KR19990025954A - Prevention and treatment of stroke containing gut extract - Google Patents

Abstract

The present invention relates to a composition for preventing and treating stroke containing Lumbricus rubellus extract. Sump extract used as an active ingredient in the composition of the present invention significantly reduces the area of cerebral infarction in the experimental cerebral infarction animal model, significantly reduces the mortality due to subarachnoid hemorrhage in the experimental subarachnoid hemorrhage model, inhibits the cerebrovascular spasm, In addition, it can be usefully used as an agent for preventing and treating stroke by showing effects such as preventing cerebrovascular contraction.

Description

Stroke preventive and therapeutic agent composition containing Gumi extract

The present invention relates to a composition for preventing and treating stroke containing Lumbricus rubellus extract.

In recent years, various circulatory diseases such as hypertension, arteriosclerosis, hyperlipidemia, stroke, and myocardial infarction are increasing rapidly due to various factors such as changes in eating habits, excessive stress, and pollution. Most of these circulatory diseases can be attributed to the formation of blood clots. In particular, stroke, the most fatal disease among the circulatory disorders, is one of bouts of cerebral artery, which is constricted or blocked by arteriosclerosis or blood clots, causing brain damage due to blockage of cerebral blood flow. It is a disease that leads to. Vascular diseases, such as stroke, are rapidly increasing 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, recombinant-tissue type plasminogen activator (r-tPA), alprostidyl, streptokinase, etc. In terms of treatment, there is no sufficient satisfactory treatment yet.

Therefore, the present inventors conducted extensive research to find an effective drug for the treatment of stroke, and used the job as its subject. Job offer has been prescribed for example in traditional Chinese medicine as a treatment for adult diseases such as arteriosclerosis, hypertension and diabetes, especially in the pharmacopoeia of the People's Republic of China (1977 edition, Part 1, pages 197-188). There are two types of job openings , Lumbricus Kwang tungesis and Lumbricus nativus , which are antipyretic, anti- cursive , blood-promoting, semi-anhydrous, joint, urination, bronchial asthma, and hypertension. It is described as effective.

Based on the effects of such recruitment, the present inventors have conducted various studies on recruitment, and as a result, the extracts can effectively prevent and treat stroke by inhibiting cerebral infarction and inhibiting spasm and contraction of cerebrovascular vessels. It confirmed and completed this invention.

1 is a graph showing the infarct area in each of the cerebral coronal sections of the experimental cerebral infarction animal model according to the pretreatment period by the job extract [○ control group (7 cases), ▲ job extract extract 7 days pretreatment group (4 cases), Job extract 14 days pretreatment group (6), Job extract 28 days pretreatment group (6)].

Figure 2 is a graph showing the comparison of the total cerebral infarct size in the experimental cerebral infarct animal model according to the pretreatment period by the Job extract.

Figure 3 is a comparison of the micrographs of the coronary section 1 (B) and the control coronary section 1 (A) of the experimental cerebral infarct animal model pretreated with the salivary extract 14 days.

Figure 4 shows a comparison of the micrographs of the coronary section 2 (B) and the control coronary section 2 (A) of the experimental cerebral infarct animal model pre-treated with the salivary extract 14 days.

FIG. 5 shows the comparison of micrographs of the coronal section 3 (B) and the control coronal section 3 (A) of the control cerebral infarction animal model pretreated with 14 days.

Figure 6 shows a comparison of the micrographs of the coronary section 4 (B) and the control coronary section 4 (A) of the experimental cerebral infarct animal model pretreated with the salivary extract 14 days.

FIG. 7 shows a comparison of micrographs of the coronal segment 5 (B) and the control coronal segment 5 (A) of the experimental cerebral infarct animal model pretreated with a globular extract for 14 days.

FIG. 8 shows a comparison of photomicrographs of the coronary section 6 (B) and the control coronary section 6 (A) of the control cerebral infarct animal model pretreated with a globular extract for 14 days.

