Ira Thabrew

A comparison of the cytotoxic potential of standardized aqueous and ethanolic extracts of a polyherbal mixture comprised of Nigella sativa (seeds), Hemidesmus indicus (roots) and Smilax glabra (rhizome)

Background: A decoction (hot-water extract) comprised of Nigella sativa (seeds), Hemidesmus indicus (roots), and Smilax glabra (rhizome) has been reported to prevent chemically-induced hepatocarcinogenic changes in rats and to exert significant cytotoxic effects on human hepatoma (HepG2) cells. However, the decoction used in previous studies to determine cytotoxicity was not standardized. Further, during preparation of pharmaceuticals for clinical use, it is more convenient to use an ethanolic extract. Therefore this study was carried out to (a) develop standardized aqueous and ethanolic extracts of the plant mixture (N. sativa, H. indicus, and S. glabra) used in the preparation of the original decoction, and (b) compare the cytotoxic effects of these two extracts by evaluating cytotoxicity to the human hepatoma (HepG2) cell line. Methods: Aqueous and ethanolic extracts have been standardized by evaluating organoleptic characters, physicochemical properties, qualitative and quantitative analysis of chemical constituents, and analysis of High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) profiles. Cytotoxic potentials of the above standardized extracts were compared by evaluating their effects on the survival and overall cell activity of HepG2 cells by use of the 3-(4, 5-dimethylthiazol-2yl) -2, 5 – biphenyl tetrazolium bromide (MTT) and Sulphorhodamine B (SRB) assays. Results: Results from MTT and SRB assays demonstrated that both extracts exerted strong dose-dependent in vitro cytotoxicity to HepG2 cells. The standardized aqueous extract showed a marginally (though significantly, P<0.05) higher cyotoxic potential than the ethanolic extract. Thymoquinone, an already known cytotoxic compound isolated from N. sativa seeds was only observed in the standardized ethanolic extract. Thus, compounds other than thymoquinone appear to mediate the cytotoxicity of the standardized aqueous extract of this poly-herbal preparation. Conclusion: It may be concluded that results obtained in the present study could be used as a diagnostic tool for the correct identification of these aqueous or ethanolic extracts and would be useful for the preparation of a standardized pharmaceutical product that may be used in the future for clinical therapy of hepatocellular carcinoma.

Modulation of apoptosis in human hepatocellular carcinoma (HepG2 cells) by a standardized herbal decoction of Nigella sativa seeds, Hemidesmus indicus roots and Smilax glabra rhizomes with anti- hepatocarcinogenic effects

BackgroundA standardized poly-herbal decoction of Nigella sativa seeds, Hemidesmus indicus roots and Smilax glabra rhizomes used traditionally in Sri Lanka for cancer therapy has been demonstrated previously, to have anti-hepatocarcinogenic potential. Cytotoxicity, antioxidant activity, anti-inflammatory activity, and up regulation of p53 and p21 activities are considered to be some of the possible mechanisms through which the above decoction may mediate its anti-hepatocarcinogenic action. The main aim of the present study was to determine whether apoptosis is also a major mechanism by which the decoction mediates its anti-hepatocarcinogenic action.MethodsEvaluation of apoptosis in HepG2 cells was carried out by (a) microscopic observations of cell morphology, (b) DNA fragmentation analysis, (c) activities of caspase 3 and 9, as well as by (d) analysis of the expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins associated with cell death.ResultsThe results demonstrated that in HepG2 cells, the decoction can induce (a) DNA fragmentation and (b) characteristic morphological changes associated with apoptosis (nuclear condensation, membrane blebbing, nuclear fragmentation and apoptotic bodies). The decoction could also, in a time and dose dependent manner, up regulate the expression of the pro-apoptotic gene Bax and down regulate expression of anti-apoptotic Bcl-2 gene (as evident from RT-PCR analysis, immunohistochemistry and western blotting). Further, the decoction significantly (p < .001) enhanced the activities of caspase-3 and caspase-9 in a time and dose dependent manner.ConclusionsOverall findings provide confirmatory evidence to demonstrate that the decoction may mediate its reported anti-hepatocarcinogenic effect, at least in part, through modulation of apoptosis.

