IN VITRO SCREENING OF CYTOTOXIC EFFECT AND ANTIOXIDANT ACTIVITY OF Pereskia bleo ...

115 Pages·2013·1.66 MB·English

IN VITRO SCREENING OF CYTOTOXIC EFFECT AND
ANTIOXIDANT ACTIVITY OF Pereskia bleo AND Centella asiatica
CRUDE EXTRACTS

By
VIMALAN RENGGANATEN

A project report submitted to the Department of Biomedical Science
Faculty of Science
Universiti Tunku Abdul Rahman
In partial fulfillment of the requirements for the degree of
Bachelor of Science (Hons) Biomedical Science
May 2013
ABSTRACT

IN VITRO SCREENING OF CYTOTOXIC EFFECT AND
ANTIOXIDANT ACTIVITY OF Pereskia bleo AND Centella asiatica
CRUDE EXTRACTS

Vimalan Rengganaten

Pereskia bleo from the family of Cactaceae and Centella asiatica from the
family of Umbelliferae are two well known medicinal plants. These plants are
often used traditionally as anti-inflammatory, antioxidant and anti-rheumatic.
In this study, the cytotoxic effect and antioxidant activity of these plants were
evaluated. Possible synergistic interaction between these two plants was
studied at ratio 1:1, 3:7 and 7:3. The whole plants were extracted using cold
solvent extraction to produce ethanolic and hexane crude extracts. The
cytotoxic effect of the crude extracts was measured using MTT assay. The
ethanolic and hexane crude extracts of Pereskia bleo and Centella asiatica
possessed high cytotoxic effect against K-562 cells upon 48 hours of treatment.
The Pereskia bleo crude yielded IC values of 21.5 µg/mL and 32.5 µg/mL,
50
and the Centella asiatica crude yielded IC values of 30.0 µg/mL and 32.0
50
µg/mL for ethanolic and hexane extracts, respectively. The highest cytotoxic
effect was observed in the mixture ratio of 3:7 of hexane crude extracts with
IC value of 21.0 µg/mL. This suggests that possible synergism activity may
50
exist at this ratio between these two plant samples. The antioxidant activity
was measured using DPPH assay. The highest antioxidant activity was
observed in the ethanolic crude extract of Pereskia bleo, with IC value of
50
475 µg/mL. Lower antioxidant activity was yielded among the mixture ratio of
crude extracts, suggesting a possible antagonism interaction. The crude
extracts of Pereskia bleo and Centella asiatica could be potential cytotoxic
and antioxidant agents. The possible synergistic interaction could exist
between the plant samples. Further studies should investigate the synergistic
interaction using purified compounds from Pereskia bleo and Centella
asiatica.

ii
ACKNOWLEDGEMENTS

This project would not been possible without the help and guidance of all the
people around me. I would like to take this opportunity and express my
deepest gratitude to my supervisor, Ms Sangeetha Arullappan. With her
excellent guidance, knowledge and patience, I successfully overcame many
obstacles and learned a lot. Her understanding, advices and encouragement
made my journey of completion of this project much smoother. Thank you for
everything you have done for us.

Next, I would like to thank my family for their endless moral support and
understanding. Knowing the importance of this project, they encouraged me to
never give up whenever things turn bad. Thank you Ma, my sisters and my
brother for being there for me throughout this project.

Next I would like to extend my gratitude to my team members, Cheng Hui
Yan, Kausalyaa Darmaseelan, Ng Lee Ping, Pavethra Iyer, Tan Hui Hua and
Uthaya Kumar for all their supports, encouragement, patience and team work.
They shared their knowledge and had each other’s back. Thank you for the
memories and the experience. Lastly, I would like to thank all my friends who
have helped me with this project. Nic Lee Wei Quan, Khor Foong Vai, Ng
Wei Wen, Thachayani, Wong Mei Yan and many more, they have been giving
moral support and encouragement throughout this project. Thank you for your
understanding and your support.
iii
DECLARATION

I hereby declare that the project report is based on my original work except for
quotations and citations which have been duly acknowledged. I also declare
that it has not been previously or concurrently submitted for any other degree
at UTAR or other institutions.

