CN101816667B - Application of forsythiaside A in preparation of drugs for preventing and treating cardiovascular and cerebrovascular diseases - Google Patents

Abstract

The invention proves that forsythiaside A has the function of inhibiting platelet aggregation and simultaneously has the obvious anticoagulation role by studying the impacts of the forsythiaside A on ADP-induced platelet aggregation in vitro of rats, the impacts on plasma prothrombin time (PT) of the rats and the impacts on plasma thrombin time (TT) of the rats. The forsythiaside A can be used for preparing drugs for preventing and treating cardiovascular and cerebrovascular diseases. The forsythiaside A can also be used as a food additive for preparing functional foods for preventing and treating the cardiovascular and cerebrovascular diseases.

Description

Application of forsythiaside A in the preparation of drugs for preventing and treating cardiovascular and cerebrovascular diseases

technical field

The invention relates to forsythiaside A, specifically the application of forsythiaside A in the preparation of drugs for preventing and treating cardiovascular and cerebrovascular diseases.

Background technique

Forsythoside A (Forsythoside A) can be extracted from Forsythia Suspensa (Thunb.) Vahl. The structural formula is:

Molecular formula C ₂₉ H ₃₆ O ₁₅ , molecular weight 624, light yellow powder compound, easily soluble in water, ethanol, methanol, etc.

It is reported that forsythiaside A has antiviral, antibacterial, antipyretic, analgesic, hepatoprotective, antioxidative, anti-inflammatory and anti-endotoxin activities. However, there has never been any report on the preparation of drugs for the prevention and treatment of cardiovascular and cerebrovascular diseases.

Contents of the invention

The purpose of the present invention is to expand the use of forsythiaside A and use it to prepare medicines for preventing and treating cardiovascular and cerebrovascular diseases.

Cardiovascular and cerebrovascular diseases are mainly caused by the formation of thrombus in blood vessels, which poses a serious threat to human health. Studies have shown that there are three factors of thrombus formation: (1) vascular endothelial injury; (2) platelet adhesion, aggregation and release reactions; (3) increased coagulation activity, increased blood viscosity, and slow blood flow. Preventing blood clotting or inhibiting platelet function can prevent blood clots from forming. Therefore, substances with the functions of thrombolysis, anticoagulation, lowering blood viscosity, protecting vascular endothelial cells, and inhibiting platelet aggregation can prevent and treat cardiovascular and cerebrovascular diseases such as myocardial infarction and cerebral infarction.

The research of the present invention shows that: forsythiaside A has the function of inhibiting platelet aggregation, and also has obvious anticoagulant effect. Therefore, forsythiaside A can be used to prepare drugs for preventing and treating cardiovascular and cerebrovascular diseases.

Forsythiaside A can be extracted from Forsythia husk or Forsythia leaf according to conventional methods, and purified by column chromatography. The forsythiaside A obtained above can be further mixed or embedded with a carrier to prepare a pharmaceutical composition containing a therapeutically effective amount of forsythiaside A, which can be in the form of tablets, injections, suspensions or solutions. Said carrier can be lactose, sucrose, sorbitol, mannitol, starch, polyethylene glycol, gelatin, cyclodextrin. Lubricants, wetting agents, flavoring agents, suspending agents, and preservatives can also be added to the composition. The content of forsythiaside A in the composition should be more than 50%, preferably more than 90%. These compositions can be used as medicines for preventing and treating cardiovascular and cerebrovascular diseases.

Forsythiaside A can also be used as a food additive to prepare functional foods for the prevention of cardiovascular and cerebrovascular diseases.

The invention explores new medical application of the known compound forsythiaside A and opens up new application field. Forsythiaside A has strong pharmacological effects and can be used to prepare drugs and functional foods for the prevention and treatment of cardiovascular and cerebrovascular diseases.

Detailed ways

The following pharmacological tests show that forsythiaside A (purity greater than 98%) has the effect of inhibiting platelet aggregation and anticoagulation.

