1) The document describes an experiment that evaluated the anxiolytic effects of a polyherbal preparation called Aq-WS&Al-OC in rats.
2) The preparation was tested in elevated plus maze, light/dark box, and open field tests, and showed significant anxiolytic effects at doses of 10-20mg/kg, as demonstrated by increases in open arm exploration, light chamber exploration, and decreases in immobility.
3) Chronic administration of the polyherbal preparation also produced significant anxiolytic effects in the behavioral tests, indicating it may be effective at reducing anxiety over extended use.
This document describes an enzymatic assay kit for quantitatively measuring acetylcholine concentration in biological samples. The kit uses acetylcholinesterase to hydrolyze acetylcholine to choline, which is then oxidized to produce a product that reacts with a dye to generate a color (colorimetric assay) or fluorescence (fluorimetric assay). The intensity is directly proportional to the acetylcholine concentration and can be used to calculate the amount in samples. The kit provides a simple, direct, and high-throughput method for acetylcholine detection with applications in drug discovery and studies of acetylcholine metabolism.
This document summarizes the bioassay of adrenocorticotropic hormone (ACTH). ACTH is a polypeptide hormone secreted by the pituitary gland that stimulates the adrenal glands to produce cortisol. The bioassay involves administering varying doses of a standard or test ACTH preparation to hypophysectomized rats and measuring the resulting depletion of ascorbic acid in the adrenal glands after 3 hours, using the depletion levels to determine the potency of the test preparation relative to the standard. The procedure involves preparing serial dilutions of the standard and test ACTH, administering doses to groups of hypophysectomized rats, removing and analyzing their adrenal glands for ascor
This document describes a spectrofluorimetry method for determining nalidixic acid concentrations in human plasma. The method involves extracting nalidixic acid from plasma samples using chloroform and analyzing the samples using a spectrofluorimeter. The method was used to analyze plasma samples from a study comparing the bioavailability of a new generic nalidixic acid tablet formulation to a standard brand name formulation. Results of in vitro tests like dissolution and assay showed the new generic formulation was comparable to the standard. Pharmacokinetic analysis of plasma concentration data from subjects who received the formulations found no significant differences between the products.
Expt. 5 Study of anti ulcer activity of a drug using nsaid induced ulcer modelVISHALJADHAV100
This document describes an experiment to study the anti-ulcer activity of drugs using a NSAID-induced ulcer model in rats. The experiment involves pretreating rats with cimetidine or ranitidine before administering ulcerogenic NSAIDs like aspirin or indomethacin. The rats are then sacrificed and their stomachs examined for ulcers. Parameters like gastric volume, acidity, and ulcer index are measured and compared between treated and untreated groups to determine the antisecretory and ulcer protective effects of cimetidine and ranitidine. The results show that cimetidine and ranitidine reduce gastric acid secretion and inhibit ulcer formation in NSAID-treated rats.
Expt. 1 Bioassay of serotonin using rat fundus strip by three point bioassayVISHALJADHAV100
This document describes an experiment to determine the unknown concentration of serotonin using a three-point bioassay with an isolated rat fundus strip preparation. The experiment involves constructing dose-response curves for a serotonin standard and test sample, selecting doses that elicit submaximal responses in a 1:2 ratio, and determining the test concentration using the measured responses. Rat fundus tissue is sensitive to serotonin and contracts in a concentration-dependent manner when exposed to increasing doses of the drug. The experiment aims to precisely and reliably estimate the concentration of an unknown serotonin sample through this validated bioassay method.
Expt. 6 Study of effect of drugs on gastrointestinal motilityVISHALJADHAV100
Objective
Principle
Requirements
Preparation of Tyrode solution
Procedure
Kymograph recording of contractions
Observation table
Result and Interpretation
Bioassy of insulin according to Indian pharmacopoeiaSONALPANDE5
This document summarizes several methods for bioassaying insulin, including preparation of standard and test solutions, rabbit, mouse, rat diaphragm, and rat epididymal fat pad methods. It also describes the radioimmunoassay method, which uses radiolabeled insulin and antibodies to determine insulin concentration in test samples by comparing to standard curves.
Expt. 2 Bioassay of acetylcholine using rat ileum by four point bioassayVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of ACh stock and standard solutions
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
This document describes an enzymatic assay kit for quantitatively measuring acetylcholine concentration in biological samples. The kit uses acetylcholinesterase to hydrolyze acetylcholine to choline, which is then oxidized to produce a product that reacts with a dye to generate a color (colorimetric assay) or fluorescence (fluorimetric assay). The intensity is directly proportional to the acetylcholine concentration and can be used to calculate the amount in samples. The kit provides a simple, direct, and high-throughput method for acetylcholine detection with applications in drug discovery and studies of acetylcholine metabolism.
This document summarizes the bioassay of adrenocorticotropic hormone (ACTH). ACTH is a polypeptide hormone secreted by the pituitary gland that stimulates the adrenal glands to produce cortisol. The bioassay involves administering varying doses of a standard or test ACTH preparation to hypophysectomized rats and measuring the resulting depletion of ascorbic acid in the adrenal glands after 3 hours, using the depletion levels to determine the potency of the test preparation relative to the standard. The procedure involves preparing serial dilutions of the standard and test ACTH, administering doses to groups of hypophysectomized rats, removing and analyzing their adrenal glands for ascor
This document describes a spectrofluorimetry method for determining nalidixic acid concentrations in human plasma. The method involves extracting nalidixic acid from plasma samples using chloroform and analyzing the samples using a spectrofluorimeter. The method was used to analyze plasma samples from a study comparing the bioavailability of a new generic nalidixic acid tablet formulation to a standard brand name formulation. Results of in vitro tests like dissolution and assay showed the new generic formulation was comparable to the standard. Pharmacokinetic analysis of plasma concentration data from subjects who received the formulations found no significant differences between the products.
Expt. 5 Study of anti ulcer activity of a drug using nsaid induced ulcer modelVISHALJADHAV100
This document describes an experiment to study the anti-ulcer activity of drugs using a NSAID-induced ulcer model in rats. The experiment involves pretreating rats with cimetidine or ranitidine before administering ulcerogenic NSAIDs like aspirin or indomethacin. The rats are then sacrificed and their stomachs examined for ulcers. Parameters like gastric volume, acidity, and ulcer index are measured and compared between treated and untreated groups to determine the antisecretory and ulcer protective effects of cimetidine and ranitidine. The results show that cimetidine and ranitidine reduce gastric acid secretion and inhibit ulcer formation in NSAID-treated rats.
