Effect of luteolin on the gene level expression of apoptosis-associated speck-like protein containing a caspase recruitment domain of NLRP3 inflammasome and NF-κB in rats subjected to experimental pancreatitis - influence of HSP70.

OBJECTIVES: Nod-like receptor pyrin domain containing 3 (NLRP3) is one of the well characterized inflammasome that controls the maturation of pro-inflammatory cytokines and thereby the inflammation in pancreas which could be a promising target for anti-inflammatory drugs. The present study is aimed to explore whether luteolin can target the NLRP3 inflammasome and modulate its activity through the signaling protein, HSP70 in the ethanol-cerulein model of experimental pancreatitis. METHODS: Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats received normal diet. Groups 3 and 4 rats received isocalorically adjusted diet containing ethanol for 5 weeks and cerulein (20 µg/kg body weight i.p., thrice weekly for the last 3 weeks of the experimental period). Additionally, group 2 and 4 rats received 2 mg/kg body weight of luteolin orally from third week. RESULTS: Luteolin co-administration decreased the serum levels of HSP70, oxidative stress markers, myeloperoxidase, GSH/GSSG and GST with concomitant downregulation in the mRNA expression of HSP70, caspase-1, ASC-NLRP3 and NF-κB. Spearman's rank correlation test showed that serum HSP70 has positive correlation with the expression of ASC-NLRP3, caspase-1, NF-κB and 4-hydroxynonenal and negative correlation with GSH:GSSG ratio. CONCLUSIONS: The modulating effect of luteolin on the expression of HSP70, NF-κB and thereby on ASC-NLRP3 complex may be claimed for its pancreato-protective activity.

Effect of Morus alba root bark extract on gene-level expression of inflammatory markers in rats subjected to ethanol and cerulein induced pancreatitis- influence of heat shock protein 70.

Background Chronic pancreatitis (CP) is a persistent inflammation of the pancreas clinically presented with severe abdominal pain, progressive fibrosis, and loss of exocrine and endocrine functions. Inflammasomes, cytosolic multiprotein complexes which regulate the formation of proinflammatory cytokines, are influenced by various factors including heat shock proteins (HSPs). Morus alba L., or white mulberry root bark is a valued traditional Asian medicine with a diverse array of phytochemicals. The aim of this investigation was to define the modulatory action of methanolic extract of Morus alba root bark (MEMARB) on NLRP3 inflammasome, and HSPs in pancreas subjected to inflammatory insult. Methods Pancreatitis was induced in male albino Wistar rats by ethanol (0-36%) and cerulein (20 µg/kg b.wt., i.p.) for 5 weeks with or without MEMARB administration. Serum lipase/amylase (L/A) ratio, oxidative stress index (OSI) and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in the pancreas were evaluated. Levels of serum HSP70 was quantified by ELISA. NF-kappa B, NLRP3-ASC, caspase-1, IL-1ß, IL-18, and HSP70 gene expression was quantified by quantitative real-time polymerase chain reaction (qPCR). Results L/A ratio and oxidative stress determined in terms of OSI and GSH/GSSG ratio were elevated in pancreatitis-induced rats. The levels were restored in MEMARB co-administered animals. Serum level of HSP70 was increased in pancreatitis-induced animals and dropped significantly in MEMARB co-administrated rats. Pancreatitis-induced group showed increased expression of NF-kappa B, IL-1ß, IL-18, caspase-1, NLRP3-ASC and HSP70 mRNA than in MEMARB treated group. Conclusions It can be concluded that the M. alba root extract modulates the expression of HSP70 and NLRP3-ASC which might be attributed to its pancreato-protective effect.

Anti-inflammatory and preventive activity of white mulberry root bark extract in an experimental model of pancreatitis.

