Iraqi J Pharm Sci, Vol.30(2) 2021 The study of toxicological activity of rutine DOI: https://doi.org/10.31351/vol30iss2pp231-240 The Acute Toxicity of Rutin in Mice Zeena Muhammad Hamid *and Hayder B. Sahib*,1 * Department of Pharmacology, College of Pharmacy, Al-N ahrain University. Abstract Acute toxicity is a step to evaluate the toxicity of a substance. Rutin is one of the flavonoid compounds with a variety of pharmacological effects. The aim of the study is to calculate the lethal dose that affect fifty percent of the mice used in the experiment (LD50). Thirty Swiss albino male and 30 non-pregnant female mice have been divided equally and randomly into 5 treated groups and one control group (n=5) Rutin has been administered with concentrations 5, 2.5.1.25,0.625 and 0.312 g/kg administered as a single dose intraperitoneally (IP) while the control group received 1% DMSO (IP). Animals were observed for any morbidity and mortality for 14 days. After 14 days the animal blood collected for biochemical and hematological analysis then all animals are euthanized for histopathological evaluation. The results showed the LD50 was 1.51 g/kg for male mice while for female mice was1.49 g/kg. No significant changes were observed at dose of 1.25glkg (female) and 0.625, 0.312 glkg (both sexes) in body weight measurements and in biochemical or hematological assays. Moreover no significant histopathological changes were reported compared to control.. It can be concluded that Rutin is practically a non-toxic substance. Keywords: Acute toxicity, Rutin, LD50%, Intraperitoneally, histopathology, DMSO, biochemical and hematological assay. ‫دراسة السمية الحادة للروتين في الفئران‬ 1،*‫صاحب‬ ‫زينة محمد حامد * و حيدر بهاء‬ . ‫ العراق‬،‫ بغداد‬،‫ دائرة مدينة الطب‬،‫*وزارة الصحة والبيئة‬ ‫ العراق‬،‫ جامعة بغداد‬،‫ كلية الصيدلة‬،‫**فرع االدوية والسموم‬ ‫الخالصة‬ ‫ وتهدف الدراسة‬، ‫ ويعتبر الروتين أحد مركبات الفالفونويد ذات التأثيرات الدوائية المتنوعة‬، ‫تعد السمية الحادة خطوة لتقييم سمية مادة ما‬ ‫ اناث غير حوامل بالتساوي‬30 ‫ ذكر و‬30 ‫ تم تقسيم الفئران البيضاء السويسرية‬.‫ وتقييم سمية الروتين في الفئران‬٪ 50 ‫إلى حساب الجرعة المميتة‬ ، ‫ كجم جرعه واحده‬/ ‫ جم‬0.312 ‫ و‬5،2.5.1.25،0.625 ‫) بتركيز‬5 = ‫ مجموعات ُمعالجة ومجموعة سيطرة واحدة (ن‬5 ‫وبشكل عشوائي إلى‬ ‫ يوم يتم جمع الدم الجراء التحليالت الباوكيمائيه والدمويه ومن ثم يتم‬1٤‫ بعد ال‬.‫ يوم‬1٤ ‫داخل الصفاق ثم التغيرات السريريه والوفيات تسجل لمده‬ LD50% 1.51 ‫ كانت النتيجة لذكور الفئران البيضاء السويسرية‬.‫قتل جميع الحيوانات لجمع االعظاء الحيويه وارسالها الختصاص امراض انسجه‬ ‫ أظهر الفحص التشريحي المرضي أنه عند‬، ‫ كجم ووزن الجسم والمقايسة البيوكيميائية والدمية‬/ ‫ جم‬LD50%1.49 ‫ كجم بينما كانت لالناث‬/ ‫جم‬ ‫ يعتبر‬، ‫ لذلك‬.‫( مع مجموعة التحكم‬p˃0.05) ‫ كجم لكال الجنسين ال يوجد فرق معنوي‬/ ‫ جم‬0.312 ، 0.625 ‫ كجم (لإلناث) و‬/ ‫ جم‬1.25 ‫الجرعة‬ .‫ كجم لكال الجنسين‬/ ‫ جم‬0.312 ‫الروتين مادة غير سامة عمليًا مع أقل تأثير ضار ملحوظ بجرعة‬ . DMSO ‫ فحص الكيمياء الحيوية والدم‬، ‫ التشريح المرضي‬، ‫ داخل الصقاق‬، LD50% ‫ روتين‬، ‫ السمية الحادة‬: ‫الكلمات المفتاحية‬ Introduction hypertension, inflammatory bowel syndrome, hyperlipidemia, and Alzheimer's disease(8-11). The aim of this study is to investigate the acute toxicity of Rutin that can kill 50%of treated male and female Swiss albino mice intraperitoneally at different concentrations over 14 days. In last years, compounds that are derived from herbal or natural origen widely take the world interest as a complementary supplement in many diseases (1) Rutin, is one of the flavonol compounds its chemical structure consists of aglycon part (quercetin) that is linked to glycoside moiety (rhamnose or called rutinose sugar part) (2) a study conducted in 1936 