KR20210002399A - A method for preparing ethyl acetate fraction from Aruncus dioicus var. kamtschaticus and the composition comprising the same as the effective ingredient - Google Patents

Abstract

The present application relates to: a method for manufacturing an Aruncus dioicus ethyl acetate fraction; the fraction thereof; and a composition for preventing, treating or alleviating diabetes, memory and cognitive decline, dyslipidemia, liver damage, cholinergic system damage, and mitochondrial damage, which contains the fraction as an active component.

Description

A method for preparing ethyl acetate fraction and a composition containing the fraction as an active ingredient {A method for preparing ethyl acetate fraction from Aruncus dioicus var. kamtschaticus and the composition comprising the same as the effective ingredient}

The present application relates to a method for preparing an ethyl acetate fraction of Equestrian Equestrian horses, the fraction, and a composition comprising the fraction as an active ingredient.

Obesity is a disease caused by excessive accumulation of remaining energy as fat when energy intake is greater than energy consumption, and is caused by genetic or lifestyle causes. In the case of Koreans, the number of people with severe obesity (body mass index less than 30-35) and ultra-high obesity (body mass index more than 35) is rapidly increasing. According to the '2016 Obesity White Paper' published by the National Health Insurance Service, Korea's ultra-high obesity population in 2006 increased from 1,448 to 3,6343 in 2015, a three-fold increase in the new nine years. The obese population increased by 1.7 times from 2,322,146 to 4.66,060, and it is predicted that one out of 17 adults in Korea will become highly obese in 2025. Recently, obesity is one of the metabolic syndromes, and metabolic syndrome is a phenomenon in which risk factors for various cardiovascular diseases and type 2 diabetes form clusters into one disease group. Therefore, obesity acts as a risk factor for causing diseases such as type 2 diabetes, dyslipidemia, fatty liver, cardiovascular disease and chronic degenerative diseases. In addition, obesity causes symptoms of'insulin resistance', which leads to type 2 diabetes. Insulin resistance is caused by impairing the signaling system of insulin due to insulin resistance generated in cells, which leads to an increase in blood sugar, and furthermore, it is a symptom that causes various diseases. Specifically, the accumulation of visceral fat is an important factor causing cardiovascular disease, and it has been proven that blood concentration increases due to an increase in synthetic secretion along with the accumulation of visceral fat. TNF-α secreted from adipocytes decreases insulin sensitivity, resulting in diabetes mellitus. It was reported as one of the mechanisms of occurrence. Recently, some scholars have reported that peripheral insulin resistance further causes central insulin resistance, thereby damaging the central nerve and causing cognitive impairment in the form of Alzheimer's disease, and Alzheimer's disease is sometimes referred to as type 3 diabetes.

The necessity of developing and researching natural products or foods to treat, prevent, or improve cognitive decline caused by insulin resistance caused by obesity has emerged.

In academic literature related to canine horseback riding, excellent antioxidant and antibacterial activities have been reported in the antioxidative and antibacterial activity of solvent extracts of equestrian horses (Kim et al., 2011. Journal of the Korean Society of Food Science and Nutrition, 40: 47-55). In a study on ingredients (Shim Chang-min, Master's thesis, Department of Pharmacy, Daegu Catholic University, 2009), cinnamic acid derivatives, quercetin derivatives, and kaempferol derivatives with antioxidant power have been reported. On the other hand, there is Korean Patent Registration No. 10-1869353 as a patent related to equestrian horseback riding, but this relates to a novel compound and anti-inflammatory composition isolated from the equestrian equestrian extract, and Korean Registered Patent No. 10-1243220 uses Samnamul extract as an active ingredient. It relates to a composition for whitening and wrinkle improvement, including diabetes, memory and cognitive decline, dyslipidemia, liver damage, and cholinergic properties of ethyl acetate fractions of dicaffeoyl glucose isomer Ⅰ, Ⅱ as major phenolic compounds in the literature. There are no studies suggesting or initiating a composition for the prevention, improvement or treatment of cognitive damage due to systemic abnormalities, mitochondrial damage, and central insulin resistance.

Under this background, the present inventors have made intensive research efforts to develop a composition for preventing, ameliorating or treating related diseases including cognitive decline caused by obesity or high fat diet, including central insulin resistance, dicaffeoyl glucose isomer Ⅰ, Ⅱ This application was completed by confirming the function of the ethyl acetate fraction of Equestrian Equestrian horses, which is the main phenolic compound.

1: Korean Patent Registration No. 10-1869353 Registration Patent Publication, 2: Korean Patent Publication No. 10-1243220

1: Antioxidant and antimicrobial activity of solvent extracts of Equestrian Equestrian (Kim et al., 2011. Korean Food Nutrition Journal of Science, 40: 47-55) 2: A Study on the Components of Equestrian Equestrian (Shim Chang-Min, Master's Thesis, Department of Pharmacy, Daegu Catholic University, 2009)

The object of the present application is to provide a method for preparing a fraction of ethyl acetate fractions.

Another object of the present application is to provide an ethylacetate fraction for snow horseback riding.

Another object of the present application is diabetes caused by a high-fat diet containing ethyl acetate fraction as an active ingredient, decreased memory and cognitive function, dyslipidemia, liver damage, damaged cholinergic system, mitochondrial damage, central insulin resistance. It is to provide a pharmaceutical composition for preventing and treating cognitive damage.

Another object of the present application is diabetes caused by a high-fat diet containing ethyl acetate fraction as an active ingredient, decreased memory and cognitive function, dyslipidemia, liver damage, damaged cholinergic system, mitochondrial damage, central insulin resistance. It is to provide a food composition for preventing and improving cognitive damage.

Other objects and advantages of the present application will become more apparent by the following detailed description in conjunction with the appended claims and drawings. Contents not described in the present specification will be omitted because those skilled in the technical field or similar field of the present application can sufficiently recognize and infer.

