Plants have been considered as valuable sources of medicines for treating a variety of diseases and ailments . Despite the availability of modern medicine throughout all countries, herbal medicines have often remained popular for historical and cultural reasons. Previous experimental results, which were highly encouraging, revealed
The national agency for sanitary vigilance is responsible for the regulation of medications and uses protocols from international regulatory agencies to evaluate the efficacy and the safety of medications. However, no studies have specifically addressed the toxic efficacy of petroleum ether extract of
Around 2 kg of leaves of
Cytotoxicity tests were performed using the
A 1-mL aliquot of each concentration was transferred, in triplicate, into a clean incubation chamber, and the volume was increased to 50 mL by adding aerated sea water. Ten shrimp nauplii were transferred to each chamber under identical conditions and were maintained throughout the experiment period. Petroleum ether extract in purified water was used as a negative control, and potassium dichromate was used as a positive control. After 24 hours of incubation, the numbers of dead nauplii were observed, and the mortality percentage was calculated. Nauplii were considered dead only if they did not move their appendages for 10 seconds during observation. The concentration that killed 50% of the nauplii (LC50) was determined by using a statistical analysis.
Animal experiments were performed in a Wistar strain of albino female rats weighing approximately between 140 to 180 g. The rats were maintained in a standard environment (temperature: 23 ± 1℃, with 55% ± 5% humidity and a 12-hours/12-hours light/dark cycle) and were supplied with water and a standard laboratory diet. Experiments were conducted in accordance with the guidelines of Committee for Purpose of Control and Supervision of Experimental Animals, New Delhi, India (Registration No: 0367/01/C/CPCSEA), and the study was approved by the institutional ethics committee of the Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India.
The assessment of acute toxicity was performed according to the OECD guidelines NO. 423 . Healthy rats were fasted overnight, but allowed access to water ad libitum. They were randomly divided into five groups (n = 6). The first group (control group) received saline. The other four groups were orally treated with a single dose of petroleum ether extract at 5, 50, 300 and 2000 mg/kg, respectively.
All the treatments were administered by force-feeding. The treated animals were under observation for 72 hours to check the food and water intake, body weight, general behavior observations; also, mortality was checked for 14 days.
The sub-acute toxicity study was performed according to OECD test guidelines . The animals were divided randomly into four groups (n = 6). Group I served as the saline control (0.9% NaCl), and groups II-IV received petroleum ether extract (125, 250 and 500 mg/kg b.w.) daily orally by gavage for 28 days . Toxicity signs and mortality were monitored daily whereas body weight changes and changes in food and water consumption were monitored weekly.
24 hours after the last dose (i.e., 29th day) all the animals were anesthetized with diethyl ether, and cardiac puncture was used to collect blood in non-heparinized tube in order to estimate the serum biochemical parameters. Following dissection, the liver, the kidney and the pancreas were removed and weight immediately.
Blood samples were collected for biochemical analysis (Star 21 Plus, Rapid Diagnostic Pvt. Ltd., New Delhi). The biochemical parameters analyzed to evaluate liver functions were alanine aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, total cholesterol and triglyceride, and total serum protein (TP). Urea, creatinine, sodium and potassium were used to assess the effects on renal functions whereas α-amylase and glucose were used to assess pancreatic functions. Colorimetric estimates were done using commercial diagnostic kits (Span Diagnostics Ltd, Gujarat, India).
Fragments of the liver, kidney and pancreas from female rats were stored immediately in formalin 10% v/v for histopathological analysis. These tissue samples were dehydrated in alcohol, cleared with xylene, and embedded in paraffin. Later, they were sectioned (5 μm), stained with hematoxylin and eosin (H&E), and examined with light microscopy. The samples were analyzed under 40 × magnifications for general structural changes in the organs.
All the studies mentioned above were done in triplicate (except the LD50 study), and the results are expressed as means ± standard deviations (SDs), with each group containing six rats. Graph pad prism version 5.04 was used to determine statistical significance by using ANOVA, followed by Tukey’s multiple comparison tests to assess the mean differences and the significance variation.