Example 1 Effect of forsythiaside A on ADP-induced platelet aggregation in rats in vitro

Blood was collected from the abdominal aorta of rats (Wistar, clean class II), anticoagulated with 3.8% sodium citrate 1:9, centrifuged at 500 r·min ⁻¹ for 5 min to obtain platelet-rich plasma (PRP). The remaining part was centrifuged at 2500r·min ^-1 for 10min and the supernatant was taken to obtain platelet poor plasma (PPP). Dilute PRP with PPP to bring the number of platelets to 3×10 ¹¹ /L.

Take 265 μL of PRP and place it in a turbidimetric tube, add 20 μL of forsythiaside (content greater than 95%) solution (final concentration: 2.1, 2.6, 3.1, 3.6, 4.1, 6.2, 8.2 mmol·L ^-1 ), and add 20 μL to the blank control tube Equal volume of normal saline, pre-warmed at 37°C for 3 minutes, started the platelet aggregation coagulation factor analyzer, quickly added 2mmol·L ^-1 ADP15μL, recorded the platelet aggregation curve, and calculated the platelet aggregation inhibition rate of the drug with the following formula.

The results showed that: Forsythiaside A had inhibitory effect on ADP-induced platelet aggregation in rats in vitro, and had an obvious dose-effect relationship (Table 1). When the final concentration of forsythiaside was 13.4mmol·L ^-1 , the inhibition rate of platelet aggregation was 72.4%, and its IC50 value for inhibiting platelet aggregation in rats was 8.1mmol·L ^-1 , indicating that forsythiaside can inhibit platelet aggregation by The aggregation of antithrombin relieves the formation of thrombus, and has a strong effect.

The effect of table 1.Forsythiaside A on ADP-induced rat platelet aggregation in vitro ( n=6)

**P<0.01 vs NS, ***P<0.001 vs NS

Example 2 Effect of Forsythiaside A on Rat Plasma Prothrombin Time (PT)

Blood was collected from the abdominal aorta of rats, anticoagulated with 3.8% sodium citrate 1:9, centrifuged at 2500r·min ^-1 for 10min, and the supernatant was collected to obtain PPP. Pre-warm the PT reagent in the reagent pre-warming well at 37°C for more than 10 minutes, and shake well before use. Take 50 μL of PPP into a turbidimetric tube, add 30 μL of forsythiaside A solution (final concentration: 8.3, 12.5, 16.6 mmol·L ^-1 ), add an equal volume of normal saline to the control tube, pre-warm at 37°C for 180 seconds, then add 100 μL of 37 ℃ PT reagent, immediately start the instrument for automatic testing, and record the results.

The results showed that: Forsythiaside can significantly prolong the isolated plasma PT of rats, and the prolonging effect is more obvious with the increase of the dose (Table 2). Forsythiaside at a final concentration of 16.6mmol·L ^-1 prolongs PT for more than 400s, indicating that forsythiaside A has a strong anticoagulant effect.

n＝6)The influence of table 2.Forsythiaside A on rat isolated plasma PT (

n=6)

***P＜0.001 vs NS

Example 3 Effect of Forsythiaside A on Rat Plasma Thrombin Time (TT)

The PPP preparation method is the same as above, take the TT reagent out of the refrigerator and let it stand at room temperature for 10 minutes, and the temperature is equilibrated to room temperature. Take 80 μL of PPP and put it into a turbidimetric tube, add 30 μL of forsythiaside solution (final concentration: 7.1, 10.7, 14.2 mmol·L ^-1 ), add an equal volume of normal saline to the control tube, pre-warm at 37°C for 180 seconds, then add TT reagent 100μL, immediately start the instrument for automatic testing, and record the results.

