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 parameters, 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.
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 Ha Noi Medical University Thien 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 parameters, 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 mixed hemorrhoids [5; 6] Treatment of sympHemorrhoids are a very common anorectal tomatic hemorrhoids ranges from dietary disorder defined as the symptomatic enlarge- advice, lifestyle modification and pharmacol- ment and abnormally downward displacement ogical approaches to office-based procedures of anal cushions They affect millions of and radical surgery depending on their grade people around the world and represent a and severity [7] Treatment of hemorrhoids in major medical and socioeconomic problem [1; modern medicine is still in its infancy Due to 2] Hemorrhoids usually present with itching, limited modern pharmacotherapeutic options rectal pain, or rectal bleeding Patients with available for treatment, herbal medicines hemorrhoidal disease may experience any of remain the therapy of choice The Tri Thien the following symptoms: bleeding, a painful Duoc hard capsule, a polyherbal proprietary anal mass, swelling, discomfort, discharge, formulation, is aimed to treat hemorrhoids and hygiene problems, soiling and pruritus [3; 4] other related conditions Tri Thien Duoc is for- According to the dentate line, hemorrhoids can mulated from bioactive flavonoids extracted be divided into internal and external hemor- from Portulaca oleracea L and Amaranthus rhoids Some hemorrhoids are regarded as spinosus L To date, there are no systematic scientific studies to delineate its toxic effects 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 10 on experimental animals Therefore, the present study was investigated to evaluate the subchronic toxicity of the Tri Thien Duoc hard capsule in experimental animals JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH II MATERIALS AND METHODS Plant materials The Tri Thien Duoc hard capsule constitutes a mixture of extracts of Portulaca oleracea L and Amaranthus spinosus L One capsule contains two herbal materials corresponding to 0.59 g extract This capsule is dissolved in water before oral administration The Tri Thien Duoc hard capsule is provided by Thien Duoc Co.,Ltd according to the principles of Good Manufacturing Practice (GMP), Good Laboratory Practice (GLP) and Good Storage Practice (GSP) 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 period of days to laboratory conditions prior to the initiation of the study They were main- - Groups (treated group): orally administered 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, albumin, total cholesterol and creatinine All serum biochemistry was performed using biochemical analyzer Erba Chem Hematological analysis was performed using automatic hematological analyzer Exigo - Boule Medical AB tained for 12 hour light and dark cycles in a At the end of the experiment (after blood well-ventilated house, with free access to food collection), the kidney and liver were removed, and water ad libitum All animals were treated cleaned with saline solution and preserved in according to international regulation on experi- 10% formalin for histopathology examinations mental animal treatment Experimental design Statistical analysis Data were analysed using Microsoft Excel A subchronic toxicity study was carried out software (2007) The levels of significance according to guidance of World Health Organi- between the experimental groups and the con- zation and Organisation for Economic Co- trol were made using the student's t-test Data operation and Development [8; 9] are shown as mean ± standard deviation All A total of thirty Wistar albino rats were divided into three groups of ten animals: - Group (control group): orally administered ml/100g per day sterile distilled water; data were considered significant at p < 0.05 III RESULTS Effect on general status - Groups (treated group): orally adminis- No animal mortality was recorded in the tered Tri Thien Duoc at dose 0.566 g extract/ treatment groups throughout the study period kg per day No significant differences in the average body JMR 116 E3 (7) - 2018 11 JOURNAL OF MEDICAL RESEARCH weights were observed between the treated any groups and the control group (p > 0.05) None changes when compared to the control group of the animals in any treated groups showed macroscopic or gross pathological Effect on hematological parameters Table Effect of Tri Thien Duoc on total red blood cells Total red blood cells (T/l) p Group Group Group (t-test student) 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 p (before-after) > 