In vitro growth of Leishmania parasites from biopsy samples of suspected cutaneous and visceral leishmaniasis cases in Sri Lanka: An observational study.

Sri Lanka reports a large focus of Leishmania donovani caused cutaneous leishmaniasis (CL). Subsequent emergence of visceral leishmaniasis (VL) was also reported recently. Expansion of the on-going disease outbreak and many complexities indicate urgent need to enhance early case detection methods. In vitro cultivation (IVC) of parasites causing visceral leishmaniasis (VL) is important for disease confirmation and to obtain sufficient quantities of parasites required in many scientific studies. IVC is carried out as a useful second line investigation for direct microscopy negative patients with CL in this setting. Along with the emergence of VL, current study was carried out to evaluate in vitro growth of local VL parasites and to identify their differences associated with in vitro growth characteristics. Routine parasitological diagnostic methods, i.e., light microscopy (LM), polymerase chain reaction (PCR) were used for confirmation of suspected cases. Lesion samples from 125 suspected CL cases and bone marrow or splenic aspirations from 125 suspected VL patients were used to inoculate IVCs. Media M199 (about 70 µl) supplemented with 15-20% of heat inactivated fetal bovine serum was used for initial culturing procedures in capillaries. Capillary cultures were monitored daily. Total of 44 different compositions/conditions were used for evaluating in vitro growth of VL causing parasite. Daily records on parasite counts, morphological appearance (size, shape, and wriggly movements) were maintained. In vitro transformation of Leishmania promastigotes to amastigotes and outcome of the attempts on recovery of live Leishmania from culture stabilates was also compared between CL and VL parasites. Proportion of cultures showing a transformation of promastigotes were 40/45 (88.9%) and 4/10 (40.0%) for CL and VL respectively. In the transformed cultures, parasites showing typical shape, size and movement patterns were less in VL (1/4, 25.0%) compared to CL (28/40, 70.0%). CL cultures showed a growth up to mass culturing level with mean duration of two weeks while it was about five weeks for VL cultures. Proportion of cultures that reached a parasite density of 1 × 106 cells/ml (proceeded to mass cultures) was significantly low in VL (4/10, 40%) as compared to CL (28/40, 70.0%). None of media compositions/conditions were successful for mass culturing of VL parasites while all of them were shown to be useful for growing CL strains. Also in vitro transformation to amastigote form and recovering of culture stabilates were not successful compared to CL. There were clear differences between in vitro growth of Leishmania parasites causing local CL and VL. Further studies are recommended for optimization of in vitro culturing of VL parasite which will be invaluable to enhance case detection in future.

Polyphenol Mediated Suppression of Hepatocellular Carcinoma (HepG2) Cell Proliferation by Clerodendrum infortunatum L. Root.

OBJECTIVE: Clerodendrum infortunatum L. has long been used in traditional medicine in Sri Lanka for tumours, cancer, and certain skin diseases. The present study aimed to assess the anticancer properties of the aqueous extract of C. infortunatum L. root (AECIR) through the activation of the apoptotic pathway on hepatocellular carcinoma (HepG2) and thus give it a scientific validation. Further, the contribution of polyphenols in antioxidant activity and cell cytotoxicity was investigated. METHODS: Powdered plant material was boiled with water (100°C) to obtained AECIR.  The DPPH assay was used to determine the antioxidant potential. The activity of AECIR on HepG2 and normal rat fibroblast (CC1) cell growth was determined using MTT assay. The morphological changes related to apoptotic pathway was examined by Ethidium Bromide/Acridine Orange (EB/AO), Rhodamine 123 (Rh123) and DNA fragmentation assay. RESULTS: The AECIR demonstrated antioxidant potential with an EC50 of 350.2 ± 1.5 ug/mL for DPPH assay. The HO•, H2O2 and •NO free radical scavenging activity was observed with EC50 of 19.7 ± 2.3, 11.7 ± 0.1 and 273.1 ± 0.9 ug/mL, respectively. The antiproliferative effect of AECIR on HepG2 cells was observed in a time and dose dependent manner with an EC50 of 239.1 ± 1.3 µg/mL while CC1 cells showed a nontoxic effect with an EC50 1062.7 ± 3.4 µg/mL after 24hrs treatment. A significant decrease in antioxidant activity (p<0.001) and 90% HepG2 cell viability was observed with polyphenol removed AECIR compared to the polyphenol present AECIR. The EB/AO uptake, depletion of mitochondrial transmembrane potential, and DNA fragmentation assay results revealed that the apoptosis was induced by AECIR. CONCLUSION: The obtained result of the present study demonstrates that the antioxidant potential and antiproliferative activity of AECIR is attributed to the presence of polyphenols. Furthermore, the findings provide the scientific base for anti-cancer potential of AECIR.

