Structural and functional validation of a cloned parasporin from Bacillus thuringiensis isolate KAU 41 native to Western Ghats of India.

Parasporins of Bacillus thuringiensis (Bt) exhibit specific toxicity to cancer cells. PCR based mining has identified apoptosis inducing parasporin in KAU41 Bt isolate from the Western Ghats of India. The study aimed to clone and overexpress the parasporin of native KAU41 Bt isolate for determining structural and functional characteristics of the protein. Parasporin gene was cloned in pGEM-T, sequenced, sub-cloned in pET30+ and overexpressed in Escherichia coli. The expressed protein was characterized by SDS-PAGE and in silico methods. Cytotoxicity of cleaved peptide was assessed by MTT assay. SDS-PAGE displayed a 31 kDa protein (rp-KAU41) overexpressed. Upon proteinase K digestion, the protein was cleaved into 29 kDa peptide which was found to be cytotoxic to HeLa cells. The protein has a deduced sequence of 267 amino acids with ß-strands folding pattern of crystal protein. Even though rp-KAU41 shared a 99.15% identity to chain-A of non-toxic crystal protein, it only showed a less similarity to the existing parasporins like PS4 (38%) and PS5 (24%) in UPGMA analysis, emphasizing the novelty of rp-KAU41. The protein is predicted to have more similarity to the pore forming toxins of Aerolysin superfamily and an additional loop in rp-KAU41 may be contributing towards its cytotoxicity. The molecular docking with caspase 3 resulted in higher Z dock and Z rank score substantiating its role in the activation of intrinsic pathway of apoptosis. The recombinant parasporin protein, rp-KAU41 is presumed to belong to the Aerolysin superfamily. An interaction with caspase 3 substantiates its role in activating the intrinsic pathway of apoptosis in cancer cells.

Chronic exposure of industrial grade calcium carbide and ethylene glycol exert genotoxic effect in Wistar albino rats.

OBJECTIVES: Calcium carbide (CaC2) and ethylene glycol (EG) are the two commonly used fruit ripening agents. The toxic effects of these chemicals on internal organs were reported in experimental animals. Even though the adverse effects of these compounds have been investigated for many years, there are no sufficient data available with regard to genotoxic effects. The present study evaluates the genotoxic effect of chronic exposures of CaC2 and EG in Wistar albino rats. METHODS: CaC2 and EG were administered to the rats orally for 180 days. Chromosomal aberrations and micronuclei formation were analysed in bone marrow and peripheral blood cells. Comet assay was performed to analyse the DNA strand break. The toxic effects of the chemicals were analysed by MTT assay with normal human intestinal epithelial (IEC-6) cells. RESULTS: Upon chronic exposure, CaC2 and EG caused chromosomal aberrations, micronuclei formation and  DNA strand breaks extensively in bone marrow and peripheral blood cells. In MTT assay, the chemicals were found to be toxic to IEC-6 cells with IC50 values at 160 and 200 µg/mL for CaC2 and EG, respectively. CONCLUSIONS: The results show that these chemicals have a potential to cause genomic level of toxicity which may lead to carcinogenic event at a chronic level exposure. The study warns to reinforce the administrative measures against the use of CaC2 and EG for fruit ripening process.

Cardioprotective effect of Justicia gendarussa on doxorubicin induced toxicity in mice.

Justicia gendarussa Burm.f, belonging to the family Acanthaceae, is widely used for various ailments traditionally. Antioxidant, anti-arthritic, anti-inflammatory, analgesic, anticancerous, properties of the plant have been widely reported. The present study analyzed the cardioprotective effect of J. gendarussa on doxorubicin (DOX) induced toxicity in mice. Ethanolic extract of J. gendarussa was administered orally for 7 consecutive days. The alterations in oxido-reduction status, biochemical and histopathological parameters were analyzed in heart tissue. DOX increased superoxide dismutase (SOD) and catalase activities to 3.4 ± 0.5 and 3.68 ± 1 from their normal values 2.43 ± 0.8 and 2.72 ± 0.88, respectively. The increased activities of both the enzymes were found reduced to 3.12 ± 0.24 and 3.41 ± 0.65 by the treatment of the extract. Similarly, DOX elevated glutathione peroxidase (GPx) activity to 44.6 ± 3.71 from the normal level 32.33 ± 3.41. DOX decreased the glutathione (GSH) level to 15.66 ± 2.51 from the normal values 31.66 ± 4.05. Upon treatment, GPx activity and GHS level found restored. The increased lipid peroxidation 2.53 ± 0.25 of DOX was also decreased to 2.0 ± 0.34 by the extract. Histopathology observations substantiate the protective effect of J. gendarussa extract. In conclusion, DOX-induced disturbance of oxido-reduction status and histopathology of heart attenuated closer to the normal indicating the protective effect of J. gendarussa against DOX-induced toxicity in cardiomyocytes.

