KR20030079105A - Mixture of herbal extracts for memory enhancement - Google Patents

Abstract

PURPOSE: Provided are a mixture of herbal extracts for memory enhancement, and a pharmaceutical composition for prevention and treatment of diseases associated with memory dysfunction. CONSTITUTION: A mixture of herbal extracts for memory enhancement is prepared by extracting a medicinal herb selected from the group consisting of steamed Rehmanniae Radix, fresh Rehmanniae Radix, dried Rehmanniae Radix, Lycium chinense MILL., Angelica uchiyamana, and Schrophulariaceae, with Dioscorea batatas DECNE., Lycium chinense Mill., and Cornus officinalis Sieb. et Zucc., using alcohol or alcohol solution as an extraction solution.

Description

Mixture of herbal extracts for memory enhancement

The present invention relates to a mixed herbal medicine extract, a method for preparing the same, and a pharmaceutical composition for preventing or treating a memory impairment disease containing the extract as an active ingredient, and more specifically, ripening sulfur, raw sulfur, dried egg sulfur, longan meat, donkey and Hyunsam Mixed herb extract extracted from the herb, consisting of medicinal herb selected from the group consisting of goji medicinal herbs, goji medicinal herbs and cornus oil, alcohol or an aqueous alcohol solution, a method for producing the mixed herbal medicine extract and a memory containing the mixed herbal medicine extract as an active ingredient The present invention relates to a pharmaceutical composition for preventing or treating a disability disorder or a health food for improving memory ability.

Recently, due to the rapid increase in the elderly population, the development of senile dementia related to aging has become a serious social problem. Molecular genetic studies and efforts have been made in various fields to determine the cause of dementia disease, but the cause of the disease is not known precisely, so it is very difficult to develop a therapeutic agent, but the primary symptom that appears is the main symptom. Efforts to control the symptoms of decay have been active until recently.

Memory is the storing and recalling of the results of experiences or information obtained from the environment. The process of memory is divided into three stages: encoding, reinforcement, and withdrawal. These processes include short-term memory as well as exchange of chemical substances in the neuronal connections (Princples of Neural Science, 4th eth, Mc Graw-Hill, 2000). It is also known that structural changes in the brain occur during the transformation into long-term memory ( Peptides, 21 (3), 407-411, 2000).

Memory is divided into explicit memory and implicit memory. In general, memory refers to external memory, which refers to remembering a person's face or the contents of an event. The hippocampus and its surrounding areas have been found to play an essential role. The hippocampus is a cerebral region that is the backbone of memory reinforcement, and memory reinforcement is a path that must pass through short-term memory to long-term memory. In particular, narrative memory is the signal received at the periphery through the cortical cortex associated with the signal, which in turn is strengthened by the CA1 or CA3 circuit of the hippocampus and stored long-term in the cortex. At this time, the greater the complexity and intensity of the signal, the stronger the strengthening occurs, and the memory stored in the cortex is also a part of the memory is longer and clearer (Princples of Neural Science, 4th eth, Mc Graw-Hill, 2000; Chinese Academy of Commerce, Beijing, Beijing Science Press, 224-230, 1991). For this reason, neurological and molecular biological studies related to general memory are focused on the hippocampus in the cerebrum.

However, there are no specific studies on the drugs that enhance memory and the mechanism of action. However, studies of factors related to memory have made significant progress, with Ca ²⁺ -stimulated adenyryl cyclase ( International Journal of Developmental Neuroscience , 19 , 387-394, 2001; Cell , 68 , 479-489, 1992; Learning & Memory , 7 , 18-31, 2000), S100-beta ( Neurobiology of Learning & Memory , 75 , 230-243, 2001), BDNF ( Neuroreport , 8 , 779-782, 1997; Behavioural Brain Research , 92, 21-30, 1998), NMDA receptors ( Behavioural Brain Research , 66 , 225-230, 1995; Cell 87, 1327-1338, 1996), CREB ( Rivista di Biologia , 90 , 371-384, 1997; European Journal of Neuroscience , 13 , 1453-1458, 2001; Cell , 103 , 595-608, 2000) have been reported to have a direct effect on memory.

In addition, it has been found that dementia disease is closely related to impaired function of cholinergic nervous system and impaired cognitive function. Therefore, it inhibits the activity of acetylcholinesterase (AchE), an enzyme of acetylcholine, in brain to maintain acetylcholine concentration. Tacrin (Tacrin, Cognex Capsule) and Donepezil (Aricept Tablet) have been developed to improve cognitive function. However, tacrin has side effects such as hepatotoxicity, dizziness, ataxia, insomnia, gastrointestinal disorders, confusion, blood pressure disorders, anorexia, fever or nervousness, and donepezil causes muscle cramps, fatigue, insomnia. Disorders of the mental nervous system, such as dizziness and extrapyramidal disorders such as resin tremors and movement disorders or digestive system disorders such as diarrhea, nausea and vomiting.

Since the hypothesis that Reactive Oxygen Species (abbreviated as "ROS") may be the cause of aging was proposed around 1956, a lot of research has been done on their role, production and pathways, and Alzheimer's disease. The development of antioxidants as a therapeutic agent for various neurodegenerative diseases including Parkinson's disease has been of great interest ( J Gerontl , 298-300, 1956). ROS refers to any oxygen-containing substance that has unpaired electrons on its outer shell, and in severe cases, damages its structure and function by reacting with proteins, nucleic acids, etc. Substances that may cause developmental cell death include, for example, 1) free radicals and 2) non-radical but toxic substances.