FIG. 9 shows a comparison of micrographs of the coronary section 7 (B) of the experimental cerebral infarct animal model pretreated with the extract for 14 days and the coronary section 7 (A) of the control group.

Figure 10 is a graph showing the effect of subarachnoid hemorrhage (SAH) surgery and job extract administration on the cerebrovascular relaxation response by CGRP [○ control, ● SAH surgical group, ■ Job extract + SAH group, A: Basal artery, B: posterior cerebral artery].

Figure 11 is a graph showing the effect of the administration of SAH surgery and job extract extract on the cerebrovascular relaxation response by sodium nitroprusside [○ control, ● SAH surgery group, ■ Job extract + SAH group, A: brain Basal artery, B: posterior cerebral artery].

12 is a graph showing the effect of SAH surgery and the administration of the job extract extract on cerebrovascular relaxation response by diltiazem [○ control group, ● SAH surgery group, ■ Job extract + SAH group, A: cerebral basal artery , B: posterior cerebral artery].

Therefore, the present invention relates to a composition for preventing and treating stroke, which contains the extract of the essence as an active ingredient.

Sump extract used as an active ingredient in the composition of the present invention may be used by any of the methods commonly used in the preparation of the extract, but preferably Korean Patent Application No. 93, the first application of the applicant Use was made by the method described in -14717 (Name of the Invention: Method of Producing Gut Extract). According to the method described in Patent Application No. 93-14717, the gut extract was lyophilized by self-decomposing the gut, repeatedly freezing and thawing the resulting solution to precipitate impurities, and then lyophilizing the supernatant obtained by centrifugation. Obtained in powder form. The globular extracts prepared by this method provide very good benefits in terms of biologic efficacy and stability of globular.

The testosterone extract prepared according to the above method significantly reduced cerebral infarct area in an experimental cerebral infarct animal model, and significantly reduced mortality due to subarachnoid hemorrhage in an experimental subarachnoid hemorrhage model, as demonstrated by the experimental example described below. It suppresses the spasm of blood vessels and prevents cerebrovascular contraction induced after subarachnoid hemorrhage. Therefore, the pharmaceutical composition containing such a sap extract can be usefully used as a preventive and therapeutic agent for stroke.

When the composition of the present invention is administered to the patient in a single dose or in separate doses when administering the composition for clinical purposes, the total daily dose is preferably in the range of 0.2 mg to 20 mg per 1 kg of body weight. Levels may be appropriately determined depending on the weight, sex, health status, diet, time of administration, method of administration, rate of excretion, purpose of administration and severity of the disease of the individual patient.

The compositions of the present invention can be administered in injectable and oral formulations as desired.

Injectable preparations, such as 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 solid dosage form for oral administration may be in the form of capsules, tablets, pills, powders and granules, and particularly, capsules and tablets are useful. Tablets and pills are preferably prepared with enteric agents. Solid dosage forms can be prepared by mixing the gut extract with one or more inert diluents, such as sucrose, lactose, starch, and the like, and carriers such as lubricants, disintegrants, binders, and the like, such as magnesium stearate.

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

1 kg of washed Lumbricus rubellus with water was added to 1 L of an aqueous solution containing 200 mM CaCl ₂ and completely self-decomposed while stirring slowly at 30 to 40 ° C. for 8 hours. After complete autolysis, the mixture was warmed at 60 ° C. for 30 minutes. The resulting solution was frozen by rapid cooling to 0 ° C., thawed by standing at −20 ° C. for one day and then thawing again by rising to room temperature. After the layers were separated, the supernatant was collected and centrifuged at 5000 rpm for 1 hour. The supernatant was subjected to diatomaceous earth filtration and 0.45 μm membrane filter filtration successively to obtain a sterile extract, which was then frozen in a freeze dryer at −30 to −40 ° C. and then dried under 0.2 mm Hg vacuum for 20 hours, at 0 ° C. It was dried for 15 hours, 20 hours at 20 ℃ and finally 5 hours at 35 ℃ to give 95g of the lyophilized powdery extract.

