Efficacy evaluation of Shenlan oral liquid in the treatment of experimental thrombosis and hyperlipidemia

GU Li-wei, LIU Dan-dan, TIAN Jing-zhuo, ZHANG Hong-bing, ZHAI Xiao-ru, YANG Qian, LI Yong-dong, LI Jin, LI Xin, SHEN Shuo, DU Mao-bo✉

1.Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

2.Shanxi Huawei Pharmaceutical Co.,Ltd., Jinzhong 030600, China

3.Artemisinin Research Center, China Academy of Chinese Medical Sciences, Beijing 100700, China

4.Qingdao Women and Children's Hospital, Qingdao 266000, China

ABSTRACT Objective: To explore the therapeutic effect of Shenlan oral liquid on thrombosis rats and hyperlipidemia mice.Methods: Male Wistar rats were divided into four groups randomly:the control group,the Shenlan oral liquid groups ( high dosage and low dosage),and the positive control group.Rats in the control group were treated with dd water intragastriclly; in the positive control group, rats were treated intragastric with aspirin.Rats were treated with different drugs intragastric for continuous 22 d.In the hyperlipidemia experiment, mice were fed with high-fat diet to induce hyperlipidemia model mice, then randomly divided into five groups; the control group,the model group, the atorvastatin calcium positive control group,and the Shenlan oral liquid groups ( high dosage and low dosage).The serum lipid and body weight changes of mice were observed after 6 weeks.Animals in Shenlan oral liquid high and low dosage were treated intragastric with six times and double the clinical dose.Results:After treatment, the mix blocking rate and average blocking rate of the vascular after electrical stimulation were obviously reduced in aspirin group, and Shenlan oral liquid high and low dose groups(P＜0.05 or P＜0.01).The maximum aggregation rate was significantly lower than that of control group (P＜0.05 or P＜0.01), and the normal coagulation function was not affected.In the treatment of hyperlipidemia, the effects of Shenlan oral liquid were measured on the total cholesterol (TC), triacylglycerol (total triglyceride, TG), low density lipoprotein(LDL-C) and high-density lipoprotein (HDL-C) with the hyperlipidemia mice.After 2 weeks administration,the serum TG level of Shenlan oral liquid high-dose group decreased compared with the model group.The TG level of serum in Shenlan oral liquid high-dose group significantly decreased compared with the model group from the fourth week (P＜0.05).After 6 weeks administration, Shenlan oral liquid high-dose and low-dose group and positive drugs reached to the same therapeutic effect.Compared with model group, serum TG level was significantly decreased (P＜0.05 or P＜0.01).Conclusion: Shenlan oral liquid can decrease the thrombosis formation in rats and inhibit the lipid level in blood in hyperlipidemia mice.

Keywords:

