Extraction, Evaluation And Toxicology Study On The Anticoagulant Activity Material From Whitmania Pigra

Objective:Whitmania pigra is a kind of traditional Chinese medicine which has been used as a remedy for blood stasis and traumatic injury since hundred years. Hirudin was regarded and extensively documented as the anticoagulants material in leeches in curing cardiovascular and cerebrovascular diseases. Three types of dry leeches, namely, Hirudo nipponica Whitman, Whitmania pigra whitman and W. acranulata whitman. were documented in Chinese pharmacopoeia(2010version). Though whitmania pigra was broadly used in the pharmacy, the leech bioactivity ingredients were few reported. Therefore, whitmania pigra was taken as the experiment subject, the extraction, evaluation and toxicology study on the anticoagulant activity material from whitmania pigra was carefully observed.These experiments can provide useful information both for industrial production and clinical application.Methods:1. Study on the extraction technology of anticoagulant activity constituents from leechAfter analyzing and comparing the anticoagulant activity and the plasma recalcification time of crude extraction from Whitmania pigra with3different processes, which are water extraction and ethanol precipitation method, ethanol extraction and water precipitation method, acetone precipitation methods, water extraction and ethanol precipitation method was selected as best extraction method. And the extracting technology was optimized by central composite design and response surface methodology.2. Separation and purification of crude extract of leech using gel filtration chromatographyDEAE Sephadex A-50and Sephadex G-50gel were used to purify the active substances extracted from leech, then the anticoagulant substances of A1and G1were obtained. Subsequently, the anticoagulant activity of leech’s crude extraction, A1and G1were tested.3. The kjeldahl method of the productNitrogen content (protein content) of crude extract, A1and G1were determined by the kjeldahl nitrogen determination method.4. Study on the antithrombotic pharmacodynamics of leechThe effect of anticoagulant activity of substance G1was tested by rats in vivo. A series of the antithrombotic experiments were done, these experiments included as follows:the influence on thrombus formation on rat arteriovenous by pass experimental thrombosis, effect on thrombus formation in vitro, effect on platelet, cholesterol and plasma specific viscosity, influence on plasma clot and euglobulin lysis of mice, effect on the permeability of rat brain capillaries and the platelet aggregation.5. Study on chronic toxicity experimentsIn this study, anticoagulant activity substance G1’s chronic toxicity have been tested carefully. G1were given to SD rats for60days by gastric lavage, the animals’general health index, weight change, routine blood test (WBC、NEU、LYM、NEU、LYM、RBC、 HGB、HCT、MCV、MCHC、PLT), the function of liver and kidney(SGPT、SGOT、 BUN、Cr), blood biochemical index (Blood glucose, cholesterol, total protein, albumin, alkaline phosphatase) and important organ coefficient of heart, liver, spleen, lung, kidney, adrenal gland, stomach, pancreas, brain, testis, uterus and thymic tissue were observed and recorded to evaluate the anticoagulant substances G1’s chronic toxicity.6. Study on acute toxicity of leech 200times of clinical dosage diet of G1was given to NIH mice for7days,activity, toilet and death of the animals were carefully observed and recorded.Results:1. Study on the extraction technologyThe crude extract from whitmania pigra with water extraction ethanol precipitation and ethanol extraction water precipitation methods showed stronger anticoagulant activity compared with the acetone alcohol precipitation. Considering the convenience and fees, water extraction ethanol precipitation method was regarded as the best extraction method for whitmania pigra. The optimum extraction process of crude extract of leech was further optimized by central composite design and response surface methodology. The optimum parameters were as follows:extracting3times,1.5h each time, the amount of water is10times(v/w), the alcohol concentration is50%for precipitation for20h, the drugs needs to be soaking4h before extraction. Verification test is done under this condition which showed deviation rate was small demonstrating our model was fit and the process conditions had good reproducibility.2. Separation and purification of crude extract of leech using gel filtration chromatographyAnion exchange agent DEAE Sephadex A-50was used as the column chromatography to purify the crude extraction from leech and three active ingredients, A1, A2, A3, were obtained respectively. After comparing their anticoagulant activity, A1(166.52±0.21U/g), showing the strongest anticoagulant activity, was selected for the further purification with Sephadex G-50gel, a substance with higher anticoagulant activity(225.28±1.03U/g) was obtained and named as G1. The anticoagulant activity of G1was much higher than what required in the Chinese Pharmacopoeia. Data showed the obtained substances after boiling water extraction, concentration, drying and other high temperature treatment of leech extract still had obvious anti coagulation activity, indicating there should be other anticoagulant components besides hirudin for hirudin was sensitive to the high temperature.3. Determination of protein content by the Kjeldahl method The total nitrogen concentration was determined by the Kjeldahl method. Data showed that A1and G1’s Nitrogen concentration was lower compared with the crude extract of leech, but the anticoagulant activity was opposite:A1,G1showed much more stronger anticoagulant activity than crude extract of leech. The relation between the nitrogen concentration and anticoagulant activity should be further studied.4. Study on the antithrombotic pharmacodynamics of leechPharmacodynamics experiments showed that G1could inhibit the thrombus formation on rat arteriovenous by pass experimental thrombosis. G1could also significantly reduce the number of platelet and inhibit the platelet aggregation; Besides, Gi could reduce plasma viscosity, shorten the whole plasma clot lysis time and euglobulin lysis time and increase the rats’Cerebral vascular permeability of incomplete cerebral ischemia.5. Study on chronic toxicity experimentsChronic toxicity experiments showed that rats with the dose20,10,5mg/kg of G1were in a good condition With the normal stool forming, behavior, eating, weight increasing. There were no significant differences (P>0.05) between the experimental group and the control group about routine blood test, the function of liver and kidney, blood biochemical index and important organ coefficient either the rats administered for60days and15days after withdrawal. Together with the same histopathological observation of each group rats’organs, Gi showed no obvious damage on rats.6. Study on acute toxicity of leechMice were selected to be studied on acute toxicity of leech. With the dose of50mg/kg of Gi (equal to30g/kg of the leech) were given to the mice.No death occurred in mice within the experiment periods and the animals histopathological observation showed no abnormality.Conclusion:DEAE Sephadex A-50and Sephadex G-50was used to purify the crude extraction from whitmania pigra and2active ingredients, namely, A1, G1were obtained. The purification method was simple, convenient showing a bright future in production of G1. G1had stronger anticoagulant activity which could be widely used in clinical practice. Though A1and G1’s Nitrogen concentration was much lower than the crude extract of leech, A1and G1had much stronger anticoagulant activity. The relation between the total protein concentration and anticoagulant activity should be more studied. Besides the small molecule protein, there are other non-nitrogen compounds which showed anticoagulant activity. Pharmacodynamics experiments and chronic toxicity experiments showed whitmania pigra were safe without obvious side effect.