Evaluation of subchronic toxicity of tri thien duoc hard capsule in experimental animals – Nguyen Thi Thanh Loan

10 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH EVALUATION OF SUBCHRONIC TOXICITY OF TRI THIEN DUOC HARD CAPSULE IN EXPERIMENTAL ANIMALS Nguyen Thi Thanh Loan1, Pham Thi Van Anh1, Vu Thi Ngoc Thanh1, Nguyen Thi Ngoc Tram2 1Ha Noi Medical University 2Thien Duoc Limited Company Tri Thien Duoc, a formulation of Portulaca oleracea L. and Amaranthus spinosus L., is aimed to treat hemorrhoids and other related conditions; however, the safety of this product’s long-term consumption has never been reported. The present study evaluated the subchronic toxicity of Tri Thien Duoc hard capsule in experimental animals. Subchronic toxicity was studied in Wistar rats based on the guidance of World Health Organization and Organisation for Economic Co-operation and Development. The rats were treated with the doses of 0.566 g extract/kg per day and 1.698 g extract/kg per day for a period of 90 consecutive days. Tri Thien Duoc hard capsule caused no significant dose-related changes in general status, hematological pa- rameters, renal and hepatic function tests; in addition, it did not cause any change in histology of the liver and kidney of the rats. Our finding obtained that the Tri Thien Duoc hard capsule did not cause subchronic toxicity in experimental animals. Keywords: Tri Thien Duoc, subchronic toxicity, experimental animals I. INTRODUCTION Hemorrhoids are a very common anorectal disorder defined as the symptomatic enlarge- ment and abnormally downward displacement of anal cushions. They affect millions of people around the world and represent a major medical and socioeconomic problem [1; 2]. Hemorrhoids usually present with itching, rectal pain, or rectal bleeding. Patients with hemorrhoidal disease may experience any of the following symptoms: bleeding, a painful anal mass, swelling, discomfort, discharge, hygiene problems, soiling and pruritus [3; 4]. According to the dentate line, hemorrhoids can be divided into internal and external hemor- rhoids. Some hemorrhoids are regarded as Corresponding author: Nguyen Thi Thanh Loan, Department of Pharmacology, Hanoi Medical University Email: nguyenthanhloan@hmu.edu.vn Received: 10/7/2018 Accepted: 15/11/2018 mixed hemorrhoids [5; 6]. Treatment of symp- tomatic hemorrhoids ranges from dietary advice, lifestyle modification and pharmacol- ogical approaches to office-based procedures and radical surgery depending on their grade and severity [7]. Treatment of hemorrhoids in modern medicine is still in its infancy. Due to limited modern pharmacotherapeutic options available for treatment, herbal medicines remain the therapy of choice. The Tri Thien Duoc hard capsule, a polyherbal proprietary formulation, is aimed to treat hemorrhoids and other related conditions. Tri Thien Duoc is for- mulated from bioactive flavonoids extracted from Portulaca oleracea L. and Amaranthus spinosus L. To date, there are no systematic scientific studies to delineate its toxic effects on experimental animals. Therefore, the pre- sent study was investigated to evaluate the subchronic toxicity of the Tri Thien Duoc hard capsule in experimental animals. JMR 116 E3 (7) - 2018 11 JOURNAL OF MEDICAL RESEARCH II. MATERIALS AND METHODS 1. Plant materials The Tri Thien Duoc hard capsule consti- tutes a mixture of extracts of Portulaca oleracea L. and Amaranthus spinosus L. One capsule contains two herbal materials corre- sponding to 0.59 g extract. This capsule is dissolved in water before oral administration. The Tri Thien Duoc hard capsule is pro- vided by Thien Duoc Co.,Ltd according to the principles of Good Manufacturing Practice (GMP), Good Laboratory Practice (GLP) and Good Storage Practice (GSP). 