 from Azadirachta indica, 36 from Emblica officinalis) were procured from the
Department of Microbiology, PAU. Ethyl acetate extracts of the culture supernatants from
starch casein broth were prepared in vacuo (-130°C). A total of 25.58% extracts diffused the
zone of clearance produced by alpha-amylase on starch agar medium. Out of these, 76.36 and
74.54% extracts inhibited the activity of α-amylase and α-glucosidase respectively. Out of 53
selected extracts, forty-one, forty-four, ten, twenty-four and forty-five extracts displayed
scavenging of diphenyl-picryl-hydrazyl, hydroxyl, nitric, superoxide and linoleate free
radicals respectively. Reduction of Fe3+ to Fe2+ was carried out by 88.67% extracts. Total
phenol content was measured in terms of catechol and gallic acid equivalents/gram extract.
On the basis of above findings, five potential extracts (AZS96, C-3, C-4, J-7 and J-8) were
identified for further study. Their 1000µg/ml concentrations inhibited the diffusion of glucose
across the semipermeable membrane within 24 hours. Michaelis-Menten kinetics indicated
the modes of α-amylase and α-glucosidase inhibition to be pure or near competitive types.
Liquid Chromatography-Mass Spectrometric analysis of C-4P-α-Amy and J-7P-α-Amy extracts
confirmed the presence of phenolic compounds. In vitro inhibition against Staphylococcus
aureus, Aspergillus fumigatus, Candida albicans, Klebsiella sp., Escherichia coli and
Yersinia sp. was displayed by n=10, 4, 3, 3, 2, 1 isolates of S. cumini respectively. Out of
these fifteen, eight different isolates were able to hydrolyze chitin and casein. Six of the
isolates degraded carboxy-methyl-cellulose present in the culture medium. Thirteen, fourteen
and fifteen of the S. cumini isolates exhibited amidase, amylase and lipase activity
respectively. Staphylococcus aureus sample treated with J-17 culture supernatant subjected to
Scanning Electron Microscopy, indicated swelling of cells and disruption of cell wall and cell
membrane. Out of 66 isolates, 57.57% belonged to Streptomyces sp., followed by
Micromonospora sp. (16.66%), Microbispora sp. (10.60%), Actinopolyspora sp. (9.09%),
Saccharopolyspora sp. (4.54%) and Rhodoccocus sp. (1.51%). On the basis of 16S rDNA
sequencing, AZS96 isolate was found to be Rhodococcus qingshengii strain BJC15-A38 and
isolate C-3 showed similarity with Streptomyces koyangensis, strain B025.

Keywords: Antidiabetic, Antioxidant, Endophytic actinomycetes, Liquid Chromatography-
Mass Spectrometry, Medicinal plants.

____________________ ___ _______________________
Signature of Major Advisor Signature of the Student
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CONTENTS
CHAPTER TITLE PAGE NO.
I. INTRODUCTION 1-4
II. REVIEW OF LITERATURE 5-51
III. MATERIALS AND METHODS 52-70
IV. RESULTS AND DISCUSSION 71-183
V. SUMMARY 184-189
REFERENCES 190-228
APPENDICES i-xii
SUBMITTED/ACCEPTED/PUBLISHED
RESEARCH ARTICLES
VITA
LIST OF TABLES
Table Title Page
No. No.
2.1 Bioactive compounds from Azadirachta indica, Aegle marmelos, 18-21
Emblica officinalis, Murraya koenigii and Syzygium cumini
2.2 Important bioactive secondary metabolites from endophytic 22, 23
actinomycetes obtained from medicinal trees
2.3 Diseases related to oxidative stress 31
3.1 Sources of Aegle marmelos, Murraya koenigii and Syzygium cumini tree 52
samples
3.2 Conditions for LC-MS/MS 64
3.3 Antibiotic discs used in antibiotic resistance sensitivity test 69
4.1 Occurrence and distribution of endophytic actinomycetes from Aegle 72
marmelos, Murraya koenigii and Syzygium cumini

