Preethi Soysa

Extraction Kinetics of phytochemicals and antioxidant activity during black tea (Camellia sinensis L.) brewing

BackgroundTea is the most consumed beverage in the world which is second only to water. Tea contains a broad spectrum of active ingredients which are responsible for its health benefits. The composition of constituents extracted to the tea brew depends on the method of preparation for its consumption. The objective of this study was to investigate the extraction kinetics of phenolic compounds, gallic acid, caffeine and catechins and the variation of antioxidant activity with time after tea brew is made.MethodsCTC (Crush, Tear, Curl) tea manufactured in Sri Lanka was used in this study. Tea brew was prepared according to the traditional method by adding boiling water to tea leaves. The samples were collected at different time intervals. Total phenolic and flavonoid contents were determined using Folin ciocalteu and aluminium chloride methods respectively. Gallic acid, caffeine, epicatechin, epigallocatechin gallate were quantified by HPLC/UV method. Antioxidant activity was evaluated by DPPH radical scavenging and Ferric Reducing Antioxidant Power (FRAP) assays.ResultsGallic acid, caffeine and catechins were extracted within a very short period. The maximum extractable polyphenols and flavanoids were achieved at 6–8 min after the tea brew is prepared. Polyphenols, flavanoids and epigallocatechin gallate showed a significant correlation (p < 0.001) with the antioxidant activity of tea.ConclusionThe optimum time needed to release tea constituents from CTC tea leaves is 2–8 min after tea is made.

Optimized enzymatic colorimetric assay for determination of hydrogen peroxide (H2O2) scavenging activity of plant extracts.

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Evaluation of phytochemical composition and antioxidant capacity of a decoction containing Adenanthera pavonina L. and Thespesia populnea L.

Background: A decoction prepared with barks of Adenanthera pavonina and Thespesia populnea is a herbal formulation which has been prescribed in Sri Lanka in the treatment of cancer patients for many years. This study was designed to investigate its phytochemical and antioxidant properties. Materials and Methods: The total phenolics and flavonoids were determined using Folin–Ciocalteau and aluminum chloride colorimetric methods, respectively. Gallic acid content in the decoction was determined using high-performance liquid chromatography. The antioxidant properties were evaluated by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical assay, nitric oxide scavenging assay, deoxyribose method, and the reducing power of the decoction. Results: The concentration of total phenols, flavonoids, and gallic acid of the decoction were 34.13 ± 3.54 w/w % gallic acid equivalents, 41.37 ± 0.57 w/w % of (-)-Epigallocatechin gallate equivalents, and 0.58 ± 0.24 mg/g, respectively. The EC50 for DPPH, nitric oxide scavenging, and deoxyribose assays were 7.24 ± 0.50, 14.02 ± 0.66, and 53.21 ± 2.82 μg/ml, respectively. Reducing power of the decoction increased with the concentration. Conclusion: These investigations suggested that the decoction prepared with A. pavonina and T. populnea can be a potential source of nutraceuticals with antioxidant activity.

Chitosan-Alginate Nanoparticle System Efficiently Delivers Doxorubicin to MCF-7 Cells

A chitosan-alginate nanoparticle system encapsulating doxorubicin (DOX) was prepared by a novel ionic gelation method using alginate as the crosslinker. These nanoparticles were around 100 nm in size and more stable with higher positive zeta potential and had higher % encapsulation efficiency (95%) than DOX loaded chitosan nanoparticles (DOX Csn NP) crosslinked with sodium tripolyphosphate (STPP). FTIR spectroscopy and thermogravimetric analysis revealed successful loading of DOX. In vitro drug release showed an initial release phase followed by slow release phase with higher cumulative release obtained with DOX loaded chitosan-alginate nanoparticles (DOX Csn-Alg NP). The in vitro cytotoxicity of DOX released from the two nanoparticle systems showed a notable difference on comparison with that of free DOX on the MCF-7 cell line. The SRB assay, AO/EB staining, and fluorescence uptake study indicated that free DOX only showed dose dependent cytotoxicity, whereas both dose and time dependency were exhibited by the two sets of NPs. While both systems show sustained release of DOX, from the cell viability plots, DOX Csn-Alg NPs showed their superiority over DOX Csn NPs. The results obtained are useful for developing DOX Csn-Alg NPs as a sustained release carrier system for DOX.

Evidence for Seroprevalence in Human Localized Cutaneous Leishmaniasis Caused by Leishmania donovani in Sri Lanka

Visceral leishmaniasis (VL) is considered as a major health threat in the Indian subcontinent. Leishmania donovani, a usually visceralizing species, causes cutaneous leishmaniasis (CL) in Sri Lanka. However, visceralizing potential of the local L. donovani is not yet fully understood. This project studied the seroprevalence of local CL by using an in-house ELISA. An IgG-based ELISA using crude Leishmania antigen (Ag) was developed and validated. A total of 50 laboratory confirmed cases of locally acquired CL were examined using the newly developed ELISA. According to the optimized ELISA, seroprevalence of anti-Leishmania IgG antibodies in the study group was 34.0% (n = 17/50). Majority of seropositive individuals were males (n = 13/17), representing 76%. Nearly half of the seropositive individuals were young adults (20–40 years, n = 9/17, 53%). Higher proportions of single lesions, large lesions, and nodular lesions were associated with a seroconversion. A proportion of local L. donovani infections leading to CL have the ability to raise an antibody response in the host. This may indicate early systemic involvement as one possibility. Study of a large number of patients with adequate follow-up would be useful.

