KR19990025952A - Vascular relaxant composition containing Lambert brookinase - Google Patents

Abstract

The present invention relates to a vasodilator composition containing a Lumbrokuinase which is a proteolytic enzyme extracted from Lumbricus rubellus . More specifically, the present invention relates to a vasoconstrictor composition containing, as an active ingredient, a Lumbrokinase active fraction that is separated from a subject through a series of processes.

Description

Vascular relaxant composition containing Lambert brookinase

The present invention relates to a vasodilator composition comprising, as an active ingredient, Lumbu- kinase, which is a proteolytic enzyme isolated from Lumbricus rubellus . More specifically, the present invention relates to a vasodilator composition comprising a room brominase active fraction that has been purified from a subject through a series of processes.

Stroke is a type of aphasia, which causes stenosis or obstruction of the cerebral artery by atherosclerosis or thrombosis resulting in cerebral damage caused by blockage of cerebral blood flow, causing hemiplegia or speech disorder, and even leading to death. Such a stroke and other vascular diseases have been on the rise in modern times and one of the greatest signs is that modern medicine needs to be solved. To date, urokinase, ticlopidine, r-tPA (recombinant-tissue plasminogen activator), alprostadil, and streptokinase have been commonly used for the treatment of stroke. There is no satisfactory treatment yet.

Therefore, the present inventors conducted extensive studies to find out effective drugs for the treatment of stroke, and used it as a subject. It has been prescribed as a treatment for geriatric diseases such as arteriosclerosis, hypertension and diabetes in traditional oriental medicine. Especially in the Pharmacopoeia of the People's Republic of China (1977 edition, Part I, pp. 197-188) Lumbricus Kwang tungesis and Lumbricus nativus are two types of drugs which are used as antipyretics, antipyretics, antipyretics, joint treatments, urinarys, bronchial asthma treatments and hypertension treatments. It is described as being effective. It is known that the effect of such a job is due to the action of proteolytic enzyme called lumbrokinase contained in the job. In particular, Lumbrokuinase, which is a candidate, has activity of inhibiting fibrin formation and inhibiting platelet aggregation as well as fibrinolytic activity compared to conventional thrombolytic enzymes. In contrast to the in vitro test, the activity in fibrinogen is very low in vivo, And it is also reported to be effective against hyperlipemia and cerebral thrombosis.

The applicant of the present invention intensively conducted researches on this type of robu- kinase to find out the method for purely isolating and purifying robu- kinase from the crude extract of rosacea and identifying the cDNA gene encoding the thrombolytic enzyme (Korean Patent Application No. 95-67279).

The present inventors have continuously studied the activity of purely purified and purified Roombrokinase by the above-mentioned method. As a result, it has been confirmed that Roombrokinase exhibits a vasodilatory relaxation effect, Thereby completing the present invention.

FIG. 1 is a graph showing fibrinolytic activity fractions FI, FII and FIII obtained as a result of performing anion exchange chromatography using a DEAE-Toyopearl 650 column for crude extracts of human beings. Absorbance at 280 nm), ○ - ○ fibrin degrading activity (㎟)].

Fig. 2 is a graph showing the room brominase active fractions F1 and F2 obtained as a result of hydrophobic chromatography using a phenyl-toeo pearl column against fibrin degrading active fraction FI [● - ● Absorbance at 280 nm ), ○ - ○ fibrin degradation activity (㎟)].

Fig. 3 is a graph showing the room bromokinase active fraction F3 obtained as a result of performing hydrophobic chromatography using a phenyl-toyoferal column against the fibrin degrading activity fraction FII [● - ● protein (absorbance at 280 nm) ○ - ○ Fibrin degradation activity (㎟).

FIG. 4 is a graph showing the results of affinity chromatography performed on a fibrin degrading active fraction FIII using a phenyl-toyoferal column and affinity chromatography using a benzamidine sepharose 6B column to obtain a room bromokinase active fraction F4 , F5 and F6 [● - ● Protein (absorbance at 280 nm), ○ - ○ Fibrin degrading activity (㎟)].

5 shows the results of 10% SDS polyacrylamide gel electrophoresis of the Rum brokinase fractions F1, F2, F3, F4, F5, and F6 of Example 2 isolated according to Example 2 (lane 1: 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 the robe brookinase active fractions F1, F2, F3, F4, F5 and F6 on the thoracic aorta and mesenteric artery extracted from the rat (1): thoracic aorta, (2) Mesenteric artery,?: 0.1 占 퐂 / ml,?: 1 占 퐂 / ml,?: 10 占 퐂 / ml).