Expt. 1 Bioassay of serotonin using rat fundus strip by three point bioassayVISHALJADHAV100
This document describes an experiment to determine the unknown concentration of serotonin using a three-point bioassay with an isolated rat fundus strip preparation. The experiment involves constructing dose-response curves for a serotonin standard and test sample, selecting doses that elicit submaximal responses in a 1:2 ratio, and determining the test concentration using the measured responses. Rat fundus tissue is sensitive to serotonin and contracts in a concentration-dependent manner when exposed to increasing doses of the drug. The experiment aims to precisely and reliably estimate the concentration of an unknown serotonin sample through this validated bioassay method.
Expt. 6 Study of effect of drugs on gastrointestinal motilityVISHALJADHAV100
Objective
Principle
Requirements
Preparation of Tyrode solution
Procedure
Kymograph recording of contractions
Observation table
Result and Interpretation
Bioassy of insulin according to Indian pharmacopoeiaSONALPANDE5
This document summarizes several methods for bioassaying insulin, including preparation of standard and test solutions, rabbit, mouse, rat diaphragm, and rat epididymal fat pad methods. It also describes the radioimmunoassay method, which uses radiolabeled insulin and antibodies to determine insulin concentration in test samples by comparing to standard curves.
Expt. 2 Bioassay of acetylcholine using rat ileum by four point bioassayVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of ACh stock and standard solutions
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
Expt. 11 Determination of acute eye irritation corrosion of a test substanceVISHALJADHAV100
This document outlines the procedure for conducting an acute eye irritation/corrosion test of a substance using albino rabbits. The objective is to determine the degree of eye irritation or corrosion caused by a test substance. A single dose of the substance is applied to one eye of each rabbit, while the other eye serves as a control. Lesions to the conjunctiva, cornea, and iris are observed and graded at various time intervals over 72 hours. Based on the grading scores of the test eye compared to the control eye, the sensitization potential of the substance can be determined.
Expt. 5 Bioassay of oxytocin using rat uterine horn by interpolation methodVISHALJADHAV100
Experiment No. 5 was a bioassay to determine unknown concentrations of oxytocin using isolated rat uterine horns through an interpolation bioassay. Rat uteri were isolated and mounted in an organ bath containing De Jalon solution. A concentration response curve was established for a standard oxytocin solution and responses to test solutions were recorded. The test responses were selected such that they fell on the linear portion of the standard curve. The height of contractions were measured and used to calculate the unknown oxytocin concentrations by interpolating the test responses on the standard curve graphically. The unknown concentrations were determined to be [value 1] μg/ml from one method of calculation and [value 2] μg/ml from the other method.
This document describes biological assays used to test insulin, including a hypoglycemic seizure test in mice and a rabbit sugar method. The hypoglycemic seizure test involves injecting varying doses of insulin samples and a reference solution into mice that have been food deprived, then observing them for seizures within 1.5 hours. The rabbit sugar method involves preparing dilutions of an insulin standard and sample solutions, injecting them into fasted rabbits, then testing their blood sugar levels 5 minutes later to quantitatively compare the solutions. The goal of these assays is to measure insulin potency and identify samples through chemical analysis and comparison to reference standards.
Expt. 7 Bioassay of acetylcholine using rat ileum by four point bioassayVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of ACh stock and standard solutions
Preparation of frog ringer solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
Assessment of Anodyne Activity of Different Leaf Extracts of Celastrus Panicu...IJSTA
This document summarizes a study that assessed the analgesic (pain-relieving) potential of different leaf extracts of Celastrus paniculatus. The study found that all test extracts (petroleum ether, ethyl acetate, and methanol) exhibited significant analgesic activity in a mouse model of pain. The methanol extract showed the most potent effect, followed by the ethyl acetate and petroleum ether extracts, respectively. This suggests that C. paniculatus leaf contains chemical constituents with marked analgesic properties.
This document provides details on bioassay methods for several compounds including vasopressin, digitalis, d-tubocurarine, histamine, and 5-hydroxytryptamine (5-HT).
It describes two common bioassay methods for vasopressin - the first uses rats to measure changes in blood pressure, the second uses rats and measures anti-diuretic activity. For digitalis, it outlines guinea pig and pigeon bioassays measuring the lethal dose. The d-tubocurarine bioassay uses rabbits to measure head drop or isolated frog muscle to measure contraction reduction. Histamine is assayed using guinea pig ileum or other tissues and measuring contraction. Finally, 5
This document summarizes the bioassay method used to test the potency of heparin preparations. The method involves comparing the clotting time of plasma incubated with the test heparin sample to the clotting time of plasma incubated with a reference standard heparin preparation. Several dilutions of both the standard and test sample are tested to identify the dilution for each that results in equivalent clotting time. The potency of the test sample can then be estimated based on its equivalent dilution relative to the standard. The bioassay follows specific procedures for obtaining and preparing plasma, making standard and test solutions, conducting the clotting time assays, and calculating potency results.
Expt. 6 Bioassay of histamine using guinea pig ileum by matching methodVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of histamine standard solution
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
This presentation is on the bioassay of heparin which helps to know the potency of new heparin drug or heparin conc in individual suffering from heparin resistance diseases.
This was made by my friend Naailah and me. Hope it helps.
Bioassays are used to determine the potency of drugs when chemical assays are not available or sufficient. They involve comparing the biological effects of a test drug to a reference standard. Three common bioassay methods include end point assays which measure the threshold dose, matching assays which bracket test doses between standard doses, and graphical assays which plot dose-response curves. Important drugs assayed include digitalis, adrenaline, acetylcholine, and antagonists. Assays use tissues like cat blood pressure, guinea pig ileum, rat uterus to measure relevant pharmacological effects like changes in blood pressure or muscle contraction.