Example 2

1 kg of Lumbricus rubellus bodies washed with water were put in 1 L of distilled water and wet-pulverized with a homomixer, and then self-decomposed for 5 hours while keeping the temperature constant at 50 ° C. The resulting solution was frozen by rapid cooling to 0 ° C., then left at −20 ° C. for one day, and then again thawed by raising the temperature to room temperature. After the layers were separated, the supernatant was collected and centrifuged at 5000 rpm for 1 hour. The supernatant was subjected to diatomaceous earth filtration and 0.45 μm membrane filter filtration in succession to obtain a sterile extract, which was then frozen to -30 ° C. and then 20 hours under 0.2 mmHg vacuum, 15 hours at 0 ° C., 12 hours at 20 ° C. and Finally, dried at 35 ° C. for 5 hours to obtain 100.5 g of a lyophilized powdery oak extract.

Experimental Example 1: anti-stroke effect test

Way :

The effect of job extract as a stroke treatment was tested by preparing an experimental animal model of cerebral infarction according to the method of Girona et al. (Enrique Zea Longa, et al., Stroke 20: 84-91, 1989). This model is a widely used middle cerebral artery occlusion model that is made by inserting a nylon suture into the internal carotid artery to close the initiation of the middle cerebral artery and calculate the extent of occlusion. Specifically, Sprague-Dawley rats of 300-450 g body weight were raised at room temperature, and the sap extract was dissolved in water at a dose of 300 mg / kg / day, and 0, 3, 7, 14 and After ingestion for 28 days, surgery was performed in the manner described above to cause middle cerebral artery occlusion. After anesthesia, the patient was allowed to recover in the warming box until the anesthesia awoke, and then moved to the normal breeding box to consume the food and water at room temperature. 24 hours after the operation, a lethal dose of cecobarbital (100 mg / kg) was administered intraperitoneally. Then, the head was lethal and the entire brain was removed from the skull to make 7 coronal sections at intervals of 2 mm from the frontal lobe. It was immersed in% 2,3,5-triphenyltetrazolium chloride solution and treated for 30 hours at room temperature and then fixed in 10% neutral formalin buffer. The cerebral infarction site of the fixed brain tissue section was measured by area meter (KP-21, koizumi, Japan). All experimental values were expressed as the standard error of the mean ± mean, and the significance between each group was analyzed by ANOVA and post test by Turkish test (Tukeys test) to determine that the P value was less than 0.05.

result :

The results measured by the test method are shown in FIGS. 1 to 9.

Figure 1 shows the comparison of the infarct area of each of the cerebral coronal sections of rats according to the pretreatment period of the sap extract. As shown in FIG. 1, the cerebral infarct area of each section was decreased compared to the control group according to the pretreatment period of the salivary extract, and in particular, the cerebral infarct area of the coronary sections 6 and 7 of the group pretreated with the sap extract for at least 14 days was the control group. It was significantly decreased compared with that of P0.05 respectively. This effect can be clearly seen from the photographs of Figs. 3 to 9, which show the comparison of each of the control sections of the control group and each of the three sections of the control group pretreatment with the extract for 15 days. In addition, as shown in FIG. 2, the size of the total cerebral infarction was also significantly reduced in proportion to the pretreatment period by the saline extract. In particular, the incidence of whole cerebral infarction in the sachet extract pretreatment group over 14 days was statistically significant as compared to the control group. Decreases (P0.05 respectively).

From the above results, it can be seen that the experimentally induced cerebral infarction by the pretreatment of the sap extract can be significantly suppressed.

