KR20020078850A - Extract of Angelicae Gigantis Radix having neuroprotective effects and pharmaceutical composition containing the same - Google Patents

Abstract

The present invention relates to a Angelicae Gigantis Radix extract having a neuroprotective action and a pharmaceutical preparation for treating and preventing stroke containing the same.

Angelica extract of the present invention and pharmaceutical preparations containing the same are not only excellent in inhibiting the death of nerve cells by various causes but also have no toxicity for the purpose of preventing and treating stroke caused by the death of nerve cells. Very effective when used.

Description

Extract of Angelicae Gigantis Radix having neuroprotective effects and pharmaceutical composition containing the same}

The present invention relates to a pharmaceutical preparation for treating and preventing cerebral diseases containing the Angelica extract having a neuroprotective action.

Angelica (當歸) is the root of the perennial plant of exhaust gas Angelica (Angelica gigas NAKAI), Angelica sinen System (A. sinensis DIELS), Angelica Aqua butyl Rover (A. acutiloba KITAG.) And dongsok closely related plants that belong to the dispersion type (Umbelliferae) As a dry, it is known to have the effect of blood donation blood (補血 和 血), landscaping pain (윤), yunjojangjang (潤燥 滑腸). Root has sedation, jingyeong, blood pressure lowering, etc. When the aqueous solution of root essential oil is used for arthritis, it is said to have analgesic and anti-inflammatory effects. Used for swelling

So far, known components of the Angelica are coumarine-based decursin, decursinol, decursinol, nodakenetin, umbelliferon, nodakenin, and beta. -Β-sitosterol, Angelan-I, Angelan-II, etc., and their pharmacological studies showed that ether extract of Angelica gigas showed excitatory effects on the rabbit's harvested intestine and uterus, and increased blood pressure and respiration. Has been reported. Isolated Dekerin and Decosinol have been shown to paralyze the rabbit intestinal tract, inhibiting the frog's extraction heart, inhibiting rabbit breathing and lowering blood pressure, and inhibiting the spontaneous action of mice. Journal 1, No. 1, pp. 25-32, 1970). Recently, Deckerin not only shows anti-cancer activity in vitro, but also enhances the activity of protein kinase C, and pectin polysaccharides Angelan-I and Angelan-II are effective as anticancer agents or immune enhancers. (Korean Patent No. 252194).

Various uses have been studied on the basis of the pharmacological action of the above-mentioned donkey, Korean Patent No. 252194 used Angelica as an anticancer agent and an immune enhancer. It describes the effect. In addition, Korean Patent Publication No. 2000-26683 used the decansin obtained from Angelica in the composition for inhibiting renal toxicity, and Korean Patent Publication No. 2000-0071673 used Angelica Extract as a composition for preventing and treating dementia. It was. However, there are no known examples of using Angelica extract as a preventive and therapeutic agent for stroke.

Accordingly, the present inventors have conducted extensive research, it was confirmed that the donkey has an effect of inhibiting neuronal necrosis, the present invention was completed based on this.

Accordingly, it is an object of the present invention to provide a Angelica extract having a stroke prevention and treatment effect.

Another object of the present invention is to provide a pharmaceutical agent having an effect on the prevention and treatment of stroke containing the Angelica extract as an active ingredient.

Angelicae extract, Angelicae Gigantis Radix to achieve the above object is prepared by extraction, concentration and lyophilization with water, lower alcohol, hexane, ethyl acetate or a mixed solvent thereof.

1 is a graph showing changes in body temperature when administration of the Angelica extract according to the present invention after ischemia induction and reperfusion.

Figures 2a and b is a microscopic picture of the coronal sections of the dorsal hippocampus of the normal control (sham) rats that did not induce whole cerebral ischemia and stained with Cresyl violet.

Figures 2c and d show the coronal sections of the dorsal hippocampus of control rats treated with physiological saline after 7 days of induction of cerebral ischemia for 10 minutes, followed by microscopic staining with Cresyl violet. Picture taken with.

Figures 2e and f show the coronal sections of the dorsal hippocampus of rats treated with Angelica extract after 10 days of induction of cerebral ischemia for 10 minutes and then stained with Cresyl violet. The picture was taken with a microscope.

Figure 3 shows the results of the measurement of the cerebral hippocampal CA1 region cell number of the control group rats treated with Angelicae Gigantis Radix extract, the control group administered physiological saline 7 days after induction of whole cerebral ischemia for 10 minutes after the normal control group It is a graph.

Hereinafter, the present invention will be described in more detail.