on citrus fruits and listed as one of vitamins ( vitamin P) (3) Rutin is present in many vegetables, fruits, plant such as Ruta Graveolens, Eucalyptus spp. , Sophora Japonica and buckwheat (4,5) . Rutin has many biological and pharmacological effects such as anti-inflammatory, antioxidant, antiangiogenic (6,7) moreover it has been found effective in many chronic diseases such as Diabetes Mellitus, Materials and Methods Acute toxicity study was done according to the guidelines of the World Health Organization (WHO) (12) .Thirty male and 30 non-pregnant female Swiss albino mice with an average weight between 25-30 g (purchased from the National center for drug control and research) that is randomly and equally divided into five treated groups and one control group (n=5) for each male and female mice groups. 1 Corresponding author E-mail: haider_bahaa@yahoo.com Received: 6/4/2021 Accepted: 20/6 /2021 Published Online First: 2021-12-12 Iraqi Journal of Pharmaceutical Science 231 Iraqi J Pharm Sci, Vol.30(2) 2021 The study of toxicological activity of rutine collection, all animals have been euthanized by cervical dislocation to collect vital organs such as ( liver, kidney, heart, lung and sex organs)to make histopathological examinations after preserving vital organs in 4% formalin (21,22). All mice have been kept in cages for 1 week in the Pharmacology laboratory at Al-Nahrain University/Collage of Pharmacy; where the study is done for environmental accommodation and all conditions such as temperature, food, and water have been adjusted. Rutin (CAS number 153-18-4 /USP has been purchased from Sigma-Aldrich) prepared freshly of 5, 2.5, 1.25, 0.625 and 0.312 g/kg. (13,14) different doses of rutin have been injected intraperitoneally (IP) as a single dose according to the bodyweight while control group has been received 1%DMSO as a negative control. The animals were observed for any sign of mortality or morbidity for 15, 30, 60. 120 and 240min for the first 24hrs after the administration of rutin; and the observation last for 14 days (13,15). Weighting all mice at zero (before injection), seven and 14 days after administration of Rutin. (16) after 14 days, blood was collect from the jugular vein (17,18) The collected blood was centrifuged at 4000 rpm for 10 min for biochemical analysis such as Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), bilirubin, creatinine, urea, and albumin serum level. While for hematological analysis blood collected in a tube containing anticoagulant such as Ethylenediaminetetraacetic acid (EDTA) for measuring White Blood Cell (WBC) count and hemoglobin(HGB) level (19)(20). After blood Statistical analysis The results were introduced as (as meansˍ+SD standard deviation) one-way analysis of variance (ANOVA) and SSPS statistical software version (20) has been used in statistical analysis of data and the level of significance was set as P>0.05a significant, highly significant (23). Results After administration of rutin at different concentration IP, the percentage of mortality of both male and female mice at dose 5 and 2.5g/kg were 100% and male at dose 1.25 also 100% while for female at the same dose were 60% other next doses there is variation in the percentage of mortality between male and female mice where at dose 0.625 g/kg the percentage of mortality for both sex were 60% while at dose 0.312 g/kg the percentage were 0% and 20% for male and female respectively. However, the sign observed can be summarized in table (1) and table (2). Where (+), (++) and (+++) represent a degree of acute toxicity as slight, moderate and severe respectively. Table 1.Sign observed after rutin administration to male mice dose