1. The anti-cancer and antioxidant properties of organic and aqueous
extracts of various types of tea.
AD van Eyk, N Dahan-Farkas, B Mokgetle, A Moussana, P Maloba, S Sibanda
Division of Pharmacology, Department of Pharmacy and Pharmacology, Faculty of Health Sciences,
Johannesburg, South Africa
Introduction
Materials & Methods
Results
Conclusions
Objectives
Results
Tea is one of the most common beverages consumed world
wide. The top leaves and buds of the plant Camellia sinensis
are used for the preparation of black, green, oolong and white
tea. Different processing procedures of the leaves and buds
results in the four different teas from this plant.1,2 The leaves
of Aspalathus linearis (rooibos plant) are harvested and dried
for use as rooibos tea, while the dried flowers of either
German chamomile (Matricaris retutica) or Roman chamomile
(Chamaemelum nobile) are utilised for chamomile tea. Ginger
tea is prepared from the dried rhizomes of the ginger plant
(Figure 1).3-7
Both normal tea as well as herbal tea are associated with
health benefits including protection against various cancers,
cardiovascular diseases and neurodegenerative diseases.
Other health benefits include anti-tumour, anti-diabetic, anti-
inflammatory, antiviral, antibacterial and anti-ageing
properties. The health benefits of the various teas are related
to the polyphenolic compounds present in tea. These
compounds are responsible for the high antioxidant activities
related to various types of tea preparations. Catechins are the
major polyphenols present in green tea, while thearubigins
and theaflavins are present in black tea. Apigenin is the major
active compound in chamomile tea, while gingerols and
zerumbone in ginger tea and aspalathin in rooibos tea are the
major active constituents.1-7
The aim of this study is thus to compare the antioxidant
activities as well as the anticancer effects against the Caco-2
human colon cancer cell line of aqueous and organic extracts
of green, black, chamomile, ginger and rooibos tea so as to
identify which tea extracts have the highest activities.
1. To compare the antioxidant activities of aqueous and
organic extracts of two brands of each of green, black,
rooibos, chamomile and ginger tea bag contents by using
the DPPH assay.
2. To compare the anti-proliferative effects as well as
alterations of cell morphology caused by abovementioned
tea bag extracts against the Caco-2 human colon cancer
cell line by using the MTT cell viability assay and phase
contrast microscopy, respectively.
3. To compare the preliminary phytochemical screening and
HPLC chemical profiles of abovementioned tea bag extracts
by using standard chemical tests and HPLC analysis,
respectively.
4. To compare the total phenolic and flavonoid content of the
abovementioned extracts by using standard colorimetric
assays.
Dry yields for extracts ranged from 5.3-48%, with the aqueous
extracts yielding mostly higher amounts. Preliminary
phytochemical screening indicated the predominant presence
of flavonoids and terpenoids in most extracts. HPLC chemical
profiles indicated many compounds present in the crude
extracts. Rooibos extracts indicated the highest antioxidant
activity with the organic extracts of all the teas showing
inhibition of Caco-2 cell growth as: Chamomile tea>Rooibos
tea>Green tea>Ginger tea>Black tea. Changes in morphology
and cell growth inhibition were also observed using phase
contrast microscopy. Total phenolic content (GAE) was higher
in black and green tea, while total flavonoid content was the
lowest in green tea.
References
Acknowledgements
The authors would like to thank the University of the Witwatersrand Faculty Research Committee for financial
support.
Zingiber officinale
(Ginger plant)
Preparation of plant extracts
Tea bag contents (2.4g) from two different brands of each of
the following teas (green: 5 Roses and Lipton; black: 5
Roses and Pick ’n Pay; rooibos (5 Roses and Pick ’n Pay;
ginger and chamomile: EVE) were extracted with 200ml
either boiling water or dichloromethane:methanol (1:1) for
15min at room temperature while stirring. After filtration, the
extracts were dried, weighed and solubilised in methanol
(organic) and water (aqueous) to 10mg/ml. The extracts were
stored in dark containers at 4oC after filtration through
0.45µm syringe filters.