The document summarizes the bioassay of heparin sodium. The bioassay involves comparing the concentration of a standard heparin preparation needed to prevent clotting of plasma to that of the test heparin solution. Prepared plasma is mixed with increasing dilutions of both the standard and test heparin solutions and the degree of clotting is observed after 1 hour and used to determine the potency of the test sample relative to the standard. Heparin is an anticoagulant drug extracted from animal tissues and used to treat and prevent blood clots and thrombosis. Its potency is typically measured using clotting-based biological assays.
This document describes an experiment to estimate the unknown concentration of an acetylcholine solution using hen's ileum tissue in an interpolation bioassay. The experiment involves mounting hen's ileum tissue in an organ bath, exposing it to solutions of known acetylcholine concentrations to generate a standard concentration-response curve, and then exposing it to the test solution to interpolate its concentration based on the curve. Key steps include tissue stabilization, adjusting basal tension, exposing tissues to different acetylcholine concentrations to create the standard curve, exposing to the test solution, measuring responses, plotting the curve, and calculating the concentration of the unknown based on its response. The aim is to estimate unknown drug concentrations using a reliable and less time-consuming interpolation bioassay
The document discusses bioassay, which is the estimation of the concentration or potency of a substance by measuring its biological response over a biological system under standard conditions. It describes the purposes of bioassay such as determining drug potency and specificity. The principles and types of bioassay techniques like graded response, quantal, and multi-point assays are explained. Examples of bioassays for important drugs like insulin are provided. The document highlights the applications, uses, advantages, and disadvantages of bioassay.
Expt. 4 Study of anti ulcer activity of a drug using pylorus ligand (SHAY) ra...VISHALJADHAV100
This document describes an experiment to study the anti-ulcer activity of drugs like ranitidine and cimetidine using a pylorus ligation rat model. Rats were divided into control, cimetidine, and ranitidine groups. The pylorus of rats were ligated for 4 hours to induce ulcers. Drugs were administered before ligation. After ligation, gastric contents were analyzed for volume, pH, and acidity. Stomachs were examined for ulcers. Cimetidine and ranitidine showed reduced gastric acid secretion and fewer ulcers compared to controls, demonstrating their antiulcer activity.
Diuretic activity of ethanolic extracts of ficus carica l fruits ijrpppharmaindexing
This document summarizes a study that evaluated the diuretic activity of ethanolic extracts of Ficus carica L. fruits in rats. Rats were treated with ethanol extracts at doses of 100 mg/kg and 200 mg/kg or with furosemide as a reference drug. The extracts produced a significant increase in urine volume as well as sodium, potassium, and chloride ion excretion compared to the control group. No toxicity was observed for the extracts at doses up to 2000 mg/kg. The 200 mg/kg extract dose produced the best results for urine output. The findings support the traditional use of F. carica as a diuretic agent.
1) The study evaluated the antidepressant activity of the ethanolic extract of Ocimum sanctum (tulsi) leaves in mice.
2) In the forced swim test, Ocimum sanctum showed significant antidepressant activity at a dose of 8.5 mg/kg compared to the control.
3) The study indicates the potential antidepressant-like effects of Ocimum sanctum, though the exact mechanism is still unknown. Further research is needed to elucidate the mechanism of action.
Gallic acid, found in plants like sumac and tea leaves, was studied for its potential anxiolytic effects in rats. Rats were given various doses of gallic acid or diazepam daily for 10 days and then tested in two models - the elevated plus maze and bright/dark arena. In both tests, gallic acid produced behaviors indicating reduced anxiety, such as increased time in open/lit areas, at doses comparable to diazepam. The study suggests that gallic acid has anxiolytic properties, possibly through effects on GABA and nitric oxide systems in the brain. Further research is needed to understand the mechanism and potential for human use.
Expt. 11 Determination of acute eye irritation corrosion of a test substanceVISHALJADHAV100
This document outlines the procedure for conducting an acute eye irritation/corrosion test of a substance using albino rabbits. The objective is to determine the degree of eye irritation or corrosion caused by a test substance. A single dose of the substance is applied to one eye of each rabbit, while the other eye serves as a control. Lesions to the conjunctiva, cornea, and iris are observed and graded at various time intervals over 72 hours. Based on the grading scores of the test eye compared to the control eye, the sensitization potential of the substance can be determined.
Expt. 5 Bioassay of oxytocin using rat uterine horn by interpolation methodVISHALJADHAV100
Experiment No. 5 was a bioassay to determine unknown concentrations of oxytocin using isolated rat uterine horns through an interpolation bioassay. Rat uteri were isolated and mounted in an organ bath containing De Jalon solution. A concentration response curve was established for a standard oxytocin solution and responses to test solutions were recorded. The test responses were selected such that they fell on the linear portion of the standard curve. The height of contractions were measured and used to calculate the unknown oxytocin concentrations by interpolating the test responses on the standard curve graphically. The unknown concentrations were determined to be [value 1] μg/ml from one method of calculation and [value 2] μg/ml from the other method.
This document describes biological assays used to test insulin, including a hypoglycemic seizure test in mice and a rabbit sugar method. The hypoglycemic seizure test involves injecting varying doses of insulin samples and a reference solution into mice that have been food deprived, then observing them for seizures within 1.5 hours. The rabbit sugar method involves preparing dilutions of an insulin standard and sample solutions, injecting them into fasted rabbits, then testing their blood sugar levels 5 minutes later to quantitatively compare the solutions. The goal of these assays is to measure insulin potency and identify samples through chemical analysis and comparison to reference standards.
Expt. 7 Bioassay of acetylcholine using rat ileum by four point bioassayVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of ACh stock and standard solutions
Preparation of frog ringer solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
Assessment of Anodyne Activity of Different Leaf Extracts of Celastrus Panicu...IJSTA
This document summarizes a study that assessed the analgesic (pain-relieving) potential of different leaf extracts of Celastrus paniculatus. The study found that all test extracts (petroleum ether, ethyl acetate, and methanol) exhibited significant analgesic activity in a mouse model of pain. The methanol extract showed the most potent effect, followed by the ethyl acetate and petroleum ether extracts, respectively. This suggests that C. paniculatus leaf contains chemical constituents with marked analgesic properties.
This document provides details on bioassay methods for several compounds including vasopressin, digitalis, d-tubocurarine, histamine, and 5-hydroxytryptamine (5-HT).
