Purification Of The Crude EFE (Earthworm Fibrinolytic Enzyme), Preparation Of The Cream EFE In Eisenia Foelida And The Clinic Curative Effects

PrefaceEarthworm fibrinolic enzynme(EFE)which is also called dilong is a kind of fibrinolysis-active proteolytic enzyme extracted from earthworm.Earthworm,as a kind of ancient herbal medicine,is widely used in the study of medicine in our country.It is early recorded in the BENJING,BIELU and Outline Treatise of Medical Herbs that earthworm has the effect of clearing heat to stop convulsion,calming liver to suppress wind,dredging and activating meridians,relieving asthma and promoting diuresis and is often used in surgery of curbuncle and sore.It is recorded in the latest edition of TRADITIONAL CHINESE MEDICINE DICTIONARY that the earthworm has the following pharmacological effects:a.the anticoagulant effect;b.the anticancer effect;c. the anti-inflammatory,analgesic and promoting wound healing effect;d.other effects. The third pharmacologic action proved by animal experiments was that EFE had the effect of anti-inflammatory,and whose action was similar to that of 50mg/kg of cortisone acetous eye drops.Based on the above research results,we mainly identified whether EFE had the positive curative effect,and primarily observed which dermatologic diseases were improved by it and which ones were not.Purposes1.Purify the crude EFE2.Prepare the cream of the active extract form EFE for the external application and the mass detection. 3.Observe the clinic curative effects of the cream EFE on five dermatologic diseases.Materials and methods1.Three steps of chromatography using AKTA purifier 100 system which is produced by GE com.of the USA.a.Gel filtration Chromatography(GFC)of the EFE crude on Sephadex G-75. In the end,we gained D5-peak with the highest activity,the densest concentration and the least dopant.And the D5-peak included 3 components whose Mr.were 30KD,33KD,41KD respectively.b.Iron exchange Chromatography of D5 from Sephadex G-75 colume on DEAE Sepharose Fast Flow.At last,we obtained one active peak(D5-S2)which contained 2 components,and their Mr.were 33KD,41KD.c.Hydrophobic interaction Chromatography of D5-S2 from DEAE Sepharose Fast Flow on Phenyl-Sepharose Fast Flow.2.The preparation of the active extract of EFE for the external application and the mass detection.a.Making the cream of EFE with the general prescription.b.The experiment of skin stimulus was performed with the back of cavia cobayas and the nasal cavity of rabbits as the objects.c.The experiment of drug stability:first,put the cream of EFE and the EFE crude at the general temperature for 6 months.Second,observe the appearance of the cream and measure the activity of EFE.3.The clinic curative effects of the cream EFE on five dermatologic diseasesThe clinic data:75 cases of chronic eczema,syndrome of rhagades on hands and feet,dermatitis caused by sebaceous flux,dermatitis caused by confused nerve and dermatitis caused by hypersensitiveness.Therapeutic program:applied the cream EFE on the damaged of the skin for about 1 or 2 weeks. According to the relative diagnostic criteria,analyze the clinic effective power.Results1.After the three steps of chromatography,eventually,we achieved one material whose Mr.is 33KD(the concentration of protein was 0.1266mg/ml and at the same time its activity is 226.67usp).2.The product does have No skin stimulus and have good stability.3.EFE had obvious effect on chronic eczema,syndrome of rhagades on hands and feet and dermatitis caused by sebaceous flux but had less effect on dermatitis caused by confused nerve and had no effect on hypersensitiveness.The significance of this researchThis subject triggers further researches on pharmacologic action of EFE and helps explore a new type of anti-inflammatory drug.Conclusions1.set up the technological flow-sheet of purification the EFE crude which contained gel filtration chromatography,iron exchange chromatography and hydrophobic interaction chromatography.2.EFE had obvious effect on chronic eczema,syndrome of rhagades on hands and feet and dermatitis caused by sebaceous flux but had less effect on dermatitis caused by confused nerve and hypersensitiveness.