Acute and subacute oral toxicity of Litsea elliptica Blume essential oil in rats

Acute and subacute oral toxicity of Litsea elliptica Blume essential oil in rats 1 first-author 2 1 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:hida@medic.ukm.my †E-mail:hida@medic.ukm.my hida@medic.ukm.my Biomedical Science Programme, School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, 50300 Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia Biomedical Science Programme, School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, 50300 Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia Department of Environmental Health, Faculty of Health Sciences, Universiti Teknologi MARA, Jalan Meranek, 94300 Kota Samarahan, Sarawak, Malaysia Department of Environmental Health, Faculty of Health Sciences, Universiti Teknologi MARA, Jalan Meranek, 94300 Kota Samarahan, Sarawak, Malaysia Litsea elliptica Blume has been traditionally used to treat headache, fever, and stomach ulcer, and has also been used as an insect repellent. The acute and subacute toxicities of L . elliptica essential oil were evaluated orally by gavage in female Sprague-Dawley rats. For the acute toxicity study, L . elliptica essential oil was administered in doses from 500 to 4 000 mg/kg (single dose), and in the subacute toxicity test, the following doses were used: 125, 250, and 500 mg/kg, for 28 consecutive days. In the acute toxicity study, L . elliptica essential oil caused dose-dependent adverse behaviours and mortality. The median lethal dose value was 3 488.86 mg/kg and the acute non-observed-adversed-effect level value was found to be 500 mg/kg. The subacute toxicity study of L . elliptica essential oil did not reveal alterations in body weight, and food and water consumptions. The haematological and biochemical analyses did not show significant differences between control and treated groups in most of the parameters examined, except for the hemoglobin, mean cell hemoglobin concentration, mean cell volume, mean cell hemoglobin, serum albumin, and serum sodium. However, these differences were still within the normal range. No abnormalities or histopathological changes were observed in the liver, pancreatic islet of Langerhans, and renal glomerulous and tubular cells of all treated groups. In conclusion, L . elliptica essential oil can be classified in the U group, which is defined as a group unlikely to present an acute hazard according to World Health Organization (WHO) classification. Litsea elliptica Acute toxicity Subacute toxicity Median lethal dose (LD 50 ) Natural insecticide Non-observed-adversed-effect level (NOAEL) Introduction 1 The widespread application of insecticides in household products and public health programs creates a major concern due to their effects on human and environmental health. Certain synthetic pesticides provide a broad range of toxic effects (Isman and Machial, 2006 2006 ). Insecticides have caused significant negative effects on the non-target organisms; therefore, a toxicity evaluation is important (Celik and Suzek, 2008 2008 ). Proliferations of research were carried out to produce insecticides from natural products as alternatives to synthetic insecticides in order to reduce their negative health impacts. The use of natural products as insecticides is environmentally desirable and economically profitable (Mittal and Subbarao, 2003 2003 ). As such, the use of plants for insecticidal purpose is becoming more popular due to virtually non-existent adverse effects. Litsea elliptica (Family: Lauraceae) Blume is a well-known tropical tree used in herbal or traditional medicine in the South East Asia region (Jiwajinda et al., 2002 2002 ). Crushed leaves of L . elliptica are applied around the forehead for treatment of headache (Grosvenor et al., 1995 1995 ), and also can be used as an herbal medicine to treat stomach ulcers and fever (Jiwajinda et al., 2002 2002 ). The extract of its leaves has also been proven to have chemopreventative activities, reducing