Anti-inflammatory activity of Trichosanthes cucumerina Linn. in rats

AIM OF THE STUDY Trichosanthes cucumerina Linn. (Family: Cucurbitaceae) is one of the medicinal plants that is often used in Sri Lankan traditional systems of medicine. One of its uses is the treatment of inflammatory conditions. However, validity of the anti-inflammatory activity has not been scientifically investigated so far. Therefore, the aim of this study was to investigate the anti-inflammatory potential of Trichosanthes cucumerina hot water extract (HWE) and its fractions. MATERIALS AND METHODS The anti-inflammatory activity of Trichosanthes cucumerina was evaluated by use of the carrageenan-induced paw oedema model in Wistar rats. In addition, the mechanism/s by which Trichosanthes cucumerina is mediated the anti-inflammatory activity was assessed by determining its effects on (a) membrane stabilizing activity and (b) nitric oxide inhibitory activity. RESULTS Apart from the lowest dose of the HWE, other tested doses (500, 750, 1000 mg/kg) produced a significant (P ≤ 0.05) inhibition of the inflammation, most pronounced at 5h after the injection of carrageenan. The anti-inflammatory effect induced by 750 mg/kg, was comparable to that of the reference drug, indomethacin at 4 and 5h. Inhibition of nitric oxide (NO) production and membrane stabilization activities are probable mechanisms by which Trichosanthes cucumerina mediates its anti-inflammatory actions. Among the tested fractions, methanol fraction (MEF) and aqueous fraction (AQF) at a dose of 75 mg/kg exhibited marked inhibition against carrageenan-induced hind paw oedema. The anti-inflammatory effect induced by MEF, was comparable to that of the reference drug, indomethacin and as well as to the 750 mg/kg of HWE at 4 and 5h. CONCLUSIONS (a) These findings rationalize the traditional usage of this plant as an anti-inflammatory agent and (b) membrane stabilizing properties and NO inhibitory activity are possible mechanisms through which Trichosanthes cucumerina mediates its anti-inflammatory action.

A study of the potential anticancer activity of Mangifera zeylanica bark: Evaluation of cytotoxic and apoptotic effects of the hexane extract and bioassay-guided fractionation to identify phytochemical constituents

The present study investigated the potential anticancer activity of the bark of Mangifera zeylanica, an endemic plant in Sri Lanka that has been traditionally used for cancer therapy. Cytotoxic and apoptotic effects were investigated in vitro using sulphorodamine assay, acridine orange and ethidium bromide staining, caspase-3 and −7 activity, DNA fragmentation and reverse transcription-quantitative polymerase chain reaction in estrogen receptor positive MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines, SKOV-3 ovarian cancer cell line and MCF-10A normal mammary epithelial cells. Hexane extract demonstrated increased levels of cytotoxicity in cancer cells (IC50, 86.6–116.5 µg/ml) compared with normal cells (IC50, 217.2 µg/ml). Chloroform extract demonstrated increased cytotoxicity to normal cells (IC50, 92.9 µg/ml) compared with cancer cells (IC50, 280.1–506.5 µg/ml). Exposure to the hexane extract led to morphological changes characteristic of apoptosis and DNA fragmentation in the three cancer cell lines. Caspase-3 and −7 were significantly activated in MDA-MB-231 and SKOV-3 cells, indicating the occurrence of caspase-dependent apoptosis in these cells, and caspase-independent apoptosis in MCF-7 cells. Furthermore, upregulation of proapoptotic Bcl-2-associated X protein occurred in the three cancer cell lines, and antiapoptotic survivin was downregulated in MCF-7 and SKOV-3 cells; by contrast, tumor protein p53 was upregulated only in MCF-7 cells, suggesting p53-mediated apoptosis in MCF-7 cells and p53-independent apoptosis in the remaining cancerous cell lines. In addition, fraction M1 obtained from bioactivity-guided fractionation of the hexane extract demonstrated increased cytotoxicity in cancer cells (IC50, 15.4–38.7 µg/ml) compared with normal cells (IC50, 114.6 µg/ml), with the highest cytotoxicity observed in MDA-MB-231 triple-negative breast cancer cells. The hexane extract of M. zeylanica bark contained polyphenols and flavonoids, and caused free radical scavenging activity. Its gas chromatography-mass spectrometry profile revealed the presence of long-chain hydrocarbons, including β-sitosterol and β-amyrin. Fraction M1 contained seven unknown compounds and a small number of known non-cytotoxic compounds. Collectively, results obtained in the present study indicate that the hexane extract of M. zeylanica bark mediates cytotoxic activities through induction of apoptosis in three cancer cell lines; thus, the hexane extract may be used to isolate novel anti-cancer compounds.

New halogenated constituents from Mangifera zeylanica Hook.f. and their potential anti-cancer effects in breast and ovarian cancer cells.

