KR20020087723A - Extract of Rheum palmatum L. having neuroprotective effects and pharmaceutical composition containing the same - Google Patents

Abstract

PURPOSE: A pharmaceutical composition which contains an extract of rhizome of Rheum palmatum L. as an effective ingredient is provided. Therefore, the composition is used in prevention and treatment of cerebral disease selected from the group consisting of cerebral apoplexy, Parkinson's disease, senile dementia and Huntington's chorea caused by neuronal death. CONSTITUTION: Rhei Rhizoma is extracted in water, lower alcohol, hexane, ethyl acetate or a mixture thereof, concentrated and then freeze dried, wherein the lower alcohol is selected from the group consisting of methanol, ethanol and butanol. For example, 1kg Rhei Rhizoma is extracted with 2L methanol aqueous solution with a sonicator for 15min two times to produce 3L extract, which is filtered, concentrated at 45deg.C, at a pressure of 0.98atm, resulting in an average yield of 5% or more. The extract exhibits excellent neuronal death inhibitory activity and is administered at a daily doses of 10 to 5,000mg/kg.

Description

Extract of Rhubarb with Neuroprotective Effects and Pharmaceutical Formulations Containing the same {Extract of Rheum palmatum L. having neuroprotective effects and pharmaceutical composition containing the same}

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

Rhubarb is the dried root and root of Rheum palmatum L., a perennial herb belonging to Polygonaceae , and related myomas. It is known to have the efficacy of verbatim pain. In oriental medicine, it is used for the treatment of thyroid sarcoma, hepatic degenerative degeneration, hepatic vein congestion, constipation, jaundice, acute enteritis, acute conjunctivitis, upper respiratory tract, cholangiocarcinoma, etc. Used. Recently, it has been found that rhubarb has a similar action to interferon.

Rhubarb having the above-mentioned efficacy is senoside A, B, C, D, E, F (sennoside A, B, C, D, E, F), anthraquinone derivative, chrysophanol, emodine (emodin), aloe-emodin, lanein, rhein, physcion, citreorosein and others torachrysone 8-glucoside, 6-hydro It has the components of 6-hydroxymusizin, calcium hydroxide, cinnamic acid ester, resin, starch, etc. The main component is an anthraquinone derivative.

Various studies have been performed based on the pharmacological action of rhubarb as described above. In Korean Patent Publication No. 2000-61327, a composition for preventing and treating liver disease, and Korean Patent No. 195886 were used as a component of a composition for treating diabetes. In Korean Patent Publication No. 1988-1296, as an ingredient of an antidote composition, in Korean Patent Publication No. 1997-223, as an inhibitor of cholesterol biosynthesis, and in Korean Patent No. 3840, as a drug for treating gastrointestinal diseases, Korean Patent Publication No. 1999- In 53083 it was used as a component of the antimicrobial composition. However, to date, there is no known example of using rhubarb extract for the prevention and treatment of brain diseases such as stroke.

Accordingly, the inventors have conducted extensive research to confirm that rhubarb has an effect of inhibiting neuronal necrosis, and the present invention has been completed based on this.

Therefore, it is an object of the present invention to provide a rhubarb extract having a brain disease prevention and treatment effect.

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

Rhubarb extract for achieving the above object is prepared by extraction, concentration and lyophilization of Rheum palmatum L. with water, lower alcohol, hexane, ethyl acetate or a mixed solvent thereof.

Figures 1a and b is a microscopic picture of the coronal sections of the dorsal hippocampus of the normal control rats (sham) that did not cause ischemia is stained with Cresyl violet (microscopy).

1C and D show physiological saline after 1 hour and 30 minutes after inducing whole cerebral ischemia for 10 minutes, and after 7 days, coronal sections of the dorsal hippocampus of control rats were made of Cresyl violet. After dyeing with), it is taken with a microscope.

Figures 1e and f induce whole cerebral ischemia for 10 minutes, and then receive rhubarb extract after 1 hour and 30 minutes, and after 7 days, coronal sections of the dorsal hippocampus of rats treated with cresyl violet (Cresyl). It was stained with violet, and photographed under a microscope.

FIG. 2 is a graph showing the results of measurement of cerebral hippocampal CA1 region cells in each group of rats after administration of physiological saline or rhubarb extract after 1 hour and 30 minutes after induction of whole cerebral ischemia for 10 minutes with a normal control group.

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

Rhubarb 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, wherein the lower alcohol is selected from the group consisting of methanol, ethanol, and butanol, Methanol is preferred because of its high yield.

In the preparation of the rhubarb extract, the extraction process 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.

Rhubarb 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 action of inhibiting the death of neurons 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 neuronal protective activity of the rhubarb 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 those of human paralysis, and are considered to be the preferred animal models for paralytic 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.

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, in order to determine whether rhubarb extract of the present invention inhibits delayed neuronal death in the CA1 region of rat brain hippocampus, rats arechemia-induced intraperitoneally and rhubarb extract is administered intraperitoneally. Hippocampal tissue sections were prepared one week after reperfusion, at which point the damage occurred almost completely (see FIGS. 1A, 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 rhubarb extract after induction of cerebral ischemia, but was not found in the group that received the rhubarb extract. It was confirmed that it was defended from apoptosis, formed a normal pyramidale, and maintained its junction with surrounding cells.

Next, in the present invention, the effect of inhibiting neuronal cell death of Sukchangpo extract was observed while artificially maintaining normal body temperature after cerebral ischemia induction and administration of Sukchangpo extract. As a result of observing hippocampal tissue sections of the rats tested under the above conditions, it was confirmed that the cell survival rate was increased by about 7 to 10 times higher than the case where no rhubarb extract was administered.

