KR100233873B1 - Vasodilation composition containing lumbrokinase - Google Patents

Abstract

The present invention relates to a vascular muscle relaxant composition containing rombrokinase, which is a proteolytic enzyme extracted from lubricus rubellus. More specifically, the present invention relates to a relaxant composition containing, as an active ingredient, an active fraction of a lombrokinase which is separated from a job through a series of processes.

Description

Vascular Muscle Relaxant Composition Containing Lombrokinase

The present invention relates to a vascular muscle relaxant composition containing rombrokinase, which is a proteolytic enzyme extracted from lubricus rubellus. More specifically, the present invention relates to a vascular muscle relaxant composition containing an active fraction of lombrokinase which is purified and purified purely through a series of processes.

Stroke is a disease of cerebral artery that causes constriction or occlusion of cerebral artery vessels due to arteriosclerosis or blood clots, leading to brain injury due to blockage of cerebral blood flow, resulting in paraplegia or speech impairment, and even death. Vascular disorders such as strokes are increasing rapidly in modern times, which is one of the biggest signs of modern medicine. To date, stroke has been generally used for the treatment of urokinase, ticlopidine, r-tPA (recombinant-tissue type plasminogen activator, recombinant tissue plasminogen activator), alprostadyl, stoletokinase, etc. In terms of treatment, there is still no satisfactory treatment.

Therefore, the present inventors conducted extensive research to find an effective drug for the treatment of stroke, and used the job as his subject. Job offer has traditionally been prescribed as a therapeutic agent for adult diseases such as arteriosclerosis, hypertension and diabetes in traditional Chinese medicine, especially in the pharmacopoeia of the People's Republic of China (1977 Edition, Part 1, pp. 197-188). There are two types of job openings, Lumbricus Kwang tungesis and Lumbricus nativus, which are antipyretic, anti-cursive, blood-promoting, anti-involuntary, joint, urination, bronchial asthma, and hypertension. It is described as effective. The effect of this job is known to be due to the action of a protease called lumbrokinase contained in the job. In particular, the lobbrokinase of gut has not only fibrinolytic activity but also fibrin formation inhibition and platelet aggregation inhibitory activity compared to conventional thrombolytic enzymes. Unlike in vitro experiments, in vivo experiments have very low activity against fibrinogen and fibrin Has been reported to be effective against hyperlipidemia and cerebral thrombosis.

Applicants have conducted intensive research on the lobbrokinase of such antigens to find a method for purely separating and purifying rombrokinase from crude extracts of the vesicles, and to identify and identify cDNA genes encoding thrombolytic enzymes of these antigens. Patent application has been filed (see Korean Patent Application No. 95-67279).

The present inventors continued to study the activity of purely isolated and purified lombrokinase by the method as described above, and as a result, it was confirmed that rombrokinase exhibited an angiomustic relaxation effect not known at all until now. This invention was completed.

FIG. 1 is a graph showing the fibrolytic activity fractions FI, FII, and FIII obtained from anion exchange chromatography using DEAE-Toyopearl 650 column on crude extracts of globules. (Absorbance at 280 nm), ○-○ fibrin degradation activity (mm 2)].

FIG. 2 is a graph showing the rombrokinase active fractions F1 and F2 obtained by performing hydrophobic chromatography on the fibrin decomposing activity fraction FI using a phenyl-Toyopearl column. ), ○-○ fibrin degrading activity (mm 2)].

FIG. 3 is a graph showing the rombrokinase activity fraction F3 obtained by performing hydrophobic chromatography on a fibrin decomposing activity fraction FII using a phenyl-Toyopearl column [●-● protein (absorbance at 280 nm), ○-○ fibrin degradation activity (mm 2)].

FIG. 4 shows the hydrobromine activity fraction F4 obtained from the hydrophobic chromatography using the phenyl-Toyopearl column and the affinity chromatography using the benzamidine Sepharose 6B column for the fibrin digestion activity fraction FIII. , F5 and F6 [?-? Protein (absorbance at 280 nm),?-? Fibrin degrading activity (mm 2)].

FIG. 5 shows the results of electrophoresis of 10% SDS polyacrylamide gels on the lobbrokinase fractions F1, F2, F3, F4, F5 and F6 of gut isolated according to Example 2 (lane 1: molecular weight labeling protein, Lane 2: fraction F1, lane 3: fraction F2, lane 4: fraction F3, lane 5: fraction F4, lane 6: fraction F5, lane 7: fraction F6).