Camellia sinensis
(Tea plant)
Aspalathus linearis
(Rooibos plant)
Matricaris retutica/
Chamaemelum nobile
(chamomile plant)
MTT cell viability assay and cell morphology
Cell viability was determined for each extract using the
standard MTT cell viability assay containing 2x PBS
washing steps before MTT addition. Initial screening at
1000µg/ml was performed. Controls: negative control:
untreated cells, positive control: camptothecin (100µM),
blank: DMSO. Morphology was observed under a phase-
contrast microscope at 10x magnification.10
HPLC chemical profiles
Chemical profiling was performed on both the aqueous and
organic tea extracts utilising reverse-phase High
Performance Liquid Chromatography (RP-HPLC). A binary
Flexar Perkin Elmer HPLC system (UV-Vis detector) was
used for all analyses. Running conditions: Brownlee
Spheri-5 RP-18, 5 µ, 100 x 4.6 mm, 10 µl, 1 ml/min, 25 °C,
260 nm. A linear gradient was used consisting of A
(methanol) and B (0.1% (v/v) formic acid in water). The
gradient consisted of the following steps: 0–10 min, 1–5%
A; 10–15 min, 5–15% A; 15–20 min, 15–20% A; and 20–40
min, 20–60% A. Rutin and Gallic acid standards were used
to construct standard curves to determine the content of
these compounds in the extracts.
Total phenolic and flavonoid content
Total phenolic content was estimated (as gallic acid
equivalent units mg/g, standard curve: 10 - 500µg/ml) using
the Folin-Ciocalteu reagent with absorbance read at 690nm.
Total flavonoid content was estimated (as rutin equivalent
units mg/g, standard curve: 10 - 500µg/ml) using the assay
involving flavonoid-aluminium complex formation with the
absorbance read at 450nm.11
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Table 1. Yield, % antioxidant activity and % cell inhibition results.
Figure 3. HPLC Chromatograms of the different tea extracts (5 mg/ml) at
260 nm. G: Gallic acid, RT 6.6 min; R: Rutin, RT 34.6 min.
Figure 1. Plants used for the preparation of tea.
DPPH antioxidant assay
Antioxidant activity of each extract was determined using
the standard DPPH assay. After initial screening at
1000µg/ml, IC50 values were determined using extracts in
the range of 10 – 1000µg/ml. Controls : negative control:
DPPH 100%, colour controls: all assay ingredients except
DPPH, positive control: ascorbic acid (1000ug/ml), blank:
methanol.8
Phytochemicals Tea Extracts
Green tea Black tea Rooibos tea Ginger tea Chamomile tea
5 Roses Lipton 5 Roses PnP 5 Roses PnP EVE EVE
O A O O A O A O A O A O A O A
Tannins +++ - +++ - +++ +++ +++ +++ + + + + - + - ++
Saponins + - + - - +++ ++ +++ +/- +/- +/- +/- - + - +
Flavonoids +++ + +++ + +++ +++ +++ +++ ++ + ++ + + ++ ++ +++
Glycosides - - - - - - - - - + - + +/- ++ + ++
Terpenoids +++ ++++ +++ ++++ ++ +++ +++ ++ - + - + ++++ +++ ++ ++
Alkaloids - - - - ++ - - - + + + + +++ - - -
Anthraquinones + - + - - +++ - +++ + + ++ + + - + ++
Coumarans - ++ - ++ +++ + +++ - + - ++ - ++ - + -
Green Tea
5Roses
Aqueous
Lipton
Organic
Lipton
Aqueous
Black Tea
5Roses
Organic
5Roses
Aqueous
P’nP
Organic
P’nP
Aqueous
Rooibos Tea
5Roses
Organic
5Roses
Aqueous
P’nP
Organic
P’nP
Aqueous
Ginger Tea
EVE
Organic
EVE
Aqueous
Chamomile Tea
EVE
Organic
EVE
Aqueous
A
Table 2. Preliminary phytochemical screening results.
Preliminary phytochemical analysis
Phytochemical screening was performed on both organic
and aqueous tea extracts using standard methods to test for
alkaloids, anthraquinones, coumarins, flavonoids, tannins,
saponins and sterols.9
Untreated Camptothecin
Figure 2. Morphology of Caco-2 cells after 72 hours treatment with
1000µg/ml tea extract. A: Black tea; B: Green tea; C: Rooibos tea; D:
Ginger tea; E: Chamomile tea. Org: Organic extract; Aqu: Aqueous
extract.
A
B
C
D
Org 5Roses Org Pick’nPay Aqu 5Roses Aqu Pick’nPay
Org 5Roses Org Lipton Aqu 5Roses Aqu Lipton
Org 5Roses Org Pick’nPay Aqu 5Roses Aqu Pick’nPay
Org EVE E D EOrg EVE Aqu EVE Aqu EVE
A