Pancreatitis is characterized by highly morbid inflammation in the pancreas. Currently, there is no specific drug available for pancreatitis except supportive medicines. The present study assessed the pancreato-protective effect of Morus alba root bark extract by using alcohol and cerulein-induced model of pancreatitis. The study also investigated the phytochemical profile through GC-MS and HPLC. Methanolic extract of Morus alba root bark extract (MEMARB) was subjected to GC-MS and HPLC studies. Male albino Wistar rats were administered ethanol (0%-36%) and cerulein (20 µg/kg b.wt. i.p.) with or without MEMARB. Serum lipase, amylase, caspase-1, lipid peroxidation products, glutathione and enzymatic antioxidants were determined. Histological changes in the pancreas were assessed. Cudraflavone B in MEMARB was quantified by HPLC. Significant amount of Cudraflavone B was detected by quantitative HPLC. Marked increase in the levels of serum amylase, lipase, caspase-1, IL-18 and IL-1ß were observed in ethanol and cerulein administered rats than in MEMARB co-administered rats. In MEMARB co-administered rats, the antioxidant status was restored to near normal levels. Histological examinations showed that MEMARB significantly reduced the inflammatory and fibrotic changes. The results reveal the potent pancreato-protective effects of Morus alba root bark. The anti-inflammatory effect of Morus alba root bark extract might be due to the presence of various phytonutrients including Cudraflavone B.

A study on the GC-MS analysis of bioactive components and pancreato-protective effect of methanolic extract of Brassica oleracea L. var. botrytis.

The ever-increasing problem of pancreatitis due to alcohol abuse demands evaluation of novel drugs of plant origin. This study explores the therapeutic effects of the methanolic extract of Brassica oleraceae (MEBO) on ethanol and cerulein induced pancreatitis in rats. The MEBO was subjected to GC-MS and HPLC analysis. Male albino Wistar rats were divided into various groups, fed with alcohol (36% of total calories for 5 weeks) and cerulein (20 µg/kg b.wt i.p, weekly thrice for last three weeks) with or without MEBO (40 mg/kg b.wt). Serum lipase, amylase, IL-1ß, IL-18, caspase-1, lipid peroxides, oxidative stress index and antioxidant status were assessed in pancreas. Six compounds were identified in GC-MS analysis. Co-administration of MEBO reduced the pancreatic marker enzymes in serum, IL-1ß, IL-18 and caspase-1 and increased the antioxidant status of pancreas. The pancreato-protective effect of Brassica oleraceae may be attributed to well-known anti-inflammatory flavonoids, luteolin, quercetin and myricetin.

Effect of hydroalcoholic fruit extract of Persea americana Mill. on high fat diet induced obesity: A dose response study in rats.

The fruits of Persea Americana Mill., commonly known as Avocado, are traditionally consumed for various health benefits including weight reduction. Here, we studied the effect of hydroalcoholic fruit extract of Persea americana (HAEPA) on high fat diet (HFD) induced obesity in rats. Obesity was induced in male Sprague Dawley rats by feeding HFD for 14 wk. The hypolipidemic effect was evaluated by co-administering 25, 50, 100 and 200 mg/kg body wt. of HAEPA. There was a significant increase in weight gain, body mass index (BMI), blood lipids, low density lipoproteins (LDL), lipid peroxides (LPO) and serum transaminases in HFD fed rats. HFD+HAEPA fed rats showed a significant decrease in blood lipids, LPO, liver lipids and increase in antioxidant status when compared to HFD control rats. The activity of lipid metabolic key enzymes such as fatty acid synthase and HMG CoA reductase in liver were also found to be decreased significantly in HAEPA co-administered rats. Lipoprotein lipase activity was found increased in HFD+HAEPA rats. Among the 4 doses studied, 100 mg of HAEPA/kg body wt. exhibited optimum hypolipidemic activity. Histopathological observations in liver and visceral adipose tissue added more evidence for the lipid lowering effect of HAEPA. It can be concluded that avocado fruit extract can act as hypolipidemic agent probably by modulating the activities of HMG CoA reductase and fatty acid synthase in liver.

Effect of Pithecellobium dulce (Roxb.) Benth. fruit extract on cysteamine induced duodenal ulcer in rats.