FIG. 6 is a graph showing the relaxation effects of lombrokinase active fractions F1, F2, F3, F4, F5 and F6 on the thoracic aorta and the mesenteric artery extracted from rats [(1): thoracic aorta, (2): Mesenteric artery, □: 0.1 µg / ml, ▨: 1 µg / ml, ■: 10 µg / ml].

FIG. 7 is a graph showing the relaxation effects of lombrokinase active fractions F1, F2, F3, F4, F5, and F6 on thoracic aorta and mesenteric artery extracted from rabbits [(1): thoracic aorta, (2): mesentery Artery, □: 0.1 µg / ml, ▨: 1 µg / ml, ■: 10 µg / ml].

FIG. 8 is a graph showing the relaxation effect of the rombrokinase active fraction F1 on the cerebral pericardium of rats.

FIG. 9 is a graph showing the relaxation effect of lombrokinase active fraction F2 on the cerebral pericardium of rats.

FIG. 10 is a graph showing changes in cerebral local blood flow due to femoral vein administration of rombrokinase active fraction F1 [A: response with time after F1 administration,-○-: local cerebral blood flow (%),-●- : Average arterial pressure (%), B: Maximum response after F1 administration, □: Average arterial pressure (%), ▧: Local cerebral blood flow (%), ** P <0.01 for baseline.

FIG. 11 is a graph showing changes in cerebral local blood flow due to femoral vein administration of rombrokinase active fraction F2 [A: response with time after F2 administration,-○-: local cerebral blood flow (%),-●- : Average arterial pressure (%), B: Maximum response after F2 administration, □: Average arterial pressure (%), ▧: Local cerebral blood flow (%), ** P <0.01 for baseline.

FIG. 12 is a graph showing changes in cerebral local blood flow due to local administration of lombarkinase active fraction F1 to the brain surface. [A: Response over time after F1 administration,-○-: Local cerebral blood flow (%),-● -: Average arterial pressure (%), B: Maximum response after F1 administration, □: Average arterial pressure (%), ▧: Local cerebral blood flow (%), ** P <0.01 for baseline.

FIG. 13 is a graph showing fluctuations in cerebral local blood flow due to topical administration of lombarkinase active fraction F2 to the brain surface [A: Response over time after F2 administration,-○-: Local cerebral blood flow (%),-● -: Average arterial pressure (%), B: Maximum response after F2 administration, □: Average arterial pressure (%), ▧: Local cerebral blood flow (%), ** P <0.01 for baseline.

Accordingly, the present invention relates to a vasomotor relaxant composition containing lombrokinase isolated from globules as an active ingredient.

More specifically, the present invention is purely separated from the crude extract of the gut through a series of separation, purification process consisting of a combination of methods such as anion exchange column chromatography, hydrophobic chromatography, gel chromatography and affinity chromatography A vascular muscle relaxant composition comprising a brokinase fraction as an active ingredient.

The rombrokinase fraction of globules used as active ingredient in the vascular muscle relaxant composition of the present invention is preferably obtained by a method similar to the method described in Korean Patent Application No. 95-67279, the applicant of the present application, The specific method is as follows. That is, the supernatant was decomposed by a conventional method, centrifuged to separate the supernatant, treated with ammonium sulfate, centrifuged to separate the precipitate, dissolved in phosphate buffer, desalted using a ultrafiltration, concentrated and frozen. After drying to obtain the crude extract of rombrokinase, the crude extract of the obtained globules was subjected to anion exchange column chromatography to separate the three fractions FI, FII and FIII, and these fractions were respectively subjected to hydrophobic chromatography and gel chromatography. And affinity chromatography to separate F1 and F2 fractions from FII, fraction F3 from FII, and fractions F4, F5, and F6 from fraction FIII, respectively, and fractions F1, F2, F3, F4, F5. And F6 can be obtained.

In the method for obtaining such a lombrokinase fraction, anion exchange column chromatography is performed using a resin such as DEAE-Toyopearl, Q-Sepharose, and the like, and hydrophobic chromatography is, for example, phenyl-. It is performed using Toyopearl, gel chromatography is generally performed using Sephacryl S-200, and affinity chromatography can generally be performed using benzamidine sepharose.