The petroleum ether extract of
For the acute toxicity tests, petroleum ether extract was given as single doses. The food and water intakes, body weights, general behavioral changes and mortalities of the animals for these tests are presented in (Table 1) Observations included changes in skin, fur, eyes, mucous membranes, the respiratory, circulatory, autonomic, and central nervous systems, somatomotor activity, and behavior pattern. Attention was also directed toward observations of tremors, convulsions, salivation, diarrhea and sleep patterns. No signs of toxicity were found. No significant variations were observed in the body weights of the rats treated with petroleum ether extract as a single dose (Table 2) Neither signs of toxicity nor death was observed throughout the study following administration of petroleum ether extract for 28 consecutive days. The treated groups did not show any significant changes in organ weights, as shown in (Table 3).
Variations of mean food and water intakes by and body weights of rats following oral administration of test extract of W. volubilis
[Fig. 1] In vitro cytotoxicity evaluation of test extract of W. volubilis administered to brine shrimp nauplus. Potassium dichromate was used as a reference standard. Percentage inhibitions of test samples are dose dependent. Each value is expressed as a mean ± standard deviation (n = 3).
Biochemical parameters such as ALT, ALP, bilirubin, total cholesterol, triglyceride, total protein, glucose, urea, creatinine, sodium, potassium and α-amylase in the treated rats were not significantly different from those in the control group, and all the values remained within the normal limits (Table 4) Histopathological changes were not related to the treatment with petroleum ether extract in any of the analyzed organs. The tissues were morphologically normal. Any changes were slight and similar to those in Wistar rats treated with the vehicle (Figs 2,4).
Effects of different oral single doses of test extract of W. volubilis in rats for acute toxicity
Organ weights of rats in the sub-acute toxicity study for the control group and the groups treated with different doses of the test extract of W. volubilis
Serum biochemical values for rats in the sub-acute toxicity study for the control group and the groups treated with different doses of the test extract of W. volubilis
Cytotoxicity studies on brine shrimps are considered to be a useful tool for preliminary assessment of
The oral route of drug administration is the most convenient and commonly used route for toxicity evaluations in pre-clinical animal models.
[Fig. 3] Histopathological examinations of rat kidney sections from the control rats and from the test rats treated with extract of W. volubilis. G, glomerulus; BS, Bowman space; PT, proximal tubule; DT, distal tubule; IV, interstitial vessel; ET, epithelium of tubules; NE, normal epithelium.
[Fig. 4] Histopathological examinations of rat pancreas sections from the control rats and from the test rats treated with extract of W. volubilis. EG, exocrine gland; IP, interlobular septae; IL, Islets of Langerhans; AI, unatrophied islets; EI, endocrine islets; VR, vascular regeneration.
Based on the acute toxicity of the petroleum ether extract of
Assessments of liver function are made by estimating the activities of elevated serum alanine transaminase, alkaline phosphatase and bilirubin, because these molecules leak into the blood stream in compliance with the extent of liver damages . Alanine transaminase, which mediates the conversion of alanine to pyruvate and glutamate, is specific to the liver and is a suitable indicator of hepatic injuries. Increased levels of this enzyme are an indicator of cellular infiltration and functional disturbance of liver- cell membranes. Alkaline phosphatase is membrane bound, and its alteration is likely to affect the membrane’s permeability and produce derangement in the transport of metabolites. On the other hand, bilirubin is associated with the function of hepatic cells. In the current study, no significant elevations in markers of liver injury (ALT, ALP and bilirubin) were observed in the rats treated with petroleum ether extract of
Inefficient scavenging of reactive oxygen species might be implicated in the oxidative inactivation of enzymes, especially the deleterious effects, due to accumulation of superoxide radicals. The generation of hydroxyl radicals from the superoxide-anion Haber-Weiss reaction, resulting in peroxidation of membrane lipids and in protein glycation, leads to oxidative damage in biomolecules such as carbohydrates, proteins, DNA and cellular organelles. A variety of derangements in metabolic and regulatory mechanisms due to insulin deficiency - elevated free fatty acids levels decrease insulin sensitivity because of pancreatic lipotoxicity/inhibition of insulin - stimulated glucose transport (glucose transport plays an essential role in free fatty acid gene expression for carbohydrates and lipids metabolism) - are also responsible for accumulation of lipids, and this may be due to a higher activity of cholesterol biosynthesis enzymes (HMG-COA reductase) or high levels of lipolysis.
Due to lipolysis, the visceral adipose deposition of elevated free fatty acids from adipose tissues to non-adipose tissues (liver, skeletal muscle) may lead to excessive endogenous glucose production. Triglyceridemia is also associated with the metabolic consequences of hyperinsulinemia, insulin resistance and glucose tolerance.