2. Experimental animals Normal healthy Wistar albino rats weighting between 160 g and 200 g were obtained from the animal center of Dan Phuong, Ha Noi. The animals were allowed an acclimatization pe- riod of 7 days to laboratory conditions prior to the initiation of the study. They were main- tained for 12 hour light and dark cycles in a well-ventilated house, with free access to food and water ad libitum. All animals were treated according to international regulation on experi- mental animal treatment. 3. Experimental design A subchronic toxicity study was carried out according to guidance of World Health Organi- zation and Organisation for Economic Co- operation and Development [8; 9]. A total of thirty Wistar albino rats were di- vided into three groups of ten animals: - Group 1 (control group): orally adminis- tered 1 ml/100g per day sterile distilled water; - Groups 2 (treated group): orally adminis- tered Tri Thien Duoc at dose 0.566 g extract/ kg per day - Groups 3 (treated group): orally adminis- tered Tri Thien Duoc at dose 1.698 g extract/ kg per day Tri Thien Duoc was orally administered daily for a period of 90 consecutive days by oral gavages. Blood with EDTA was used immediately for determination of hematological parameters (total red blood cells, hematocrit, hemoglobin concentration, total white blood cells and platelet count). Standardized diagnostic kits of Hospitex Diagnostics (Italy) and DIALAB GmbH (Austria) were used for determination of the following biochemical parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, albu- min, total cholesterol and creatinine. All serum biochemistry was performed using biochemi- cal analyzer Erba Chem. Hematological analy- sis was performed using automatic hemato- logical analyzer Exigo - Boule Medical AB. At the end of the experiment (after blood collection), the kidney and liver were removed, cleaned with saline solution and preserved in 10% formalin for histopathology examinations. 4. Statistical analysis Data were analysed using Microsoft Excel software (2007). The levels of significance between the experimental groups and the con- trol were made using the student's t-test. Data are shown as mean ± standard deviation. All data were considered significant at p < 0.05. III. RESULTS 1. Effect on general status No animal mortality was recorded in the treatment groups throughout the study period. No significant differences in the average body 12 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH weights were observed between the treated groups and the control group (p > 0.05). None of the animals in any treated groups showed any macroscopic or gross pathological changes when compared to the control group. 2. Effect on hematological parameters Table 1. Effect of Tri Thien Duoc on total red blood cells Days Total red blood cells (T/l) p (t-test student) Group 1 Group 2 Group 3 Before 7.31 ± 0.76 7.10 ± 0.78 6.99 ± 0.43 > 0.05 After 30 days 7.57 ± 0.54 7.32 ± 0.62 7.32 ± 0.30 > 0.05 p (before-after) > 0.05 > 0.05 > 0.05 After 60 days 7.05 ± 0.78 6.86 ± 0.76 6.90 ± 0.48 > 0.05 p (before-after) > 0.05 > 0.05 > 0.05 After 90 days 6.94 ± 0.89 7.03 ± 0.80 7.09 ± 0.65 > 0.05 p (before-after) > 0.05 > 0,05 > 0.05 After 90 days of treatment, repeated daily oral administration of Tri Thien Duoc at oral doses of 0.566 g extract/kg/day and 1.698 g extract/kg/day did not cause significant changes (p > 0.05) when comparing the treated groups to the control. Figure 1. Effect of Tri Thien Duoc on hemoglobin concentration Figure 2. Effect of Tri Thien Duoc on hematocrit As shown in figures 1 and 2, there is no significant difference in hematocrit, hemoglobin con- centration between the treated groups and the control group (p > 0.05). As summarized in table 2, white blood cell values of groups that were treated with Tri Thien Duoc showed no difference when comparing the treated groups to the control group (p > 0.05). JMR 116 E3 (7) - 2018 13 JOURNAL OF MEDICAL RESEARCH Table 2. Effect of Tri Thien Duoc on total white blood cells Days Total white blood