Experimental Example 2: Test of the oral administration effect of Sump extract in experimental subarachnoid hemorrhage model

Way:

As one of the methods to test the hematopoietic improvement effect on the cerebrovascular disorders, we observed subarachnoid hemorrhage (SAH) in rats. The experimental animals were treated with a control group (control 10 animals), SAH surgical group (SAH 8 rats), and sap extract for 2 weeks (200 mg / kg / day, oral administration), and SAH surgery group (sump extract + SAH group, 8 animals). Subarachnoid hemorrhage was induced in the following ways. First, sterilized spinal needles (22 guage) were inserted into the control (cisterna magna) in rats weighing 200-250 g, and cerebrospinal fluid was removed about 0.2-0.5 ml through the needle, and then autologous venous blood was slowly injected into the control. After 48 hours of blood injection, the same procedure was performed and recovered, and the animals were observed for 7 days after the first blood injection and counted the number of dead animals. Only the animals survived after the 7-day observation period was over. After sacrificing the experimental animals, the basal artery, middle cerebral artery and posterior cerebral artery of the brain were extracted, and the diameter (inner diameter) of the vascular muscle was measured and compared.

result:

During the 7-day observation period, no experimental animals died in the control group, but only 5 of the 8 surgical cases survived and 3 died within 1 to 2 days in the SAH surgical group, resulting in a 62.5% survival rate. On the other hand, in the group that was pretreated for 2 weeks, only 1 of the 8 surgical cases died on the 3rd day and showed 87.5% survival rate. Therefore, long-term administration of job extract significantly reduced the mortality rate from experimental subarachnoid hemorrhage. Reduction could be observed.

In addition, the diameter of the vascular muscle measured in each experimental animal group is shown in Table 1.

Cerebral artery Diameter μm Control group (10) SAH Surgery Group (5) Job extract ± SAH group (7) Cerebral artery 382 ± 36 324 ± 19 344 ± 47 Middle cerebral artery 274 ± 19 216 ± 21 ^* 258 ± 6 ^** Posterior cerebral artery 258 ± 34 205 ± 14 ^* 240 ± 12 ^** Note) Vascular diameter was measured by internal diameter and experimental values were expressed as mean ± standard error. * P0.05 vs control; ** P0.0 vs SAH. · Numbers in parentheses are experimental Jesus (number of surviving animals after 7 days observation period).

As shown in Table 1, the internal diameter of each vessel was significantly decreased in the SAH surgical group compared to the control group, and in the group in which oral administration of job extracts was orally administered for 2 weeks (SAE group), the basal artery, middle cerebral artery and The internal diameters of the posterior cerebral arteries were all significantly increased compared to the SAH surgical group. Therefore, it can be seen that the extracts have the effect of inhibiting cerebrovascular spasm caused by SAH, and by these effects, the extracts can effectively treat cerebrovascular spasm, which is a major cause of death or neuropathy or sequelae caused by SAH. It is expected to be available as a useful drug.

Experimental Example 3: Effect of Oral Administration of Sump Extract on Cerebrovascular Spasm after Experimental Subarachnoid Hemorrhage

Way:

In this experiment, we observed the preventive effect of the sputum extract on cerebrovascular contraction, which is a major cause of death and cerebral ischemia and neurodegeneration, after subarachnoid hemorrhage in rats with experimental subarachnoid hemorrhage (SAH). In this experiment, the extract was orally administered daily at a dose of 500 mg / kg / day for 2 weeks before the SAH surgery.

