Nutritional, phytochemical, antioxidant and anticancer properties of Eurycoma longifolia Jack and Clinacanthus nutans Lind. herbal teas
Camellia sinensis tea is popularly consumed due to its nutraceutical and pharmaceutical values. Its consumption triggers the attention towards other medicinal plants in search for more promising drinks called herbal teas. However, many processing factors influence the tea and herbal tea quality by a...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2018
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| Subjects: | |
| Online Access: | https://eprints.ums.edu.my/id/eprint/38196/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/38196/2/FULLTEXT.pdf |
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| Summary: | Camellia sinensis tea is popularly consumed due to its nutraceutical and pharmaceutical values. Its consumption triggers the attention towards other medicinal plants in search for more promising drinks called herbal teas. However, many processing factors influence the tea and herbal tea quality by affecting their nutrient and bioactive compounds. The aim of this study was to investigate the effect of fermentation, drying techniques, water temperature and steeping time towards the nutritional and phytochemical content as well as the antioxidant and anticancer potentials of herbal teas developed from leaves of Eurycoma longifolia Jack and Clinacanthus nutans Lind. Two local commercial products of C. sinensis (BOH green tea and SABAH black tea) were used as comparison throughout the study. The plants were processed into unfermented and fermented leaves before freeze dried and microwave-oven dried. Their infusions were prepared by steeping the dried leaves in (i) different water temperature (50, 60, 80 and 100 °C) for 10 min and (ii) 100 ° C water for different steeping time (1, 2, 5, 10, 15 and 20 min). The first part of the study was to evaluate the physical appearance and proximate compositions in the dried leaves, as well as the colour, pH value, mineral and vitamin C content in their infusions. For the evaluation of phytochemical, antioxidant and anticancer properties, their infusions were tested. All data were analysed using GraphPad Prism version 5.01 and expressed as means ± standard deviation (S.D.) of five replicate analyses in five independent experiments. Oneway analysis of variance (ANOVA) followed by Tukey's multiple range test was carried out to determine the significance between means. The statistical significant level was set at P ≤ 0.05. In the nutritional content determination, the results showed that commercial teas of C. sinensis and herbal teas of £ longifolia and C. nutans were physically different in their dried leaf colour and surface morphology, as well as the infusion colour and pH value. Proximate composition showed that C. nutans dried leaves contained the highest percentage of ash, moisture and crude protein; while E longifolia dried leaves contained the highest crude fibre. Crude lipid in dried leaves was relatively similar between £ longifolia and C. nutans. Higher total carbohydrates were observed in £ longifolia dried leaves compared to that of C. nutans. High contents of elements were detected in dried leaves of E longifolia and C. nutans, but low in their respective infusions. Infusions of C. nutans showed promising Ca, K, Mg and Na sources as these elements were significantly higher compared to the commercial tea infusions. Vitamin C also was detected in both E longifolia and C. nutans infusions. In the phytochemical content studies, the highest TPC was recorded in unfermented freeze dried leaf of E longifolia infusion (635.36 ± 57.16 mg TAE/L) when steeping the leaves in 100 °c boiling water for 20 min, while the highest FC was recorded in unfermented freeze dried leaf of E longifolia infusion (168.17 ± 2.89 mg CE/L) when the leaves was steeped in 100 °c boiling water for 10 min. At least 5 individual phenolics were detected in E longifolia infusions (gallic acid, chlorogenic acid, vanillic acid, ECG and EGCG), whilst, 8 individual phenolics were detected in C. nutans infusions (gallic acid, chlorogenic acid, p-coumaric acid, caffeic acid, ferulic acid, EC, ECG and EGCG). The effects of fermentation and drying techniques towards those phenolic compounds were varied, depending on their chemical structures. In the antioxidant activity determination, E longifolia infusions possessed higher FRAP values compared to those of C. nutans. While in ABTS and DPPH assays, similar strength of free radical scavenging activity was observed for E longifolia infusions and the commercial teas. The unfermented freeze dried leaf of E /ongifolia infusion had the highest antioxidant activity in all three assays when steeping the leaves in 100 ° c boiling water for 10 min or longer. The phenolic content was strongly correlated with the antioxidant activity of the commercial tea and herbal tea infusions. In the anticancer activity evaluation of herbal extracts, the unfermented freeze dried leaf of E long1folia extract was the most potent anticancer source due to its high cytotoxicity effect towards the non-hormone dependent breast cancer cell line MDA-MB-231, hormone dependent breast cancer cell line MCF-7 and colon cancer cell line HT-29 (IC5o =45.0 ± 3.5 μg/ml, 69.3 ± 17.2 μg/ml and 97.9 ± 1.8 μg/ml, respectively) in dose-dependent manners. The protective effect against hydrogen peroxide-induced damage towards normal cells NIH-3T3 and synergistic effect with drug tamoxifen against the breast cancer cell lines were also displayed. There was a presence of orange colour or green-orange colour which indicates the apoptotic cells when stained with acridine orange/propodium iodide. The typical characteristics of apoptotic cell including shrinkage of cell, bleb of membrane, condensed chromatin and fragmented nuclear were observed under high magnification of fluorescence microscope. Multiple DNA bands were formed on MDA-MB-231 and MCF-7 treated cell lines; while single DNA band formed on HT-29 treated cell line. In cell death mode analysis, the cells were arrested at G2/M for both HT-29 and MCF-7 cell lines; while observed at 5-phase for MDA-MB-231 cell line. The number of apoptotic cell of MCF-7 and MDA-MB-231 were increased from 24h to 48h but slightly decreased at 72h of treatment. Treated HT-29 cells have showed gradual increase number of apoptotic cells from 24 h to 72 h of treatment. In possible anticancer mechanism analysis, HT-29 demonstrates the downregulation of Bcl-2 and up-regulation of Bax expressions with p53 independent activity. The treated HT-29 cells also displayed the exclusion of caspase 3 and 8 activation and release of cytochrome c. The up-regulation of Bax showed in increase manner from 24 h to 48 h of treatment for MDA-MB-231 cells with downregulation of Bcl-2 expression in same period. This expression also might responsible for the expression of cytochrome c and caspase 3 which caused the apoptosis cell death. The expression of caspase 8 and cytochrome c were detected in the treated MCF-7 cells, with up-regulations of Bax and p53 and also downregulation of Bcl-2 expression. Those various mechanisms of action were displayed by cancer cells when treated with the extract have confirming the apoptotic cell death and the involvement of Bcl-2 family regulation. In short, among the herbal teas of E longifolia and C. nutans, the unfermented freeze dried leaf of E Jongifolia infusion displayed good nutritional values with the highest total phenolic and flavonoid contents as well as the most effective antioxidant and anticancer sources. As a conclusion, the fermentation, drying technique, water temperature and steeping time clearly did affect those properties in commercial teas and herbal teas tested. However, the effect was differently as it might depend on the nature or chemical structure of nutrient constituents and the phytochemicals that responsible for the antioxidant and anticancer properties of herbal teas; hence create a difficult decision to determine the exact best way of leaves processing and infusion preparation. |
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