KR100526404B1 - Composition for preventing neuronal cell death caused by cerebral ischemia continiaing extract from astragali radix root - Google Patents

Abstract

 The present invention relates to a composition for preventing cerebral ischemic nerve cell damage, including an extract of Astragalus. In particular, the present invention provides a composition for preventing cerebral ischemic nerve cell damage, which is harmless to the human body and does not cause side effects, and can be used as a food or a drug to prevent diseases caused by nerve cell damage.

Description

Composition for preventing cerebral ischemic neuron damage, including Astragalus extract {COMPOSITION FOR PREVENTING NEURONAL CELL DEATH CAUSED BY CEREBRAL ISCHEMIA CONTINIAING EXTRACT FROM ASTRAGALI RADIX ROOT}

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a composition for preventing neuronal cell death by cerebral ischemia comprising an extract of Astragalus, and more particularly, to a composition for preventing Astragalus extract and effectively preventing cerebral ischemic disease using the same in the hippocampal tissue CA1 region. will be.

[Private Technology]

Recently, cerebrovascular disease has emerged as a national problem among various diseases in Korea. Cerebrovascular disease is on the rise with increasing incidence of elderly population and is the leading cause of death.

Cerebrovascular disease can be classified into hemorrhagic cerebrovascular disease and ischemic cerebrovascular disease. Ischemic cerebrovascular disease can be subdivided into thrombosis, em bolism, transient ischemic attack, and infarction. Pathological abnormalities have occurred in the blood vessels that supply blood flow. When brain ischemia, supply of cell energy and oxygen to brain tissue is not smooth and neural function abnormality and neural cell necrosis occurs.

Therapeutic drugs for cerebral ischemic diseases include platelet aggregation inhibitors or antithrombin agents such as ticlopidine, cilostazole and prostacycline. These drugs often have side effects, such as headaches, palpitations, and burden on the liver, which limits their use.

In Korea, although the effects of preventing and treating cerebral neuronal cell damage caused by cerebral ischemia have not been proven, DHEA and melatonine, which are aging-related health foods, are sometimes abused.

There is a need for the development of drugs that can effectively prevent neuronal damage caused by cerebral ischemia without side effects.

In order to solve the problems of the prior art, an object of the present invention is to provide a plant extract that is harmless to the human body and can prevent nerve cell damage caused by cerebral ischemia.

It is another object of the present invention to provide a composition for preventing cerebral ischemic nerve cell damage.

In order to achieve the above object, the present invention provides a composition for preventing cerebral ischemic nerve cell damage, which comprises the extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for preventing nerve cell damage comprising an extract of Astragalus which can effectively prevent cerebral ischemic nerve cell damage.

Astragali Radix is a perennial herb that belongs to the legumes (Leguminosae) plant, peeled and dried. It grows in the northern and northern regions of Korea. Actions, diuretic effects, etc. have been reported.

Astragalus extract of the present invention is provided by extracting with water, alcohol or organic solvent. Preferably, the dried supernatant is immersed in at least one solvent selected from the group consisting of water, an alcohol and an organic solvent to obtain a supernatant, and then powdered by evaporation or lyophilization. Ethanol, methanol, butanol or propanol may be used as the alcohol, and alcohol may be used as a 100% stock solution, but 90 to 50% alcohol may be used. The organic solvent may be hexane, chloroform or ethyl acetate. However, the solvents are not limited to the above examples, and all known solvents may be used. A specific extraction method is to immerse by adding a solvent 1: 5 to 6 weight ratio in the dried sulfuric acid, then filtered it and then depressurized, concentrated and lyophilized.

In one embodiment of the present invention, Astragalus extract was prepared using a solvent 70% ethanol, it was confirmed that the Astragalus extract can significantly prevent neuronal damage caused by cerebral ischemia.

Accordingly, the present invention provides a composition for preventing cerebral ischemic nerve cell damage, which includes the extract of Astragalus as an active ingredient.

The composition for preventing cerebral ischemic nerve cell damage may include only extract of Astragalus, but may further include a pharmacologically acceptable carrier or excipient according to the formulation and the method of use. In this case, the content of Astragalus extract in the composition for preventing cerebral ischemic nerve cell damage is preferably 0.001 to 50% by weight. When the content of the Astragalus extract is less than 0.001% by weight, a large amount of administration may be necessary for effective efficacy, and when it exceeds 50% by weight, the efficacy may be constant compared to the amount of use, which may be uneconomical. However, most preferably, according to the method of use and purpose of using the composition for preventing cerebral ischemic nerve cell damage, it is preferable to appropriately adjust the content of Astragalus extract.

The pharmacologically acceptable carrier or excipient may use a conventional material depending on the formulation, and representative examples thereof include water, dextrin or saline.

Ischemic nerve cell damage preventing composition can be used as a drug or food, and when used as a drug, the administration method can be oral or parenteral. The formulation of the composition may vary depending on the method of use, but may include PLASTERS, GRANULES, LOTIONS, POWDERS, Syrups, LIQUIDS AND SOLUTIONS, and Aerosols. AEROSOLS, OINTMENTS, FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, INFUSIONS, TABLETS, INJECTIONS, CAPSULES AND PILLS Or the like.

