The antioxidant and cellular effects of Withania somnifera alone and in combination with selenium

140 Pages·2013·7.05 MB·English

Withania somnifera
The antioxidant and cellular effects of
alone and in combination with selenium

Claire Lynne Venter

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Withania somnifera
The antioxidant and cellular effects of
alone and in combination with selenium

by

Claire Lynne Venter

Submitted in fulfilment of the requirements for the degree

MASTER OF SCIENCE

in the

FACULTY OF HEALTH SCIENCES

Department of Anatomy
University of Pretoria

May 2013

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The antioxidant and cellular effects of Withania somnifera alone
and in combination with selenium

by

Claire Lynne Venter

Supervisor: Prof MJ Bester
Department: Anatomy
Degree: MSc Cell Biology

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Abstract

The respiratory system is a target for environmental oxidants from airborne pollution and
other contaminants. Chronic exposure may result in inflammatory conditions such as
allergies and asthma. The ability of the body to counteract oxidation is dependent on the
oxidative status of the individual, and can also benefit from the use of medicinal plant
preparations such as Withania somnifera (WS), which is accredited with antioxidant and anti-
inflammatory properties. Selenium is an important cofactor of antioxidant enzymes such as
glutathione peroxidase. A deficiency of Se will result in decreased antioxidant enzymes
levels and a poor response to oxidative damage. The aim of this study was to determine the
antioxidant content and activity of WS, as well as its in vitro effects with seleno-L-methionine
(Se-Met), the major component of dietary selenium, in a physiologically relevant cell model
system. Little is known about the combinational effects of non-enzymatic and enzymatic
antioxidants i.e., WS in combination with Se-Met, and these effects were also quantified.

The antioxidant content (total phenolic (TPC) and flavonoid content (TFC)) and activity (2,2-
diphenyl-2-picrylhydrazyl (DPPH), trolox equivalent antioxidant capacity (TEAC)) and oxygen
radical absorbance capacity (ORAC) of a water extract of WS, as traditionally prepared and
used, was determined. The intracellular and total protection of WS alone (0-33.33 mg/ml)
and in combination with Se-Met (0-33.33 pg/ml) (range: ½-100x RDA) was evaluated in a
standard cell line, the SC -1 fibroblast cell line and in physiologically relevant primary cultures
of chick embryo lung cells (CELC) using the 2’,7’-dichlorofluorescein diacetate (DCFH-DA)
assay.

Water extracts of WS, as traditionally consumed, were found to possess significant
antioxidant content and activity. For TPC and TFC, WS was found to contain 4.84 g CE/100
g DW, and 355 mg CE/100 g DW respectively. For DPPH, the IC of WSwas 56.34 mg/ml,
50
and the antioxidant activity of WS was 274.8 µmol TE/g. For TEAC, the IC of WSwas 60.3
50
mg/ml, and the antioxidant activity of WS was 2.2 mmol TE/g. Using ORAC, the antioxidant
activity of WS was found to be 15.05 µM TE/g.

WS and Se-Met exhibited both pro-oxidant and antioxidant effects in the DCFH-DA assay
and this may be a function of concentration, as well as combinations of WS and Se-Met
ratios. Data indicates that both WS and Se-Met, alone and in combination, elicit protection
via the intracellular (at all concentrations) and extracellular (at higher concentrations)

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compartment of the SC-1 cell. A synergistic interaction is observed extracellularly between
WS and Se-Met, with mostly additive effects occurring intracellularly.

For CELC, WS and Se-Met alone both demonstrated total protective effects at almost all
concentrations, and intracellular protective effects at higher concentrations. The combination
of WS and Se-Met provided total and intracellular protection to the CELC at the highest
concentration combination. Antagonism between WS and Se-Met was observed at low
concentrations in the total protection assay, with weak synergism and antagonism occurring
at higher concentrations. Synergism between WS and Se-Met was observed at low
concentrations intracellularly, with antagonistic interactions occurring at higher
concentrations.

In conclusion, WS and Se-Met, alone and in combination at non-cytotoxic concentrations,
displayed significant cellular protection against oxidative damage and this was a function of
cell line, concentration and ratios between WS and Se-Met.

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Declaration

I, Claire Venter, declare that the dissertation, which I hereby submit for the degree MSc Cell
Biology at the University of Pretoria, is my own work and has not previously been submitted
by me for a degree at this or any other tertiary institution.

Signed:

Date:

Department of Anatomy, School of Medicine, Faculty of Health Sciences, University of
Pretoria

South Africa

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Acknowledgements

I would like to thank the following people for their contribution to this journey:

Firstly, my supervisor, Prof Megan Bester, for your guidance, motivation and patience. I have
learnt so much from your leadership and abilities.

A special word of thanks to June Serem, who was always happy and willing to help me with
new laboratory techniques and statistics.

Prof Bosman, Liefie, Natalie, Albert, and all my other friends and colleagues in the
Department of Anatomy for your support during this extra-long journey, you all deserve a
medal.

Mom, Dad and Leigh, you have always supported me in all my endeavours, and given me
every opportunity to succeed in life.

To all my friends – you know who you are!

And last, but certainly no t least, my husband Dolf. Thank you for the unwavering emotional
support, love and encoura gement, and for always believing in me (and for all the coffee!)

