KR100552995B1 - The extracts of Pueraria thunberginia which is effective on improvement of brain function and the methods for its manufacture - Google Patents

Abstract

The present invention relates to a gallium extract having a brain function improving effect and a method for producing the same, more particularly, extracts obtained from gallium ( Pueraria thunberginia ) inhibit the caspase-3 activity in vitro experiments. In vivo experiments significantly reduce memory loss caused by 192-saporin, which destroys neurons, and acetylcholine esterase (AchE) and choline acetyltransferase. By enhancing the activity of ChaT), it shows the effect of improving brain function, and in particular, it can be used as an anti-aging and senile dementia prevention and treatment medicament or health food, and also relates to a method of preparing a browning extract.

Aging, senile dementia, browning extract, caspase-3, 192-saporin, acetylcholine esterase (AchE), choline acetyltransferase (ChaT)

Description

The extracts of Pueraria thunberginia which is effective on improvement of brain function and the methods for its manufacture}             

Figure 1 shows the antioxidant efficacy of gallium extract.

Figure 2 shows the effect of inhibiting apoptosis of the browning extract.

Figure 3 shows the effect of reducing the memory, learning ability induced by the 192-saprine (saporine) of the gallium extract.

Figure 4 shows the efficacy of enhancing the cholineacetyl transferase activity of the gallium extract.

Figure 5 shows the efficacy of enhancing the acetylcholine esterase activity of the gallium extract.

The present invention relates to a browning extract having a brain function improvement (brain cell protection) effect and a method for producing the same, more particularly, extracts obtained from browning ( Pueraria thunberginia ) caspase-3 (caspase-) in vitro experiments. 3) has the effect of inhibiting activity, in vivo significantly reduced memory loss by 192-saporin (192-saporin) that destroys nerve cells, acetylcholine esterase (AchE) and choline acetyl By enhancing the activity of the transferase (Choline acetyltransferase (ChaT), it shows the effect of improving brain function, and in particular, can be used as an anti-aging and senile dementia prevention and treatment medicament or health food, and also relates to a method for producing a gallium extract.

Recently, due to the rapid increase in the elderly population, the development of aging and Senile Dementia associated with aging has emerged as a serious social problem. Despite the high incidence, the disease is currently devoid of significant treatments and preventive agents, causing massive socioeconomic losses.

Alzheimer's disease can be caused by a variety of causes, but Alzheimer's disease is the most common senile dementia, and the main cause of this problem is the accumulation of beta amyloid (Ab1-42) in the brain and its neurotoxicity. And Selkoe, Annu. Rev. Neurosci., 17, 489-517 (1994)] and other factors. After all, the root cause of dementia is the deactivation and killing of neurons by various intracellular toxicity that occurs with aging. However, tacrine (Cognex; Donepezil, Aricept), known to date, promotes the action of acetylcholine, a neurotransmitter, which may alleviate symptoms of dementia, but prevents neuronal cell death. I can't. In addition, various side effects (liver toxicity, vomiting, diarrhea, etc.) are known as problems. In the case of dementia, acetylcholine neurons are initially damaged, and at the same time, many neurons, such as glutamate neurons, are damaged. Thus, enhancing the activity of acetylcholine has only a temporary effect on early patients.

Neurons are killed by apoptosis, excitatory neurotoxicity, and oxidative stress caused by free radicals. Aging and degenerative brain diseases are associated with the cause of such cell death. In particular, in aging and senile dementia, brain cell death by free radicals and cell death has been observed. In addition, it is reported that an enzyme called caspase-3 is activated as a major mechanism causing cell death.

Therefore, in order to prevent and improve aging and senile dementia, many attempts have been made to ultimately prevent neuronal death, and more specifically, inhibiting activation of major factors and enzymes in mechanisms that induce brain cell death. It is the best strategy.