gm/kg 5 T/M The period at sign observed 5 mint_15 mint 5/5 15mint_4hrs 4hrs_6hrs 2.5 5/5 5 mint_15 mint 15mint_4hrs 4hrs_6hrs 24-48 hrs. 5min_15 min 1.25 5/5 15mint_4hrs 4hrs_6hrs 24-48 hrs. sign of toxicity Clonic convulsion, loss of gait, miosis, muscular fasciculation , tachycardia, dyspnea, lacrimation, death(+++) hypoactivity, asthenia, abdominal contract, diarrhea, atypical locomotion, back limbs failling, bardycardia, dyspnea, death(++) bradycardia, atypical locomation, piloerection, asthenia ,dyspnea and death(+) clonic convulsion, loss of gait, miosis, muscular fasculation, tachycardia, dyspnea ,lacrimation, death(+++) hypoactivity, asthenia ,abdominal contract, diarrhea, atypical locomotion ,back limbs failling , bardycardia, dyspnea, death(++) bradycardia,atypical locomation,piloerection,asthenia,dyspnea and death(+) Hypoactivity clonic convulsion, loss of gait,miosis,muscular fasculation,tachycardia,dyspnea,lacrimation,death(++) hypoactivity, asthenia, abdominal contract ,diarrhea, atypical locomotion, back limbs failling, bardycardia, dyspnea, death(++) bradycardia, atypical locomotion, piloerection, asthenia, dyspnea, and death(+) hypoactivity, sunken eye 232 No.of mice dead at this period 2 2 1 2 1 1 1 0 0 1 4 Iraqi J Pharm Sci, Vol.30(2) 2021 The study of toxicological activity of rutine Continued table 1. dose gm/kg 0.625 T/M 5/3 The period at sign observed 10_15 mint 15mint_4hrs 6 _24 hrs 0.312 5/0 24-48 hr 1_6 hr 24 hrs_14 days sign of toxicity loss of gait, muscular fasciculation, miosis, tachycardia (+) asthenia, atypical locomotion(back limbsfalling, abdominal control, hypoactivity, hyperventilation ++ asthenia, hypoactivity, piloerection, atypical locomotion (back limbs failing) and pale food pads, and pale ear hypoactivity, sunken eye tachycardia, hypoactivity, stool in the anus no sign of toxicity, no death Table 2. Sign observed after rutin administration to female mice: dose T/M The period sign of toxicity gm/kg at sign observed 5min-15 min 5 5/5 15min-4 hrs 4hrs-6hrs 2.5 5/5 5 mint_15 mint 15mint_4hrs 4hrs_6hrs 24-48 hrs 5min_15 min 1.25 1 1 2 1 1 1 0 0 24-48 hrs 1_6 hr Hypoactivity tachycardia, hypoactivity, stool in the anus 1 0 24 hrs_48hr hypoactivity, bluish mucous membrane of the mouth, and tail 1 48hr-14 days no sign of toxicity 0 10_15 mint 15mint_4hrs 6 _24 hrs 5/1 No.of mice dead at this period 2 loss of gait, muscular fasculation,miosis,tachycardia (+) asthenia, atypical locomotion(back limbs falling, abdominal control, hypoactivity, hyperventilation ++ asthenia, hypoactivity, piloerection, atypical locomotion (back limbs failing) and pale food pads, and pale ear 6-24hrs 0.312 1 0 0 0 4hrs_6hrs 5/3 2 clonic convulsion, loss of gait, miosis, muscular fasciculation, tachycardia, dyspnea, lacrimation , death(+++) Hypoactivity ,asthenia ,abdominal contract, diarrhea, atypical locomotion , back limbs failing , bardycardia , dyspnea, death(++) bradycardia,atypical locomation, piloerection, asthenia, dyspnea and death(+) hypoactivity, bluish mucous membrane of the mouth, and tail 5/3 15mint_4hrs 0.625 clonic convulsion, loss of gait, miosis ,muscular fasculation, tachycardia, dyspnea, lacrimation, death(+++) hypoactivity, asthenia, abdominal contract, diarrhea, atypical locomotion, back limbs failing, bradycardia, dyspnea, death(++) Bradycardia, atypical locomotion, piloerection, asthenia, dyspnea, and death(+) Clonic convulsion, loss of gait, miosis, muscular fasciculation, tachycardia, dyspnea, lacrimation, death(+++) hypoactivity, asthenia, abdominal contract, diarrhea, atypical locomotion, back limbs failling,bardycardia, dyspnea, death(++) bradycardia, atypical locomotion, piloerection, asthenia, dyspnea, and