FIG. 7 is a graph showing the relaxation effects of the robe brookinase active fractions F1, F2, F3, F4, F5 and F6 on the thoracic aorta and mesenteric artery extracted from rabbit. [(1): thoracic aorta, (2) Mesenteric artery,?: 0.1 占 퐂 / ml,?: 1 占 퐂 / ml,?: 10 占 퐂 / ml).

Fig. 8 is a graph showing the relaxation effect of the room bromokinase active fraction F1 on the brain flap artery of rats. Fig.

FIG. 9 is a graph showing the relaxation effect of the room bromokinase active fraction F2 on the brainstem artery of the rat. FIG.

FIG. 10 is a graph showing changes in brain local blood flow due to intravenous femoral vein administration of Rum brokinase active fraction F1. [A: Reaction over time after administration of F1, - - -: local cerebral blood flow (% : Mean arterial pressure (%), B: peak response after F1 administration, □: mean arterial pressure (%), ▧: local cerebral blood flow (%), ** P0.01 for baseline.

FIG. 11 is a graph showing changes in brain local blood flow by the intravenous femoral vein administration of Rum brokinase active fraction F2. [A: Reaction over time after administration of F2, - - -: local cerebral blood flow (% : Mean arterial pressure (%), B: highest response after F2 administration,:: mean arterial pressure (%), ▧: local cerebral blood flow (%), ** P0.01 for basal value.

FIG. 12 is a graph showing changes in brain local blood flow due to topical administration of the Ruminal Brokinase active fraction F1 on the brain surface [A: reaction with time elapsed after F1 administration, - - -: local cerebral blood flow (%), -: mean arterial pressure (%), B: maximum response after F1 administration, □: mean arterial pressure (%), ▧: local cerebral blood flow (%), ** P0.01 for baseline.

FIG. 13 is a graph showing changes in brain local blood flow due to topical administration of the active ingredient Fragment II of Rum Virokinae [A: reaction over time after administration of F2, - - -: local cerebral blood flow (%), - -: mean arterial pressure (%), B: peak response after F2 administration, □: mean arterial pressure (%), ▧: local cerebral blood flow (%), ** P0.01 for baseline.

Accordingly, the present invention relates to a vasodilator composition comprising as an active ingredient a rovatory kinase isolated from a subject.

More specifically, the present invention relates to a method for purifying a pure, purified, purified or purified compound from a crude extract of a human body by a series of separation and purification processes, which comprises a combination of methods such as anion exchange column chromatography, hydrophobic chromatography, gel chromatography and affinity chromatography, The present invention relates to a vasodilator composition containing as an active ingredient a room-borne kinase fraction.

The Rumen brookinase fraction used as an active ingredient in the vasodilator composition of the present invention can be preferably obtained by a method similar to that described in Korean Patent Application No. 95-67279, The concrete method is as follows. That is, the solution was digested by a conventional method and centrifuged to separate the supernatant, followed by treatment with ammonium sulfate, centrifugation to separate the precipitate, dissolution in phosphate buffer, desalting using an ultrafilter, Dried to obtain a crude extract of Lambert brokinase, and then the crude extract of the obtained extract was subjected to anion exchange column chromatography to separate three fractions FI, FII and FIII, and these fractions were subjected to hydrophobic chromatography, gel chromatography F2, F3, F4, F2, F3, F4, F5, F5 and F6 from fractions F1, F2 and F3, respectively, F5 and F6 can be obtained.

In the method for obtaining such a robu- kinase fraction, anion exchange column chromatography is carried out using a resin such as DEAE-Toyopearl, Q-Sepharose, etc., and hydrophobic chromatography is carried out by, for example, The gel chromatography is generally carried out using Sephacryl S-200, and the affinity chromatography can be generally carried out using benzamidine sepharose.

The purified Lumbrokinase fractions F1, F2, F3, F4, F5, and F6 obtained by this method can be used for the treatment of chest aorta, mesenteric aorta, carotid artery, renal artery, ileal artery, femoral artery, It has excellent vasodial relaxation effect on muscles of various blood vessels such as cerebral palsy artery and the like, and thus can be usefully used as a vascular muscle relaxant.