Hereinafter, the content of the present application will be described in detail. On the other hand, the description and embodiment of one embodiment disclosed in the present application may be applied to the description and embodiment of other aspects with respect to common matters. In addition, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the specific description described below.

In order to achieve the object of the present application, the present application provides a method for preparing a snow horseback riding extract and a snow horseback riding ethyl acetate fraction. The preparation method of the ethyl acetate fraction of the snow horseback riding,

Mixing and extracting the freeze-dried snow horseback riding with ethanol to obtain a snow horseback riding extract;

Filtering and concentrating the extract;

Dissolving the concentrated extract in tertiary distilled water and fractionating using n-hexane, chloroform and ethyl acetate solvents in the same ratio to obtain each fraction;

The ethyl acetate fraction may be concentrated and freeze-dried to obtain a step.

Aruncus dioicus var.kamtschaticus used in this experiment can be purchased at Taebaek Ssamchae Village, located in Taebaek City, Gangwon-do, and can be used in any part of the Equestrian Horse, but specifically, freeze-dried using young leaves and stems, and frozen. After mixing 20 g of dried snow horses in 0.5 to 2 L of 50% to 80% ethanol, specifically 1 L, extractor including a reflux cooling device at 30 to 50°C, specifically at 40°C for 2 hours and 30 minutes It can be extracted using. The extracted extract may be concentrated by filtering with a filter device, specifically, No. 2 filter paper (Whatman Inc, Kent, UK) to obtain a snow horse extract. The concentrated snow horseback riding extract is dissolved in an organic solvent, specifically in tertiary distilled water, and n-hexane, chloroform, and ethyl acetate solvents are mixed or separated in various ratios, and can be fractionated by sequentially or simultaneously. Specifically, fractionation may be performed by using the solvent in the same ratio, or ethyl acetate fraction may be preferably used. Each fraction was concentrated, freeze-dried, and stored at -20°C.

To achieve the purpose of the present application, ultra-performance liquid-quadrupole-time-of flight mass spectrometry (UPLC-QTOF/MS ² ) can be used based on chromatographic analysis to separate the main compounds of the Equestrian Equestrian ethyl acetate fraction. have. That is, a sample dissolved in methanol was filtered through a 0.2 μm filter, and UPLC separation of the phenolic compound was performed in an ACQUITY UPLC BEH C ₁₈ column (2.1 x 100 mm, 1.7 μm particle size, Waters Corp, Milford, MA, USA). And, it can flow at 40℃ at a flow rate of 0.4 mL/min. The mobile phases used in the analysis are solvent A (distilled water with 0.1% formic acid) and solvent B (ACN with 0.1% formic acid), and data can be obtained using suitable solvent gradient conditions. The following conditions were used in this application: 0-0.5 min, 0% B; 0.5-8 min, 100% B; 8-8.5 min 100% B; 8.5-10 min 0% B; And 10-11 min 0% B. To obtain MS ² data, the ionization can be operated in negative electron spray (ESI) mode and analyzed using ionization conditions suitable for the experiment. The conditions used in this application are as follows: lamp impact energy, 20-45 V; Capillary voltage, 3 kV; Desolvent temperature, 350°C; Pressure in the nebulizer, 40 psi; fragmentor, 175 V; cone voltage, 40V; Mass range, 50-1, 200 m/z; Oven temperature, 40°C. All MS data can be analyzed using MarkerLynx software (Waters MassLynx™, Waters, Milford, MA, USA), but is not limited thereto.

In order to achieve the object of the present application, the present application provides a pharmaceutical composition for the prevention and treatment of metabolic diseases caused by a high fat diet or a food composition for prevention and improvement, comprising an ethyl acetate fraction of equestrian horse riding as an active ingredient. The metabolic disease may be any one or more selected from the group consisting of diabetes, memory and cognitive decline, dyslipidemia, liver damage, cholinergic system damage, mitochondrial damage, and cognitive impairment due to central insulin resistance.

In order to achieve the object of the present application, the present application provides a pharmaceutical composition for preventing and treating diabetes caused by a high fat diet or a food composition for preventing and improving, comprising an ethyl acetate fraction as an active ingredient. The effect of preventing, treating or improving diabetes can be confirmed through measurement of glucose tolerance by methods including, but not limited to, fasting blood sugar and IPGTT.

In order to achieve the object of the present application, the present application is a pharmaceutical composition for preventing and treating memory loss and cognitive decline caused by a high fat diet containing ethyl acetate fraction as an active ingredient, or a food composition for prevention and improvement Provides. In the present invention, the term "prevention, treatment or improvement of memory loss" refers to all actions in which the symptoms of memory loss, one of the symptoms of dementia, are improved or beneficially changed by the administration of the pharmaceutical composition or food composition according to the present invention. do. In addition, in the present invention, the term "prevention, treatment or improvement of cognitive decline" means that all symptoms of cognitive decline, one of the symptoms of dementia, are improved or beneficially changed by the administration of the pharmaceutical composition or food composition according to the present invention. Means action.

In addition, in the present invention, "prevention" refers to diabetes, memory and cognitive decline, dementia, dyslipidemia, liver damage, damaged cholinergic system caused by a high fat diet by administration of the pharmaceutical composition or food composition according to the present invention , Mitochondrial damage, refers to any action that inhibits or delays the onset of cognitive damage due to central insulin resistance, and "treatment" refers to any action that improves or beneficially alters symptoms caused by dementia by administration of the pharmaceutical composition. it means. Dementia is a syndrome that accompanies various symptoms of acquired cognitive dysfunction such as decreased memory, speech and behavioral disorders, due to the irreversible destruction of the cranial nerves due to atrophy of the brain, reduction of nerve cells and the appearance of senile plaques. Refers to. Alzheimer's Disease (Alzheimer's Disease) is the most important cause of dementia and is classified into familial type and sporadic type, and more than 90% of all diseases are sporadic type that occurs mainly in the elderly over 65 years of age. In the familial type, mutations in the amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2) genes are known as etiologies. In the case of the sporadic type, aging and diversity of ApoE4 alleles were reported as etiologies. Aggregation of Aβ (β-amyloid) is a pathological phenomenon common in both familial and sporadic types. Aggregated Aβ generates free radicals and induces inflammatory reactions to induce neuronal cell death.

