antioxidant activity of pluchea indica less. extract after in vitro digestion and absorption by caco-2

107 Pages·2015·1.26 MB·English

ANTIOXIDANT ACTIVITY OF PLUCHEA INDICA LESS.
EXTRACT AFTER IN VITRO DIGESTION AND ABSORPTION
BY CACO-2 CELL LINE

KRISADAPORN POLSIRI

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE MASTER DEGREE OF
SCIENCE
IN BIOLOGICAL SCIENCE
FACULTY OF SCIENCE
BURAPHA UNIVERSITY
AUGUST 2015
COPYRIGHT OF BURAPHA UNIVERSITY

ACKNOWLEDGEMENT

In the success of this thesis, I would like to express my sincere gratitude and
deep appreciation to my principal advisor, Dr. Chatchawin Petchlert for support,
attention, motivation, technical assistance, helpful suggestion and comment,
encouragements and support throughout my study.
I would like to thank Dr. Nattida Chotechuang as an external examiner from
Department of Food Technology, Faculty of Science, Chulalongkorn University and
Dr. Salil Chanroj as an examiner from Faculty of Science, for all of their guidance,
valuable advice through this thesis.
Great appreciation is also given to Biological Science Graduate Program,
Department of Biochemistry for laboratory facilities. I would like to thanks Professor
Dr. Nateetip Krishnamra from Department of Physiology, Faculty of Science,
Mahidol University for providing the Caco-2 cells in this research.
I would like to thank Assistant Professor Dr. Klaokwan Srisook for technical
assistance in Cell culture. I would like to thank Dr. Somchart Maenpuen for helpful
suggestion and comment, encouragements and support in this research. I would like to
thank Dr. Panata Wanichwatanadecha for helpful suggestion in this research.
I also would like to thank Burapha University and Faculty of Science for the
financial support financially supported. I would like to thank research grant 2014
from Burapha University (supporting grant from National Research Council).
Finally, a great respect is brought to my parents and my family for their
love, takecaring, attention, encouragement, guidance and supporting throughout my
study. I specially thank to all lecturers of Department of Biochemistry and all of my
friends for their kindness, help, suggestion, encouragement, solidarity, and
togetherness.

Krisadaporn Polsiri
THE RELEVANCE OF THE RESEARCH WORK TO THAILAND

The main purposes of this research are to know the basic knowledge of the
antioxidant activity of Pluchea indica Less. tea extracts both before and after in vitro
digestion including absorption by Caco-2 cell lines. This research project provided
supporting data for the use of the plants and its compound for the well-being of people
with a help of science. This research project provided support the utilization of this tea.
It may be used to consume as a healthy food. Providing the scientific evidence to
justify the use of P. indica leaves in traditional medicine.
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53910187: MAJOR: BIOLOGICAL SCIENCE; M.Sc. (BIOLOGICAL
SCIENCE)
KEYWORDS: PLUCHEA INDICA LESS./ IN VITRO DIGESTION/ DPPH/
REDUCING POWER/ TOTAL PHENOLIC CONTENT/ TOTAL
FLAVONOID CONTENT/ CACO-2 CELL MONOLAYERS
KRISADAPORN POLSIRI: ANTIOXIDANT ACTIVITY OF
PLUCHEA INDICA LESS. EXTRACT AFTER IN VITRO DIGESTION AND
ABSORPTION BY CACO-2 CELL LINE. ADVISORY COMMITTEE:
CHATCHAWIN PETCHLERT, Ph.D. 94 P. 2015.

Pluchea indica Less. is an evergreen large shrub found abundantly in salt
marshes and mangrove swamps in India, Bangladesh, Myanmar, China, the
Philippines, Malaysia, Thailand and Australia. P. indica Less. is a plant with anti-
inflammatory and antioxidant medicinal properties. However, antioxidant activity of
P. indica tea extract (PITE) after in vitro digestion have not ever been studied. This
study aimed to investigate antioxidant activity of PITE both pre and post-in vitro
digestion including absorption by Caco-2 cells. Such activities were performed using
DPPH radical scavenging assay, reducing power, total phenolic and total flavonoid
contents. Pre-digestion of PITE at 0.1 mg/ml showed a strong scavenging activity on
DPPH by 84.77%. When IC was considered to indicate the potential of this activity,
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the value in the intestinal digestion had the lowest value with 0.0187±0.008 mg/ml
when compared to all phases. DPPH radical scavenging activity of PITE has been
studied across Caco-2 cell monolayers. The results showed that PITE might be
accessible via the epithelial cells that resulted in the reduction of percent scavenging
activity in the basolateral side of transwell. However, P. indica tea was still effective
for this activity. Reducing power pre-digestion of PITE was 137.82±0.07 mg QE/g
extract whereas this power after oral and intestinal digestion was not detectable. In
addition, total phenolic and total flavonoid contents of PITE in pre-digestion showed
high amount were 196.56±0.02 and 243.75±0.01 mg QE/g extract, respectively. Total
phenolic content of PITE in post-digestion decreased nearly seven folds. Total
flavonoid content of PITE was attenuated in oral and gastric digestion, but it increased
after intestinal digestion (286.58±0.05 mg QE/g extract). Then total phenolic and total
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flavonoid content of PITE has been studied across Caco-2 monolayers. PITE could
pass through the Caco-2 cells to basolateral side. However, total phenolic and total
flavonoid contents were significantly decreased in the apical and basolateral sides. In
conclusion, P. indica Less. tea may be considered as an alternative healthcare to
consumers.
CONTENTS