Free radicals are produced by human exposure to radiation or internal enzyme reactions. They react with the -SH group of proteins to cause loss of enzyme activity, promote crosslinking, damage DNA, RNA, enzymes, and cell membranes, resulting in cell necrosis. cause. That is, the free radical theory (free radical theory) is superoxide generated in the metabolic process (superoxide anion, O ^2-), hydroxy radical (hydroxy radical, OH ^-) and peroxyl radicals (peroxyl radical, RO ^2-), etc. Free radicals act on cells and tissues to produce harmful substances, and the accumulation of these radicals is a fundamental cause of aging and chronic degenerative diseases. Enzymes such as superoxide dismutase (SOD). In addition, a radical, but ROS is toxic to the biological material is hydrogen peroxide, sodium hypochlorite (hypochlorous acid, HOCI), singlet oxygen (singlet oxygen), peroxy Age Treatment (peroxynitrite, ONOO ^-) there are and the like, which normal metabolic processes Since they are always produced in the body, antioxidants are simultaneously present in vivo to remove them. Representatives of antioxidants include trace amounts of antioxidants and superoxide dismutase, catalase, glutathione peroxidase (GSH-Px), and trace amounts of antioxidants present in cells such as ascorbate and alpha-tocopherol. Antioxidant enzymes).

Superoxide dismutase, glutathione peroxidase and catalase, which are representative antioxidant enzymes present in the living body, are known to have complementary functions such as increased activity of other enzymes when one enzyme is insufficient. Among these three enzymes, superoxide dismutase is rarely present in neurons and is known to be hardly changed in the damage caused by hydrogen peroxide. Catalase is an enzyme that directly converts hydrogen peroxide into oxygen and water, and it is found that it is distributed in various tissues in vivo, and the content of each tissue is different. That is, the largest amount of catalase present in the peroxisome, an organelle within the cell, is the liver, and a very small amount is known to exist in the brain. Glutathione peroxidase converts hydrogen peroxide to water as a secondary reaction, oxidizing glutathione (reduced form). Unlike catalase, glutathione peroxidase requires a substrate called glutathione, and not only simple enzymatic activity but also glutathione concentration, glutathione reductase activity, and NADPH, which are converted to reduced form by glutathione reductase. Are affected by a number of factors. It is widely distributed in liver tissues of rats and is reported to be present in liver, kidney and leukocytes in humans. While catalase and glutathione peroxidase have similar functions, they differ in their distribution, and are known to be complementary to each other in the manner that glutathione peroxidase acts at low concentrations of hydrogen peroxide and catalase at high concentrations (Free radicals in biology and medicine 3rd edition, Oxford university press, New York, 142, 1999).

Sukji Hwang (Steamed Rehmannia Radix, Rehmannia glutinosa (G _AERTNER ) L _IBOSCHITZ ) has pharmacological effects such as hypoglycemic action, circulatory system, _cardiac and diuretic action, liver function protection and antibacterial action.

Gojija (枸杞 子; Lycium Fructus, Lycium chinense M _ILL .) Has a non-specific immune boosting effect, which significantly increases the phagocytosis of the reticuloendothelial system, and regulates the effects of water, which is a blood stimulating function and a biostimulant. When mixed with the feed of the egg will greatly increase the weight and number of action. In addition, it has a pharmacological action such as lowering blood pressure and blood sugar, lowering cholesterol level, anti-fatty liver action, promoting growth, inhibiting cancer cells, and increasing uterine weight.

Medicinal herbs (山藥; Discoreae Rhizoma, Discorea batatas D _ECNE .) _Lowers blood sugar, acts on the digestive system, acts remarkably on the coordination of the intestinal tract, and has anti-aging effects and pharmacological effects.

Cornus Fuctus ( Cornus officinalis S _IEB . Et Z _UCC. ) Has hypoglycemic action, diuretic action, hypotensive action, staphylococcus aureus, dysentery, inhibition of ascites cancer cells, lymph cell proliferation in immune system. It has pharmacological effects such as the action of inhibiting platelet aggregation and enhancing the contractile force of the myocardium.

Baekbokryeong (白 茯 笭; Hoelen, Poria cocos (F _R. ) W _OLF ) has a pharmacological action, such as diuretic action, hypoglycemic action, heart contractility increase and immune enhancer action.

Moutan Cortex Radicis, Paeonia suffruticosa A _NDR has pharmacological effects such as analgesic, sedation, antipyretic, antispasmodic, anti-inflammatory, antithrombotic, antiallergic and gastric juice secretion.

_Alixmatis Rhizoma, Alisma canaliculatum A. B _R. et B _OUCHE has pharmacological effects such as diuresis, tinnitus and decreased vision.

Accordingly, the present inventors have tried to develop a new memory capacity disorder prevention and treatment agent with low side effects and good efficacy and even causative treatment. 1) Selected from the group consisting of Sukjihwang, rawjihwang, driedjihwang, longan meat, Angelica and Hyunsam The present invention has been completed by revealing the effect of enhancing the predominant memory ability of the mixed herbal medicine extract consisting of herbal medicine, 2) gojija, 3) powder and 4) cornus.

It is an object of the present invention to provide a mixed herbal extract, a method for preparing the same, and a composition for preventing and treating a memory disorder disorder containing the extract as an active ingredient.

1 is a graph of the delay time of the passive avoidance of rats administered the mixed herbal medicine extract of the present invention,

Control group: no treatment (n = 10),

Ginkgo leaf extract: group treated with 2.6 mg / day ginkgo leaf extract for 10 days (n = 10),

Nature Made: Group treated with 10 mg / day Nature Made Extract for 10 days (n = 10),

Mixed herbal medicine extract: group treated with 400mg / day mixed herbal medicine extract for 10 days (n = 12),

Figure 2 is a graph comparing the degree of hybridization of mRNA isolated from the hippocampus of rats to which the mixed herbal medicine extract of the present invention was compared with the control group (Cy5 and Cy3 signal values are shown on a log-log scale),

Cy3 signal value: means the degree of hybridization of the control group,

Cy5 signal value: Signal value normalized by the internal control group, meaning the degree of hybridization of the mixed herbal medicine extract administration group,

Figure 3 is a graph comparing the genes significantly differently expressed in the rats administered the mixed herbal medicine extract of the present invention with the control group (genes that are considered to be meaningful BDE is more than two times),

Genes: The number of all genes considered to be meaningful,

Annotated genes: the number of all genes whose function is found out of meaningful genes,

Unannotated genes: the number of all genes whose function is unknown among the meaningful genes,

YMT_02 an. : Genes abundant in mixed herbal extracts.Number of all genes with known function.