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Table of contents

Chapter 1: Introduction ...................................................................................................... 1

Chapter 2: Literature review ............................................................................................... 4
Introduction ....................................................................................................................... 4
Oxidative stress ................................................................................................................. 4
Antioxidants ....................................................................................................................... 5
The role of antioxidants in lung disease ............................................................................. 7
Withania somnifera ............................................................................................................ 8
Active biochemical constituents of Withania somnifera .................................................. 9
Non-enzymatic antioxidants: Plant-derived antioxidants related to Withania somnifera ... 12
Selenium ......................................................................................................................... 13
Seleno-methionine ....................................................................................................... 14
The role of selenium in lung disease ............................................................................... 15
Interactions between antioxidants types .......................................................................... 15
Aims ................................................................................................................................ 16
Hypotheses ..................................................................................................................... 17

Chapter 3: Antioxidant c ontent and capacity of Withania somnifera and selenium
alone and in combinatio n ................................................................................................. 18
3.1 Introduction ........................................................................................................... 18
3.2 Materials ............................................................................................................... 19
Preparation of extracts of Withania somnifera .............................................................. 19
Preparation of seleno-methionine ................................................................................ 20
Reagents, equipment and disposable plastic ware ....................................................... 20
Laboratory facilities ...................................................................................................... 20
3.3 Methods ................................................................................................................ 20
Total phenolic content .................................................................................................. 20
Total flavonoid content ................................................................................................. 21
2,2-Diphenyl-1-picrylhydrazyl radical-scavenging assay .............................................. 21
Trolox equivalent antioxidant capacity assay ............................................................... 22
Oxygen radical absorbance capacity assay ................................................................. 22
Data management and statistical analysis ................................................................... 23
3.4 Results and discussion ......................................................................................... 23
Total phenolic content .................................................................................................. 23
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Total flavonoid content ................................................................................................. 26
2,2-Diphenyl-1-picrylhydrazyl radical-scavenging assay .............................................. 27
Trolox equivalent antioxidant capacity assay ............................................................... 34
Oxygen radical absorbance capacity assay ................................................................. 39
3.5 Summary .............................................................................................................. 41

Chapter 4: The effects of Withania somnifera and selenium, alone and in combination,
on the SC-1 permanent cell line ....................................................................................... 42
4.1 Introduction ........................................................................................................... 42
4.2 Materials ............................................................................................................... 43
SC-1 fibroblasts ........................................................................................................... 43
Withania somnifera and seleno-methionine .................................................................. 43
Media, supplements, reagents and plastic ware ........................................................... 44
Laboratory facilities ...................................................................................................... 44
4.3 Methods ................................................................................................................ 44
Cultivation, maintenance and preservation of the SC-1 fibroblast cell line .................... 44
Preparation of Withania somnifera extracts and seleno-methionine solutions .............. 45
The cellular effects of Withania somnifera and seleno-methionine on the SC-1 cell line
.................................................................................................................................... 46
Neutral Red assay: lysosomal membrane integrity ................................................... 46

Crystal Violet assay: cell number .............................................................................. 46

Phase contrast and light microscopy ........................................................................ 47
Oxidative/antioxidant effects of effect of Withania somnifera and seleno-methionine
alone and in combination on the SC-1 cell line ............................................................. 47
Total protective effects ............................................................................................. 47
Withania somnifera ............................................................................................... 48
Seleno-methionine ................................................................................................ 48
Withania somnifera and seleno-methionine .......................................................... 48
Intracellular protective effects ................................................................................... 48
Withania somnifera ............................................................................................... 49
Seleno-methionine ................................................................................................ 49
Withania somnifera and seleno-methionine .......................................................... 49
Interactions between Withania somnifera and seleno-methionine ................................ 50
Data management and statistical analysis ................................................................... 51
4.4 Results and discussion ......................................................................................... 51
Effect of Withania somnifera and seleno-methionine on the SC-1 cell line ................... 51
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Neutral Red and Crystal Violet assays ..................................................................... 51
Phase contrast and light microscopy ........................................................................ 56
Total protective effect of Withania somnifera and seleno-methionine, alone and in
combination, against oxidative damage in the SC-1 cell line ........................................ 59
Intracellular protective effect of Withania somnifera and seleno-methionine, alone and in
combination, against oxidative damage in the SC-1 cell line ........................................ 60
Withania somnifera ................................................................................................... 61
(A) Total protection ............................................................................................... 61
(B) Intracellular protection ..................................................................................... 62
Seleno-methionine ................................................................................................... 65
(A) Total protection ............................................................................................... 65
(B) Intracellular protection ..................................................................................... 65
Withania somnifera and seleno-methionine in combination ...................................... 68
(A) Total protection ............................................................................................... 68
(B) Intracellular protection ..................................................................................... 69
Interactions between Withania somnifera and seleno-methionine ................................ 71
(A) Total protection ................................................................................................... 71
(B) Intracellular protection ........................................................................................ 72
4.5 Summary .............................................................................................................. 73

Chapter 5: The effect of Withania somnifera and selenium, alone and in combination,

on chick embryo lung primary cultures .......................................................................... 75
5.1 Introduction ........................................................................................................... 75
5.2 Materials ............................................................................................................... 76
Chick embryo lung primary cultures ............................................................................. 76
Withania somnifera and seleno-methionine .................................................................. 76
Media, supplements, reagents and plastic ware ........................................................... 76
Laboratory facilities ...................................................................................................... 76
5.3 Methods ................................................................................................................ 77
Establishment of primary chick embryo lung cell cultures ............................................. 77
The cellular effects of Withania somnifera and seleno-methionine on the CELC .......... 77
Oxidative/antioxidant effects of Withania somnifera and seleno-methionine, alone and in
combination, on the CELC ........................................................................................... 78
Interactions between Withania somnifera and seleno-methionine ................................ 78
Data management and statistical analysis ................................................................... 78
5.4 Results and discussion ......................................................................................... 78
Establishment of the chick embryo lung primary culture ............................................... 81
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