Chemistry of carbon compounds – Miscellaneous organic carbon compounds – C-metal
Patent
1980-07-20
1982-09-21
Rosen, Sam
Chemistry of carbon compounds
Miscellaneous organic carbon compounds
C-metal
424 98, 424177, A23J 100, C09H 100, A61K 3700
Patent
active
043506259
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION
1. Technical Field
This invention relates to a new substance which is obtained from venom of venomous snakes belonging to the family of trimeresurus taxonomically and which has fibrinolytic activity to mammals, and a method of manufacturing the same and its applications in medicine.
2. Background Art
In general, animal toxin such as venom of snakes is known to involve various proteinic toxin factors, some of which may endorse the presence of materials showing various biochemical actions such as, for example, hemorrhagic toxin, neurotoxin, hemolytic constituent, hemopexis constituent, etc. Recent years have witnessed biochemical studies of these animal toxins which are well in progress.
Taking note of the action of the venom of Trimeresurus which is feared to have very often caused fatalities to human life and cattle, the present inventor has long been engaged in studying the action of protein toxin, and their effects on the blood coagulation system, fibrinolytic system and plastocyte function. He has discovered that a substance obtained by a special treatment of crude venom of Trimeresurus has a strong fibrinolytic activity and, as the result of further investigation, he has succeeded in isolating such substance singly and moreover, found that it has an extensive range of clinical uses as a fibrinolytic agent.
DISCLOSURE OF INVENTION
The principal object of the present invention resides in providing a new singly isolated substance, a method of manufacturing the same and its applications in medicine, said new substance being obtained by fractionating a solution of crude venom of Trimeresurus by means of so-called molecular sieve chromatography as the main procedure using a stationary phase of material having a molecular sieve effect, in combination with ion exchange chromatography using a stationary phase of ion exchange material so as to collect fractions of the largest activity of fibrinolysis each time in the aforesaid chromatographic procedure. The present invention will be explained in detail centering on preferred embodiments hereinafter.
Crude venom of Trimeresurus for use as the material in the present invention is defined and is to be understood as toxin which can be obtained from venomous snakes belonging to the family of Trimeresurus Crotalidae by classification, for example, Trimeresurus flavoviridis, Trimeresurus tokarensis, Trimeresurus okinavensis, Trimeresurus mucrosquamatus, Trimeresurus elegans, Trimeresurus stejnegsi, Trimeresurus tinkami, Trimeresurus moticola, and Trimeresurus gracilis.
A freezedried powder of crude venom obtained from one or more of these venomous snakes, is dissolved in a suitable solvent, for example, distilled water, physiological saline or a buffer solution of pH within the range of 6 to 8. This solution is fractionated by the so-called molecular sieve chromatography using a stationary phase of material having a molecular sieve effect. For example, a suitable gel filtration agent such as Sephadex (G-75 or G-100) (Trade name) or Biogel (Trade name) is used as a column filler in which said solution is adsorbed and permeated and subsequently, the resulting solution thus adsorbed and permeated is eluted in a buffer solution of suitable electrolyte of pH, about 6-8, thereby enabling it to be fractionated into 4 fractions, P-I, P-II, P-III and P-IV. Each of these fractions is tested by the standard fibrin plate method so that the fraction P-II can be ensured to have an activity of fibrinolysis.
This fraction, P-II is subjected to dialysis in distilled water and the internal solution after dialysis is freezedried. Subsequently, the fraction P-II thus freezedried is dissolved in a buffer solution of pH around 6 such as sodium acetate buffer solution and the resulting solution is fractionated by ion exchange chromatography. Namely, it is adsorbed in a column filled with CM cellulose as a cation exchange body at low temperature and the portion thus adsorbed is eluted in a buffer solution by slowing increasing salt concentration in the buffer solution so that
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