Galhwa (Pueraria thunberginia) is a plant listed in the Korean Food Standard and Korean Pharmacopoeia (Medicinal Herbs) Standard Collection, and it corresponds to the bud of Pueraria thunbergiana Bentham, and it is known to have the main effect of main poisoning in traditional Chinese medicine prescription. It has been used to relieve hangovers or improve gastrointestinal dysfunction after drinking. In recent years, as an active ingredient in gallium, gastric ulcer, duodenal ulcer therapeutic agent (Domestic Patent Registration No. 341146), osteoporosis, arthralgia, arthritis treatment agent (Domestic Patent Publication No. 2001-67023), and oral bad breath removal, gum disease treatment, Patents have been applied for the purpose of removing waste products in the body and enhancing liver function (Korean Patent Publication No. 2001-107032). However, there are no studies on the protective effect of brain cells, the memory, and the improvement of learning ability.

Thus, the present inventors conducted a study to find a material that can be usefully used as a brain function improving agent in natural herbal medicine that has been traditionally widely used for securing safety and minimizing side effects. Extract of brown flower ( Pueraria thunberginia ), which has an antioxidant effect and inhibits the death of brain cells, has strong antioxidant power in neurons and also inhibits the death of brain cells, and memory and learning ability in animal disease (dementia) models The present invention was completed by confirming the effect of the improvement.

Therefore, an object of the present invention is to provide a gallium extract having an effect of improving brain function, medicines and health foods containing the same.

In addition, the present invention has another object to provide a method for producing the gallium extract.

The present invention is characterized by a gallium extract that has an effect of improving brain function.

In addition, it includes medicines and health foods containing the gallium extract.

Referring to the present invention in more detail as follows.

The present invention has the effect of inhibiting the apoptosis (apoptosis and oxidative toxicity) of brain neurons in vitro experiments obtained from brown flowers ( Pueraria thunberginia ), and inhibits caspase-3 activity In vivo experiments significantly reduced memory loss caused by 192-saporin, which destroys neurons, and significantly reduced acetylcholine esterase (AchE) and choline acetyltransferase (ChaT). By enhancing the activity, it shows the effect of improving brain function, and in particular, can be used as an anti-aging and senile dementia prevention and treatment medicament or health food, and is also characterized by a method of preparing a browning extract.

First, a simple extraction from the above-mentioned browning, or look at in more detail the process of efficiently separating the more excellent browning extract as follows.

After grinding the above-mentioned grinding finely finely, the ground grinding is put into water, ethanol, methanol or acetone (acetone) aqueous solution and the solvent is extracted 2 to 3 times while reflux cooling at 30 to 70 ℃ for 1 to 4 hours. At this time, the usage-amount of aqueous solution is suitable about 3 to 5 times the volume of a gallium.

The extract extracted above is concentrated by evaporation under conditions of 30 to 70 ° C., and then heated or lyophilized to make the extract liquid. In addition, the extract powder is dissolved in water or dimethyl sulfoxide (DMSO) and used directly for experiments, or the acetone extract powder is obtained by dividing the double acetone concentrate or powder into methanol and n-hexane, respectively. The fractions are each fractionated with acetate to make an extract.

As described above, the extract, concentration, and purification were performed to test the antioxidant effect, apoptosis inhibitory effect, and the like in the neuronal cell culture. Both extracts and acetone extracts showed excellent antioxidant and inhibitory effects on cell death. In particular, acetone extract showed the best effect, water fraction of the acetone extract powder showed the best effect. Also, remember about each extract. The underwater maze test for learning ability test and immunohistochemical test for measuring activity of acetylcholine neurons showed excellent effects.

On the other hand, the present invention can be used as anti-aging and senile dementia treatment medicines and health foods containing the gallium extract obtained by the extraction method as an active ingredient.

When prepared as a pharmaceutical, the gallium extract of the present invention may be administered orally or parenterally during clinical administration and may be provided in the form of a general pharmaceutical formulation.

The gallbladder extract of the present invention may be administered in various oral or parenteral formulations during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used Are prepared using.

Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium styrene talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilizers, suppositories. As the non-aqueous and suspending solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The content in the preparation of the active ingredient according to the present invention can be appropriately selected depending on the absorbency, inactivation rate, excretion rate, age, sex and condition of the user in the body. In the case of the gallium extract of the present invention, it is 20 to 300 mg / kg, preferably 50 to 100 mg / kg, and can be administered 1-3 times a day.

