IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS) e-ISSN:2278-3008, p-ISSN:2319-7676. Volume 11, Issue 3 Ver. IV (May - Jun.2016), PP 108-117 www.iosrjournals.org Chronic toxicity studies: Effects of Urtica urens Linn. on hematological, biochemical and histo-pathological parameters in albino rabbits. Farah-Saeed1and Mansoor Ahmad2 1Department of Pharmacognosy, Dow College of Pharmacy, Dow University of Health Sciences, Karachi, Pakistan. 2Research Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Karachi, Karachi-75270, Pakistan. Abstract: The purpose of this research work was to explore chronic toxicity data on Urticaurens Linn (U. urens). To achieve this purpose 25 mg/kg/day dose of U. urens was given orally to test group albino rabbits (both sexes; UM: male test group treated with U. urens; UF: female test group treated with U. urens) for threemonths. The blood was collected by cardiac puncture and evaluation of hematological, blood and urine biochemistry of male and female test groups were carried out by comparing with their standard control groups respectively. Histo-pathological examination of heart, liver, kidney and stomach tissues of male test group (UM) was carried out with reference to the male control group. Platelet count was found significantly raised (p0.01) in both sexes test groups. Urea level was (p0.01) lowered in female test group while (p0.01) raised in male test group as compared to their respective control groups. Significant elevation and depression (p0.01) of cardiac enzymes and lipid profile parameters were observed in both test groups on comparison with their control groups. SGOT and alkaline phosphatase levels were (p0.05) lowered in female test group whereas; (p0.01) depressed in male test group as matched with their control groups. Gamma GT was found (p0.01) elevated in female test group and (p0.05) lowered in male test group. No noteworthy changes were observed in urine parameters of male and female test groups except for the presence of blood in male test group urine. No significant histo-pathological changes were found in stomach, heart and liver tissues; while acute tubular necrosis was observed in male test group in comparison to male control group. Our results affirm the safe and effective use of U. urens if taken in prescribed dose and for recommended time period. Keywords:Biochemical evaluation,burning nettle,hematological parameters, histo-pathological evaluation I. Introduction Urtica urens L. (Urticaceae) is commonly known as small nettle or burning nettle. Stinging hairs are the characteristic feature of this plant.It was incorporated in European Pharmacopeia 7th edition, under the heading of Urticaefolium monograph [1-2]. Traditionally, the herb was used for the treatment of diabetes mellitus, arthritis, rheumatism of joints and muscles. In general,Urtica urens is used in allergic conditions, blood cleansing, inflammation reduction, relieving of pain, to stop hair fall, increase in urine flow, stop bleeding, dilating blood vessels, lowering blood pressure and healing wounds [3]. The medicinal uses of U. urens are attributed to its rich chemical constitution. Its known chemical constituents are formic acid, histamine, serotonin, choline, amino acids, lecithin, kaempferol, sterols and vitamins, scopoletin, sterols, fatty acids, isolectin and carbohydrates [4]. Sterols and phenolcarboxylic acids are the main active constituent [2]. Reported chemical constituents are shown below. DOI: 10.9790/3008-110304108117 www.iosrjournals.org 108 | Page Chronic toxicity studies: Effects of Urtica urens Linn. on hematological, biochemical and… DOI: 10.9790/3008-110304108117 www.iosrjournals.org 109 | Page Chronic toxicity studies: Effects of Urtica urens Linn. on hematological, biochemical and… DOI: 10.9790/3008-110304108117 www.iosrjournals.org 110 | Page Chronic toxicity studies: Effects of Urtica urens Linn. on hematological, biochemical and… The purpose of this research work was to carry out chronic toxicity studies by administering low dose of U. urens L. (25 mg/kg/ml) for the period of three months in both sexes of rabbits (UM and UF) and then determining their hematological, blood biochemistry and urine biochemistry parameters with reference to their respective control groups. While histo-pathological studies were carried out only on male control and treated (UM) groups after taking out blood samples for above stated tests. II. Materials And Method 2.1 Chemicals Ethanol, acetic acid, formalin, diagnostic kits, xylene, paraffin wax, eosin, hematoxylin and canada balsam were purchased from Merck, Germany. All the chemicals were of analytical grade. 