The total daily dose to be administered to a patient in a single dose or in separate doses upon administration of the angiostatic relaxant composition of the present invention for clinical purposes is preferably in the range of 0.2 mg to 20 mg per kg of body weight, The level can be appropriately determined depending on the kind of the room bromokinase active fraction to be used, the body weight, sex, health condition, diet, administration time, administration method, excretion rate and severity of the disease.

The Lumbroku kinase active fractions of the present invention may be formulated into oral preparations and administered, but it is preferable to formulate them into injectable preparations, especially intravenous preparations.

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

The present invention will be described more specifically by the following examples and experimental examples, but the scope of the present invention is not limited in any way by them.

Example 1: Preparation of goat's crude extract

5 kg of the product was pulverized with a homomixer, followed by addition of the same amount of 10 mM sodium phosphate buffer solution (pH 7.4), followed by extraction 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 the supernatant in an amount of 30%, stirred at 4 ° C for 6 hours or more, and centrifuged at 4500 rpm for 30 minutes to recover only the supernatant. Ammonium sulfate (195 g / l) was dissolved in the supernatant at a concentration of 60%, and the mixture was further stirred for 6 hours or longer, followed by centrifugation. The supernatant was tilted and the precipitate was dissolved in 10 L of a 20 mM sodium phosphate buffer solution (pH 7.4) and filtered through a 0.45 μm filter membrane. The filtrate was desalted using a supernatant of molecular weight 10,000 and concentrated. The extract was lyophilized to give ammonium sulfate 20 g of a room bromokinase crude extract was obtained as a fraction.

Example 2: Isolation and Purification of the Room Brookinase Fraction

Anion Exchange Column Chromatography: To a column (5.0 x 35 cm) filled with DEAE-Toyopearl 650 resin and previously equilibrated with 20 mM sodium phosphate buffer (pH 7.4), 20 g of the crude extract obtained in Example 1 was added to a membrane filter , Followed by washing with 20 mM sodium phosphate buffer (pH 7.4), followed by sequential elution with a linear gradient using 0-0.5M NaCl. The adsorbed protein was eluted at a rate of 2.0 ml / min. The enzymatic activity of the eluted fractions was measured by Fibrin plate assay (Haemostasis, 13, 301-315, 1983) to obtain three fractions representing the fibrin cleavage site, and each fraction was analyzed by FI, FII and FIII fractions (See Fig. 1).

Treatment of Fraction FI (Separation and Purification of Fractions F1 and F2): The fraction FI thus isolated was subjected to hydrophobic chromatography using a phenyl-tosopearl column (2.0 x 15 cm), washed with 1 M ammonium sulfate, Two peaks representing the fibrin cleavage site were isolated during the elution with an ammonium descending gradient (1 M-0 M) (1.5 ml / min) (see FIG. 2). This was treated by gel chromatography using Sephacryl S-200 and affinity column chromatography using benzamidine sepharose 6B, respectively, to separate purely purified fractions F1 and F2.

Treatment of fraction FII (separation and purification of fraction F3): The fraction FII isolated above was subjected to hydrophobic chromatography using a phenyl-tosopearl column (2.0 x 15 cm), washed with 1 M ammonium sulfate, Peak indicating the fibrin cleavage site was isolated (see Fig. 3) by elution with a gradient (1M-0M) (1.5 ml / min), and this was separated by gel chromatography using Sephacryl S-200 and benzamidine And purified by affinity column chromatography using OZ 6B to isolate pure F3 fraction.

Treatment of fraction FIII (separation and purification of fractions F4, F5 and F6): The fraction FIII isolated above was subjected to hydrophobic chromatography using a phenyl-toeo Pearl column and then to a benzamidine sepharose 6B column (2.0 × 25 cm), washed with 20 mM sodium phosphate buffer solution (pH 7.4), washed with 0.1 M acetic acid buffer solution (pH 5.0), eluted with an arginine solution concentration gradient (0 M-1 M) (1.2 ml / min), three active peaks were isolated (see Fig. 4). This was treated respectively by phenyl-tosopearl column chromatography and gel chromatography using Sephacryl S-200 to separate the purely purified fractions F4, F5 and F6.