ETHNOPHARMACOLOGICAL RELEVENCE Mangifera zeylanica Hook.f. (Anacardiaceae) is a plant endemic to Sri Lanka. Its bark has been used in traditional and Ayurvedic medicine for the treatment of various diseases including some cancers. AIM OF THE STUDY This study was planned to isolate and identify potentially cytotoxic compounds from the bark of M. zeylanica, which may have contributed to its ethno pharmacological use in the treatment of cancer. MATERIALS AND METHODS The chloroform extract of M. zeylanica bark which is cytotoxic to breast and ovarian cancer cells was fractionated using column chromatography and preparative reversed phase high performance liquid chromatography to isolate four compounds. Structures of the isolated compounds were elucidated by means of (1)H- and (13)C NMR spectroscopy, and mass spectrometric techniques. Cytotoxic potential of the isolated compounds was tested in MDA-MB-231 (triple negative breast cancer), MCF-7 (estrogen receptor positive breast cancer), SKOV-3 (ovarian epithelial cancer) and MCF-10A (normal mammary epithelial) cells by SRB assay. Human cancer drug target real-time PCR array was carried out to analyze regulation of possible cancer drug target genes in compound 2 treated triple negative breast cancer cells. DPPH radical scavenging and caspase 3 and 7 induction in response to isolated compounds were also studied. RESULTS Two new halogenated compounds, bromomangiferic acid (1), and chloromangiferamide (2) along with two known compounds quercetin (3), and catechin (4), were isolated from the bark of Mangifera zeylanica for the first time. Interestingly, chloromangiferamide showed cytotoxicity only to triple negative breast cancer cells [IC50:73.19±0.87µM (24h), 56.29±0.86µM (48h)] with no cytotoxicity to other two cancer cell lines or to normal mammary epithelial cells. Quercetin and catechin were cytotoxic to all three cancer cell lines while bromomangiferic acid had no effect. Chloromangiferamide significantly regulated expression of genes associated with apoptosis, drug metabolism, cell cycle, receptor tyrosine kinase signaling, protein kinases, histone deacetylases, growth factors and receptors, topoisomerases, PI-3 kinases and phosphatases in triple negative breast cancer cells. CONCLUSION Selective cytotoxic activity in triple negative breast cancer cells and regulation of some cancer drug target genes by chloromangiferamide indicate that it can be used to develop a potential chemotherapeutic agent for triple negative breast cancer cells.

Isolation of a new resorcinolic lipid from Mangifera zeylanica Hook.f. bark and its cytotoxic and apoptotic potential

Mangifera zeylanica is a plant endemic to Sri Lanka and its bark has been used in traditional medicine to treat some cancers. This study was aimed to isolate potentially cytotoxic compound/s from the hexane extract of the bark of M. zeylanica by bio-activity guided fractionation. The structure of the isolated compound (1) was elucidated using 1H, 13C NMR and mass spectrometric techniques. Compound 1 was identified as a new resorcinolic lipid (5-((8Z, 11Z, 14Z)-hexatriaconta-8, 11, 14-trienyl) benzene-1,3-diol). Apoptotic potential of the isolated compound was determined only in MCF-7 (estrogen receptor positive) breast cancer cells to which it was more cytotoxic than to normal mammary epithelial cells. Oxidative stress markers [reactive oxygen species (ROS), glutathione levels (GSH) and glutathione-S-transferase (GSH)] were also determined in MCF-7 cells treated with compound 1. Treatment with compound 1 led to an increase in caspase 7 activity, morphological features of apoptosis and DNA fragmentation in MCF-7 cells. Furthermore, it also led to an increase in ROS and GST levels while depleting GSH levels. Results of this study suggest that isolated new resorcinolic lipid can induce apoptosis in MCF-7 cells, possibly via oxidative stress mechanism.

In vitro and in vivo evaluation of antioxidant activity of Trichosanthes cucumerina aerial parts.

The present study was conducted to determine whether aerial parts of Trichosanthes cucumerina extracts can exert significant antioxidant activity. The antioxidant activity of a hot water extract (HWE) and a cold ethanolic extract (CE) of T. cucumerina aerial parts was evaluated by assessing its (a) radical scavenging ability and prevention effect of lipid peroxidation in vitro, and (b) effects on lipid peroxidation and antioxidant enzyme activities, in vivo.In vitro antioxidant assays (DPPH, TBARS and carotene-linoleic acid assays) clearly demonstrated the antioxidant potential of HWE and CEE. Moreover, HWE increased SOD: by 91.2% and GPX by 104.4% while CEE increased SOD: by 115.5% and GPX by 96.4%) in CCl4-induced rats. Treatments with HWE and CE prevented the accumulation of lipid peroxidation products by 30.5% and 33.8%, respectively, in liver tissues compared to the rats exposed only to CCl4. In conclusion, the present investigation demonstrates for the first time that components in T. cucumerina aerial parts can exert significant antioxidant activity in vivo and in vitro.