It describes two common bioassay methods for vasopressin - the first uses rats to measure changes in blood pressure, the second uses rats and measures anti-diuretic activity. For digitalis, it outlines guinea pig and pigeon bioassays measuring the lethal dose. The d-tubocurarine bioassay uses rabbits to measure head drop or isolated frog muscle to measure contraction reduction. Histamine is assayed using guinea pig ileum or other tissues and measuring contraction. Finally, 5
This document summarizes the bioassay method used to test the potency of heparin preparations. The method involves comparing the clotting time of plasma incubated with the test heparin sample to the clotting time of plasma incubated with a reference standard heparin preparation. Several dilutions of both the standard and test sample are tested to identify the dilution for each that results in equivalent clotting time. The potency of the test sample can then be estimated based on its equivalent dilution relative to the standard. The bioassay follows specific procedures for obtaining and preparing plasma, making standard and test solutions, conducting the clotting time assays, and calculating potency results.
Expt. 6 Bioassay of histamine using guinea pig ileum by matching methodVISHALJADHAV100
Objective
Principle
Requirements
Experimental specifications (conditions)
Preparation of histamine standard solution
Preparation of Tyrode solution (PSS)
Procedure
Kymograph recording of contractions
Observation table
Calculation
Result and interpretation
This presentation is on the bioassay of heparin which helps to know the potency of new heparin drug or heparin conc in individual suffering from heparin resistance diseases.
This was made by my friend Naailah and me. Hope it helps.
Bioassays are used to determine the potency of drugs when chemical assays are not available or sufficient. They involve comparing the biological effects of a test drug to a reference standard. Three common bioassay methods include end point assays which measure the threshold dose, matching assays which bracket test doses between standard doses, and graphical assays which plot dose-response curves. Important drugs assayed include digitalis, adrenaline, acetylcholine, and antagonists. Assays use tissues like cat blood pressure, guinea pig ileum, rat uterus to measure relevant pharmacological effects like changes in blood pressure or muscle contraction.
The document summarizes the bioassay of heparin sodium. The bioassay involves comparing the concentration of a standard heparin preparation needed to prevent clotting of plasma to that of the test heparin solution. Prepared plasma is mixed with increasing dilutions of both the standard and test heparin solutions and the degree of clotting is observed after 1 hour and used to determine the potency of the test sample relative to the standard. Heparin is an anticoagulant drug extracted from animal tissues and used to treat and prevent blood clots and thrombosis. Its potency is typically measured using clotting-based biological assays.
This document describes an experiment to estimate the unknown concentration of an acetylcholine solution using hen's ileum tissue in an interpolation bioassay. The experiment involves mounting hen's ileum tissue in an organ bath, exposing it to solutions of known acetylcholine concentrations to generate a standard concentration-response curve, and then exposing it to the test solution to interpolate its concentration based on the curve. Key steps include tissue stabilization, adjusting basal tension, exposing tissues to different acetylcholine concentrations to create the standard curve, exposing to the test solution, measuring responses, plotting the curve, and calculating the concentration of the unknown based on its response. The aim is to estimate unknown drug concentrations using a reliable and less time-consuming interpolation bioassay
The document discusses bioassay, which is the estimation of the concentration or potency of a substance by measuring its biological response over a biological system under standard conditions. It describes the purposes of bioassay such as determining drug potency and specificity. The principles and types of bioassay techniques like graded response, quantal, and multi-point assays are explained. Examples of bioassays for important drugs like insulin are provided. The document highlights the applications, uses, advantages, and disadvantages of bioassay.
Expt. 4 Study of anti ulcer activity of a drug using pylorus ligand (SHAY) ra...VISHALJADHAV100
This document describes an experiment to study the anti-ulcer activity of drugs like ranitidine and cimetidine using a pylorus ligation rat model. Rats were divided into control, cimetidine, and ranitidine groups. The pylorus of rats were ligated for 4 hours to induce ulcers. Drugs were administered before ligation. After ligation, gastric contents were analyzed for volume, pH, and acidity. Stomachs were examined for ulcers. Cimetidine and ranitidine showed reduced gastric acid secretion and fewer ulcers compared to controls, demonstrating their antiulcer activity.
Diuretic activity of ethanolic extracts of ficus carica l fruits ijrpppharmaindexing
This document summarizes a study that evaluated the diuretic activity of ethanolic extracts of Ficus carica L. fruits in rats. Rats were treated with ethanol extracts at doses of 100 mg/kg and 200 mg/kg or with furosemide as a reference drug. The extracts produced a significant increase in urine volume as well as sodium, potassium, and chloride ion excretion compared to the control group. No toxicity was observed for the extracts at doses up to 2000 mg/kg. The 200 mg/kg extract dose produced the best results for urine output. The findings support the traditional use of F. carica as a diuretic agent.
1) The study evaluated the antidepressant activity of the ethanolic extract of Ocimum sanctum (tulsi) leaves in mice.
2) In the forced swim test, Ocimum sanctum showed significant antidepressant activity at a dose of 8.5 mg/kg compared to the control.
3) The study indicates the potential antidepressant-like effects of Ocimum sanctum, though the exact mechanism is still unknown. Further research is needed to elucidate the mechanism of action.
Gallic acid, found in plants like sumac and tea leaves, was studied for its potential anxiolytic effects in rats. Rats were given various doses of gallic acid or diazepam daily for 10 days and then tested in two models - the elevated plus maze and bright/dark arena. In both tests, gallic acid produced behaviors indicating reduced anxiety, such as increased time in open/lit areas, at doses comparable to diazepam. The study suggests that gallic acid has anxiolytic properties, possibly through effects on GABA and nitric oxide systems in the brain. Further research is needed to understand the mechanism and potential for human use.