YMT_02 unan. : Genes abundant in mixed herbal extracts.Number of all genes whose function is unknown.

Control an. : Genes abundant in the control group, the number of all genes with known function,

Control unan. : Genes abundant in the control group, the number of all genes whose function is not known,

Figure 4 is a graph of the protective effect of the cell damage induced by hydrogen peroxide caused by the concentration of the mixed herbal medicine extract of the present invention, ie cell proliferation,

Figure 5 is a graph of the active oxygen species removal ability for the active oxygen species of PC12 cells induced by hydrogen peroxide according to the concentration of the mixed herbal medicine extract of the present invention,

Figure 6 is a graph of the active oxygen species removal ability per cell number for the active oxygen species of PC12 cells induced by hydrogen peroxide according to the concentration of the mixed herbal medicine extract of the present invention.

In order to achieve the above object, the present invention is 1) medicinal herb selected from the group consisting of Sookjihwang, rawjihwang, dried jihwang, longan meat, Angelica and Hyunsam, 2) gojija, 3) powder and 4) cornus It provides a mixed herbal medicine extract having.

In addition, the present invention provides a method for preparing the mixed herbal medicine extract.

In addition, the present invention provides a pharmaceutical composition or health food for the prevention or treatment of memory impairment diseases containing the mixed herbal medicine extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention is 1) improving the memory capacity extracted from the herbal medicine consisting of medicinal herb selected from the group consisting of sulfuric acid, raw sulfur, dried sulfur, longan, yonggi, tangui and Hyunsam, 2) gojija, 3) acid and 4) acid and lactation It provides a mixed herbal extract having the activity.

In the present invention, the mixed herbal medicines preferably include _{ripening sulfur} , goji berry, medicinal herbs and cornus _oil , and the alternative herbal medicines with ripening sulfur are raw Rehmannia Radix, Rehmannia glutinosa (G _AERTNER ) L _IBOSCHITZ ) Dried Rehmannia Radix, Rehmannia glutinosa (G _AERTNER ) L _IBOSCHITZ ), Longanae Arilus, Euphoria longan (L _OUR .) S _TEUD .), Korean Angelica, Angelica sinensis (O _LIV .) D _IELS ) and _Hyunsam (玄 蔘 Scrophulariae Radix, Scrophularia buergeriana M _IQ .).

The composition ratio of the mixed herbal medicine in the present invention is 1) herbal medicine selected from the group consisting of Sookjihwang, rawjihwang, dried jihwang, longan meat, Angelica and Hyunsam: 2) Gugijagi: 3) Medicinal herbs: 4) Cornus 5-90: 5-90: It is preferable that it is 5-90: 5-90 weight ratio (w / w), and it is more preferable that it is 30-40: 30-40: 10-2-20: 10 weight ratio (w / w).

In addition, the mixed herbal medicine in the present invention provides a mixed herbal medicine extract further comprising one or two or more herbal medicines selected from the group consisting of Baekbokryeong, Seokchangpo, halved, dermis, cheonma, horsewood skin and taxa.

Herbal medicine further added in the present invention is preferably one or more herbal medicines selected from Baekbokryeong, Mokdanpi and Taxa, and as a substitute herbal medicine with Baekbokryeong Seokchangpo (石菖蒲; herbal name Acori Graminei Rhizoma, scientific name Acorus gramineus S _OLAND ) , contrary (半夏;.. saengyakmyeong Pinellia Tuber, scientific name _{Pinellia ternata (T HUNB) B RETT} ), the dermis (陳皮; saengyakmyeong Unshiu Citrus Peel, scientific name Fraxinus rhynchophylla H _ANCE) and Pegasus (天麻; saengyakmyeong Gastrodiae Rhizoma, scientific name Gastrodia elata B _L ) and the like.

The composition ratio of the mixed herbal medicine in the present invention is 1) a herbal medicine selected from the group consisting of Sookjihwang, rawjihwang, dried jihwang, longan meat, tangui and Hyunsam: 2) gojija: 3) pesticides: 4) cornus milk: 5) baekbokyeong, Seokchangpo, Banha It is preferable that one or two or more herbal medicines selected from the group consisting of dermis, dermis, horse skin, and taxa are 5-90: 5-90: 5-90: 5-90: 5-90 weight ratio (w / w) It is more preferable that it is 30-40: 30-40: 10-10-20: 10-20: 5-10 weight ratio (w / w).

The present invention is extracted with an alcohol or an aqueous alcohol solution to the mixed herbal medicine, the alcohol or an aqueous alcohol solution may be selected from the group consisting of 5 to 100% ethyl alcohol and 5 to 100% methyl alcohol, 70-100 It is preferable to use the ethyl alcohol of%, wherein the extraction method may be by cooling, reflux or ultrasonic method, and preferably ultrasonic extraction.

Since the mixed herbal medicine extract of the present invention has memory enhancing activity, it can be usefully used for the prevention or treatment of memory impairment diseases such as dementia or Alzheimer's disease.