The edible fruits of Pithecellobium dulce (Roxb.) Benth. are traditionally used for various gastric complications in India. Here, we investigated the antiulcer activity of hydroalcoholic fruit extract of P. dulce (HAEPD) by applying cysteamine induced duodenal ulcer model in rats. Duodenal ulcer was induced in male albino Wistar rats by oral administration of cysteamine @ 420 mg/kg body wt. as a single dose. The rats were pre-administered orally with HAEPD @ 200 mg/kg body wt. for 30 days prior to ulcer induction. Rats pre-administered with ranitidine @ 30 mg/kg body wt. served as reference drug control. Ulcer score, thiobarbituric acid reactive substances (TBARS), glycoproteins, superoxide dismutase, catalase and glutathione peroxidase and reduced glutathione levels were measured in the duodenum. Rats pre-administered with the HAEPD showed significantly reduced ulcer score comparable to that of ranitidine pretreated rats. The co-administration of HAEPD lowered the TBARS level and also restored the levels of glycoproteins, enzymatic and non-enzymatic antioxidants. Histopathological observations confirmed the presence of inflammation, necrosis and hemorrhagic spots in the duodenum of ulcer control rats which were significantly reduced due to HAEPD treatment. No abnormal alterations were observed in normal rats treated with HAEPD at the dosage studied. The results demonstrated antioxidant and cytoprotective nature of P. dulce, and thereby its significant anti ulcer property.

The modulating effect of Persea americana fruit extract on the level of expression of fatty acid synthase complex, lipoprotein lipase, fibroblast growth factor-21 and leptin--A biochemical study in rats subjected to experimental hyperlipidemia and obesity.

BACKGROUND: Obesity is a multifactorial disorder which is closely associated with hyperlipidemia. Avocados are edible fruits traditionally consumed for various health benefits including body weight reduction. HYPOTHESIS/PURPOSE: To determine the hypolipidemic and anti-obesity effect of hydro-alcoholic fruit extract of avocado (HFEA) in rats fed with high fat diet (HFD). STUDY DESIGN: Male Sprague Dawley rats were divided into four groups. Groups 1 and 2 rats were fed with normal diet. Groups 3 and 4 rats were fed with HFD for 14 weeks. In addition, Groups 2 and 4 rats were co-administered with 100 mg/kg body weight of HFEA from 3rd week onwards. METHODS: The HFEA was subjected to HPLC to quantify the major phytonutrients. Body mass index (BMI), adiposity index (ADI), total fat pad mass (TFP), blood lipid levels were determined in all the groups of rats. The mRNA expression of fatty acid synthase (FASN), lipoprotein lipase (LPL), fibroblast growth factor 21 (FGF21) and leptin was also assessed. RESULTS: HFEA was found to contain flavonoids: rutin-141.79, quercetin-5.25, luteolin-165, phenolic compounds: gallic acid-198.57, ellagic acid-238.22, vanillic acid-4.79 and phytosterols: betasitosterol-70, stigmasterol-12.5 (mg/100 g). HFEA reduced BMI, ADI, TFP, blood cholesterol, triglycerides, and LDL in rats fed with HFD. Serum leptin was found reduced in HFEA co-administered rats. The mRNA expression of FASN, LPL, and leptin in subcutaneous and visceral adipose tissue was found to be significantly reduced in HFEA co-administered rats. The gene expression of fibroblast growth factor-21 (FGF21) was found to be significantly increased in HFEA treated rats when compared to HFD control rats. CONCLUSION: The hypolipidemic effect of HFEA may be partly due to its modulating effect on endogenous fat synthesis and adiponectin formation through the transcription factor FGF21. The results also show that avocado fruit extract has profound influence on leptin activity, which controls satiety and hunger to regulate the food intake.

Urine proteome analysis to evaluate protein biomarkers in children with autism.

BACKGROUND: Autism is a complex developmental disability for which no specific diagnostic markers have been identified so far. The present study aimed to evaluate whether there is any abnormal protein(s) excreted in the urine of autistic children by proteome analysis which may act as diagnostic marker. METHODS: Urine proteome analysis was carried out in first void urine samples of autistic and normal children (n=30) in the age group of 4-12 years by 2D-PAGE followed by MALDI-TOF-MS analysis. RESULTS: Comparison of 2D-PAGE gels revealed that many urinary proteins are expressed differentially in autistic children. Total numbers of spots observed were 250 and 159 in autism and normal samples respectively, out of which 95 matches were observed. In addition, 3 spots of abnormally expressed peptides were selected, excised and analyzed. Peptide sequence with significant match score was for kininogen-1 (KNG-1)-50 (spot-1), IgG1 heavy chain variable region-35(spot-2) and mannan-binding lectin serine protease-2 isoform-2 precursor-45(spot-3). All the autistic children showed significant increase (p<0.001) in urinary kininogen level measured quantitatively by ELISA, when compared to normal children. CONCLUSION: Increased urinary kininogen-1 level in all the autistic children and the possibility of this protein as a diagnostic marker need further investigation.