In the present invention, the toxicity of the rhubarb extract was confirmed through oral administration and intraperitoneal administration to rats, and it was confirmed that there was no toxicity.

The inhibitory effect of the rhubarb extract confirmed by 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 91.8% of the rhubarb measured according to the present invention. In Chinese medicine, 17.8% (1,200 mg / kg) and 15.6% (600 mg / kg) of Cheonma and 18.4% (1,000 mg / kg) and 16.6% (500 mg / kg) of Daeseunggi-tang. (Kg) is known to have an inhibitory effect on neuronal cell death (Kor. J. Herbology vol. 14, No. 1, 1999), but is also lower than rhubarb extract.

Pharmaceutical formulations for the treatment and prevention of brain diseases of the present invention containing rhubarb extract as an active ingredient can 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 such solid preparations 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 rhubarb 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, rhubarb extract of the present invention was found to exhibit excellent neuronal cell death inhibitory activity, it can be used as a therapeutic and / or prophylactic agent for brain disease patients in the clinic.

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 Rhubarb Extract

Rhubarb was purchased from Jeongsan and used after it was verified by the Department of Herbal Medicine, College of Oriental Medicine, Kyung Hee University.

2 L of an 85% methanol aqueous solution was added to 1 kg of dried rhubarb, and extracted twice with ultrasonic waves 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 rhubarb suspension

The rhubarb extract obtained in Example 1 was suspended in 100 ml of water, and then fractionated sequentially according to polarity by solvent separation 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. The aneurysm clip was removed from the total carotid arteries of the four rats finally selected 5, 10, 20 and 30 minutes after ischemia induction to stop 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 in delayed 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

Neuroprotective Activity of Rhubarb Extracts

In order to observe delayed neuronal death in the CA1 region of hippocampus, rhubarb is induced in rats for 10 minutes in the same manner as in Example 3, followed by 90 minutes of rhubarb prepared in Example 1. The extracts were dissolved in 0.89% saline and then injected into the abdominal cavity in amounts of 1, 10, and 100 mg / kg, respectively, 2.0 ml per rat body weight. 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. 1A, 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 tissue of rats injected with physiological saline instead of rhubarb extract was used as the control group.

As a result, hippocampal neurons were observed normally in sham operated (see FIGS. 1A and B), and in the absence of control neurons, cells were released from surrounding cells as tissues relaxed, unlike normal neurons. It could be observed that the cell body also lost its original pyramidal form and was condensed to form a single cell. In addition, it was confirmed that the cell death (apoptosis) was achieved by the concentration of nuclear chromatin and the collapse of the nuclear membrane (see Fig. 1c, d).

Neurons in the rhubarb extract showed similar morphology to normal cells, and were easily distinguished due to their protruding perikaryon and centrally located round nuclei, and in a relatively healthy state, surrounding neutrophils. Is clearly distinguished from. In addition, it was confirmed that the majority of cells defended against apoptosis, formed a normal pyramidale shape, and continued to maintain junctions with surrounding cells (see FIGS. 1E and F).

Example 5

Neuroprotective Effect of Rhubarb Extracts

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 the rhubarb extract prepared in Example 1 in 0.89% saline, and the volume of 2.0 ml / kg and the doses of 1, 10 and 100 mg / kg, respectively, were 0 minutes after induction of cerebral ischemia. Rats were intraperitoneally injected at 90 minutes.

In order to maintain 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 kept at least 6 cm in the rectum into the rectum. 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 values are 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. 2), and the results are shown in Table 1 below. It was.

Neuroprotective Effect of Rhubarb Extract Normal control No addition control Rhubarb extract 1 (mg / kg) 10 (mg / kg) 100 (mg / kg) Average cell count 308 27 28 195 285 Standard Deviation 6.6 6.1 7.1 28.6 4.4 Number of measurements 11 7 2 4 2 p-value 0.000 0.500 0.482 0.000 0.000 Neuronal cell protection rate (%) 0.0 0.3% 59.7% 91.8%

p-value: confidence interval

Nerve cell protection rate (%) = (number of rhubarb extract administration group neurons-no control group neuron) / (normal control group neuron-no control group neuron) × 100

As shown in Table 1, in the normal control (sham) body temperature adjusted to 37 ℃ the average number of cells per 1 mm 2 × width × length was 308 ± 6.6, whereas in the no-added control group was 27 ± 6.1, neuronal death It could be confirmed that a lot happened.

In the experimental group to which rhubarb extract was administered 1, 10 and 100 mg / kg, the cell number averaged 28 ± 7.1 cells / mm, 195.9 ± 28.6 cells / mm and 285 ± 4.4 cells / mm, respectively, compared to the no-added control group, respectively. 0.3%, 59.7% and 91.8% of the neurons were confirmed that a very good neuronal cell protection effect was confirmed (p <0.01; see Figure 2).

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 4 groups of 10 animals each, and the rhubarb 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 rhubarb 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

Rhubarb extract of Example 1 100 mg

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

Rhubarb extract of Example 1 200 mg

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

Rhubarb extract of Example 1 100 mg

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

Rhubarb 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.

Rhubarb extract of the present invention and pharmaceutical preparations containing the same 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 (4)

Rhubarb extract prepared by extraction, concentration and lyophilization of Rheum palmatum L. with water, lower alcohol, hexane and ethyl acetate or a mixed solvent thereof. The rhubarb extract according to claim 1, wherein the lower alcohol is selected from the group consisting of methanol, ethanol and butanol. Pharmaceutical formulation for the treatment and prevention of brain diseases, containing Rheum palmatum L. extract as an active ingredient. The pharmaceutical formulation of claim 3, wherein the brain disease is selected from the group consisting of stroke, Parkinson's and senile dementia, and chorea.