The mixed herbal medicine extract of the present invention was confirmed through a manual avoidance test whether the memory enhancement effect. As a method of evaluating memory ability, the evaluation of spatial perception using an underwater T maze, the Morris underwater maze, etc., and the evaluation of event memory using a manual evasion test using a round-trip box are known. The avoidance test was used to observe the effect of memory enhancement. Manual avoidance tests test the rats' ability to remember unpleasant stimuli over 24 hours. That is, the rats are placed in a room with dark compartments, a strong incandescent light is applied to the dark compartments, and an electric shock is applied through the metal plate on the floor. The rats are removed from the test apparatus and tested again for their ability to remember electrical shock after 24 hours. Ginkgo bilba or nature made, which is known to improve memory ability, was also evaluated under the above conditions. . As a result, the mixed herbal medicine extract showed an excellent memory enhancement effect than ginkgo biloba or nature made for sale as a conventional health supplement (see Fig. 1 ).

In addition, the present inventors performed a cDNA microarray using the hippocampus, which is the backbone of the memory-enhancing mechanism, to identify the genetic mechanism by which the mixed herbal extract of the present invention improves memory capacity. MRNA was purified by dissection of the hippocampus of the mixed herbal medicine extract administration group or control group, labeling using oligo-dTprimer, and then applying the gene to the incited rat GEMRE 2 cDNA microarray. Was compared and reviewed. The combination of transthyretin, Axl receptor tyrosine kinase, and neuron-specific protein PEP-19, which are specifically expressed in the mixed herbal extract group, It can be seen that the mixed herbal extracts enhance memory capacity by promoting neuronal growth or inhibiting degeneration (see FIGS . 2 and 3 ).

In addition, the antioxidant effect of the mixed herbal medicine extract of the present invention was examined.

The protective effect against cell damage induced in PC12 cells pretreated with the mixed herbal medicine extract of the present invention was observed by MTS assay on cell viability. Induction of cell damage is superoxide (O ^2-), hydrogen peroxide (H ₂ O _2), and the hydroxy radical (OH ^-) are used and the like. As a result, the mixed herbal extract decreased cell viability at low concentration, but increased cell viability as concentration increased (see FIG. 4 ).

In addition, the protective effect against cellular damage induced by PC12 cells pretreated with mixed herbal extracts was observed as inhibition of reactive oxygen species. Induction of cell damage is superoxide (O ^2-), hydrogen peroxide (H ₂ O _2), and the hydroxy radical (OH ^-) are used and the like. As a result, in the mixed herbal medicine extract, the generation of reactive oxygen species decreased with concentration compared to the control group (see FIGS . 5 and 6 ).

Such antioxidant effects are in line with the results of cDMA microarrays, and the mixed herbal extracts can be seen to improve memory capacity by inhibiting aging as well as neuronal degeneration.

Taken together, the mixed herbal medicine extract of the present invention can be seen that the effect of improving the memory ability is significantly superior to the ginkgo bilba (Ginko bilba) or nature made (nature made) on the market as a dietary supplement.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of memory impairment diseases containing a mixed herbal extract as an active ingredient.

The pharmaceutical composition for preventing or treating a memory disorder-related disease of the present invention contains the mixed herbal medicine extract as an active ingredient. The mixed herbal extract may be administered in various formulations, oral and parenteral, in actual clinical administration. Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose in mixed herbal extracts. And gelatin etc. are mixed and prepared. In addition to simple excipients, glidants such as magnesium stearate, talc and the like 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 are available. Etc. may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and the like. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. Dosage units may contain, for example, one, two, three or four times the individual dosage, or they may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.

In the pharmaceutical composition for preventing or treating a memory disorder-related disease, an effective dose of the mixed herbal medicine extract is 100 to 800 mg / kg, preferably 200 to 400 mg / kg, and may be administered 1-6 times a day. However, the dosage level for a particular patient may vary depending on the weight, age, sex, health status, diet, time of administration, administration method, excretion rate, severity of the disease, and the like of the patient.

In particular, when the mixed herbal medicine extract according to the present invention is administered to the human body, there is no concern of side effects as compared to other synthetic medicines because it is a natural extract, and in fact, these herbal medicines are used as a food additive, so the stability is secured.

In addition, the present invention provides a health food for improving the memory area containing the mixed herbal medicine extract as an active ingredient.

When the extract of the present invention is used as a food, the extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment). In general, the extract of the present invention may be added in the amount of 0.1 to 1% by weight, preferably 0.2 to 0.6% by weight based on the raw materials in the manufacture of food or beverage. An effective dose of the extract of the present invention may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control, Since there is no problem in terms of safety, it is certain that it can be used in an amount above the above range.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Mixed Herbal Extracts

The present inventors crushed 500 g of Sukji sulfur, wolfberry, medicinal herbs, cornus, baekbokyeong, neck peel and talc each into a small mortar and mortar and then put in a flask containing 1 liter of 70% ethyl alcohol (10 liters) at room temperature with an ultrasonic shaker for 10 minutes. The supernatant was collected by extraction. The precipitate was extracted in the same manner using 80% ethyl alcohol, 90% ethyl alcohol and 100% ethyl alcohol, and then the supernatant was mixed. The filtrate was concentrated with a vacuum condenser and lyophilized with a freeze-drier to obtain lyophilized products of individual herbal medicines.The recovery rates were 19.83% for Sugi, 20.05% for Goji berry, 10.22% for kochujang, 41.64% for cornus oil, 1.11% for Baekbokyeong, 21.45% Tax was 20.92%.

The individual lyophilisates were added by the ratio as shown in Table 1 on the basis of the weight before extraction to obtain a mixed herbal mixture, dissolved in distilled water at a concentration of 1 g / mL and centrifuged at 10,000 g for 15 minutes to remove insoluble materials, and then 0.22 μm. Prepared by passing through a filter.