Rutin modulates ASC expression in NLRP3 inflammasome: a study in alcohol and cerulein-induced rat model of pancreatitis.

Inflammasomes are protein complexes formed in response to tissue injury and inflammation to regulate the formation of proinflammatory cytokines. Nod-like receptor pyrin domain containing 3 (NLRP3) is one such inflammasome involved in pancreatic inflammation. Caspase activation recruitment domain (CARD) is an interaction motif found in all the major components of NLRP3 inflammasome such as apoptosis associated speck-like CARD containing protein (ASC) and procaspase-1. NLRP3 activates procaspase-1 with the concerted action of CARD domain of ASC. In the present study, the effect of rutin, a natural flavonoid on the expression of ASC of NLRP3, was investigated in rats treated with ethanol (EtOH) and cerulein (Cer). Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats were fed normal diet, whereas groups 3 and 4 rats were fed EtOH (36 % of total calories) containing diet for a total period of 5 weeks and also administered Cer (20 µg/kg body weight i.p.) thrice weekly for the last 3 weeks. In addition, groups 2 and 4 rats received daily 100 mg/kg body weight of rutin from third week. Rutin co-administration significantly decreased the level of pancreatic marker enzymes, oxidative stress markers, inflammatory markers, mRNA expression of caspase-1, cytokines, ASC-NLRP3, and protein expression of caspase-1 and ASC in rats received EtOH-Cer. The results of the study revealed that rutin can reduce inflammation in pancreas probably by influencing the down regulation of ASC-NLRP3 which might result in the reduced activation of caspase-1 and controlled cytokine production.

Rutin rich Emblica officinalis Geart. fruit extract ameliorates inflammation in the pancreas of rats subjected to alcohol and cerulein administration.

BACKGROUND: The modulating effect of methanolic extract of Emblica officinalis (MEEO) on ethanol (EtOH)- and cerulein (Cer)-induced pancreatitis in rats was investigated in this study. METHODS: Male albino Wistar rats were divided into four groups. Group 1 and 2 rats served as control and fed normal diet. Group 3 and 4 rats were fed isocalorically adjusted diet containing EtOH (36% of total calories) for 5 weeks and also subjected to intraperitoneal injection of Cer 20 µg/kg b.wt. thrice weekly for the last 3 weeks of the experimental period. In addition, group 2 and 4 rats received 200 mg/kg b.wt. of MEEO from 15th day till the experimental period. Serum levels of lipase (L), amylase (A), cytokines IL-1ß, IL-18, caspase-1 and oxidative stress index (OSI) were determined. Levels of fecal trypsin, total collagen, caspase-1, myeloperoxidase (MPO), antioxidants and mRNA expression of caspase-1, IL-1ß and IL-18 were determined in the pancreas. RESULTS: HPLC analysis showed the presence of rutin in MEEO. We observed a significant elevation in serum L/A ratio, IL-1ß, IL-18, caspase-1, OSI, collagen, MPO activity and the mRNA expression of IL-1ß, IL-18 and caspase-1 and significant reduction in fecal trypsin and antioxidant status in EtOH- and Cer-administered rats. The inflammatory markers were found to be reduced and the antioxidant status of pancreas was maintained in MEEO-coadministered rats. CONCLUSIONS: The rutin rich nature of E. officinalis can be claimed for its anti-inflammatory and pancreato protective effects.

Effect of thymoquinone on ethanol and high fat diet induced chronic pancreatitis--a dose response study in rats.

A significant increase in serum lipase, amylase, capase-1 and myeloperoxidase activities, oxidative stress index (OSI), IL-1beta and IL-18 was observed in rats receiving ethanol (EtOH) and high fat diet (HFD). Thymoquinone (TQ) supplementation along with EtOH and HFD significantly decreased the levels of serum lipase, amylase, capase-1, myeloperoxidase, OSI and maintained the antioxidant status when compared to untreated EtOH and HFD fed rats. Among the 4 doses, 100 mg of TQ/kg body weight was found to provide optimum protective effect on pancreas against EtOH and HFD induced abnormal changes. Histological observations added more evidence for the anti-inflammatory effect of TQ.

Abnormal circadian rhythm and cortisol excretion in autistic children: a clinical study.