IOSRPHR(www.iosrphr.org) IOSR Journal of Pharmacyiosrphr_editor
This study investigated the effects of the aldosterone antagonist spironolactone on pain and anxiety in mice. Spironolactone was administered orally to mice in varying doses. Thermally induced pain was assessed using hot plate and tail immersion tests, where spironolactone decreased pain threshold in a dose-dependent manner. Visceral pain induced by acetic acid was increased, as spironolactone reduced the number of writhes. In behavioral tests, spironolactone increased immobilization time and urination while decreasing ambulation, rearing and grooming. The study suggests spironolactone modulates pain response by increasing somatic pain and decreasing visceral pain, while also
IOSRPHR(www.iosrphr.org) IOSR Journal of Pharmacyiosrphr_editor
This study investigated the effects of the aldosterone antagonist spironolactone on pain and behavior in mice. Spironolactone was found to decrease pain thresholds in thermal pain tests but increase pain thresholds in a visceral pain test, indicating it modulates pain responses at spinal and supraspinal sites. In behavioral tests, spironolactone decreased ambulation and rearing but increased immobilization and urination. The results suggest spironolactone acts on mineralocorticoid receptors in the brain to modulate pain and that these receptors are involved in descending pathways regulating spinal nociception and visceral sensitivity.
Abstract— Roots of Panax notoginseng were fermented with 30 fungi respectively. Almost one-third of the products showed increasing antibacterial activity. All products could inhibit GST-CDC25 phosphatase as a potential antitumor agent. HPLC profiles proved that components of unfermented P. notoginseng and fermented P. notoginseng have obviously changes.
This document describes experiments to study the effects of various drugs on isolated frog heart, blood pressure, and heart rate in dogs. The experiments involve setting up perfused frog heart preparations and measuring the chronotropic and inotropic effects of drugs. The effects of drugs are also studied on normal and calcium-depleted hypodynamic frog hearts. In addition, the effects of sympathomimetic and parasympathomimetic drugs on mean blood pressure and heart rate are measured in anesthetized dogs using a cannulation technique.
The document describes a study that evaluated the neuroprotective effects of Cucurbita pepo (pumpkin seed) extracts against diazepam-induced amnesia and ethanol-induced cognitive impairment in rodents. Rodents were administered ethanol or diazepam to induce impairment and then treated with aqueous or ethanolic C. pepo extracts. Their learning and memory was assessed using Morris water maze and 8-arm radial maze tests. The results showed that rodents treated with C. pepo extracts had significantly reduced transfer latency times and errors compared to the impairment-induced groups, suggesting the extracts improved learning and memory. Therefore, C. pepo may have potential as a therapeutic agent for neurodegenerative diseases like
This study evaluated the analgesic efficacy of garlic shoot extract (GSE) in experimental pain models in mice. Mice were orally administered GSE at doses of 125, 250, and 500 mg/kg, and tested in the writhing, hot plate, and tail flick tests. GSE significantly reduced writhing episodes in a dose-dependent manner, and prolonged reaction times in the hot plate and tail flick tests at higher doses, comparable to standard analgesics. The study demonstrates that GSE possesses analgesic properties in both central and peripheral pain models.
EVALUATION OF ANALGESIC AND ANTI-INFLAMMATORY.pdfgynomark
Amaranthus roxburghianus is one of the traditionally well-known plants with outstanding therapeutic properties, and
is used mostly in treating different diseases in India. Thus, based on these medicinal properties, various investigations
have been undertaken in order to appraise the pharmacological activities and the chemical composition of these
species. Here, we elucidate the analgesic and anti-inflammatory activity of Amaranthus roxburghianus ethanolic
leaves extract. phytochemical screening of Amaranthus roxburghianus extract showed the presence of alkaloids,
Carbohydrates, Glycosides, Flavonoids, Tannins, Proteins, Amino Acids. the ethanolic leaves extract of Amaranthus
roxburghianus, possess peripheral and central analgesic activity in animal model. The Amaranthus roxburghianus
leaves extract shows anti-inflammatory activity in different animal model. Flavonoids and tannins are the major
constituents of Amaranthus roxburghianus leaves, which may be responsible for its Analgesic, Anti-inflammatory
activity.
KEYWORDS: Amaranthus roxburghianus, Phytochemical screening, Analgesic activity, Anti-inflammatory activity
INVESTIGATION THE EFFECT OF PENTHYLENTETRAZOLE INDUCING EPILEPSY MODEL USING ...Self-employed researcher
This study investigated the anticonvulsant effects of Epilobium hirsutum extract in a pentylenetetrazole (PTZ)-induced seizure model in mice. Mice were pretreated with 100 or 200 mg/kg of E. hirsutum extract or valproate before being injected with PTZ. The extract increased seizure onset time and decreased duration compared to the PTZ group. Neurological tests and biochemical assays also showed the extract reduced oxidative stress and improved motor function versus PTZ. The results suggest E. hirsutum has anticonvulsant properties potentially due to its antioxidant compounds like flavonoids and phenolic acids.
The document summarizes an assessment of the neuropharmacological activity of the methanolic extract of Lilium candidum. Preliminary tests found the extract contains flavonoids, tannins, steroids, terpenoids, and alkaloids. In hole cross and open field tests on mice, the extract at doses of 200 and 400 mg/kg showed significant suppression of movement from 30-90 minutes after administration, comparable to the drug diazepam. This indicates the extract has central nervous system depressant effects by decreasing exploratory behavior in a dose-dependent manner. The presence of flavonoids, alkaloids, terpenoids and tannins in the extract may account for its neuropharmacological
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
DOI: 10.21276/ijlssr.2016.2.3.16
ABSTRACT- The present research article was described about the hypotriglycerdemic activity of Withania coagulans
bud extract. Withania coagulans Dunal belonging to the family Solanaceae is a small bush which is widely spread in
South Asia. The biological activity of with anolides from Withania coagulans has antihyperglycaemic activity and the
plant is commonly called as Indian cheese maker due to the milk coagulation characteristics of the bud. The present study
was to investigate preliminary studies shows satisfactory result. The chromatographic studies like TLC, HPTLC and
HPLC show good spot. HPTLC shows maximum height and area of 18.83%.HPLC shows maximum peak at 1.867
minutes having area coverage of 87.4%.The free radical scavenging activity of chloroform fraction (CF) of a crude drug
shows 510μg/ml of scavenging activity. The IC50 value for MTT assay was found to be 84.7μg/ml. The GLUT4 study
shows significant uptake of glucose. PPAR gamma activity regulation of glucose disposal and insulin sensitivity in the
skeletal muscles shows concentration dependence response using standard Pioglitazone. The bud of Withania coagulants
will be a promising medicine for more ailments.
