Evaluation of acute and subchronic toxicity of testin ct3 in experimental animal

Nội dung text: Evaluation of acute and subchronic toxicity of testin ct3 in experimental animal

1. Journal of military pharmaco-medicine n o7-2017 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 LD 50 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: LD 50 ; Acute toxicity; Subchronic toxicity; Testin CT3; Experimental animals. INTRODUCTION used as folk medicine for male sexual Modern medicine has gained great dysfunction, sperm reduction [3, 4, 6, 7]. achievements in the treatment of sperm- To have scientific evidence on the safety, producing progress disorders, but most testin CT3 was investigated the acute and drugs have side effects due to their long- subchronic toxicity in experimental animal. lasting, relatively high cost treatment. SUBJECTS AND METHODS Therefore, in recent years, many scientists 1. Subjects and materials. tend to study traditional medicine, which shows good results, high safety, reasonable * Material: price, possible prolonged use and less - Testin CT3 remedy consists of 8 adverse effects. Testin CT3 is a medicine pharmaceutical materials: Herba et Radix developed from eight medicinal herbs, Eurycomae longifoliae 10 g, Fructus Cnidi including Herba et Radix Eurycomae 12 g, Herba Epimedii 10 g , Radix Angelicae longifoliae , Fructus Cnidi , Herba Epimedii, sinensis 14 g, Fructus Tribuli terrestris 12 g , Radix Angelicae sinensis , Fructus Tribuli Radix Astragali membranacei 14 g, Fructus terrestris, Radix Astragali membranacei , Lycii 16 g, Radix Morindae officinalis 10 g. Fructus Lycii , Radix Morindae officinalis. All of these pharmaceutical materials meet These traditional herbal medicines are 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
2. Journal of military pharmaco-medicine N o7-2017 - The aqueous extract of testin CT3 and death were observed. The oral median remedy was concentrated to ratio 4.5:1 lethal dose (LD 50 ) was calculated as the (4.5 g medicinal herbs per 1 mL). This geometric mean of dose that caused 0% extract met manufacturer's standard and and 100% mortality, respectively. was diluted in different concentrations for * Subchronic oral toxicity study: animal drank. Based on the Ministry of Health's - The doses of testin CT3 were calculated regulations on safety and effectiveness of by gram of medicinal herbs. traditional medicines [1, 2]. * Study subjects: - Rabbits were randomly distributed Healthy Swiss mice, body weight of into 3 groups of 8 animals per group. 20.0 ± 2.0 g, 6 weeks old, both sex, were + Group 1 (control group): animals used for acute oral toxicity study. Healthy received normal saline. rabbits, body weight 1.8 - 2.1 kg, both sex, + Group 2 (treated 1 group): animals were used for subchronic toxicity study. received testin CT3 with dose of 5.88 g/kg Animals were obtained from the Section body weight/24 hours (equivalent expected of Breeding (Military Medical Academy), dose in humans). housed in a standard environmental condition of 12/12 h light/dark cycle, fed + Group 3 (treated 2 group): animals with standard rat pellet and water ad libitum. received testin CT3 with dose of 17.64 They were allowed to acclimatization for g/kg body weight/24 hours (3 times of 7 days to the laboratory conditions before dose used in treated group 1). the experiment. Rabbits were taken the medication continuously for 42 days, once a day in 2. Methods. the morning, in volume of 10 mL per * Acute oral toxicity study: animal per one time. Based on the Ministry of Health's - Assessment criteria: Monitor general regulations on safety and effectiveness of condition, natural activities, eating, faeces, traditional medicines [1, 2]. The experiment urine, body weight. Hematology: red blood was conducted in Swiss mice. The mice cells count, hemoglobin, white blood cell were divided randomly into 9 groups of 10 count, platelet count. Biochemical mice per group. After an overnight