In Vitro Anticancer Effect of Gedunin on Human Teratocarcinomal (NTERA-2) Cancer Stem-Like Cells

Gedunin is one of the major compounds found in the neem tree (Azadirachta indica). In the present study, antiproliferative potential of gedunin was evaluated in human embryonal carcinoma cells (NTERA-2, a cancer stem cell model) and peripheral blood mononuclear cells (PBMCs), using Sulforhodamine (SRB) and WST-1 assays, respectively. The effects of gedunin on expression of heat shock protein 90 (HSP90), its cochaperone Cdc37, and HSP client proteins (AKT, ErbB2, and HSF1) were evaluated by real-time PCR. Effects of gedunin on apoptosis were evaluated by (a) apoptosis associated morphological changes, (b) caspase 3/7 expression, (c) DNA fragmentation, (d) TUNEL assay, and (e) real-time PCR of apoptosis related genes (Bax, p53, and survivin). Gedunin showed a promising antiproliferative effect in NTERA-2 cells with IC50 values of 14.59, 8.49, and 6.55 μg/mL at 24, 48, and 72 h after incubations, respectively, while exerting a minimal effect on PBMCs. Expression of HSP90, its client proteins, and survivin was inhibited and Bax and p53 were upregulated by gedunin. Apoptosis related morphological changes, DNA fragmentation, and increased caspase 3/7 activities confirmed the proapoptotic effects of gedunin. Collectively, results indicate that gedunin may be a good drug lead for treatment of chemo and radiotherapy resistant cancer stem cells.

Protective Effects of Six Selected Dietary Compounds against Leptin-Induced Proliferation of Oestrogen Receptor Positive (MCF-7) Breast Cancer Cells

Background: Obesity is considered as one of the risk factors for breast cancer. Leptin has been found to be involved in breast cancer progression. Therefore, novel approaches to antagonize biological effects of leptin are much needed. The objective of this study was to evaluate the protective effects of six dietary compounds (quercetin, curcumin, gallic acid, epigallocatechin gallate (EGCG), ascorbic acid and catechin) and assess the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in leptin-stimulated MCF-7 breast cancer cells in vitro. Methods: MCF-7 cells were exposed to leptin, leptin and compound and compound alone for 48 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT and fluorometric assays after 48 h incubation. Phosphorylation of ERK1/2 was quantified by ELISA. Results: Only quercetin, curcumin and EGCG showed significant protective effects against leptin-induced proliferation of MCF-7 cells. Increase in ERK1/2 phosphorylation in response to leptin was reduced by the addition of quercetin, curcumin and EGCG. Conclusions: Considering the high prevalence of obesity, this observation provides a rationale for use of curcumin, quercetin and EGCG as antagonists of leptin in the treatment of obese breast cancer patients.

Screening of Fifteen Mangrove Plants Found in Sri Lanka for in-vitro Cytotoxic Properties on Breast (MCF-7) and Hepatocellular Carcinoma (HepG2) Cells

Aims: Evaluation of cytotoxic potential on the leaves and stem bark extracts of 15 mangrove plants grown in Sri Lanka on breast cancer (MCF -7) and hepatocellular carcinoma (HepG2) cells. Place and Duration of Study: At the Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo between 1 of February 2014 to April 2015. Methodology: Leaves and stem barks of 15 mangrove plants were extracted with hexane, chloroform, ethyl acetate and methanol. Resulting extracts were screened for cytotoxic activity against MCF-7 and HepG2 cells using the Sulforhodamine B (SRB) assay. Original Research Article Samarakoon et al.; EJMP, 14(4): 1-11, 2016; Article no.EJMP.26107 2 Results: Phoenix paludosa, Avicennia officinalis and Scyphiphora hydrophyllacea showed highest cytotoxic properties on cancer cells. Chloroform extract of stem bark of S. hydrophyllacea, Bruguiera gymnorrhiza (chloroform, ethyl acetate and methanol extracts of leaves), hexane and ethyl acetate extracts of leaves of Aegiceras corniculatum, methanol extracts of leaves and stem bark of Nypa fruticans and Rhizophora mucronata, methanol extract of stem bark of Sonneratia alba and Rhizophora apiculata and methanol extract of bark of A. officinalis exerted selective cytotoxicity to HepG2 cells. The hexane extract of leaves of B. gymnorrhiza, chloroform extract of leaves of N. fruticans, ethyl acetate extract of stem bark of Lumnitzera littorea, chloroform extract of leaves of Rhizophora apiculata and chloroform extract of leaves of Pemphis acidula showed selective cytotoxic effects against MCF-7 cells. Out of the 116 mangrove extracts tested, 82 extracts showed no significant cytotoxic effects (IC50>100 μg/mL) against MCF 7 or HepG2 cells. Conclusion: The cytotoxic activities demonstrated by some of the solvent extracts of some mangrove plants provide scientific evidence for their therapeutic potentials and further studies are needed to identify active compounds responsible for cytotoxic effects.