Key-words- Withania coagulants, Hypotriglycerdemic, HPLC, HPTLC, GLUT-4, MTT assay
Isolation of Alkaloid from a Medical Plant (A Case Study of Morinda Lucida)iosrjce
The isolation and detection of alkaloids content of Morinda lucida (Ezeogwu) from Rubiceace family,
a medicinal plant was carried out using solvent extraction process. The dried powdered leaves of the plant were
divided into batches. Different solvents were used on them. After 6 days of occasional shaking, it was filtered.
The filtrates were used for testing the presence of alkaloids in Morida Lucida. Mayer’s reagents Wagner and
Lugol’s reagents and 5m sodium hydroxide were used as detecting reagents. Mayer’s reagent yields cream
precipitate in both acidic and alkaline extracts. Wagner and Lugol’s reagents yield reddish brown precipitate in
both acidic and alkaline extracts. 5ml sodium hydroxide gave white swirling precipitate. Other coloured
precipitate like orange and pale orange was gotten as a result of difference in solvents used for isolation. The
presence of the above precipitate indicates the presence of alkaloids in Morinda Lucida.
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gouthami 5-9-26
1. ANTIANXIETY ACTIVITY OF Aq-WS&Al-OC,
A POLYHERBAL PREPARATION IN RATS
UNDER THE GUIDANCE OF
Mr.MD.GAYASUDDIN MOUID., M.Pharm.,
HEAD OF DEPARTMENT PHARMACOLOGY
SMT.SAROJINI RAMULAMMA COLLEGE OF PHARMACY
PRESENTED BY
A.GOUTAMI GODAVARI
B.PHARM –IVyr,
05-09-26
3. INTRODUCTION
Although many drugs are available in allopathic medicine to
treat anxiety disorders they produce various systemic side
effects where as ayurvedic medicine products are free from
side effects and less toxic.
Aq-WS&Al-OC, a polyherbal product containing
aq.extracts of Withania somnifera and shilajit and alcoholic
extracts of Ocimum sanctum and Camellia sinensis has
antianxiety and antidepressant properties[1] .
But there is lack of experimental and clinical evidence.
Therefore this study was under taken to evaluate effects of
this polyherbal preparation on anxiety like behaviour in
rats by using experimental models like elevated plus maze
and light and dark box.
Anxiety is a normal emotional behaviour . When it is severe
/chronic it becomes pathological and can cause cardiovascular
and psychiatric disorders
.
4. SCOPE AND PLAN OF WORK
SCOPE:
To study the anxiolytic-like activity of Aq-WS & Al-OC, a
polyherbal product in rats.
PLAN OF WORK:
To evaluate the effects of polyherbal preparation by using
experimental modals like elevated plus maze, light and dark box
and openfield.
5. MATERIALS AND METHODS
1.ANIMALS:
Male wistar albino rats weighing 150 to 180g (90
to 110days old) were used for this study.
They were housed in clean, clear, polypropylene
cages in groups of four and maintained at 24±2˚C
with 12h light and dark cycle.
Each rat was used only once.
Fig1: Albino rat and
polypropylene cage
2.DRUGS:
The standard anxiolytic drug- diazepam (0.5 and
1mg/kg)
The Test drug- dry powder of Aq-WS&Al-OC (5,10
and 20mg/kg) were suspended in 1%gumacacia solution.
6. DRUG PROFILE
Aq-WS&Al-OC (polyherbal preparation)
Category: anxiolytic.
Table1:Phytochemical screening:
Composition and use:
• For Withania somnifera and Ocimum sanctum- antistress, adaptogenic and
immunomodulatory ,antioxidant.
• Camellia sinensis- CNS stimulant.
• Shilajit (herbomineral product)-strengthen the nervous system,treatment of
nervous disorders like mentalstress, depression.
Route of administration: oral route
s.no Plant name Active constituents Chemical test
1 Withania somnifera Alkaloids +
2 Ocimum sanctum Tannins&urosolicacid +
3 Camellia sinensis Methyl xanthines +
7. METHODS
Drug solution was prepared freshly just before the administration using
aq.extraction by distillation method and alcoholic extraction by soxhlet
apparatus administered orally.
Locomotor activity was assesssed with open field test.
Where as in elevated plus maze and light and dark box unfamiliar, non-
protective and bright environmental stress provokes inhibition of normal
behavior.
As in elevated plus maze open arms are more fear provoking than the closed
arm.
Control group of animals received appropriate volume of vehicle, 1% gum
acacia solution.
In acute study: drugs /vehicle were administered 60mins prior to experiment.
In chronic study : drugs /vehicle were administered once daily for ten days
and the last dose was given on the tenth day, 60mins prior to experiment.
8. APPARATUS
1.ELEVATED PLUS MAZE
The wooden maze consisted of
two open arms (length 50 cm X
breadth 10 cm) and two closed arms
of the same size (height 40 cm).
The arms of the same type were
opposite to each other, with a central
square of 10 cm.
The maze was elevated to a height
of 50 cm above the floor.
Fig2: Elevated plus maze
9. 2.LIGTH AND DARKBOX:
The apparatus consisted of an open top wooden box.
Two distinct chambers, a black chamber painted black
and illuminated with dimmed red light and a bright
chamber painted white and brightly illuminated with 100
W white light source, were located 17 cm above the box.
The two chambers were connected through a small open
doorway (7.5 X 5 cm) situated on the floor level at the
centre of the partition.
3.OPEN FIELD:
The apparatus consisted of a large rectangular box (100 X
80 cm) with 60 cm high walls.
The floor was made of wire mesh and divided into
twenty-five squares (outer 16 and central 9).
The box was illuminated with 100 W bulb placed 60 cm
above the centre of the field.
Fig3:light and dark box
Fig4:open field
10. OPERATING PROCEDURE
1) IN ELEVATED PLUS MAZE :-
Each animal was placed in the centre square of plus maze,
facing one of the open arms.
The number of entries into and the time spent in open and
closed arms and the number of rears In each arm in a 5min
period was noted.
2) IN LIGHT AND DARK BOX:-
The animal was placed at the centre of the brightly lit arena
in the Light and dark box.