death(+) hypoactivity No.of mice dead at this period 0 0 233 1 1 1 2 Iraqi J Pharm Sci, Vol.30(2) 2021 The study of toxicological activity of rutine From the calculation of both percentage of mortality and dose giving for both male and female mice, a Dose-response curve has been drawn to calculate LD50% as shown in figure (1) and (2) for male and female respectively where lethal dose was 1.51 g/kg for male mice while female mice were 1.49 g/kg. Figure 3. Body weight changes of treated groups of male mice receiving 0.625 g/kg and 0.312 g/kg of rutin at day 0, 7 and 14 where CMD0, RMBD0, RMCD0, CMD7, RMBD7, RMCD7, CMD14, RMBD14, and MCD14 represent control group, group of mice receive 0.625 g/kg 0.312 g/kg at day 0, control group, group of mice receive 0.625 g/kg, 0.312 g/kg at day 7, control group, group of mice receive 0.625 g/kg, 0.312 g/kg at day 14 respectively. Figure 1. dose-response curve of rutin in male mice. Figure 2.Dose-Response curve of rutin in female mice. By measuring the body weight at day 0, 7 and 14 to all treated and control group the bodyweight tend to increase gradually with time to all treated and control groups of male and female mice, However, There is no significant difference (p˃0.05) in body weight changes by comparing treated male and female mice groups that have been received 1.25, 0.625 and 0.312g/kg of rutin with the control group at day 0, 7 and 14 as shown in figure (3) and (4) of treated and control male and female mice respectively. Figure 4.Body weight changes between control and treated groups of female mice receiving 1.25, 0.625 and 0.312 g/kg of rutin at day 0, 7 and 14 where CFD0, RFAD0, RFBD0 , RFCD0, CFD7, RFAD7, RFBD7,RFCD7, CFD14, DFA14, RFBD14, RFCD14 represent control group, group of mice receive 1.25 g/kg, 0.625g/kg 0.312 g/kg at day 0, control group, group of mice receive 1.25 g/kg, 0.625g/kg 0.312 g/kg at day 7, control group, group of mice receive 1.25 g/kg, 0.625g/kg 0.312 g/kg at day 14 respectively. Biochemical and hematological analysis of treated mice that have been received 1.25, 0.625 and 0.312g/kg for both sex revealed that there is no significant difference (p˃ 0.05) when comparing with control groups. As shown in table (3). 234 Iraqi J Pharm Sci, Vol.30(2) 2021 The study of toxicological activity of rutine Table 3. serum and blood profile of treated male mice receiving 0.652, 0.312 g/kg rutin and female mice receiving 1.25, 0.652, 0.312 g/kg and control mice groups. Serum and blood profile AST U/L ALT U/L ALP U/L Bilirubin T mg/dl Albumin g/dL Creatinin e mg/dl Urea mg/dl WBC ×10⁹/L HGB g/dl Cm (mean± SD) CF (mean± SD) Male 0.625 (mean± SD) Male 0.312 (mean± SD) Female 1.25 (mean± SD) 219.5±10.60 Female 0.625(mean± SD) 217.5±9.19 Female 0.312(mean ± SD) 217±4.24 263.7±8.34 216.25±6.57 266.55±9.12 264.6±1.27 53.7±3.81 60±2.82 57±4.24 55±4.24 64.5±4.94 62.5±0.70 61.5±3.53 74±8.48 68±2.82 77±4.24 76.5±10.60 71±4.24 69±5.65 68.8±1.83 0.1±0.14 0.1±0.14 0.15±0.07 0.05±0.07 0.2±0.14 0.15±0.07 0.1±0.14 3.05±0.21 3.2±0.14 3.1±0.14 3± 0.28 3.15±0.21 3.05±0.35 2.9±0.28 0.4±0.14 0.4±0.28 0.5±0.14 0.45±0.35 0.55±0.35 0.5±0.14 0.45±0.07 30.5±2.12 31.45±2.75 31.5±3.53 31±1.41 32.6±1.27 31.6±2.68 31.5±2.12 6±0.28 4.95±0.35 6.45±0.77 6.3±0.28 5.95±0.07 5.2±0.28 5.05±0.35 14.25±0.77 13.7±0.84 13.9±0.14 14±1.41 13.35±0.49 13.4±0.56 13.5±0.70 tissue (p˃0.05) of rutin treated mice at dose 1.25, 0.625 and 0.312 g/kg for male and female mice when compare with the tissue of control group as shown in the following figures: The results of Histopathological examination of vital organs after staining with Hematoxylin and eosin (H and E) stain revealed there are no significant