Hwang Ji Hwang ( Steamed Rehmannia Radix (Korea)) 16 g Wolfberry ( Lycii fructus (Korea)) 16 g Potion ( Discoreae Radix (Korea)) 8 g Cornus ( Corni Fuctus (Korea)) 8 g Baek Bok Ryeong ( Hoelen (Korea)) 4 g Mountain Cortex Radicis (Korea) 4 g Taxi ( Alismatis Radix (Korea)) 4 g Sum 60 g

Comparative Example Passive Avoidance Test

In order to confirm the memory effect of the mixed herbal medicine extract prepared in Example 1, the inventors performed a manual avoidance test in comparison with an extract known to have an effect of improving memory capacity. Specifically, the mixed herbal medicine extract of the present invention was administered to Sprague-Dawley male rats around 180 g in weight, and the comparison group was a ginkgo leaf which is a health supplement known to improve the existing memory ability. (Ginko biloba) and Nature Made were administered by manual evasion test.

Ginko biloba (Ginko biloba) is a freeze-dried product of Ginkgo biloba. Its ingredients are 24% ginkgo flavone glycosides and 6% terpene lactones. It is on sale and the adult daily dose is 160 mg. In addition, Nature Made Nutritional Products (USA) is a dietary supplement containing 437 mg of Soya lecithin, a basic ingredient of 144 mg of phosphatidyl choline, and 600 mg / tablet. It is known that the adult daily dose is 600 mg.

Passive avoidance box (Shuttle box) manufactured for manual avoidance test is manufactured by changing the size to 60 ㎝ × 20 ㎝ × 20 ㎝ based on the model of GEMINI avoidance system (San Diego Instruments, San Diego, CA) It was. The box is divided into two rooms of the same size, with acrylic plates on the wall, and the walls that divide the two rooms have guillotine-type doors that are 10 cm wide and 10 cm wide, allowing them to be opened and closed from the outside up and down. The lid of the left chamber was fitted with a very bright (250 W) sodium bulb to create a very bright environment that the animals do not like. At the bottom of the right room, an electric shock device was installed on the bottom to apply electric shock to the soles of the animals. A 2.0 mA electric shock was applied using an AC variable power electric shock generator. To increase the brightness of the left room, the walls of the left room were coated with aluminum foil to maximize light reflection and the right room was painted matt black. A small translucent window 1 cm high was made to look inside to see which room the animal was in.

The control group, ginkgo biloba administration group, Nature Made administration group, mixed herbal medicine extract administration group was divided into four groups and each experiment was carried out 10, 10, 10, 12 animals. The control group was supplied with sufficient water and feed to the rats in the steady state without any special treatment, and the learning and memory test were performed. The mixed herbal medicine extract group was weighed 100 g of the test animal for 10 days before the memory test was conducted to the rat in the steady state. The lyophilizate of the mixed herbal medicine extract was diluted and consumed in drinking water so that 400 mg of the mixed herbal medicine extract was administered and continued to be administered during the learning and memory test. On the other hand, the ginkgo biloba administration group and the Nature Made administration group were diluted in drinking water so that a 2.6 mg aqueous solution of Ginkgo biloba per 100 g body weight and a 10 mg Nature Made aqueous solution per 100 g were administered to the rats in a steady state for 10 days prior to the memory test. Intake was continued during the study and memory test.

Manual avoidance test was performed by applying the method of Da Cunha (Braz. J. Med. Biol. Res. 23 : 301-306, 1990). The study was conducted for 2 days and the animals were transferred to the behavior observation room and stabilized 1 hour before the start of the experiment. On the first day of study, the left room light of the shuttle box was turned on, the door blocking the left right room was opened, and the test animal was placed with the tail toward the door. The experiment animal instinctively moves to the relatively dark right room. When moving to the right room, the right room was searched for 1 minute, and the experimental animals were taken out. The method was repeated twice to learn the relative comfort of the right chamber. In addition, on the second day of learning, turn on the light in the left room of the shuttle box and place the test animal with the tail facing the door, and then move the test animal directly to the right room. At this time, a 2.0 mA electric shock was applied to the sole of the experimental animal for 5 seconds using the electric shock device installed on the bottom of the right room. If the door is opened 4 seconds after the electric shock is applied, the test animal moves to the left room immediately. In this way, we learned that there is a strong electrical stimulation in the right chamber.

The retention test was performed 24 hours after the second day of study. The left room of the shuttle box was turned on, the door was opened, and the tail of the test animal was placed toward the door. The better the experimental animal's memory, the more likely it is to remember the strong electrical stimulation experienced on the second day of learning and to avoid entering the room on the right. The time for entering both the front and back feet into the right chamber was measured. The measurement time was recorded in seconds, and if more than 600 seconds elapsed, it was recorded as 600 seconds.

As a result, as can be seen in Table 2 and Figure 1 , the mixed herbal medicine extract of the present invention remembers the fear of the electrical stimulation of the right room (retention latency) hesitant to go to the right room (gtention leaf administration group and nature comparison group) Significantly longer than the maid administration group. Therefore, it was confirmed that the excellent memory capacity enhancement effect of the mixed herbal medicine extract.

Number of rats Control Ginkgo leaf administration group Nature Maid Administration Mixed Herbal Extract Administration Group Delay in seconds One 194 496 600 110 2 48 600 0 600 3 13 233 84 596 4 19 600 454 54 5 312 23 600 559 6 600 171 114 389 7 224 16 377 553 8 600 24 450 600 9 362 169 11 600 10 103 600 0 600 11 600 12 600 Mean ± mean error (sec) 238.1 ± 72.40 293.2 ± 80.26 268.9 ± 79.43 487.6 ± 57.42