Purified lombrokinase fractions F1, F2, F3, F4, F5 and F6 obtained by this method were characterized by thoracic aorta, mesenteric aorta, carotid artery, renal artery, ileal artery, femoral artery, as evidenced by the experimental examples below. It exhibits an excellent vascular muscle relaxation effect on the muscles of various blood vessels, such as the ductal artery, and thus can be usefully used as a vascular muscle relaxant.

In the present invention, the total daily dose to be administered to a patient in a single dose or in separate doses when administering the vascular muscle relaxant composition for clinical purposes is preferably in the range of 0.2 mg to 20 mg per 1 kg of body weight, but is specific for a particular patient. The level may be appropriately determined according to the type of lombrokinase activity fraction to be used, the weight of the individual patient, sex, health condition, diet, time of administration, method of administration, rate of excretion and the severity of the disease.

The rombrokinase active fractions of the present invention may be formulated in oral formulations, but are preferably formulated in injectable formulations, particularly intravenous formulations.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be prepared using suitable dispersing agents, wetting agents, or suspending agents according to known techniques. Pharmaceutically acceptable solvents that can be used include water, Ringer's solution and isotonic NaCl solution and sterile fixed oils are conventionally employed as a solvent or suspending medium. Any non-irritating fixed oil may be used for this purpose, including mono-, diglycerides, and also fatty acids such as oleic acid are used in the preparation of injectables.

The present invention is explained in more detail by the following examples and experimental examples, but the scope of the present invention is not limited in any way by these.

Example 1

[Production of Job Crude Extracts]

5 kg of fresh bulbs were pulverized with a homomixer and the same amount of 10 mM sodium phosphate buffer solution (pH 7.4) was added and extracted at 45 ° C. for 4 hours, followed by centrifugation at 3500 rpm for 1 hour to obtain a supernatant. . Ammonium sulfate (176 g / L) was added to this supernatant to 30%, and after stirring at 4 ° C. for 6 hours or more, the supernatant was recovered by centrifugation at 4500 rpm for 30 minutes. Ammonium sulfate (195 g / L) was dissolved in the supernatant at a concentration of 60%, stirred for at least 6 hours, and then precipitated, followed by centrifugation. The supernatant was decanted and the precipitate was dissolved in 10 l of 20 mM sodium phosphate buffer (pH 7.4), filtered through a 0.45 μm filter membrane, the filtrate was desalted using an ultrafiltration device having a molecular weight of 10,000, concentrated and lyophilized to ammonium sulfate. As a fraction, 20 g of lombarkinase crude extract was obtained.

Example 2

[Isolation and Purification of Lombrokinase Fraction]

Anion Exchange Column Chromatography: Membrane filter 20 g of the crude extract obtained in Example 1 was packed into a column (5.0 × 35 cm) filled with DEAE-Toyopearl 650 resin and pre-equilibrated with 20 mM sodium phosphate buffer (pH 7.4). After filtration and washing with 20 mM sodium phosphate buffer (pH 7.4), the elution protein was eluted at a rate of 2.0 ml / min by elution in a linear gradient using 0-0.5 M NaCl. The enzymatic activity of each of the eluted fractions was measured by Fibrin plate assay (Haemostasis, 13, 301-315, 1983) to obtain three fractions showing fibrin cleavage titers, and the FI, FII and FIII fractions, respectively. (See FIG. 1).

Treatment of Fraction FI (Isolation and Purification of Fractions F1 and F2): The fraction FI separated above was subjected to hydrophobic chromatography using a phenyl-Toyopearl column (2.0 × 15 cm), washed with 1M ammonium sulfate, and then sulfuric acid. Two peaks showing fibrin cleavage titers were separated during elution (1.5 mL / min) through ammonium descent (1 M-OM) (see FIG. 2). This was treated by gel chromatography using Sephacryl S-200 and affinity column chromatography using benzamidine Sepharose 6B to separate the purely purified fractions F1 and F2.

Treatment of Fraction FII (Isolation and Purification of Fraction F3): The fraction FII separated above was subjected to hydrophobic chromatography using a phenyl-toyopearl column (2.0 × 15 cm), washed with 1M ammonium sulfate, and then lowered to ammonium sulfate. During elution (1.5 mL / min) via gradient (1M-OM), peaks showing fibrin degradation titers were separated (see FIG. 3). This was treated by gel chromatography using Sephacryl S-200 and affinity column chromatography using benzamidine Sepharose 6B to separate the purely purified fraction F3.