A Comparison of Mesenchymal Stem Cell-derived Hepatocyte-like Cells and HepG2 Cells for Use in Drug-Induced Liver Injury Studies.

Liver cell lines obtained from hepatomas, for example, HepG2 cells, are commonly used in drug toxicity studies. However, functional hepatocyte-like cells derived from mesenchymal stem cells (MSCs) could be a better option for use in the study of drug metabolism and toxicity. Overdose of acetaminophen (APAP) and excess alcohol consumption are common causes of liver damage. The objective of the present study was to investigate the use of MSC-derived hepatocyte-like cells (MSCdH) in the assessment of drug-induced liver injury (by using APAP and ethanol), and to compare the toxic effects observed in the MSCdH with those exhibited by HepG2 cells. MSCs were isolated from umbilical cord and their functionality confirmed by their ability to differentiate into adipocytes, osteocytes and hepatocyte-like cells. It was shown that the MSCs successfully differentiated into hepatocyte-like cells, and these cells were further characterised by using various enzyme assays and by assessing albumin secretion and urea synthesis. Cytotoxicity was evaluated in the HepG2 and MSCdH after exposure to ethanol and APAP, with cell viability being determined by using the MTT assay. After exposure to ethanol and to APAP, cell viability decreased in a concentration-dependent manner for both types of hepatocytes. The respective EC50 values of ethanol-induced toxicity for HepG2 and MSCdH cells were 2.5% and 1.3% v/v (p < 0.001); for APAP-induced toxicity they were 19.1 mM and 12.6 mM (p < 0.001). These findings show that there is a distinct difference between the two types of hepatocytes in terms of APAP-induced and ethanol-induced liver injury.

The Effectiveness of the Nimali Variety of Sri Lankan Punica granatum L. Fruit Extracts on Rhabdomyosarcoma (RD) Cells Concerning the Apoptotic Signaling Pathway.

OBJECTIVE: Pomegranate ,a polyphenol-rich fruit, has been considered as one of the ancient fruits with anticancer effect. Cell cycle arrest is considered as an ordinary factor in human cancer, and apoptosis is the frequent drug target. This study aimed to evaluate the effectiveness of the Nimali variety of Sri Lankan Punica granatum L. fruit extracts on rhabdomyosarcoma (RD) cells concerning the apoptotic signaling pathway. METHODS: Antiproliferative activity of aqueous extracts of pomegranate peel, pericarp, was assessed using multiple extraction methods (sonication, microwaving, sonication followed by microwaving, keeping in a waterbath, and boiling at 100ºC). Total protein content, nitric oxide production, LDH, and caspase-8 and caspase-3 activities were analyzed in peel extracts prepared by sonicated or microwave methods. RT-qPCR was performed with intact RNA to explore the apoptotic pathway and gene expression. RESULTS: Peel extracts expressed minimum cell viability in a dose-dependent manner, induced cell death on RD cells. However, sonicated peel extract (SPL) indicated the lowest IC50 of 14.8±2.2 µg/mL comparative to healthy VERO cells (>1,000 µg/mL). A decrease of nitrite content in the supernatant was visualized in the graph plotted against concentration. Furthermore, SPL upregulated caspase-8 and caspase-3 signaling pathways and expression of p21 and p53 genes. CONCLUSION: The findings highlighted the promising therapeutic potential of SPL to inhibit RD growth and progression and to modulate the caspase-8 and caspase-3, p53, and p21 dependent pathway.