Shenlan oral liquid

Thrombosis

Hyperlipidemia

Blood-activating and stasis-removing

1.Introduction

With the development of social economy and the continuous improvement of people's living standards, the lifestyle gradually shifted to western diet, and the prevalence of thrombosis and hyperlipidemia increased sharply in our country.There isn’t disease name of hyperlipidemia in traditional Chinese medicine, and it is believed that hyperlipidemia is closely related to phlegm dampness,stasis blocking channels, spleen deficiency.Clinical dialectical is mostly attributed to phlegm dampness turbidity blocking, liverdepression and spleen-deficiency or qi stagnation and blood stasis,and most of the syndromes are stomach heat stagnation spleen syndrome, phlegm turbidity blocking syndrome or liver-depression and spleen-deficiency syndrome.Traditional Chinese medicine refers to the "coronary heart disease" general referred to as "chest Bi", belongs to the "chest pain, true pain" category, the etiology and pathogenesis of the main manifestations of blood stasis, cold coagulation, Qi stagnation, phlegm turbidness, Yang deficiency, etc.,mostly for the essential empty and out solid, intermingled deficiency and excess.[1].The "coronary heart disease" in western medicine refers to atherosclerotic lesions of coronary arteries or dynamic vasospasm caused by blood vessel stenosis or blockage, resulting in myocardial ischemia, angina (heart oxygen) or myocardial necrosis and other heart diseases.Traditional Chinese medicine plays an irreplaceable role in the early stage of coronary heart disease, occult coronary heart disease, coronary heart disease with poor treatment effect of Western medicine, and coronary heart disease after surgery.Shenlan oral liquid mainly contains Chuanxiong (Ligusticum),Danshen (Salvia miltiorrhiza), Shuizhi (Leech), Jiaogulan(Gynostemma), etc.It is an important Chinese patent medicine for the effective treatment of coronary heart disease with blood stasis.Ligusticum Chuanxiong is warm and bitter in taste, with meridian tropism in liver, gallbladder and pericardium, known as "Qi medicine in the blood", can not only promote blood stasis, but also Qi pain relief[2].Salvia miltiorrhiza, as a commonly used Chinese medicine for promoting blood circulation and removing blood stasis,relieving pain through menstruation, tastes bitter and is slightly cold,with meridian tropism in heart, liver.Moreover, clearing the heart and removing vexation, cooling the blood to eliminate carbuncle,used for chest pain, abdominal pain, lump accumulation.Danshen-Ligusticum Chuanxiong pair is the most commonly used compound Chinese patent medicine combination in the clinical application of cardiovascular and cerebrovascular diseases[3], Modern pharmacological studies have shown that Danshen-Chuanxiong can play a good role in the treatment of coronary heart disease,angina pectoris, cerebral thrombosis and other cardiovascular and cerebrovascular diseases by reducing vascular endothelial damage, anti-atherosclerosis, anti-thrombosis, anti-inflammatory and other mechanisms[4-6].Leeches, is natured, taste hot, salty,bitter, have small poison, with meridian tropism in liver.It has the effect of breaking blood, removing blood stasis and stimulate the menstrual flow.It also belongs to the category of activating blood circulation and removing blood stasis.In recent years, the advantage of leeches in cardiovascular and cerebrovascular diseases has gradually emerged, and has become one of the research hotspots in the treatment of cardiovascular and cerebrovascular diseases[7].Gynostemma is cold and bitter in taste, with meridian tropism in lung, spleen and kidney channels.It has the function of clearing heat and detoxifying, relieving cough and removing phlegm, and has the function of ginseng tonic effect[8].Four kinds of drugs used together,supplement Qi and activating blood circulation, both through the meridians and tonic.This study aims to investigate the effects of Shenlan oral Liquid on experimental thrombus formation in rats and serum total cholesterol (TC), triglyceride (TG) and other indexes in hyperlipidemia mice, so as to provide evidence for clinical use.

2.Materials and methods

2.1 Experimental material

2.1.1 animal

Male Wistar species rats, 150 g ～ 200 g, Male C57BL/6 mice aged 4-5 weeks, weight 17 g ～ 20 g.All the growth, breeding and highfat feeds for rats and mice were purchased from Beijing Huafukang Biotechnology Co., LTD.The animal qualification certificate number is SCXK (Beijing) 2019-0008.

2.1.2 Pharmaceutical reagents and instruments

Shenlan Oral Liquid is provided by Shanxi Huawei Pharmaceutical Co., LTD.Batch number: 20121201,20121101.

Positive control drug aspirin enteric-coated tablets purchased from Bayer HealthCare Manufacturing S.r.l..Atorvastatin calcium is purchased from Pfizer Ireland Pharmaceuticals, after grinding with double steam water, prepare the solution for reserve.Reagents were provided and tested by Beijing Beijian Xinchuangyuan Biotechnology Co., LTD.The contents of serum total cholesterol TC (LOT: B2002), TG (LOT: B2001), HDL-C (LOT: B2003)and LDL-C (LOT: B2004) were determined by Toshiba 120 total brake biochemical analyzer; Prothrombin Time Assay kit(Coagulation method, LOT: STY-50101-35-1), thrombin time assay kit (coagulation method, LOT: STY-50301-33-6), Activated partial thrombin time Assay kit (coagulation method,LOT:STY-50201-42-6), Platelet aggregation function test kit (ADP-activated turbidimetric method, Shandong Tailixin Medical Technology Co.,LTD.).Fibrinogen assay kit (Clauss method, LOT: STY-50401-35-1)was purchased from Taizhou Zhongqin Shidi Biotechnology Co.,LTD..The relative situation of thrombosis was measured by LS-14B small animal thrombosis instrument, STEELLEX INSTRUMENT Platelet aggregation coagulation factor analyzer measures clotting function, OLYMPUS the morphology and structure of liver tissue were observed by fluorescence microscope (BX51).

