Induction of apoptosis by eugenol in human breast cancer cells.

In the present study, potential anticancer effect of eugenol on inhibition of cell proliferation and induction of apoptosis in human MCF-7 breast cancer cells was investigated. Induction of cell death by eugenol was evaluated following MTT assay and monitoring lactate dehydrogenase released into the culture medium for cell viability and cytotoxicity, giemsa staining for morphological alterations, fluorescence microscopy analysis of cells using ethidium bromide and acridine orange and quantitation of DNA fragments for induction of apoptosis. Effect of eugenol on intracellular redox status of the human breast cancer cells was assessed by determining the level of glutathione and lipid peroxidation products (TBARS). Eugenol treatment inhibited the growth and proliferation of human MCF-7 breast cancer cells through induction of cell death, which was dose and time dependent. Microscopic examination of eugenol treated cells showed cell shrinkage, membrane blebbing and apoptotic body formation. Further, eugenol treatment also depleted the level of intracellular glutathione and increased the level of lipid peroxidation. The dose dependent increase in the percentage of apoptotic cells and DNA fragments suggested that apoptosis was involved in eugenol induced cell death and apoptosis might have played a role in the chemopreventive action of eugenol.

Effect of Hemidesmus indicus R.Br. root extract against Salmonella enterica serovar Typhimurium-induced apoptosis in murine macrophage cell line (P388D1).

BACKGROUND & OBJECTIVE: Previous studies on natural products had mainly dealt with their antimicrobial activity and studies on the interference of these bioactive compounds with host-bacterial interaction is limited. The present study was undertaken to investigate the effect of the sterols and fatty acids present in the chloroform fraction of crude methanol extract of Hemidesmus indicus root (CHI) on Salmonella enterica serovar Typhimurium (S. Typhimurium) mediated apoptosis in a murine macrophage cell line (P388D1). METHODS: Bacterial sensitivity test was carried out with different concentrations of CHI and the optimum dose was fixed as 100 mug/ml for CHI, which was safe on host cells as the CD(50) (50% of cell death) dose of CHI was determined to be 500 mug/ml in the P388D1 cell line. RESULTS: The CHI-treated bacteria had negligible cytotoxicity and were less potent to invade and proliferate intracellularly. Murine macrophages infected with wild bacteria, stained with Hoechst 33258, had swollen and damaged morphology with characteristic apoptotic bodies whereas macrophages infected with treated bacteria had comparative normal architecture. Immunofluorescence and transmission electron micrographs both confirmed that CHI-treated bacteria were defective and smaller than the wild bacteria. Ultrastructures of P388D1 cells infected with wild bacteria showed many ingested bacteria and characteristic Salmonella-containing vacuoles (SCV). Some cells had condensed or fragmented nuclei with swollen mitochondria, whereas most of the cells infected with treated bacteria were normal in morphology and a few had internalized bacteria, but the typical bacteria laden SCV was not observed in cells infected with CHI-treated S. Typhimurium. INTERPRETATION & CONCLUSION: Our results showed that the choloroform fraction of H. indicus root blocked the cytotoxic activity of S. Typhimurium in a macrophage cell line. More studies need to be done to elaborate and confirm our findings.

Effect of Hemidesmus indicus R.Br. root extract against Salmonella enterica serovar Typhimurium-induced apoptosis in murine macrophage cell line (P388D1).

Background & objectives: Previous studies on natural products had mainly dealt with their antimicrobial activity and studies on the interference of these bioactive compounds with host-bacterial interaction is limited. The present study was undertaken to investigate the effect of the sterols and fatty acids present in the chloroform fraction of crude methanol extract of Hemidesmus indicus root (CHI) on Salmonella enterica serovar Typhimurium (S. Typhimurium) mediated apoptosis in a murine macrophage cell line (P388D1). Methods: Bacterial sensitivity test was carried out with different concentrations of CHI and the optimum dose was fixed as 100 μg/ml for CHI, which was safe on host cells as the CD50 (50% of cell death) dose of CHI was determined to be 500 μg/ml in the P388D1 cell line. Results: The CHI-treated bacteria had negligible cytotoxicity and were less potent to invade and proliferate intracellularly. Murine macrophages infected with wild bacteria, stained with Hoechst 33258, had swollen and damaged morphology with characteristic apoptotic bodies whereas macrophages infected with treated bacteria had comparative normal architecture. Immunofluorescence and transmission electron micrographs both confirmed that CHI-treated bacteria were defective and smaller than the wild bacteria. Ultrastructures of P388D1 cells infected with wild bacteria showed many ingested bacteria and characteristic Salmonella-containing vacuoles (SCV). Some cells had condensed or fragmented nuclei with swollen mitochondria, whereas most of the cells infected with treated bacteria were normal in morphology and a few had internalized bacteria, but the typical bacteria laden SCV was not observed in cells infected with CHI-treated S. Typhimurium. Interpretation & conclusions: Our results showed that the choloroform fraction of H. indicus root blocked the cytotoxic activity of S. Typhimurium in a macrophage cell line. More studies need to be done to elaborate and confirm our findings.