the occurrence of stomach cancer in Thailand (Bhamarapravati et al., 2003 2003 ) and Nakahara et al. ( 2002 2002 ) also reported that its leaves have a significant antimutagenic activity. L . elliptica was reported to have potential insecticidal activities (Rohani et al., 1997 1997 ). An aqueous cream with 1/3 active compound from L . elliptica has a protective role up to 96.6% against mosquito bites (Ibrahim and Zaridah, 1998 1998 ). The methanol extract of L . elliptica leaves has been shown to control the vector of dengue fever (Hidayatulfathi et al., 2003 2003 ). Significant insecticidal activities have been found against larvae of Aedes aegypti and Aedes albopictus (Hidayatulfathi et al., 2003 2003 ) and adults of A . aegypti (Hidayatulfathi et al., 2004 2004 ). Based on previous studies, the L . elliptica essential oil could be useful as an insecticide, thereby reducing the use of synthetic pesticides. Prior to effective formulation of L . elliptica essential oil as mosquito control, evaluation of its toxic effects is required. The toxicity evaluation is based on the duration of exposure and, according to Environmental Protection Agency (EPA), 14 d and 28 d are recommended for acute and subacute toxicity studies, respectively (EPA, 2000 2000 ; 2002 2002 ). According to EPA ( 2002 2002 ), the main purpose of the acute toxicity study is to determine the median lethal dose (50% death) LD 50 and non-observed-adversed-effect level (NOAEL) value by evaluating the mortality rate and signs of toxicity. According to EPA ( 2000 2000 ), the suggested dose for subacute toxicity study is based on the NOAEL. Therefore, the chosen dose in subacute toxicity study should be NOAEL, 1/2NOAEL, and 1/4NOAEL. The present work evaluated the acute and subacute oral toxicities of L . elliptica essential oil in female Sprague-Dawley (SD) rats and the results obtained from this study will provide the safety information of this extract before its commercialization as a natural product pesticide. Materials and methods 1 Plant materials and extraction 2 The leaves of L . elliptica were obtained from Bangi Forest Reserve, Selangor, Malaysia. The voucher specimen (FRI41999) was deposited at the Herbarium of the Forest Research Institute Malaysia, Kepong. Then, the leaves were dried at room temperature (25–28 °C), and ground to produce fine particle. The extraction of essential oil was done using Clevenger apparatus (Duran and Favorit, Germany) by a water steam distillation method. The extraction was done for at least 8 h and the temperature was adjusted to maintain the boiling conditions. Sodium sulphate dehydrates (Na 2 SO 4 ) was added to remove the remaining water in the essential oil to obtain 100% purity with the density of 860 mg/ml. Animals 2 Female SD rats weighing 180–220 g were obtained from the Laboratory Animal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia. The animals were kept in plastic cages in environmental conditions (22–24 °C, 12 h:12 h dark/light cycle), fed a mouse pellet diet (mouse pellet 702 P, Gold Coin Feedmills (M) Sdn. Bhd., Pelabuhaan Utara, Malaysia), and allowed to drink water ad libitum without distraction. All the animal handling protocols were approved by the Animal Ethics Committee of Laboratory Animal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia. Acute toxicity 2 The assessment of acute toxicity was performed according to the EPA ( 2002 2002 ) test guidelines. Healthy rats were fasted overnight, but allowed access to water ad libitum, and were randomly divided into seven groups ( n =10). The first group (control group) received distilled water only. The other six groups were orally treated with a single dose of L . elliptica essential oil at 500, 1 000, 2 000, 2 500, 3 000, or 4 000 mg/kg, respectively. The doses in this acute toxicity study were based on the results from a range-finding study, where the observations on mortality and toxicity signs were made. All the treatments were administered by force-feeding. Animals were