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.

In Vitro Dengue Virus Inhibition by Aqueous Extracts of Aegle marmelos, Munronia pinnata and Psidium guajava.

Dengue is an arboviral (insect-transmitted) infection of global concern. Currently, there are still no specific dengue antiviral agents to treat the disease. Plant extracts have been used in traditional medicine for treating various viral infections - thus, in the present study, aqueous extracts of dried flowers of Aegle marmelos (AM), whole plant of Munronia pinnata (MP) and leaves of Psidium guajava (PG) were investigated for their potential capacity to inhibit dengue virus infection of Vero cells. The maximum non-toxic dose (MNTD) and the 50% cytotoxic concentration (CC50) were determined by using the MTT assay. A plaque reduction antiviral assay was carried out with dengue virus types 1 (DV1), 2 (DV2), 3 (DV3) and 4 (DV4), in order to calculate the half-maximum inhibitory concentration (IC50). AM extract inhibited all four virus serotypes tested; MP extract inhibited DV1, DV2 and DV4, but not DV3; PG extract inhibited DV1, DV2 and DV4, but not DV3. Thus, the results suggest that AM is a promising candidate for the pan-serotype inhibition of dengue viral activity.

Measuring the sero-prevalence of Leishmania donovani induced cutaneous leishmaniasis: A method comparison study.

An in-house enzyme-linked immunosorbent assay (ELISA) based on crude antigen of Leishmania reported a high sero-prevalence (82.0%) in Leishmania donovani induced cutaneous leishmaniasis (CL) in Sri Lanka. ELISA was further compared with established serological tools to identify a suitable point of care diagnostic tool. Sero-prevalence of 100 CL samples were analyzed using in-house ELISA, Indian dipstick test and rK39 strip test. Results obtained were further compared with direct agglutination test (DAT) for 40 CL. Test performance was evaluated using Kappa index value. Clinico-epidemiological characteristics of patients were analyzed using SPSSv25.0. Cost analysis of tests was carried out. ELISA showed a high sero-positivity of 81.0% (n = 81/100) while DAT (57.5%,n = 23/40), Indian dipstick test (22.0%,n = 22/100) and rK39 test (15.0%,n = 15/100) showed a comparatively less sero-positivity. According to Kappa index values, there were no perfect agreement between tests. Among ELISA positive patients (n = 81/100), DAT, Indian dipstick test and rK39 demonstrated sero-positivity rates of 61.3% (n = 19/31), 25.9% (n = 21/81) and 16.0% (n = 13/81) respectively. Among ELISA negative patients (n = 19/100), three assays demonstrated sero-positivity rates of 44.4% (n = 4/9), 5.3% (n = 1/9) and 10.5% (n = 2/19) respectively. DAT can be used as an alternative test when ELISA is not available or negative. Clinico-epidemiological profiles of patients that showed sero-positivity by each assay were different. Cost per patient was approximately 5.5 USD for DAT and 3.0 USD for each other tests. Established serological tests demonstrated different and relatively lower detection rates. Species, strain and antigen heterogeneity, inconsistency in amount of used antigens, sera, antibody expression patterns and testing methodologies could be responsible. This study highlighted the importance of designing an in-house serological assay based on local parasite.

Serological studies on rK39 negative Visceral Leishmaniasis in an endemic focus of Leishmania donovani induced Cutaneous Leishmaniasis.

Sri Lanka reports a focus of L. donovani induced cutaneous leishmaniasis (CL). Our more recent parasite and clinical studies and historical evidence point towards long term existence of Leishmania in the country, indicating a possible evolution leading to antigenic heterogenicity as well. In-house enzyme-linked immunosorbent assay (ELISA) that was developed during phase 1 study indicated >80% sero-positivity in local CL, while visceral leishmaniasis (VL) remained very rare with majority being negative when tested with rK39 assay. A novel serological tool was developed and sero-positivity of VL was assessed for the first time. The assay showed 100.0% sensitivity and 98.3% specificity for detection of VL. Samples were showed less positivity with established direct agglutination test (DAT) and rK39 strip test. The assay was less expensive than that of established rapid diagnostic tests (RDTs), culture and PCR assays. This assay may be useful in diagnosing clinical VL infections, detection of light microscopy (LM) negative patients, tracking post treatment stages, field screening of asymptomatic cases and in further serological studies.

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.

First Serological Study Revealing High Humoral Response and Evidence for Antigenic Heterogeneity in Leishmania donovani Induced CL in Sri Lanka.