4.2 Alpha-amylase percent inhibitory activity of ethyl acetate extracts of 77, 78
endophytic actinomycetes from medicinal trees
4.3 Alpha-glucosidase percent inhibitory activity of ethyl acetate extracts of 82, 83
endophytic actinomycetes from medicinal trees
4.4 Diphenyl picryl hydrazyl radical scavenging activity (%) of ethyl acetate 88, 89
extracts of endophytic actinomycetes from medicinal trees
4.5 Scavenging effects (%) of the ethyl acetate extracts from endophytic 91, 92
actinomycete isolates on hydroxyl radicals
4.6 Scavenging effects (%) of the ethyl acetate extracts from endophytic 95
actinomycete isolates on nitric oxide free radical
4.7 Relative nitrite content of the ethyl acetate extracts at various 97
concentrations
4.8 Percent inhibition of superoxide anions by various actinomycete extracts 100
from medicinal trees
4.9 Beta carotene photobleaching inhibitory potential (%) of ethyl acetate 103, 105
extracts
4.10 Reductive ability of ethyl acetate extracts and butylated hydroxy toluene 109, 110
at different concentrations (100-1000 µg/ml)
4.11 Total phenol content of ethyl acetate extracts of endophytic 114, 115
actinomycetes from medicinal trees
4.12 Relative functionality traits of potential ethyl acetate extracts 117
4.13 Effect of ethyl acetate extracts (100-1000 µg/ml) on the diffusion of 120
glucose out of dialysis membrane upto 24 hr
4.14 Anti-carbohydrate digesting activity of the purified fractions in vitro 122
4.15 Michaelis-Menten constants and maximum velocities of the purified 123
extracts and their respective controls
4.16 LC-MS/MS data representing tentatively identified phytochemicals in 148
purified ethyl acetate extract (C-4P-α-Amy) with retention time, mass,
molecular formulae and reported bioactivity
4.17 LC-MS/MS data representing tentatively identified phytochemicals in 149
purified ethyl acetate extract (J-7P-α-Amy) with retention time, mass,
molecular formulae and reported bioactivity
4.18 Antimicrobial activity of endophytic actinomycetes isolated from S. 150
cumini expressed as mean of inhibition factor (MIF) and percent
inhibition
4.19 Proteolytic and chitinolytic potentials of the actinomycete isolates 152
4.20 Enzyme production potential of the actinomycete isolates 155
4.21 Number of endophytic actinomycetes showing various bioactive 159
potentials
4.22 Antibiotic sensitivity spectra of various endophytic actinomycetes 161, 162
obtained from medicinal trees
4.23 Occurrence of endophytic actinomycetes in medicinal trees 167
4.24 Morphological and biochemical characterization of endophytic 168, 172
actinomycetes isolated from five medicinal trees
4.25 Sequence producing significant alignments (AZS96) 180
4.26 Alignment view using combination of NCBI GenBank (C-3) 183
LIST OF FIGURES
Figure Title Page
No. No.
2.1 Five native medicinal trees traditionally used in Indian systems of medicine 7
2.2 Bioactive compounds obtained from five selected traditional medicinal trees of 15
India
2.3 Bioactive compounds reported from endophytic actinobacteria associated with 17
medicinal trees
2.4 Reaction taking place during the scavenging of DPPH free radical 33
2.5 Hydroxyl radical scavenging mechanism 35
2.6 Nitric oxide radical scavenging mechanism 37
2.7 Superoxide radical scavenging mechanism 38
2.8 Steps during β-carotene photobleaching in the presence of linoleate radical 40
3.1 Preparation of the ethyl acetate extract 54
3.2 Effects of C-3 ethyl acetate extract on in vitro inhibitory glucose diffusion at 59
different concentrations (100, 250, 500, 750 and 1000 µg/ml)
3.3 Preparation of the extracts for silica gel column chromatography 60
4.1 Isolation and purification of endophytic actinomycetes 73
4.2 Alpha amylase inhibitory activities of few potential ethyl acetate extracts in 76
relation to commercial drug Acarbose
4.3 Alpha glucosidase inhibitory activity of few potential ethyl acetate extracts in 81
relation to commercial drug Voglibose
4.4 Diphenyl picryl hydrazyl radical scavenging effects exhibited by certain ethyl 86
acetate extracts in relation to vitamin C
4.5 Hydroxyl radical scavenging effects exhibited by some of the ethyl acetate 93
extracts in relation to vitamin C
4.6 Nitric oxide scavenging effects exhibited by some of the ethyl acetate extracts in 96
relation to vitamin C
4.7 Superoxide radical scavenging effects exhibited by potential extracts in relation 99
to vitamin C
4.8 Antioxidant potential of some ethyl acetate extracts in relation to vitamin C 104
4.9 Reducing power exhibited by potential extracts in relation to butylated hydroxy 108
toluene (BHT)
4.10 Relative content of catechol and gallic-type phenolic compounds in ethyl acetate 112
extracts
4.11 Determination of antioxidant properties and phytochemical composition of the 116
endophytic actinomycete extracts