Example 2 cDNA microarray

As a result of the memory test, rats with the shortest delay time and one of the longest rats were excluded, and the rat with the second shortest retention latency from the control group and the rat with the second longest memory potential in the mixed herbal extract group. Was selected. In order to prevent unnecessary brain changes and trauma during the sacrifice of the selected rats, a single strong impact was sacrificed to the spine without anesthesia and the hippocampus was quickly collected and stored at -70 ° C. The collected sample was isolated from the RNA by an experimental method provided by the manufacturer using TRIzol reagent (Gibco BRL, Life Technologies, USA). RNA isolated above was quantified with a spectrophotometer (DU500, Beckman. Inc., USA), and then mRNA was purified using an oligotex mRNA mid kit (Qiagen GmbH, Hilden, Germany). mRNA was quantified by spectrophotometer and treated with 600 ng (50 ng / μl) dry ice and labeled with Cy5-dUTP (mixed herbal extract dose) and Cy3-dUTP (control) (Incyte Genomics (Palo Alto, California, USA)) Incyte rat GEM 2 microarray (Incyte Genimics, Inc., USA) hybridization (hybridization) to obtain the first data. The Incyte rat GEM 2 microarray is a cDNA chip containing genes from the central nervous system of SD rats, which contains a total of 8,478 genes, including 7,747 specific genes (5,087 genes whose function has been identified and function is unknown). 2,660 genes).

Results Data analysis and plotting were performed using GEMTools software (Incyte Genimics, Inc., USA), and gene expression levels of two cDNAs were expressed by calculating the BDE (balanced differential expression) as shown in Equation 1. At this time, the value of the mixed herbal medicine extract administration group is used after normalizing by using an internal control (internal control).

The cDNA microarray scan results of the control group and the mixed herbal medicine extract group as shown in FIG. 2 are shown in the log-log scale scatter plot . In the linear regression analysis of FIG. 2 , the slope was 0.9628 ± 0.004 and the R ² was 0.836. Usefulness has been shown to be sufficient. In FIG. 2 (-) point having a point value are having a lot of the expressed genes in a mixed saengyakjae extract administration group, (+) values means a lot of the expressed gene in the control group. BDE | 2 | A total of 27 genes showed a difference in expression levels, 19 of which were found to have dual functions, and 8 of which had no function. The genes that were found to be significant among the specifically expressed genes were commonly used. Table 3 shows the list of genes whose function was found to be greater than or equal to 2 and showed significant differences in expression.

gene BDE Gin bank Rat Prialbumin (Transtyretin) -3.2 K03252 Mouse mRNA for type II 57 kd keratin -2.2 X03491 Rat Phosphotidyl Ethanolamine N-Methyltransferase -2.2 L14441 Rat Neuron-Specific Protein PEP-19 -2.1 M24852 Mouse Purkinie Cell Protein-4 (Pcp-4) -2.1 M96359 Rat Axl Receptor Thyroxine Kinase -2.0 AF046886 Mouse mRNA for type I 47 kd keratin -2.0 X03492 Sprag-Daury (Clone LRB9) RAB15 mRNA 2.0 M83679 Rat AMPA Type Glutamate Receptor Subunit 2 (GLUR2) mRNA 2.0 M85035 Mouse p19 mRNA, complete cds 2.0 U17259 Rat CDK108 mRNA 2.0 Y17328 Rat Neuron Allfactorome-Related ER Local Protein (D2Sut1e) 2.1 U03414 Mouse A-X Actin 2.1 J04181 Rat alpha-actin cardiac protein 2.1 X80130 Rat Calcium / Calmodulin-dependent Protein Kinase Type II Alpha-Subunit 2.3 J02942 Membrane Glycoprotein M6 2.3 S65735 Rat spectrin-like protein GTRAP41 2.3 AF225960 Rat Neuron Pentraxine Precursor 2.4 U18772 Rat Substrate Binding Subunit of Type II 5'-Diodinase D2p29 2.7 AF245040

If they are again divided into genes expressed in each of the control group and the mixed herbal medicine extract administration group is shown in FIG . Of the genes found specifically in the mixed herbal extract extract group ( Table 4 ), the most interesting are transthyretin, Axl receptor tyrosine kinase and neuron-specific, also called prealbumin. Protein PEP-19 (neuron-specific protein PEP-19). Among these, Axl receptor thyroxine kinase regulates thyroxine by regulating the growth of thyroid cells ( Thyroid , 9 , 563-567, 1999), and transthyretin is a transport protein that plays a role in the transfer of thyroxine from blood to the brain. known and - of (Brain Research Brain Research Reviews, 17 , 109-138, 1992), so thyroxine are known to increase the expression of nerve growth factor (Journal of Neurochemistry 63, 6-332, 1994) transthyretin nerve cells It may be inferred that it will indirectly affect growth. The neuron-specific protein PEP-19 is a calmodulin-regulatory protein that not only inhibits the apoptotic process of PC12 cells but also inhibits neuronal degeneration by inhibiting neuronal oxidation. Known ( Journal of Neuroscience , 20 , 2860-2866, 2000).

gene BDE Characteristics Prialbumin (transtyretin) -3.2 Transport protein for thyroxine and retinol Transfer hormone transport from blood to brain Phosphotidyl Ethanolamine N-methyltransferase -2.2 Promoting Synthesis of Phosphatidylcholine from Phosphatidyl Ethanolamine Neuron-Specific Protein PEP-19 -2.1 Calmodulin-regulated protein, resistance to apoptosis in PC12 cells, and inhibition of calmodulin-dependent neuronal nitric oxide synthase Mouse Purkinie Cell Protein-4 (Pcp-4) -2.1 Homologous Encoding of Rat Brain-Specific Antibody PEP-19 Axl receptor thyroxine kinase -2.0 Regulate thyroid cell growth and differentiation

Among the genes found specifically in the control group, attention was drawn to neuronal pentraxin and CDK108. Pentraxin is known to be expressed in lesions of Alzheimer's disease or degenerative central nervous system disease. It can be seen as an indicator of degeneration ( Table 5 ). CDK108 is a gene that synthesizes mu-crystalin. Mu-crystallin is known as a stress protein. Taken together, these cDNA microarray results suggest that mixed herbal extracts enhance neuronal capacity by promoting neuronal growth or inhibiting degeneration.

