Aflatoxin B1-induced DNA damage in Labeo rohita: protective effect of an antioxidant supplement, Amrita Bindu.

The present study was undertaken to investigate the effect of potent hepatocarcinogen aflatoxin B1 in adduct formation and DNA damage in Labeo rohita. Also, the salubrious efficacy of an antioxidant supplement Amrita Bindu (based on Indian system of Medicine) was investigated. Fish weighing 175-250 g were administered intraperitoneally a single dose of 100 microg aflatoxin B1/100 g body wt. and another group was given 20% solution of Amrita Bindu along with aflatoxin B1 at 100 microg/100 g body wt. On the 3rd and 6th day, the liver tissue was analyzed for aflatoxin concentration, aflatoxin-DNA adduct formation and DNA damage measured in terms of single strand breaks. The fishes administered with aflatoxin B1 showed elevated concentration of aflatoxin along with a parallel increase in the DNA adduct when compared with the controls. While the fish co-administered with Amrita Bindu showed 34% and 24% reduction in aflatoxin deposition (accumulation) and aflatoxin-DNA adduct formation respectively on the 3rd day, a further reduction by around 41% and 33% in aflatoxin deposition and DNA adduct formation respectively was observed on the 6th day. Furthermore, the increased single strand breaks (measured by alkaline single cell gel assay) and fragmentation observed in agarose gel electrophoresis in aflatoxin B1 administered fish were significantly reduced by Amrita Bindu co-administration. In conclusion, this is the first report to show aflatoxin B1-induced DNA adduct formation and DNA damage in one of the major Indian culturable fish, Labeo rohita. Also, our observations show that the antioxidant supplement, Amrita Bindu, has a potential role in ameliorating the aflatoxin B1-induced DNA damage thus suggesting its applicability in preventing the vital macromolecule DNA.

Antioxidant activity of a salt-spice-herbal mixture against free radical induction.

A combination of spices (Piper nigrum, Piper longum and Zingiber officinale), herbs (Cyperus rotundus and Plumbago zeylanica) and salts make up Amrita Bindu. The study was focused to evaluate the antioxidant property of individual ingredients in Amrita Bindu against the free radical 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The analysis revealed the antioxidant potential of the ingredients in the following order: Piper nigrum>Piper longum>Cyperus rotundus>Plumbago zeylanca>Zingiber officinale. Two different experiments were designed. In experiment I, rats were fed with normal diet whereas in experiment II rats were given feed mixed with Amrita Bindu for 3 weeks (4 g/kg of feed). Rats from both experimental groups were challenged against a single intraperitonial injection of phenylhydrazine (PHZ) (7.5 mg/kg body weight). At the end of 24 and 72 h, blood was analysed for free radicals and antioxidant levels. It was interesting to note that rats with Amrita Bindu pretreatment showed significantly lower levels of free radicals, lipid peroxidation and protein carbonyls along with significantly higher levels of antioxidants when compared with rats without Amrita Bindu pretreatment on PHZ administration. These results reveal that Amrita Bindu, a salt-spice-herbal mixture exerts a promising antioxidant potential against free radical induced oxidative damage.

Oxidative damage to lipids and proteins induced by aflatoxin B(1) in fish (Labeo rohita)-protective role of Amrita Bindu.

In the present study, fish (Labeo rohita) were treated with a single intraperitoneal administration of aflatoxin B(1) (AFB(1)) (100µg/100gBW). The resultant oxidative damage to lipids (measured as conjugated diene and lipid peroxidation (LPO)) and proteins (protein carbonyl) in liver, kidney and brain at the end of 3rd and 6th day was assessed. Our results showed that AFB(1) induced a significant increase in conjugated diene formation and LPO not only in liver but also in kidney and brain. A parallel increase in protein carbonyl level was observed in these tissues. When 1:1 mixture of 20% solution of Amrita Bindu (a salt-spice-herbal mixture based on Indian system of medicine) was co-administered along with 100µg AFB(1), the AFB(1) induced increase in conjugated diene, LPO and protein carbonylation were minimised to a greater extent. These results led to conclusion that (i) AFB(1) not only induces oxidative damage to the primary target organ-liver in L. rohita, but also in kidney and brain, (ii) co-administration of Amrita Bindu confers protection to lipids and protein against the AFB(1) induced oxidative damage in all the three tissues.

Amrita Bindu--a salt-spice-herbal health food supplement for the prevention of nitrosamine induced depletion of antioxidants.