In order to achieve the object of the present application, the present application provides a pharmaceutical composition for preventing and treating dyslipidemia caused by a high fat diet, or a food composition for prevention and improvement, comprising an ethylacetate fraction of equestrian horse riding as an active ingredient. Dyslipidemia is a blood vessel caused by an increase in total cholesterol, low-density lipoprotein-cholesterol (LDL-Cholesterol), triglyceride, or a decrease in high-density lipoprotein-cholesterol (HDL-Cholesterol), such as hyperlipidemia and arteriosclerosis. It refers to the disease, and refers to symptoms such as blood circulation disorders in which blood supply to various organs and extremities is not smooth as the inner surface of the artery becomes narrow due to the deposition of cholesterol on the inner wall of the artery. Dyslipidemia may cause various diseases according to changes in blood lipid concentration. In particular, it is known to cause arteriosclerosis, high blood pressure, heart disease, and cerebral hemorrhage.

In order to achieve the object of the present application, the present application provides a pharmaceutical composition for preventing and treating liver damage caused by a high fat diet, or a food composition for prevention and improvement, comprising an ethylacetate fraction of equestrian horse riding as an active ingredient. Liver damage includes, but is not limited to, hepatitis B, hepatitis C, alcoholic liver disease, cirrhosis, and various damages to liver tissues or cells due to liver cancer. The dyslipidemia and liver damage were measured in serum glutamic oxaloacetic transaminase (GOT), glutamine pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatine (CRE), lactate dehydrogenase (LDH), total cholesterol (TCHO), It can be measured through triglyceride (TG) and high density lipoprotein cholesterol (HDLC) concentrations. In addition, low density lipoprotein cholesterol (LDLC) can be calculated by the calculation formula, LDLC (mg/dL) = TCHO-(HDLC + TG/5) according to Friedewald's method, and HTR, the ratio of HDLC to TCHO, is HTR (%). = (HDLC/TCHO) x 100 can be calculated.

In order to achieve the object of the present application, the present application provides a pharmaceutical composition for the prevention and treatment of damaged cholinergic system caused by a high fat diet, or a food composition for prevention and improvement, comprising an ethyl acetate fraction as an active ingredient. . The cholinergic system uses acetylcholine (ACh) as a neurotransmitter, and acetylcholine is a neurotransmitter commonly found in neuromuscular junctions. There are two cholinergic diffusion control systems in the brain, one of which is the Basal Forebrain Complex. The cell body of cholinergic neurons is located in the basal forebrain nucleus, especially the Basal nucleus of Meynert, and the cell body is also distributed in the medial septal nuclei projected to the hippocampus. In the minor basal ganglia, acetylcholine spreads to almost all areas of the cortical region and can affect the overall aspects of neurological and mental function. For the analysis of the cholinergic system, MDA, SOD, GSH, ACh levels and AChE activity experiments of brain tissue may be performed, but the analysis index is not limited thereto.

In order to achieve the object of the present application, the present application provides a pharmaceutical composition for preventing and treating mitochondrial damage, or a food composition for preventing and improving, comprising an ethyl acetate fraction of equestrian horse riding as an active ingredient. Diseases due to mitochondrial dysfunction include swelling due to mitochondrial membrane potential abnormalities, dysfunction due to oxidative stress caused by reactive oxygen species, or free radicals, dysfunction due to genetic factors, and oxidative phosphorylation for mitochondrial energy production. Diseases due to defects of the disease may include, and diseases caused by the above causes are multiple sclerosis, encephalomyelitis, cranial neuromyositis, peripheral neuropathy, Reye's syndrome, Friedrich ataxia, Alper syndrome, MELAS, migraine, psychosis, depression, seizures and dementia. , Parathyroid episode, optic nerve atrophy, optic neuropathy, retinal pigmentation, cataract, hyperaldosteronemia, hypoparathyroidism, myopathy, muscle atrophy, myoglobinuria, muscle tone inhibition, muscle pain, decreased motor tolerance, tubulopathy, kidney failure, liver failure , Hepatic insufficiency, hepatomegaly, iron blood cell anemia, neutropenia, hypothrombocytopenia, diarrhea, villi atrophy, multiple vomiting, dysphagia, constipation, sensorineural hearing loss, epilepsy, mental retardation, epilepsy, Alzheimer's, Parkinson, Huntington Disease and the like may occur. In order to confirm the function of mitochondria, Mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and Adenosin triphosphate (ATP) can be measured, and p-JNK, BAX, Cytochrome to analyze factors related to mitochondrial-mediated apoptosis pathway. C, p-Akt, p-tau, TNF-α, and p-NK-κB may be measured using Western blot, but the index is not limited thereto.

In order to achieve the object of the present application, the present application is a pharmaceutical composition for the prevention and treatment of cognitive damage caused by central insulin resistance caused by a high-fat diet containing ethyl acetate fraction as an active ingredient or a food composition for prevention and improvement Provides. When insulin resistance is caused in the central nervous system, when insulin and insulin receptors are bound, the serine residue of the insulin receptor substrate (IRS) is phosphorylated, thereby reducing the activity of Akt. As hyperphosphorylation progresses, leading to nerve fiber entanglement, which can lead to neurotransmission disturbance and apoptosis, inhibition of hyperphosphorylation of IRS and tau, recovery of AMPK phosphorylation, increase in IDE expression and decrease in beta-amyloid plaque formation are recognized due to insulin resistance. This is an indicator that can confirm that damage-related mechanisms are suppressed. Cognitive damage due to central insulin resistance can be confirmed through the measurement of each factor p-IRS, p-Akt, p-tau, p-AMPK, IDE, and Amyloid β, but the indicator is not limited thereto.