cells (G/l) p (t-test student) Group 1 Group 2 Group 3 Before 7.39 ± 0.90 6.93 ± 1.31 7.35 ± 1.25 > 0.05 After 30 days 7.14 ± 1.52 7.44 ± 1.75 7.51 ± 1.42 > 0.05 p (before - after) > 0.05 > 0.05 > 0.05 After 60 days 7.70 ± 1.13 7.28 ± 1.90 7.66 ± 2.08 > 0.05 p (before - after) > 0.05 > 0.05 > 0.05 After 90 days 7.61 ± 1.15 7.72 ± 1.33 7.25 ± 0.93 > 0.05 p (before - after) > 0.05 > 0.05 > 0.05 Table 3. Effect of Tri Thien Duoc on platelet count Days Platelet count (G/l) p (t-test student) Group 1 Group 2 Group 3 Before 529.10 ± 107.92 566.80 ± 82.17 520.60 ± 132.02 > 0.05 After 30 days 494.70 ± 113.60 511.90 ± 136.01 560.20 ± 126.55 > 0.05 p (before - after) > 0,05 > 0,05 > 0,05 After 60 days 506.30 ± 112.32 535.80 ± 102.31 503.30 ± 94.36 > 0.05 p (before - after) > 0.05 > 0.05 > 0.05 After 90 days 514.50 ± 108.88 495.00 ± 133.80 493.40 ± 113.51 > 0.05 p (before - after) > 0.05 > 0.05 > 0.05 As observed in table 3, no significant difference in the platelet count was observed between groups that were treated with Tri Thien Duoc at dose 0.566 g extract/kg/day and 1.698 g extract/ kg/day with the control group (p > 0.05). 3. Effect on liver damage As shown in figures 3 and 4, there was no significant difference in AST and ALT values between the treated groups and the control group. 14 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH Figure 3. Effect of Tri Thien Duoc on aspartate amino transferase Figure 4. Effect of Tri Thien Duoc on alanine aminotransferase 4. Effect on liver function Table 4. Effect of Tri Thien Duoc on total bilirubin, albumin and total cholesterol Parameters Days Group 1 (1) Group 2 (2) Group 3 (3) Total bilirubin (mmol/l) Before (a) 13.23 ± 0.68 13.45 ± 0.71 13.38 ± 0.42 After 30 days (b) 13.33 ± 0.65 13.32 ± 0.83 13.41 ± 0.59 After 60 days (c) 13.35 ± 0.45 13.24 ± 0.79 13.34 ± 0.53 After 90 days (d) 13.47 ± 0.56 13.56 ± 0.48 13.39 ± 0.50 p2-1 > 0.05, p3-1 > 0.05, pb-a > 0.05, pc-a > 0.05, pd-a > 0.05 Albumin (g/ dL) Before (a) 3.99 ± 0.23 3.78 ± 0.26 3.82 ± 0.28 After 30 days (b) 3.84 ± 0.34 3.69 ± 0.29 3.76 ± 0.32 After 60 days (c) 3.65 ± 0.34 3.81 ± 0.29 3.69 ± 0.32 After 90 days (d) 3.74 ± 0.18 3.71 ± 0.34 3.81 ± 0.28 p2-1 > 0.05, p3-1 > 0.05, pb-a > 0.05, pc-a > 0.05, pd-a > 0.05 Total choles- terol (mmol/l) Before (a) 1.33 ± 0.24 1.20 ± 0.16 1.30 ± 0.23 After 30 days (b) 1.18 ± 0.23 1.27 ± 0.15 1.27 ± 0.37 After 60 days (c) 1.27 ± 0.16 1.16 ± 0.14 1.37 ± 0.33 After 90 days (d) 1.19 ± 0.17 1.25 ± 0.20 1.33 ± 0.26 p2-1 > 0.05, p3-1 > 0.05, pb-a > 0.05, pc-a > 0.05, pd-a > 0.05 As shown in table 4, serum levels of total bilirubin, albumin and total cholesterol of the treated groups using Tri Thien Duoc at dose 0.566 g extract/kg/day and 1.698 g extract/kg/day were not statistically different when compared to the control group (p > 0.05). JMR 116 E3 (7) - 2018 15 JOURNAL OF MEDICAL RESEARCH 5. Effect on kidney function The effect of subchronic oral administration of Tri Thien Duoc on the creatinine of the control and treated groups is shown in the table 5. Repeated daily oral administration of Tri Thien Duoc at oral doses of 0.566 g extract/kg/day and 1.698 g extract/kg/day did not cause significant changes (p>0.05) when comparing the treated groups to the control. Table 5. Effect of Tri Thien Duoc on creatinine Days Creatinine (mg/dl) p (t-test student) Group 1 Group 2 Group 3 Before 1.06 ± 0.08 1.06 ± 0.07 1.06 ± 0.07 > 0.05 After 30 days 1.07 ± 0.07 1.05 ± 0.08 1.05 ± 0.10 > 0.05 p (before - after) > 0.05 > 0.05 > 0.05 After 60 days 1.06 ± 0.10 1.05 ± 0.08 1.06 ± 0.07 > 0.05 p (before - after) > 0.05 > 0.05 > 0.05 After 90 days 1.05 ± 0.07 1.05 ± 0.05 1.06 ± 0.11 > 0.05 p (before - after) > 0.05 > 0.05 > 0.05 6. Histopathological examination Histopathological examination of the control group and the Tri Thien Duoc treated rats with 0.566 g extract/kg/day and 1.698 g extract/kg/day showed normal structure and absence of any