Safety Evaluation of Tongchui Baihua Capsule

Kaicong FU, Mingjun CUI, Jian LUO, Lihong FU, Kai NI, Min GAO

1. Pu’er National Institute of Ethnic Traditional Medicine, Pu’er 665000, China; 2. Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; 3. Yunnan Institute of Traditional Chinese Medicine, Kunming 650000, China

Abstract [Objectives] The study aimed to evaluate the safety of Tongchui Baihua capsule. [Methods] Long-term toxicology experiment was adopted, the rats were randomly divided into control group and administration group according to the gender of male and female; the drug administration group was given different doses of Tongchui Baihua capsules, and the drug was administered by continuous gavage for 13 weeks; the general conditions, haematological indexes, blood biochemical indexes and visceral coefficients of the rats were detected at the 13th week and 4 weeks after the drug withdrawal respectively, and the toxic symptoms were observed. [Results] The rats were given Tongchui Baihua capsule in different doses by gavage for 13 weeks, and no obvious toxicity was observed in weight, haematological indexes, blood biochemical indexes and histopathology. [Conclusions] Under the condition of long-term toxicity experiment, Tongchui Baihua capsule was safe for rats.

Key words Tongchui Baihua capsule; Long-term toxicology experiment; Safety evaluation

1 Introduction

Lumbar disc herniation refers to a series of clinical symptoms, such as low back pain, lower extremity radiation pain, numbness of skin, cooling, drop of skin temperature, prickly urination and so on, which are caused by the external force of the lumbar disc, the nucleus pulposus comes out from the rupture and compresses the nerve root of the lumbar spine; the disease is more common in the population, due to the lack of effective treatment, once suffering, it will seriously affect the normal life of patients[1-2]. In order to find an effective treatment method, Pu’er National Institute of Ethnic Traditional Medicine dug out a special prescription from Hani folk people, and developed it into a hospital preparation of Tongchui Baihua capsule by Pu’er National Hospital, which was clinically proved to have special effect on lumbar disc herniation and lumbocrural pain[3].

The prescription is mainly composed of 19 herbs, such as ethnic medicine all-grass of beautiful spotflower, whiteflower leadword root, and traditional Chinese medicine manchurian wildginger, root of pseudo-ginseng, centipede, scorpion,etc., the 19 kinds of medicinal herbs are reasonable in collocation, complement each other, enhance the curative effect, and play a role in healing, relieving pain and dredging channels and collaterals. The prescription contains 4 kinds of toxic medicinal herbs, such as manchurian wildginger, centipede, scorpion and ground beetle, this ethnic medicine prescription has been used clinically in Pu’er National Hospital for more than 5 years, and 125 cases of clinical observation have been collected successively, and no clinical adverse reactions have occurred in the patients. In order to further explore the safety of Tongchui Baihua capsule, Pu’er National Hospital and Yunnan Institute of Traditional Chinese Medicine carried out a long-term toxicity experiment of Tongchui Baihua capsule on rats, providing a reference for the clinical safety application of Tongchui Baihua capsule, which is an ethnic medicine preparation.

2 Materials and Methods

2.1 Materials

2.1.1Instruments. SQP electronic analytical balance(Sartorius, Germany); Mouse intragastric device(Dongxiyi (Beijing) Technology Co., Ltd.); RM2235 microtome(Shanghai Leica Instrument Co., Ltd.); SYSMEX XT-1800i automatic animal blood analyzer(Japan Sysmex Corporation); Qlympus Au5400 automatic biochemical analyzer(Olympus Co., Ltd.).

2.1.2Materials. Tongchui Baihua capsule, 0.45 g per capsule, 5 capsules once, 3 times a day, daily dosage was 13.65 g (Pu’er National Institute of Ethnic Traditional Medicine); drinking water(Kunming Zhenming Food Co., Ltd.); kits(Nanjing Jiancheng Bioengineering Institute); rat feed and corncob padding(Double Lion Experimental Animal Feed Technology Co., Ltd.).