Ayurveda, practised in India, identified a large number of plant components to be used in the diet for the prevention or the delayed development of degenerative disorders. They include some of the commonly used spices, namely pepper and ginger. The Materia Medica includes both naturally occurring and artificially produced salts, as a partial substitute for common salt. Health promoting herbs and spices which are classified pharmacologically as rejuvenating, nourishing, invigorating, cleansing, wound-healing, etc., are used as food additives. Amrita Bindu is a salt-spice-herbal mixture based on these principles and was tested for its effect in maintaining antioxidant defense systems in blood and liver when exposed to a carcinogenic nitrosamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Amrita Bindu supplementation prevented MNNG induced depletion of the antioxidant enzymes and the scavenger antioxidants glutathione and vitamins A, C and E. Amrita Bindu provides protection against free radical and reactive oxygen species induced tissue lipid peroxidation and the resultant tissue degeneration.

Brahmighritham, an Ayurvedic herbal formula for the control of epilepsy.

Brahmighritham is an Ayurvedic herbal recommended for the control of epilepsy. The preparation of the drug is described. It was tested for its oral effectiveness in controlling pentylenetetrazole-induced seizures in male albino rats and was compared with benzdiazepam. Alterations in the EEG pattern and gross neurological function were measured or rated 60 min after pentylenetetrazole administration. Thirty-day pretreatment with both Brahmighritham and benzdiazepam served to make the rats more insensitive to epileptogenic events.

Double-blind cross-over study of modified Anna Pavala Sindhooram in patients with hyperlipidemia or ischemic heart disease.

The plasma lipid lowering effect of modified Anna Pavala Sindhooram (APSm) was tested in 30 patients with plasma lipid abnormalities and with symptomatic ischemic heart disease (IHD) in a double-blind cross-over design. HDL2 cholesterol and total HDL cholesterol were increased during APSm therapy leading to a reduction in the coronary risk index. Plasma cholesterol, LDL and VLDL cholesterol, triglycerides, phospholipids and free fatty acids were significantly reduced during APSm therapy while withdrawal of the drug led to a slow increase in these lipids. No adverse side effects were noticed. Some patients were able to reduce their daily doses of antihypertensive and vasodilator drugs during APSm therapy.

Possible regeneration of the islets of Langerhans in streptozotocin-diabetic rats given Gymnema sylvestre leaf extracts.

Two water soluble extracts, GS3 and GS4, obtained from the leaves of Gymnema sylvestre, were tested in streptozotocin treated rats for their effects on blood glucose homeostasis and pancreatic endocrine tissue. In the diabetic rats, fasting blood glucose levels returned to normal after 60 days of GS3 and after 20 days of GS4 oral administration. Blood collected during the conduct of oral glucose tolerance tests was used to assay for serum insulin. GS3 and GS4 therapy led to a rise in serum insulin to levels closer to normal fasting levels. In diabetic rat pancreas, both GS3 and GS4 were able to double the islet number and beta cell number. This herbal therapy appears to bring about blood glucose homeostasis through increased serum insulin levels provided by repair/regeneration of the endocrine pancreas.

Use of Gymnema sylvestre leaf extract in the control of blood glucose in insulin-dependent diabetes mellitus.

GS4, a water-soluble extract of the leaves of Gymnema sylvestre, was administered (400 mg/day) to 27 patients with insulin-dependent diabetes mellitus (IDDM) on insulin therapy. Insulin requirements came down together with fasting blood glucose and glycosylated haemoglobin (HbA1c) and glycosylated plasma protein levels. While serum lipids returned to near normal levels with GS4 therapy, glycosylated haemoglobin and glycosylated plasma protein levels remained higher than controls. IDDM patients on insulin therapy only showed no significant reduction in serum lipids, HbA1c or glycosylated plasma proteins when followed up after 10-12 months. GS4 therapy appears to enhance endogenous insulin, possibly by regeneration/revitalisation of the residual beta cells in insulin-dependent diabetes mellitus.

Antidiabetic effect of a leaf extract from Gymnema sylvestre in non-insulin-dependent diabetes mellitus patients.

The effectiveness of GS4, an extract from the leaves of Gymnema sylvestre, in controlling hyperglycaemia was investigated in 22 Type 2 diabetic patients on conventional oral anti-hyperglycaemic agents. GS4 (400 mg/day) was administered for 18-20 months as a supplement to the conventional oral drugs. During GS4 supplementation, the patients showed a significant reduction in blood glucose, glycosylated haemoglobin and glycosylated plasma proteins, and conventional drug dosage could be decreased. Five of the 22 diabetic patients were able to discontinue their conventional drug and maintain their blood glucose homeostasis with GS4 alone. These data suggest that the beta cells may be regenerated/repaired in Type 2 diabetic patients on GS4 supplementation. This is supported by the appearance of raised insulin levels in the serum of patients after GS4 supplementation.