Angelica extract according to the present invention can be obtained by extraction, concentration and lyophilization with water, lower alcohol, hexane, ethyl acetate or a mixed solvent thereof, the lower alcohol is selected from the group consisting of methanol, ethanol, and butanol, Methanol is preferred because of its high yield.

The extraction process during the production of the Angelica extract may be used by ultrasonic extraction, filtration, reflux extraction and / or concentrated under reduced pressure, in addition to the conventional extraction method may be used. Concentration and freezing may also be used in a variety of commonly known methods.

Angelica extract of the present invention can be used as an agent for the treatment and prevention of brain diseases caused by neuronal death because of its excellent effect of inhibiting the death of nerve cells by causes such as cerebral ischemia.

Therefore, the present inventors used a 4-vessel occlusion model developed by Pulsinelli in 1979 for the purpose of observing the effect of stroke on the neuronal cells of the Angelica extract. The model is a representative animal model of neuronal death due to cerebral ischemia, which uses the principle that nerve cells in the hippocampus are damaged when the blood vessels supplying blood to the rat's brain are temporarily blocked and then reperfused. About 5-7 days after blocking, cell damage occurs and the cell damage is similar to apoptosis, which is called delayed cell necrosis. Drugs that exhibit neuronal necrosis in this model are known to be effective in animal models of ischemia similar to human palsy, and are considered to be a desirable animal model for palsy experiments because they can secure significant clinical effects. have. Among the above models, the forebrain ischemia model has been in the spotlight recently. It is a method of causing 4-vessel occlusion in rats, and the blood flow of the posterior brain is not affected. It is widely used because it does not affect the body circulation.

In the present invention, first, the decrease in body temperature during cerebral ischemia exhibits a neuroprotective effect by preventing nerve cell damage in the hippocampus (Busto et al., J Cereb Blood Flow Metab 1987; 7: 720-738). After inducing cerebral ischemia in rats, the Angelica extract was administered to observe changes in body temperature. As a result, it was confirmed that the body temperature of about 1.8 ~ 2.5 ℃ compared to the normal control according to the dose of Angelica extract (see Figure 1).

The most susceptible to rat-induced cerebral ischemia is the CA1 pyramidal neuron of the hippocampus, which begins to die 72 hours after reperfusion (Pulsinelli WA et al., Ann Neurol 1982; 11: 491-498). Therefore, to determine whether the Angelica extract of the present invention inhibits delayed neuronal death in the CA1 region of the rat hippocampus, induced cerebral ischemia of the rat and intraperitoneally administering the Angelica extract to neurons. Hippocampal tissue sections were prepared one week after reperfusion, at which point the damage almost occurred, and observed under light microscopy (see Figures 2a, b, c, d, e, and f).

In the case of apoptosis induced by any external stimulus or internal stimulus, the cells contract first and lose their intrinsic shape, and the contraction destroys the connection with the surrounding cells, thereby interacting with the cells. This is stopped. When the contraction progresses to a certain extent, the cell membrane forms a blister and forms a necrotic body (apoptotic body). The necrosis was found in the hippocampal tissue sections of the group that did not receive the Angelica extract after induction of cerebral ischemia, but the group was not found in the group where the Angelica extract was administered. It was confirmed that it was defended from apoptosis, formed a normal pyramidale, and maintained its junction with surrounding cells.

Next, the present invention was observed while artificially maintaining normal body temperature after cerebral ischemia induction and administration of the Angelica extract to determine whether the inhibitory effect of the Angelica extract is associated with a decrease in body temperature. As a result of observing hippocampal tissue sections of rats tested under the above conditions, it was confirmed that the cell survival rate was increased by about 71.0 to 88.7% compared to the case where the donkey extract was not administered.

In the present invention, whether or not the toxicity of the Angelica extract was confirmed through oral administration and intraperitoneal administration to the rat, it was confirmed that there is no toxicity.

The neuronal cell death inhibition effect of the Angelica extract confirmed through the above results is very excellent. The evidence is based on the drug that has been studied so far: LY231617 25-30%, L-NAME 23%, 3-bromo-7-nitroindazole 20%, MK-801 (2 mg) / Kg, ip) 24%, eliprodil (20 mg / kg, ip) 25%, NBQX (30 mg / kg, ip) 42%, 7-NI 17.5%, GYKI52466 11% And LY300168 is known to have a neuronal killing effect of 23% (O'Neill MJ et al., Eur J Pharmacol 1996; 310), which is lower than 56.0% of the results of the measurement of the Angelica gigas according to the present invention. For Chinese herbal medicine, rhubarb was 16.8% (1,000 mg / kg) and 16.3% (500 mg / kg), Cheonma was 17.8% (1,200 mg / kg) and 15.6% (600 mg / kg), It is known that Hubak showed 13.7% (1,000 mg / kg), Daeseunggi-tang showed 18.4% (1,000 mg / kg) and 16.6% (500 mg / kg) neuronal cell death inhibitory effect. (Kor. J. Herbology vol. 14, No. 1, 1999), but this is also lower than Angelica extract.

