KR100509440B1 - Pharamceutical preparation comprising extract of Yak-kong (Rhynchosia nolubilis) for Alzheimer's disease, dementia, amyosthenia of smooth muscle, and glaucoma - Google Patents

Abstract

The present invention relates to inhibitors of acetylcholinesterase (hereinafter also referred to as AChE) containing rat bean extract and pharmaceutical preparations and foods comprising the same.

The rat eye extract of the present invention is effective in treating and preventing various diseases caused by a decrease in the concentration of acetylcholine in the body, such as Alzheimer's disease, senile dementia, myasthenia gravis, asthenia of the gastrointestinal tract and bladder smooth muscle, and glaucoma without toxicity or side effects. When used in addition to foods and food supplements, it provides excellent effects such as memory facilitation, mental concentration, and prevention of dementia.

Description

Treating or preventing senile dementia including Alzheimer's disease containing rat bean extract, myasthenia gravis, asthenia of the gastrointestinal tract and bladder smooth muscle, and glaucoma disease, and pharmaceutical preparations comprising the same Alzheimer's disease, dementia, amyosthenia of smooth muscle, and glaucoma}

The present invention relates to inhibitors of acetylcholinesterase (hereinafter also referred to as AChE) containing rat bean extract and pharmaceutical preparations comprising the same, more specifically various diseases caused by a decrease in the concentration of acetylcholine in the body, namely Alzheimer's disease. A pharmaceutical agent for treating or preventing senile dementia, myasthenia gravis, asthenia of the gastrointestinal tract and bladder smooth muscle, and glaucoma disease, and a food comprising the same.

Acetylcholinesterase (AChE) is an enzyme that hydrolyzes acetylcholine, one of the neurotransmitters that mediate the activity of parasympathetic nerves in the body, to choline and acetate. Its function is most distributed in the cholinergic nerve and its surroundings, especially in the muscle nerve junction, and is an important enzyme found in plasma, liver and other tissues.

In general, the acetylcholine is secreted into a gap called synaptic cleft in the axon-end membrane of nerve cells, and binds to receptors on post-synaptic neuronal cell membranes to transfer action potentials. After delivering excitability, it is known to be degraded into choline and acetate by the enzyme AChE, which is then absorbed into cells and reused.

Active research has shown that many physical and mental disorders are deeply linked to abnormal levels of acetylcholine, which are associated with acetylcholine synthesis and degradation mechanisms.

In Alzheimer's disease, a typical senile dementia related to acetylcholine, the brain of the patient mostly dies of neuronal neurons at the base of the forebrain, and the neuronal dominant synapses and the cerebral cortex are degenerated. The death of these neurons has been reported to significantly reduce the production of acetylcholine, a neurotransmitter that plays an important role in memory.

As part of the treatment of Alzheimer's disease, maintaining high levels of acetylcholine in the brain significantly improves the memory of patients. Currently, in the clinical field, acetylcholine is reversibly inhibited by the degradation of acetylcholine as a treatment for Alzheimer's disease. The use of anti-cholinesterase (anti-AChE) to maintain the concentration of is preferred. Therefore, currently, acetylcholine degrading enzyme inhibitors such as tacrine and aricept are mainly used for the treatment of Alzheimer's disease. However, although some of these drugs are recognized, long-term high-dose administration often requires discontinuation of treatment due to side effects such as muscle pain, nausea due to gastrointestinal disorders, vomiting, diarrhea and loss of appetite, and liver toxicity. Problems such as this have been pointed out.

According to another pathological study of the choline nervous system, gastrointestinal motility is regulated by nerve stimulation, and acetylcholine has been reported to promote this gastrointestinal motility. Therefore, neostigmine, which is a type of anticholinesterase, may be used for the treatment of postoperative bloating due to intestinal obstruction or difficulty urination due to inability of bladder detrusor.

In addition, myasthenia gravis is a neuromuscular disorder caused by the destruction of acetylcholine receptors in the muscle membranes due to autoimmune diseases and the inability of acetylcholine released from the nerve endings to the muscle membranes. As part of the treatment, acetylcholine degradation inhibitors such as mestinone and neostigmine are used.