Example 3: Molecular weight determination of the room bromokinase fraction

The molecular weights of each of the Lumbroku kinase fractions F1, 2, F3, F4, F5 and F6 obtained in Example 2 were measured according to the Laemmli method (Laemmli, UK, (1970) Nature, 227: 680) - polyacrylamide gel electrophoresis. The electrophoresis was carried out using 12% acrylamide. After electrophoresis, the cells were stained with 0.05% Coomassie briliant blue R-250 and decolorized with a mixed solution of 10% methanol and 10% acetic acid. As a result, it was found that there was almost no difference in molecular weight because the positions of the bands were almost the same (see FIG. 5). From the results shown in Fig. 5, it was estimated that the molecular weights of F1, F2, F3, F5 and F6 were about 24,000 Da, 25,000 Da, 30,000 Da, 24,500 Da, 35,000 Da, 36,000 Da, respectively.

Experimental Example 1: Vasodilatory relaxation test of the room bromokinase (in vitro test)

Way :

The relaxin action of vascular smooth muscle of Rum brokinase active fractions F1, F2, F3, F4, F5 and F6 isolated according to the present invention was measured by the following method.

In this study, rabbits (2.5-3 kg) and Sprague-Dawley rats (250-300 g) were used as test animals without distinction of sex and thoracic aorta (TA), superior mesemtric artery (MA) The effect of vascular smooth muscle on carotid artery (CA), renal artery (RA), ileal artery (IA), and femoral artery (FA) After each blood vessel was extracted, the connective tissues and fat around the blood vessel muscle were removed under a dissecting microscope, and the blood vessel was cut to a length of 2-3 mm. Each of these three vascular rings was immersed in physiological saline solution saturated with 95% O ₂ -5% CO ₂ at 37 ° C, connected to a force-displacement transducer, and then placed in a physiological saline solution And the stabilization tension was maintained at 0.5-2 g according to the tissue for 90 minutes. F2, F3 and F4 according to the present invention when the muscle contraction is maintained at a constant height after causing muscle contraction with phenylephrine (0.3-3 μM in the case of the rat and 0.3-3 mM in the rabbit) , F5 and F6 were administered at intervals of 3 minutes, respectively, so that the final accumulation concentration in the organ bath was 0.1, 1 or 10 占 퐂 / ml. The dose-dependence curve for the test drug in each experiment was generated by repeating the test at 60-minute intervals.

result :

The relaxation effects of the Rum brochinase active fractions F1, F2, F3, F4, F5, and F6 on the thoracic aorta and mesenteric artery from rat and rabbit were graphically shown in FIGS. 6 and 7, respectively. In addition, the relaxation effects of representative robu- kinase active fractions F1 and F2 on various arterial vascular muscle tissues extracted from the rat and rabbit are separately shown in Tables 1 and 2, respectively. As can be seen from the results shown in Table 1, when the blood vessels were contracted with phenylephrine, when F1 and F2 were administered at the concentrations of 0.1, 1, and 10 μg / ml, Dependent relaxation. In addition, as can be seen from the results shown in Table 2, the active fractions of Rum brokinase F1 and F2 showed slight degree of difference but showed excellent relaxation effect in all of the tested vascular muscles as in the rat. Therefore, it can be seen that the Rum Virokinae active fractions F1 and F2 exhibit excellent vasodilator relaxation effect regardless of the species. The effects of the room-borne kinase active fractions F1 and F2 on the vasomotor relaxation of rats and rabbits are summarized in Table 3 below. From these results, the relaxation effects of F1 and F2 on the vasomotor muscles were observed in conduit vessels such as the aorta, (RA), which is smaller than the diameter of the renal artery (RA).

Relaxation of F1 and F2 in rat vasculature F1, 占 퐂 / ml F2, 占 퐂 / ml 0.1 One 10 0.1 One 10 TA (6) 5.90 + - 3.22 38.97 + - 7.85 81.80 ± 4.02 3.67 ± 2.33 36.13 + - 11.42 74.47 ± 9.19 MA (6) 5.35 ± 3.39 39.08 ± 5.86 83.70 +/- 7.50 5.35 ± 3.39 39.08 ± 5.86 83.70 +/- 7.50 CA (3) 2.23 ± 2.23 35.43 + - 3.40 71.23 + - 5.13 4.73 ± 2.47 28.00 ± 8.26 72.37 + - 7.19 RA (3) 0.00 ± 0.00 14.77 + - 8.66 61.17 ± 9.13 3.33 ± 3.33 18.90 ± 1.10 68.33 + - 10.14 IA (5) 2.00 ± 2.00 8.12 ± 3.37 40.62 + - 4.92 2.50 ± 2.50 10.63 ± 7.10 29.05 + - 9.72 FA (3) 0.00 ± 0.00 4.17 + 4.17 24.07 ± 0.93 0.00 ± 0.00 0.00 ± 0.00 25.00 ± 0.00 Note) Relaxation rate was calculated as 100% of 0.3-3 μM phenylephrine, and () is experimental.