Treatment of Fraction FIII (Isolation and Purification of Fractions F4, F5, and F6): The fraction FIII separated above was subjected to hydrophobic chromatography using a phenyl-toyopearl column, followed by a benzamidine Sepharose 6B column (2.0). Affinity column chromatography using x25 cm), washed with 20 mM sodium phosphate buffer (pH 7.4), washed with 0.1 M acetic acid buffer (pH 5.0), and eluted with an arginine solution concentration gradient (OM-1M). Three active peaks were separated in the course of (1.2 ml / min) (see Figure 4). This was processed by gel chromatography using phenyl-Toyopearl column chromatography and Sephacryl S-200 to separate the purely purified fractions F4, F5 and F6.

Example 3

[Measurement of Molecular Weight of Lombrokinase Fraction]

The molecular weight of each of the lombrokinase fractions F1, F2, F3, F4, F5 and F6 obtained in Example 2 was determined by Laemmli method [Laemmli, U, K., (1970) Nature, 227: 680]. According to the SDS-polyacrylamide gel electrophoresis was measured. 12% acrylamide was used for this electrophoresis, and after electrophoresis, stained with 0.05% Coomassie briliant blue R-250 and decolorized with a mixed solution of 10% methanol and 10% acetic acid to analyze the band. As a result, it was found that the positions of the bands were almost similar, so that there was almost no difference in molecular weight (see Fig. 5). From the results in FIG. 5, the respective molecular weights were estimated to be about 24,000 Da in F1, 25,000 Da in F2, 30,000 Da in F3, 24,500 Da in F4, 35,000 Da in F5, and 36,000 Da in F6.

Experimental Example 1

[Method of Vascular Muscle Relaxation Test (In Vitro Test) of Lombrokinase]

Relaxation of lobbrokinase activity fractions F1, F2, F3, F4, F5, and F6 on vascular smooth muscle of the isolates according to the present invention was measured by the following method.

In this test, the rabbits (2.5-3 ㎏) and rats (Sprague-Dawley rat, 250-300 g) were used without any gender discrimination. Thoracic aorta (TA) and mesenteric artery (MA) The effects of carotid artery (CA), renal artery (RA), ileal artery (IA) and femoral artery (FA) on vascular smooth muscle were measured. After each blood vessel was removed, the connective tissue and fat around the vascular muscle were removed under a dissecting microscope and cut into 2-3 mm lengths. Each of these fine vessels was immersed in a saline solution at 37 ° C. saturated with 95% O ₂ -5% CO ₂ , connected to a force-displacement transducer, and then muscled with saline solution. It was fixed indoors and the equilibrium tension was maintained for 90 minutes at 0.5-2g depending on the tissue. After causing muscle contraction with phenylephrine (0.3-3 μM in rats, 0.3-3 mM in rabbits), when the muscle contraction is maintained at a constant height, the lomobrokinase fractions F1, F2, F3, F4 , F5 and F6 were administered every 3 minutes so that the final accumulated concentration in the organ bath was 0.1, 1 or 10 μg / ml. The dose-dependent curve for the test drug in each experiment was generated by repeating the test at 60 minute intervals.

[result]

The relaxation effects of the rombrokinase fractions F1, F2, F3, F4, F5, and F6 on the thoracic aorta and mesenteric artery extracted from rats and rabbits are shown graphically in FIG. 6 and FIG. In addition, the relaxation effects of the representative rombrokinase active fractions F1 and F2 on various arterial vascular muscle tissues extracted from rats and rabbits are shown separately in Tables 1 and 2, respectively. As can be seen from the results shown in Table 1, when tissues were contracted with phenylephrine and F1 and F2 were administered at concentrations of 0.1, 1, and 10 µg / ml, respectively, the concentrations of the tissues were different from each other, but dose-dependent. Phosphorus relaxation was shown. In addition, as can be seen from the results shown in Table 2, the Lombrokinase activity fractions F1 and F2 showed a slight relaxation degree, but showed an excellent relaxation effect as in the rat in all the vascular muscles tested. Therefore, it can be seen that the rombrokinase active fractions F1 and F2 exhibit excellent vascular muscle relaxation effects regardless of species. The effects of rombrokinase fractions F1 and F2 on the relaxation of vascular muscle in rats and rabbits are summarized in Table 3 below. From these, the vascular muscle relaxation effects of F1 and F2 are more effective than conduit vessels such as the aorta. The renal arteries, such as renal artery (RA), were found to be larger in resistant angioplasty.