Mestinone, which is most commonly used in the treatment of myasthenia gravis, also intends to increase the concentration of acetylcholine in the body by inhibiting the action of acetylcholinesterase, which breaks down the neurotransmitter acetylcholine, so that acetylcholine stays on muscle receptors for a long time. As a therapeutic agent developed in, there is an advantage that the effect appears within a short time when taking, but often complain of abdominal pain or diarrhea depending on the dose of the drug. In addition, AChE inhibitors used to treat myasthenia gravis, including mestinone, lack the selective discrimination of nicotine and muscarinic acetylcholine receptors, resulting in side effects from the excitation of muscarinic receptors in patients with drugs. However, the absorption rate is very low, and the therapeutic effect may be partially, so the treatment range is narrow.

In addition, it has been reported that AChE inhibitors effectively lower intraocular pressure by promoting waterproof resorption in glaucoma, an ocular disease that causes visual impairment due to optic nerve damage due to increased intraocular pressure. However, currently developed AChE inhibitors require special care because they are at risk of developing cataracts when used for 6 months or longer to treat glaucoma.

In order to effectively treat and prevent various diseases caused by the decrease in the level of the neurotransmitter acetylcholine, alternative methods of promoting the synthesis of acetylcholine by administering acetylcholine precursors such as choline have been suggested. Due to the drawback of failing to cross the cerebrovascular barrier and ending in a short time, drug treatments that currently inhibit the hydrolytic action of acetylcholine using an acetylcholinesterase inhibitor such as tacrine, as described above, are mainly used. It is used. However, currently developed and commercially available acetylcholine degrading enzyme inhibitors are reported to have side effects such as liver disorders caused by toxicity, gastrointestinal disorders such as abdominal pain or diarrhea, and muscle pain.

Accordingly, the present inventors have continued the scientific research on a new substance having reduced toxicity or side effects compared to conventional acetylcholinease inhibitors, and found an inhibitory activity of acetylcholinesterase in rat eye extract. To complete.

The present invention provides a mouse eye extract that can be used as an inhibitor of acetylcholine degrading enzyme in order to effectively inhibit the activity of acetylcholinesterase to treat and prevent various diseases related to the concentration of acetylcholine in the body without toxicity or side effects. For the purpose of

In addition, the present invention is useful for treating and preventing various diseases caused by a decrease in the concentration of acetylcholine in the body, that is, senile dementia including Alzheimer's disease, myasthenia gravis, ataxia of the gastrointestinal tract and bladder smooth muscle, and glaucoma without toxicity or side effects. It is to provide a pharmaceutical formulation comprising a rat bean extract as a component.

Furthermore, the present invention is to provide a food comprising rat bean extract that can be ingested for the purpose of preventing the disease, promoting memory, and the like by inhibiting the decomposition of acetylcholine.

The present invention relates to an inhibitor of acetylcholinesterase (AChE) containing rat bean extract and pharmaceutical preparations comprising the same, more specifically various diseases caused by a decrease in the concentration of acetylcholine in the body, namely Alzheimer's disease, including A pharmaceutical agent for treating or preventing senile dementia, myasthenia gravis, asthenia of the gastrointestinal tract and bladder smooth muscle, and glaucoma disease, and a food comprising the same.

Rat eye extract is a solution extracted from various kinds of organic solvents, such as methanol, ethanol and acetone, of Seomoktae (Rhynchosia nolubilis), a legume (Leguminosae) plant Processed material.

Rat eye beans are superior to any food and have high fiber, which contributes to the activation of intestines. Especially, they are known as medicinal beans because of their excellent medicinal properties. They are known as medicinal beans. Research into the composition and efficacy of the trend is ongoing.

As a characteristic ingredient not found in general foods, the rat eye bean contains a lot of saponins, fats, vitamins, minerals, etc., together with genistein and daidzein belonging to isoflavones.

