In Vitro Chemopreventive Properties of Green Tea, Rooibos and Honeybush Extracts in Skin Cells
Abstract
:1. Introduction
2. Results
2.1. Effect of Green Tea and Herbal Tea Extracts on Cell Proliferation
2.2. Induction of Pro-Apoptotic Caspase-3 Activity
2.2.1. Relationship between Pro-Apoptotic Effect and Cell Viability
2.2.2. Morphological Alteration in Normal Cells Associated with Induction of Apoptosis
2.2.3. Characterisation of Rooibos-Induced Apoptosis in Normal Cells by Flow Cytometry
2.3. Differences in Polyphenol Content and Specific Ratios between Methanol and Aqueous Extracts
3. Discussion
4. Materials and Methods
4.1. Reagents and Assay Kits
4.2. Preparation of Extracts and Polyphenols Analyses
4.3. Cell Culture
4.4. Modulation of Cell Proliferation and Apoptosis
4.4.1. Cell Proliferation Assay
4.4.2. Cell Viability and Apoptosis Assays
4.4.3. Hoechst Stain
4.5. Characterization of Pro-Apoptotic Activity of Rooibos by Flow Cytometry
4.5.1. Caspase-3 Activity
4.5.2. Membrane Depolarisation
4.5.3. DNA Fragmentation
4.6. Statistical Analysis
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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- Sample Availability: Samples of the compounds are not available from the authors.
Cell Type | Extract | Green Tea and Herbal Teas (mg/mL) | |||
---|---|---|---|---|---|
C. sinensis | A. linearis | C. intermedia | C. subternata | ||
Premalignant cells | MeOH | 0.035 ± 0.003 cA * | 0.037 ± 0.005 cA | 0.350 ± 0.077 aA | 0.190 ± 0.019 bA |
Aq | 0.045 ± 0.007 cB * | 0.068 ± 0.011 cB | 0.202 ± 0.037 aB | 0.164 ± 0.039 bA | |
Normal cells | MeOH | 0.063 ± 0.009 cA | 0.058 ± 0.014 cA | 0.151 ± 0.013 bA | 0.200±0.025 aA |
Aq | 0.154 ± 0.021 bB | 0.208 ± 0.038 aB | 0.091 ± 0.022 cB | 0.098 ± 0.019 cB | |
Cancer cells | MeOH | 0.035 ± 0.012 cA | 0.016 ± 0.003 dA * | 0.150 ± 0.046 bA | 0.223 ± 0.030 aA |
Aq | 0.124 ± 0.025 aB | 0.048 ± 0.022 bB | 0.143 ± 0.037 aB | 0.158 ± 0.039 aB |
Tea/Herbal Tea | Cell Type | Units | Methanol Extract | Aqueous Extract | ||||
---|---|---|---|---|---|---|---|---|
C. sinensis | Premalignant cells | Extract (mg/mL) | 0.080 | 0.050 | 0.035 | 0.170 | 0.114 | 0.038 |
Caspase-3_Fold | 5.75 ± 1.26 a | 3.54 ± 1.00 a | 1.14 ± 0.22 c,* | 6.88 ± 1.65 a | 4.94 ± 2.43 a,b | 1.57 ± 0.76 c,* | ||
% ATP production | 36.12 ± 6.90 c | 49.08 ± 12.55a | 79.19 ± 7.79a | 47.49 ± 5.88 c | 67.63 ± 3.81b | 83.67 ± 8.18 a | ||
Normal cells | Extract (mg/mL) | 0.230 | 0.154 | 0.051 | 0.340 | 0.228 | 0.075 | |
Caspase-3_Fold | 4.33 ± 0.68 a | 1.98 ± 0.36 b | 1.22 ± 0.18 c,* | 5.58 ± 2.01 a | 3.20 ± 1.47 b | 1.07 ± 0.15 c,* | ||
% ATP production | 28.58 ± 4.22 c | 50.11 ± 1.50 b | 82.37 ± 5.04 a | 42.69 ± 14.23 c | 60.64 ± 15.10 b | 95.57 ± 26.22 a | ||
Cancer cells | Extract (mg/mL) | 0.210 | 0.141 | 0.047 | 0.410 | 0.275 | 0.091 | |
Caspase-3_Fold | 2.13 ± 0.25 a | 1.79 ± 0.20 a | 1.26 ± 0.37 b,* | 1.55 ± 0.22 a | 1.35 ± 0.12 a | 1.13 ± 0.17 b,* | ||