This application is for diabetes, memory and cognitive decline, dyslipidemia, liver damage, cholinergic system abnormality, mitochondrial damage, central It provides a pharmaceutical composition effective in the prevention and treatment of cognitive damage caused by insulin resistance.

The pharmaceutical composition including the fraction of the present application may further include an appropriate carrier, excipient, and diluent commonly used in the preparation of pharmaceutical compositions. The pharmaceutical composition comprising the fraction according to the present application is in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method. It can be formulated and used. Carriers, excipients, and diluents that may be included in the composition containing the fraction include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate. , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient, such as starch, calcium carbonate, and sucrose, in the fraction. ) Or lactose (lactose), gelatin, etc. are mixed to prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include water and liquid paraffin, which are simple diluents commonly used for suspensions, liquid solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweetening agents, fragrances, and preservatives. have. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. The composition of the present application may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular. The preferred dosage of the fractions of the present application varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. Administration may be administered once a day, or may be divided several times.

The composition comprising the fraction of the present application provides a health functional food composition for the prevention and improvement of cognitive damage caused by diabetes, memory and cognitive decline, dyslipidemia, liver damage, cholinergic system abnormality, mitochondrial damage, central insulin resistance do. Foods to which the present extract can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, and health supplement foods. In addition, the fractions of the present application may be added to food or beverages for the purpose of preventing cognitive damage due to diabetes, memory and cognitive decline, dyslipidemia, liver damage, cholinergic system abnormality, mitochondrial damage, and central insulin resistance. . At this time, the amount of the extract in the food or beverage may be added in an amount of 0.01 to 15% by weight of the total food weight, and the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. have. The health functional food of the present application includes forms such as tablets, capsules, pills, and liquids. The health functional beverage composition of the present application is not particularly limited to other ingredients other than containing the extract as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as an additional ingredient, such as a conventional beverage. Examples of the natural carbohydrates described above include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides such as conventional sugars such as dextrin, cyclodextrin, and the like, and xylitol, sorbitol, erythritol, and the like. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present application In addition to the above, the composition of the present application includes various nutrients, vitamins, minerals (electrolytes), and synthetic flavors. Flavoring agents and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols , Carbonated beverages, etc. In addition, the compositions of the present application may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages These components may be independently or in combination. The proportion of these additives is not so important, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the composition of the present application.

Equestrian ethyl acetate fraction of the present application is excellent in preventing, treating or improving diabetes, memory and cognitive decline, dyslipidemia, liver damage, damaged cholinergic system, mitochondrial damage, and cognitive damage caused by central insulin resistance.

1 is a graph measured by comparing (A) fasting blood sugar, (B) glucose tolerance (IPGTT), and (C) AUC with a control group and a high-fat diet group of mice administered with an ethylacetate fraction of equestrian horse riding.
Figure 2 is a control group of (A) spatial cognition (Y-maze), (B) short-term memory (Passive avoidance), (C) long-term memory (Morris water maze) of rats administered with ethyl acetate fraction for equestrian riding, This is a graph measured compared to the high fat diet group.
Figure 3 is a graph measured by comparing the (A) MDA, (B) SOD, (C) GSH levels of the rats administered with the Equestrian Equestrian Equestrian Fraction with the control group and the high fat diet group.
Figure 4 is a comparison of the Western blot measurement of (A) acetylcholine (ACh), (B) acetylcholinesterase (AChE), (C) AChE in mice administered with the ethyl acetate fraction of the equestrian horseback riding with the control group and the high fat diet group. This is the graph shown.
Figure 5 is a graph measured by comparing the (A) MMP, (B) ROS, (C) ATP levels of the rats administered with the Equestrian Equestrian Ethyl Acetate fraction compared with the control group and the high fat diet group.
Figure 6 is a graph measured by comparing the (A) MMP, (B) ROS, (C) ATP levels of the rats administered with the Equestrian Equestrian Ethyl Acetate fraction with the control group and the TMT group.
Figure 7 is the p-JNK, BAX, Cytochrome C, p-Akt, p-tau, TNF-α, p-NK-κB indicators of the rats administered with the ethyl acetate fraction of equestrian horses compared to the control group and the TMT group. Western blot pictures and graphs of the results.
Figure 8 is a comparison of the (A) p-IRS, p-Akt, p-tau, (B) p-AMPK, IDE, β-amyloid levels of the rats administered with the Equestrian Equestrian Equestrian Fraction with the control group and the high fat diet group. It is a measured graph.
FIG. 9 is a graph showing (A) UPLC chromatography and dicaffeoylglucose molecular structure of the Equestrian Equestrian Equestrian Equestrian Fraction, (B) UPLC-QTOF/MS ² results of dicaffeoylglucose isomer I, and (C) UPLC-QTOF/MS ² results of dicaffeoylglucose isomer ^II. to be.

Hereinafter, the present invention will be described in more detail through examples according to the present application. However, the following example of the present application is only an example of the present application. These examples are intended to illustrate the present invention in more detail, and it is obvious to those of ordinary skill in the art that the scope of the present invention set forth in the appended claims is not limited by these examples. something to do.

Example 1. Equestrian Equestrian Extract and Equestrian Equestrian Equestrian Fraction

Aruncus dioicus var. kamtschaticus used in this experiment was purchased from Taebaekssamchae Village located in Taebaek City, Gangwon-do. Snow dog riding was verified by the National Institute of Forest Science as snow dog riding. It was freeze-dried using young leaves and stems of snow horses, and 20 g of freeze-dried snow horses were mixed with 1 L of 80% ethanol, and extracted using a reflux cooling device at 40°C for 2 hours and 30 minutes The resulting extract was filtered through No.2 filter paper (Whatman Inc, Kent, UK) and concentrated to obtain a snow horse extract.

The concentrated snow horseback riding extract was dissolved in tertiary distilled water, and fractionated using the same ratio of n-hexane, chloroform, and ethyl acetate solvents. Each fraction was concentrated, freeze-dried, and stored at -20°C. Of the fractions, an ethyl acetate fraction was used.