Effect of methacrylonitrile on membrane bound enzymes of rat brain.

Methacrylonitrile (MeAN) is a plastic monomer. Its effect on membrane bound enzymes like Na+K+ -ATPase, Ca2+ -ATPase, Mg2+ -ATPase, NADH dehydrogenase, alkaline phosphatase (ALP) and various elements like sodium (Na+), potassium (K+), and calcium (Ca2+) in rat brain were studied. Administration of 50 mg/kg body weight/day (0.25 LD50) and 100 mg/kg body weight/day (0.5 LD50) by gavage to rats for 7 days resulted in a significant decrease in activities of Na+K+ -ATPase, Ca2+ -ATPase, Mg2+ -ATPase, and NADH dehydrogenase. A significant reduction in calcium content, potassium content and a significant increase in sodium content and alkaline phosphatase activity in MeAN treated animals were observed. Inhibition of membrane bound enzymes occurred due to either direct effect of MeAN or indirect effect of changes in ionic homeostasis in MeAN treated animals.

Antibacterial and antidiarrhoeal effects of alkaloids of Holarrhena antidysenterica WALL.

The alkaloids from the ethanolic extract of H. antidysenterica seeds were evaluated for their antibacterial activity against clinical isolates of enteropathogenic Escherichia coli (EPEC) in vitro, and their antidiarrhoeal activity on castor oil-induced diarrhoea in rats, in vivo. The plasmid DNA, whole cell lysate and outer membrane protein profile of a clinical isolate of EPEC was determined in presence of alkaloids of H. antidysenterica. The disc diffusion and agar well diffusion methods were used to evaluate the antibacterial efficacy. The alkaloids showed strong antibacterial activity against EPEC strains. In castor oil-induced diarrhoea, alkaloids reduced the diarrhoea with decrease in the number of wet faeces in pretreated rats at a dose of 200-800 mg/kg. The loss of plasmid DNA and suppression of high molecular weight proteins were observed on alkaloids treatment. Taking into account the multiple antibiotic resistance of EPEC, the results suggest usefulness of alkaloids of H. antidysenterica seeds as antibacterial and antidiarrhoeal agents.

Antidiarrhoeal effects of methanolic root extract of Hemidesmus indicus (Indian sarsaparilla)--an in vitro and in vivo study.

Methanolic extract of H. indicus root (MHI) was screened for its antimicrobial activity against S. typhimurium, E. coli and S. flexneri, in vitro and in experimentally induced diarrhoea in albino rats, in vivo. MHI had an anti enterobacteriae effect as evident from agar well diffusion method and decrease in CFU/ml in MHI treated LB broth culture. MHI inhibited the castor oil induced diarrhoea in rats as judged by a decrease in the amount of wet faeces in MHI-pretreated rats at a dose of 500-1500 mg/kg. The results indicated that MHI was more active than standard antidiarrhoeal drug, lomotil. Phytochemical tests revealed the main constituents as tannins, steroids, triterpenoids and carbohydrates. Present findings suggested that MHI might elicit an antidiarrhoeal effect by inhibition of intestinal motility and by its bacteriocidal activity.

Effect of eugenol and tincture of crataegus (TCR) on in vitro oxidation of LDL + VLDL isolated from plasma of non-insulin dependent diabetic patients.

The present study was carried out to study the effect of antioxidants on oxidised LDL + VLDL and found that vitamin E, eugenol and tincture of crataegus (antioxidants) inhibited oxidation of (LDL + VLDL) similar to standard antioxidant (butylated hydroxy toluene). Vitamin C acted as an antioxidant at lower concentration, and prooxidant at higher concentration.

Antioxidant effect of eugenol in rat intestine.

The effect of eugenol on the antioxidant status of the rat intestine after short and long term (15 days and 90 days respectively) oral administration of 1000 mg/kg.b.wt (a dosage which has been reported to be highly hepatoprotective) was studied. The level of lipid peroxidation products (TBARS) and the activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) were found to be near normal on eugenol treatment. The level of glutathione (GSH) did not show any change on 15 days of eugenol treatment, but it was increased significantly on 90 day eugenol treatment. The activity of glutathione-S-transferases (GSTs) was increased significantly in both 15 day eugenol treated and 90-day eugenol treated groups. The results suggest that eugenol is nontoxic, protective and induces glutathione-S-transferases (GSTs) and thereby it may facilitate the removal of toxic substances from the intestine.

Methacrylonitrile induced oxidative stress in rat liver.

MeAN administration (40mg/kg body wt/day (i.e. 1/5 of LD50) resulted in increased levels of lipid peroxidation products, conjugated dienes and lipofuscin-like substances in rat liver. Significant decrease in GSH and a decreased activity of hepatic SOD, CAT and GPx were observed. There was also an increase in glutathione S-transferase and G6PD activities, decreased plasma ceruloplasmin and vitamin C implying oxidative stress caused by MeAN.