All of the gallbladder extracts of the present invention are herbal medicines (natural substances) listed in the food industry, the safety of which has already been proven, there are no side effects, and in fact, the gallbladder extract according to the present invention is administered in an excessive amount (2 to 5 g / kg). The results of the study showed no effect on mortality and behavior, and the internal organs were analyzed to show no change.

In addition, the present invention includes a health food for the treatment of diseases caused by the abnormal increase and activation of inflammatory cells caused by the abnormal increase and activation of cells with the gallium extract as an active ingredient.

The health food is a food prepared by adding the gallium extract to a general food, or encapsulating, powdering, and suspension, and when ingesting it, has a specific effect on health. As a result, there are no side effects that may occur during long-term use of the drug.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1: Preparation of Gallium Extract

Distilled water, 70% aqueous ethanol solution, 50% aqueous methanol solution or 40% acetone aqueous solution was added to about 5 times the dry weight of finely pulverized grinding, and extracted twice with reflux cooling at 60 ° C. for 2 hours. The extracts thus obtained were concentrated by evaporation at 45 ° C., and some of the concentrates were powdered by heating or lyophilization. The acetone concentrate or powder was partitioned into methanol and n-hexane, respectively, and the methanol fractionation layer was partitioned into water and ethyl acetate, respectively.

Example 2: Determination of Antioxidant Activity and Apoptosis Inhibitory Effect in Primary Neuronal Cell Culture

The gallium aqueous solution extract obtained in Example 1 was dissolved in distilled water or dimethyl sulfoxide (DMSO) (5 mg / ml), and antioxidant efficacy was measured in primary neuronal cell culture. Primary neuronal cells were cultured for 12 days in a 5% CO ₂ incubator with cerebral cortex isolated from the fetus of 14 days pregnant ICR mice. Iron chloride (FeCl ₂ , 100 μM) or butionine sulfoxamine (DL-Buthionine-Sulfoximine, BSO, 20 μM), which are mainly used to measure antioxidant efficacy, was used. By treating the neurons with the reagents and the gallium extract at the same time, the ability to inhibit neuronal cell death by oxidative stress was measured. Analysis of neuronal cell death may be performed using tryphan blue staining to exclude dead neurons, and then count the number of surviving cells with a reverse phase contrast microscope. LDH measurement was used. As a result, the gallium extract was excellently inhibited oxidative damage by iron chloride (FeCl ₂ , 100 μM) or butionine sulfoxamine (DL-Buthionine-Sulfoximine, BSO, 20 μM) at 30 ㎍ / ㎖. This was similar to trolox (vitamin E analog), which is often used as a positive control (see Fig. 1).

To measure the efficacy of inhibiting apoptosis, neurons were treated with 50-100 nM of staurosporine, or 10-20 μM of cyclosporine A, together with the browning extract, The ability to inhibit cell death was measured. As a result, cell death by 50-100 nM of staurosporin and 10-20 μM of cyclosporin A was excellently inhibited at 10-30 μg / ml. This was similar to CHX (cycloheximide) which is frequently used as a positive control (see FIG. 2).

The concentration of the browning extract used in this experiment was set to 10 to 120 µg / ml. Analysis of apoptosis by oxidative toxicity and apoptosis was repeated three or more times (n = 12 or more) and the significance of the results was assayed by a one-way ANOVA test.

Example 3: Caspase-3 Activity Measurement

Neurons induce apoptosis by the activation of an enzyme called caspase-3 by intracellular genetic signals. Therefore, the mechanism of inhibiting apoptosis was investigated through the activity of caspase-3, a key enzyme that induces apoptosis. After completing the assay for inhibition of apoptosis of the gallium extract in the same manner as in Example 2, the protein was extracted from neurons and Western blotting (Western Blotting) to measure the activation of caspase-3. As a result, when apoptosis was induced by staurosporin, the amount of activated caspase-3 increased as shown in FIG. 2 (C). Inhibited.