% ATP production | 62.27 ± 8.70 c | 75.41 ± 7.10 b | 96.70 ± 8.59 a | 58.92 ± 4.58 c | 73.75 ± 6.09 b | 88.35 ± 7.24 a | ||
A. linearis | Premalignant cells | Extract (mg/mL) | 0.130 | 0.087 | 0.029 | 0.140 | 0.094 | 0.031 |
Caspase-3_Fold | 4.67 ± 0.63 a, | 3.06 ± 0.28 a | 1.32 ± 0.40 b,* | 4.89 ± 0.78 a | 2.33 ± 0.59 b | 1.24 ± 0.25 c,* | ||
% ATP production | 41.74 ± 5.09 c | 68.12 ± 3.27 b | 83.04 ± 4.51 a | 50.41 ± 5.35 b | 69.76 ± 7.86 b | 76.36 ± 6.61 a | ||
Normal cells | Extract (mg/mL) | 0.260 | 0.174 | 0.058 | 0.290 | 0.194 | 0.060 | |
Caspase-3_Fold | 2.91 ± 0.29 a | 2.46 ± 0.30 a | 1.57 ± 0.21 b,* | 1.80 ± 0.22 a | 1.71 ± 0.33 a | 1.58 ± 0.44 a,* | ||
% ATP production | 38.68±5.91 b | 54.29 ± 9.58 b | 89.76 ± 10.73 a | 41.09 ± 6.74 b | 54.11 ± 6.55 b | 78.65 ± 15.47 a | ||
Cancer cells | Extract (mg/mL) | 0.310 | 0.163 | 0.016 | 0.260 | 0.154 | 0.048 | |
Caspase-3_Fold | 1.98 ± 0.17 a | 1.56 ± 0.19 b | 1.30 ± 0.24 b,* | 2.22 ± 0.25 a | 1.86 ± 0.26 a,b | 1.46 ± 0.39 b,* | ||
% ATP production | 54.53 ± 6.51 c | 72.00 ± 5.23 b | 90.60 ± 15.84 a | 51.72 ± 2.39 c | 63.19 ± 5.56 b | 87.05 ± 6.65 a |
Herbal tea | Cell Type | Units | Methanol Extract | Aqueous Extract | ||||
---|---|---|---|---|---|---|---|---|
C. intermedia | Premalignant cells | Extract (mg/mL) | 0.720 | 0.360 | 0.180 | 0.520 | 0.260 | 0.130 |
Caspase-3 fold | 1.12 ± 0.34 a,* | 1.34 ± 0.29 b,* | 1.17 ± 0.25 a,* | 3.40 ± 0.95 a | 1.81 ± 0.26 b | 1.32 ± 0.18 c,* | ||
% ATP production | 60.05 ± 12.31 b | 72.21 ± 11.72 b | 94.66 ± 4.73 a | 39.08 ± 2.50 c | 60.23 ± 8.09 b | 78.90 ± 10.09 a | ||
Normal cells | Extract (mg/mL) | 1.370 | 0.760 | 0.150 | 0.500 | 0.295 | 0.091 | |
Caspase-3 fold | 1.09 ± 0.11 a,* | 1.10 ± 0.18 a,* | 0.98 ± 0.26 a,* | 1.86 ± 0.61 a | 1.42 ± 0.31 a,b | 1.16 ± 0.26 b | ||
% ATP production | 39.33 ± 4.46 c | 61.95 ± 11.40 b | 89.50 ± 20.60 a | 38.13 ± 12.42 b | 54.03 ± 11.80 b | 80.35 ± 20.17 a | ||
Cancer cells | Extract (mg/mL) | 1.290 | 0.84 | 0.38 | 0.440 | 0.291 | 0.143 | |
Caspase-3 fold | 0.65 ± 0.17 c | 0.94 ± 0.14 b,* | 1.40 ± 0.23 a | 1.69 ± 0.32 a | 1.51 ± 0.13 a | 1.32 ± 0.20 b,* | ||
% ATP production | 49.57 ± 5.00 c | 66.6 ± 7.97 b | 95.48 ± 9.11 a | 48.65 ± 8.56 b | 63.32 ± 8.60 b | 92.17 ± 8.98 a | ||
C. subternata | Premalignant cells | Extract (mg/mL) | 0.468 | 0.234 | 0.117 | 0.417 | 0.209 | 0.104 |
Caspase-3 fold | 1.73 ± 0.20 a | 1.46 ± 0.12 a | 1.10 ± 0.25 b,* | 3.27 ± 1.00 a | 2.03 ± 0.44 b | 1.45 ± 0.22 c,* | ||
% ATP production | 52.72 ± 10.24 c | 71.09 ± 7.65 b | 87.17 ± 9.40 a | 50.33 ± 7.21 b | 61.50 ± 8.53 b | 75.80 ± 6.51 a | ||
Normal cells | Extract (mg/mL) | 1.080 | 0.640 | 0.200 | 0.370 | 0.230 | 0.098 | |
Caspase-3 fold | 1.25 ± 0.27 a,* | 1.39 ± 0.37 a,* | 1.14 ± 0.16 a,* | 2.11 ± 0.61 a | 1.77 ± 0.37 a,b | 1.45 ± 0.27 a,* | ||
% ATP production | 40.17 ± 6.89 c | 64.71 ± 5.99 b | 84.98 ± 8.28 a | 49.96 ± 6.09 c | 63.28 ± 7.45 b | 80.42 ± 9.88 a | ||