observed for signs of toxicity, body weight, food consumption, and water intake as well as mortality for a period of 14 d after treatment. The toxicity signs and symptoms were observed in individual cages during the first 3 h after the essential oil administration, and subsequently monitored daily throughout the duration of the study (Konan et al., 2007 2007 ). According to the mortality of rats observed within 14 d, the LD 50 value was calculated. At Day 15, all surviving animals were sacrificed, internal organs were excised, and organ weights were measured. Subacute toxicity 2 The subacute toxicity study was performed according to the EPA ( 2000 2000 ) test guidelines. The animals were divided randomly into four groups with 10 rats per group. L . elliptica essential oil was administered orally by gavage with doses 125, 250, or 500 mg/kg for 28 consecutive days, while the control rats received distilled water only. The chosen doses were based on the dose of NOAEL that was obtained from the acute toxicity study, which dose being 500 mg/kg. Toxicity signs and mortality were monitored daily, whereas body weight changes, and food and water consumptions were monitored weekly. At the end of the study, animals were fasted overnight, anesthetized with diethyl ether, and cardiac puncture was done to obtain blood samples. The heparinised blood samples were used for determining haematological parameters. Meanwhile, the non-heparinized tube was used for blood chemistry analysis and a fluoride tube was used for blood glucose determination. Following dissection, the liver, the pancreas, the heart, the kidneys, and the spleen were removed and weighed immediately. Hematological and biochemical analyses 2 An automatic hematology analyzer (ABC vet. Isolab Sdn. Bhd., France) was used to analyze haematological parameters, including red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin (Hb), haematocrit (Hct), mean cell hemoglobin (MCH), mean cell volume (MCV), mean cell hemoglobin concentration (MCHC), and platelet (Plt) count. For biochemical analysis, blood was centrifuged at 1 500× g for 10 min to obtain serum, and stored at −40 °C. Blood urea nitrogen (BUN), creatinine, potassium, sodium, chloride, calcium, phosphorus, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, and albumin were assayed using automated chemical analysis: Hitachi 902 (Roche Diagnostic Sdn. Bhd., Japan). Enzymatic glucose-oxidase kits (Catalogue No. TR 15 104, Trace Scientific, Melbourne, Australia) were used to analyze plasma glucose levels. Morphological analysis 2 The macroscopic external features of the selected organs were performed to detect any abnormal signs. All the organs were perfused with 0.9% (9 g/L) saline solution, and fixed in 10% buffered formalin solution at room temperature. The organs were processed according to Ochei and Kolhatkar ( 2000 2000 ), enclosed with paraffin and subjected to haematoxylin-eosin (H & E) staining for microscopic histological examination under 40× magnification by histopathological experts. Statistical analysis 2 All studies mentioned above were done in triplicate except for the LD 50 study. The LD 50 was calculated using probit analysis (SPSS 11.5). All values were expressed as mean±standard error of the mean (SEM) and were analyzed by one-way analysis of variance (ANOVA) followed by Scheffe post hoc test, and statistically significant findings were considered those in which P < 0.05. Results 1 Acute toxicity study 2 The acute toxicity of L . elliptica essential oil given as single doses orally is presented in Table 1 1 . The NOAEL of L . elliptica essential oil was 500 mg/kg. The rats showed the signs of toxicity such as hypoactivity, lacrimation, and piloerection, in a dose-dependent manner, and these signs remained until death. Mortality was observed in the groups receiving 2 500, 3 000, and 4 000 mg/kg with one, five, and six deaths, respectively (Table 1 1 ). From the acute toxicity data, the calculated LD 50 obtained was 3 488.86 mg/kg. All treated