Virgin coconut oil complements with its polyphenol components mitigate sodium fluoride toxicity in vitro and in vivo.

Virgin coconut oil (VCO), prepared from fresh coconut kernel without any chemical refining, is an emerging functional food. The pharmacological benefits of VCO are believed to be due to the natural combination of phenolics. Although cell culture studies have demonstrated the antioxidant activity of VCO under different oxidative stress conditions, a valid in vivo demonstration of the antioxidant activity of VCO is yet to come. Sodium fluoride (NaF), an environmental pollutant, is widely used to induce oxidative stress in cell culture models and rodents to test the antioxidant potential of several compounds. Herein, VCO and its polyphenolic (VCOP) and non-phenolic oil fraction (VCOF) were individually tested in fluoride-exposed normal intestinal cells (IEC-6) and mice to address their contribution to the documented antioxidant potential. It was found that pretreatment of VCOP (40 µg/mL) was effective in mitigating the fluoride-induced cell death when compared to VCO (200 µg/mL) and VCOF (160 µg/mL). Further, exposure to fluoride (10 mM), increased the intracellular ROS measured based on the dichlorofluorescein (DCF) fluorescence, and this, in turn, was significantly reduced when the cells were supplemented with VCOP. Oral administration of VCO (2 mL/kg bwt) reversed the drop in the hepatic catalase and SOD activities to near normal with a minimal level of lipid peroxidation in fluoride intoxicated mice. However, VCOP and VCOF were less effective in lowering the fluoride-induced increase in hepatic oxidative stress markers. It is reasoned that the oil components of VCO complement the natural antioxidant molecules resulting in an overall increase in their bioavailability.

Chronic exposure of industrial grade calcium carbide and ethylene glycol alter histological architecture of systemic organs by disrupting redox balance in rat.

OBJECTIVES: The threat to human health or the surroundings by the use of artificial fruit ripening agents has become a global concern. Calcium carbide (CaC2) and ethylene glycol (EG) are the two widely using ripening agents. The present study evaluates the toxic effect of chronic exposures of CaC2 and EG in rats. METHODS: CaC2 and EG were administered to the rats for 180 days orally. The alterations in oxido-reduction status, haematological, biochemical and histopathological parameters were analysed. Arsenic content in CaC2 and animal samples were detected by atomic absorption spectrometer and phosphorus by molybdate-UV method. RESULTS: At chronic doses, there were no significant alterations in haematological and biochemical parameters except in creatinine level especially by EG. However, histological details revealed microvesicular fatty change in liver, corpuscles degeneration in kidney and lymphocytes infiltration in various tissues. In intestine, the mucosal lesion scoring was found high (p<0.01). SOD and CAT activities and GSH level was reduced significantly by CaC2 administration (p<0.01). Arsenic and phosphorus detected is above the toxic level: 7.222 and 13.91 mg/dL in CaC2, 1.634 and 6.22 mg/dL in blood and 0.563 and 6.99 mg/dL in liver, respectively. CONCLUSIONS: The study suggests that the industrial grade CaC2 and EG induce systemic toxicity to rats and the liver is the most susceptible organ. The CaC2 and EG toxicity is mediated through the upset of redox balance and subsequent inflammatory responses. This could be due to the presence of arsenic and phosphorus contents that detected above the normal level in the industrial grade CaC2.

Acute and subacute toxicity evaluation of calcium carbide and ethylene glycol in Wistar albino rats.