Example 4 Animal Experiments in SD Rats

The rats used in this experiment were Sprague-Dawley-based males aged 5 weeks (220 ± 10 g) and purchased males (Sam taco, Korea) and maintained a solid environment for rats while maintaining an environment of temperature 23 ± 3 ℃ and relative humidity 50 ± 10%. (Samyang Feed, Korea) and water were supplied and used for 1 week after being adapted to the laboratory environment. Isolation of the experimental group was performed on the 192-sapolin-damaged group, which damages acetylcholinergic neurons in rats treated with sham (untreated), medial septum (memory of brain function, neurons and brain structures). (lesion), 192-sapolin damage + browning extract administration group, and each group was subjected to behavioral test after 1 week of treatment, and biopsy was performed after the last behavioral test.

Example 5 Induced Impairment of Memory and Learning in SD Rats

The experimental group was anesthetized with sodium pentobarbital (50 mg / kg, ip) in rats and then mediated septum (AP: -0.2, L: ± 0.3, using stereotaxic technique). 1 μg of 192-saporin (ATS) was injected at both H: -6.2) positions. Microinjection was connected to a 1 ml gas-tight glass syringe [Hamilton, Reno, NV] by using a polyethylene tubing, and a perfusion pump (Pump 22, Harvard Apparatus, South Natick, MA) was used for 5 minutes after injection at a flow rate of 0.2 μl / min, and then the syringes were removed. Drug treatment for each injured group was performed the next day. The simulated group was infused with artificial CSF made of 145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl ₂ , and 1.0 mM MgCl ₂ in the same manner.

Example 6: Morris-water maze test

The water tank used as an aquatic labyrinth was a circular barrel of 180 cm in diameter and 50 cm in height, filled with 30 cm of water at a temperature of 22 ± 2 ° C. The perimeter of the underwater maze kept spatial cues constant, including a video camera, a bench, and a device for temperature control on the bench. The sheath was attached to the round transparent acrylic with a diameter of 12 cm and placed 1.5 cm below the water surface. The underwater labyrinth is divided into four identical quadrants, which are divided into northeast (NE), northwest (NW), southeast (SE) and southwest (SW), with the evacuation zone in the center of the northeast quadrant, one of which is the starting point. Was used. The rats were trained four days a day for seven days (acquisition test), and at the end of the eighth day of the last test, a free swim test (retention test) was performed. The animals were allowed to swim for 60 seconds with the shelter removed. The behavior of all animals was recorded with a video camera. The training session measured the time taken from the start to climb into the shelter. As a result, 192-saporin-induced learning, memory deterioration effectively inhibited in the group administered with the gallium extract in the acquisition test (acquisition test) and the retention test (retention test) [Fig. 3].

Example 7: Measurement of Choline-acetyl trasferase (ChAT) activity

Immediately after all behavioral experiments, animals were anesthetized with sodium pentobarbital (100 mg / kg, ip) and perfused through the heart with 900 ml of 4% formalin solution (100 ml) prepared with 100 ml saline followed by phosphate buffer (PBS). It was. Then, the brain was taken out and fixed with the same fixative for 2-3 hours, and placed in PBS containing 20% sucrose and stored at 4 ° C. for 1 day. The next day, after freezing the brain, brain tissue was grown to a thickness of 30 μm at the hippocampus and medial septum of the dorsal and ventral. Tissues were washed several times with PBS and primary sheep polyclonal ChAT antibody (Cambridge Research Biochemicals, Wilmington, DE) was used. The primary antibody was 2% rabbit serum and 0.1% sodium azid in PBST with 0.3% Triton X-100 in PBS [Sigma, St. Louis, MO] was prepared by diluting 2000 times. Brain tissues were cultured in primary antiserum with continuous shaking at 4 ° C. for 72 hours. The tissues were then washed three or more times with PBST and biotinylated anti-sheep secondary antibodies diluted 200-fold in PBST containing 2% rabbit serum at room temperature for 2 hours [Vector Laboratories, Burlingame , CA]. The tissue was then washed three or more times with PBST and then biotinylated anti-rabbit secondary with biotin bound rabbit diluted 200-fold in PBST containing 2% normal goat serum at room temperature for 2 hours. antibody) [Vector Laboratories, Burlingame, CA]. After washing three times with PBST, brain tissue was soaked in Vectastain Elite ABC reagent (Vector) for 2 hours at room temperature. After rinsing with PBS, the tissues were fortified with nickel chloride and expressed using diaminobenzadine as a colorant.