gross pathological lesions in the liver and kidney. Group 1. Control group (HE x 400) Normal structure Group 2. Treated group, Tri Thien Duoc at dose 0.566 g extract/kg/day Normal structure Group 3. Treated group, Tri Thien Duoc at dose 1.698 g extract/kg/day Normal structure 16 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH Group 1. Control group Normal structure Group 2. Treated group, Tri Thien Duoc at dose 0.566 g extract/kg/day Normal structure Group 3. Treated group, Tri Thien Duoc at dose 1.698 g extract/kg/day Normal structure IV. DISCUSSION Currently, the use of herbal medicines in the treatment of hemorhhoids has expanded rapidly in both developed and developing countries. Data concerning toxicity of plants are important as a baseline before exploring the therapeutic potential of a new herbal medi- cation. A subchronic toxicity study provides information on the effects of repeated oral ex- posure and can indicate the need for further longer term studies [9; 10]. Body weight changes serve as a sensitive indication of the general health status of animals [10]. Weight gains were observed in all animals administered with Tri Thien Duoc. In addition, none of the animals in any treated groups showed any macroscopic or gross pathological changes when compared to the control group. It can be stated that Tri Thien Duoc did not interfere with the normal metabolism of animals as corroborated by the non-significant difference from animals in the control group. The hematopoietic system is one of the most sensitive targets of toxic compounds and is an important index of the physiological and pathological status in animals [8; 9]. After 90 days of treatment, there was no significant difference in total red blood cells, hematocrit, hemoglobin concentration, total white blood cells and platelet count between groups that were treated with Tri Thien Duoc and the con- trol group; therefore, it can be concluded that the administration of Tri Thien Duoc did not affect the hematological profile and blood for- mation process. Similarly, Bhande Satish re- ported the extract of Amaranthus spinosus did not lead to any deleterious effects on hemato- logical parameters in the animals treated at 125 and 250 mg/kg per day. The absence of significant changes may suggest that Tri Thien Duoc does not have toxic effects at these dose regimens in albino rats [11]. The liver plays a key role in many meta- bolic process of not only itself but of other tis- sues as well. Severe hepatic injury, as a result of the metabolism of some of the toxic phyto- chemicals found in medicinal plants and failure of the metabolic products to be eliminated by the liver may be associated with marked distortion of these functions. Total bilirubin, albumin and total cholesterol are useful indi- JMR 116 E3 (7) - 2018 17 JOURNAL OF MEDICAL RESEARCH ces of the excretory function of the liver. Be- sides, ALT and AST are useful indices for identifying inflammation and necrosis of the liver. Accordingly, the liver releases AST, ALT and an elevation in plasma concentration is an indicator of liver damage [9]. In view of the serum biochemical parameters of the animals treated with Tri Thien Duoc, the non-significant changes in ALT, AST, total bilirubin, albumin and total cholesterol in both male and female rats at all doses indicates that Tri Thien Duoc had no deleterious effect on liver function and liver damage. Furthermore, histopathological examination of the liver of the control group and all treated groups did not reveal any mor- phological differences. This is consistent with the levels of AST, ALT and creatinine of groups that treated Tri Thien Duoc were not significantly different to the control group. Kidney function analysis is very important in the toxicity evaluation of drugs and plant extracts. The biochemical analyses were done to evaluate the possible alterations in renal functions influenced