histopathological changes in Figure 5. represent section of liver x400 that stain with hematoxyline and eosin stain (H&E) where section of liver look like normal histological structure apperance in which central vein and the hepatocyte cell arranged as thread around it. There are no fibrosis or hepatocyte inflammation or necrosis where A,B,C,D,E,F represent groups of female that have been 1.25 , 0.625, 0.312 g/kg of rutin ,control group, groups male mice reciving 0.625,0.312 g/kg rutin respectively.no significant difference when compare all male and female mice treated at all dose with control dose. 235 Iraqi J Pharm Sci, Vol.30(2) 2021 The study of toxicological activity of rutine Figure 6. represent section of kidney x400 that stain with hematoxyline and eosin stain (H&E) that show there is no infilteration of inflammatory cell in interstitial space , normal tubular vascular and glomerular A,B,C,D,E,F represent groups of female that have been receiving 1.25 , 0.625, 0.312 g/kg of rutin ,control group, groups male mice reciving 0.625,0.312 g/kg rutin respectively. no significant difference when compare all male and female mice treated at all dose with control dose. Figure7. represent section of heart x 400 that stain with hematoxyline and eosin stain (H&E) that show normal structure of heart with no infilteration of inflammatory cell in perivascular or interstitial regions , no injury or necrosis, where A,B,C,D,E,F represent groups of female that have been receiving 1.25 , 0.625, 0.312 g/kg of rutin ,control group, groups male mice reciving 0.625,0.312 g/kg rutin respectively. no significant difference when compare all male and female mice treated at all dose with control dose. 236 Iraqi J Pharm Sci, Vol.30(2) 2021 The study of toxicological activity of rutine Figure 8.Represent section of lung x10 that stain with hematoxyline and eosin stain (H&E) that show normal structure of lung that consist of normal alveoli, no damage in the alveolar septa or alveolar cell necrosis , there is no inflammatory cell or edema in interstitial space or intra-alveolar region, there is no congestion or hemorrhage in capillary or alveolar duct where A,B,C,D,E,F represent groups of female that have been receiving 1.25 , 0.625, 0.312 g/kg of rutin ,control group, groups male mice receiving 0.625,0.312 g/kg rutin respectively. .no significant difference when compare all male and female mice treated at all dose with control dose. Figure 9. represent Section of testis and ovary where testis show normal structure of seminiferous tubules that inside it consist of numerous sperm and normal spermatogenesis, no atrophy in leyding cells or tubules or injury of basement membrane of tubules. While ovary consist of normal primary follicle and graafian follicle with no change and normal ovarian stroma A,B,C,D,E,F,G represent groups of female that have been receiving 1.25 , 0.625, 0.312 g/kg of rutin ,control group female,control group male, groups male mice 237 Iraqi J Pharm Sci, Vol.30(2) 2021 The study of toxicological activity of rutine receiving 0.625,0.312 g/kg rutin respectively. No significant difference when compare all male and female mice treated at all dose with control dose. protein diet (40). Creatinine and urea consider as a Discussion marker of kidney function (32). The capability of a substance to make harmful, toxic effect or even death after single short Hematological analysis was done to exposure can be named acute toxicity study (11). In evaluate the risk of hematological changes due to it the present study, male and female Swiss Albino considers important and sensitive parameter where injected as a single dose intraperitoneally at a toxic material or