Apoptotic Effects of Hibiscus Tea Extract on HeLa Cells

R. Mozal, A. Stieve, K. Wilhoite

Butler University, Indianapolis, Indiana
Abstract Results
The purpose of this study was to investigate the anti-cancer effects of hibiscus leaf
extract and sigma tea extract with a positive control of colchicine and negative control
of DMSO on HeLa cells. Two methods of measurement were used, trypan blue staining
to determine viable and non-viable HeLa cells as well as immunofluorescence to
determine apoptotic and non-apoptotic nuclei of HeLa cells. Cells were counted by hand
across multiple Butler University Students and data was pooled. We had contradictory
data with our methods of measurement which resulted in inconclusive results about the
effects of hibiscus leaf extract on HeLa cells.

Introduction
● Chakrabarty (2011) reported that sigma black tea is rich in a polyphenol, theaflavin,
that depolymerizes microtubules in HeLa cancer cells.
● Hibiscus leaf extract is rich in polyphenols including catechin and ellagic acid which
are related to signal transduction regulation. (Lin et al., 2011)
● These extrinsic death signals prepare the cell the cell for suicide, apoptosis.
(Ashkenazi & Dixit, 1998)
● These properties of hibiscus tea extract could potentially prevent the growth and
division of HeLa cancer cells similarly to sigma black tea.
● A major hibiscus tea polyphenol epigallocatechin-3-gallate (EGCG) suppresses cell
proliferation blocking the cell cycle at the G1 phase. (Lin et al., 1999)
● There are no direct reports about the effects of hibiscus leaf extract on cytoskeletal
protein in cervical carcinoma cells.
● Based on these results, we hypothesized that introducing HeLa cells to hibiscus leaf
extract in vitro it will induce apoptosis through AIF with the activation of p53,
which suppresses the growth of tumor cells.
Figure 1. Treated HeLa Cells stained with DAPI under Fluorescence. Cultures of HeLa cells were
treated with 4 solutions and stained with DAPI for fluorescence microscopy. Cells were counted based on
apoptotic and non apoptotic characteristics. The error bars represent a standard deviation between the
means of the data ±1.6. A t-test was conducted between DMSO control and hibiscus leaf treatment, a
p-value of 0.5 was obtained to show no statistical significance with the anticancer effects of hibiscus tea.
A. B. C.
Figure 2. Trypan Blue Stain on HeLa Cells. Cultures of HeLa cells were treated with 4 solutions and
stained with trypan blue. White cells were counted as viable and blue cells were not viable. The error bars
represent a standard deviation between the means, of ±6.29. A t-test was conducted between the DMSO
control and our hibiscus leaf treatment, a p-value of 0.022 was obtained showing a statistically significant
difference between the viability of DMSO and hibiscus treated cells.
Figure 3. Non-Apoptotic HeLa Cells Under Fluorescence
Microscopy Three slides of HeLa cells were stained with DAPI
(A), green fluorescence (B), and phalloidin respectively (C).
These cells were all treated with our negative control DMSO.
Figure 4. Apoptotic HeLa Cells Under Fluorescence
Microscopy These slides of HeLa cells were stained with DAPI
(D), green fluorescence (E), and phalloidin (F) respectively.
These cells were all treated with our negative control colchicine.

D. E. F.

Materials and Methods Discussion References

● Prepare the solutions of DMSO , colchicine, sigma tea extract, and hibiscus extract.
● Place 3mL of DMEM to a 15mL conical tube for each of the treatments, then add the
treatments and mix
● Dispense 1.5 mL of treatment into the corresponding wells, 2 wells receiving each
treatment
● Store the plates in the incubator until lab period
● Harvest the cells
● Remove 100uL of each treatment and place it in a corresponding 1.5mL
microcentrifuge tube
● Add 100uL of trypan blue to each tube and mix
● Prepare the hemocytometers with 10uL of the mixtures
● Count the number of viable and non-viable cells
● Do the calculations to determine percent viability
● Figure 1 shows count of normal vs apoptotic cells, it has a p-value of 0.05, it is not statistically
1. Ali, Özmen. “Cytotoxicity of Hibiscus Rosa-Sinensis Flower Extract.” Caryologia, vol.
significant meaning we should reject the null-hypothesis, that HTE would induce apoptosis.
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● Figure 6 shows viability of Hela cells in different solutions, a p-value of 0.022, it is statistically
2. Lin, Hui-Hsuan, et al. “Hibiscus Sabdariffa Leaf Induces Apoptosis of Human Prostate
significant meaning that we should accept the null-hypothesis, that HTE would induce apoptosis.
Cancer Cells in Vitro and in Vivo.” Elsevier Food Chemistry, vol. 132, no. 2, 30 Aug.
● With the countering results we can conclude that further testing needs to be done to see if the
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HTE would induce apoptosis in cells. Since the current results are inconclusive we will reject the
3. Chakrabarty, Subhendu, et al. “Theaflavins Depolymerize Microtubule Network through
null hypothesis, that HTE induces apoptosis in HeLa cells.
Tubulin Binding and Cause Apoptosis of Cervical Carcinoma HeLa Cells.” Journal of
● Since the hypothesis was rejected if we look at similar experiments we find that the apoptosis
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induced by the hibiscus tea extract was seen in breast cancer cell tissue (Nguyen, Hibiscus)
doi:10.1021/jf104231b.
where it induced apoptosis in cells using high concentrations of hibiscus extract.
4. Lin, Jen-Kun, et al. “Cancer Chemoprevention by Tea Polyphenols through Mitotic Signal
● Another study done showed that hibiscus leaf extract helped to inhibit prostate cancer cell
Transduction Blockade.” Biochemical Pharmacology, vol. 58, no. 6, 1999, pp. 911–915.
growth(Chiu et al.). This means that there could be further trials to try to see if there could be
5. Ashkenazi, Avi, and Vishva M Dixit. “Death Receptors: Signaling and Modulation .”
things that need change or altering to help prove the null-hypothesis true.
American Association for the Advancement of Science, vol. 281, no. 5, 28 Aug. 1998, pp.
● If we were do another experiment using higher amounts of the hibiscus tea extract combined
1305–1308.
with a factor that will increase the effectiveness of hibiscus tea extract to induce apoptosis in
6. Nguyen, Christopher, et al. “Hibiscus Flower Extract Selectively Induces Apoptosis in
cells, like taxol or cisplatin (Nguyen, Hibiscus ).
Breast Cancer Cells and Positively Interacts with Common Chemotherapeutics.” BMC
● An alternative experiment to try out is if it works well with certain types of cancer cells. It was
Complementary Medicine and Therapies, BioMed Central, 6 May 2019,
shown that it helped prevent the further growth of prostate cells (Chiu et al.) we could try and
bmccomplementmedtherapies.biomedcentral.com/articles/10.1186/s12906-019-2505-9#ci
test the effectiveness of the extract in inducing apoptosis or inhibiting cell growth in different
teas.
types of cancer cells, then compare the results and use the information to find similarities.