Background Naturally ripened fruits play a vital role in human nutrition. Under certain conditions, synthetic chemicals like calcium carbide (CaC2) and ethylene glycol (EG) are being freely used illegally in India and other countries for fruit ripening without serious concern on its toxic effects. This preclinical study evaluated the toxicity on different organs after the exposure of industrial-grade CaC2 and EG to the rats. Methods Acute toxicity was induced by the oral administration of a single dose of chemicals to the rats, and their morbidity and mortality were monitored. For subacute study, different organs of animals were analyzed biochemically and histologically after the exposure of low doses of chemicals for 30 days. Results At an acute dose of 5 mg/kg body weight of CaC2, 85% of the animals were found dead within 14 days; however, no mortality was observed following EG administration. At subacute doses, RBC and hemoglobin levels were found to be declined (p < 0.01), whereas total WBC and platelet counts, especially lymphocytes, were elevated remarkably (p < 0.01). Total protein, albumin, and urea were also found to be increased (p < 0.01). Histopathological observations support the toxicity in rats at higher doses of CaC2 and EG. Conclusions The study revealed that the artificial fruit-ripening agents like CaC2 and EG cause toxic effects on the internal organs of rats. The subsequent inflammatory response might have weakened the immune system. This in turn suggests the requisite for urgent measures to regulate the use of harmful synthetic agents in fruit ripening.

A parasporin from Bacillus thuringiensis native to Peninsular India induces apoptosis in cancer cells through intrinsic pathway.

Parasporins, a class of non-insecticidal crystal proteins of Bacillus thuringiensis (Bt) are being explored as promising anticancer agents due to their specific toxicity to cancer cells. The present study has identified 25 Bt isolates harbouring parasporin genes from Western Ghats region, the hotspot of biodiversity in India. Among these, the isolate, KAU 41 (Kerala Agricultural University isolate 41) contained non-hemolytic homogenous crystals showing specific cytotoxicity towards cancer cells. SDS-PAGE analysis of this crystal, isolated by aqueous biphasic separation, revealed a 31 kDa sized peptide. The N-terminal sequence deciphered in BLAST analysis showed homology to a hypothetical Bt protein. Upon proteolysis, a 29 kDa active peptide was generated which exhibited heterogenic cytotoxic spectrum on various cancer cells. HeLa cells were highly susceptible to this peptide with IC 50 1 lg/mL and showed characteristics of apoptosis. RT-qPCR analysis revealed the overexpression of APAF1, caspase 3 and 9 by 14.9, 8 and 7.4 fold, respectively which indicates the activation of intrinsic pathway of apoptosis. However, at higher concentrations of peptide (greater than 3 lg/mL), necrotic death was prominent. The results suggest that the 31 kDa protein from Bt isolate, KAU 41 is a parasporin that may have high therapeutic potential.

Non-enzymatic conversion of primary oxidation products of Docosahexaenoic acid into less toxic acid molecules.

Docosahexaenoic acid (DHA) is long chain omega-3 fatty acid with known health benefits and clinical significance. However, 4-hydroxy hexenal (HHE), an enzymatic oxidation product of DHA has recently been reported to have health-damaging effects. This conflict raises major concern on the long-term clinical use of these fatty acids. Even though the enzymatic and non-enzymatic conversion of HHE to nontoxic acid molecules is possible by the aldehyde detoxification systems, it has not yet studied. To address this, primary oxidation products of DHA in lipoxidase system were subjected to non-enzymatic conversion at physiological temperature over a period of 1 week. The reaction was monitored using HPLC, IR spectroscopy and biochemical assays (based on the loss of conjugated dienes, lipid peroxides aldehydes). Short term and long term cytotoxicity of the compounds generated at various time points were analyzed. IR and HPLC spectra revealed that the level of aldehydes in the primary oxidation products reduced over time, generating acids and acid derivatives within a week period. In short term and long term cytotoxicity analysis, initial decomposition products were found more toxic than the 1-week decomposition products. Further, when primary oxidation products were subjected to aldehyde dehydrogenase mediated oxidation, it generated products that are also less toxic. The study suggests the possible non-enzymatic conversion of primary oxidation products of DHA to less cytotoxic acid molecules. Exploration of the physiological roles of these acid molecules may explain the biological potential of omega-3 fatty acids.

Kingiodendron pinnatum, a pharmacologically effective alternative for Saraca asoca in an Ayurvedic preparation, Asokarishta.