rats were carefully examined for up to 14 d after the dose for toxicity signs and lethality. D/T: number of dead rats/number of treated rats. None: no toxicity symptoms observed during the observation period L. elliptica essential oil (mg/kg) D/T Symptom  500 0/10 None 1 000 0/10 Hypoactivity 2 000 0/10 Hypoactivity 2 500 1/10 Hypoactivity 3 000 5/10 Hypoactivity, lacrimation, piloerection 4 000 6/10 Hypoactivity, lacrimation, piloerection The body weight of surviving animals is shown in Table 2 2 . On Day 7, there was a significant body weight decrease in group 3 000 mg/kg compared to the control ( P < 0.05). On Day 14, there were significant decreases in body weights in groups 2 000, 2 500, and 3 000 mg/kg compared to the control ( P < 0.05), and in group 3 000 mg/kg compared to the 500 and 1 000 mg/kg groups ( P < 0.05). Group 4 000 mg/kg was not included, since there were too few surviving animals in this group. As for selected organs weights, there were significant changes only in the renal and the heart for absolute and relative weights in all L . elliptica treated groups, except the 500 mg/kg group (Table 2 2 ). Data are expressed as mean±SEM, n =10 for each group Significant at P < 0.05 as compared with control only Significant at P < 0.05 as compared with the control and 500 mg/kg Significant at P < 0.05 as compared with the control, 500 and 1 000 mg/kg Significant at P < 0.05 as compared with 1 000 and 2 000 mg/kg Significant at P < 0.05 as compared with 2 500 mg/kg 2 Parameter 2 Control 5 Treatment of Litsea elliptica essential oil 500 mg/kg 1 000 mg/kg 2 000 mg/kg 2 500 mg/kg 3 000 mg/kg Body weight loss (%)  Day 7 5.98±0.08 5.03±0.07 4.80±0.02 3.63±0.05 2.44±0.04 0.98±0.02  Day 14 10.29±0.13 7.91±0.09 7.43±0.05 5.32±0.06 a a 3.90±0.04 a a 2.46±0.04 c c Absolute weight (g)  Liver 7.81±1.04 7.84±1.29 8.67±0.90 8.41±1.29 8.35±0.77 8.52±0.78  Renal 1.34±0.06 1.34±0.07 1.03±0.05 b b 1.01±0.08 b b 1.28±0.10 d d 1.51±0.11 b b d d e e  Spleen 0.44±0.10 0.48±0.06 0.45±0.08 0.48±0.16 0.50±0.11 0.49±0.18  Heart 0.32±0.03 0.33±0.03 0.35±0.03 0.36±0.03 0.39±0.05 b b 0.39±0.07 b b Relative weight (%)  Liver 3.40±0.48 3.49±0.58 3.87±0.38 3.86±0.56 3.92±0.40 4.09±0.26  Renal 0.58±0.04 0.60±0.04 0.46±0.02 b b 0.46±0.03 b b 0.60±0.05 d d 0.73±0.06 b b d d e e  Spleen 0.19±0.04 0.27±0.03 0.20±0.03 0.22±0.07 0.23±0.05 0.24±0.10  Heart 0.14±0.01 0.15±0.02 0.15±0.01 0.16±0.02 0.18±0.02 a a 0.19±0.03 a a Subacute toxicity study 2 Neither toxicity signs nor death was observed throughout the study following administration of L . elliptica essential oil for 28 consecutive days. L . elliptica essential oil did not cause any significant changes in body and organ weights, as shown in Table 3 3 . The food and water consumptions also revealed no differences among treated and untreated groups (statistical analysis was not done since two animals were kept per cage). Data are expressed as mean±SEM, n =10 for each group. No statistical difference was found between the control and L . elliptica essential oil treated groups ( P > 0.05) 2 Parameter 2 Control 3 Treatment of Litsea elliptica essential oil 125 mg/kg 250 mg/kg 500 mg/kg Body weight (g)  Day 0 200.00±2.89 197.00±2.13 198.00±1.87 197.00±2.00  Day 7 209.00±4.33 208.00±2.50 207.00±2.90 201.50±3.42  Day 14 212.50±4.30 212.00±2.71 211.50±3.66 204.50±2.93  Day 21 214.50±4.68 213.00±3.67 213.00±3.51 210.50±4.68  Day 28 217.50±4.90 217.00±3.35 214.00±4.58 212.50±3.75 Absolute weight (g)  Liver 5.53±0.86 5.98±0.93 5.69±0.80 6.09±0.38  Renal 1.29±0.15 1.37±0.13 1.34±0.21 1.35±0.12  Spleen 0.38±0.07 0.44±0.07 0.45±0.07 0.45±0.10  Heart 0.75±0.09 0.72±0.07 0.75±0.08 0.71±0.11  Pancreas 1.16±0.32 1.01±0.23 1.02±0.17 1.02±0.34 Relative weight (%)  Liver 2.54±0.34 2.76±0.38 2.65±0.29 2.87±0.18  Renal 0.60±0.06 0.63±0.06 0.63±0.07 0.64±0.04  Spleen 0.18±0.03 0.21±0.04 0.21±0.03 0.21±0.03  Heart 0.35±0.05 0.34±0.02 0.35±0.04 0.33±0.04  Pancreas 0.54±0.13 0.47±0.10 0.48±0.07 0.48±0.15 Hematological and biochemical parameters 2 The hematological