2.2 Plant Extract Urtica urens mother tincture (Lot # 3030909) was purchased from Willmar Schwabe, Germany suppliers. Extracts was concentrated by rota-evaporator (Buchi-Rotary Evaporator, Switzerland, model # B490) at 40C. The extracts obtained were stored in cool, dry place for further studies. 2.3 Experimental Animals Twenty-four male and female rabbits weighing between 1.0 and 1.2 kg were purchased from animal house of Dow University of Health Sciences, (DUHS) Karachi and kept in animal house for a period 15 days to acclamized in separate cages. They were fed commercial feed and water ad libitum. Their weights were checked on random basis. Blood (6 ml) was collected from rabbits for evaluation of hematological and biochemical parameters by cardiac puncture at the end of three month.Blood samples collected into clean non-heparinized bottles were allowed to clot and serum was separated from the clot and centrifuged according to groups into clean bottles for the biochemical analysis.After the collection of blood samples as well as urine sample for urine analysis; histopathology was carried out by carefully dissecting out liver, kidney, heart and stomach organs of male rabbits. Animal studies were carried out according to the NIH guide for the care and use of laboratory animals [5]. 2.4 Animal Grouping And Drug Dosing Four groups were made (male control – 6 rabbits), (female control – 6 rabbits), (male test (UM) – 6 rabbits) and (female test (UF) – 6 rabbits). Male and female control groups were given distil water, while test groups UM and UF were given 25mg/kg U. urens. All the administrations were given orally. The treatment continued for 90 days. Blood (6 ml) was collected by cardiac puncture with 10 ml sterile syringe using 1mg/1ml EDTA as anti-coagulant for the determination of blood and biochemical parameters. Voided urine samples were collected for each group animals. After collecting blood samples, male control and treated (UM) groups’ animals were sacrificed and their heart, stomach, liver and kidneys were extracted out for carrying out their histo-pathology. 2.5 Hematological Evaluation Hematological examination of the collected blood samples was performed according to standard procedures listed as follow. Total erythrocyte counts were counted using a Neubar chamber under a light microscope at 40 x 10 magnifications. Blood samples were diluted to 200 times by Hayem’s reagent before counting. Blood hemoglobin concentration was determined using a Sahli’shemometer. Micro wintrobe hematocrit tubes and hematocrit centrifuge were used to determine the (PCV). Total leucocyte counts were detected using a Neubar chamber under a light microscope at 10 x 10 magnification after diluting blood samples to 10 times with Turk’s solution. Mean erythrocyte volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) for particular blood samples were also calculated[6-10]. 2.6 Biochemical Evaluation Serum samples were obtained by centrifugation of blood at 1300 x g for 15 min. The Menariniclassic chemistry analyzer was used to determine the calcium (Ca), phosphorus (P), blood urea, creatinine, total bilirubin, total protein, albumin, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine phosphokinase (CPK), cholesterol, glucose, amylase, and gamma- glutamyltransferase (GGT). The globulin concentration was determined by subtracting the albumin concentration from the total protein concentration [11-12]. 2.7 Urine Analysis Voided sample of urine was collected byplacing a clean, empty box in the site where the animals usually urinates[13]. DOI: 10.9790/3008-110304108117 www.iosrjournals.org 111 | Page Chronic toxicity studies: Effects of Urtica urens Linn. on hematological, biochemical and… 2.8 Histo-Pathological Analysis After blood collection, the liver, kidney heart and stomach of the male control and test group (UM) were carefully dissected from the abdominal region and were immediately fixed in 10% neutral buffered formalin. Fixed samples were trimmed and processed for paraffin embedding. Sections (5–7 μm) were cut and the tissues were dehydrated with alcohol of graded concentrations and allowed to dry. The sample slides were subsequently stained in haematoxylin-eosin and examined under a light microscope; photomicrographs of the samples were recorded[14-16]. 2.9 Statistical Analysis All the results were presented as a mean plus or minus standard error of mean (M ± SEM). Differences between control and treatment groups were analyzed by student t-test[17]. III. Results 3.1 Effects of U. urens on complete blood countof treated male (UM) and female (UF) rabbits. Hemoglobin and hematocrit levels were p0.05 lowered, while platelet count was found raised (p0.01) in female test group (UF) as compared to the female control group.