Pharmaceutical preparations for the treatment and prevention of brain diseases of the present invention containing Angelica extract as an active ingredient may be administered in various oral or parenteral formulations during actual clinical administration, and when formulated, commonly used fillers and extenders , Diluents or excipients such as binders, wetting agents, disintegrants, surfactants and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose ( Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of the Angelica extract according to the present invention may vary depending on the sex, age, weight, and degree of disease of the patient, but in general, 10 to 5000 mg / kg may be administered in 1 to 3 times a day. , And 100-250 mg / kg is preferable.

As a result, the Angelica extract of the present invention was found to exhibit excellent neuronal cell death inhibitory activity, and thus can be used as a therapeutic and / or prophylactic agent for stroke patients in clinical practice.

Hereinafter, the present invention will be described in more detail with reference to Examples and Preparation Examples, but the scope of the present invention is not limited thereto.

Example 1

Preparation of Angelica Extract

Dongguk was purchased from Tokyo Mulsan and used after it was verified by the Department of Herbology, College of Oriental Medicine, Kyung Hee University.

2 L of an aqueous 85% methanol solution was added to 1 kg of dried Angelica, and then extracted twice using ultrasonication for 15 minutes using a sonicator to obtain 3 L of an extract. The extract obtained above was filtered through filter paper and concentrated under reduced pressure at 0.98 atmosphere. The obtained concentrate was lyophilized and used as a sample, and the average yield was 5% or more.

Example 2

Fraction of Angelica Suspension

The Angelica extract obtained in Example 1 was suspended in 100 ml of water, and then fractionated sequentially according to polarity by a solvent separation method to obtain n-hexane, ethyl acetate, and water fractions. The obtained fractions can be used in clinical experiments and mechanism studies in the following examples in the same manner as the extract of Example 1 above.

Example 3

Rat Cerebral Ischemia Induction

To determine the most suitable ischemic induction time in rats, first, eight weeks-old male rats (SLC, Japan) of about 170 g were adapted to the experimental environment for one week. Rats were anesthetized with 5% isoflurane contained in a mixture of nitrogen and oxygen (70% nitrogen, 30% oxygen), and then the head was lowered horizontally to the stereotaxic apparatus. Fixed at 30 °. The nose and around the mouth were connected to an anesthesia (Ohameda V.M.C./Boc Health Care, Cyprane, UK) with a plastic cone, and the anesthesia was kept at 1.5% isoflurane.

The tail was fixed on the operating table and the cervical spine was extended. First of all, in order to operate the throat, a ring was made of silicone tube in the carotid artery to induce ischemia and reperfusion. In addition, trachea, esophagus, external jugular vein, and common carotid arteries are located in the back to block the circulation of microvessel during ischemic induction. (cervical) and paravertebral muscles were penetrated and then wound closed with surgical clips.

Then, the rat was turned to make a midline incision, and the first cervical spine at the bottom of the occipital bone was operated to prevent muscle injuries using a surgical magnifier, and a small electrical acupuncture of less than 1 mm The vertebral artery was cauterized by inserting it into the tunnel through which the vertebral artery passed through. A surgical microscope was used to confirm that the vertebral artery present in the tunnel passing into the bone was completely cauterized and closed and then closed with a surgical clip. 24 hours after closure, surgical clips were removed and the carotid artery was tightened with aneurysm clips for 5, 10, 20 and 30 minutes respectively to induce ischemia. If the specular reflection was not lost within 1 minute, the neck was sutured tightly. At this time, the rats that did not lose the specular reflection were excluded because they did not cause complete bilateral parallel CA1 nerve damage. They also excluded. 5 minutes, 10 minutes, 20 minutes and 30 minutes after ischemia induction, the aneurysm clip was removed from the total carotid artery of the four rats finally selected to stop ischemia and reperfusion.