and biochemical profiles of the control and treated groups are shown in Tables 4 4 and 5 5 , respectively. The RBCs, WBCs, Hct, and Plt values did not show any differences among treated and control groups. L . elliptica essential oil significantly increased the Hb concentration in all doses, though decreased the MCH and MCHC levels in 125 and 250 mg/kg of L . elliptica essential oil groups ( P < 0.05). L . elliptica essential oil at a dose of 250 mg/kg significantly decreased the level of MCV, in contrast with control group ( P < 0.05). However, all the haematological values in all treated groups were still within the normal range. For the biochemical profile, all the data showed no statistical differences among treated and control groups. Data are expressed as mean±SEM, n =10 for each group. RBC: red blood cell; WBC: white blood cell; Hb: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; PLT: platelets Significant difference compared with the control group ( P < 0.05) 2 Parameter 2 Control 3 Treatment of L . elliptica essential oil 125 mg/kg 250 mg/kg 500 mg/kg RBC (×10 12 L −1 ) 7.82±0.15 7.92±0.11 8.27±0.18 8.16±0.16 WBC (×10 9 L −1 ) 10.23±0.74 12.09±1.20 11.53±0.72 12.64±1.39 Hb (g/dl) 13.45±0.32 14.62±0.22 * * 14.71±0.21 * * 14.82±0.27 * * HCT (l/l) 0.40±0.01 0.39±0.01 0.40±0.01 0.41±0.01 MCV (fl) 51.60±0.65 49.80±0.39 48.80±0.63 * * 50.30±0.47 MCH (pg) 17.18±0.18 18.48±0.13 * * 17.80±0.25 18.19±0.16 * * MCHC (g/dl) 33.26±0.41 37.14±0.15 * * 36.46±0.13 * * 36.14±0.27 * * PLT (×10 9 L −1 ) 655.90±63.72 432.50±141.44 491.90±119.00 424.40±70.82 Data are expressed as mean±SEM, n =10 for each group Significant difference compared with the control group ( P < 0.05) 2 Parameter 2 Control 3 Treatment of L. elliptica essential oil 125 mg/kg 250 mg/kg 500 mg/kg Urea (mmol/L) 7.09±1.17 6.48±0.62 7.01±1.68 7.68±2.01 Creatinin (mmol/L) 0.07±0.01 0.07±0.00 0.07±0.01 0.07±0.01 Sodium (mmol/L) 142.79±1.74 141.66±2.54 139.84±2.34 * * 139.07±1.49 * * Potassium (mmol/L) 5.74±0.66 5.53±0.33 5.51±0.70 5.38±0.65 Calcium (mmol/L) 3.03±0.12 2.91±0.09 2.94±0.17 2.87±0.09 Cloride (mmol/L) 100.76±1.68 99.58±1.84 99.92±1.69 99.69±4.76 Phosphate (mmol/L) 1.72±0.20 1.97±0.21 1.94±0.29 1.98±0.29 ALT (U/L) 38.98±10.98 38.34±8.38 37.33±7.86 39.79±11.77 AST (U/L) 102.08±18.83 114.29±20.63 114.63±24.03 115.41±29.57 Albumin (g/L) 44.49±3.97 40.87±2.75 40.18±3.39 39.66±4.21 * * Total protein (g/L) 75.16±6.91 72.33±3.07 72.85±6.06 74.44±5.98 Glucose (mmol/L) 6.90±0.25 6.82±0.26 7.55±0.22 7.84±0.41 Morphological analysis 2 Gross pathological examination revealed no detectable abnormalities in the selected organs. In addition, histopathological examination found no detectable alteration neither in the control nor the treated groups (Fig. 1 1 ). H&E staining of control (a) and treated groups of 125 (b), 250 (c), and 500 mg/kg (d). No abnormality was found in all treated or control groups Discussion 1 In this toxicity study, using probit analysis, LD 50 of L . elliptica essential oil was estimated to be 3488.86 mg/kg, thus the essential oil can be classified in the U group as unlikely to present an acute hazard in normal use by World Health Organization (WHO, 2005 2005 ) recommending classification guidelines of pesticides. Administration of a single oral dose of the L . elliptica essential oil had produced some toxicity symptoms, which probably were the result of disturbances on the function of autonomic nervous system (ANS) and central nerves system (CNS). Lacrimation is one of the toxic symptoms shown to be a result of the muscarinic effect of cholinergic poisoning (Liang, 1996 1996 ) and exposure to organophosphate insecticides (Costa, 2006 2006 ). According to Gotoh et al., ( 2006 2006 ), hypoactivity was suggested to be due to a decrease in locomotor activity controlled by the CNS. Acute oral toxicity study showed that L . elliptica essential oil possessed a similarity in some toxicity symptoms produced by organophosphate insecticides such as malathion, diazinon, and chlorpyrifos, which