gene BDE Characteristics Substrate Binding Subunit of Type II 5'-Diodinase D2p29 2.7 cAMP-Induced Active Protein Type II Iodotyronine 5'-Diodinase, a Membrane-binding Enzyme, Promotes Biosynthesis of Thyroid Hormone in the Brain Neuron pentraxine precursor 2.4 Localized in Alzheimerdiseases neurofibrillary tangles Spectrin-Protein GTRAP41 2.3 Interacts with the carboxy terminal region of intracellular EAAT4 and modulates its glutamate transport activity Membrane Glycoprotein M6 2.3 Main CNS Myelin Protein PLP / DM20 Alpha-Actin Cardiac Protein 2.1 Associated with contractile structure in various muscles Mouse A-X Actin 2.1 Indicators to determine the prognosis and level of tumor progression of pigment cell tumors Neuron Allfactorome-Related ER Local Protein (D2Sut1e) 2.1 Neuronal protein with unknown function CDK108 2.0 Mu-crystallin coding gene, a stress protein Mouse p19 2.0 Feature not known AMPA type glutamate receptor subunit 2 (GLUR2) 2.0 Ca ²⁺ Permeability Regulation of Heteromic AMPA Receptor Complex RAB15 2.0 Low molecular weight GTP-binding protein nerve endings in synaptic capillary flow regulation Inhibitory GTPase during early endocytictrafficking

Example 3 Antioxidant Activity Test by MTS Essay

Measurement of cell proliferation and cytotoxicity cell proliferation assay kit; was carried out using (CellTiter 96 AQueous One Solution Cell Proliferation Assay kit Promega, UK?). Temperature and humidity using RPMI 1640 medium (Gibco BRL, Life Technologies, USA) containing 10% FBS (fetal bovine serum; Hyclone, USA) and 1% penicillin-streptomycin (Gibco BRL, Life Technologies, USA) In the 37 ℃ incubator maintained at 95% of air and 5% CO ₂ mixed gas 12 PC × cultured in a 96 well plate 1 × 10 ⁵ cells / ㎖ was dispensed for 24 hours at 37 ℃ 5% CO _The culture was carried out in _two incubators (Nuaire, USA). The culture solution was removed and the mixed herbal medicine extract was diluted in the medium so as to have a final concentration of 20, 40, 60, 80, 100 mg / ml, and treated with cells and pretreated at 37 ° C. for 24 hours. Cells in the same medium 24 hours after once washed with water, and 250 μM hydrogen peroxide for 30 minutes in a medium containing (Sigma, USA), and put into the MTS solution of 20 ㎕ per 100 ㎕ sepoaek to each well in 37 ℃ CO ₂ incubator After the reaction for 1 hour, UV absorbance was measured at 490 nm using a microplate reader (Molecular Device, USA).

As a result, as shown in FIG. 4 , the protective effect, that is, the antioxidant effect of the mixed herbal medicine extract against PC12 cell damage induced by hydrogen peroxide, was compared with the control group, and the cell viability decreased at low concentration and increased with increasing concentration. The 80 mg / ml and 100 mg / ml groups showed significant increases at P <0.05 and P <0.01, respectively.

<Example 4> Antioxidant activity test by active oxygen species removal ability

Free radical species (ROS) measurement was performed by applying the method of Jang et al. (Neuroscience Letters, 292: 41-44, 2000). 1 × 10 ⁵ cells / ml of PC12 cells were dispensed into 96 well plates, incubated in a 37 ° C. CO ₂ incubator for 24 hours, and then cultured for 24 hours under the same conditions. The cells were washed once with FBS-free medium and treated with hydrogen peroxide for 30 minutes, and H2DCFDA (Molecular Probes, USA) dissolved in N ', N-dimethyl formamide (amresco, USA) was brought to a final concentration of 50 μM. The mixture was reacted for 1 hour in a 37 ° C water bath. The generated fluorescence was measured at excitation 485 nm / emission 538 nm using Fluoroscan Ascent FL (Thermo Labsystem, Finland).

As a result, as shown in FIG. 5 , ROS generation was significantly suppressed depending on the concentration in the mixed herbal medicine extract administration group, and P <0.01 was significantly suppressed at all concentrations. In addition, the inhibitory effect of ROS generation per cell number of the mixed herbal medicine extract administration group on hydrogen peroxide-induced PC12 cell damage was determined by dividing the amount of ROS generated in PC12 cells by the number of cells shown in MTS . Same as The ROS generation per cell number tended to decrease depending on the concentration in the mixed herbal extract group, and there was a significant decrease of P <0.01 at all concentrations.

Preparation Example 1 Preparation of Tablet

The present inventors homogeneously mixed 100.0 mg of the mixed herbal medicine extract prepared according to Example 1, corn starch 90.0 mg, lactose 180 mg, L-hydroxypropyl cellulose 18.0 mg, polyvinylpyrrolidone 5.0 mg and ethanol appropriate amount Granulated by the wet granulation method, 1.8 mg of magnesium stearate was added and mixed, and the tablets were compressed to 400 mg.

Preparation Example 2 Preparation of Soft Capsule

Mixed herbal medicine extract prepared according to Example 1 100.0 mg, soybean oil 180.0 mg, lead 40.0 mg, coconut oil 128.0 mg, soybean phospholipid 20.5 mg, gelatin 212.0 mg, glycerin (specific gravity 1.24) 50.0 mg, d- sorbitol 76.0 MG, paraoxybenzoic acid methyl 0.54 mg, paraoxybenzoic acid propyl 0.90 mg, methyl vanillin 0.56 mg and yellow 203 were appropriately prepared to be contained in one capsule according to the method of soft capsule in the Pharmacopeia General Regulations.

