Studies On Anticoagulant Constituents Of Leech And Leech Injection (Lyophilized Powder)

Leech is a kind of Traditional Chinese Medicine of anticoagulation, and lots of studies are investigated on their chemical composition, pharmacological action and clinical application. Those provided theoretical basis for their further research. In the past, it was thought that only the leech sucking blood have anticoagulant activity and anticoagulants only exist in fresh saliva of those leeches. But plenty of clinical facts proved that the activity of Whitmania pigra was also very strong, which only eated spiral shell or clam. Furthermore, the dried body of Whitmania pigra was the leading genus in leech market and clinical application in China. However, the chemical components of this kind of leech were unknown for scientists and the preparation of it was not very deep. It’s worth researching what exactly is the anticoagulation constituent and developing a new preparation.In this paper, three aspects of the leech Whitmania pigra had been researched: anticoagulant constituents, extracting technology and leech injection (lyophilized powder).First of all, we summarized several aspects of leech in detail: chemical constituents, pharmacology, preparation and clinical application, etc.Using plasma recalcification time (PRT) as index and following up the anticoagulant activity, isolation and purification anticoagulant components from the leech Whitmania pigra had been completed: Firstly, the best extraction solvent, solvent I—water (40%, v/v), was found out from five classic solutions. Then two kinds of sediments were got by using 60% and 85% ethanol. The latter was proved to be the active part of this kind of leech. At last, the best separation condition was found out: the deposition of 85% ethanol was subjected to an Anion-exchange chromatography column, and gel permeation chromatography on Sephadex G-25 and Sephadex LH-20 columns and then RP-HPLC successively. Finaly, three active polypeptides were isolated and purified. Because compound 1 and 2 can transfered each other in natural conditions, we can only determine their molecular weight: 7100 Da and 5531 Da. Compound 3 was identified as a single pure substance by RP-HPLC and SDS-PAGE. Its molecular weight was determined as 8608 Da by MALDI-TOF-MS and its amino acid composition was also determined. It was proved to be a novel anticoagulant. Because it was isalated from Whitmania pigra and had 40% ancticoagulant activity of W. pigra, it was named whitmanin.Using the anticoagulant component whitmanin as the index element and via orthogonal experiment, four factors were reviewed: concentration of solventⅠ, dosage of solventⅠ, soak duration and soak times, and then the technology of extracting active component from medicinal materials was optimized: extract with 40% solventⅠ(four times of materials, v/m) for 4 times, and every time maintain 24 hours. The refine technology was also confirmed: after impurity was deposited with 60% ethanol, the anticoagulant substance was obtained from the deposition of 85% ethanol, and then water solution of the latter was filtrated with a microporous filter membrane (0.22μm), at last, the active part of leech (LI) was obtained.The antitrombotic effect of LI was studied by means of thrombus in vitro, fibrin plate and whole blood-gore. The anticoagulation effect of LI was determined by means of plasma recalcificatic time, thrombon time and prothrombon time. All of three dosages (high, middle and low) of LI could not only dissolve the thrombus in vitro (p＜0.001), normal or heated fibrin plate (p＜0.001) and whole blood-gore (remarkably), but also prolong the plasma recalcificatic time, thrombon time and prothrombon time (p＜0.001). Which prove that LI has potent antitrombotic and anticoagulation activities. Furthermore, safety trial showed that intervenous injection of LI is of low toxicity, it has no allergic reaction in the guinea pigs, no haemolysis in vitro, and no trend to bleed, which prove that LI is of no evident side effect.The recipe of leech injection was optimized by comparing different kinds of supports (support-MR, support-ML, support-GS, support-ME and support-MHP), dosages of supports and freezing time, which have different effects on appearance, color, resolution and diaphaneity of leech injection (lyophilized powder). Finally, 4～8% support-ML was confirmed as the best support; To insure the injection to keep stable in different pH, 0.9% support-ME was used as the protector. Use the content of whitmanin as index, the influence of supports and methods of sterilization were reviewed, and the recipe and technology of preparation were confirmed.The content of whitmanin in the raw material, leech injection and the active part was mensured by RP-HPLC. Based on the results of examining several batches, whitmanin was chosen as the quality index to evaluate the quality of raw material, leech injection (lyophilized powder) and the active part. To make sure the deposited condition of leech injection, the stability tests of leech injection were developed too, including influencing factor tests, accelerated tests and observed samples for a period of time.