0.05 > 0.05 > 0.05 After 90 days 6.94 ± 0.89 7.03 ± 0.80 7.09 ± 0.65 p (before-after) > 0.05 > 0,05 > 0.05 Days > 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 Effect of Tri Thien Duoc on hemoglobin concentration Figure Effect of Tri Thien Duoc on hematocrit As shown in figures and 2, there is no significant difference in hematocrit, hemoglobin concentration 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) 12 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH Table Effect of Tri Thien Duoc on total white blood cells Total white blood cells (G/l) p Group Group Group (t-test student) 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 > 0.05 > 0.05 > 0.05 7.70 ± 1.13 7.28 ± 1.90 7.66 ± 2.08 > 0.05 > 0.05 > 0.05 7.61 ± 1.15 7.72 ± 1.33 7.25 ± 0.93 > 0.05 > 0.05 > 0.05 Days p (before - after) After 60 days p (before - after) After 90 days p (before - after) > 0.05 > 0.05 Table Effect of Tri Thien Duoc on platelet count Platelet count (G/l) p Days Group Group Group (t-test student) 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 > 0,05 > 0,05 > 0,05 506.30 ± 112.32 535.80 ± 102.31 503.30 ± 94.36 > 0.05 > 0.05 > 0.05 514.50 ± 108.88 495.00 ± 133.80 493.40 ± 113.51 > 0.05 > 0.05 > 0.05 p (before - after) After 60 days p (before - after) After 90 days p (before - after) > 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) Effect on liver damage As shown in figures and 4, there was no significant difference in AST and ALT values between the treated groups and the control group JMR 116 E3 (7) - 2018 13 JOURNAL OF MEDICAL RESEARCH Figure Effect of Tri Thien Duoc on Figure Effect of Tri Thien Duoc on aspartate amino transferase alanine aminotransferase Effect on liver function Table Effect of Tri Thien Duoc on total bilirubin, albumin and total cholesterol Parameters Total bilirubin (mmol/l) Days Group (1) Group (2) Group (3) 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 dL) (g/ 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 cholesterol (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) 14 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH 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 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 Effect of Tri Thien Duoc on creatinine Creatinine (mg/dl) p Days Group Group Group (t-test student) 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 > 0.05 > 0.05 > 0.05 1.06 ± 0.10 1.05 ± 0.08 1.06 ± 0.07 > 0.05 > 0.05 > 0.05 1.05 ± 0.07 1.05 ± 0.05 1.06 ± 0.11 > 0.05 > 0.05 > 0.05 p (before - after) After 60 days p (before - after) After 90 days p (before - after) > 0.05 > 0.05 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 Control group (HE x 400) Normal structure JMR 116 E3 (7) - 2018 Group Treated group, Tri Group Treated group, Tri Thien Duoc at dose 0.566 g extract/kg/day Normal structure Thien Duoc at dose 1.698 g extract/kg/day Normal structure 15 JOURNAL OF MEDICAL RESEARCH Group Control group Normal structure Group Treated group, Group Treated group, Tri Thien Duoc at dose 0.566 g extract/kg/day Normal structure Tri Thien Duoc at dose 1.698 g extract/kg/day Normal structure IV DISCUSSION pathological status in animals [8; 9] After 90 Currently, the use of herbal medicines in days of treatment, there was no significant the treatment of hemorhhoids has expanded difference in total red blood cells, hematocrit, rapidly in both developed and developing hemoglobin concentration, total white blood countries Data concerning toxicity of plants cells and platelet count between groups that are important as a baseline before exploring were treated with Tri Thien Duoc and the con- the therapeutic potential of a new herbal medi- trol group; therefore, it can be concluded that cation A subchronic toxicity study provides the administration of Tri Thien Duoc did not information on the effects of repeated oral ex- affect the hematological profile and blood for- posure and can indicate the need for further mation process Similarly, Bhande Satish re- longer term studies [9; 10] ported the extract of Amaranthus spinosus did Body weight changes serve as a sensitive not lead to any deleterious effects on hemato- indication of the general health status of logical parameters in the animals treated at animals [10] Weight gains were observed in 125 and 250 mg/kg per day The absence of all animals administered with Tri Thien Duoc significant changes may suggest that Tri Thien In addition, none of the animals in any treated Duoc does not have toxic effects at these groups showed any macroscopic or gross dose regimens in albino rats [11] pathological changes when compared to the The liver plays a key role in many meta- control group It can be stated that Tri Thien bolic process of not