fast, parameters: alanine aminotransferase each group received from 90, 120, 150, (ALT), aspartate aminotransferase (AST), 180, 210, 240, 270, 300 and 330 g/kg urea, creatinine. Histopathology: on the body weight and was administered orally day 42, the rabbits were operated to in volume of 0.3 mL sterile distilled water. observe the whole organ of the body, The animals were observed for signs of compared with the control group. In each toxicity and mortality for the first critical group, 50% of rabbits were randomly 72 hours and thereafter daily for 7 days. taken to get liver, spleen and kidney as Signs of toxicity included paw-licking, specimen to assess the lesions at the stretching, respiratory distress, diarrhoea microscopic level. 36
3. Journal of military pharmaco-medicine n o7-2017 - Time for testing: Assessment criteria were expressed as mean ± standard error were checked on the first day (D 0), at the mean (SEM). Data were assessed by end of day 14 (D 14 ) and day 42 (D42 ). one-way ANOVA followed by Newman- * Data processing method: Keuls multiple comparison test. Values for Data were analysed using SPSS 22.0 which p < 0.05 was considered to be for Windows. The experimental results statistically significant. RESULTS 1. 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. Dose Number of Number of mice dead (after Percentage cumulative of Group (g/kg body weight) mice (n) 72 hours) mice dead 1 90 10 0 0 2 120 10 1 10 3 150 10 1 10 4 180 10 2 20 5 210 10 4 40 6 240 10 4 40 7 270 10 6 60 8 300 10 9 90 9 330 10 10 100 From the above results, using the computer Excel method, calculate the LD 50 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 LD 50 = 250.13 g/kg (210.2 - 297.45 g/kg). 37
4. Journal of military pharmaco-medicine N o7-2017 2. 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 (1) Treated 2 (2) Control (3) p First day (D 0) (a) 2.04 ± 0.02 2.03 ± 0.02 2.03 ± 0.02 p2-1 > 0.05 Day 14 (D 14 ) (b) 2.08 ± 0.03 2.04 ± 0.06 2.01 ± 0.01 p3-1 > 0.05 p3-2 > 0.05 Day 42 (D 42 ) (c) 2.15 ± 0.06 2.11 ± 0.09 2.13 ± 0.03 - 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. 2. 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 (1) Treated 2 (2) Control (3) p Red blood cells (x10 12 /l) First day (D 0) (a) 5.67 ± 0.16 5.42 ± 0.05 5.95 ± 0.27 p2-1 > 0.05 Day 14 (D 14 ) (b) 5.51 ± 0.13 5.32 ± 0.15 5.38 ± 0.15 p3-1 > 0.05 Day 42 (D 42 ) (c) 5.61 ± 0.15 5.69 ± 0.35 5.56 ± 0.11 p3-2 > 0.05 p (before - after) pb-a > 0.05; p c-a > 0.05; p c-b > 0.05 Haemoglobin (G/l) First day (D 0) (a) 115.25 ± 2.80 112.50 ± 1.97 117.88 ± 2.67 p2-1 > 0.05 Day 14 (D 14 ) (b) 110.14 ± 2.67 109.25 ± 3.49 109.75 ± 3.15 p3-1 > 0.05 p3-2 > 0.05 Day 42 (D 42 ) (c) 110.88 ± 3.00 114.38 ± 6.91 110.00 ± 3.78 p (before - after) pb-a > 0.05; p c-a > 0.05; p c-b > 0.05 White blood cells (G/l) First day (D 0) (a) 9.28 ± 1.19 6.59 ± 0.49 7.61 ± 0.81 p2-1 > 0.05 Day 14 (D 14 ) (b) 8.00 ± 0.76 6.21 ± 0.55 7.84 ± 1.56 p3-1 > 0.05 p3-2 > 0.05 Day 42 (D 42 ) (c) 9.31 ± 1.17 7.83 ± 0.94 10.9 ± 1.25 p (before - after) pb-a > 0.05; p c-a > 0.05; p c-b > 0.05 38
5. Journal of military pharmaco-medicine n o7-2017 Platelet (G/l) First day (D 0) (a) 509.38 ± 54.20 438.50 ± 46.50 495.75 ± 84.57 p2-1 > 0.05 Day 14 (D 14 ) (b) 424.57 ± 81.01 513.00.± 85.90 543.12 ± 123.1 p3-1 > 0.05 p3-2 > 0.05 Day 42 (D 42 ) (c) 453.50 ±.75.04 431.25 ± 52.52 551.50 ± 56.45 p (before - after) pb-a > 0.05; p c-a > 0.05; p c-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. 3. 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 (1) Treated 2 (2) Control (3) p AST (UI/l) First day (D 0) (a) 24.88 ± 3.09 25.63 ± 5.26 27.12 ± 7.07 p2-1 > 0.05 Day 14 (D 14 ) (b) 44.38 ± 13.24 50.14 ± 12.13 28.13 ± 5.09 p3-1 > 0.05 p3-2 > 0.05 Day 42 (D 42 ) (c) 52.75 ± 14.87 39.75 ± 9.15 37.63 ± 10.13 p (before - after) pb-a > 0.05; p c-a > 0.05; p c-b > 0.05 ALT (UI/l) First day (D 0) (a) 48.88 ± 4.19 52.25 ± 5.03 46.37 ± 8.86 p2-1 > 0.05 Day 14 (D 14 ) (b) 57.38 ± 3.88 62.63 ± 4.29 60.00 ± 8.11 p3-1 > 0.05 p3-2 > 0.05 Day 42 (D 42 ) (c) 58.50 ± 3.58 58.00 ± 4.68 58.25 ± 7.42 p (before - after) pb-a > 0.05; p c-a > 0.05; p c-b > 0.05 Urea (mmol/L) First day (D 0) (a) 3.89 ± 0.59 3.23 ± 0.84 3.74 ± 0.91 p2-1 > 0.05 Day 14 (D 14 ) (b) 3.38.