As can be seen from the results described in the above table, the lobbrokinase active fractions isolated according to the present invention are excellent in vascular relaxation regardless of species, and thus, may be usefully used as vascular muscle relaxants.

Experimental Example 2

[Relaxation test (in vivo experiment) method of lobe kinase fraction to cerebral pericardium artery]

Rats (Sprague-Dawley rat, 250-300 g) were anesthetized with urethane (1 g / 1 kg, intraperitoneal) and fixed to an animal rack equipped with a heating pad (hearting pad) to maintain a constant body temperature. After tracheostomy, ventilation was performed by connecting to a respirator (Harvard, Model 683). A blood pressure was measured by inserting a PE50 polyethylene tube into the left femoral artery and connecting it to a Statham P23D pressure transducer. A polyethylene tube was inserted into the left carotid artery to allow the arterial blood to be bled or the blood was reinfused. Meanwhile, arterial blood was collected through the left carotid artery before and after installing the two intestines, and gas and pH were measured (NOVA Biomedicals, STAT profile 3).

The mean values of blood gas and pH during the experiment were as follows: pH 7.38 ± 0.04; Paco ₂ 33.3 ± 2.8 mmHg; Pao ₂ 101.3 ± 5.1 mmHg. Anal body temperature was measured continuously and kept constant (37 ± 0.05 ° C.).

Cerebral periosteal artery is described by Hong et al. [Amer. J. Physiol. 266, H11-H16, 1994], two cranial windows were installed. The specific experimental method is briefly described as follows. The rat is fixed in the stereotactic apparatus (Storelting) in the prone position, and the cranial window is cut in a square (5 × 5 mm) in the head of the head (mainly on the right side). ) The incision was carefully made and the brain area was covered with warm mineral oil during surgery. A wall of bone wax was made around the two openings, and an inlet and an outlet were placed thereon, and the upper lid was transparent so that the perfusion solution was fixed by the rebase so that the perfusion solution did not leak when the nutrient solution was perfused into the two openings. The cerebral blood vessels were equilibrated for 60 minutes after the two openings were installed. Two cranial infusions were perfused with mock cerebrospinal fluid (CFS) at a rate of 0.3 ml / min. The outer diameter of the cerebral pericardium was selected to be 2-30㎛, and the image was captured using a CCD video camera (Sanyo, VDC 3900) connected to a stereo microscope (Nikon, SMZ-2T) installed above the two windows. It could be grabbed and viewed directly on a television monitor. On the other hand, from the 480x magnified image, the cerebrovascular diameter was automatically measured using a butterfly analyzer (Width Analyzer, C3161, Hamamatsu). The motion of the vessel diameter was shown on the Polygraph (grass) through the analog output. The results were also stored in a video cassette recorder VT-S 730, Hitachi. The pressure in the cranial window was kept constant at 5-6 mmHg during the experiment by adjusting the height of one end of the plastic tube connected to the exit of the window. In this experiment, the lombrokinase active fractions F1 and F2 were topically administered through two incisions at 5 minute intervals at doses of 0.1, 1 and 10 μg / ml, respectively, after the cerebrovascular equilibrium was maintained. Cerebral periosteal artery relaxation was observed for 5 minutes at 0.2 minute intervals after the active fraction was administered. Indicated. The composition of the cerebrospinal fluid used in this experiment is as follows: 125 mM NaCl, 3.5 mM KCl, 1.3 mM CaCl ₂ , 1.1 mM MgCl ₂ and 25 mM NaHCO ₃ ).

[result]

The relaxation effect of the periosteal artery by administration of the lobrokinase active fractions F1 and F2 is shown in FIGS. 8 and 9 and Table 4, respectively. From these results, no expansion of the cerebral pericardium was observed when the active fraction F1 was administered at a dose of 0.1 μg / ml, but a consistent expansion of the pericardial artery was observed when the dose of 1 μg / ml was administered. The vasodilator effect lasted 2-3 minutes, and the vasodilator response lasted more than 5 minutes at 10 µg / ml. As can be seen from the results shown in Table 4, the maximum vasodilator effect of the active fraction F1 was increased by 36.6% at 1.6 minutes when administered at a dose of 1 µg / ml, and when administered at 10 µg / ml. Maximal at 4 minutes after administration, an increase of 62.5%.