One week after induction of ischemia, the rat was anesthetized with chloral hydrate (35.0 mg / kg, ip), and the incision was made by injecting a needle into the left ventricle and injecting a heparinized 5% sodium nitrate saline solution 10 Minutes, perfusion to the heart and perfusion with 4.0% formalin fixative. Thereafter, the brains of the rats were removed and post-fixed in 0.1 M phosphate buffered formalin fixative for 2 hours, and then immersed in 30% sucrose solution and left overnight at 4 ° C. In the fixed brain, a coronal block in the dorsal hippocampus between -2.5 mm and -4.0 mm is incised, frozen at -70 ° C, and frozen using a sliding microtome. Tissue sections containing hippocampus were prepared by cutting at intervals.

Sections prepared by the above method were stained and fixed in cresyl violet and then the most susceptible to delayed neuronal death of dorsal hippocampal CA1 (Crain BJ et al., Neuroscience 1988; 27: 387-402) The number of damaged neurons present in the 1,000 μm portion of the middle zone was observed. The number of cells was observed by three observers at 6 magnifications of two different left and right sides of three different tissue sections in one brain tissue. Values were averaged.

Example 4

Body Temperature Changes in Cerebral Ischemia Rats

The purpose of this study was to investigate the changes in body temperature of rats with administration of Angelica extract.

In the same manner as in Example 3, rats with cerebral ischemia were induced and reperfused for 10 minutes, and only rats having a loss of consciousness of 20 ± 5 minutes were selected. 0 and 90 minutes after induction of ischemia, the donkey extract prepared in Example 1 was dissolved in 0.89% saline solution, and then 2.0 ml per rat body weight, 100, 250, and 500 mg, respectively. The amount of / kg was injected intraperitoneally, respectively. As a control group, rats administered with 0.89% saline only were used.

Body temperature was monitored at 30-minute intervals up to 6 hours after ischemia induction, and body temperature was measured by inserting a probe into the rectum at least 6 cm (Miyazawa T et al. , J. Cereb. Blood Flow Metat. 1992; 12: 817-822).

As a result, it was observed that the body temperature did not drop in the group administered with Angelica extract.

Example 5

Observation of nerve cell protection of Angelica gigas extract

In order to observe delayed neuronal death (CA) in the CA1 region of hippocampus (hippocampus) induces cerebral ischemia in rats for 10 minutes in the same manner as in Example 3, and then the donkey prepared in Example 1 after 90 minutes The extracts were dissolved in 0.89% saline and then injected intraperitoneally with 2.0 ml per rat body weight, respectively 100, 250, and 500 mg / kg. Next, the rats were killed one week later, and hippocampal tissue sections were prepared in the same manner as in Example 3, and the prepared tissues were observed under an optical microscope (see FIGS. 2A, B, C, D, E, and F). . The brain hippocampal tissue of rats that did not induce cerebral ischemia was used as a normal control group, and the brain hippocampal tissues of rats that were injected with physiological saline instead of donkey extract in the above-described procedure.

As a result, hippocampal neurons were observed normally in the normal control (sham operated) (see FIGS. 2A and B), and in the absence of control neurons, the cells were released from the surrounding cells while the tissues were relaxed unlike the normal neurons. It could be observed that the cell body also lost its original pyramidal form and condensed to form a single cell. In addition, the nuclear chromatin was concentrated and the collapse of the nuclear membrane was observed to confirm that apoptosis (apoptosis) was achieved (see Fig. 2c, d).

Neurons in the group administered with Angelica extract showed similar morphology to normal cells, and were easily distinguished due to their protruding perikaryons and the centrally located round nucleus and the surrounding neutrophils in a relatively healthy state. Is clearly distinguished from. In addition, the majority of cells were defended against apoptosis to form a normal pyramidale form and continued to maintain junctions with surrounding cells.

Example 6

Neuroprotective Effect of Angelica vulgaris Extract

To determine whether the neuroprotective effect of Angelica gigas extract was related to the decrease in body temperature, the effects of Angelica gigas extract were observed while maintaining normal body temperature.

First, rats were divided into groups for the experiment. The first group was the sham group, and the procedure and physiological saline administration were the same, but the brain ischemia was not caused, and the second group was the no additive control group. After induction of cerebral ischemia in rats in the same manner as in Example 3, only 2.0 ml / kg of saline was injected intraperitoneally after 0 and 90 minutes. Groups 3, 4, and 5 were dissolved in 0.89% saline, and the volume of 2.0 ml / kg and the doses of 100, 250, 500 mg / kg, respectively, were 0 minutes after induction of cerebral ischemia. Rats were intraperitoneally injected at 90 minutes.