Isoflavones are known to be effective in chronic diseases such as osteoporosis, kidney failure and heart disease in addition to anti-cancer effects. Rat eye also has a lipid-lowering effect in blood, which is said to be effective in preventing cardiovascular diseases such as hypertension and arteriosclerosis. Loss-inhibiting effects have been reported, and there are ongoing researches on new efficacy of rat eye extract in various fields.

The inventors of the present invention have also completed the present invention, confirming that the substance exhibits the action of inhibiting the activity of acetylcholinesterase in the body during extensive studies on the efficacy of the rat bean extract.

Acetylcholinesterase is an enzyme that hydrolyzes acetylcholine, one of the neurotransmitters in the body, to choline and acetate, and is closely related to the increase and decrease of acetylcholine in the body. Therefore, in order to treat and prevent various diseases caused by abnormal concentration of acetylcholine, namely Alzheimer's disease, senile dementia, myasthenia gravis, weakness of gastrointestinal tract and bladder smooth muscle, and glaucoma, a substance which inhibits the action of acetylcholinesterase as a therapeutic agent I use it.

Acetylcholinesterase inhibitory activity against rat eye extract of the present invention was determined using acetylcholinesterase extracted from cultured PC12 cell line, as described in Chung YK, Heo HJ, Kim EK, Kim HK, Huh TL, Lim. Y, Kim SK, Shin DH. Mol Cells 2001; 11: 137].

Homogenation was carried out with a sufficient amount of 10 mM Tris-HCl (pH 7.2) containing 1M NaCl, 50mM MgCl ₂ and 1% Triton X-100 in a culture PC12 cell line, followed by centrifugation to remove acetylcholinestera from the cell line. The extract was used as an enzyme and 0.5mM acetylthiocholine and 1mM 5,5'-dithio-bis (2-nitrobenzoic acid) were used as substrates. Each enzymatic reaction was carried out on tacrine samples which are widely used.

After incubating the reaction solution in which the substrate solution was added to the enzyme for 30 minutes, the absorbance was measured at a wavelength of 405 nm, and the enzyme activity inhibition was determined from the following equation (1):

% Enzyme activity inhibition = {1-[(SR-SC) / (ER-EC)]} x 100

Where

SR is the absorbance value measured in the sample reaction with enzyme inhibitor,

SC is the absorbance value using a buffer instead of enzyme as a control of the sample reaction,

ER is the absorbance value measured in the enzyme reaction without adding a sample,

EC shows absorbance values using buffer instead of enzyme as a control of the enzyme reaction.

As a result, it was possible to obtain a graph of the enzyme activity inhibition for rat eye extract as shown in FIG. According to this graph, the IC ₅₀ value of the rat bean extract was found to be about 1.9 mg / ml, and the concentration of 10 mg / ml was found to inhibit the activity of the enzyme by 80% or more.

In addition, as an acute toxicity test for the rat eye extract of the present invention, seven Sprague-Dawley-based rats were administered daily 500 mg per kg, but was tested daily, 7 There was no death by day.

As can be seen from the above test results, the acetylcholinesterase inhibitory activity against the rat bean extract of the present invention shows a lower value than that of tacrine, which is currently mainly used as an anticholinesterase substance. Since the rat eye extract is a natural physiologically active substance as is known, it is relatively safe in terms of side effects or acute toxicity when administered to humans or animals, unlike conventional acetylcholinesterase inhibitors such as tacrine and aricept. As a component, it can be said to be an excellent material that can be safely used without problems substantially even in a large dose.

Accordingly, the present invention provides an acetylcholinesterase inhibitor containing rat bean extract.

In addition, the present invention, in the treatment of various diseases caused by abnormal concentration of acetylcholine, namely Alzheimer's disease, senile dementia, myasthenia gravis, asthma of the gastrointestinal tract and bladder smooth muscle, and glaucoma, the active ingredient together with a pharmaceutically acceptable carrier It provides a pharmaceutical formulation comprising a rat bean extract.