Experimental Example 1. Fasting blood glucose and glucose tolerance measurement

Statistical processing of experimental animals and experiments was prepared and performed by the following method.

Four-week-old C57BL/6 male mice were purchased from a laboratory animal supplier (Samtako Bio Korea, Korea). The experimental animals maintained a constant temperature (22±2℃) and humidity (55%), and were supplied with a sufficient amount of drinking water and feed under 12 hours of light and shading conditions. Each group was divided into 4 groups of 8 animals and adapted to a regular diet (protein (20 kcal%/g), carbohydrate (70 kcal%/g), fat (10 kcal%/g) included, 3.85 kcal/g) for one week. All experimental groups, except for the normal diet, included a high fat diet, including protein (20 kcal%/g), carbohydrate (20 kcal%/g) and fat (60 kcal%/g), 5.24 After inducing obesity for 15 weeks with kcal/g), samples (20, 40 mg/kg of body weight (EFAD20 and EFAD40 group)) were orally administered with a high fat diet from 16 weeks to 19 weeks. During the experiment period, the condition of the experimental animals was confirmed through observation of body weight and dietary intake once a week.

All experiments were expressed as mean and standard deviation (mean±SD) through repeated experiments, and statistical significance was verified by analysis of variance (ANOVA) using SAS ^® version 9.1 (SAS institute, Cary, NC, USA). Was performed, and the significant difference between each sample was verified at the 5% level by Duncan's multiple range test.

The following method was used to measure fasting blood glucose and glucose tolerance of the experimental animals. Fasting blood glucose from 15 weeks until the obesity induction period to 3 weeks after the sample diet was measured using an Accu-Chek glucose meter (Roche Diagnostics Australia Pty. Ltd., Castle Hill, Australia) by collecting blood from venous vessels of the rat tail. Intraperitoneal gluose tolerance test (IPGTT) was performed at week 19, and the experimental animals were intraperitoneally injected with glucose (2 g/kg of body weight) after 12 hours fasting. Blood glucose was measured using an Accu-Chek glucose meter for 0, 15, 30, 60, 90 and 120 minutes through blood obtained from venous vessels of rat tail.

As a result of the experiment, it was confirmed that the fasting blood sugar of the rats fed a high fat diet was very high compared to the rats fed the regular diet through the fasting blood sugar measured at week 15. Accordingly, the fasting blood sugar in the sample diet groups after feeding the sample It was confirmed that it was effectively reduced (Fig. 1(A)). In addition, through the IPGTT results to determine glucose tolerance, it was confirmed that in the high-fat diet group, blood sugar soared rapidly after glucose injection, and it took a long time to recover after that, and the increase in blood sugar was rapid in the group with high concentration in the sample group. It was not possible to confirm the tendency for blood sugar to gradually recover (Figs. 1(B), (C)). Accordingly, it was confirmed that the sample was effective in preventing, treating, and improving diabetes, as it had excellent ability to suppress the increase in blood sugar caused by high fat and impaired glucose tolerance.

Experimental Example 2. Behavioral Analysis Experiment

Statistical processing of experimental animals and experiments was prepared and performed by the method described in Experimental Example 1. Behavioral analysis experiments were performed by the following methods: Y-maze, Passive Avoidance, and Morris water maze tests.

Y-maze test

After IPGTT measurement, a Y-maze experiment was performed to test the spatial memory and innate tendency of mice for a short period without learning. The maze is made of white plastic, and each arm is 33 cm long, 15 cm high and 10 cm wide, and has an angle of 120°C. In this experiment, immediate spatial preference was recorded by SMART video tracking system (SMART v3.0, Panalb SL, Energia, Barcelona, Spain) for 8 minutes, and spontaneous spatial preference behavior was calculated as a percentage.

Passive Avoidance test

After the Y-maze experiment, a passive avoidance experiment was performed to evaluate the short-term memory capacity of mice formed by instantaneous electrical stimulation for two days. In the experimental equipment, two spaces are separated by a door, one space has very bright lights, and the other space is configured to allow electricity to flow through the metal grid floor. On the first day, leave the rat in a bright room for 1 minute for 1 minute, open the door between the two spaces, and close the door when the rat moves into the dark space. Then, an electric shock (0.5 mA, 3 s) was applied to the foot. On the second day, the rat was placed in a bright room and the step-through latency time it took to move to a dark place was measured (step through latency maximum testing time: 300 s).

Morris water maze test

After the passive avoidance experiment, the Morris water maze experiment was performed to evaluate the long-term spatial memory capacity through repetitive learning in rats. The experimental set-up is a circular stainless steel pool (90 cm in diameter) consisting of randomly divided quadrants (E, N, S and W) with visual symbols on the sides. The pool was filled with water (22?2?C) dissolved in white powdered milk to a height of 30 cm. The platform (6 cm in diameter) did not change position during training and was placed in the middle of the N zone. Four exercises (E, N, S and W) placed in each quadrant to reach the platform were conducted over four days. The SMART video tracking system measured the waiting time until reaching the hidden platform for a maximum of 60 seconds for 4 days. After 4 days of training, the platform was removed and how long it stayed in the N area where the platform was located was measured for 60 seconds, and the experimental results are as follows.

Figure 2 (A), (B), (C) is the result of the Y-maze experiment to see the exploration ability of innate rats, it was confirmed that there was no difference in the motor ability between the rats of each group, and the shift behavior The result was very low in the high fat diet group, and it was confirmed that the spatial cognitive ability of the rats was reduced. Accordingly, it was confirmed that spatial perception was improved in the sample group.

2D is a result of a passive avoidance experiment to see short-term memory performance through electric shock, and it was confirmed that short-term memory capacity was decreased in the high fat diet group, and thus showed excellent improvement in the sample group.

Figure 2 (E), (F), (G) is a Morris water maze experiment to see the long-term memory capacity of rats through 4 days of repetitive learning, rats of each group for 4 days recognize the platform As a result of confirming the degree of staying in the N zone where the platform existed after removing the platform, it was found that the high-fat diet group lacks the ability to recognize the platform. It was confirmed that such impaired long-term memory was improved. Through each experiment, it was confirmed that the impaired cognitive ability and memory caused by the high fat diet were effectively improved through the sample diet.