2.1.3Experimental animals. There were 162 SPF rats of 60-80 g, half male and half female, which were provided by Hunan SJA Laboratory Animal Co., Ltd.

2.2 Methods

2.2.1Animal grouping and drug administration. 162 rats were randomly divided into four groups: drinking water control group, low [3 g/(kg·d)], medium [6 g/(kg·d)] and high-dose [12 g/(kg·d)] groups of Tongchui Baihua capsule; except 30 rats in the control group, 44 rats were in each dose group of Tongchui Baihua capsule, half male and half female. After continuous gavage of Tongchui Baihua capsule for 3 months, 20 rats in the control group were randomly selected at the 13th week in accordance with half male and half female, and 26 rats in each dose group of Tongchui Baihua capsule were selected for autopsy.

2.2.2General observation. The general state of rats in the control group and Tongchui Baihua capsule group was observed every day, the body weight and material consumption were measured once a week, and the rest animals were dissected after 4 weeks of discontinuation. The toxicity of Tongchui Baihua capsule was observed during the drug administration and convalescence period, the weight and average food intake of rats were weighed once a week, and the average food intake of each animal was recorded as the average.

2.2.3Blood examination. At the end of drug administration and convalescence period, the hemoglobin content, the number of red blood cells, the average volume of red blood cells, the average hemoglobin concentration of red blood cells, the number of platelets and the number of white blood cells in the control group and Tongchui Baihua capsule group were measured.

2.2.4Biochemical indexes of blood. At the end of drug administration and convalescence period, the liver function indexes, such as glutamic-pyruvic transaminase(GPT), glutamic-oxalacetic transaminase(GOT), total protein(TP), total bilirubin(TBil), renal function indexes such as urea, biochemical indexes such as glucose and cholesterol were measured in the control group and Tongchui Baihua capsule group.

2.2.5Pathological examination. At the end of drug administration and convalescence period, the rats in control group and Tongchui Baihua capsule group were dissected to observe the wet weight of liver, lung and kidney and other major organs of male and female rats and calculate the organ coefficient; the organs of rats were fixed with 10% neutral formalin solution, then paraffin embedded sections and microscopic examination were carried out.

2.2.6Data processing method. The data were processed by SPSS software 16.0,Ttest was used for measurement data confor-ming to normal distribution and homogeneous variances, whileT’ test was used for heterogeneous variances; rank sum test was used for measurement data that did not conform to the normal distribution, each group was compared with the control group, andP<0.05 indicated that the data had statistical significance.

3 Results and Analyses

3.1 The effect on the general situationDuring the experiment, no abnormal behavior was observed in the control group and the group of Tongchui Baihua capsule, and the rats’ mental state and dietary activities were normal. At the end of drug administration and the end of recovery period, the food intake of Tongchui Baihua capsule group was slightly reduced compared with the blank control group of drinking water, but there was no significant difference (P>0. 05), which belonged to the normal category, this was shown in Table 1. Compared with the control group, the body weight of male and female animals in the low-, middle- and high-dose groups of Tongchui Baihua capsule increased slowly, but the food intake and body weight growth returned to normal in the recovery period, this can be seen in Table 2.

Table 1 Effects of drug administration on food intake of SD rats g

Table 2 Effects of drug administration on the weight of SD rats (g,

3.2 Hematological examinationAs can be seen in Table 3, there were no statistically significant differences on the white blood cell count, hemoglobin count and activated partial thromboplastin time between rats in the group of Tongchui Baihua capsule and the blank control group at the end of the 13th week of gavage(P>0.05); while there was a statistically significant increase or decrease on the other hematological examination indexes of rats in the Tongchui Baihua capsule group compared with those of the control group, and there was no significant dose-time dependence of the hematological indexes of rats in the dose groups of Tongchui Baihua capsule. Based on the above results, it was believed that the individual differences of animals were the cause[4-5]; at the end of the convalescence period, there was no statistical difference on the hematological indexes between each dose group and the control group. Therefore, the continuous administration of Tongchui Baihua capsule by gavage did not affect the hematological indexes of rats.