In addition, the present invention provides a food or food supplement comprising rat bean extract for effectively inhibiting the decomposition of acetylcholine in the body, thereby preventing the disease and promoting memory.

When prepared in the form of a pharmaceutical preparation according to the present invention, the rat eye extract is combined with a pharmaceutically acceptable conventional carrier and, depending on the purpose of administration, tablets, hard or soft capsules, chewing tablets, powders, solutions or It may be formulated in the form of preparations for oral administration such as suspensions or preparations for parenteral administration such as injectable solutions or suspensions.

In the case of formulating the active compounds of the present invention into tablets, capsules, chewing tablets, powders, solutions, suspensions and the like for the purpose of oral administration, binders such as gum arabic, corn starch, microcrystalline cellulose or gelatin , Excipients such as dicalcium phosphate or lactose, disintegrants such as alginic acid, corn starch or potato starch, lubricants such as magnesium stearate, sweeteners such as sucrose or saccharin and flavors such as peppermint, methyl salicylate or fruit flavor In the case where the dosage unit form is a capsule, a liquid carrier such as polyethylene glycol or fatty oil may be included in addition to the above components.

In addition, injections in the form of solutions or suspensions for parenteral administration can be administered parenterally, eg, subcutaneously, intravenously, intramuscularly, or intraperitoneally. Generally, an injectable solution or suspension is an effective amount in a pharmaceutically acceptable liquid carrier such as water, saline, aqueous dextrose and related sugar solutions, nonvolatile oils, glycols such as ethanol, glycerin, polyethylene glycol, propylene glycol, and the like. It can be prepared by homogeneously mixing the active compounds of. In addition, if necessary, auxiliary agents such as antibacterial agents, chelating agents, buffers, and preservatives may be included.

As the above-mentioned pharmaceutically acceptable carrier, any adjuvant can be used which is pharmaceutically pure, substantially non-toxic, and which does not inhibit the action of the active ingredient.

The acetylcholinesterase inhibitory effective amount referred to herein is a dose necessary to sufficiently achieve the effect of inhibiting the activity of the enzyme acetylcholinesterase, and the type of disease and the severity of the disease that the patient suffers from Depending on the sex, age, weight and health of the patient, mode of administration, frequency and time of administration, the use of concomitant medications and other relevant circumstances, an appropriate range may be determined at the discretion of the patient's physician.

The pharmaceutical preparation containing the rat eye extract of the present invention as an active ingredient is 1 to 500 mg, preferably 50 to 200 mg per 1 kg of body weight per day for oral administration once to several times a day, preferably once to several times. In the case of injection administration, in the case of injection administration, 0.5 to 250 mg, preferably 10 to 100 mg per kg of body weight, may be administered once to several times a day, preferably once to three times. have.

In addition to the pharmaceutical preparations described above, the mouse eye extract of the present invention may be formulated in a suitable amount to conventional beverages, such as soft drinks, mineral water, alcoholic beverages, chewing gum or caramel products, candy, ice cream, confectionary, or the like, or include vitamins or minerals. It can also be prepared in the form of a food or food supplement by incorporating it into a dietary supplement or food additive.

In the case of the food containing the rat eye extract, it is possible to obtain the effect of improving the memory when ingested continuously for a long time and preventing the disease such as preventing dementia.

Hereinafter, the acetylcholinesterase inhibitory activity test and specific formulation of the rat eye extract of the present invention will be described in more detail based on the following examples.

However, these examples are provided to illustrate and specifically describe the present invention, and the scope of the present invention should not be understood as being limited thereto.

Example 1 Preparation of Rat Eye Extract

1 kg of dried rat bean was selected and 2 liters of ethanol aqueous solution was added, and it was divided into a predetermined amount and ultrasonically pulverized for 15 minutes or more to extract the active ingredient from the rat bean.

After ultrasonic extraction, each was combined and filtered, and then the organic solvent was distilled off and concentrated under reduced pressure. The obtained concentrate was freeze-dried to obtain a rat bean extract in a yield of 5% or more, which was used as a sample and active ingredient of the following Example 2-7.