_________________________________
VIMALAN RENGGANATEN

iv
APPROVAL SHEET

This project report entitled “IN VITRO SCREENING OF CYTOTOXIC
EFFECT AND ANTIOXIDANT ACTIVITY OF Pereskia bleo AND
Centella asiatica CRUDE EXTRACTS” was prepared by VIMALAN
RENGGANATEN and submitted as partial fulfillment of the requirements for
degree of Bachelor of Science (Hons) in Biomedical Science at Universiti
Tunku Abdul Rahman.

Approved by:

_______________________
(Ms SANGEETHA ARULLAPPAN) Date: ………………………….
Supervisor
Department of Biomedical Science
Faculty of Science
Universiti Tunku Abdul Rahman
v
FACULTY OF SCIENCE
UNIVERSITI TUNKU ABDUL RAHMAN

Date: ____________________

PERMISSION SHEET

It is hereby certified that VIMALAN RENGGANATEN (ID No:
09ADB07208) has completed this final year project entitled “IN VITRO
SCREENING OF CYTOTOXIC EFFECT AND ANTIOXIDANT
ACTIVITY OF Pereskia bleo AND Centella asiatica CRUDE EXTRACTS”
supervised by Ms. Sangeetha Arullappan from the Department of Biomedical
Science, Faculty of Science.

I hereby give permission to my supervisor to write and prepare manuscripts of
these research findings for publishing in any form, if I do not prepare it within
six (6) months from this date, provided that my name is included as one of the
authors for this article. The arrangement of the name depends on my
supervisor.

Yours truly,

_______________________________________
(VIMALAN RENGGANATEN)
vi
TABLE OF CONTENTS

PAGE

ABSTRACT i
ACKNOWLEDGEMENTS iii
DECLARATION iv
APPROVAL SHEET v
PERMISSION SHEET vi
TABLE OF CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xii
LIST OF ABBREVIATIONS xiv

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 4
2.1 Pereskia bleo 4
2.1.1 General Description 4
2.1.2 Bioactive Compounds 6
2.1.3 Previous Investigations 8

2.2 Centella asiatica 10
2.2.1 General Description 10
2.2.2 Bioactive Compounds 12
2.2.3 Previous Investigations 13

2.3 Cancer 15

2.4 Cytotoxic Assays 16

2.5 Antioxidant Assays 18

2.6 Synergistic Interaction 19

3 MATERIALS AND METHODS 21
3.1 Materials 21
3.1.1 Plant Materials 21
vii
3.1.2 Cancer Cell Line 21
3.1.3 Chemicals and Solvents 22
3.1.4 Equipments and Apparatus 23

3.2 Methodology 24
3.2.1 Preparation of Plant Materials 24
3.2.2 Plant Extraction 24
3.2.3 Preparation of Plant Stocks 25
3.2.4 Preparation of Complete Medium 26
3.2.5 Preparation of Positive and Negative 26
Controls
3.2.6 Preparation of Reagents for Bioassays 27
3.2.7 Preparation of Freezing Medium 27
3.2.8 Culture and Subculture of K-562 Cells 28
3.2.9 Cryopreservation of K-562 Cells 29

3.3 Cell Quantification 30

3.4 Bioassays 30
3.4.1 MTT Assay 30
3.4.2 DPPH Assay 32

3.5 Data Analysis 32

4 RESULTS 33
4.1 Percentage Yield of Pereskia bleo and Centella 33
asiatica Extracts

4.2 MTT Assay 36
4.2.1 K-562 Cancer Cells 36
4.2.2 Positive Controls 48

4.3 DPPH Assay 51
4.3.1 Positive Control 60

5 DISCUSSION 63
5.1 Plant Extraction 63

5.2 Bioassay Results 68
5.2.1 Cytotoxic Assay 68
5.2.2 DPPH Assay 76
viii
5.3 Study Limitations 80

5.4 Future Studies 81

6 CONCLUSIONS 84

REFERENCES 85

APPENDICES 94

ix