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

BACKGROUND: Semecarpus 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. METHODS: The 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. RESULTS: S. 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. CONCLUSION: The 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.

A traditional poly herbal medicine "Le Pana Guliya" induces apoptosis in HepG2 and HeLa cells but not in CC1 cells: an in vitro assessment.

"Le Pana Guliya" (LPG) is a polyherbal formulation which is used to treat different types of cancers in traditional medicine. In this study we describe in vitro efficacy and mechanism of action of LPG on two cancer cell lines (HepG2 and HeLa) compared with a normal cell line CC1. The MTT, LDH assays and protein synthesis were used to study antiproliferative activity of LPG while NO synthesis and GSH content were assayed to determine the oxidative stress exerted by LPG. Rhodamine 123 staining, caspase 3 activity, DNA fragmentation and microscopic examination of cells stained with ethidium bromide/acridine orange were used to identify the apoptosis mechanisms associated with LPG. The LPG showed the most potent antiproliferative effect against the proliferation of HepG2 and HeLa cells with an EC50 value of 2.72 ± 1.36 and 19.03 ± 2.63 µg/mL for MTT assay after 24 h treatment respectively. In contrast, CC1 cells showed an EC50 value of 213.07 ± 7.71 µg/mL. Similar results were observed for LDH release. A dose dependent decrease in protein synthesis was shown in both cancer cell types compared to CC1 cells. The reduction of GSH content and elevation of cell survival with exogenous GSH prove that the LPG act via induction of oxidative stress. LPG also stimulates the production of NO and mediates oxidative stress. Rhodamine 123 assay shows the mitochondrial involvement in cell death by depletion of Δψ inducing downstream events in apoptosis. This results in increase in caspase-3 activity eventually DNA fragmentation and LPG induced apoptotic cell death. In conclusion the present study suggested that the LPG exerted an anticancer activity via oxidative stress dependent apoptosis. Therefore present study provides the scientific proof of the traditional knowledge in using LPG as an anticancer agent.

Cytotoxic effects of ergone, a compound isolated from Fulviformes fastuosus.

BACKGROUND: Mushrooms 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. METHODS: Bioactivity 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. RESULTS: The 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. CONCLUSION: The 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.

Polyphenols contribute to the antioxidant and antiproliferative activity of Phyllanthus debilis plant in-vitro.

BACKGROUND: Phyllanthus debilis (Elapitawakka) is a medicinal plant used in traditional systems of medicine in Sri Lanka. Present study was carried out to evaluate in-vitro anti-oxidant and anti-proliferative activity of the water extracts of aerial parts (AP) and roots (RP) of P.debilis plant and the role of polyphenolic compounds in view of its medicinal use. METHOD: Total polyphenols, flavonoids and proanthocyanidin content of the extracts were quantified. DPPH, hydroxyl radical, nitric oxide and hydrogen peroxide scavenging potentials and the total antioxidant capacity, ferric ion reducing power were determined to evaluate antioxidant capacity. Anti-proliferative activity was assessed with MTT assay for Human Rhabdomyosarcoma (RD) and normal rat liver cells (CC1) after 24 h exposure to the plant extracts. DPPH and MTT assays were carried out for AP and RP extracts after removal of polyphenols to assess the contribution of polyphenols on antioxidant and anti-proliferative activity of Phyllanthus debilis. RESULTS: Flavonoid content of the AP extract was significantly lower than that of RP (P < 0.001) while no significant difference was observed in polyphenolic as well as in proanthocyanidin contents. All the assays except for phosphomolybdate assay demonstrated that the RP extract had higher antioxidant capacity (p < 0.001) compared to AP. Further, antioxidant capacity and anti-proliferative activity were lower (p < 0.001) in AP and RP in the absence of polyphenols compared to the crude extract. CONCLUSION: Root contains higher levels of flavonoids than the aerial part. Moreover, the presence of polyphenols is required for antioxidant and anti-proliferative activities of both AP and RP.