AIM: To determine the circadian rhythm alteration of cortisol excretion and the level of corticosteroids in children with different grades of autism severity. METHODS: The study included 45 children with different grades of autism severity (low [LFA], medium [MFA], and high functioning autism [HFA]), 15 in each group, and 45 age/sex-matched children with typical development. The urinary levels of free cortisol (at three phases of 24-hour cycle), corticosteroids, vanilylmandelic acid, and 5-hydroxyindole acetic acid were determined. RESULTS: Alteration in the pattern of cortisol excretion (Phases I, II, and III) was observed in children with LFA (Phase I: 43.8 ± 4.43 vs 74.30 ± 8.62, P=0.000; Phase II: 21.1 ± 2.87 vs 62 ± 7.68, P<0.001; Phase III: 9.9 ± 1.20 vs 40 ± 5.73, P<0.001) and MFA (Phase I: 43.8 ± 4.43 vs 52.6 ± 7.90, P<0.001; Phase II: 21.1 ± 2.87 vs 27.4 ± 4.05, P<0.001; Phase III: 9.9 ± 1.20 vs 19 ± 2.50, P<0.001) compared to the control group. The corticosteroids excretion levels were higher in all the groups of children with autism than in the control group. The level of 5-hydroxyindole acetic acid was significantly higher in children with LFA (8.2 ± 1.48 vs 6.8 ± 0.85, P<0.001) and MFA (8.2 ± 1.48 vs 7.4 ± 0.89, P=0.001) and not significantly higher in children with HFA than in the control group. The changes were correlated with degrees of severity of the disorder. CONCLUSION: These data suggest that altered cortisol excretion pattern and high level of corticosteroids in urine may probably be a consequence of altered hypothalamic-pituitary-adrenal axis function, which may contribute to the pathogenesis and affect the severity of autism.

Antiulcerogenic activity of hydroalcoholic fruit extract of Pithecellobium dulce in different experimental ulcer models in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The ethnopharmacological importance of Pithecellobium dulce is evidenced by its traditional use for gastric complications. The aim of the study is to evaluate the gastroprotective activity and the mechanism of action of hydroalcoholic fruit extract of P. dulce (HAEPD) in rats by using chemical and stress induced ulcer models. MATERIALS AND METHODS: Gastric ulcer was induced by administering alcohol (or) acetylsalicylic acid (or) hypothermic restraint stress to rats pretreated with HAEPD (200 mg/kgbwt for 30 day). Volume of gastric fluid, pH, acidity, activities of pepsin, H(+), K(+)-ATPase, myeloperoxidase, mucin content, nucleic acids, glycoproteins and prostaglandin E(2) (PGE(2)) levels were assessed in gastric tissues. RESULTS: Ulcer score was significantly minimized in HAEPD administered animals. pH and acidity of gastric fluid were significantly minimized and the mucin, PGE(2) levels were significantly maintained in drug pre administered animals. The activities of H(+), K(+)- ATPase and myeloperoxidase were found to be significantly elevated in ulcer control animals and found to be decreased in drug pretreated animals. The cell proliferation was found to be enhanced in drug received animals. The total protein bound carbohydrate to total protein ratio was found to be significantly maintained by HAEPD. The effects were found to be comparable with that of standard drug omeprazole. CONCLUSION: It is concluded that HAEPD possess a potent antiulcer activity probably by acting as cytoprotective and antiacid secretory agent.

Acute and sub-acute toxicity study of hydroalcoholic fruit extract of Pithecellobium dulce.

This study was carried out to evaluate the acute and sub-acute toxicity profile of the hydroalcoholic fruit extract (HAEPD) of Pithecellobium dulce (Leguminosae). Albino rats were treated orally with 100, 200 and 500 mg kg(-1) bodyweight (BW) of HAEPD for 90 days to assess its sub-acute toxicity. HAEPD at single doses of 100, 500, 1000, 2000 and 4000 mg kg(-1) BW was also administered to rats to assess its acute toxicity. The rats were observed for physical discomfort, BW change and feeding habits. Pithecellobium dulce did not cause any abnormal changes in haematological or biochemical parameters. Pathologically, no gross abnormality in the tissue architecture was observed. The LD(50) was found to be 3916 mg kg(-1) BW and potential effective doses for efficacy studies are 100 and 300 mg kg(-1) BW as the minimum and maximum doses, respectively. It is concluded that HAEPD can be used safely for experimental and clinical trials.

