Experimental Study on Relieving Physical Fatigue of Mice by Qixue Shuangbu Tinctura

Lin-Wei Chen, Bao-Chang Cai, Nan Chen, Qin Wang, De-Xi Peng, Hua Wang,Cheng-Ye Yuan, Chun-Hua Cao

1Taizhou Hospital Affiliated to Nanjing University of Chinese Medicine, Taizhou, Jiangsu,China.

2Nanjing University of Chinese Medicine, Nanjing, Jiangsu,China.

Abstract Objective: To explore the effect of Qixue Shuangbu Tinctura on alleviating the physical fatigue of mice.Method: Male ICR mice were randomly divided into 6 groups,high dose, middle dose and low dose group, liquor control group, positive control group and negative control (saline)group. According to "Health Food Test and Evaluation Technical Specifications" (2003 edition) on the effect of alleviating physical fatigue experimental study, the weight-bearing swimming test of mice was carried out after gavage for 30 days.Result: Qixue Shuangbu Tinctura can effectively prolong the time of weight-bearing swimming in mice, reduce the production of blood lactic acid in vivo, and timely eliminate serum urea nitrogen, and promote the reserve of hepatic glycogen in mice after weight-bearing swimming exercise.Conclusion: Qixue Shuangbu Tinctura has the function of relieving physical fatigue.

Key words: Qixue Shuangbu Tinctura, Physical fatigue, Weightbearing swimming

Introduction

W ith the acceleration of people's complex lifestyle and its rhythm, the increase of stress at work and the impact of environmental pollution, it has led to an increase in hidden risks to the body's health. Various geriatric diseases, and chronic diseases are becoming more prevalent among a younger age group. The population of sub-health is rising at an alarming rate. According to the research statistics, 3/4 people in the world have some sub-health problems. However, fatigue has become the most important factor that causes the sub-health state of the body. The mechanism of fatigue is very complex, and is affected by many factors. How to effectively alleviate physical fatigue has become one of the urgent problems in contemporary society.

Medicinal liquor has a long history in China. Early in Qin and Han Dynasty of China, Huangdi Neijing recorded taking medicinal liquor to strengthen the body [1].Qixue Shuangbu Tinctura is a clinical prescription of Professor Meng Jingchun, a prominent TCM doctor of Jiangsu Provincial Hospital of traditional Chinese medicine. Qixue Shuangbu Tinctura has the functions of replenishing Qi and blood, tonifying Yang and dispersing knot, and so on [2-3]. The compound is composed of Ginseng Radix et Rhizoma,Astragali Radix, Angelicae Sinensis Radix, Paeoniae Radix Alba, Citri Reticulatae Pericarpium, Polygoni Multiflori Radix, Polygonati Odorati Rhizoma, Polygonati Rhizoma and Lycii Fructus, 9 kinds of medicine and food homologous Chinese medicine composition.Extraction technology, chemical composition and trace elements of Qixue Shuangbu Tinctura have been studied [4-9]. In order to further verify its efficacy in alleviating physical fatigue, ICR male mice were used to carry out weight-bearing swimming test.

1 Materials and instruments

1.1 Experimental animals

There were 180 male ICR mice, all of which were clean grade. The average weight of the mice was 20 ± 2 g. The experimental animals were supplied by Nantong University Laboratory Animal Center (license No.:SCXK (Su) 2017-0001, certificate number: 0036354.Feeding temperature: 20 ℃, humidity 40%～70%.Experimental animals were fed with regular feed, free feeding and drinking water.

1.2 Laboratory samples

According to the contents of "Health Food Inspection and Evaluation Technical Specification"(2003 edition) about reducing physical fatigue by alcohol, the concentration of ethanol in Qixue Shuangbu Tinctura was reduced to 15%, and the sample volume was concentrated to a certain multiple before the experiment.

1.3 Experiment reagent

Urea nitrogen kit (specification: 50T/48 sample,production lot number: 201612245), whole blood lactic acid detection kit (specification is 50T/48 sample, product production batch number: 20161216)and liver glycogen test kit (specification is 50T/48 sample, production lot number: 201612240) were purchased from Nanjing completed Bioengineering Institute. Heparin sodium injection (2 mL: 12500 unit,production lot number: 140812) was purchased from Changzhou Qianhong biochemical Pharmaceutical Co., Ltd.. Normal saline (product production lot number) : 1402022705) was purchased from Chenxin Pharmaceutical Co., Ltd.. Picric acid (production lot number: 160401) was purchased from Shanghai First Biochemical Pharmaceutical Co., Ltd.. Concentrated sulphuric acid (AR) (production lot number: 160615)was purchased from Nanjing Chemical Reagent Co.,Ltd.. China Jinjiu (batch number: 20160801) was purchased from Jinpai Co., Ltd..