Simple isocratic method for simultaneous determination of caffeine and catechins in tea products by HPLC.

Tea is a popular beverage almost all over the world. Many studies show that tea consumption is closely associated with positive health impact. Most of the HPLC methods used for the determination of tea constituents include gradient elution systems which involve expensive instrumentation. The objective of this study was to develop a simple, rapid precise and low cost HPLC method for the separation and quantification of catechins and caffeine in tea (Camellia sinensis). The method utilizes a phenyl column (2.1 × 150 mm) with a UV-detector (280 nm) where excellent chromatographic separation of tea components i.e. gallic acid (GA), caffeine (Caf), epicatechin (EC) and (-)-epigallocatechin gallate (EGCG) was achieved. The isocratic elution system of acetonitrile, glacial acetic acid and deionized water (8:1:91 v/v/v) at a flow rate of 0.5 mL/min was involved. This method produced excellent accuracy and precision. Within run and between run precision was less than 7.5 %. The equations for calibration curves were y = 0.117 (±0.010)x + 0.173 (±0.024), y = 0.100 (±0.003)x + 0.045 (±0.019), y = 0.016 (±0.001)x + 0.006 (±0.004), y = 0.025 (±0.001)x-0.025 (±0.007) for GA, Caf, EC and EGCG respectively. The method validation parameters prove that the method is efficient, a simple and adequate for the quantitative determination of principal components in tea samples.

Evaluation of anticancer properties of a decoction containing Adenanthera pavonina L. and Thespesia populnea L.

BACKGROUND: A decoction composed of Adenanthera pavonina L. and Thespesia populnea L. is currently being used in the treatment of cancer patients. METHODS: Lactate Dehydrogenase (LDH) release, (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) MTT, and Sulforhodamine B (SRB) assays were carried out to study cytotoxicity and anti-proliferative activity against the HEp-2 cells, 24 h post-treatment with the decoction. RESULTS: The mean (± SD) values of EC50 were 195.50 (±40.68), 120.02 (±29.82) and 77.06 (±8.80) µg/ml for LDH, MTT, and SRB assays respectively. These results strongly correlate the morphological changes observed in cells treated with the decoction. Induction of apoptosis was visualized by fluorescence microscopy stained with ethidium bromide/acridine orange dye mix. In addition, brine shrimp lethality assay showed an EC50 value at a higher concentration (1.96 mg/mL). CONCLUSIONS: These results suggest that the decoction prepared with Adenanthera pavonina L. and Thespesia populnea L. exhibits anti-proliferative activity and induces apoptosis on the HEp-2 cancer cells but no toxicity against Artemia salina.

Antioxidant potential, in vitro cytotoxicity and apoptotic effect induced by crude organic extract of Anthracophyllum lateritium against RD sarcoma cells.

