Evaluation of antibacterial, antioxidant and toxicological activities of crude extracts of Calotropis

59 Pages·2011·13.61 MB·English

II
Evaluation of antibacterial, antioxidant and toxicological activities
of crude extracts of Calotropis gigantean, Mimosa pudica,
Achyranthes aspera and Hibiscus rosa-siensis variety: Kalyani from
Faridpur, Bangladesh
A dissertation submitted to the department of Mathematics and Natural Sciences of
BRAC University
in partial fulfillment of the requirements for the degree of
Master of Science in Biotechnology
Suhmitted hy:
Submitted to:
Nayeema Nushrat Department ofMNS
Registration No. 09376003
BRAC University
Mohakhali, Dhaka
Evaluation of antibacterial, antioxidant and toxicological activities of
crude extracts of Calotropis gigantean, Mimosa pudica, Achyranthes
aspera and Hibiscus rosa-siensis variety: Kalyani from Faridpur,
Bangladesh
A Thesis
By
Nayeema Nushrat
MASTIi:R OF SCffiNCIi:
IN
BIOTECHNOLOGY
BRAC UNIVERSITY
66 MOHAKHALI, DHAKA 1212, BANGLADESH
July, 2011
APPROVAL CERTIFICATE
This thesis entitled "Evaluation of antibacterial, antioxidant and toxicological activities
of crude extracts of Caiotropis gigantean, Mimosa pudica, Achyranthes aspera and
Hibiscus rosa-siensis variety: Kalyani from Faridpur, Bangladesh" is submitted by
Nayeema Nushrat in partial fulfillment of the requirement for the degree of Master of Science
in Biotechnology, Department of Mathematics and Natural Sciences, BRAC University,
Dhaka. This work was performed at Laboratories of the MNS department, BRAC University,
and department of Microhiology, University of Dhaka and the department of pharmacy,
University of Asia Pacific, Dhaka, Bangladesh.
SUPERVISOR SUPERVISOR
N .
~~
Ib·O -=r.u;o I
Dr. Chowdhury Rafiqul Ahsan Dr. N::t;:m Choudhury
Professor Co-ordinator
Department of Microbiology Department of MNS
Dhaka University BRAC University
Dhaka Mohakhali, Dhaka
TTl
DEDICATION
I dedicate this dissertation to my dear parents, for encouraging my curiosity and teaching me
that the key to success is perseverance and hard work.
IV
ACKNOWLEDGEMENTS
All praises are due to the Almighty Allah for his mercy and blessings which enabled the
author for successful completion of this dissertation. The author obtained help from many
individuals and institutions during conducting and completing this research work and as such
she wishes to acknowledge them with gratitude and appreciation.
J am very grateful to Professor Naiyyum Choudhury for providing me with opportunity to
work on this project. I am thankful to Professor Choudhury Rafiqur Ahsan for helping me
understand the logic hehind the suhject and through his research program T learned different
research skills throughout my masters research programme.
I appreciate help and assistance of Mohammad Shahriar, Assistant Professor, University of
Asia Pacific and allowing me to use his lab equipment. I am very thankful to the BRAC
University, University of Dhaka and University of Asia Pacific, Dhaka, Bangladesh for all
out cooperation during the course of my work.
Finally, I would like to express all my love and gratitude to my beloved family for their
support, understanding and encouragement throughout my life, especially my dear parents,
M. Rahmatuzzaman and Khodeza Begum, who are always my source of inspiration.
CONTENTS
DEDTCA TTON ........................................................................................................................ TV
ACKNOWLEDGEMENTS ...................................................................................................... V
CONTENTS ............................................................................................................................ VI
LIST OF FIGURES ................................................................................................................ IX
TJST OF TART.ES .................................................................................................................... x
Abbreviations .......................................................................................................................... XI
ABSTRACT ............................................................................................................................. 12
CHAPTER 1 ........................................................................................................................ 13
INTRODUCTION and LITERATURE REVlEW. .......................................................... J:l
1.1 Plants: A therapeutic goldmine .............................................................................. 13
1.2 Medicinal plants and their applications to healthcare ............................................ 14
1.3 Hypothesis .............................................................................................................. 17
1.4 Justification ofthe study ........................................................................................ 17
1.5 Aims and objectives of study ................................................................................. 18
1.6 Plants Selected in the study .................................................................................... I 8
1.6.1 Taxonomy of the four piants ........................................................................... 19
1.6.2 Habit and distribution of the plants ................................................................. 20
1.6.2.1 Habit and distribution of akonda .............................................................. 20
1.6.2.2 Habit and distribution oflajjaboti lata ..................................................... 21
1.6.2.3 Habit and distribution ofbilai akra/apang ................................................ 21
1.6.2.4 Habit and distribution of holudzoba ......................................................... 2 I
1.6.3 Ethnomedical uses of the screened plants ....................................................... 21
1.7 Antioxidants ........................................................................................................... 22
1.7.1 Categories of antioxidants and their mechanisms of action ............................ 22
1.7.2 Synthetic Antioxidants .................................................................................... 23
"T
1.7.2.1 Butylated Hydroxytoluene (BHn. ........................................................... 24
1.7.2.2 Propyl Gallate ........................................................................................... 24
1. 7.3 Potential sources of natural antioxidants ........................................................ 24
1.7.4 Superiority of natural antioxidants over synthetic .......................................... 25
1.7.5 Plants with Antioxidant Activity ..................................................................... 25
1. 8 Microbial resistance .............................................................................................. 25
1.8.1 Microbial cell targets ...................................................................................... 25
1.8.2 Chromosomal mutations and acquisition of new genes .................................. 26
1.8.3 Microbial mechanisms of resistance ............................................................... 26
1.9 Naturally-occurring antimicrobial substances ....................................................... 26