The number of entries into and the time spent in the bright
Arena, the number of rears in the bright and dark arenas
And the duration of immobility were noted for 5mins.
In above procedures hand operated counters and
Stop watches were used to score the behaviour of animals.
BEHAVIOURALASSESSMENT
Fig6: rat towards open arm
Fig5: rat in centre square
11. 3) IN OPEN FIELD:-
Each animal was placed in one of the peripheral corner squares of the box
and the number of peripheral and central squares crossed, the time spent in
central square and the number of rears were observed for a 5mins period.
Fig7:Rat in peripheral squares
In statistical analysis the data were analysed by one-way ANOVA.
12. RESULTS
ELEVATED PLUS MAZE:
Diazepam(0.5 and 1mg/kg) treated rats showed a significant increase in the number of
entries,time spent and number of rears in the open arms as well as percentile ratio of open
arm to total arm entries and reduction in time spent in the closed arms in acute study but
reduction in time spent in closed arms not significant in chronic study
ACUTE STUDY:
Aq-WS&Al-OC(10 and 20mg/kg) administration increased number of entries, the time spent
and the rears in open arms of elevated plus maze model.
TABLE2:Effects of diazepam and Aq-WS&Al-OC on behaviour of rats in elevated plus maze (acute study)
Treatment n Dose Number of arm entries Percentile ratio
of open/total
Open Total arm entries
Time spent in squares (sec)
Open arms closed arm
Number of
rears
1% Gum acacia 8 10ml 1.50±0.50 6.38±1.37 21.18±5.09 16.13±4.93 221.5±14.58 1.0±0.45
Diazepam 6 o.5ml 3.67±0.42 8.17±0.83 44.53±1.34” 45.50±9.82 184.3±10.9 4.17±0.6’
Diazepam 6 1mg 4.50±0.22’ 10.5±0.67 43.37±2.42” 69.5±10.5 158.83±10.5’ 3.0±0.62’
Aq-WS&Al-OC 6 5mg 2.33±0.80 7.00±1.57 31.33±4.71 67.1±21.09’ 90.0±18.36” 1.0±0.67
Aq-WS&Al-OC 6 10mg 5.17±1.62” 11.83±2.22 40.73±4.39” 80.83±20.44’ 72.33±13,93” 1.5±0.62
Aq-WS&Al-OC 6 20mg 5.67±0.84” 14.17±1.33” 40.84±4.04” 117.33±17.89” 40.17±19.95” 3.16±0.4
One-Way F 3.87 4.50 3.42 5.85 3.17 5.82
ANOVA P <0.05 <0.05 <0.05 <0.01 <0.01 <0.05
Values are mean±SEM. df=5,32. *P<0.05, **P<0.001, compared to respective vehicle treated control group. One-way ANOVA followed by
Dunnett’s test.
13. CHRONIC STUDY:
Aq-WS&Al-OC(5,10 AND 20mg/kg)Administration increased the no. of entries,
the time spent and rears in open arms.
It reduces time spent in closed arms
Treatment n Dose Number of arm entries Percentile ratio
of open/total
Open Total arm entries
Time spent in squares (sec)
Open arms closed arm
Number
of rears
1% Gum acacia 8 10ml 1.620.49 5.50±1.05 24.58±5.94 30.75±12.7 207.87±21.87 1.87±0.6
Diazepam 6 o.5ml 9.0±1.23” 13.66± 1.11” 64.6±4.87” 101±6.05 155.1±12.32 5.66±0.7’
Aq-WS&Al-OC 6 5mg 6.83±1.35” 11.33±1.33 57.71±5.89” 104±27.35” 117.8±20.06” 7.16±2.0
Aq-WS&Al-OC 6 10mg 7.33±1.22 12.1±1.227” 40.73±6.59” 152.3±26.7” 96.9±1.84” 6.3±0.88
Aq-WS&Al-OC 6 20mg 3.66±0.66” 6.83±0.30” 55.8±8.56” 148.3±22.0” 134.83±22.2” 2.5±0.4
TABLE3:Effects of diazepam and Aq-WS&Al-OC on behaviour of rats in elevated plus maze (chronic study)
One-Way F 11.23 11.13 8.33 6.76 4.61 5.82
ANOVA P <0.01 <0.01 <0.01 <0.05 <0.001 <0.05
Values are mean±SEM. df=4,27. *P<0.05, **P<0.001, compared to respective vehicle treated control. One-way ANOVA
followed by Dunnett’s test
14. LIGHT AND DARK BOX
DIAZEPAM(0.5 and 1mg/kg) both in acute and chronic study there was significant increase
in the time spent and rears in light arena and decreased duration of immobility.however it did
not alter the no. of entries into light chamber to any significant extent.
ACUTE STUDY:
Aq-WS&Al-OC at higher doses (10 and 20mg/kg) increased the time spent and the number
of rears and decreased the duration of immobility .
TABLE4:Effects of Aq-WS&Al-OC on behaviour of rats in bright and dark arena paradigm (acute study)
Treatment n Dose Number of
entries into
light chamber
Time spent in
squares (sec)
Number of rears in
chamber
Light Dark
Duration of
immobility(sec)
1% Gum acacia 8 10ml 2.0±0.25 7.6±1.48 2.0±0,37 9.67±0.7 78.8±11.40
Diazepam 6 o.5ml 3.3±0.42 40.8±8.8 8.16±1.3” 15.5±2.2 14.66±5.44’
Diazepam 6 1mg 2.1±0.4 19.6±7.8 5.6±0.7’ 12.1±2.0 62.0±20.3
Aq-WS&Al-OC 6 5mg 1.66±0.3 11.3±1.2 3.17±0.8 6.6±1.64 62.33±23.63
Aq-WS&Al-OC 6 10mg 2.3±0.49 27.5±5.6 3.8±0.79 16.3±2.5 59.50±23.61
Aq-WS&Al-OC 6 20mg 2.66±0.3 26.3±2.2 5.16±1.0’ 17.0±2.4 9.83±4.13’
One-Way F 2.20 5.61 6.94 2.25 2.65
ANOVA P NS <0.05 <0.01 <0.05 <0.05
Values are mean±SEM. df=5,23. *P<0. 05 , **P<0.001, compared to respective vehicle treated control group. One-way ANOVA followed
by Dunnett’s test.