Relaxation of F1 and F2 in the rabbit's vasculature F1, 占 퐂 / ml F2, 占 퐂 / ml 0.1 One 10 0.1 One 10 TA (9) 17.15 + - 4.38 26.44 + - 4.62 65.42 ± 4.96 24.28 + - 7.05 37.30 ± 6.44 77.12 + - 4.74 MA (9) 23.61 + - 4.65 28.64 + - 4.98 64.12 + - 5.26 30.84 + 8.96 34.84 + - 9.98 64.93 + - 6.60 CA (4) 10.88 ± 1.75 21.00 ± 3.86 84.55 + 2.84 11.13 ± 1.17 22.33 ± 3.17 61.58 + - 8.56 RA (4) 9.90 + - 3.53 18.93 + - 8.84 5.757 ± 10.16 14.83 + - 3.46 28.88 ± 6.19 59.10 + - 7.24 Note) Relaxation rate was calculated as 100% of 0.3-3 μM phenylephrine, and () is experimental.

Comparison of the EC ₅₀ values of the relaxation of F1 and F2 in the vasculature of rabbit and rat rabbit Rat F1 (× 10 ^-6 M) F2 (× 10 ^-6 M) F1 (× 10 ^-6 M) F2 (× 10 ^-6 M) TA 4.89 ± 1.25 2.03 + - 0.54 2.98 + - 0.64 8.50 + - 2.55 MA 4.63 ± 1.20 4.39 ± 2.12 3.96 ± 1.48 1.58 ± 0.20 CA 2.85 0.20 4.41 ± 0.90 2.82 ± 0.75 3.60 ± 1.47 RA 3.64 ± 1.14 4.65 ± 2.01 4.13 ± 0.97 5.62 ± 2.21 FA - - ND ND Note) 1. ND: Not measured (EC ₅₀ can not be obtained because maximum shrinkage height has not reached 50%) 2. All values are expressed as mean ± standard error of mean.

As can be seen from the results shown in the above table, the Ruminal brookina active fractions isolated according to the present invention are excellent in vasodilatory action irrespective of the species, and thus can be usefully used as a vasodilator.

EXPERIMENTAL EXAMPLE 2: Relaxation Test on the Brain Smock Artery of the Room Brookinase Fraction (In Vivo Test)

Way:

Sprague-Dawley rats (250-300 g) were anesthetized with urethane (1 g / kg, intraperitoneal) and fixed on an animal fixation pad equipped with a heating pad to keep the body temperature constant. After performing a tracheostomy, it was connected to a respirator (Harvard, Model 683) and ventilated. The left femoral artery was anesthetized with a PE50 polyethylene tube and connected to a Statham P23D pressure transducer to measure blood pressure. The left carotid artery was intubated with a polyethylene tube to infuse arterial blood or re - inject blood. The arterial blood was collected from the left carotid artery before and after the two windows were installed and the gas and pH were measured (NOVA Biomedicals, STAT profile 3). The average values of blood gas and pH during the experiment were as follows: pH 7.38 ± 0.04; Paco ₂ 33.3 + - 2.8 mm Hg; Pao ₂ 101.3 ± 5.1 mmHg. Anal temperature was continuously measured and maintained constant (37 ± 0.05 ° C).