In addition, the composition for preventing ischemic nerve cell damage may be used for the purpose of protecting nerve cells caused by cerebral ischemic disease. The dosage of the composition is preferably determined in consideration of the age, sex, condition, absorption of the active ingredient in the body, inactivation rate and the drug used in combination, and 50 mg / kg (based on the daily Astragalus extract) Body weight) to 500 mg / kg body weight.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1: Preparation of Astragalus Extract

After grinding finely dried 100 g of Astragalus, 600 ml of 70% alcohol for drinking ethanol was added thereto, followed by immersion at 50 ° C. for 2 hours. The extract was concentrated using a vacuum rotary evaporator to completely remove ethanol. Thereafter, the extract was completely removed (9.8 g) for 48 hours using a freeze dryer. One-tenth of the extract thus obtained was dissolved in 10 ml of water as a stock solution (98 mg extract / ml water) and used for the experiment.

Example 2: Verification of neuroprotective effect of Astragalus extract during cerebral ischemia

The method of measuring the prevention and improvement effect of neuronal cell death by cerebral ischemia is anesthesia of gerbil and the ligation of the common carotid a. The method of measuring the lactate dehydrogenase concentration according to the damage is the most widely accepted, and the above two methods to verify the efficacy of the Astragalus extract.

2-1. Lactate dehydrogenase ( in vitro )

PC12 cells were used to measure neurite growth and expression of growth factors over time.

In addition, lactate dehydrogenase (LDH) concentration is secreted from the cultured cells into extracellular cultures to induce neuronal damage through hypoxic environment by administering COCl ₂ to PC12 cells. Was measured. This was measured using a microplate reader by taking a cell culture solution from 20-24 hours when the secretion was almost completed from damaged and destroyed cells.

Astragalus extract prepared in Example 1 was treated with 0, 10, 50, 100, 500 and 1000 ㎍ / ㎖ in PC12 cells before or after hypoxic environment induction, respectively. PC12 cells were cultured at 37 ° C. for 20 to 24 hours to obtain a cell culture medium. The concentration of lactate dehydrogenase in the culture medium was measured using an Beckman DU-640 Absorbance Spectrometer using Zhong Sheng Biotech standard reagent. dynamic) method.

The result is as shown in FIG. In the case of pretreatment of the Astragalus extract before induction of hypoxic environment in FIG. Thus, Astragalus extract significantly inhibits neuronal death.

2-2. Animal testing

Animal experiments confirmed the neuronal cell death inhibitory effect of Astragalus extract.

end. Breeding and Administration of Laboratory Animals

As test animals, 60 male and female Mongolian gerbils ( Meriones unguiculatus ) weighing 65-75 g were used.

Experimental animals were bred at a temperature of 23 ± 2 ℃ and a relative humidity of 55 ± 10% under constant light and dark cycles from 7 am to 7 pm. Food and water were taken freely.

I. Experiment

In the experiment, 0.5 ml oral was administered orally using an esophageal needle 30 minutes before ischemia induction or 30 minutes after ischemia induction. No substance was administered as a control.

The experimental animals were subjected to general anesthesia with 3% isoflurane (isoflurane, Baxtor, USA) in a gas mixture of nitrogen and oxygen 7: 3, and the operation was performed while maintaining anesthesia with 2.5% isoflurane in the same gas. After cutting and disinfecting the hair of the neck, the incision was made to expose both common carotid arteries, ligation was carried out using an aneurysm clip (aneurysm clip, Staelting, USA) for 5 minutes, and then the clip was removed. Reperfusion was performed and normal group underwent sham operation. At this time, each experimental group confirmed the complete occlusion of the entire artery by observing the blood circulation of the central artery of retina using an ophthalmoscope. During induction of ischemia, a rectal thermometer was inserted to measure body temperature, and the body temperature was kept constant at a normal body temperature of 37 ± 0.3 ° C. using a thermal pad automatically adjusted according to the temperature of the experimental animal.

The control group and each experimental group were anesthetized by intraperitoneal injection of thiopental sodium at 40 mg / kg body weight four days after induction of cerebral ischemia, and containing 1000 IU of heparin per 1,000 ml. Physiological saline at 4 ° C. was injected into the left ventricle and washed perfusion. After the perfusion wash, the animals were immediately perfused with 4% paraformaldehyde (in 0.1 M phosphate buffer; PB, pH 7.4) at 4 ° C.

After perfusion fixation, the brain was removed by using a bone cutter to extract the brain, and then fixed in the same fixative after 4-6 hours. The post-fixed brain was soaked in 30% sucrose solution (in 0.1 M phosphate buffer) until it sank to the bottom. Thereafter, the tissue was cut into a slide microtome (Sliding microtome, Reichert-Jung, Germany) to a thickness of 30 μm and placed in a 6 well plate containing a storage solution and stored at 4 ° C. until use.