Glucose is the primary and an essential source of energy for the cells in the body. The blood-glucose level in the body is tightly regulated and is maintained at approximately 5 mmol/L. Not maintaining this level leads to chronically high (hyperglycemia) or low (hypoglycemia) glucose levels. Selective destruction of the β-cells of the pancreas by generating excess reactive oxygen species in order to break DNA bases by alkylation leads to diabetogenic activity. Increased total cholesterol and triglyceride levels represent a displayed lipid profile known as an atherogenic profile, uptake of oxidized form of lipids by cellular macrophages results in the formation of foam cells, and vascular endothelial cholesterol accumulation, which promotes the development of characteristic fatty streaks found in atherosclerotic lesions, leads to the development of coronary heart disease. During oxidative stress, reactive oxygen species have a strong oxidizing ability to damage cellular proteins, which causes loss of body weight due to increased muscle destructions and loss of protein content in the tissue, and subsequently changes in serum levels. The reduction is attributed to the damage produced locally in the endoplasmic reticulum, which results in the loss of cytochrome P 450 and leads to its functional failure, resulting in decreased protein synthesis and increased accumulation of triglycerides during glycation of lipoprotein lipase deficiency and possibly contributing to a significant elevation of triglycerides. This action could be due to microproteinuria and increased protein catabolism, which are important clinical markers in essential organ failure [25, 26]. These results raised and confirmed a number of interesting issues with petroleum ether extract of
The reduction is attributed to the damage produced locally in the endoplasmic reticulum, which results in the loss of cytochrome P 450 and leads to its functional failure, resulting in decreased protein synthesis and increased accumulation of triglycerides during glycation of lipoprotein lipase deficiency and possibly contributing to a significant elevation of triglycerides. This action could be due to microproteinuria and increased protein catabolism, which are important clinical markers in essential organ failure [25, 26]. These results raised and confirmed a number of interesting issues with petroleum ether extract of
The kidneys are the primary means for eliminating waste products of metabolism that are no longer needed by the body; these products, non-protein nitrogen’s, include urea (from the metabolism of amino acid) and creatinine (from muscle creatinine). The concentrations of these, particularly of urea, can be as high as 10 times normal for 1 to 2 weeks, and this total condition is called uremia/total renal failure. It would be reasonable to suspect that decreasing the number of functional nephrons, which reduces the glomerular filtration rate (GFR), would also cause major decreases in renal excretion of water and solutes. A reduction in the number of nephrons leads to electrolyte and fluid retention, and death usually ensues when the number of nephrons falls below 5 to 10 percent of normal. In contrast to the electrolytes, many of the waste products of metabolism, such as urea and creatinine, accumulate almost in proportion to the number of nephrons that have been destroyed. The reason for this is that substances such as urea and creatinine depend largely on glomerular filtration for their excretion, and they are not reabsorbed as avidly as the electrolytes. Therefore, if the GFR decreases, the creatinine excretion rate also transiently decreases, causing accumulation of creatinine in the body fluids and raising the plasma concentration until the excretion rate of creatinine returns to normal. However, this normal rate of creatinine excretion occurs at the expense of an elevated plasma creatinine concentration. Effects of petroleum ether extract of
Excretions of excessive cellular inorganic ions (sodium and potassium) are common biological parameters in cases of diabetic ketoacidosis, which leads to tissue dehydration and abnormal levels of electrolytes. Excretion of elevated levels of serum inorganic ions from intra and extra cellular spaces occurs for normal electrolyte equilibrium, but does not in cases of insulin deficiency, hypertonicity and acidemia . Increased fatty acid oxidation is a characteristic of starvation and of diabetes mellitus, leading to ketone body production by the liver (ketosis). Ketone bodies are acidic, and when produced in excess over long periods, as in diabetes, cause ketoacidosis, which is ultimately fatal. In the present study, no significant elevations in cellular inorganic ions were observed with petroleum ether extract of
Elevated levels of pancreatic amylase are common in diabetic ketoacidosis (DKA). Pancreatic acinar cells are responsible for secretion of pancreatic amylase, which is a key enzyme in the digestive system and catalyses the hydrolysis of starch to a mixture of smaller oligosaccharides such as maltose, maltotriose, and oligoglucans . Pancreatic amylase levels were in normal range in the rats treated with petroleum ether extract of
This is the first study to investigate the toxicity of petroleum ether extract of