15. CHRONIC STUDY:
Chronic administration of test drug(Aq-WS&Al-OC)at all doses(5,10 and 20
mg/kg) increased the time spent and number of rears in bright chamber and
decreased duration of immobility.
At low doses(5 and 10 mg/kg) the test compound increased the number of entries
into bright chamber.
Treatment n Dose Number of
entries into light
chamber
Time spent in
Light chamber
(sec)
Number of rears in chamber
Light Dark
Duration of
immobility(sec)
1% Gum acacia 8 10ml 1.39±0..18 13.2±2.0 2.7±0.65 5.75±0.95 164.1±21.4
Diazepam 6 o.5ml 2.66±0.3 22.6±4.6 6.83±1.0” 8.83±1.1 69.16±8.19’
Aq-WS&Al-OC 6 5mg 2.83±0.5 25.0±4.8 7.3±0.8 15.1±2.7 11.3±5.48
Aq-WS&Al-OC 6 10mg 3.0±0.73 36.±10.4 7.5±1.7 20.6±3.96 18.16±7.17
Aq-WS&Al-OC 6 20mg 2.66±0.2 35.6±5.7 6.5±0.8” 14.1±2.4” 6.66±3.53
Table 5: Effects of Aq-WS&Al-OC on behaviour of rats in bright and dark arena (Chronic study)
One-Way F 3.32 3.54 3.84 6.28 28.03
ANOVA P <0.05 <0.05 <0.05 <0.05 <0.01
Values are mean±SEM. df=4,27. *P<0.05 , **P<0.001, compared to respective vehicle treated control group. One-way ANOVA followed
by Dunnett’s test.
16. LOCOMOTOR ACTIVITY-OPEN FIELD TEST
DIAZEPAM(0.5 and 1mg/kg) in acute study increase the time spent in inner squares without
any significant change in crossing of inner squares but in chronic total no. of squares crossed was
increased.
ACUTE STUDY:
Locomotor activity in open field test was not effected at all by the doses.
Aq-WS&Al-OC (20mg/kg ) increase both the no. of inner squares crossed and time spent .
Treatment n Dose Number of squares crossed
---------------------------------
Peripheral central Total
Time spent in squares
(sec)
Number of rears
1% Gum acacia 8 10ml 59.62±5.5 3.75±0.94 63.37±4.83 3.50±1.21 6.75±0.90
Diazepam 6 o.5ml 68.16±7.06 3.50±1.08 71.66±9.00 4.16±1.92 12.66±1.35
Diazepam 6 1mg 59.66±7.06 6.50±1.14 66.16±7.32 9.33±2.78 11.83±1.70
Aq-WS&Al-OC 6 5mg 69.5±9.15 2.83±1.36 72.16±10.1 1.83±0.79 14.16±2.89
Aq-WS&Al-OC 6 10mg 61.7±7.02 5.16±1.62 66.33±8.38 5.33±1.38 18.83±1.62”
Aq-WS&Al-OC 6 20mg 75.0±5.90 11.00±1.91” 86.0±6.55 9.33±0.84 22.16±4.06”
TABLE6:Effects of diazepam and Aq-WS&Al-OC on behaviour of rats in open field (acute study)
One-Way F 3.90 3.50 6.32
ANOVA P <0.01 <0.05 <0.01
Values are mean±SEM. df=5,32. *P<0.05, **P<0.001, compared to respective vehicle treated control. One-way ANOVA
followed by Dunnett’s test.
17. Treatment n Dose Number of squares crossed
---------------------------------
Peripheral central Total
Time spent in squares (sec) Number of
rears
1% Gum acacia 8 10ml 56.25±5.32 3.25±1.19 59.5±5.97 5.75±2.98 8.75±1.84
Diazepam 6 o.5ml 82.16±3.42 10.0±2.26 92.16±3.68 14.0±1.98 21.16±2.49”
Aq-WS&Al-OC 6 5mg 85.0±10.48 10.66±1.68 95.66±9.25
’
8.83±1.70 19.00±3.17’
Aq-WS&Al-OC 6 10mg 83.85±8.68” 12.33±1.96 96.16±10.24 14.33±1.82 18.50±2.94
Aq-WS&Al-OC 6 20mg 65.16±7.25 4,66±1.54 69.83±8.24 6.83±1.51 13.83±2.75
TABLE7:Effects of diazepam and Aq-WS&Al-OC on behaviour of rats in open field (chronic study)
One-Way F 3.48 5.68 5.21 3.77 4.04
ANOVA P <0.05 <0.05 <0.01 <0.05 <0.05
Values are mean±SEM. df=4,31. *P<0.05 , **P<0.001, compared to respective vehicle treated control group. One-way ANOVA followed by
Dunnett’s test
CHRONIC STUDY
Aq-WS&Al-OC(5 and 10mg/kg)not only increase the total no. of squares(peripheral and inner)
crossed but also time spent in inner squares and rears.
At the highest dose(20mg/kg) test drug failed to alter any of the parameter. However on
repeated administration the test drug increased the locomotor activity .
In statistical analysis the data were analysed by one-way ANOVA was significant.
18. DISSCUSION
All the results suggest decreased fear, an increased exploratory behavior and
the disinhibitory effect of the test drug Aq-WS&Al-OC are similar to those
induced by the standard anxiolytic.
Anxiolytic activity is due to different constituents of polyherbal preparation.
Withania somnifera with GABA mimetic activity and bioactive compounds
of root extracts reduce the enhanced brain levels of tribulin,5HT and
corticotrophin, are linked with anxiety state.
Ocimum sanctum have cortisol sparing immunomodulatory activity
contribute to the behavioural disinhbitory activity.
Camellia sinensis contain methyl xanthines are CNS stimulant.
Shilajit reduces the turnover of serotonin contributes to anxiolytic activity.
19. CONCLUSION
Aq-WS&Al-OC exhibited anxiolytic-like activity comparable and
similar to that of diazepam.
Thus, drug combination in Aq-WS&Al-OC polyherbal preparation
ensures synergism and helps to over come the side effects of other
drugs.
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