Objectives: To investigate the acute and subchronic toxicity of testin CT3. Method: Acute administration of testin CT3 was done as single dose from 90 g to 330 g of testin CT3 per kg/bodyweight in mice and subchronic toxicity study for 42 days was done by daily oral administration of testin CT3 at doses of 5.88 and 17.64 g/kg bodyweight in rabbits.
Journal of military pharmaco-medicine no7-2017 EVALUATION OF ACUTE AND SUBCHRONIC TOXICITY OF TESTIN CT3 IN EXPERIMENTAL ANIMAL Nguyen Thi Phuong Thao*; Nguyen Hoang Ngan** Vu Manh Hung**; Vu Van Dien***; Tran Cong Truong**** SUMMARY Objectives: To investigate the acute and subchronic toxicity of testin CT3 Method: Acute administration of testin CT3 was done as single dose from 90 g to 330 g of testin CT3 per kg/bodyweight in mice and subchronic toxicity study for 42 days was done by daily oral administration of testin CT3 at doses of 5.88 and 17.64 g/kg bodyweight in rabbits Results: The acute toxicity study showed the LD50 of testin CT3 was 250.13 g/kg (210.20 - 297.45 g/kg) The subchronic toxicity study suggested that testin CT3 with doses of 5.88 g/kg/24 days and 17.64 g/kg/24 days during 42 days, did not affect the increasing rabbit’s body weight; did not change the haematological parameters, ALT, AST, urea and creatinine levels; did not cause damage to liver, kidney and spleen histopathology Conclusion: Our results suggest that the testin CT3 is relatively safe when administered orally in mice and rabbits * Keywords: LD50; Acute toxicity; Subchronic toxicity; Testin CT3; Experimental animals INTRODUCTION Modern medicine has gained great achievements in the treatment of spermproducing progress disorders, but most drugs have side effects due to their longlasting, relatively high cost treatment Therefore, in recent years, many scientists tend to study traditional medicine, which shows good results, high safety, reasonable price, possible prolonged use and less adverse effects Testin CT3 is a medicine developed from eight medicinal herbs, including Herba et Radix Eurycomae longifoliae, Fructus Cnidi, Herba Epimedii, Radix Angelicae sinensis, Fructus Tribuli terrestris, Radix Astragali membranacei, Fructus Lycii, Radix Morindae officinalis These traditional herbal medicines are used as folk medicine for male sexual dysfunction, sperm reduction [3, 4, 6, 7] To have scientific evidence on the safety, testin CT3 was investigated the acute and subchronic toxicity in experimental animal SUBJECTS AND METHODS Subjects and materials * Material: - Testin CT3 remedy consists of pharmaceutical materials: Herba et Radix Eurycomae longifoliae 10 g, Fructus Cnidi 12 g, Herba Epimedii 10 g, Radix Angelicae sinensis 14 g, Fructus Tribuli terrestris 12 g, Radix Astragali membranacei 14 g, Fructus Lycii 16 g, Radix Morindae officinalis 10 g All of these pharmaceutical materials meet Vietnamese Pharmacopoeia IV standards * Thainguyen University of Medicine and Pharmacy ** Vietnam Military Medical University *** Hanoi University of Pharmacy **** Military Institute of Traditional Medicine Corresponding author: Nguyen Hoang Ngan (nguyenhoangngan@yahoo.com) Date received: 12/06/2017 Date accepted: 03/09/2017 35 Journal of military pharmaco-medicine No7-2017 - The aqueous extract of testin CT3 remedy was concentrated to ratio 4.5:1 (4.5 g medicinal herbs per mL) This extract met manufacturer's standard and was diluted in different concentrations for animal drank - The doses of testin CT3 were calculated by gram of medicinal herbs * Study subjects: Healthy Swiss mice, body weight of 20.0 ± 2.0 g, weeks old, both sex, were used for acute oral toxicity study Healthy rabbits, body weight 1.8 - 2.1 kg, both sex, were used for subchronic toxicity study Animals were obtained from the Section of Breeding (Military Medical Academy), housed in a standard environmental condition of 12/12 h light/dark cycle, fed with standard rat pellet and water ad libitum They were allowed to acclimatization for days to the laboratory conditions before the experiment Methods * Acute oral toxicity study: Based on the