Preparation Example 3 Preparation of Capsule

100.0 mg of the mixed herbal extracts prepared according to Example 1, 83.0 mg of corn starch, 175.0 mg of lactose and 2.0 mg of magnesium stearate were homogeneously mixed and filled to contain 360 mg in one capsule.

Preparation Example 4 Preparation of Food and Beverage

The present inventors prepared a food or beverage composition containing the mixed herbal medicine extract prepared according to Example 1 as an active ingredient as follows.

<4-1> Preparation of Chewing Gum

Chewing gum was prepared using a conventional method using the composition and content of 0.24 to 0.64% of the mixed herbal medicine extract prepared according to Example 1, 20% of gum base, 76.36 to 76.76% of sugar, 1% of fruit flavor, and 2% of water. It was.

<4-2> Preparation of Ice Cream

Mixed herbal medicine extract prepared according to Example 1 0.24 ~ 0.64%, milk fat 10.0%, nonfat milk solids 10.8%, sugar 12.0%, starch syrup 3.0%, emulsifying stabilizer (span, span) 0.5%, flavor (strawberry) 0.15 % And water of 63.31 to 62.91%, an ice cream was prepared using a conventional method.

<4-3> Preparation of Beverage

Mixed herbal extracts prepared according to Example 1 0.48 to 1.28 mg, honey 522 mg, thioctoamide amide 5 mg, nicotinic acid amide 10 mg, riboflavin sodium 3 mg, pyridoxine hydrochloride 2 mg, inositol 30 mg, orthoic acid 50 Drinks were prepared using conventional methods in the composition and content of mg and 200 ml of water.

As described above, the mixed herbal medicine extract of the present invention exhibits a very high memory capacity enhancing effect, and the mixed herbal medicine, which is a raw material of the mixed herbal medicine extract, is a natural medicine that is harmless to humans and has a good absorption rate, so it is associated with memory disorders such as dementia or Alzheimer's disease. It can be usefully used as a prophylactic or therapeutic composition.

Claims (14)

1) a herbal medicine selected from the group consisting of Sookjihwang, rawjihwang, Guhwanghwang, longan meat, Angelica and Hyunsam, 2) gojija, 3) acid and 4) acid and lactation, the herbal medicine extracted from alcohol or aqueous solution Having a mixed herbal extract. The composition of claim 1, wherein the composition ratio of the mixed herbal medicine is 1) herbal medicine selected from the group consisting of Sookjihwang, rawjihwang, dried jihwang, longan meat, Angelica and Hyunsam: 2) Gugija: 3) Medicinal herbs: 4) Cornus 5-90: 5-90: 5-90: 5-90 Mixed herb extract, characterized in that the weight ratio (w / w). The composition of claim 2, wherein the composition ratio of the mixed herbal medicine is 1) herbal medicine selected from the group consisting of Sookjihwang, rawjihwang, dried jihwang, longan meat, Angelica and Hyunsam: 2) Gugija: 3) Medicinal herbs: 4) Cornus 30-40: 30-40: 10-20: 10-20 mixed herbal extracts, characterized in that the weight ratio (w / w). The mixed herbal medicine extract of claim 1, wherein the mixed herbal medicine further comprises one or two or more herbal medicines selected from the group consisting of Baekbokryeong, Seokchangpo, Banja, Dermis, Cheonma, Peel Skin and Taxa. The composition of claim 4, wherein the composition ratio of the mixed herbal medicine is 1) a herbal medicine selected from the group consisting of Sookjihwang, rawjihwang, dried jihwang, longan meat, Angelica and Hyunsam: 2) gojija: 3) pesticides: 4) cornus: 5) Baekbokyeong, One or two or more herbal medicines selected from the group consisting of Seokchangpo, Hanja, Dermis, Cheonma, Bark skin and Taxa are 5-90: 5-90: 5-90: 5-90: 5-90 weight ratio (w / w) Mixed herbal medicine extract, characterized in that. The composition of claim 5, wherein the composition ratio of the mixed herbal medicines is 1) herbal medicines selected from the group consisting of Sookjihwang, rawjihwang, dried jihwang, longan meat, Angelica and Hyunsam: 2) gojija: 3) pesticides: 4) cornus lactation: 5) Baekbokyeong, 30-40: 30-40: 10-20: 10-20: 5-10 weight ratio (w / w) of one or two or more herbal medicines selected from the group consisting of Seokchangpo, Banja, Dermis, Cheonma, Bark skin and Taxa Mixed herbal medicine extract, characterized in that. The mixed herbal medicine extract according to claim 1, wherein the alcohol or aqueous alcohol solution is selected from the group consisting of 5 to 100% ethyl alcohol and 5 to 100% methyl alcohol. The method of claim 1, wherein the extract increases gene expression of transthyretin, Axl receptor tyrosine kinase, or neuron-specific protein PEP-19. Mixed herbal medicine extract, characterized in that. The mixed herbal extract according to claim 1, wherein the extract has an antioxidant action. 1) A manufacturing method for extracting the herbal medicine consisting of a medicinal herb selected from the group consisting of sulfuric acid sulfur, raw sulfur, dried sulfur, longan meat, Angelica and Hyunsam, 2) gojija, 3) powdered acid and 4) acid and lactation. The method of claim 10, wherein the mixed herbal medicine further comprises one or two or more herbal medicines selected from the group consisting of Baekbokryeong, Seokchangpo, Banja, Dermis, Cheonma, Bark skin and Taxa. A pharmaceutical composition for treating or preventing memory disorders, comprising the mixed herbal medicine extract of claim 1 as an active ingredient. The pharmaceutical composition of claim 12, wherein the memory disorder disorder is dementia or Alzheimer's disease. The health food for memory enhancement containing the mixed herbal medicine extract of claim 1 as an active ingredient.