From each tissue section, tissues with well-formed hippocampal formation were selected, and washed three times with 0.01 M PBS for 10 minutes to remove the preservatives from the tissues. It was placed on gelatinized slides and dried sufficiently at 37 ° C. After immersion in distilled water for a while and stained for 2 minutes in a 2% cresyl violet acetate (Sigma, USA) solution. The tissue is washed thoroughly with running water to remove excess dye from the slide, briefly immersed in distilled water, and then dehydrated and excess cresyl through 50%, 70%, 80%, 90%, 95% and 100% solution. Violet was washed. After confirming that the tissue was visible in the Nissle body (Nissle body), it was immersed in xylene (Junsei, Japan) and made transparent, and then sealed with Canada Balsam (Kanto, Japan).

Each organization photographed the CA1 area 1000 times with an Axioplan microscope (Carl Zeiss, Germany) with a digital camera attached. The part stained in purple was selected using an image analyzer (Optimas 6.5, USA) program to count neurons. One-way ANOVA test was performed to verify the significance of each group. The most common part of each group was selected and photographed using an Axiophot microscope (Carl Zeiss, Germany).

All. Experiment result

In animal experiments, the hippocampal tissues of the non-surgical negative control group were stained in FIG. 2A and FIG. And the same staining pattern as D. In FIG. 2, each of C and D is an enlarged photograph of a CA1 region of A and B at 400 times.

In the negative control group, neurons were observed in the CA1 region, whereas in the positive control group, cells dyed by ischemia and almost no stained cells were observed.

Looking at the results in the experimental group administered the Astragalus extract is as shown in FIG. In FIG. 3, A and B were treated with Astragalus extract 30 minutes before the induction of cerebral ischemia, C and D were treated with Astragalus extract 30 minutes after cerebral ischemia induction, A and C were male, B and D were female induced cerebral ischemia 4 After one day, the animal was sacrificed to observe neurons in the hippocampus. As a result, when Astragalus extract was administered before induction of cerebral ischemia in both males and females, cell death inhibitory effect was observed, so that the cells of CA1 region were stained darkly. . All pictures were taken 25 times.

In the results of FIG. 3, only the CA1 region of the hippocampus was magnified 400 times and shown in FIG. 4. In FIG. 4, the pre-cerebellar ischemia-treated group was observed to be similar to the negative control group, and it was confirmed that the cytoprotective effect was excellent.

In addition, the results of confirming the degree of neuronal cell death according to the application and the method of applying the Astragalus extract after ischemia-reperfusion was shown in FIG. 5. 5 is a negative control group (NC), positive control group (PC), pre-group treated with Astragalus extract before ischemia-reperfusion and post-group treated with Astragalus extract after ischemia-reperfusion. The cell number was divided by the number of neurons counted in the negative control group. Asterisks indicate efficacy at the 99% confidence level. In FIG. 6, a survival rate of about 11.2% was observed in the positive control group compared to the negative control group, but a higher neuronal survival rate was observed in the experimental group administered with Astragalus extract than in the positive control group. In particular, the survival rate was the highest in the experimental group administered with Astragalus extract before induction of cerebral ischemia, which showed about 90% survival rate. This pattern was not significantly different in females and males. In addition, when considering that the cell survival rate of 50-60% by Eselen, a comparative drug of cell survival rate, it can be seen that the neuroprotective effect of the Astragalus extract is remarkably excellent.

That is, in the above in vivo experiments, it was confirmed that the neuroprotective effect of the Astragalus extract was very excellent when cerebral ischemia occurred. Therefore, continuous administration of Astragalus extract will effectively prevent stroke.

As described above, the present invention confirms that the Astragalus extract can effectively prevent cerebral ischemic nerve cell damage, and provides it as a composition for preventing cerebral ischemic nerve cell damage. In addition, the present invention reveals a new use of Astragalus to increase the income of farmers producing Astragalus, can greatly contribute to public health.

Figure 1 confirms the neuronal cell death effect of Astragalus extract by lactate dehydrogenase measurement experiment.

2 is a photograph of hippocampal tissue staining of negative controls (A and C) and positive controls (B and D) when ischemia-reperfusion was performed on experimental animals.

Figure 3 is a photograph staining the hippocampus tissue of the experimental animal after administration of the Astragalus extract 30 minutes before ischemia-reperfusion (A and B) or 30 minutes after (C and D).

FIG. 4 is an enlarged photograph of only CA1 region 400 times in the hippocampus tissue observed in FIG. 3.

5 is a graph showing the survival rate of neurons after administration of the Astragalus extract to the experimental animals 30 minutes before or 30 minutes after ischemia-reperfusion.

Claims (5)

Ischemic cerebrovascular disease prevention composition comprising the extract as an active ingredient. The composition of claim 1, wherein the Astragalus extract is extracted with an ethanol aqueous solution. The composition of claim 1, wherein the Astragalus extract is a powder. The composition of claim 1, wherein the composition is a medicament. The ischemic cerebrovascular disease composition of claim 1, wherein the ischemic cerebrovascular disease is selected from thrombosis, embolism, transient ischemic attack and small infarction.