1.10 Plant secondary metabolites ................................................................................. 27
1.11 Significance of Toxicological Assay ................................................................... 28
CHAPTER 2 ........................................................................................................................ 29
MATERIALS and METHODS ........................................................................................ 29
2.1 Plant materials ........................................................................................................ 29
2.1.1 Collection of plant materials and identification .............................................. 29
2.1.2 Plant storage ....................................................................................................2 9
2.1.3 Preparation of plant materials ......................................................................... 29
2.2 Extraction of plant materials through soxhlet extraction method ..........................2 9
2.2.1 Chemicals .........................................................................................................2 9
2.2.2 Equipment~ ...................................................................................................... 29
2.2.3 Extraction of plant materials with chloroform ................................................ 30
2.2.4 Extraction of plant materials with methanol ................................................... 30
2.3 Assessment of antimicrobial activity of selected plants by disk diffusion meth0d32
2.3.1 Microorganisms and media preparation .......................................................... 32
2.3.2 Inoculum preparation ...................................................................................... 32
2.3.3 Agar disk diffusion .......................................................................................... 33
VII
2.4 Antimicrobial activity of selected plants by agar well diffusion method ............. .33
2.4.1 Microorganisms .............................................................................................. 34
2.4.2 Inoculum preparation ...................................................................................... 34
2.4.3 Agar well Diffusion. ........................................................................................ 34
2.5 Minimum inhibitory concentration determination by broth microdilution ............ 35
2.5.1 Microorganisms ............................................................................................. .35
2.5.2 Inoculum preparation ...................................................................................... 35
2.5.3 Microtiter plate assay ..................................................................................... .35
2.6 Antioxidant activity of methanol extracts of selected plants ................................. 37
2.6.1 Chemicals ........................................................................................................ 37
2.6.2 Determination of antioxidant activity of methanol extracts of selected plants
with the DPPH free radical method ........................................................................ .3 7
2.7 Toxicological Evaluation of Four plants ................................................................ 38
2.7.1 Cell Culture ..................................................................................................... 38
2.7.2 Morphological Study ....................................................................................... 38
CHAPTER 3 ........................................................................................................................ 39
RESULTS ........................................................................................................................ 39
3.1 Results .................................................................................................................... 39
3.1.1 Collection and extraction of plant material... .................................................. 39
3.1.2 Antimicrobial activity ..................................................................................... 39
3.1.3 Radical scavenging activity ............................................................................ .40
3.1.4 Cytotoxic activity ............................................................................................ 40
CHAPTER 4 ........................................................................................................................ 52
Discussion ........................................................................................................................ 52
APPENDIX ............................................................................................................................. IX
VIII
LIST OF FIGURES
Figure 2.1 Schematic Presentation of Process involved in Study ........................................... .31
Figure 2.2: Illustration of the microtitre plate .......................................................................... 36
Figure 3.1 Free radical scavenging activity of the investigated plants .................................... 49
Figure 3.2 Pictures of collected plants ..................................................................................... 50
Figure 3.3 Micrographs of MDCK cultures treated with methanol extracts of different
concentrations .......................................................................................................................... 51
LIST OF TABLES
Table 1.1 Systematic classification (taxonomy) of Akonda .................................................... 19
Table 1.2 Systematic classification (taxonomy) ofLaiiaboti Lata .......................................... 19
Table 1.3 Systematic classification (taxonomy) of Bilai akra ................................................ 20
Table 1.4 Systematic classification (taxonomy) of Holudzoba .............................................. 20
Table 1.5: List of plants screened in the study ......................................................................... 21
Table 1.6 Examples of some disorders and diseases associated with free-radical pathology .. 22
Table 1.7 Mechanisms of antioxidant activity (pokorny et aI., 2001) ..................................... 23
Table 1.8 Some Potential Sources of Natural Antioxidants from Plants ................................. 24
Table 3.1 Plant Collection Record ........................................................................................... 44
Table 3.2 Percent yield of different plant material extraction ................................................ .45
Tahle 3.3 Antimicrohial activity of plant extract~ ohserved in disk diffusion a~say expressed
in terms of zone of inhibition (mm) ........................................................................................ .46
Table 3.4 Antimicrobial activity of plant extracts observed in agar well diffusion assay
expressed in terms of zone of inhibition (mm) ........................................................................ 47
Table 3.5 MlC of methanol extracts of investigated plants .................................................... .48
Table 3.6 Free radical scavenging activity of methanol extracts of investigated plants ......... .48