Saraca asoca (Fabaceae) is a prime ingredient in Asokarishta, a well-known Ayurvedic preparation for gynecological ailments. Due to scarcity, adulteration or substitution of related raw drugs is a common practice in its preparation. The bark of Kingiodendron pinnatum (Roxb. ex DC.) Harms, morphologically similar to S. asoca (Asoka) is a widely used substitute. The present study aimed to evaluate the pharmacological effectiveness of K. pinnatum as an alternative for S. asoca in Asokarishta by determining the inhibitory effect of estrogen induced uterus endometrial thickening in immature female rats. Arishta was prepared using S. asoca and with the substitute, K. pinnatum as per Ayurvedic Pharmacopeia. Uterus endometrial thickening was induced by the administration of estradiol (20 µg/kg b. wt, i.p) to 8-day-old rats for 5 alternate days. On day 16, following estradiol administration, the serum estrogen level was found elevated to 156.5 ± 8 pg/ml from the normal value 32.4 ± 5 pg/ml and consequently increased the thickness of uterus endometrium from 16.7 ± 1.4 to 75.2 ± 15.3 µm. Upon oral administration of 400 µl/kg b. wt Asokarishta (ASA) and Arishta made with K. pinnatum (AKP), the thickening was reduced to 42.5 ± 12.7 and 47.1 ± 10.5 µm and the estrogen level diminished to 102.6 ± 10 and 97.3 ± 8 pg/ml, respectively. Arishta also reduced the chronic/acute inflammations in mice and improved the antioxidant status of rats. No toxic symptom was observed in the animals by the treatment of Arishta. The study supports the use of K. pinnatum as an alternative to S. asoca in Asokarishta and gives a scientific validation for Asokarishta in gynecological ailments.

Ameliorative effect of Apodytes dimidiata on cisplatin-induced nephrotoxicity in Wistar rats.

Context Nutraceuticals possessing antioxidant potential have been used to alleviate side effects exerted by many chemotherapeutics, including cisplatin. Since Apodytes dimidiata E. Mey. Ex Arn. (Icacinaceae) shows antioxidant potential, it may possess significant chemoprotective effects. Objectives The study investigated whether A. dimidiata could attenuate cisplatin-induced renal damage. Materials and methods Nephrotoxicity was induced by cisplatin (single i.p., 16 mg/kg b wt.) in Wistar rats. Methanolic leaf extract of A. dimidiata (AMF) was administered at a dose of 250 mg/kg b. wt. orally for 5 consecutive days before/after cisplatin administration. Blood and renal parameters were analysed. Total phenolic and flavonoid content in AMF and its NO scavenging effect was determined. Results Significant protective effect of AMF on cisplatin-induced nephrotoxicity was observed in pre-treated animals. The reduction of urea, creatinine and lipid peroxidation was 58.31%, 42.19% and 60%, respectively, and the increase in haemoglobin and leucocyte count was 28.25% and 42.91%, respectively. The increase calculated for GSH, GPx, SOD and catalase was 35.64%, 18.14%, 74.42% and 35.46%, respectively. Tissue architecture of kidney was almost normal in AMF treated animals. The results were comparable to the standard drug, silymarin. AMF contained high level of polyphenols and flavonoids and was found to scavenge NO radicals (IC50 121.8 µg/mL). Discussion and conclusion AMF can effectively counteract cisplatin mediated renal acute toxicity possibly by scavenging reactive oxygen and nitrogen species. Accordingly, the study suggests that AMF can ameliorate free radical-induced damage associated with chemotherapeutic drugs.

Protective effect of Scutellaria species on AAPH-induced oxidative damage in human erythrocyte.