Example 2 Inhibitory Activity of Acetycholine Esterase from Rat Extract

In this example, the following experiment was conducted to demonstrate the inhibitory activity of acetylcholinesterase of rat eye extract.

Acetylcholinesterase experiments to determine the inhibitory activity against acetylcholine degradation of enzymes are described in Chung YK, Heo HJ, Kim EK, Kim HK, Huh TL, Lim Y, Kim SK, Shin DH. Mol. Cells 2001; 11: 137].

Acetylcholinesterase enzyme was extracted from PC12 cell (obtained from the Korean Cell Line Bank) culture and used as a comparative substance for enzyme inhibitory activity with the rat eye extract obtained in Example 1 as an enzyme inhibitor sample. Tacrine, the most commonly used anticholinesterase substance in the clinical field, was purchased from Sigma Co., USA.

First, a 10 mM Tris-HCl (pH 7.2) solution containing 1 M NaCl, 50 mM MgCl ₂ and 1% Triton X-100 was added to a culture in which PC12 cell line was incubated in Dulbecco's Modified Eagle Medium (DMEM) medium for 7 days. Enzymes were prepared by adding 5 times the amount of PC12 cells, homogenizing homogeneously, centrifuging at 10,000 g for 30 minutes and separating the supernatant.

 Rat eye extract is 0.1, 0.2, 0.3, 0.6. It was dissolved in distilled water so as to have a concentration of 1.3, 2.5, 5 and 10 mg / ml, and tacrine used as a comparative substance was dissolved in distilled water at a concentration of 1, 10, 100, 1,000, 10,000 nM.

To each well of a 96-well plate, 30 μl of 50 mM phosphate buffer (pH 8.0), 10 μl of the mouse bean extract solution of this concentration and 10 μl of enzyme AChE were added as a sample, followed by 0.5 mM acetylthio as a substrate solution for the enzyme. 50 μl of 50 mM phosphate buffer (pH 8.0) containing choline (acetylthiocholine) and 1 mM 5,5′-dithio-bis (2-nitrobenzoic acid) [5,5'-dithio-bis (2-nitrobenzoic acid)] 100 μl of the reaction solution was prepared by sample concentration, and the reaction solution was incubated for 30 minutes in an incubator at 37 ° C.

After incubation, the absorbance was measured at an wavelength of 405 nm with an ELISA reader to obtain an SR value, that is, an absorbance value measured after the reaction of the sample to which the enzyme inhibitor was added.

As a control for the sample reaction, after incubating the buffer instead of the enzyme, the absorbance was measured in the same manner to obtain an SC value.

Subsequently, in order to determine the enzyme reactivity when no enzyme inhibitor was added, a buffer solution was added instead of the sample to obtain the absorbance ER value. As a control, a buffer solution was added instead of the sample and the enzyme, and then the absorbance EC value was obtained.

As a result of the enzyme activity inhibition calculated by the above formula (1) from the absorbance values SR, SC, ER and EC values measured above, a graph as shown in FIG. 1 was obtained.

According to the results, the IC ₅₀ value of the rat bean extract was found to be about 1.31 mg / ml, and the concentration of 5 mg / ml was found to inhibit the activity of the enzyme by 60% or more.

As for the comparative substance of enzyme inhibitory activity, tacrine was also tested for enzyme activity inhibition by the same method and procedure as that performed in the rat bean extract. As a result, the IC ₅₀ value of tacrine was about 130nM.

Acetylcholinesterase inhibitory activity against the rat eye extract of the present invention shows a lower value than that of tacrine, but the mouse eye extract is a bioactive substance with excellent biosafety and little toxicity as it is known. Considering the serious side effects of conventional AChE inhibitors such as tacrine, liver disorders due to toxicity, gastrointestinal disorders such as abdominal pain and diarrhea, and muscle pain, the acetylcholinesterase inhibitory activity of the rat eye extract of the present invention is very high. It is a meaningful figure.