Cancer cells | Extract (mg/mL) | 1.140 | 0.680 | 0.223 | 0.430 | 0.294 | 0.158 | |
Caspase-3 fold increase | 0.97 ± 0.26 a,* | 1.04 ± 0.12 a,* | 1.15 ± 0.06 a,* | 1.45 ± 0.33 a,* | 1.55 ± 0.35 a,* | 1.28 ± 0.23 a,* | ||
% ATP production | 36.09 ± 1.31 c | 60.68 ± 3.46 b | 76.79 ± 0.70 a | 39.68 ± 2.28 c | 66.38 ± 1.77 b | 82.00 ± 8.73 a |
Tea/Herbal Tea | Methanol Extracts | Aqueous Extracts | ||||
---|---|---|---|---|---|---|
Premalignant Cells | Normal Cells | Cancer Cells | Premalignant Cells | Normal Cells | Cancer Cells | |
C. sinensis | −0.881 | −0.888 | −0.800 | −0.742 | −0.878 | −0.672 |
(<0.0001) | (<0.0001) | (<0.0001) | (<0.0001) | (<0.0001) | (<0.0001) | |
A. linearis | −0.881 | −0.802 | −0.769 | −0.745 | −0.547 | −0.724 |
(<0.0001) | (<0.0001) | (<0.0001) | (=0.0001) | (=0.0002) | (<0.0001) | |
C. subternata | −0.841 | −0.588 | - | −0.783 | −0.864 | −0.554 |
(<0.0001) | (=0.0004) | - | (<0.0001) | (<0.0001) | (=0.0027) | |
C. intermedia | −0.527 | - | 0.479 | −0.819 | −0.699 | −0.599 |
(=0.0005) | - | (=0.0015) | (<0.0001) | (<0.0001) | (<0.0001) |
Tea/Herbal Extracts | Polyphenols * | Concentration * (µg/mg Extract) | TP/FLAVA Ratio | ||
---|---|---|---|---|---|
Methanol | Aqueous | Methanol | Aqueous | ||
C. sinensis | TP | 256.5 ± 36.9 A | 161.0 ± 20.8 B | 1.94 | 2.07 |
FLAVA | 132.3 ± 3.4 A | 77.6 ± 1.5 B | |||
EGCG | 111.9 ± 3.0 A | 46.1 ± 1.5 B | |||
Total flavanols | 200.8 ± 6.2 A | 100.0 ± 2.8 B | |||
A. linearis | TP | 350.7 ± 35.0 A | 250.5 ± 16.4 B | 12.92 | 13.92 |
FLAVA | 27.1 ± 0.8 A | 18.0 ± 0.6 B | |||
DHC | 151.8 ± 1.7 A | 100.6 ± 1.8 B | |||
Total_ mono | 209.1 ± 1.8 A | 138.1 ± 3.6 B | |||
C. intermedia | TP | 172.1 ± 4.1 A | 164.5 ± 11.3 B | 15.23 | 9.19 |
FLAVA | 11.3 ± 0.9 B | 17.9 ± 0.9 A | |||
Xanthones | 87.8 ± 2.9 A | 54.0 ± 0.8 B | |||
Hesperidin | 88.8 ± 11.6 A | 7.3 ± 0.6 B | |||
Total_ mono | 186.2 ± 13.0 A | 64.0 ± 0.6 B | |||
C. subternata | TP | 220.5 ± 14.5 A | 175.0 ± 24.1 B | 16.96 | 7.64 |
FLAVA | 13.0 ± 0.6 B | 22.9 ± 0.9 A | |||
Xanthones | 78.5 ± 1.3 A | 30.7 ± 4.4 B | |||
Hesperidin | 63.1 ± 8.6 A | 8.0 ± 0.2 B | |||
Total_mono | 174.4 ± 9.0 A | 51.3 ± 4.5 B |
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Magcwebeba, T.U.; Swart, P.; Swanevelder, S.; Joubert, E.; Gelderblom, W.C.A. In Vitro Chemopreventive Properties of Green Tea, Rooibos and Honeybush Extracts in Skin Cells. Molecules 2016, 21, 1622. https://doi.org/10.3390/molecules21121622
Magcwebeba TU, Swart P, Swanevelder S, Joubert E, Gelderblom WCA. In Vitro Chemopreventive Properties of Green Tea, Rooibos and Honeybush Extracts in Skin Cells. Molecules. 2016; 21(12):1622. https://doi.org/10.3390/molecules21121622
Chicago/Turabian StyleMagcwebeba, Tandeka U., Pieter Swart, Sonja Swanevelder, Elizabeth Joubert, and Wentzel C. A. Gelderblom. 2016. "In Vitro Chemopreventive Properties of Green Tea, Rooibos and Honeybush Extracts in Skin Cells" Molecules 21, no. 12: 1622. https://doi.org/10.3390/molecules21121622