The brain smectic artery is obtained by the method of Hong et al. (Amer. J. Physiol. 266, H11-H16, 1994). A concrete experimental method will be briefly described as follows. The rat was placed in a prone position on a stereotaxic apparatus (Stoelting) and the skull was incised in a square (5 × 5 mm) window. The dura mater was carefully dissected and the brain area covered with warm mineral oil during surgery. The walls were made of bone wax around the two windows, the entrance and exit were placed on the wall, and the upper lid was made transparent so that the nutrient solution was perfused through the two windows and fixed with a rebase so as not to leak the perfusion solution. The brains were equilibrated for 60 minutes after the two windows were installed. Mock cerebrospinal fluid (CFS) was perfused through the two windows at a rate of 0.3 ml / min. The external diameter of the brain parenchyma was 2-30 μm and the images were imaged using a CCD video camera (Sanyo, VDC 3900) connected to a stereomicroscope (Nikon, SMZ-2T) And made it possible to observe directly on a TV monitor. On the other hand, the diameter of the cerebral blood vessels was automatically measured from this image enlarged 480 times using a width analyzer (C3161, Hamamatsu). The movement of the vessel diameter is 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 two windows 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 room brominase active fractions F1 and F2 were administered topically through two chambers at intervals of 5 minutes at 0.1, 1 and 10 / / ㎖ after the equilibration of the cerebral blood vessels, respectively. Brain flap artery relaxation was observed for 5 minutes at intervals of 0.2 minute after the administration of the active fraction. The effect was evaluated as the increase and decrease rate (%) of the diameter after the administration of the active fraction, based on the diameter of the brainstem artery before the administration of the active fraction Respectively. The composition of artificial 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 effects of the brain pseudoaneurysm by administration of the Room Brookinase active fractions F1 and F2 are shown in FIGS. 8 and 9 and Table 4, respectively. From these results, it was observed that when the active fraction F1 was administered at a dose of 0.1 μg / ml, dilatation of the brain parenchymal artery was not observed, but when the dose of 1 μg / ml was administered, These vasodilating effects lasted for 2-3 min, and vasodilatation reaction persisted for more than 5 min at 10 μg / ml. As can be seen from the results shown in Table 4, the maximum vasodilating effect of the active fraction F1 was increased by 36.6% at the dose of 1 μg / ml at 1.6 minutes of administration, and at 10 μg / And the maximum value was increased by 62.5% at 4 minutes after administration.

The vasodilatation response was observed at 1 μg / ml and 10 μg / ml at the time of administration of the room brookinase active fraction F2, and vasodilatation reaction was more transient than F1, but vasodilatory effect was more remarkable 9). That is, when the active fraction F2 was administered at a dose of 1 μg / ml, it increased by more than 60% at 0.4-0.6 minutes after topical administration and increased by 90% at 1 minute at 10 μg / ml.

From these results, it can be seen that the Rum Virokinae active fractions F1 and F2 extracted from Candidate cause a direct vascular degeneration action on the cerebral blood vessels.

Time (minutes) MS, 0.1 [mu] g / ml MS, 1 [mu] g / ml MS, 10 [mu] g / ml F1 F2 F1 F2 F1 F2 0 0 0 0 0 0 0 0.2 -10 -12.0 15.4 55.8 -7.7 73.2 0.4 7.7 0 19.0 65.5 -7.7 73.2 0.6 -3.9 0 6.0 59.7 -12.0 82.8 0.8 6.0 0 10.0 50.1 0 90.5 1.0 -15.4 3.9 0 30.8 3.9 80.9 1.2 -2.0 -3.9 2.0 7.7 12.0 48.2 1.4 12.0 -7.7 29.0 -3.1 17.3 36.6 1.6 2.0 -10.0 36.6 7.7 17.3 25 1.8 6.0 2.0 7.7 11.6 25.0 17.3 2.0 2.0 -3.9 4.0 25.1 36.6 7.7 2.2 -2.0 -6.0 -10.0 30.8 28.9 9.6 2.4 3.9 -7.7 -10.0 19.3 40.4 11.6 2.6 0 -3.9 -12.0 9.6 37.7 38.5 2.8 13.5 -13.5 -12.0 28.9 34.7 25 3.0 10.0 -12.0 -15.4 19.3 42.4 42.4 3.2 3.9 -12.0 -3.9 19.3 40.4 36.6 3.4 -3.9 -12.0 2.0 25.1 44.3 75.1 3.6 0 -3.9 3.9 17.3 42.4 42.4 3.8 0 -2.0 5.8 21.2 63.5 32.7 4.0 13.5 0 0 61.6 65.5 80.9 4.2 3.9 0 -5.0 55.8 44.3 69.3 4.4 6.0 30.0 3.9 77.0 34.7 63.5 4.6 12.0 19.3 -3.9 73.2 32.7 52 4.8 10.0 23.1 2 82.8 34.7 34.7 5.0 2.0 55.8 3.9 80.8 38.5 32.7 2) Time (minutes) was the time after topical application of the room bromine kinase active fractions F1 and F2 to the brain membrane via two windows, and was observed at every 0.2 minute intervals. 3) The diameters of brain parenchymal arteries were expressed as% increase and decrease in% based on the pre-injection extract administration.