± 0.35 4.08 ± 0.22 3.55 ± 0.27 p3-1 > 0.05 p3-2 > 0.05 Day 42 (D 42 ) (c) 3.35 ± 0.54 3.81 ± 0.41 3.64 ± 0.37 p (before - after) pb-a > 0.05; p c-a > 0.05; p c-b > 0.05 Creatinin (mmol/L) First day (D 0) (a) 80.13 ± 5.19 73.38 ± 5.08 82.38 ± 1.93 p2-1 > 0.05 Day 14 (D 14 ) (b) 78.38 ± 4.38 81.75.±.2.53 87.13 ± 2.30 p3-1 > 0.05 p3-2 > 0.05 Day 42 (D 42 ) (c) 85.88 ± 4.92 81.63 ± 1.25 85.50 ± 1.45 p (before - after) pb-a > 0.05; p c-a > 0.05; p c-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
6. Journal of military pharmaco-medicine N o7-2017 4. 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 1 group; (b): Treated 1 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
7. Journal of military pharmaco-medicine n o7-2017 DISCUSSION LD 50 was 250.13 g/kg (210.20 g/kg - Traditional medicine has maintained 297.45 g/kg), 110.63 times higher than greater popularity all over developing the effect dose in mice. Also, testin CT3 countries and its use is rapidly on the orally was safe in the doses of 5.88 increase. Despite the fact, the safety of g/kg/24 hours and 17.64 g/kg/24 hours herbal medicine has recently been during 42 consecutive days. questioned due to reports of illness and fatalities (Park et al, 2010 [5]); or REFERENCES hepatotoxicity and nephrotoxicity. So, it is 1. Do Trung Dam . Methods of toxicity nessesary to evaluate the acute and determination of drugs. Medical Publishing subchronic toxicity of herbal medicine. House. 2014, pp.101-112. In acute toxicity of testin CT3, the result 2. Viet Nam Ministry of Health . Decision showed that the LD 50 of testin CT3 was No. 01/2007/QD-BYT promulgating the regulation 250.13 g/kg. From traditional using, one on clinical trials of medicines. 2007. remedy of testin CT3 including 98 gram of 3. Vo Van Chi . Dictionary of medicinal medicinal herbs was expected using for plants in Vietnam. Medical Publisher. 2012. one person (50 kg) per day. Therefore, the expected dose of testin CT3 in humans 4. Jameel Mohd, Ansari Javed Akhtar, Ali were 98/50 = 1.96 g/kg/day. The converted Abuzer, Ahamad Javed, Ali M, Tamboli Ennus . dose (with ratio dose of mice/person is Pharmacological scientific evidence for the 12) in mice was 23.52 g/kg/day. The LD 50 promise of Tribulus terrestris. International of testin CT3 (250.13 g/kg) was 10.63 Reseach Journal of Pharmacy. 2012, 3 (5), times higher than the effect dose in mice pp.403-406. (23.52 g/kg). This result showed that the 5. Park M, Choi H, Kim J, Lee H, Ku S. 28 safe treatment range of testin CT3 was good. days repeated oral dose toxicity test of In chronic toxicity study, testin CT3 aqueous extracts of Mahwangyounpae-tang, a with doses of 5.88 g/kg/24 hours and polyherbal formula. Food Chem Toxicol. 2010, 17.64 g/kg/24 hours, which was orally 48, pp.2477-2482. administered for 42 consecutive days, 6. Shaheed Ur Rehman, Kevin Choe and showed no affect on rabbit’s weight Hye Hyun Yoo . Review on a traditional herbal growth, hematological indexs, biochemical medicine. Eurycoma longifolia Jack (Tongkat parameters (ALT, AST, urea, creatinin). It Ali): Its traditional uses, chemistry, evidence- also did not cause any damages to the based Pharmacology and Toxicology. liver, kidney or spleen of the rabbits. Molecules. 2016, 21 (3), 331; These results indicated that testin CT3 doi:10.3390/molecules21030331. was safe in the doses and period of oral administration time. 7. Yonggang Chen, Xiaohan Liu, Lin Li, Jinhu W.U, Diling Chen . Protective effect of an CONCLUSION Oligosaccharide extracted from radix morindae Testin CT3 was safe and good when officinalis on senile dementia in rats. Lat Am J test oral acute toxicity in mice, with the Pharm. 2013, 32 (8), pp.1152-1157. 41