also have some neurotoxic symptoms (Kwong, 2002 2002 ). The acute oral toxicity study of L . elliptica essential oil also caused a significant decrease in body weight at higher doses, and might be associated with the toxic symptoms that occurred, which lead the rats to become anorectic. According to Lansdown ( 1993 1993 ), changes in body and organ weights are probably due to the toxic effects of the xenobiotic. Furthermore, the changes in organ-to-weight ratio or the relative weight could also be due to organ injury as a result of exposure to the toxic material (Wang et al., 2007 2007 ). In the present study, an increase in absolute and relative weights of the kidneys and the heart is most probably due to oedema (Costa-Silva et al., 2008 2008 ). However, the acute oral administration of L . elliptica essential oil did not induce any significant toxicity symptoms at the dose concentration of 500 mg/kg and this dose has been suggested to be the NOAEL for this study. Based on the acute toxicity study of the L . elliptica essential oil, the doses to be evaluated in subacute toxicity study for 28 d repeated dose given were found to be 125 (1/4NOAEL), 250 (1/2NOAEL), and 500 mg/kg (NOAEL). The 28-d toxicity test has been accepted in practice for a subacute oral toxicity study. Subacute oral toxicity study has been applied in safety assessment studies to provide safety information prior to the commercialization of a certain product (Arts et al., 2004 2004 ; Bautista et al., 2004 2004 ). In the subacute toxicity study, no toxicity signs were detected throughout the experiments; therefore, the present results suggest that L . elliptica essential oil administered orally is non-toxic to rats. The haematological system is sensitive to toxic chemicals and can be used as an important index to monitor the physiological changes in human and animal (Li et al., 2010 2010 ). Therefore, toxic chemicals risk evaluation involved the analysis of blood parameters. The synthetic insecticides such as α-cypermethrin, carbendazim, and chlorpyrifos at various doses given are reported to cause anemia in rats (WHO, 1995 1995 ). However, the hematological parameters obtained from this study showed that the L . elliptica essential oil did not influence hematological parameter values (Sanderson and Philips, 1981 1981 ); therefore, there is potential for future utilization of L . elliptica essential oil. Generally, all biochemical parameters observed in the subacute toxicity study did not show any significant changes compared with the control group and all the values were still within the normal ranges (Petterino and Argentino-Storino, 2006 2006 ). L . elliptica essential oil did not cause alterations in the values of transaminase enzymes, which are good biomarkers predicting possible toxicity of the liver (EI Hilaly et al., 2004 2004 ). Similarly, no alteration was observed in the glucose or creatinine level, which reflects normal pancreas or renal function, respectively. 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Joint FAO/WHO WHO (World Health Organization) The WHO Recommended Classification of Pesticides by Hazards and Guidelines to Classification 2004 , 2005 . : Geneva, Switzerland International Programme on Chemical Safety , . The WHO Recommended Classification of Pesticides by Hazards and Guidelines to Classification 2004 We wish to thank the staff of Faculty of Health Sciences, Universiti Kebangsaan Malaysia, particularly the Dean, for providing research facilities, Assoc. Prof. Dr. Khairul OSMAN, Assoc. Prof. Dr. Ahmad Rohi bin GHAZALI, and all the individuals, who directly or indirectly support this research. 10.1631/jzus.B1100021 Project supported by the Science Fund, Ministry of Science, Technology and Innovation, Malaysia (No. 02-01-02-SF0205), and the Universiti Kebangsaan Malaysia (No. UKM-OUP-TKP-21-101/2011) 2011-01-17 2012-09-23 2011-11-30 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2012 2012 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 13