A biochemical study on the level of proteins and their percentage of nitration in the hair and nail of autistic children.

BACKGROUND: Autism is a complex disorder which is heterogeneous in nature with varying degrees of severity for which no specific biological marker has been identified. Several studies are focused on the hair and nail protein pattern as a means to identify specific markers for the diagnosis of many childhood disorders like mental retardation, dyslexia, trichorrhexis nodosa, trichothiodystrophy, etc. The present study is one such approach in investigating the electrophoretic pattern of proteins in hard keratins and their percentage of nitration since nitric oxide production and nitration of tyrosine residues in proteins of autistic children are the emerging topic of research. METHODS: We extracted and quantified the proteins from hair and nail samples of autistic children with different grades of severity, [low functioning autism (LFA), medium functioning autism (MFA), and high functioning autism (HFA)] and also from age- and sex-matched normal children. Protein pattern was evaluated by one-dimensional SDS-PAGE and the separated proteins were made to cross react with anti-nitro tyrosine antibody by Western blot analysis. Blood levels of TBARS, NO, GSH, vitamins A and C, SOD and GPx were also determined. RESULTS: In the autistic groups, decreased concentration of protein in both hair and nail samples was observed. The SDS-PAGE analysis revealed that there was a significant decrease in both high and low sulfur proteins in the hair and nail extracts of autistic children and the Western blot analysis showed increased percentage of nitration of low sulfur proteins in autistic children when compared with normal children. Decreased levels of enzymatic and non-enzymatic antioxidants and increased concentration of TBARS and NO were also observed in the blood of autistic children. The LFA group showed more significant alteration (p<0.001) in the concentration of proteins (in hair and nail) and percentage of nitration when compared with HFA and controls. CONCLUSION: Lower protein content and higher percentage of nitration in hair and nail of autistic children correlated with their degrees of severity.

Level of trace elements (copper, zinc, magnesium and selenium) and toxic elements (lead and mercury) in the hair and nail of children with autism.

Autism is a multi-factorial pathology observed in children with altered levels of essential and elevated levels of toxic elements. There are also studies reporting a decrease in nutritional trace elements in the hair and nail of autistic children with healthy controls; moreover, bioelements have been shown to play an important role in the central nervous system. Therefore, the purpose of the present study was to assess the levels of trace elements like copper (Cu), zinc (Zn), magnesium (Mg), and selenium (Se) and toxic elements like mercury (Hg), and lead (Pb) in the hair and nail samples of autistic children and to evaluate whether the level of these elements could be correlated with the severity of autism. The subjects of the study were 45 autistic children with different grades of severity (low (LFA), medium (MFA), and high (HFA) functioning autism) according to Childhood Autism Rating Scale, n = 15 children in each group and 50 healthy children (age and sex matched). The boys and girls ratio involved in this study was 4:1, and they were 4-12 years of age. The study observed a valid indication of Cu body burden in the autistic children. The children with different grades of autism showed high significance (p < 0.001) in the level of copper in their hair and nail samples when compared to healthy controls. The level of Cu in the autistic children could be correlated with their degree of severity (more the Cu burden severe is autism). The study showed a significant elevation (p < 0.001) in the levels of toxic metals Pb and Hg in both hair and nail samples of autistic children when compared to healthy control group. The elevation was much pronounced in LFA group subjects when compared among autistic groups MFA and HFA. The levels of trace elements Mg and Se were significantly decreased (p < 0.001) in autistic children when compared to control. The trace element Zn showed significant variation in both hair and nails of LFA group children when compared to control group and other study groups. The significant elevation in the concentration of Cu, Pb, and Hg and significant decrease in the concentration of Mg and Se observed in the hair and nail samples of autistic subjects could be well correlated with their degrees of severity.

Concomitant production, partial purification and characterization of a serine protease and a proteolysis-resistant metallolipase from Bacillus pumilus SG2.