Posing a threat to the ongoing leishmaniasis elimination efforts in the Indian subcontinent, L. donovani-induced cutaneous leishmaniasis (CL) has been recently reported in many countries. Sri Lanka reports a large focus of human cutaneous leishmaniasis (CL) caused by Leishmania donovani, a usually visceralizing parasite. Enhanced case detection, early treatment, and in-depth understanding of sequalae are required to contain the spread of disease. Visceralizing potential of dermotropic strains has not been fully ruled out. Sri Lankan strains have shown a poor response to established serological assays. The present concern was to develop an in-house serological assay and to determine the seroprevalence of CL for identifying visceralizing potential and its usefulness in enhancing case detection. Crude cell lysate of dermotropic L. donovani promastigotes-based indirect enzyme-linked immunosorbent assay (ELISA) was previously optimized. Assay was evaluated using sera from 200 CL patients, 50 endemic and 50 nonendemic healthy controls, 50 patients with other skin diseases, and 50 patients with other systemic diseases. Seroprevalence and clinicoepidemiological associations were analyzed. Assay was compared with light microscopy (LM) and in vitro culturing (IVC). Cost comparison was carried out. Seroprevalence of CL was 82.0%. The assay had 99.5% specificity, and all healthy controls were negative at 0.189 cut-off. Positive and negative predictive values were 99.4% and 84.7%, respectively. Positivity obtained in ELISA was comparable to LM and higher than that of IVC. Cost per patient was 3.0 USD for both ELISA and LM and 6.0 USD for IVC. Infections occurring in all age groups and both genders demonstrated >75.0% of seropositivity. Patients had lesions with different durations/types/sizes showed >70.0% of seropositivity. Study identified a high seroprevalence of L. donovani-induced CL for the first time, indicating potential for visceralization or transient serological response. This can be used as a second line test in LM-negative CL cases to enhance clinical case detection. Further studies are warranted to examine in-depth correlations, antigen profiles, comparison with other established serological tools, and usefulness in the detection of asymptomatic cases. (National patent LK/P/1/19697).

Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis.

Human leishmaniasis which is considered a neglected tropical parasitic disease presents in three main clinical forms (i.e., cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL)) that are mainly determined by its causative species. Leishmania donovani, the most virulent and visceralizing parasite, is increasingly reported to cause CL in many countries in the world. Although CL is generally not considered to evoke a humoral immune response except for a nonrobust and a variable response in minority of cases, VL is associated with a clear strong humoral response. However, humoral response in L. donovani-induced CL has not been well evaluated before. A suitable serology-based assay is an essential primary step in such a study. An indirect enzyme-linked immunosorbent assay (ELISA) based on Leishmania promastigote crude antigen (Ag) was designed and optimized in order to utilize in further serological studies on this new clinical entity. Optimization included quantification of crude Ag, checkerboard titration method for determination of optimal concentrations for coating Ag, human sera and secondary antibody (Ab) with suitable coating buffer, blocking buffer, and incubating temperatures. The selected coating buffer was 0.02 M phosphate buffer, pH 6.8, and the blocking buffer was 2% fetal bovine serum with 0.01 M phosphate-buffered saline. At least 1 µg of crude Ag was required for coating the ELISA plate, while 1 : 1000 serum was used as primary Ab. The optimized concentration of secondary Ab was 1 : 64000 which might be altered according to manufacturer recommendations. The assay specificity was pre-evaluated using sera (n = 20 from each category) from confirmed CL patients and controls (other skin diseases which mimic CL, other systemic diseases that mimic VL, nonendemic healthy controls, and endemic healthy controls). This procedure described an optimization procedure of an ELISA technique for detection of anti-Leishmania antibodies in patients with L. donovani caused CL.

Quantification of Soluble or Insoluble Fractions of Leishmania Parasite Proteins in Microvolume Applications: A Simplification to Standard Lowry Assay.

Protein quantification is often an essential step in any research field that involves proteins. Although the standard Lowry assay and its modifications are most abundantly used in protein quantification, the existing methods are rigid or often demonstrate nonlinearity between protein concentration and color intensity. A method for fast and accurate qualitative and/or quantitative determination of total soluble/insoluble proteins or micro-well plate immobilized proteins isolated from Leishmania parasites in microvolumes was described in the current study. Improvements in cost-effective techniques are necessary to increase the research outputs in resource-limited settings. This method is a modification to the established Lowry assay for protein quantification. Concentrations of unknown samples were calculated using a standard curve prepared using a standard series of bovine serum albumin (BSA). The optimized reagents were 2 N NaOH (sodium hydroxide), 2% Na2CO3 (sodium carbonate), 1% CuSO4 (copper sulfate), 2% KNaC4H4O6 (potassium sodium tartrate), and 2 N Folin and Ciocalteu's phenol. This modified protein assay was sensitive for quantifying Leishmania proteins in a total crude extract or in a soluble fraction within the approximate range of 10-500 µg/ml (1-50 µg/assay) and showed a linearity between color intensity and concentration of the protein. This is an easier, fast, and accurate method for quantifying proteins with microvolumes in a cost-effective manner for routine use in research laboratories in resource-limited settings.