Page
ABSTRACT........................................................................................................ iv
CONTENTS........................................................................................................ vi
LIST OF TABLES............................................................................................... x
LIST OF FIGURES............................................................................................. xi
CHAPTER
1. INTRODUCTION..................................................................................... 1
1.1 Objectives...................................................................................... 2
1.2 Contribution to knowledge............................................................ 3
1.3 Scope of the study.......................................................................... 3
2. LITERATURE REVIEWS........................................................................ 4
2.1 Free radicals.................................................................................... 4
2.1.1 Types of free radicals............................................................. 4
2.1.2 Formation of free radicals...................................................... 7
2.1.3 Steps involving free radical generation……………………... 8
2.1.3.1 Initiation..................................................................... 8
2.1.3.2 Propagation................................................................. 9
2.1.3.3 Termination................................................................. 9
2.1.4 Molecular damage induced by free radicals............................ 11
2.1.4.1 Lipids........................................................................... 11
2.1.4.2 Carbohydrates............................................................. 11
2.1.4.3 DNA............................................................................ 11
2.1.4.4 Proteins....................................................................... 12
2.2 Antioxidant..................................................................................... 12
2.2.1 Types of antioxidants.............................................................. 13
2.2.1.1 Enzymatic antioxidants................................................ 13
2.2.1.2 Nonenzymatic antioxidants.......................................... 14
2.3 Determination of antioxidant activity............................................. 20
2.3.1 2, 2-diphenyl- 1-picryl-hydrazyl (DPPH) scavenging assay... 20
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CONTENTS (CONTINUED)

Chapter Page
2.3.2 Reducing power..................................................................... 21
2.3.3 Total phenolic content............................................................ 21
2.3.4 Total flavonoid content........................................................... 22
2.4 Human digestive system................................................................. 22
2.4.1 Component of the digestive system........................................ 23
2.4.1.1 The Mouth ………………........................................... 23
2.4.1.2 The Esophagus.............................................................. 24
2.4.1.3 Stomach........................................................................ 24
2.4.1.4 Small Intestine.............................................................. 25
2.4.1.5 Large Intestine.............................................................. 26
2.4.1.6 Pancreas, Liver, and Gall bladder................................. 27
2.4.1.7 The Rectum................................................................... 28
2.4.1.8 The Anus........................................................................ 28
2.5 Absorption ………………………………………………………... 28
2.6 Caco-2 cell monolayer model.......................................................... 29
2.7 Background of Pluchea indica Less................................................ 31
2.7.1 Properties ……………………................................................ 32
2.7.2 Parts used................................................................................. 32
2.7.3 Medicinal use........................................................................... 32
2.7.4 Studies..................................................................................... 33
3. RESEARCH METHODOLOGY…………............................................... 38
Part I: Preparation of plant extracts....................................................... 38
3.1 Chemicals and equipments............................................................... 38
3.1.1 Chemical................................................................................... 38
3.1.2 Equipments............................................................................... 38
viii
CONTENTS (CONTINUED)

Chapter Page
3.2.1 Preparation of plant extract……………….................................. 38
Part II: Determination of antioxidant activity of Pluchea indica Less. 39
3.1 Chemicals and equipments ………………………………………. 39
3.1.1 Chemical ………...………………………………………….. 39
3.1.2 Equipments.............................................................................. 39
3.2 Method.…………..…………………….…………………………. 39
3.2.1 DPPH radical scavenging assay.............................................. 39
3.2.2 Reducing power assay............................................................. 40
3.2.3 Total phenolic content ………………………………………. 41
3.2.4 Total flavonoid content ……………………………………... 41
3.2.5 Statistical analysis.................................................................... 41
Part III: In vitro digestion………..…………………………………… 42
3.1 Chemicals and equipments.............................................................. 42
3.1.1 Chemical.................................................................................. 42
3.1.2 Equipments.............................................................................. 42
3.2 Method …………………................................................................ 42
3.2.1 In vitro digestion of Pluchea indica Less…………………… 42
Part IV: Antioxidant activity of P. indica Less. tea extract after
treatment with Caco-2 cells.………………………………… 44
3.1 Chemicals and equipments............................................................... 44
3.1.1 Chemical.…………..…………………….………………….. 44
3.1.2 Equipments.............................................................................. 44
3.2. Method............................................................................................ 44
3.2.1 Cell culture ………………………………………………….. 44
3.2.2 Cell counts using a hemacytometer ………………………… 45
3.2.3 Caco-2 cells treatment............................................................. 46
4. RESULTS.................................................................................................. 48
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CONTENTS (CONTINUED)

Chapter Page
4.1 Yield of crude extracts from Pluchea indica Less. leaves………… 48
4.2 Antioxidant activities of P. indica Less. tea extract before and
after in vitro digestion……………………………………………. 48
4.2.1 DPPH radical scavenging assay……………………………. 48
4.2.2 Reducing power assay ……………………………………... 51
4.2.3 Total phenolic content……………………………………… 52
4.2.4 Total flavonoid content…………………………………….. 52
4.3 Absorption of P.indica Less. tea extract after in vitro digestion by
Caco-2 cell 53
4.3.1 DPPH radical scavenging assay…………………………….. 54
4.3.2 Total phenolic content ……………………………………... 55
4.2.4 Total flavonoid content…………………………………….. 57
5. DISCUSSION, CONCLUSIONS AND PERSPECTIVE.……………… 60
REFERENCES…………………………………………………………….. 68
APPENDIX………………………………………………………………… 81
Appendix 1……………………………………………………………. 82
Appendix 2……………………………………………………………. 87
Appendix 3………………………….…………………………………. 91
BIOGRAPHY……………………………………………………………… 94