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.

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.

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.

The present study investigated the anti-inflammatory effects of a polyherbal decoction comprised of Nigella sativa, Hemidesmus indicus, and Smilax glabra in order to justify its claimed antihepatocarcinogenic activity. Activation of hepatic nuclear factor-kappa B (NF-κB), IκB kinase (IKK α/β) proteins, and TNFα and IL-6 expression was investigated in diethylnitrosamine- (DEN-) induced C3H mice-bearing early hepatocarcinogenic changes. Acute phase inflammatory response was evaluated by carrageenan-induced rat paw edema formation. Anti-inflammatory mechanisms were also assessed by determining effect on (a) membrane stabilization, (b) nitric oxide (NO) inhibitory activity, and (c) inhibition of leukocyte migration. A significant inhibition of the paw edema formation was observed in healthy rats as well as in rats bearing early hepatocarcinogenic changes with oral administration of the decoction. As with the positive control, indomethacin (10 mg/kg b.w.) the inhibitory effect was pronounced at 3rd and 4th h after carrageenan injection. A notable IKK α/β mediated hepatic NF-κB inactivation was associated with a significant hepatic TNFα downregulation among mice-bearing hepatocarcinogenic changes subjected to decoction treatment. Inhibition of NO production, leukocyte migration, and membrane stabilization are possible mechanisms by which anti-inflammatory effect is mediated by the decoction. Overall findings imply that anti-inflammatory activity could be one of the mechanisms by which the decoction mediates its antihepatocarcinogenic effects.

Context:Scyphiphora hydrophyllacea is a shrub mangrove plant of the family Rubiaceae and not yet been studied for anti-hepatocarcinogenic effects.Objectives:We investigated possible in vitro anti-hepatocarcinogenic and antioxidant properties of S. hydrophyllacea.Materials and Methods:Dried leaves of S. hydrophyllacea were sequentially extracted into hexane, chloroform, ethyl acetate, and methanol and tested for cytotoxicity on HepG2 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and sulforhodamine B assays, and for antioxidant activities by the free radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assays. Total phenolic and flavonoid contents were estimated in all four extracts. The hexane and chloroform extracts were tested for pro-apoptotic properties in HepG2 cells, and bioactive components were identified by gas chromatography–mass spectrometry (GC-MS) analysis.Results:The hexane and chloroform extracts showed dose-dependent and time-dependent cytotoxic effects. Morphological changes observed under fluorescence microscope related to apoptosis, and significant (P < 0.001) increases in caspase 3 and 9 levels were observed in hexane and chloroform extract-treated cells. Slight DNA fragmentation was observed only in response to the chloroform extract. mRNA expressions of p53 and Bax were significantly upregulated by low doses of hexane and chloroform extracts. Highest antioxidant activity was observed in the methanol extract. GC-MS profiles identified 24 and four major compounds in the hexane and chloroform extracts, respectively. These included some known anticancer compounds such as lupeol.Conclusion:Cytotoxicity, antioxidant effects, and apoptosis-related changes exerted by hexane and chloroform extracts of S. hydrophyllacea concluded that these two extracts are good source for isolation of possible anticarcinogenic compounds.SUMMARY The hexane and chloroform extracts of Scyphiphora hydrophyllacea showed dose-dependent and time-dependent cytotoxic effects.Morphological changes related to apoptosis and significant (P < 0.001) increases in caspase 3 and 9 levels were observed in hexane and chloroform extract-treated cells.mRNA expressions of p53 and Bax were significantly upregulated by low doses of hexane and chloroform extracts.Highest antioxidant activity was observed in the methanol extract.GC-MS profiles identified 24 and four major compounds in the hexane and chloroform extracts, respectively. Abbreviation used: DPPH: 1,1-diphenyl-2-picryl-hydrazyl, ABTS: 2,2’-azinobis-3-ethylbenzthiazoline-6-sulfonic acid, GC-MS: gas chromatography–mass spectrometry, DNA: deoxyribonucleic acid, HCC: Hepatocellular carcinoma, GAE: gallic acid equivalents, SRB: sulforhodamine B, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, AO/EB: acridine orange/ethidium bromide, GAPDH: Glyceraldehyde 3-phosphate dehydrogenase, IC50: half maximal inhibitory concentration; QE: quercetin equivalents, HE: hexane extract, CE: chloroform ex...

Purpose: To evaluate in vitro (using human hepatoma HepG2 cells) and in vivo (using mouse liver with diethlynitrosamine (DEN)-induced hepatocarcinogenesis) effect of a standardized decoction on the expression of p53 (tumour suppressor) and p21 (cyclin kinase inhibitor) genes with the long-term goal of developing the formulation into a globally acceptable therapy for hepatocellular carcinoma (HCC). Methods: The effect of the decoction on (a) mRNA and (b) protein expression of p53 and p21 genes in HepG2 cells and mouse livers with DEN-induced early hepatocarcinogenesis were evaluated by (a) reverse transcription PCR (RT-PCR) and (b) immunohistochemical and Western blot analysis, respectively. Results:The results demonstrated that the decoction significantly (p < 0.001) enhanced the expression of p53 and p21 genes in a time-and dose-dependent manner in HepG2 cells. A dose of 75 µg/ml significantly increased p53 mRNA at 24 and 48 h and p21 mRNA at 12, 24, 48 h of incubation with the decoction (p < 0.01). Induction of hepatocarcinogenesis in mice significantly increased hepatic expression of both p53 and p21 compared to distilled water control (p < 0.001), while treatment with the decoction further enhanced expression of both genes in DEN-induced hepatocarcinogenesis (p < 0.01). Conclusion:Overall, the findings demonstrate that the decoction may mediate its reported antihepatocarcinogenic effect, at least in part, through the modulating activities of genes involved in tumour suppression and cell cycle arrest.

Y chromosome anomalies are closely associated with non-obstructive azoospermia (NOA), a major etiology in male infertility. Klinefelter syndrome (KS) and Y chromosome microdeletions are some of the well-identified genetic defects in this regard, while Y chromosome aneuploidies have been reported to be susceptive. We report the rare case of a patient presenting with three complex genetic defects: mosaic Y chromosome aneuploidy; loss of the heterochromatin region in the q arm of the Y chromosome (Yqh-); and azoospermia factor C subregion (AZFc) microdeletion. The patient reported he had been subfertile for five years. Semen analysis confirmed total azoospermia along with an unaffected hormonal profile for serum follicle stimulating hormone (FSH), luteinizing hormone (LH), and prolactin levels. Since the microdeletion analysis of azoospermia factor (AZF) region revealed the presence of three microdeletions in the AZFc region, the patient was offered intracytoplasmic sperm injection (ICSI) upon the retrieval of sperm by testicular sperm extraction (TESE) as the best possible assisted reproductive treatment (ART) option. It was further suggested to carry out pre-implantation genetic screening (PGS) in order to facilitate the transfer of only female embryos, thus preventing the dissemination of Y chromosomal anomalies.

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