Experimental Example 3: In vivo experiment on brain local blood flow

Way:

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

The animals were fixed on a stereotactic frame and the scalp was incised along the midline to expose the bones of the bregma. The anterior part of the bregma was located 4 to 6 mm laterally, the anterior part of the bregma was 2 to 1 mm in diameter, Craniotomy was performed. At this time, the thickness of the skull was left thin to prevent epidural hemorrhage. A needle probe (diameter 0.8 mm) for laser-Doppler flowmeter (Transonic Instrument, USA) was used with a stereotactic micromanipulator to be perpendicular to the surface of the cerebral parietal cortex And carefully approximated to the brain parenchymal artery. After stabilization for a certain period of time, the robu- kinase active fractions F1 and F2 isolated from the stomachs were intravenously injected into the femoral vein at a dose of 0.1, 1, and 10 μg / kg, respectively, or 0.1, 1, / Ml and the cerebral blood flow was measured according to the experimental protocol. The measured changes in cerebral blood flow were expressed as 100% before the administration of the candidate extract-active substance. All the test values were expressed as mean ± standard error, and the significance between the groups was analyzed by the Student's t-test to determine that the P value was less than 0.05.

result :

The results of intravenous injection of femoral vein of room bromokinase active fractions F1 and F2 in the anesthetized rats and fluctuation of cerebral blood flow by local administration to the brain surface are shown in Figs. 10, 11, 12 and 13, These are summarized in Table 5. As shown in FIG. 10, regional cerebral blood flow (rCBF) was increased to 50% at 0.1, 1, and 10 mg / ml by intravenous administration of fraction F1, and this increase was continued for 30 minutes. However, mean arterial pressure (MAP) was not affected by intravenous administration of fraction F1. Intravenous administration of F2 also resulted in a dose-dependent increase in cerebral blood flow by intravenous injection of 0.1, 1, and 10 mg / ml, and this increase in cerebral blood flow increased by up to 70% and lasted for more than 20 minutes (See FIG. 11).

Also, as shown in FIG. 12, cerebral blood flow was dose-dependently increased by the local administration (0.1, 1, 10 占 퐂 / ml) on the surface of the brain surface of the room bromokinase active fraction F1 without fluctuation of mean arterial pressure And this increase was manifested by Danshi Hyosung. On the other hand, when the local administration effect of the room bromine kinase active fraction F2 on the surface of the meniscus was observed, the brain blood flow was not changed when 0.1 μg / ml was administered. However, when 10 μg / ml was administered, baseline), and this increase reaction lasted more than 20 minutes (Fig. 13).

Changes in cerebral blood flow by intravenous injection of F1 and F2 and topical administration to the brain surface in rats F1, 占 퐂 / ml F2, 占 퐂 / ml 0.1 One 10 0.1 One 10 Intravenous injection (4) 101.88 ± 1.89 107.99 + - 4.39 151.95 ± 8.78 ^** 103.63 + - 3.63 113.96 + - 4.25 159.57 ± 3.24 ^** Topical administration (6) 110.42 ± 3.79 114.82 ± 3.45 169.86 ± 14.69 ^** 102.88 + - 0.94 118.18 ± 1.72 ^** 225.29 ± 21.64 ^** 1) Values in parentheses indicate the experimental values. 2) All the values were expressed as mean ± standard error. 3) ** P0.05 vs control baseline value 4) .

From these results, it can be seen that F1 and F2, which are active fractions of Rumbrinokinase in a small amount, have an effect of increasing cerebral blood flow within a range not causing fluctuation of peripheral blood pressure. Therefore, it is expected that it can be used as a remedy for cerebral blood flow disorder in cerebral blood flow disorder disease (stroke cerebral infarction, etc.).

Claims (5)

The present invention relates to an angiotensin-releasing agent composition comprising a rhubarb kinase active fraction of the present invention. 3. The method according to claim 1, wherein the Ruminal Broken kinase active fraction of Form A is separated from the crude extract of Rumen by a combination of anion exchange column chromatography, hydrophobic chromatography, gel chromatography and affinity chromatography, F2, F3, F4, F5 and F6. 3. An anginal muscle relaxant composition according to claim 2, wherein the roumbina kinase active fraction of the progeny is the robu- kinase fraction F1 or F2 of the progeny. The composition of claim 1, wherein the composition is formulated as a injectable preparation using a pharmaceutically acceptable carrier. 5. The composition according to claim 4, wherein the injection agent is an intravenous agent preparation.