1.4 Experiment instruments

UV-1800 UV-Vis spectrophotometer was purchased from Daojin Enterprise Management (China) Co., Ltd.;WH-2 Vortex oscillator (Vortex Mixer) was purchased from Changzhou Huao Instrument Manufacturing Co., Ltd.. CP224C Electronic analysis balance was purchased from Orhaus Electronic balance Co., Ltd..HWS-24 Digital display constant temperature Water Bath Pot was purchased from Phaseology Instrument(Shanghai) Co., Ltd.. TDL80-2B centrifuge was purchased from Shanghai Anting Scientific instrument Factory. Swimming box (50×50×40 cm), mouse gavage needle, capillary tube, colorimetric dish, volumetric bottle, measuring tube, glass tube, centrifuge tube,surgical scissors test tube holder, stopwatch, filter paper, lead skin, beaker, filter paper, micro fluid remover and tweezers were used.

2 Experimental method

2.1 Grouping and handling of experimental animals

A total of 180 ICR male mice (average body weight:20 ± 2 g) of clean grade were divided into 6 groups randomly, high dose (HD), middle dose (MD), low dose (LD) and liquor control group (LC), the positive control group (Chinese Jinjiu ) (PC) and the negative control group (normal saline group) (NC). Mice were administrated by means of intragastric administration once a day for 30 days. During the experiment, mice were given free feeding and drinking water. According to the technical specification of health food, the mice in LD, MD, HD groups were given 8.5 mL/kg, 16.7 mL/kg, 33 mL/kg Qixue Shuangbu Tinctura respectively.The mice in LC group were given 10mL/KG/d distilled liquor with 48 % vol, whcih was raw wine used for extracting compound formulas of traditional Chinese medicine. The PC and NC groups were given Chinese Jinjiu with 15% vol and saline at the dosage of 10 ml/kg/d, respectively.

2.2 Determination of body weight growth rate and viscera index in mice

Sixty male ICR mice of clean grade were randomly divided into six groups and fed by gavage for one month according to the different administration methods mentioned above. The body weight was measured once every 2 days, and the mice were killed after 30 min of oral administration. The viscera of the mice including liver, spleen and thymus were separated and weighed. The viscera index and the weight gain value and body weight increase value of the mice at 30 days were calculated.

2.3 Weight-bearing swimming test in mice

Sixty male ICR mice of clean grade were randomly divided into six groups. Different groups of mice were fed continuously for one month. After 30 min of administration on the 30th day, the swimming experiment was carried out on mice with 5% lead wire at the tail end. The swimming pool was 50 × 50 × 40 cm. The depth of water was more than 30 cm, and the temperature of water was 25 ℃± 1.0 ℃. The time from swimming to drowning was observed.

2.4 Determination of whole blood lactic acid

2.4.1 Modeling of high blood lactic acid (BLA)

Sixty male ICR mice of clean grade were randomly divided into six groups. The mice were fed continuously for one month. After 30 min of administration on the 30th day, the mice did not swim under the tail load, but were swimming in a swimming box with 30 ℃water for 10 min and then stopped; the model of high BLA was established.

2.4.2 Preparation of blood samples and determination of BLA and blood urea nitrogen

20 min before swimming, immediately after swimming and rest after swimming, 20μL of blood was collected from the eyes of the model mice by capillary tube. The blood samples were stored in refrigerator at 4 ℃. And the serum was separated at 2000 rpm / 15 min, after coagulation. BLA and blood urea nitrogen in each stage was determined by the kit.

2.5 Determination of liver glycogen (anthrone method)

After the blood lactic acid experiment, the mice were killed. Their liver organs were taken out, rinsed with normal saline, and sucked dry by filter paper.The pretreatment of the sample before determination:the sample of the same liver of the model animal was rinsed with normal saline, then the moisture was absorbed by filter paper and weighed by weight. Add a certain amount of lye in the test tube and mix well,then boil in boiling water for 20 min, to cool the water.The hydrolysate of glycogen was prepared to form the detection solution of glycogen.

2.6 Data statistics

SPSS19.0 statistical software was used to analyze and process the experimental data. The mean values of all the data were expressed by X ± s, in addition, the difference between groups was significant (P ＜ 0.05).

3 Experimental results

3.1 Effect of Qixue Shuangbu Tinctura on body weight of mice

The mice in each group had normal spirit and no abnormal manifestation. There was no significant difference in body weight between high-middle-low dose group and the LC and NC groups (P ＞ 0.05) , and it also showed that there was no harm to the body of the mice. The results are shown in Table 1.

3.2 Effect of Qixue Shuangbu Tinctura on various organs of Mice

There was no significant difference in viscera (pleen,thymus and liver) /body mass indexs between highmiddle-low group and LC and NC groups (P ＞ 0.05).In the review, it can be concluded that the Qixue Shuangbu Tinctura has no damage to the organs. The results are shown in Table 2.