Effect of patulin on the kinetic properties of the enzyme aldolase studied in rat liver.

The toxic nature of the secondary metabolite has been studied in rats. Changes in the concentration of a few key enzymes in carbohydrate metabolism have also been studied. In this, liver aldolase concentration was found to be significantly lowered. Since aldolase is one of the important bifunctional enzymes of glycolysis, it has been isolated and purified and studied on its kinetic properties were made. The kinetic studies did not show any significant variations in the properties of liver aldolase of normal and patulin treated animals. These results suggest that most probably, patulin toxicosis inhibits the biosynthesis of liver aldolase.

Effect of patulin on some enzymes of carbohydrate metabolism studied in rats.

The toxic nature of the secondary metabolite of Penicillium patulum has been studied in rats. Liver, Kidney and Intestine of the experimental animals showed derangement in carbohydrate metabolism. Changes in the concentration of a few key enzymes in carbohydrate metabolism have also been studied. Glycogen phosphorylase is found to be markedly increased while the glycolytic enzymes like hexokinase and aldolase are significantly lowered. Gluconeogenesis is stimulated and this is evidenced by increased glucose-6-phosphatase and fructose-1,6-diphosphatase activity. Our results revealed that, patulin, the secondary metabolite of Penicillium patulum showed toxicity in all the organs studied.

Control of alcohol addiction by SKV therapy--its action on water, food intake, brain function and cell membrane composition.

Alcohol being easily permeable through cell membrane causes toxic damage to many tissues. Rats drinking aqueous ethanol (25% v/v) for 120 days and 240 days showed an initial rise in body weight. The reduced rate in weight gain in chronic alcoholism is associated with a fall in food intake. Ethanol ingesting animals showed slow response to stimuli and increase in blood ethanol and serum GGTP levels. Liver plasma membrane, kidney brush-border membrane and pancreatic plasma membrane from alcoholic rats showed significant alterations in cholesterol/phospholipid molar ratio and membrane ATPases. Water retention with the enlargement of liver and kidney associated with increased fluid consumption are also seen during alcoholism. SKV by breaking alcohol dependence reduces drinking, lowers blood ethanol level and fluid intake without developing withdrawal symptoms. Restriction of ethanol intake by SKV therapy resulted in the reversal of organ enlargement and membrane composition in alcoholics.

Effect of an isolate from gymnema sylvestre, R. Br. In the control of diabetes mellitus and the associated pathological changes.

Gymnema sylvestre, R. Br., popularly known as Meshashringi in Sanskrit and Sarkaraikolli in Tamil, was investigated for the control of type I (insulin dependent) diabetes in experimental animals. The hypoglycaemic extract was found to bring about blood glucose homeostasis, by increasing serum insulin levels. The islets of langerhans appear to be restored or regulated by the herbal extract. Increased glycoprotein, which is the major metabolic abnormality in diabetes mellitus and the resultant nephropathy, retinopathy and micro and macroangiopathy, is brought under control by the administration of the leaf extract.

Studies on brain structure and neurological function in alcoholic rats controlled by an Indian medicinal formula (SKV).

An Indian herbal brew known in Ayurvedic pharmacy as asavam (SKV) was tested for its effectiveness in controlling addiction to ethanol in rats. Rats on SKV therapy with free access to 15% ethanol showed a marked reduction in voluntary ethanol intake. Their performance in simple neurological tests improved and a reversal of ethanol-induced changes in the electroencephalogram and electrocardiogram were also recorded. SKV treatment appeared to correct the fatty changes in liver and the signs of haemorrhage, demyelination and spongiosis seen in the brain of ethanol-fed rats.

An Indian herbal formula (SKV) for controlling voluntary ethanol intake in rats with chronic alcoholism.

Chronic ethanol ingestion in rats showed metabolic and physiological changes similar to alterations reported in human alcoholics. There was a lowering of blood glucose concentration, urea and plasma proteins and elevated concentrations of serum gamma-glutamyl transpeptidase. Administration of SKV, an Ayurvedic formula produced by fermentation of cane sugar with raisins and 12 herbal ingredients brought down voluntary ethanol ingestion in the rats and increased food intake. ECG and EEG studies in alcoholic rats showed cardiac depression, augmentation of frequency and amplitude of the alpha, delta and theta waves and weakness in the beta waves. These changes were reversed during SKV-induced voluntary alcohol restriction. The involvement in the ECG and EEG wave patterns was associated with improvement in blood glucose, plasma protein levels and reduction in gamma glutamyl transpeptidase activities. SKV appeared to have no adverse reaction with ethanol (it contains 1-2% ethanol) and appears to be a promising way to combat alcoholism.