ANTIOXIDANT, DNA DAMAGE PROTECTIVE AND HEPATOPROTECTIVE ACTIVITIES OF AMORPHOPHALLUS CAMPANULATUS

Objective: Amorphophallus campanulatus (AC) is commonly known as Suran. It is used to cure piles, deranged digestion and liver diseases. The present study was carried out to evaluate AC plant as a new source of natural bioactive molecules having antioxidant potential and their protective role against hepatotoxicity induced by t-BHP.Methods: Ethanolic extracts of AC leaf and fruit were subjected to assessment of its antioxidant potential by superoxide anion radical scavenging activity (SARSA), free radical scavenging activity (FRSA), ferrous ion chelation activity (FIC) and reducing power (RP). The study was further carried out to assay the DNA damage and hepatoprotective activity of AC leaf extract.Results: Leaf extract of AC showed lower IC50 for SARSA, FRSA and FIC (111.67 mg/ml, 11.58 mg/ml and 8.56 mg/ml, respectively) than fruit extract. HPLC analysis of leaf showed the presence of chlorogenic acid, caffeic acid, rutin, quercetin and kaempherol. Leaf extract showed potential to protect hydroxyl radical-induced DNA damage. Hepatoprotective effects of AC leaf was shown by increasing superoxide dismutase (SOD), glutathione content (GSH), catalase (CAT) and protein content and decreasing nitric oxide (NO) and MDA formation, when t-BHP treated hepatocyte cells were exposed to different concentration (1.0, 2.5, 5.0 and 7.5 µg) of extract.Conclusion: The results suggest that AC leaf has antioxidant, cytoprotective and hepatoprotective properties which may be due to the presence of important bioactive constituents; hence it may be used in the form of dietary component and also in formulations against liver diseases.Keywords: Hepatoprotective activity, Glutathione, Antioxidant enzyme, Lipid Peroxidation, Hepatocyte

ROLE OF VICIA FABA FRUIT EXTRACT AGAINST CYTOTOXICITY INDUCED BY ACETAMINOPHEN IN PRIMARY CULTURED RAT HEPATOCYTES

Objective: In the present study, Vicia faba fruit extract was screened for their hepatoprotective activity against cytotoxicity induced by acetaminophen in primary cultured rat hepatocytes.Methods: Vicia faba fruit, seed and leaf were subjected to assessment of its total phenolic content, reducing power, free radical scavenging activity, superoxide anion radical scavenging activity and lipid peroxidation. HPLC analysis, DNA damage protection and hepatoprotective activities of V. faba fruit extract were also analysed.Results: Among the tested extracts, V. faba fruit extract exhibited highest total phenolic content (104.90 mg gallic acid equivalents (GAE)/g), reducing power (0.83 ascorbic acid equivalents (ASE)/ml), free radical scavenging activity (IC50= 31.27 μg/ml), superoxide anion radical scavenging activity (IC50= 33.52 μg/ml) and LPO (IC50= 621.75 μg/ml). HPLC of V. faba fruit extract showed presence of polyphenols i.e. gallic acid (70.81 mg/100g) and catechin (49.63 mg/100g) and showed a significant reduction in the formation of nicked calf thymus DNA against either Fenton's reagent or UV radiation. Supplementation of V. faba fruit extract conferred significant protection against cytotoxicity induced by acetaminophen in primary cultured rat hepatocytes in comparison to standard hepatoprotectant silymarin.Conclusion: V. faba fruit extract possesses significant antioxidant, DNA damage protective and hepatoprotective activities and may be used for management of drug induced liver injury

Natural Terpenes Prevent Mitochondrial Dysfunction, Oxidative Stress and Release of Apoptotic Proteins during Nimesulide-Hepatotoxicity in Rats

Nimesulide, an anti-inflammatory and analgesic drug, is reported to cause severe hepatotoxicity. In this study, molecular mechanisms involved in deranged oxidant-antioxidant homeostasis and mitochondrial dysfunction during nimesulide-induced hepatotoxicity and its attenuation by plant derived terpenes, camphene and geraniol has been explored in male Sprague-Dawley rats. Hepatotoxicity due to nimesulide (80 mg/kg BW) was evident from elevated SGPT, SGOT, bilirubin and histo-pathological changes. Antioxidants and key redox enzymes (iNOS, mtNOS, Cu/Zn-SOD, Mn-SOD, GPx and GR) were altered significantly as assessed by their mRNA expression, Immunoblot analysis and enzyme activities. Redox imbalance along with oxidative stress was evident from decreased NAD(P)H and GSH (56% and 74% respectively; P<0.001), increased superoxide and secondary ROS/RNS generation along with oxidative damage to cellular macromolecules. Nimesulide reduced mitochondrial activity, depolarized mitochondria and caused membrane permeability transition (MPT) followed by release of apoptotic proteins (AIF; apoptosis inducing factor, EndoG; endonuclease G, and Cyto c; cytochrome c). It also significantly activated caspase-9 and caspase-3 and increased oxidative DNA damage (level of 8-Oxoguanine glycosylase; P<0.05). A combination of camphene and geraniol (CG; 1∶1), when pre-administered in rats (10 mg/kg BW), accorded protection against nimesulide hepatotoxicity in vivo, as evident from normalized serum biomarkers and histopathology. mRNA expression and activity of key antioxidant and redox enzymes along with oxidative stress were also normalized due to CG pre-treatment. Downstream effects like decreased mitochondrial swelling, inhibition in release of apoptotic proteins, prevention of mitochondrial depolarization along with reduction in oxidized NAD(P)H and increased mitochondrial electron flow further supported protective action of selected terpenes against nimesulide toxicity. Therefore CG, a combination of natural terpenes prevented nimesulide induced cellular damage and ensuing hepatotoxicity

Protective role of morin, a flavonoid, against high glucose induced oxidative stress mediated apoptosis in primary rat hepatocytes.

Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM) of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor) & Endo-G (endonuclease-G) from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress

Naringenin accords hepatoprotection from streptozotocin induced diabetes in vivo by modulating mitochondrial dysfunction and apoptotic signaling cascade

Diabetic complications cause noticeable liver damage, which finally progresses to diabetic hepatopathy. Nutritive antioxidants not only reduce the liver damage, but also prevent it by modulating the release of various proteins involved in apoptotic signaling cascades. This study explores the molecular mechanisms underlying diabetes-induced liver damage and its modulation by naringenin. Antioxidant status, liver & kidney biomarker enzymes, reactive oxygen species (ROS) generation, mitochondrial membrane potential, expression of apoptotic proteins like Bax (bcl-2 associated X), Bcl-2 (b-cell Lymhoma-2), Caspase-3, Caspase-9, AIF (Apoptosis inducing factor) and Endo-G (Endonuclease-G) were studied in streptozotocin induced diabetic rats. Significant hyperglycemia, disturbed antioxidant status, altered carbohydrate metabolizing enzymes, increased ROS and lipid peroxidation; decreased mitochondrial membrane potential and enhanced release of AIF and Endo-G were observed. Hyperglycemia also affected apoptosis and its related genes at both transcriptional and translational level (Caspase-3 & 9, Bax and Bcl-2) in the liver of diabetic rats. Naringenin, a flavonone, exerted anti-hyperglycemic effect and was able to prevent oxidative stress and resultant apoptotic events caused due to diabetes-induced hepatotoxicity. Thus, our study shows, a protective effect of naringenin against diabetes induced liver damage and redox imbalance, which could further be exploited for the management of diabetic hepatopathy

Modulation of hyperglycemia induced DNA damage, nuclear condensation and apoptosis by morin.

<p>(a) Intra-nucleosomal DNA fragmentation in control, high glucose treated (40 mM) and morin treated (pre, co and post-treated) stressed cells. Genomic DNA was isolated and analyzed on 1.8% agarose gel. Lane1 DNA Marker 100 bp: Lane 2: Control DNA, Lane 3: DNA from 40 mM Glucose treated hepatocytes, Lane 4: Morin pre-treatment, Lane 5: Morin Co-treatment, Lane 6: Morin Post treatment. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041663#s3" target="_blank">Results</a> are representative of three separate experiments. (b) Rat hepatocytes were stained with Hoechst 33258 and visualized at 10× and 40× magnifications to observe chromatin condensation. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041663#s3" target="_blank">Results</a> are representative of three separate experiments. (c) Effect of morin on hyperglycemia induced changes in cell cycle. Control, high glucose treated (40 mM) and morin treated (pre, co and post-treated) stressed cells were stained with propidium iodide and kept in dark for 30 minutes. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041663#s3" target="_blank">Results</a> expressed as the % of sub-G1 population. The propidium iodide fluorescence was measured using flow cytometer (Beckton Dickinson-LSR) with FL-2 filter. S.D. was below ±5% in all the cases. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041663#s3" target="_blank">Results</a> are representative of three separate experiments.</p

Effect of morin on m-RNA level of apoptotic genes.

<p>(a) m-RNA level of apoptotic genes such as caspase-3 and caspase-9 were amplified by RT-PCR and analyzed by 1.8% agarose gel electrophoresis followed by ethidium bromide staining. GAPDH was used as internal control. Graph shows fold change in m-RNA level. (b) Caspase -3 enzymatic activity of control and stressed cells. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041663#s3" target="_blank">Results</a> are shown as mean ± S.E. # denotes significant difference compared with control values and *P<0.05, **P<0.01 and ***P<0.001 denotes significant difference compared with 40 mM glucose.</p

Effect of morin on antioxidant status, MMP and transcriptional level of Bax and Bcl-2.

<p>(a) Transcriptional level of antioxidant genes. m-RNA of MnSOD, Cu Zn-SOD, GPx and GR were amplified by RT-PCR and analyzed by electrophoresis on 1.8% agarose gel and ethidium bromide staining. GAPDH was used as internal control. (b) Bar graphs show fold change in expression of MnSOD, Cu Zn-SOD, GPx and GR. (c) Induction of mitochondrial membrane collapse in hepatocytes cultured with high glucose. MMP was assessed in control, high glucose treated (40 mM) and morin treated (pre, co and post-treated) stressed cells. Mitochondrial membrane potential of treated and control cells was assessed by fluorescent spectrophotometer using JC-1. Decrease in the red (polarized)/green (depolarized) fluorescence ratio reflects increased number of depolarized mitochondria. (d) m-RNA level of Bcl-2 and Bax, were amplified by RT-PCR and analyzed by 1.8% agarose gel electrophoresis followed by ethidium bromide staining. GAPDH was used as internal control. Graph shows fold change in m-RNA level. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041663#s3" target="_blank">Results</a> are shown as mean ± S.E. # denotes significant difference compared with control values and *P<0.05, **P<0.01 and ***P<0.001 denotes significant difference compared with 40 mM glucose.</p