In the case of a drop in body temperature, the experiment was performed by separating the case of neglecting the drop of body temperature and the case of blocking the protective effect of neurons due to hypothermia while partially preventing the decrease in body temperature. In order to reduce body temperature, the thermostatic heater using the rectal temperature reflecting the brain temperature was maintained for 12 hours using a method of fixing at 37 ° C. The body temperature was allowed to enter the probe into the rectum at least 6 cm. Measured by insertion (Miyazawa T et al. , J. Cereb. Blood Flow Metat. 1992; 12: 817-822).

Next, the hippocampal tissue sections of the rats were prepared in the same manner as in Example 3, and the neuroprotective ability was improved by obtaining the average value of normal CA1 vertebral neuronal cell numbers within 1 mm of the sections. Measured. The measured value is the mean ± standard deviation, and the data values for each group were analyzed by Student's t-test comparing the control group and each sample group (** p <0.01; see FIG. 3), and the results are shown in Table 1 below. It was.

Neuroprotective Effect of Angelica Extract. Normal control No addition control Angelica Extract 100 (mg / kg) 250 (mg / kg) 500 (mg / kg) Average cell count 287 32 213 258 244 Standard Deviation 8.2 9.7 13.7 11.1 6.7 Number of measurements 5 5 4 3 2 p-value 0.000 0.000 0.000 Neuronal cell protection rate (%) 0.0 71.0 88.7 83.3

p-value: confidence interval

As shown in Table 1, in the normal control (sham) body temperature adjusted to 37 ℃ the average number of cells per 1 mm 2 × height × 287 ± 8.2, whereas in the no-added control group was 32 ± 9.7 showed neuronal apoptosis It could be confirmed that a lot happened.

In the experimental group administered 100, 250 and 500 mg / kg, the mean number of cells was 213 ± 13.7 cells / mm, 258 ± 11.1 cells / mm and 244 ± 6.7 cells / mm, respectively. By confirming that 71.0%, 88.7% and 83.3% of the neurons are alive, it was confirmed that they show a very good neuronal protective effect (p <0.01; see FIG. 3).

Example 7

Acute Toxicity Experiment

1. Oral administration

ICR-based mice (25 ± 5 g, central laboratory animals) and SD mice (Sprague Dawley) were divided into four groups of 10 animals each, and the oral extract of the present invention was orally administered at doses of 500, 725, 1000 and 5000 mg / kg, respectively. Administered. Two weeks after oral administration, toxicity was not observed in all four groups, and no apparent symptoms were found in the control group.

2. Intraperitoneal administration

ICR-based mice (25 ± 5 g, central laboratory animals) and SD mice (Sprague Dawley) were divided into four groups of 10 animals each, and then the Angelica extract of the present invention was intraperitoneally administered at doses of 25, 250, 500 and 725 mg / kg, respectively. Administered. Toxicity was observed for 24 hours after intraperitoneal administration. None of the four groups died and no symptoms were observed.

Preparation Example 1

Angelica extract 100 mg of Example 1

Sodium Metabisulfite3.0 mg

Methylparaben0.8 mg

Propylparaben0.1 mg

Sterile Distillation Amount for Injection

In the usual manner, the above ingredients are mixed to prepare a final volume of 2 ml, and then filled in ampoules and sterilized to prepare an injection.

Preparation Example 2

Angelica extract 200 mg of Example 1

Lactose 100 mg

Starch 100 mg

Magnesium stearate proper amount

A tablet is prepared by mixing and tableting the above components according to a conventional tablet manufacturing method.

Preparation Example 3

Angelica extract 100 mg of Example 1

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Preparation Example 4

Angelica extract 1000 mg of Example 1

20 g of sugar

Isomerized sugar 20 g

Lemon flavor

Add 100 ml of purified water

According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Angelica extract and pharmaceutical preparations containing the same according to the present invention are not only excellent in inhibiting the death of nerve cells by the causes of cerebral ischemia, but also have no toxicity, thus preventing and treating brain diseases caused by the death of nerve cells. It is very effective when used for the purpose.

Claims (3)

Angelicae Gigantis Radix Angelicae Gigantis Radix Extracted by Angelicae Gigantis Radix with water, lower alcohol, hexane and ethyl acetate or a mixed solvent thereof, concentrated and lyophilized. The sugar extract according to claim 1, wherein the lower alcohol is selected from the group consisting of methanol, ethanol and butanol. A pharmaceutical preparation for the treatment and prevention of stroke comprising Angelicae Gigantis Radix extract as an active ingredient.