Vasodilation was also observed by administration of 1 μg / ml and 10 μg / ml, and the vasodilation response was transient compared to F1, but the vasodilation effect was more pronounced. 9 degrees).

In other words, when the active fraction F2 was administered at a dose of 1 μg / ml, it increased more than 60% at 0.4-0.6 minute after topical administration and at least 90% at 1 minute when administered 10 μg / ml.

From these results, it can be seen that the lombrokinase active fractions F1 and F2 extracted from the gut directly cause vasodilation to cerebrovascular vessels.

Experimental Example 3

[In-vivo Experimental Methods for Cerebral Local Blood Flow]

Cerebral blood flow was measured as described below by laser-Doppler flowmetry.

The experimental animal was fixed in a stereotactic frame and the scalp was cut along the midline to expose the parietal bone, followed by a 4-6 mm flank of the bregma and a 5-6 mm diameter craniotomy in the -2-1 mm front. ). At this time, the thickness of the skull was left thin to prevent epidural bleeding. Stereotactic microscopy (needle probe; 0.8mm diameter) for laser-Doppler flowmeter (Transonic Instrument, U, S, A.) is perpendicular to the cerebral (parietal) cortex surface Stereotactic micromanipulators were used to carefully approach the periosteum artery. After stabilization for a period of time, the lobbrokinase activity fractions F1 and F2 isolated from the intestine were intravenously injected into the femoral vein at doses of 0.1, 1 and 10 µg / kg, respectively, or 0.1, 1 and 10 µg / Brain doses were measured according to the experimental protocol by topical administration at a dose of ml. The fluctuation of the measured cerebrovascular flow was expressed as 100% before administration of the testosterone extract active substance. All experimental values were expressed as the standard error of the mean ± mean, and the significance between each group was analyzed by the Student's t-test method to determine that the P value was less than 0.05.

[result ]

The results of measurement of fluctuation of cerebral blood flow by intravenous injection and topical administration to the brain surface of the lobe kinase fractions F1 and F2 in anesthetized rats were measured in FIGS. 10, 11, 12, and 13 It is shown in FIG. And compared with Table 5 it was shown. As shown in FIG. 10, regional cerebral blood flow (rCBF) increased up to 50% at 0.1, 1 and 10 µg / ml by intravenous administration of fraction F1, and this increase persisted for 30 minutes. . However, mean arterial pressue (MAP) was not affected by intravenous administration of fraction F1.

Dose-induced increase in dose-dependent cerebral blood flow was observed by intravenous injection of 0.1, 1, 10 µg / ml, and the increase in cerebral blood flow increased by up to 70%. (See Figure 11).

In addition, as shown in FIG. 12, the local administration (0.1, 1, 10 µg / ml) of the lombrokinase active fraction F1 to the surface of the brain membrane caused a dose-dependent increase in cerebral blood flow without a change in mean arterial pressure. Was observed and this increase was shown to be short aging. On the other hand, when the local administration effect of the lombrokinase active fraction F2 on the surface of the brain was observed, it did not change the brain blood flow at the administration of 0.1 and 1 ㎍ / ml, but at the baseline (10 ㎍ / ml). increased by more than 100% of the baseline) and the increase lasted more than 20 minutes (Figure 13).

From these results, it can be seen that F1 and F2, which have a small amount of rombrokinase active fractions, have an effect of increasing cerebral blood flow in a range not causing fluctuations in peripheral blood pressure. Therefore, it is expected to be used as an agent for improving cerebral blood flow disorder in cerebral blood flow disorders (such as cerebral infarction after stroke).

Claims (5)

Vascular muscle relaxant composition containing the active fraction of rombrokinase. The lobbrokinase fractions F1 and F2 according to claim 1, wherein the active fraction of lobbrokinase of the gut is separated from the crude extract of the gut by a combination of anion exchange column chromatography, hydrophobic chromatography, gel chromatography and affinity chromatography. , At least one of F3, F4, F5, and F6. 3. The vascular muscle relaxant composition according to claim 2, wherein the active fraction of lobbrokinase of globular is Lombrokinase fraction F1 or F2 of globular. The vascular muscle relaxant composition of claim 1 formulated as an injectable preparation using a pharmaceutically acceptable carrier. The vascular muscle relaxant composition according to claim 4, wherein the injection is an intravenous preparation.

Images