Oxalate binding protein from the kidney of rat and human mitochondria: studies on properties.

The oxalate binding protein of rat and human kidney mitochondria were extracted by Triton X-100 and purified on Sephadex G-200 column followed by HPLC. Their molecular masses were found to be 62 kD and 58 kD respectively, rich with arginine and acidic amino acids, 7% of carbohydrates and 1% of inorganic ions. Antibodies raised to the rat protein inhibited the oxalate binding and cross-reacted to the human protein as well as rat liver protein. The binding of oxalate to the protein was rapid, reversible, dependent on concentration of oxalate, temperature sensitive and inhibited by oxalate analogues. The saturation reached at 175 nM oxalate for rat protein with a Kd of 33.3 nM and Bmax of 21 nmoles while for human protein the saturation reached at 183 nM oxalate and had a Kd of 41 nM and Bmax of 14 n moles. The half-saturation concentration of inhibitor (IC50) of oxalate was 0.25 microM for rat protein and 0.225 microM for human protein while the structural analogues of oxalate had higher IC50 values. Proteoliposomes showed accumulation of oxalate confirming transport function of the protein. The rat protein promoted calcium oxalate crystallization in vitro better than that of human protein and antibody inhibited the crystal growth in vitro.

Oxalate binding to rat kidney mitochondria: induction by oxidized glutathione.

Increased oxalate binding with negative correlation with reduced glutathione content was observed during lipid peroxidation in rat kidney mitochondria. In presence of oxidized glutathione (GSSG), peroxidized mitochondria lost 48% of protein-SH with concomitant 3-fold increase in oxalate binding activity while control mitochondria lost only 20% protein-SH with only 0.8 fold increase in oxalate binding activity. The GSSG-induced oxalate binding was apparently due to two-fold increased affinity of oxalate to the protein. Reduced glutathione (GSH) inhibited oxalate binding competitively with Ki, 1.4 x 10(-3) M. Urolithic rat kidney mitochondria showed 30-50% increase in oxalate binding activity along with depletion of GSH and protein-SH. These studies suggest that oxalate binding is regulated by thiol status of mitochondria.

Oxidative stress in rat liver and lung induced by furanoterpenoids isolated from Fusarium solani infected sweet potatoes.

A crude extract containing some toxic furanoterpenoids was isolated from F. solani infected sweet potatoes. Chronic administration of the crude extract to male albino rats at a dosage of 1 mg/kg body weight/day for 21 days brought about a sharp increase in the thiobarbituric acid reactive substances and a depression of glutathione levels in the lung and liver homogenates. The antioxidant defense system was affected as evident from a significant fall in the activities of the enzymes, superoxide dismutase, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase and glutathione-S-transferase. Such an alteration could be the reason for the lung and liver damage caused by these toxic furanoterpenoids.

Hypolipidemic activity of tincture of Crataegus in rats.

Tincture of Crataegus (TCR), an alcoholic extract of the berries of Crataegus oxyacantha, when administered to rats fed a hyperlipidemic diet (HLD), could prevent the elevation in plasma lipid levels. A significant decrease in lipid deposits in liver and aorta was also observed. Analysis of the plasma lipoprotein profile showed that TCR produced remarkable reduction in the increased levels of cholesterol, triglycerides and phospholipids in the low density lipoprotein (LDL) and very low density lipoprotein (VLDL) fractions in hyperlipidemic rats. Histological examination showed severe fatty vacuolation and degeneration of liver of HLD fed rats. TCR administration had an ameliorating effect on these changes. Agarose gel electrophoretic pattern of plasma lipoproteins also indicated that the drug brought down the raised levels of the atherogenic beta-lipoproteins in hyperlipidemic rats.

Lung injury by furanoterpenoids isolated from Fusarium solani infected sweet potato, Ipomea batatas.

Intraperitoneal administration (1 mg/kg body/wt./day for 21 days) of crude extract of furanoterpenoids, isolated from F. solani damaged I. batatas caused pulmonary oedema in albino rats. The elevated broncho alveolar lavage (BAL) angiotensin converting enzyme (ACE), lactate dehydrogenase (LDH) and protein levels indicated lung damage. The estimation of pulmonary extracellular surfactant phospholipids showed an alteration in various phospholipid fractions.

Effect of amiodarone, an antiarrhythmic drug, on serum and liver lipids and serum marker enzymes in rats.

Administration of amiodarone (AD) to rats leads to marked damage to liver, as evidenced by pathological changes and significant increases in activities of serum marker enzymes and levels of lipids like cholesterol and phospholipids with no alteration in the triglyceride levels. The risk factor, that is the total cholesterol/HDL cholesterol ratio, exhibited increase in the experimental animals, indicating that amiodarone treatment may lead to the development of coronary heart disease.