All treated brain tissues were fixed on gelatine-coated slide glass, covered with a cover glass, and observed under a microscope. The number of ChAT-immunoreactive neurons in the medial septum and hippocampus was measured by a magnification of 200 times using a 200 × 200 μm microscope rectangle grid [ 4]

Example 8 Measurement of Acetylcholinesterase (AChE) Expression

In 500 ml of incubation medium, sodium citrate (0.1 M, 0.735 g / 25 ml), copper sulfate anhydrous (30 mM, 0.374 g / 25 ml), potassium (potassium, 5 mM, 0.082 / 25 mL), 250 mg of acetylthiocholine iodide / phosphate buffer 325 mL, and 50 mL of distilled water were added thereto. The solution was shaken at room temperature until it became blue. The cut tissues were placed in a fixer and stained at room temperature for 2 hours. The subsequent tissue analysis was the same as in Example 7 [Fig. 5].

Preparation Example 1 Preparation of Powder and Capsule

10 mg of the galvanized extract was mixed with 14.8 mg of lactose, 3 mg of crystalline cellulose, and 0.2 mg of magnesium stearate. The mixture was filled into No. 5 gelatin capsules using a suitable apparatus.

The components of the powder and capsules are as follows.

Gall flower extract ··············· 10 mg

Lactose ······················· 14.8 mg

Crystalline Cellulose ... 3 mg

Magnesium Stearate 0.2 mg

Preparation Example 2 Preparation of Injection Solution

Injection was prepared by mixing 10 mg of galvanized extract, 180 mg of mannitol, 26 mg of Na ₂ HPO ₄ .12H ₂ O and 2974 mg of distilled water. The solution was placed in a bottle and heated at 20 ° C. for 30 minutes for sterilization.

Gallium Extract ·············· 10 mg

Mannitol 180mg

Na ₂ HPO _{4 12} H ₂ O 26 mg

Distilled water ················· 2974 mg

Preparation Example 3 Preparation of Health Food

It was prepared by mixing 0.1 g of galvanized extract, powdered vitamin E, ferric nitrate, zinc oxide, nicotinic acid amide, vitamin A, vitamin B ₁ and vitamin B ₂ .

The components of the beverage are as follows.

Galvanized extract ················ 0.1 g

15 g of vitamin C

7.5 g of powdered vitamin E ···················

5.75 g of ferric nitrate ·················

Zinc Oxide ······ 3.5 g

Nicotinic Acid Amide · ・ ・ ・ ・ ・ ・ ・ ・ 3.5 g

0.2 g of vitamin A

Vitamin B ₁ ······················ 0.25 g

Vitamin B ₂ 0.3 g 0.3 g

Starch ····················· 18.0 g

······························

As described above, the browning extract of the present invention is excellent in the antioxidant effect in neurons, excellent in inhibiting the death of neurons, excellent in the memory, learning ability in the animal disease (dementia) model, By increasing the activity of acetylcholine neurons is expected to be useful as a health food and medicine for improving brain function.

Claims (4)

A drug for the prevention and treatment of senile dementia, characterized by containing an extract of brown flower ( Pueraria thunberginia ). delete Finely pulverizing the browning ( Pueraria thunberginia ), and then extracting the solvent 2-3 times with reflux cooling at 30 to 70 ° C. for 1 to 4 hours with an aqueous solution selected from water, ethanol, methanol, and acetone; And The extract is concentrated by evaporation at 30 ~ 70 ℃, heated or lyophilized to obtain a powdery method comprising the step of obtaining a powdered extract effective for senile dementia, characterized in that it comprises a powder. 4. The method of claim 3, wherein the acetone concentrate or powder is fractionated into methanol and n-hexane, and the methanol fraction layer is fractionated into water and ethyl acetate, respectively, to obtain a water fraction. And a method for producing a gallium extract effective for treatment.

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