Ministry of Health's regulations on safety and effectiveness of traditional medicines [1, 2] The experiment was conducted in Swiss mice The mice were divided randomly into groups of 10 mice per group After an overnight fast, each group received from 90, 120, 150, 180, 210, 240, 270, 300 and 330 g/kg body weight and was administered orally in volume of 0.3 mL sterile distilled water The animals were observed for signs of toxicity and mortality for the first critical 72 hours and thereafter daily for days Signs of toxicity included paw-licking, stretching, respiratory distress, diarrhoea 36 and death were observed The oral median lethal dose (LD50) was calculated as the geometric mean of dose that caused 0% and 100% mortality, respectively * Subchronic oral toxicity study: Based on the Ministry of Health's regulations on safety and effectiveness of traditional medicines [1, 2] - Rabbits were randomly distributed into groups of animals per group + Group (control group): animals received normal saline + Group (treated group): animals received testin CT3 with dose of 5.88 g/kg body weight/24 hours (equivalent expected dose in humans) + Group (treated group): animals received testin CT3 with dose of 17.64 g/kg body weight/24 hours (3 times of dose used in treated group 1) Rabbits were taken the medication continuously for 42 days, once a day in the morning, in volume of 10 mL per animal per one time - Assessment criteria: Monitor general condition, natural activities, eating, faeces, urine, body weight Hematology: red blood cells count, hemoglobin, white blood cell count, platelet count Biochemical parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, creatinine Histopathology: on the day 42, the rabbits were operated to observe the whole organ of the body, compared with the control group In each group, 50% of rabbits were randomly taken to get liver, spleen and kidney as specimen to assess the lesions at the microscopic level Journal of military pharmaco-medicine no7-2017 - Time for testing: Assessment criteria were checked on the first day (D0), at the end of day 14 (D14) and day 42 (D42) * Data processing method: Data were analysed using SPSS 22.0 for Windows The experimental results were expressed as mean ± standard error mean (SEM) Data were assessed by one-way ANOVA followed by NewmanKeuls multiple comparison test Values for which p < 0.05 was considered to be statistically significant RESULTS Acute oral toxicity study of testin CT3 In acute toxicity study (table 1), 100% deaths was recorded for all the animals that received 330 g/kg body weight of testin CT3 while there was no death and no serious clinical signs were observed in the animals that received 90 g/kg body weight or less of testin CT3 10% deaths were recorded for the animals that received 120 and 150 g/kg bodyweight of testin CT3 Table 1: Results of acute oral toxicity of testin CT3 Group Dose (g/kg body weight) Number of mice (n) Number of mice dead (after 72 hours) Percentage cumulative of mice dead 90 10 0 120 10 10 150 10 10 180 10 20 210 10 40 240 10 40 270 10 60 300 10 90 330 10 10 100 From the above results, using the computer Excel method, calculate the LD50 dose, then check by the Litchfield - Wilcoxon method [1], drawing interline linear correlation between doses and death Figure 1: Grap of linear correlation between doses and death in 72 hours Calculation LD50 = 250.13 g/kg (210.2 - 297.45 g/kg) 37 Journal of military pharmaco-medicine No7-2017 Sub-acute toxicity study of testin CT3 * The effect of testin CT3 on the changes of body weight in the control and treated rabbits: Table 1: The effect of testin CT3 on body weight rabbits (n = 8, M ± SE) Mean body weight (kg) Time for testing Treated (1) Treated (2) Control (3) p First day (D0) (a) 2.04 ± 0.02 2.03 ± 0.02 2.03 ± 0.02 Day 14 (D14) (b) 2.08 ± 0.03 2.04 ± 0.06 2.01 ± 0.01 Day 42 (D42) (c) 2.15 ± 0.06 2.11 ± 0.09 2.13 ± 0.03 p2-1 > 0.05 p3-1 > 0.05 p3-2 > 0.05 - Comparison of rabbits’ mean body weight in two groups taking testin CT3 with the control group at times, showing that the