Example 3 Preparation of Tablets Containing Root Bean Extract

Component content

Mouse Eye Bean Extract 200mg

Corn Starch 68mg

Lactose 90mg

Microcrystalline Cellulose 40mg

Magnesium Stearate 2mg

According to the conventional method of preparing tablets, the above ingredients were added to the indicated contents, mixed uniformly, stirred and granulated. After drying, using a tableting machine, a tablet of interest was prepared containing 200 mg of rat eye extract, an active ingredient per tablet.

Example 4 Preparation of Capsules Containing Rat Eye Extract

Component content

Mouse Eye Bean Extract 200mg

Corn Starch 68mg

Lactose 90mg

Microcrystalline Cellulose 40mg

Magnesium Stearate 2mg

According to the conventional method for preparing the capsules, the above-mentioned ingredients were added to a given content and mixed uniformly, and then, the desired capsules were prepared by filling into gelatin capsules of an appropriate size such that 200 mg of the mouse bean extract per capsule was included.

Example 5 Preparation of Chewing Chewing Tablets Containing Rat Eye Bean Extract

Component content

Mouse Eye Bean Extract 200mg

240mg granulated sugar

Fragrance (Pepamine or Lemon) 2mg

Corn Starch 60mg

Mannitol 100mg

Magnesium Stearate 20mg

Purified water

According to a conventional method of manufacturing chewing tablets, the above ingredients are mixed in an appropriate amount of water, dissolved while heating, cooled, and put into a molding machine to prepare chewing tablets of a desired shape so as to contain 200 mg of eye bean extract per one. It was.

Example 6 Preparation of Candy Containing Rat Eye Extract

Component content

Mouse Eye Bean Extract 200mg

Granule Syrup 640mg

Fragrance (Pepamine or Lemon) 2mg

Starch syrup 230mg

50% tartaric acid 20mg

Appropriate amount of purified water

The granulated sugar syrup was heated to 110 ° C. while completely dissolving in a small amount of water, and then the remaining water and syrup was dissolved, and the temperature was raised to 145 ° C. The fire was turned off, the fragrance and tartaric acid were added, mixed, cooled to 75 to 80 ° C., and then molded with a molding roller to prepare candy containing the bean sprout extract.

Example 7 Preparation of Drinks Containing Root Bean Extract

Component content

Mouse Eye Bean Extract 200mg

2 g of concentrated juice

Sucrose 12g

100 mg sodium citrate

Fragrance 70mg

Water

According to a conventional method for preparing a beverage, the above ingredients are mixed in an appropriate amount of water in a given amount, heated to dissolve, cooled, and filled into a container, which contains 200 mg of bean sprout extract per bottle of 200 ml of beverage. A beverage was prepared.

Rat eye extract according to the present invention is an effective acetylcholinease inhibitor that can inhibit the activity of acetylcholinesterase, in the form of a pharmaceutical preparation containing it as an active ingredient, senile dementia, including Alzheimer's disease, severe It can be effectively used for the treatment and prevention of various diseases caused by the decrease in the concentration of acetylcholine in the body, such as ataxia, gastrointestinal and bladder smooth muscle weakness and glaucoma.

In particular, rat eye extract is a natural active substance extracted from rat eye bean (Seomoktae, medicinal soybean), and has excellent biosafety. Therefore, rat eye extract is toxic compared to conventional acetylcholinesterase inhibitors. There is an advantage that can be safely administered to patients suffering from related diseases without the same side effects.

In addition, the rat eye extract of the present invention can be added to various foods or used as a food supplement, thereby obtaining effects such as memory promotion or mental concentration and dementia prevention by improving the function of the neurotransmitter system.

1 is a graph showing that the rat eye extract of the present invention has a concentration-dependent effect of inhibiting the activity of acetylcholine degrading enzyme.

Claims (4)

Pharmaceutical preparations for the treatment or prevention of senile dementia, myasthenia gravis, ataxia of the gastrointestinal tract and bladder smooth muscle, and Alzheimer's disease, characterized by comprising a pharmaceutically acceptable carrier, together with an extract of rats eye as an active ingredient. . delete delete delete