As control animals, Sprague-Dawley rats (200-250 g in weight, male and female) were used for 2 weeks (500 mg / kg / day, oral administration of control, SAH surgery group and salivary extracts). ) And three groups of SAH surgery (Human extract + SAH group), and animals surviving 4-7 days after SAH surgery were used for the experiments. Subarachnoid hemorrhage was performed as follows. First, sterilized spinal needles (22 guage) were inserted into the control rats weighing 200-250 g, and cerebrospinal fluid was removed about 0.2-0.5 ml through the needle, and then autologous venous blood was slowly injected into the control. After 48 hours of blood injection, the same procedure was performed and recovered. After 4-7 days of the first blood injection, the animals were sacrificed to extract the basal and posterior cerebral arteries of the brain. In order to extract the cerebral arteries, the experimental animals of the control group, the SAH surgical group and the sap extract + SAH group were anesthetized with ether, the carotid artery was cut and hemorrhagic, and the cervical spine was cut, the skull was broken, and the brain was carefully removed. Paraffin wax block containing physiological salt solution (PSS; composition (mM): NaCl 130, KCl 4.7, NaH ₂ PO ₂ 1.18, MgSO ₄ 1.17, CaCl ₂ 1.6, NaHCO ₃ 14.9, Glucose 5.5) The basal artery and the posterior cerebral artery were carefully extracted using a microsurgical instrument immersed in a paraffin wax block. The isolated artery is cleanly removed from the surrounding connective tissue and then made into rings (about 2 mm in length) to remove blood, etc., and a small-caliber vascular muscle containing PSS (37 ℃) saturated with 95% O ₂ -5% CO _2. It was suspended in the muscle chamber of a small vessel myograph (designed by Dr. Mulvany, University of Aarhus, Denmark). In the chamber containing PSS, the myosight program installed in the small-diameter vascular myograph system was set to 90 mmHg, and then equilibrated for 60 minutes. U46619 was used as a contraction-inducing substance for muscle contraction, and CGRP, sodium nitroprusside, and diltiazem each drug was used in a multiple dose of 10 ^-10 M to induce accumulation reaction. Shrinkage reaction was expressed as 100% contraction by 100mM KCl, and the contraction reaction by agonist was expressed as the percentage of shrinkage, and relaxation reaction was the degree of relaxation to the maximum contraction in%. The PSS used in the contraction-induced experiments contained 2 μM indomethacin and 0.2 μM proprenol to exclude the effects of endogenous prostaglandins and catecholamines.

result:

The measured experimental results are shown in FIGS. 10 to 12.

FIG. 10 compares the effects of SAH surgery and globular extract administration on cerebrovascular relaxation by CGRP. As shown in FIG. 10, CGRP in the basal artery caused a relaxation response by dose (10 ⁻¹⁰ to 10 ⁻⁷ M) in both the control group and the SAH group. The relaxation response in the posterior cerebral artery was more pronounced than in the basal artery. In comparison with the control response of the SAH group, the cerebrovascular response by CGRP was significantly attenuated by SAH surgery in the basal artery. The relaxation inhibitory effect of SAH was not affected by the administration of the extract. However, in the posterior cerebral artery, the inhibitory effect of SAH surgery on CGRP-induced cerebrovascular relaxation was markedly attenuated by long-term administration of job extracts.

Meanwhile, FIG. 11 compares the effects of SAH surgery and job extracts on cerebrovascular relaxation by sodium nitroprusside, which is a nitrogen oxide source. As shown in FIG. 11, in this case, the relaxation response of the posterior cerebral artery was similar to that of the basal artery. In addition, the relaxation response was markedly weakened by SAH surgery. Especially, the relaxation response curve shifted toward the normal control group. In the basal artery, it was observed that the relaxation response inhibited by SAH surgery was recovered by oral administration of the extract. .

In addition, Figure 12 compares the effects of SAH surgery and job extracts on cerebrovascular relaxation by administration of diltiazem, a calcium channel blocker. As shown in FIG. 12, both the basal and posterior cerebral arteries of the control group exhibited a dose-dependent relaxation response when diltiazem was administered. The SAH surgery did not show any change in the relaxation response of both angiomuses. However, in the basal arteries, the relaxation response by diltiazem was significantly increased by 2 weeks of long-term administration of the job extract.

From the experimental results, it can be seen that the gut extract has a prophylactic effect on cerebrovascular contraction after cerebral hemorrhage by long-term oral administration, and can be used as a therapeutic agent in combination with other vasodilators.

Claims (3)

Stroke preventive and therapeutic composition containing the extract as an active ingredient. The composition for preventing and treating stroke according to claim 1, formulated into an oral or injectable formulation in combination with a pharmaceutically acceptable carrier. The lyophilized powder according to claim 1, wherein the lyophilized powder is a lyophilized powder obtained by self-decomposing the globular, by repeatedly freezing and thawing the resulting solution to precipitate impurities, and then lyophilizing the supernatant obtained by centrifugation. Stroke prevention and treatment composition.