change was not statistically significant (p > 0.05) - Comparison between the post-versus-pre-medication times, rabbits’ mean body weight in all groups was increasing (2.15; 2.11 and 2.13 compared with 2.04; 2.03 and 2.03, respectively) The results indicate that testin CT3 at both dose levels does not affect the growth of rabbits’ normal weight The effect of testin CT3 on the changes of haematological parameters in the control and treated rabbits Table 2: Haematological parameters for rabbits treatment with testin CT3 (n = 8, M ± SE) Time for testing Treated (1) Treated (2) Control (3) p 12 Red blood cells (x10 /l) First day (D0) (a) 5.67 ± 0.16 5.42 ± 0.05 5.95 ± 0.27 Day 14 (D14) (b) 5.51 ± 0.13 5.32 ± 0.15 5.38 ± 0.15 Day 42 (D42) (c) 5.61 ± 0.15 5.69 ± 0.35 5.56 ± 0.11 p (before - after) p2-1 > 0.05 p3-1 > 0.05 p3-2 > 0.05 pb-a > 0.05; pc-a > 0.05; pc-b > 0.05 Haemoglobin (G/l) First day (D0) (a) 115.25 ± 2.80 112.50 ± 1.97 117.88 ± 2.67 Day 14 (D14) (b) 110.14 ± 2.67 109.25 ± 3.49 109.75 ± 3.15 Day 42 (D42) (c) 110.88 ± 3.00 114.38 ± 6.91 110.00 ± 3.78 p (before - after) p2-1 > 0.05 p3-1 > 0.05 p3-2 > 0.05 pb-a > 0.05; pc-a > 0.05; pc-b > 0.05 White blood cells (G/l) First day (D0) (a) 9.28 ± 1.19 6.59 ± 0.49 7.61 ± 0.81 Day 14 (D14) (b) 8.00 ± 0.76 6.21 ± 0.55 7.84 ± 1.56 Day 42 (D42) (c) 9.31 ± 1.17 7.83 ± 0.94 10.9 ± 1.25 p (before - after) 38 pb-a > 0.05; pc-a > 0.05; pc-b > 0.05 p2-1 > 0.05 p3-1 > 0.05 p3-2 > 0.05 Journal of military pharmaco-medicine no7-2017 Platelet (G/l) First day (D0) (a) 509.38 ± 54.20 438.50 ± 46.50 495.75 ± 84.57 Day 14 (D14) (b) 424.57 ± 81.01 513.00.± 85.90 543.12 ± 123.1 Day 42 (D42) (c) 453.50 ±.75.04 431.25 ± 52.52 551.50 ± 56.45 p (before - after) p2-1 > 0.05 p3-1 > 0.05 p3-2 > 0.05 pb-a > 0.05; pc-a > 0.05; pc-b > 0.05 After 14 and 42 days of treatment, there were no significant differences in hematological indexes between the groups (p > 0.05), indicating that testin CT3 was not toxic to bone marrow The effect of testin CT3 on the changes of biochemical parameters in the control and treated rabbits Table 3: Biochemical parameters for rabbit treatment with testin CT3 (n = 8, M ± SE) Time for testing Treated (1) Treated (2) Control (3) p p2-1 > 0.05 p3-1 > 0.05 p3-2 > 0.05 AST (UI/l) First day (D0) (a) 24.88 ± 3.09 25.63 ± 5.26 27.12 ± 7.07 Day 14 (D14) (b) 44.38 ± 13.24 50.14 ± 12.13 28.13 ± 5.09 Day 42 (D42) (c) 52.75 ± 14.87 39.75 ± 9.15 37.63 ± 10.13 p (before - after) pb-a > 0.05; pc-a > 0.05; pc-b > 0.05 ALT (UI/l) First day (D0) (a) 48.88 ± 4.19 52.25 ± 5.03 46.37 ± 8.86 Day 14 (D14) (b) 57.38 ± 3.88 62.63 ± 4.29 60.00 ± 8.11 Day 42 (D42) (c) 58.50 ± 3.58 58.00 ± 4.68 58.25 ± 7.42 p (before - after) p2-1 > 0.05 p3-1 > 0.05 p3-2 > 0.05 pb-a > 0.05; pc-a > 0.05; pc-b > 0.05 Urea (mmol/L) First day (D0) (a) 3.89 ± 0.59 3.23 ± 0.84 3.74 ± 0.91 Day 14 (D14) (b) 3.38.± 0.35 4.08 ± 0.22 3.55 ± 0.27 Day 42 (D42) (c) 3.35 ± 0.54 3.81 ± 0.41 3.64 ± 0.37 p (before - after) p2-1 > 0.05 p3-1 > 0.05 p3-2 > 0.05 pb-a > 0.05; pc-a > 0.05; pc-b > 0.05 Creatinin (mmol/L) First day (D0) (a) 80.13 ± 5.19 73.38 ± 5.08 82.38 ± 1.93 Day 14 (D14) (b) 78.38 ± 4.38 81.75.±.2.53 87.13 ± 2.30 Day 42 (D42) (c) 85.88 ± 4.92 81.63 ± 1.25 85.50 ± 1.45 p (before - after) p2-1 > 0.05 p3-1 > 0.05 p3-2 > 0.05 pb-a > 0.05; pc-a > 0.05; pc-b > 0.05 Most of the biochemical parameters were not also altered by the testin CT3 The lack of significant alterations in the levels of ALT, AST, urea, creatinine, which are good indicators of liver and kidney functions, suggests that subchoronic administration of testin CT3 neither alterred hepatocytes and kidneys of mice nor the normal metabolism of the animals 39 Journal of military pharmaco-medicine No7-2017 The effect of testin CT3 on histopathology of the liver, kidney and spleen - Macroscopic observation: After testin CT3 treatment, liver, kidney and spleen morphology was observed, their colors were intact in two groups taking testin CT3, not different from the control group (a) (b) (c) Figure 1: Histopathology images of experimental rabbits’ livers (HE