2.2 Establishment of an experimental thrombus model in rats

According to body weight, male Wistar rats were randomly divided into normal group, aspirin positive control group (15 mg/kg), Shenlan oral liquid low (L) and high dose groups (H), which were twice and 6 times the clinical daily dosage, respectively.Each cage of 5-6 rats was given 2 mL/100 g intragastric administration or distilled water, once a day for 22 d, and weighed twice a week.After the last administration of isoflurane, the rats in each group were given air anesthesia, the common carotid artery was separated,and the direct current was 0.8 mA to stimulate for 1 min, and the thrombosis related situation within 3 minutes was detected.The detection indexes are maximum blocking rate, time point of reaching maximum blocking rate and average blocking rate after stimulation.

2.3 Effect on coagulation system in rats

The group administration method was the same as 1.2.Anesthesia was performed after the last administration, blood was taken from abdominal aorta, and platelet aggregation was measured.The plasma was prepared, and the fibrinogen content (FIB), thrombin time (TT),prothrombin time (PT) and activated partial thrombin time (APTT)were determined by semi-automatic hemagglutination apparatus.

2.4 Effect on platelet aggregation in rats

The methods of drug administration and blood collection in groups were the same as 1.3, 3.8% sodium citrate solution 1 was used after abdominal aorta blood collection:9 Anticoagulation was placed in a blood hose, centrifuged at 1 000 rpm for 10min at room temperature,turbidity supernatant was taken to make platelet-rich plasma (PRP),and the remaining part was centrifuged at 3 500 rpm for 10 min at room temperature, clarified supernatant was taken to make plateletpoor plasma (PPP), and then platelet count was performed by a routine blood flow analyzer.PRP was adjusted by PPP to a platelet count of 400 000 /μL.The maximum platelet aggregation rate was determined by turbidimetric method.

2.5 Establishment of hyperlipidemia model in C57BL/6 mice[9]

Male C57BL/6 mice were randomly divided into 5 groups:blank group, model group, positive control atorvastatin calcium group, Shenlan oral liquid low-dose (L) and high-dose (H) groups,with 12 mice in each group.Except blank group mice were fed ordinary diet, other groups were fed high fat diet.Each mouse was given medication or distilled water intragastric at 0.2 mL/10 g.Atorvastatin calcium group of positive control was given intragastric administration according to the clinical dose of 20mg/kg, Shenlan oral liquid low dose and high dose groups were treated intragastric with six times and double the clinical dose respectively, blank group and model group were given equal volume of sterile double steaming water, continuous administration for 6 weeks.The blood lipid level was tested every two weeks, and the drug was administered until the end of the 6th week (12 h before the autopsy without water),and the blood was collected and dissected two hours after the drug administration.

Blood was collected from the orbital venous plexus of each group,the whole blood was left at room temperature for 2-3 h, centrifuged at 3500 rpm for 15 min, and the upper serum was gently absorbed to be measured.The liver was fixed during dissection, and the lipid droplet formation in the liver was detected by HE staining.

2.6 Statistical processing

Use GraphPad Prism statistical data analysis software, the data with mean ± standard deviation (x ±s), according to analyzed statistically with single factor analysis of variance, uses independent samples t test, P ＜ 0.05 represents data have significant difference.

3.Experimental result

3.1 Effect of Shenlan Oral Liquid on thrombosis in rats

No death occurred in the 4 groups after continuous administration for 22 d, and the body weight increased normally.Compared with the model control group, Shenlan oral liquid was similar to the positive drug aspirin, which significantly reduced the maximum and average vascular blockage rate after stimulation (P ＜ 0.05), indicating that Shenlan oral liquid had an inhibitory effect on thrombosis in rats.The results are shown in Table 1 and Figure 1.