only itself but of other tis- Duoc did not interfere with the normal sues as well Severe hepatic injury, as a result metabolism of animals as corroborated by the of the metabolism of some of the toxic phyto- non-significant difference from animals in the chemicals found in medicinal plants and failure control group of the metabolic products to be eliminated by The hematopoietic system is one of the the liver may be associated with marked most sensitive targets of toxic compounds and distortion of these functions Total bilirubin, is an important index of the physiological and albumin and total cholesterol are useful indi- 16 JMR 116 E3 (7) - 2018 JOURNAL OF MEDICAL RESEARCH ces of the excretory function of the liver Be- blood profiles or biochemical parameters This sides, ALT and AST are useful indices for finding was further confirmed by histopa- identifying inflammation and necrosis of the thological observations of the kidney tissue in liver Accordingly, the liver releases AST, ALT this study In another study, we evaluated the and an elevation in plasma concentration is an acute toxicity of Tri Thien Duoc hard capsules indicator of liver damage [9] In view of the in experimental animals The results demon- serum biochemical parameters of the animals strated that the maximum tolerable dose of Tri treated with Tri Thien Duoc, the non-significant Thien Duoc is 7.9 g extract/kg/day, which is changes in ALT, AST, total bilirubin, albumin five times higher than the administered dose in and total cholesterol in both male and female this study It caused no animal mortality in the rats at all doses indicates that Tri Thien Duoc treated groups throughout the 90 day follow- had no deleterious effect on liver function and up These observations indicate that Tri Thien liver damage Furthermore, histopathological Duoc at the dose level of 0.566 g extract/kg examination of the liver of the control group and 1.698 g extract/kg/day can be used for and all treated groups did not reveal any mor- further pharmacological activity 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 V CONCLUSION The present study demonstrated that the Tri Thien Duoc hard capsule at doses 0.566 g Kidney function analysis is very important extract/kg/day and 1.698 g extract/kg/day did in the toxicity evaluation of drugs and plant not produce any toxic signs or evident symp- extracts The biochemical analyses were done toms at subchronic oral toxicity to evaluate the possible alterations in renal ACKNOWLEDGMENTS functions influenced by the plant products [12] Concentration of creatinine can be used in The authors thank the Professor Le Dinh describing the function of the kidneys [9] Roanh, director of the Center for Research Creatinine level presented no significant differ- and Early Detection of Cancer, for reading ences between the control group and the histology of liver and kidney of experimental treated groups In addition, histological studies rats could present more information regarding the REFERENCES nephrotoxicity of Tri Thien Duoc The results of this study indicate that histopathological Bharat Gami (2011) Hemorrhoids - 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3209 dose oral toxicity study in rodents, Environ- Douglas MacKay (2001) Hemorrhoids and Varicose Veins: A Review of Treatment Options Alternative Medicine Review, 6(2), 126 - 140 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 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- Robert A., Ganz (2013) The evaluation thus spinosus on hematological parameters of and treatment of hemorrhoids: A guide for the wistar albino rats Journal of Experimental gastroenterologist Clinical gastroenterology Biology and Agricultural Sciences, 4(1), 117 - and hepatology, 11, 593 - 603 120 Varut Lohsiriwat (2013) Approach to 12 Olson H., Betton G., Robinson D et Hemorrhoids Current Gastroenterology Re- al (2000) Concordance of the toxicity of phar- ports 15, 332 maceuticals in humans and in animals Regu- World Health Organization (2000) Working group on the safety and efficacy of 18 latory Toxicology and Pharmacology, 32(1), 56 - 67 JMR 116 E3 (7) - 2018 ... of Tri Thien Duoc on Figure Effect of Tri Thien Duoc on aspartate amino transferase alanine aminotransferase Effect on liver function Table Effect of Tri Thien Duoc on total bilirubin, albumin... evaluated the and an elevation in plasma concentration is an acute toxicity of Tri Thien Duoc hard capsules indicator of liver damage [9] In view of the in experimental animals The results demon-... capsule contains two herbal materials corresponding to 0.59 g extract This capsule is dissolved in water before oral administration The Tri Thien Duoc hard capsule is provided by Thien Duoc Co.,Ltd