Table 3 Detection of hematological indexes during the

3.3 Biochemical examination of serumAs can be seen in Table 4, there were no significant differences in glutamic-pyruvic transaminase(GPT), blood glucose(GLU), urea and creatinine between rats in the group of Tongchui Baihua capsule and the blank control group at the end of the 13th week of gavage, showing no statistical significance(P>0.05); while there was a statistically significant increase or decrease on total bilirubin(TBil), total protein(TP) and total cholesterol(TC) of rats in the Tongchui Baihua capsule group compared with those of the control group; at the end of the convalescence period, there was no significant increase or decrease on total bilirubin(TBil), total protein(TP) and total cholesterol(TC) of rats in the Tongchui Baihua capsule group compared with those of the control group, and there was no significant dose-effect relationship between serum biochemical indexes and gavage dose of Tongchui Baihua capsule in rats. It was considered that the statistical differences were caused by individual differences of animals, which did not constitute toxicological significance, indicating that the changes of indicators were not caused by drug toxicity[6-7].

Table 4 Detection of biochemical indexes of serum during the experiment

3.4 Organ coefficients and histopathological examination of organsAt the end of drug administration and convalescence period, no changes related to the test substance were observed in the organs of stomach, brain, duodenum, skeletal muscle, testis, epididymis, uterus, ovary, prostate, adrenal gland, thyroid, esophagus, aorta, bladder, thymus, skin, bone marrow, spinal cord, trachea, heart, spleen, mesenteric lymph and other organs and tissues.

According to the data analysis in Table 5 and 6, the changes of wet weight of organs, body weight, pathological results and organ-body ratio coefficients of rats in the control group and Tongchui Baihua capsule group were analyzed; at the end of drug administration, compared with the control group, the increase or decrease of organ-body ratio coefficients of rats in Tongchui Baihua capsule group was of great biological significance, but it was lack of dose and time dependence, and no changes related to test substance were observed in organs and tissues, indicating that the change of organ indexes might not be drug-specific toxicity damage; at the end of the convalescence period, there was no significant change in the ratio of organ to body in female rats, while the coefficients of liver, brain, kidney to body ratios in male rats increased, combined with the urine examination and pathological tissue examination of rats, it could be regarded as non-toxic target organ[8].

Table 5 Effects of organ-body ratio coefficients of female rats during the experiment(%,

Table 6 Effects of organ-body weight ratio coefficients of male rats during the experiment(%,

4 Discussions

Long-term toxicity test is the main research method for preclinical safety evaluation of drugs. In this study, Tongchui Baihua capsule was repeatedly given to animals in large doses in accordance with the clinical medication route, and the toxicity reaction and toxic target tissues and organs were closely detected, the dose-effect and time-effect relationship of toxicity were obtained, and the nature and incidence rate of the possible toxicity reaction of Tongchui Baihua capsule in clinical practice were predicted, which had important

reference significance for the clinical application of Tongchui Baihua capsule.

The long-term toxicity experiment results of Tongchui Baihua capsule on rats showed that at the end of the drug administration and convalescence period, rats in the low-, medium- and high-dose groups of Tongchui Baihua capsule were generally in good condition, and their activities and feed and water intake were normal; compared with the control group, the body weight increased slowly in the partial period, but the food intake and body weight growth returned to normal in the convalescence period, no significant dose-effect relationship was observed with the gavage dose of Tongchui Baihua capsule in other clinical tests, and no significant histomomechanical changes related to drug administration were observed in all organs and tissues of rats in Tongchui Baihua capsule group. Based on the above research data, it can be concluded that under the condition of long-term toxicity experiment, Tongchui Baihua capsule was safe for rats, which provided a safety evaluation basis for the clinical application of the ethnic drug Tongchui Baihua capsule.