Tab 1 Effect of Shenlan oral liquid on thrombosis in rats(±s)

Tab 1 Effect of Shenlan oral liquid on thrombosis in rats(±s)

Note: Compared with the Model group, *P＜0.05; **P＜0.01.

Groups Number of animals Average Blockage Rate (%) Maximum blockage rate (%) Time Point of Maximum Blockage(s)Model 10 48.36±26.72 75.03±21.57 93.6±63.04 Aspirin 9 7.35±13.63** 33.45±29.54** 30.89±36.92*Low dose (L) 8 20.01±26.13* 49.45±24.16* 84.50±60.28 High dose (H) 8 10.97±19.46** 25.80±23.44** 67.00±59.11

3.2 Effect of Shenlan Oral Liquid on coagulation function in rats

The high dose of Shenlan oral liquid could reduce FIB content to some extent, but the difference was not statistically significant compared with the normal group(P＞0.05).The effect of Shenlan oral liquid on FIB, TT, PT and APTT of rats was not obvious, indicating that the normal function of the coagulation system of rats was not affected after the use of Shenlan oral liquid (See Table 2).

Tab 2 Effect of Shenlan oral liquid on coagulation function in rats(n=3,±s)

Tab 2 Effect of Shenlan oral liquid on coagulation function in rats(n=3,±s)

Note: Compared with the normal group, *P＜0.05;**P＜0.01.

Groups FIB(g/L) TT(s) PT(s) APTT(s)Control 2.32±0.79 46.73±3.99 13.33±0.85 16.63±4.08 Aspirin 1.92±0.20 46.30±5.98 12.63±0.40 15.67±1.88 Low dose(L) 1.91±0.18 48.1±1.04 13.47±0.15 17.17±1.70 High dose (H) 1.72±0.41 47.20±0.85 13.07±0.76 15.30±2.79

3.3 Effect of Shenlan Oral Liquid on platelet aggregation

After 22 d of preventive administration, platelet aggregation function test kit was used to detect the effect of Shenlan oral liquid on platelet aggregation induced by ADP (3 μM).It was found that,like positive drug aspirin, Shenlan oral liquid in both high and low dose groups significantly inhibited platelet aggregation (P ＜ 0.05 or P ＜ 0.01), and the results were shown in Figure 1.

Fig 1 Influence of Shenlan oral liquid on rats’platelet aggregation

3.4 Effect of Shenlan Oral Liquid on blood lipid

The body weight of C57BL/6 mice increased significantly after being fed high fat diet, as shown in Figure 2 (B).The high dose group and positive drug of Shenlan oral liquid reduced the body weight (P＜0.05).At the end of the experiment, it was found that the liver weight of hyperlipidemia mice increased, and the liver weight of model combination positive drug group was significantly different from that of normal group (P＜0.05), while the liver weight of Shenlan oral liquid administration group was increased, but there was no significant difference.(Table 3).

Tab 3 Effect of Shenlan oral liquid on hyperlipidemia in mice(n=12,±s)

Tab 3 Effect of Shenlan oral liquid on hyperlipidemia in mice(n=12,±s)

Note: Compared with normal group, *P＜0.05;**P＜0.01.Compared with the model group, #P＜0.05;##P＜0.01.

(g) Liver weight (g) Liver coefficient(g/100g)Normal group 12 25.31±1.16 0.99±0.103 3.96±0.411 Model group 12 27.63±2.32* 1.21±0.186* 4.36±0.400*Positive group 12 26.03±1.45# 1.18±0.119* 4.36±0.400*Low dose(L) 12 27.83±2.22* 1.09±0.130 3.91±0.353#High dose (H) 12 26.56±1.57# 1.07±0.144 4.05±0.377 group Number of animals Body weight