Experimental Example 3. Biochemical analysis of serum

Statistical processing of experimental animals and experiments was prepared and performed by the method described in Experimental Example 1. The following method was performed for the biochemical analysis of serum.

Experimental animals were anesthetized with ethyl ether and blood was collected from the heart, and the collected blood was centrifuged at 4°C for 15 minutes at 15,000 rpm, and serum was used as an analysis sample. Serum glutamic oxaloacetic transaminase (GOT), glutamine pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatine (CRE), lactate dehydrogenase (LDH), total cholesterol (TCHO), triglyceride (TG) and high density lipoprotein cholesterol ( HDLC) concentration was measured using a blood analyzer (Fuji dri-chem 4000i; Fuji film Co., Tokyo, Japan). In addition, low density lipoprotein cholesterol (LDLC) was calculated according to the method of Friedewald et al., LDLC (mg/dL) = TCHO-(HDLC + TG/5), and HTR, the ratio of HDLC and TCHO, was HTR (%) = ( HDLC/TCHO) x 100.

As a result of the experiment, it was confirmed that the levels of GOT and GPT, which are liver damage factors, were very high in the high-fat diet group, and the contents of TCHO and TG were high. Therefore, it was confirmed that liver damage and dyslipidemia were caused by a high fat diet, and GOT, GPT, TCHO, and TG were reduced in the sample group.Through this, the sample effectively reduced liver damage and dyslipidemia caused by a high fat diet. It was confirmed that it was improved (Table 1).

Experimental Example 4. Biochemical Indicator Experiments (ex vivo tests)

The experimental animals and the statistical processing of the experiment were performed by the method described in Experimental Example 1, and the brain tissue of the rat sacrificed for the biochemical indicator experiment was placed on ice, and 10 times cold phosphate buffered physiological saline (PBS) was added and then chopped. . After homogenization, the brain tissue was maintained at -80°C until measured in MDA, SOD, GSH, ACh levels and AChE activity experiments.

In order to measure the biochemical indicator of lipid peroxide, MDA (Malondialdehyde), the pretreated brain tissue was centrifuged at 2,500x g for 10 minutes to obtain a supernatant. The supernatant was mixed with 1% phosphoric acid and 0.67% TBA solution and then placed in a water bath (95°C) for 1 hour. Then, absorbance was measured at 532 nm.

In order to measure the level of superoxide dismutase (SOD), the previously pretreated brain tissue was centrifuged at 400 x g for 10 minutes at 4°C. Mix the supernatant with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt) (WST-1) working solution and enzyme solution Placed at 37°C for 20 minutes. Then, absorbance was measured at 450 nm for 10 minutes at 1 minute intervals.

In order to measure reduced glutathione (GSH) in brain tissue, a 10-fold dose of phosphate buffer was taken into the brain tissue, mixed and homogenized. After that, the homogenate was centrifuged at 10,00 0 xg for 15 minutes. Each sample was relative quantified through protein quantification in the supernatant. The supernatant was mixed with 5% metaphosphoric acid and centrifuged at 2,000 xg for 2 minutes. Tris-HCl buffer (0.26M, pH 7.8), 0.65N NaOH and OPT (1mg/mL) were added to the supernatant, and left for 15 minutes in the dark. Then, it was measured with a fluorescent microplate reader (Infinite 200, Tecan Co., San Jose, CA, USA) (excitation filter 320 nm, emission filter, 420 nm). The measured GSH levels were expressed as values for the reduced glutathione standard curve.

To measure the cholinergic system, acetylcholine (ACh) levels and acetylcholinesterase (AChE) activity were measured as biochemical indicators. Acetylcholine (ACh) is a neurotransmitter secreted at the synaptic terminal, and acetylcholinesterase (AChE) is an enzyme that degrades ACh. For the measurement of each index, the pretreated brain tissue was centrifuged at 12,000 xg for 30 minutes at 4°C. To measure the ACh level, the supernatant was mixed with an alkaline hydroxylamine reagent (2M hydroxylamine in HCl and 3.5N sodium hydroxide). After 1 minute, (0.5 N HCl and 0.37 M FeCl ₃ in 0.1 N HCl) solution was added to the mixture, and the absorbance was checked at 540 nm. To measure AChE activity, 50mM sodium phosphate buffer (pH 8.0) was added to the previous supernatant and the mixture was left at 37°C for 5 minutes. Then, the Ellman reaction mixture (1mM DTNB and 0.5mM acetylthiocholine in 50mM sodium phosphate buffer) was added. The absorbance was incubated at 37° C. for 20 minutes and then measured at 405 nm at 1 minute intervals for 10 minutes.

The experimental results were as described below.

As a result of checking the MDA level, which is a lipid peroxide in brain tissue, the high fat diet group showed a high MDA content, and the sample group showed that the MDA content corresponds to the general diet group, indicating that the sample effectively inhibited the production of MDA ( Fig. 3(A)). As a result of confirming the levels of SOD and reduced form of GSH, which play a major role in the body's antioxidant system, in the brain tissue, it was confirmed that the levels of SOD and reduced form of GSH were rapidly decreased in the high-fat diet group, indicating that the antioxidant system was damaged (Fig. 3 (B)). ),(C)). Accordingly, it was confirmed that the sample diet was excellent in effectively recovering the antioxidant system damaged by the high fat diet, as the SOD and reduced GSH levels increased in the sample group.

As a result of the cholinergic system experiment, it was confirmed that the cholinergic system was damaged as the ACh content decreased and the AChE activity increased in the high fat diet group, and thus the ACh content in the sample group was increased (Fig. 4 (A)) and AChE activity decreased. As confirming (Fig. 4 (B), (C)), it was confirmed that the damaged cholinergic system caused by the high fat diet was effectively recovered through the sample diet.