The results showed that: forsythiaside A can significantly prolong the isolated plasma TT in rats, and there is an obvious dose-effect relationship (Table 3).

n＝6)The influence of table 3 forsythiaside A on rat isolated plasma TT (

n=6)

*P<0.05 vs NS, ***P<0.001 vs NS

Embodiment 4 90 days feeding toxicity test

Select 80 healthy Wistar rats, weighing (85±10) g, and randomly divide them into 4 groups, 20 in each group, half male and half male. Administration by intragastric administration (forsythiaside A content greater than 60%), once a day, 10ml/kg body weight each time. Dose settings are: 1.8g/kg, 0.6g/kg, 0.18g/kg 3 dosage groups and a control group. The control group was given the same volume of distilled water. After the administration, the body weight was weighed once a week, and the dosage was adjusted according to the body weight. During the period of administration, observe the general performance of the animals such as food intake, activities, feces, and toxic reactions. Fast overnight (16 hours) after the last dose of medicine, kill the animals, take blood to detect hematology and blood biochemical indicators, use SYSMEX XT-1800i automatic hematology analyzer to measure hematology indicators; use glass slide method to measure coagulation time; SELECTRA-E automatic biochemical analyzer measures blood biochemical indicators. And the heart, liver, spleen, lung, kidney, uterus, ovary, and testis of each mouse were weighed, and the corresponding organ coefficient per 100 g body weight was calculated. At the same time, the heart, liver, spleen, lung, kidney, stomach, small intestine, adrenal gland, thymus, ovary, and testis of each animal were taken for histopathological examination. The measured values of each drug administration group were compared with the normal control group, and the t-value method or corrected t-value method was used for statistical analysis, and the safety was comprehensively evaluated according to the measurement results.

The results showed that: (1) During the administration period, the rats in each administration group of forsythiaside A and the normal control group had normal appearance signs, behavioral activities, food intake and feces, and none of the animals died. During the experiment, it can be seen that the water consumption of the animals in the medication group increased, and the rest had no obvious abnormalities. (2) After 13 weeks of continuous administration, the body weight of the rats increased normally, and there was no significant difference between the administration group and the normal control group. There was no significant difference between the food utilization rate administration group and the normal control group. (3) Give rats continuous medication for 13 weeks, measure red blood cell count (RBC), hemoglobin (HB), platelet count (PLT), white blood cell count (WBC) and their classification in the administration group and the normal control group: lymphocyte (LYM ), monocytes (MOV), granulocytes (Gran), coagulation time (CT). There was no significant difference in each index between the administration group and the normal control group. (4) Give rats continuous medication for 13 weeks, measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum total protein (TP), albumin (ALB), blood urea nitrogen (BUN) in the administration group and the normal control group ), creatinine (cr), triglycerides (TG), total cholesterol (CHOL), alkaline phosphatase (ALP), glucose (GLU). No biologically significant changes were found in the administration group compared with the normal control group. However, the triglyceride (TG) in the male animal treatment group was significantly reduced, and there was a dose-effect relationship; the female animal also had a similar change, but the reduction was smaller. Compared with the control group, the difference was not significant. (5) After 13 weeks of continuous drug administration to rats, the wet weight and organ coefficients of major organs such as the liver, spleen, kidney, testis, ovary, adrenal gland, and thymus between the administration group and the normal control group were compared. significant meaning. (6) There was no abnormality in gross anatomy. It shows that under the conditions of this experiment, forsythiaside was administered to Wistar rats for 90 days continuously. Compared with the control group, no toxic pathological changes related to the test substance were found in the high-dose group.

The above experimental results show that forsythiaside A has a good anticoagulant effect, and is non-toxic and safe to use. Therefore, forsythiaside A can be used to prepare medicines for preventing and treating cardiovascular and cerebrovascular diseases. It can also be used as a food additive to prepare functional food for preventing and treating cardiovascular and cerebrovascular diseases.

Claims (1)

1. Application of forsythiaside A in the preparation of anticoagulant drugs.