3.5 Dynamic effects of Shenlan Oral Liquid on four items of blood lipid

The body weight of mice increased steadily after high fat diet,but different batches of drugs had no significant effect on the body weight of mice compared with model group.In contrast, the positive drug atorvastatin calcium caused weight loss in mice (Figure 2B).After feeding high-fat diet, serum TG, TC, HDL-C and LDL-C levels of mice were detected respectively, and it was found that serum TG increased significantly 4 weeks later, and the modeling was successful, and then randomized administration was performed.The serum lipid status of mice in each group after administration was detected respectively.It was found that Shenlan oral liquid was similar to atorvastatin calcium, which could reduce TG in blood from 2 weeks after administration, and showed a statistically significant difference to 4 weeks after administration(P＜0.05),compared with the positive drug atorvastatin calcium, it has more obvious hypolipidemia effect.The positive drug atorvastatin calcium did not show significant hypolipidemic effect until the 6th week of administration (P＜0.01).

Fig 2 Influence of Shenlan oral liquid on body weight of rats and mice

Fig 3 Influence of Shenlan oral liquid on hyperlipidemia mice

3.6 Pathological morphology of liver

As shown in Figure 4, mouse livers were dissected and fixed and then stained by HE.Under light microscope observation, the structure of liver lobules in blank group was clear, the boundary was clear, the cytoplasm was uniform and plump, the nucleus was located in the center of the cell, no fat droplets were deposited, and there was no obvious lesion of liver cells.In the model group, the boundary of hepatic lobules was blurred, and the volume of some cells became significantly larger and appeared swollen (The area around the manifold is more serious)[10].There are many round lipid vacuoles of different sizes in the hepatocytes (marked by red arrows), and part of the nucleus is pushed aside.Compared with model group, the liver cell structure of Shenlan oral liquid high-low dose group and positive group was relatively complete, and lipid deposition was reduced.In the high-dose group of Shenlan oral liquid, the improvement was more significant.In the low-dose group, the steatosis was also significantly improved.Occasionally, lipid vacuoles were formed,but the volume was small.

Fig 4 Influence of Shenlan oral liquid on hyperlipidemia mice(×400)

Fig 5 Classifications, functions and meridian distributions of Shenlan oral liquid

4.Discussion

Thrombus usually forms at the site of endothelial stripping or injury and consists of insoluble fibrin, deposited platelets, accumulated white blood cells, and trapped red blood cells.In this study, direct current electric stimulation was used to stimulate the carotid artery in rats, causing damage to the carotid intima, thus stimulating and activating coagulation factors, initiating the coagulation mechanism,causing platelet and fibrin aggregation, and forming a mixed model of carotid artery thrombosis[11].The thrombus formed by this method is very close to the clinicopathological state, good repeatability,simple and practical.At the same time, platelets in patients with cardiovascular disease are overreactive and highly aggregated, which can easily induce the formation of thrombus.The experimental results showed that Shenlan oral liquid had obvious inhibitory effect on carotid artery thrombosis in rats.Shenlan oral liquid can not only reduce the platelet aggregation of rats, but also does not affect the normal coagulation function and weight gain of rats(Figure 2).The positive drug was aspirin, a common clinical drug used to prevent thrombosis and platelet aggregation.This drug mainly reduces the formation of thromboxane A2 and promotes its decomposition by inhibiting the prostaglandin synthetase in platelets, so as to promote the formation of prostacycline in the blood vessel wall and prevent platelet aggregation and thus inhibit platelet;Aspirin also inhibits prothrombin formation in the liver and is used to prevent the formation of blood clots[12].However, studies have found that highdose administration or slow drug metabolism can lead to increased concentration of aspirin, resulting in prolonged PT, APTT and TT,which greatly increases the risk of bleeding, intravascular bleeding,cerebral stroke and other serious hazards[13].

Through pharmacodynamic study, it was found that Shenlan oral liquid had significant inhibition on the occurrence, severity and time of thrombosis in rats, and significantly reduced platelet aggregation,and had no effect on normal growth and coagulation function of rats, suggesting that Shenlan oral liquid has a good possibility of antithrombotic clinical application.