Cytotoxic effects of ergone, a compound isolated from Fulviformes fastuosus

BackgroundMushrooms inspired the cuisines of many cultures and conventional medicaments for cancer. However, a substantial number of mushroom species are yet unexplored, possessing an unknown chemical, biological and pharmacological profiles. Fulviformes fastuosus is a terrestrial mushroom, which is commonly found in Sri Lankan woodlands. The current study was aimed at isolation and characterization of a potent cytotoxic compound from F. fastuosus and investigating the apoptotic effect induced by the active principle against cancer and normal cell lines.MethodsBioactivity guided isolation of active principles from the methanol extract of F. fastuosus was performed by a rapid extraction and isolation method using different chromatographic techniques. Potential cytotoxic compound was identified using one and two dimensional nuclear magnetic resonance spectroscopy and mass spectrometry. Isolated compound was screened for in vitro cytotoxicity against Hepatocellular carcinoma (HepG-2), Muscle rhabdomyosarcoma (RD) and Rat Wistar liver normal (CC-1) cell lines using 3 4, 5-(dimethylthiazol-2-yl) 2-5-diphenyl tetrazolium bromide (MTT) cell viability assay. Apoptotic features of cells were observed via microscopic examination and ethidium bromide/acridine orange fluorescent staining.ResultsThe interpretation of spectral data resulted in the identification of the chemical structure as ergosta-4,6,8 (14),22-tetraen-3-one (ergone). Ergone exhibited promising cytotoxic properties against RD cells with less cytotoxicity effect on CC-1 cells. In addition, ergone also possesses a strong cytotoxic effect against HepG-2 cells showing low toxic level for CC-1 cells. Apoptotic features of treated cells were detected via morphological characterization and ethidium bromide/acridine orange staining.ConclusionThe present study elaborates the isolation of a potent cytotoxic compound; ergone, from F. fastuosus via a rapid and efficient isolation method. Importantly, ergone has exhibited greater cytotoxic activity against RD cells with high selectivity index compared to cytotoxicity against HepG-2 cells. Ergone can be used in the development of therapeutic strategies for curbing rhabdomyosarcoma.

Development and Validation of a Simple High Performance Liquid Chromatography/UV Method for Simultaneous Determination of Urinary Uric Acid, Hypoxanthine, and Creatinine in Human Urine

Uric acid and hypoxanthine are produced in the catabolism of purine. Abnormal urinary levels of these products are associated with many diseases and therefore it is necessary to have a simple and rapid method to detect them. Hence, we report a simple reverse phase high performance liquid chromatography (HPLC/UV) technique, developed and validated for simultaneous analysis of uric acid, hypoxanthine, and creatinine in human urine. Urine was diluted appropriately and eluted with C-18 column 100 mm × 4.6 mm with a C-18 precolumn 25 mm × 4.6 mm in series. Potassium phosphate buffer (20 mM, pH 7.25) at a flow rate of 0.40 mL/min was employed as the solvent and peaks were detected at 235 nm. Tyrosine was used as the internal standard. The experimental conditions offered a good separation of analytes without interference of endogenous substances. The calibration curves were linear for all test compounds with a regression coefficient, r2 > 0.99. Uric acid, creatinine, tyrosine, and hypoxanthine were eluted at 5.2, 6.1, 7.2, and 8.3 min, respectively. Intraday and interday variability were less than 4.6% for all the analytes investigated and the recovery ranged from 98 to 102%. The proposed HPLC procedure is a simple, rapid, and low cost method with high accuracy with minimum use of organic solvents. This method was successfully applied for the determination of creatinine, hypoxanthine, and uric acid in human urine.

Water extract of Semecarpus parvifolia Thw. leaves inhibits cell proliferation and induces apoptosis on HEp-2 cells

BackgroundSemecarpus parvifolia Thw is used as an ingredient of poly herbal decoctions to treat cancer in traditional medicine. The present study aims to investigate the antiproliferative activity on HEp 2 cells by the water extract of S. parvifolia leaves and to evaluate potential mechanisms.MethodsThe plant extract was exposed to S. parvifolia for 24 hours and antiproliferative activity was quantified by Sulforhodamine B (SRB), 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Lactate dehydrogenase (LDH) assays. Morphological changes were observed after staining cells with ethidium bromide/acridine orange (EB/AO) and Giemsa dye. Comet assay was performed to evaluate the DNA damage. The toxicity of the plant extract was determined by brine shrimp lethality assay.ResultsS. parvifolia leaves reduced the cell proliferation in a dose and time dependent manner. A two fold increase in NO level was observed at higher concentrations. Morphological changes characteristic to apoptosis were observed in light microscopy, Giemsa and EB/AO stained cells. Fragmented DNA further confirmed its capacity to induce apoptosis. No lethality was observed with brine shrimps.ConclusionThe results suggest that Semecarpus parvifolia Thw induces apoptosis in HEp-2 cells through a NO dependent pathway.

Development of a Rapid and Simple Method to Remove Polyphenols from Plant Extracts

Polyphenols are secondary metabolites of plants, which are responsible for prevention of many diseases. Polyvinylpolypyrrolidone (PVPP) has a high affinity towards polyphenols. This method involves the use of PVPP column to remove polyphenols under centrifugal force. Standards of gallic acid, epigallocatechin gallate, vanillin, and tea extracts (Camellia sinensis) were used in this study. PVPP powder was packed in a syringe with different quantities. The test samples were layered over the PVPP column and subjected to centrifugation. Supernatant was tested for the total phenol content. The presence of phenolic compounds and caffeine was screened by HPLC and measuring the absorbance at 280. The antioxidant capacity of standards and tea extracts was compared with the polyphenol removed fractions using DPPH scavenging assay. No polyphenols were found in polyphenolic standards or tea extracts after PVPP treatment. The method described in the present study to remove polyphenols is simple, inexpensive, rapid, and efficient and can be employed to investigate the contribution of polyphenols present in natural products to their biological activity.