Experimental Example 5. Mitochondrial damage test (Ex vivo tests)

Statistical processing of experimental animals and experiments was performed by the method described in Experimental Example 1.

First, a method for examining mitochondrial damage and improvement due to a high fat diet is as described below. The following procedure was performed to separate mitochondria from brain tissue. Whole rat brain tissue was mixed with isolation buffer (215 mM mannitol, 75 mM sucrose, 0.1% bovine serum albumin (BSA) (Bioworld, Dublin, OH, USA), 1 mM EGTA, 20 mM HEPES sodium, and pH 7.2). Centrifuged for 5 minutes at 13,000 xg. Then, the supernatant was centrifuged at 13,000 x g for 10 minutes, and the supernatant was removed. An isolation buffer containing 0.1% digonin was added to the pellet and allowed to stand for 5 minutes. After 5 minutes, centrifugation was performed at 13,000 x g for 15 minutes, and the supernatant was removed. Then, an isolation buffer without EGTA was added to the pellet and centrifuged at 10,000 x g for 10 minutes. Finally, an isolation buffer without EGTA was added to the remaining pellets and used for later experiments.

In addition, TMT is known to cause mitochondrial damage in specific areas of the hippocampus in the brain. Therefore, using the fact that brain mitochondria obtained from mice intraperitoneally injected with TMT (trimethyltin) were confirmed to have membrane potential (MMP) damage and increase in reactive oxygen species (ROS), and that ATP production was reduced, the ethyl acetate fraction of equestrian horseback riding The following experimental method was used to confirm the effect of improving the damaged mitochondria. In ICR mice (Samtoko, Osan Korea), humidity, temperature and light intensity were each 55%, 22±2℃, using a 12h light-dark cycle, and samples (20, 40 mg/kg of body weight (EFAD20 and EFAD40 group)) ) Was administered orally for 3 weeks. TMT was administered intraperitoneally to the two groups of mice fed the two sample diets at a concentration of 7.1 µg/kg for 3 weeks, using a 0.85% sodium chloride solution. Control rats were injected with only sodium chloride solution, and mitochondria were isolated by the method described above.

In addition, p-JNK, BAX, Cytochrome C, p-Akt, p-tau, TNF-α, p-NK-κB used Western blot to analyze factors related to the mitochondrial-mediated apoptosis pathway in mice intraperitoneally injected with TMT. Each was measured.

Mitochondrial membrane potential (MMP) measurement

An experiment using JC-1 was performed to confirm the mitochondrial membrane potential (MMP), which is the driving force of mitochondrial viability. First, assay buffer (EGTA-free buffer with 5 mM pyruvate and 5 mM malate) and 1 µm JC-1 were added to the isolated mitochondria (final concentration: 0.8 mg/mL) and shaken gently. This was allowed to stand in the dark for 20 minutes and then measured with a fluorescent microplate reader (excitation filter 530/25 nm, emission filter; 590/30 nm).

Reactive oxygen species (ROS) measurement

A method using DCF-DA was performed to measure ROS present in mitochondria. First, 25 µm DCF-DA was added to the separated mitochondria (final concentration: 0.8 mg/mL) and left for 20 minutes. Subsequently, the ROS level of mitochondria was quantified with a fluorescent microplate reader (Infinite 200, Tecan Co., San Jose, CA, USA) (excitation filter, 485/20 nm, emission filter, 528/20 nm).

Adenosin triphosphate (ATP) measurement

ATP levels, a major function of mitochondria, were measured with an ATP assay kit purchased from Promega Corporation (Madison, WI, USA). ATP assay mixture solution was added to the previously isolated mitochondrial sample. It was quantified with an ATP standard quantification curve.

The experimental results are as described below.

First, in order to confirm the effect of improving mitochondrial function caused by a high fat diet, MMP, a membrane potential difference that plays a major role in mitochondrial activity in mitochondria isolated from brain tissue, was measured. As a result, MMP decreased in the high fat diet group. It was confirmed that the MMP was increased in the sample group (Fig. 5(A)). In addition, as a result of checking the ROS level inside the mitochondria, it was confirmed that the high-fat diet group showed a high ROS level and the ROS level decreased in the sample group (FIG. 5(B)). Finally, as a result of confirming the ATP levels biosynthesized in mitochondria, it was confirmed that the energy biosynthesis of mitochondria was damaged through low ATP levels in the high fat diet group, and it was confirmed that the mitochondrial biosynthetic ability was improved in the sample group (Fig. C)). Accordingly, it was confirmed that the damage of mitochondrial activity caused by a high fat diet was effectively suppressed by feeding the sample, and it was confirmed that the sample was excellent in improving mitochondrial damage caused by a high fat diet.

In addition, when mice with damaged mitochondria caused by trimethyltin (TMT) ingested the ethyl acetate fraction of equestrian horses, the improvement effect was confirmed.As a result, the group that consumed the ethyl acetate fraction of equestrian horses had improved mitochondrial function damage. That is, it was confirmed that the MMP of the rats that ingested the ethyl acetate fraction of equestrian horse riding increased, the ROS decreased, and the ATP increased (FIG. 6A,B,C). That is, it was confirmed that the ethyl acetate fraction of Equestrian Equestrian Horse showed an improvement effect on both mitochondrial damage caused by high fat diet or TMT.