3.3 Effect of Qixue Shuangbu Tinctura on the ability of swimming weight-bearing in mice

Compared with the NC and the LC control group, the weight loading swimming time in HD, MD, LD groups were longer. The results show that Qixue Shuangbu Tinctura can prolong the weight-bearing swimming time of mice, thus alleviating physical fatigue. The results are shown in Table 3.

3.4 Effect of Qixue Shuangbu Tinctura on blood lactic acid in mice

After 20 minutes of swimming rest, blood lactic acid area of the high-dose, medium-dose, and low-dose groups of Qixue Shuangbu Tinctura had a decreasing trend. Blood lactate area value of high-dose, mediumdose, and low-dose groups of Qixue Shuangbu Tinctura were lower than that of the NC and LC groups. The results are shown in Figure 1. The results showed that each dose group of Qixue Shuangbu Tinctura reduced the blood lactate content of mice after weight loading swimming. The results are shown in Table 4.

3.5 Effect of Qixue Shuangbu Tinctura on hepatic glycogen and urea nitrogen content in mice

The three doses of Qixue Shuangbu Tinctura and the positive control group had higher glycogen content than the NC and LC groups. The results showed that the three doses of Qixue Shuangbu Tinctura can increase the liver glycogen reserve of mice after weight-bearing swimming. The results are shown in Table 5.

The serum urea content of the three doses of Qixue Shuangbu Tinctura were lower than that of the NC and the LC groups. The results showed that the three doses of Qixue Shuangbu Tinctura can reduce the production of serum urea of mice after weight-bearing swimming. The production of urea is shown in Table 6.

4 Disscussion

The most powerful manifestation of whether or not to resist fatigue is whether it can improve the endurance of exercise. Exhaustive swimming time is a direct manifestation of whether exercise endurance is improved. The experimental results in Table 3 fully demonstrate that the Qixue Shuangbu Tinctura can prolong the time of weight-bearing swimming. The high-middle medium-dose, and low-dose groups of Qixue Shuangbu Tinctura have significant differences compared with the NC and LC groups. It is proved that Qixue Shuangbu Tinctura can improve the exercise endurance of mice and alleviate the physical fatigue after weight-bearing swimming.

Table 1: Effect of oral administration of Qixue Shuangbu Tinctura on body weight gain in mice

Table 2: Effect of oral administration of Qixue Shuangbu Tinctura on organ / body weight of mice

Table 3: The results of weight loading swimming time in mice

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Table 5: Effect of oral administration of Qixue Shuangbu Tinctura on hepatic glycogen in mice

Table 6: Effect of oral administration of Qixue Shuangbu Tinctura on blood urea nitrogen in mice after exercise

After exercise, the blood lactic acid content of the body directly reflects the important indicators of the body's ability to resist fatigue. The results of Table 4 showed that after weight-bearing swimming, blood lactic acid content of three doses groups of Qixue Shuangbu Tinctura and positive group were lower than those of NC and LC groups. The results showed that Qixue Shuangbu Tinctura could reduce the blood lactic acid value after weight-bearing swimming and alleviate physical fatigue.

Sugar is an important substance to maintain the life activities of the body. When the glycogen is consumed,the body will suffer from a series of discomforts such as fatigue in the center. Hepatic glycogen is an extremely important sensitive indicator of fatigue. The results of the study in Table 5 indicate that the original stock of hepatic glucose of three doses of Qixue Shuangbu Tinctura and positive control group increased after weight-bearing swimming in mice, indicating that fatigue was relieved by the reserve of liver glycogen.

Before the body's excessive exercise produces fatigue, the glycogen is exhausted, and the blood sugar concentration drops linearly. The body consumes protein and amino acids in the body, and releases urea nitrogen and other components to accelerate the body fatigue. There is a direct relationship between the urea nitrogen and the degree of adaptability of the body movement. It is of great significance to use urea nitrogen to assess fatigue. The results of Table 6 showed that the urea nitrogen content in the low-dose and middle-dose groups of Qixue Shuangbu Tinctura is higher than that of the NC and LC groups, indicating that Qixue Shuangbu Tinctura can alleviated physical fatigue by reducing the content of urea nitrogen in the body.

According to the technical provisions of the "Health Food Functional Evaluation Procedures and Test Methods", Two or more of the blood lactic acid, serum urea nitrogen and hepatic glycogen in weight-bearing swimming and weight-bearing swimming time were positive, the sample of this study can be judged to have the function of alleviating physical fatigue. This experiment showed that Qixue Shuangbu Tinctura can effectively prolong the time of weight-bearing swimming in mice, reduce the production of blood lactic acid in the body and eliminate the small amount of exercise in time. The amount of serum urea nitrogen in the rats promotes the storage of hepatic glycogen in the mice after weight-bearing swimming.

In summary, it can be determined that Qixue Shuangbu Tinctura has the function of relieving physical fatigue and can be further developed and applied.