by the plant products [12]. Concentration of creatinine can be used in describing the function of the kidneys [9]. Creatinine level presented no significant differ- ences between the control group and the treated groups. In addition, histological studies could present more information regarding the nephrotoxicity of Tri Thien Duoc. The results of this study indicate that histopathological examination of Tri Thien Duoc treated rats showed normal structure and absence of any gross pathological lesion in the kidney; there- fore, Tri Thien Duoc did not affect the kidney function. Overall, the findings of this study indicate that there are no significant differences in blood profiles or biochemical parameters. This finding was further confirmed by histopa- thological observations of the kidney tissue in this study. In another study, we evaluated the acute toxicity of Tri Thien Duoc hard capsules in experimental animals. The results demon- strated that the maximum tolerable dose of Tri Thien Duoc is 7.9 g extract/kg/day, which is five times higher than the administered dose in this study. It caused no animal mortality in the treated groups throughout the 90 day follow- up. These observations indicate that Tri Thien Duoc at the dose level of 0.566 g extract/kg and 1.698 g extract/kg/day can be used for further pharmacological activity. V. CONCLUSION The present study demonstrated that the Tri Thien Duoc hard capsule at doses 0.566 g extract/kg/day and 1.698 g extract/kg/day did not produce any toxic signs or evident symp- toms at subchronic oral toxicity. ACKNOWLEDGMENTS The authors thank the Professor Le Dinh Roanh, director of the Center for Research and Early Detection of Cancer, for reading histology of liver and kidney of experimental rats. REFERENCES 1. Bharat Gami (2011). Hemorrhoids - a common ailment among adults, causes & treatment: A review. International Journal of Pharmacy and Pharmaceutical Sciences, 3(5), 5 - 12. 2. Orit Kaidar-Person., Benjamin Per- son., Steven D Wexner (2007). Hemorrhoidal Disease: A Comprehensive Review. Journal of 18 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH the American College of Surgeons, 204(1), 201 - 117. 3. Schubert, M.C., Sridhar, S., Schade, R.R et al (2009). What every gastroenterolo- gist needs to know about common anorectal disorders. World Journal of Gastroenterology, 15, 3201 - 3209. 4. Douglas MacKay (2001). Hemorrhoids and Varicose Veins: A Review of Treatment Options. Alternative Medicine Review, 6(2), 126 - 140. 5. Brill AI., Fleshman JW., Ramshaw BJ et al (2015). Minimally invasive procedures: What family physicians need to know. The Journal of Family Practice, 54(1), S1 - S24. 6. Robert A., Ganz (2013). The evaluation and treatment of hemorrhoids: A guide for the gastroenterologist. Clinical gastroenterology and hepatology, 11, 593 - 603. 7. Varut Lohsiriwat (2013). Approach to Hemorrhoids. Current Gastroenterology Re- ports. 15, 332. 8. World Health Organization (2000). Working group on the safety and efficacy of herbal medicine. Report of regional office for the western pacific of the World Health Or- ganization. 9. Organisation for Economic Co- operation and Development (2004). Guide- lines for the testing of chemicals repeated dose oral toxicity study in rodents, Environ- mental Health and Safety Monograph Series on Testing and Assesment, 407. 10. National Research Council (2006). Toxicity testing for assessing environmental agents. Interim Report. Washington, DC, USA: National Academies Press. 11. Bhande Satish S., Wasu Yogesh H. (2016). Effect of aqueous extract of Amaran- thus spinosus on hematological parameters of wistar albino rats. Journal of Experimental Biology and Agricultural Sciences, 4(1), 117 - 120. 12. Olson H., Betton G., Robinson D et al (2000). Concordance of the toxicity of phar- maceuticals in humans and in animals. Regu- latory Toxicology and Pharmacology, 32(1), 56 - 67.