drugs targeting on it that lead to different concentration, (5 and 2.5 g/kg), of rutin detect the toxicity of drugs and physiopathological showed high mortality rate (100%). This may be due alteration in a biological system(34,10) alteration in to that Rutin have the ability to inhibit hematological analysis such as hemoglobin (HGB) Acetylcholinesterase (AChE) in dose dependent (24) level may indicate toxicity that induces hemorrhage manner this inhibition done due to OH group of (41) and hemolysis or dysfunction of renal system rutin can bind to the anionic site in the AChE and (25) While white blood cell(WBC) increase level of it inhibit its activity . This inhibitory activity of may indicate there is an activation of the immune cholinesterase (Acetylcholinesterase and system due to either injury of liver, kidney or other buterylesterase) had been studied in vitro and in vivo (26,27) tissue or infection(42) . The increase Acetylcholine activity at its Rutin treated groups in male and female in receptors (nicotinic and muscarinic) may lead to doses (1.25, 0.625, and 0.312 g/ kg) show no cholinergic crisis that is show in tables 1 and 2 such significant difference when compare with the as Clonic convulsion, loss of gait, miosis, muscular control group and all results of the biochemical fasculation, tachycardia, dyspnea, lacrimation, analysis revealed it lie in the normal range that is urination that finally lead to respiratory failure , confirmed by histopathological examination for cyanosis and death(28,29) subsequent dose show (liver, kidney, heart, lung and sex organs) that show significant difference in the mortality rate and no significant changes of treated tissue when survival rate between male and female mice this may compare with control tissue groups(43). Other studies be due to sex effect where cytochrome P450activity (11) show that rutin when administer orally for 13 weeks in female higher than in male or may be due to to rat was safe till 2000 mg/kg (44) while other study IL-6 level , after its stimulated synthesis by any show when administered orally to rats as a stimulus , its level will sustained in circulation in (30) single(acute toxicity study) and repeated male mice more than female mice . The sign of dose(subacute toxicity study) is safe till 5000 mg/kg toxicity in lower dose is less extensive and resolve (45) . In the present study, the lowest observed adverse step by step with time (31). The LD50 of Rutin in effect LOAE was seen at dose 0.312 g/kg and male 1.51 g/kg while to female 1.49 g/kg . according to” Loomis and Hayes classification” is Body weight of animals have been used as classified as a practically non-toxic substance (43) . a good indicator of the side effects of the drugs or (32) Conclusion chemical on the animal . Reduction in body Acute toxicity study of rutin revealed that weight of the mice when administered certain drugs it is a practically non-toxic substance or material over a certain time may be indicated of the harmful effect of that drugs (33)while there is no Acknowledgment significant difference in body weight gain from I would like to express my great normal diet compare with the control group with appreciation to college of pharmacy/ Al-Nahrain good water and food consumption may indicate drug University for offering all the facilities to finish this did not change the metabolic events of the tested work. treated mice groups and not affect growth rate (34). It Reference is concluded that mice treated with rutin do not show 1. Huang Y, Feng F, Jiang J, Qiao Y, Wu T, a significant difference in body weight compared Voglmeir J, et al. 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