Triglycerides and lipids (including phospholipids, glycolipids,steroids and sterols) in plasma are collectively known as lipids,which are essential substances for cell metabolism and play an extremely important role in nutrient transport and energy supply of the body.Hyperlipidemia is a disorder of overall fat metabolism caused by a combination of genetic and environmental factors.It is characterized by an excess of plasma lipids, such as cholesterol and/or triglyceride[14]，also known as hyperlipidemia，serum test results showed total cholesterol(TC)、triglyceride(TG)、low density lipoprotein cholesterol,(LDL-C), one or more levels of them were higher than normal, along with low levels of high density lipoprotein cholesterol (HDL-C)[15].

Hyperlipidemia is the result of genetic factors and environmental factors, and it is also the most important risk factor of cardiovascular and cerebrovascular diseases.A large number of studies have shown that atherosclerosis caused by hyperlipidemia is the main cause of coronary heart disease, hypertension and cerebrovascular diseases,so the control of blood lipids is a serious problem facing the whole society[16].The World Health Organization (WHO) divides common clinical hyperlipidemia into 6 types based on clinical test phenotypes, among which the phenotype of elevated triglycerides accounts for the majority[17].

In this experiment, hyperlipidemia model of C57BL/6 mice was created by simulating human high-fat diet and feeding high-fat diet.With the extension of feeding time, TG, TC and LDL-C of model mice increased significantly, indicating that the modeling was successful.After 6 weeks of administration of Shenlan oral liquid, the serum TG level of hyperlipidemia mice was significantly reduced.In this study, atorvastatin calcium (Lipitor, 20 mg/kg), the most widely used statin for hyperlipidemia, was used as positive control.Atorvastatin calcium is A hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) inhibitor that significantly reduces plasma LDL-C and TG levels by inhibiting HMG-CoA reductase and cholesterol synthesis in the liver.It also increases the uptake and catabolism of LDL-C by increasing the number of low-density lipoprotein receptors on the surface of liver cells[18].Atorvastatin calcium can treat hyperlipidemia with elevated TC, elevated LDL-C,elevated apolipoprotein B (ApoB), and elevated TG[19].We found that clinical doses of atorvastatin calcium did not start to significantly reduce serum TG levels in mice until week 6 (P＜0.01), the tested drug Shenlan oral liquid showed the effect of reducing serum TG level of mice significantly after 4 weeks of administration (P＜0.05).At the same time, Shenlan oral liquid can also reduce the weight of liver and the deposition of fat droplets in liver cells of hyperlipidemia mice.The results suggest that Shenlan oral liquid has no side effects and has a good prospect in the treatment of hyperlipidemia.In the clinical treatment of hyperlipidemia, Chinese medicine mainly uses activating blood circulation and removing blood stasis, invigorating spleen and dispelling dampness, tonifying liver and kidney, clearing heat and defecation, eliminating food and phlegm, nourishing Yin and nourishing blood.The main components of Shenlan oral liquid are Chuanxiong, Danshen, Shuizhi and Jiaogulan are commonly used Chinese medicine to reduce blood lipid[20].Among them,Danshen, Chuanxiong and Shuizhi all contain good function of promoting blood circulation and removing blood stasis(Analysis of formulation See Figure 5), both through the meridians and tonic.

The results of this study showed that Shenlan oral liquid was effective in inhibiting thrombus formation in rats, reducing serum triglyceride level in hyperlipidemia mice, and improving lipid metabolism disorder, which provided experimental basis for further expanding clinical application of Shenlan oral liquid.The disadvantage of this study is that the mechanism of Shenlan oral liquid in treating thrombus and lowering blood lipid is still unclear,which needs further study.

Author contribution description:

GU Liwei, LIU Dandan: Responsible for experiment implementation, data processing and paper writing.TIAN Jingzhuo:Responsible for pathological sample processing and data analysis.ZHANG Hongbing, ZHAI Xiaoru, YANG Qian, LI Yongdong,LI Jin, LI Xin: Responsible for collection of medicinal materials,sample preparation and experimental guidance.Shen Shuo: Provide technical guidance.DU Maobo: Responsible for experimental design, experimental guidance and paper revision and correction.

All authors declare no conflict of interest.