BACKGROUND: Scutellaria baicalensis is a well-known plant in traditional Chinese medicine. Recently, several Scutellaria species with therapeutic potential have been recognized worldwide. Scutellaria colebrookiana and Scutellaria violacea, native to the Western Ghats of India, are reported to possess free radical scavenging efficacy. At present, the protective effect of these Scutellaria spp. against 2,2' azobis (2-amidinopropane) hydrochloride (AAPH)-induced oxidative damage in human erythrocytes has been analyzed. METHODS: Oxidative stress in erythrocyte was induced by AAPH. The inhibition of hemolysis, membrane lipid peroxidation, and protein damage by chloroform extracts of Scutellaria spp. was assessed biochemically. Phytochemicals of the extracts were analyzed by Fourier transform infrared spectrophotometer (FTIR). RESULTS: Approximately 95% of erythrocytes were lysed by AAPH over 3 h of incubation. Significant reduction in hemolysis was observed by the extracts, and the IC50 values were 18.3 and 23.5 µg/mL for S. colebrookiana and S. violacea, respectively. Both the extracts were found to inhibit AAPH-induced lipid peroxidation in ghost membrane with IC50 92±2.8 and 70±5.6 µg/mL. In the analysis of the membrane proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the AAPH-induced degradation of actin was found reduced by both the extracts. The FTIR spectrum revealed the presence of polyphenols, carboxylic acids, alkanes, and aromatic compounds in extracts. In quantitative analysis, the total polyphenolic content estimated was 380±0.23 and 203.7±1.4 mg of gallic acid equivalent per gram extract of S. colebrookiana and S. violacea. CONCLUSIONS: Results indicate that S. colebrookiana and S. violacea are capable of protecting erythrocytes from oxidative damage. This cytoprotective effect of the extract is possibly by its antioxidant property.

Evaluation of Cytotoxic and Antitumour Properties of Apodytes dimidiata and Characterisation of the Bioactive Component.

Apodytes dimidiata, belonging to the family Icacinaceae, is used for treating inflammation and various gastrointestinal ailments in Zulu traditional medicine. In the present study, significant cytotoxicity was exhibited by the methanolic extract of the A. dimidiata leaf against various cancer cell lines. The extract was purified partially through silica gel column by successive elution using various solvents of increasing polarity. Among these, the active methanolic fraction was found to be the most cytotoxic with IC50 values ranging from 0.92 to 3.95 µg/mL for Ehrlich's ascites carcinoma (a carcinoma cell line), Jurkat (human T lymphocyte cell line), and SK-BR-3 (mammary tumour cell line). The treated cells showed morphological alterations characteristic of apoptosis. Upon oral administration of active methanolic fraction at a dose of 250 mg/kg body weight, the solid tumour volume in mice was significantly reduced to 55.14% and the life span of the ascites tumour-bearing mice increased to 44.65% compared to untreated control. The active fraction with Rf value 0.56 was purified from the methanolic fraction by preparative thin-layer chromatography and was subjected to high-performance thin-layer chromatography, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and nuclear magnetic resonance analysis. The iridoid glycoside genipin was identified as the active component.

Effect of Saraca asoca (Asoka) on estradiol-induced keratinizing metaplasia in rat uterus.

BACKGROUND: Estrogen-mediated uterus endometrium instability is considered as one of the etiological factors in dysfunctional uterine bleeding (DUB) and uterine cancer. Saraca asoca (Family: Fabaceae) and its fermented preparation, Asokarishta, are extensively used as uterine tonic to treat gynecological disorders in Ayurveda. The present study evaluated the effect of S. asoca (Asoka) on estrogen-induced endometrial thickening of rat uterus. METHODS: Endometrial thickening was induced by intraperitoneal injection of estradiol (20 µg/kg b.wt) to 8-day-old immature rats for alternate 5 days. Methanolic extract (200 mg/kg b. wt) from S. asoca bark was given orally along with estradiol. Uterus endometrial thickening was analyzed histopathologically and serum estrogen level by radioimmunoassay (RIA). Cyclooxygenase (COX-2) expression in rat uterus was also estimated by Western blot. Anti-inflammatory activity of the extract was analyzed by formalin- and carrageenan-elicited paw edema models in mouse. RESULTS: Uterus endometrium proliferation and keratinized metaplasia with seven to eight stratified epithelial layers on day 16 was observed in rats administered with estradiol. Treatment with S. asoca reduced the thickening to two to four layers and the serum estrogen level diminished significantly to 82.9±12.87 pg/mL compared to rats administered with estrogen alone (111.2±10.68 pg/mL). A reduction of formalin- and carrageenan-induced paw edema in mouse by S. asoca extract was observed. Lower level of lipopolysaccharides (LPS)-induced COX-2 enzyme in rat uterus by the extract further confirms its anti-inflammatory activity. CONCLUSIONS: Present study reveals the antiproliferative and antikeratinizing effects of S. asoca in uterus endometrium possibly through its anti-estrogenic and anti-inflammatory properties.