Our objective was to investigate the concomitant production of protease and lipase by a bacterial strain. A promising bacterial strain was isolated from a food-processing industrial effluent, which can produce both protease and lipase. The isolate was characterized by sequencing the 16S rRNA gene. The PCR amplified gene was subjected to analysis by BLAST to ascertain the genetic relatedness of the isolate, Bacillus pumilus SG2. The enzymes were produced and subjected to purification by ammonium sulfate precipitation and dialysis followed by gel filtration chromatography; twelve-fold purity was obtained. The lipase produced was found to be proteolysis-resistant. The partially purified enzymes were characterized for their optimum pH value, temperature, response to inhibitors, surfactants and oxidants. The relative molecular weights of protease and lipase were determined as 28 kDa and 40 kDa, respectively, by zymogram studies.

Bacoside A downregulates matrix metalloproteinases 2 and 9 in DEN-induced hepatocellular carcinoma.

Cancer metastasis is a complex multi-step process, responsible for a majority of cancer-related deaths by affecting the critical organs and causing complications in therapies. Hepatocellular carcinoma is a multi-factorial disease and is the third most common cause of cancer related mortality worldwide. Clinical and experimental studies have shown that MMP-2 and MMP-9 are involved in tumor invasion and metastases and their elevated expression has been associated with poor prognosis. Our recent studies showed a strong anti-oxidant and hepatoprotective effects of bacoside A (BA) against carcinogen. Nevertheless the effect of BA on the activities and expression of MMP-2 and MMP-9 during hepatocellular carcinoma is not yet recognized. Therefore, the present study was designed to assess the same. Results of gelatin zymography study showed that BA co-treatment significantly decreased the activities of MMP-2 and MMP-9, which is increased during hepatocellular carcinoma. Further immunoblot analysis showed decreased expression of MMP-2 and MMP-9 in rats co-treated with BA compared to DEN-induced hepatocellular carcinoma. Our results reveal that BA exerts its anti-metastatic effect against DEN-induced hepatocellular carcinoma by inhibiting the activities and expressions of MMP-2 and MMP-9.

Chemopreventive effect of bacoside A on N-nitrosodiethylamine-induced hepatocarcinogenesis in rats.

PURPOSE: Chemoprevention is an effective approach to control hepatocarcinogenesis. Bacoside A, the active constituent of Bacopa monniera Linn., is anticipated to play a role in chemoprevention of liver cancer. METHODS: In the present study, we investigated the chemopreventive effect of bacoside A against N-nitrosodiethylamine-induced hepatocarcinogenesis in an animal model. RESULTS: Administration of carcinogen showed a significant elevation in the levels of lipid peroxidation, serum tumor marker enzymes and liver injury marker enzymes with subsequent decrease in the levels of both hemolysate and liver antioxidant status. Bacoside A co-treatment maintained the N-nitrosodiethylamine-induced alterations at near normal level. Histopathological and electron microscopic study of the liver tissue also supports the above biochemical observations. CONCLUSIONS: From our findings we conclude that bacoside A is effective to prevent DEN-induced hepatocellular carcinoma by quenching lipid peroxidation and enhancing antioxidant status through free radical scavenging mechanism and having potential of protecting endogenous enzymatic and non-enzymatic antioxidant activity.

Level of nitrated proteins in the plasma, platelets and liver of patients with liver cirrhosis.

Over-expression of nitric oxide synthase (NOS) and nitric oxide (NO) formation are associated with the pathogenesis of liver cirrhosis. NO-related stress alters the functions of biomolecules, especially proteins, probably as a result of nitration. The aim of this study was to assess the level of protein nitration and its correlation with the severity of the disease. Liver cirrhosis patients with different grades of severity (grades A, B, and C according to the Child-Pugh classification) were enrolled in this study. Nitroprotein content, arginine, citrulline, NO in terms of total nitrite, nitrosothiol (RSNO) and protein carbonyls were measured in blood. Immunohistochemical detection of nitroprotein was carried out in liver sections of cirrhosis patients. A significant elevation in the levels of serum and platelet arginine, arginase, citrulline, plasma, and platelet nitroproteins, RSNO, total nitrite, protein carbonyls and also a significant amount of nitrated proteins by immunohistochemical detection in tissue were observed in cirrhosis patients. The alterations were highly significant in grade C patients with bleeding complications when compared to those of grade B and A patients. In platelets, both cytosolic and cytoskeletal proteins were found to be nitrated significantly. The level of nitrite seems to have positive correlation with the level of nitroproteins in different grades of cirrhosis. The level of nitroproteins in plasma, platelets and liver tissue can be correlated with the severity of liver cirrhosis.