x200) (a) (b) (c) Figure 2: Histopathology images of experimental rabbits’ kidneys (HE x200) (a) (b) (c) Figure 3: Histopathology images of experimental rabbits’ spleen (HE x200) (a): Treated group; (b): Treated group; (c): Control group The results showed that histopathology images of livers, kidneys and spleens in testin CT3 treated groups was normal, without lesions, similar to those in control group Thus, testin CT3 which was orally administered for 42 consecutive days did not cause any damage to the liver, kidney or spleen of the rabbits 40 Journal of military pharmaco-medicine no7-2017 DISCUSSION Traditional medicine has maintained greater popularity all over developing countries and its use is rapidly on the increase Despite the fact, the safety of herbal medicine has recently been questioned due to reports of illness and fatalities (Park et al, 2010 [5]); or hepatotoxicity and nephrotoxicity So, it is nessesary to evaluate the acute and subchronic toxicity of herbal medicine In acute toxicity of testin CT3, the result showed that the LD50 of testin CT3 was 250.13 g/kg From traditional using, one remedy of testin CT3 including 98 gram of medicinal herbs was expected using for one person (50 kg) per day Therefore, the expected dose of testin CT3 in humans were 98/50 = 1.96 g/kg/day The converted dose (with ratio dose of mice/person is 12) in mice was 23.52 g/kg/day The LD50 of testin CT3 (250.13 g/kg) was 10.63 times higher than the effect dose in mice (23.52 g/kg) This result showed that the safe treatment range of testin CT3 was good In chronic toxicity study, testin CT3 with doses of 5.88 g/kg/24 hours and 17.64 g/kg/24 hours, which was orally administered for 42 consecutive days, showed no affect on rabbit’s weight growth, hematological indexs, biochemical parameters (ALT, AST, urea, creatinin) It also did not cause any damages to the liver, kidney or spleen of the rabbits These results indicated that testin CT3 was safe in the doses and period of oral administration time CONCLUSION Testin CT3 was safe and good when test oral acute toxicity in mice, with the LD50 was 250.13 g/kg (210.20 g/kg 297.45 g/kg), 110.63 times higher than the effect dose in mice Also, testin CT3 orally was safe in the doses of 5.88 g/kg/24 hours and 17.64 g/kg/24 hours during 42 consecutive days REFERENCES Do Trung Dam Methods of toxicity determination of drugs Medical Publishing House 2014, pp.101-112 Viet Nam Ministry of Health Decision No 01/2007/QD-BYT promulgating the regulation on clinical trials of medicines 2007 Vo Van Chi Dictionary of medicinal plants in Vietnam Medical Publisher 2012 Jameel Mohd, Ansari Javed Akhtar, Ali Abuzer, Ahamad Javed, Ali M, Tamboli Ennus Pharmacological scientific evidence for the promise of Tribulus terrestris International Reseach Journal of Pharmacy 2012, (5), pp.403-406 Park M, Choi H, Kim J, Lee H, Ku S 28 days repeated oral dose toxicity test of aqueous extracts of Mahwangyounpae-tang, a polyherbal formula Food Chem Toxicol 2010, 48, pp.2477-2482 Shaheed Ur Rehman, Kevin Choe and Hye Hyun Yoo Review on a traditional herbal medicine Eurycoma longifolia Jack (Tongkat Ali): Its traditional uses, chemistry, evidencebased Pharmacology and Toxicology Molecules 2016, 21 (3), 331; doi:10.3390/molecules21030331 Yonggang Chen, Xiaohan Liu, Lin Li, Jinhu W.U, Diling Chen Protective effect of an Oligosaccharide extracted from radix morindae officinalis on senile dementia in rats Lat Am J Pharm 2013, 32 (8), pp.1152-1157 41 ... the acute and subchronic toxicity of herbal medicine In acute toxicity of testin CT3, the result showed that the LD50 of testin CT3 was 250.13 g/kg From traditional using, one remedy of testin CT3. .. administration of testin CT3 neither alterred hepatocytes and kidneys of mice nor the normal metabolism of the animals 39 Journal of military pharmaco-medicine No7-2017 The effect of testin CT3. .. oral toxicity study of testin CT3 In acute toxicity study (table 1), 100% deaths was recorded for all the animals that received 330 g/kg body weight of testin CT3 while there was no death and