BACKGROUND: Macrofungi have an established history of use in traditional oriental medicine. Anthracophyllum lateritium is a terrestrial macrofungus found in the dry zone forest reserves in Sri Lanka. Yet there are no scientific reports on bioactive properties of this species. Hence, the current study was aimed at determining the antioxidant potential, in vitro antiproliferative activity and apoptotic effect induced by crude methanolic extract of A. lateritium against RD sarcoma cell line. METHOD: The crude extract of A. lateritium was dissolved in methanol (MEFCA) and antioxidant activity was evaluated using in vitro assays: inhibition of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging, ferric ion reducing power and 2-deoxy-D-ribose degradation assay. Total phenol and flavonoid contents of MEFCA were assayed using folin Ciocalteu method and aluminium chloride colorimetric method. In vitro cytotoxicity was determined using MTT assay against RD cells after 24 h exposure to MEFCA. Ethidium bromide/ acridine orange staining, DNA fragmentation and protein synthesis experiments were used to study the apoptotic features and antiproliferative activities of the treated cells. Glutathione assay and griess nitrite assay were used to analyze the reduced glutathione content and liberation of nitric oxide from apoptotic cells. RESULTS: MEFCA showed promising antioxidant activity with EC50 values of 8.00 ± 0.35 µg/mL for DPPH scavenging and 83.33 ± 0.45 µg/mL for 2-deoxy-D-ribose degradation assay. The phenolic content was 265.15 ± 0.46 of (w/w) % of Gallic acid equivalents and flavonoid content was 173.01 ± 0.35 of (w/w) % of Epigallocatechingallate. A. lateritium showed strong in vitro cytotoxic activity with an EC50 of 18.80 ± 4.83 µg/mL for MTT assay against RD cells. Ethidium bromide/acridine orange staining and DNA fragmentation indicated the apoptotic features of treated cells. Protein levels showed a dose dependent decrease supporting the fact that A. lateritium induces apoptosis of treated cells. Glutathione content and nitric oxide content of cells exhibited a dose dependent increase suggesting the apoptosis of RD cells was mediated by both nitrie ions and nitric oxide. CONCLUSIONS: The crude extract of the A. lateritium exhibited potent antioxidant, antiproliferative activity and apoptotic effect against RD cells providing supportive evidence for the ethnopharmacological use of this fungus in control of oxidative damage and remedy of cancer.

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

BACKGROUND: Tea 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. METHODS: CTC (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. RESULTS: Gallic 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. CONCLUSION: The optimum time needed to release tea constituents from CTC tea leaves is 2-8 min after tea is made.

Evaluation of antioxidant and antiproliferative activity of Flueggea leucopyrus Willd (katupila).

BACKGROUND: Flueggea leucopyrus Willd is a shrub grown in many parts of the dry zones in Sri Lanka. The leaves of F. leucopyrus has been used for treating cancer in the traditional system of medicine in Sri Lanka. Hence, this study was performed to analyze the antioxidant and antiproliferative properties of the aqueous extract of the leaves of F. leucopyrus on HEp-2 cells. METHOD: The aqueous extract of F. leucopyrus leaves (AEFLL) was freeze dried. Total phenolic content was assayed using Folin Ciocalteu reagent. Antioxidant activities of the extracts were evaluated using in vitro assays: inhibition of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging and 2-deoxy-D-ribose degradation assay. Nitric oxide radical scavenging activity was determined by using Griess reagent. The MTT, LDH assays and protein synthesis were used to study antiproliferative and cytotoxic activities against the Hep-2 cell after 24 hour exposure. DNA fragmentation and microscopic examination of cells stained with a mixture of ethidium bromide/acridine orange were used to visualize apoptosis in HEp-2 cells treated with the AEFLL. RESULTS: The total phenolic content of the extract was 22.15 ± 1.65 (w/w) % of gallic acid equivalent. The values for EC50 were 11.16 ± 0.37, 4.82 ± 1.82 and 23.77 ± 3.16 µg/mL for DPPH radical scavenging, nitric oxide radical scavenging activity and 2-deoxy-D-ribose degradation assay respectively. The EC50 with MTT and LDH assays were 506.8 ± 63.16 and 254.52 ± 42.92 µg/mL respectively. A dose dependent decrease in protein synthesis in HEp-2 cells was shown with an EC50 value of 305.84 ± 12.40 µg/mL. DNA fragmentation and ethidium bromide/acridine assays showed that the AEFLL induces apoptosis in HEp-2 cells. These results were in conformity with the morphological changes observed in the cells treated with the AEFLL. The brine shrimp bioassay showed that the AEFLL had no lethality over the concentration range of 50-500 µg/mL. CONCLUSIONS: Aqueous extract of the leaves of F. leucopyrus extract demonstrated antioxidant activity in vitro. Further it showed antiproliferative properties and induced apoptosis in HEp-2 cells.

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.