A highly sensitive modified nested PCR to enhance case detection in leishmaniasis.

BACKGROUND: Human leishmaniasis is one of the major parasitic diseases with worldwide distribution. Sri Lanka is a recently established focus of leishmaniasis caused by a variant Leishmania donovani. Early case detection and management is a main approach identified for L. donovani control in the regional leishmaniasis elimination drive. Usefulness of light microscopy and in-vitro culture are limited in chronic, atypical or treated lesions though timely and accurate detection of all light microscopy/in-vitro culture negative cases of all forms of leishmaniasis is necessary for treatment. Timely treatment is important to minimize risk for death in visceral disease and undesired sequelae of long standing infection and illness on both patients and community. We described a 100% sensitive, Leishmania spp. specific modified version of a nested PCR (Mo-STNPCR) that also minimizes carry over and cross contaminations while facilitate investigation of light microscopy and in-vitro culture negative clinically suggestive cases of leishmaniasis. METHODS: Leishmania DNA was amplified using previously published P221: 5'-GGTTCCTTTCCTGATTTACG-3' and P332: 5'-GGCCGGTAAAGGCCGAATAG-3'outer primers followed by a nested reaction using P223: 5'-TCCCATCGCAACCTCGGTT-3' and P333: 5'-AAGCGGGCGCGGTGCTG-3' inner primers that by passes the requirement of tube handling between the two steps of the conventional nested PCR. Leishmania DNA was detected in a range of infected tissue material. Infected material from patients with cutaneous leishmaniasis (n = 30), visceral leishmaniasis (n = 10) and from a control group including patients with non-leishmanial skin diseases (n = 10), other systemic diseases (n = 10) and healthy individuals (n = 10) were examined with Mo-STNPCR. Results were further compared with those of light microscopy and in-vitro culture. RESULTS: Mo-STNPCR method was 100% sensitive and 100% specific for diagnosis of leishmaniasis. Light microscopy and in-vitro culture were positive in 75.0% (n = 30/40) and 72.5% (n = 29/40) samples respectively where combined results of them gave 87.5% (n = 35/40) sensitivity. Mo-STNPCR did not cross react with control samples. Furthermore, Mo-STNPCR reduces the risk of cross-contaminations and carry over contaminations since the full reaction is carried out without opening the tubes. Per patient cost was calculated as 22 USD while the same was 3 and 6 USD for light microscopy and in-vitro culture respectively. CONCLUSION: Mo-STNPCR method is a useful tool in detecting leishmaniasis in minority of cases that go undetected by first line investigations.

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.

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.

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 Hepatoprotective activity of Eriocaulon quinquangulare in vitro using porcine liver slices against ethanol induced liver toxicity and free radical scavenging capacity.

BACKGROUND: Production of reactive oxygen species is a common cause in alcohol induced liver diseases. Decoction prepared from the whole plant of Eriocaulon quinquingulare is prescribed to treat liver disorders. The aim of this study was to investigate the hepatoprotective activity and antioxidant capacity of the water extract of E. quinquangulare in vitro. METHOD: The aqueous extract of the whole plant of E. quinquangulare (AEQ) was investigated for its phytochemical constituents, antioxidant and membrane stabilization properties in-vitro. The antioxidant activities of AEQ were investigated using 1,1-Diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical, nitric oxide scavenging and ferric reducing antioxidant power (FRAP) assays. Membrane stabilizing effect of the extract was determined by hypotonic solution induced human erythrocyte hemolytic assay (HEHA). Further, hepatoprotective activity against ethanol induced hepatotoxicity was carried out using porcine liver slices. RESULTS: The total phenolics and flavonoids were 10.3 ± 1.6 w/w % gallic acid equivalents and 45.6 ± 3.8 w/w % (-)-epigallocatechin gallate equivalents respectively. The values of EC50 for DPPH, hydroxyl radical and nitric oxide scavenging assays were 37.2 ± 1.7 µg/ml, 170.5 ± 6.6 µg/ml and 31.8 ± 2.2 µg/ml respectively. The reducing capability of AEQ was 6.9 ± 0.2 w/w % L-ascorbic acid equivalents in the FRAP assay. For hypotonic solution induced HEHA, the IC50 was 1.79 ± 0.04 mg/ml. A significant decrease (p < 0.05) was observed in ALT, AST and LDH release from the liver slices treated with AEQ compared to the ethanol treated liver slices. A significant reduction in lipid peroxidation (p < 0.05) was also observed in liver slices treated with the plant extract compared to that of the ethanol treated liver slices. CONCLUSIONS: The results suggest AEQ possess hepatoprotective activity against ethanol induced liver toxicity of porcine liver slices which can be attributed to antioxidant properties and membrane stabilizing effects caused by the plant material.