Furthermore, p-JNK, BAX, Cytochrome C, p-Akt, p-tau, TNF-α, p-NK-κB used Western blot to analyze factors related to the mitochondrial-mediated apoptosis pathway in mice intraperitoneally injected with TMT. Each was measured. c-Jun N terminal kinase (JNK) is a factor directly related to apoptosis, and becomes active when ser-63 and ser-73 residues are phosphorylated. JNK increases the expression of Bcl-2-associated X protein (BAX), and BAX causes disruption of the mitochondrial membrane, eventually inducing the release of cytochrome C present in the mitochondria into the cytoplasm. This causes mitochondrial-mediated apoptosis. Protein kinase B (Akt) is a factor related to cell survival and is activated when Akt is also phosphorylated. When the activity of Akt decreases, apoptosis proceeds radically. Inactivation of Akt induces the activation of GSK3β, which phosphorylates tau, resulting in tau hyperphosphorylation, resulting in the formation of neurotoxic substances. Thus, mitochondrial-mediated apoptosis occurs radically, and hydrogen peroxide (H ₂ O ₂ ), which is excessively secreted by mitochondrial damage, activates the transcription of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. It leads to neuroinflammation by inducing the secretion of tumor necrosis factor alpha (TNF-α). As a result of the experiment, it was confirmed that the ethyl acetate fraction of equestrian equestrian horses inhibited the mitochondrial-mediated apoptosis pathway and eventually improved neuronal cell death and further improved cognition (FIG. 7A,B,C).

Experimental Example 6. Central Insulin Resistance Related Factor Experiment

Statistical processing of experimental animals and experiments was performed by the method described in Experimental Example 1. An experiment to determine the change in the central insulin resistance-related factor was performed in the following manner.

Brain tissue was mixed with protinE ^TM Animal cell/tissue (GeneAll Biotechnology, Seoul, Korea) containing 1% protease inhibitor cocktails (Thermo Fisher Scientific, Rockford, IL, USA) and homogenized with a bullet blender. Protein content in the sample was relatively quantified using the Bradford protein assay. Protein in the sample was separated by dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was washed after passing through a blocking process for 1 hour with 5% skim milk. After that, the membrane was put into the primary antibody solution and stirred for 12 hours. Thereafter, the membrane was washed and placed in a secondary antibody solution for 1 hour, followed by washing. Finally, after immersion in ECL reagent (Bionote, Hwaseong, Korea), luminescence was detected with ChemiDoc (iBrightTM CL1000 equipment, Invitrogen, Carlsbad, CA, USA). The density of the bands on the membrane was measured with Image-J software (National Institutes of Health, Bethesda, Maryland, USA). The density of each factor p-IRS, p-Akt, p-tau, p-AMPK, IDE, and Amyloid β was divided by the density of β-actin as the result value.

When insulin resistance is caused in the central nervous system, when insulin and insulin receptors are bound, the serine residue of the insulin receptor substrate (IRS) is phosphorylated, thereby reducing the activity of Akt. Hyperphosphorylation proceeds, leading to nerve fiber entanglement, which causes nerve transmission and apoptosis. As a result of the experiment, it was confirmed through the results of FIG. 8(A) that central insulin resistance was caused in the high fat diet group, and p-tau was increased. On the other hand, it was confirmed that the sample group effectively improved the central insulin resistance and further inhibited tau hyperphosphorylation. In addition, the imbalance of energy homeostasis caused by insulin resistance decreases the activity of AMPK, which is responsible for self-digestion and secretes insulin degradation enzyme (IDE), which degrades not only insulin but also beta amyloid plaques. . Through the results of FIG. 8(B), it was confirmed that the phosphorylation of AMPK was reduced in the high fat diet group, thereby reducing the amount of IDE expression, and further, it was confirmed that beta-amyloid plaque formation was promoted. On the other hand, in the sample group, an increase in the amount of IDE expression and a decrease in beta-amyloid plaque formation were confirmed according to the recovery of AMPK phosphorylation. Thus, by confirming that the mechanism related to cognitive damage caused by central insulin resistance caused by a high fat diet was suppressed through the sample diet, it was confirmed that the sample had an excellent effect on improving cognitive damage caused by a high fat diet.

Experimental Example 7. UPLC-QTOF/MS ² analysis

Ultra-performance liquid-quadrupole-time-of flight mass spectrometry (UPLC-QTOF/MS ² ) (Waters, Milford, MA, USA) based on chromatographic analysis was performed to separate the main compounds of the ethyl acetate fraction of Equestrian Equestrian. . The sample dissolved in methanol was filtered through a 0.2 μm filter, and UPLC separation of the phenolic compound was performed in an ACQUITY UPLC BEH C ₁₈ column (2.1 x 100 mm, 1.7 μm particle size, Waters Corp, Milford, MA, USA), It flowed at 40 degreeC at a flow rate of 0.4 mL/min. The mobile phases used in the analysis were solvent A (distilled water with 0.1% formic acid) and solvent B (ACN with 0.1% formic acid), and the solvent gradient conditions were as follows: 0-0.5 min, 0% B; 0.5-8 min, 100% B; 8-8.5 min 100% B; 8.5-10 min 0% B; And 10-11 min 0% B. To obtain MS ² data, the ionization was operated in negative electron spray (ESI) mode. The ionization conditions are as follows: lamp collision energy, 20-45 V; Capillary voltage, 3 kV; Desolvent temperature, 350°C; Pressure in the nebulizer, 40 psi; fragmentor, 175 V; cone voltage, 40V; Mass range, 50-1,200 m/z; Oven temperature, 40°C. All MS data were analyzed using MarkerLynx software (Waters MassLynx™, Waters, Milford, MA, USA).

As a result of the experiment, two main peaks were identified through the chromatogram (Fig. 9(A)), and the fragment ion tendency pattern for the main peak was confirmed to be named dicaffeoyl glucose isomer Ⅰ, Ⅱ, respectively, through references, and accordingly It was confirmed that the main phenolic compounds of the ethyl acetate fraction of Equestrian Equestrian horses were dicaffeoyl glucose isomers I and II (Figs. 9(B) and (C), respectively).

Although the formulation examples of the pharmaceutical composition and food composition containing the fractions of the extract of Equestrian Equestrian horse of the present application are described, the present application is not intended to limit it, but is intended to be described in detail.

Formulation Example 1. Preparation of powder

300 mg of ethyl acetate fraction of equestrian horseback riding in Example 1

100 mg lactose

10 mg of talc

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2. Preparation of tablet

Equestrian Equestrian Equestrian Equestrian Fraction 50 mg of Example 1

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

After mixing the above ingredients, tableting by tableting according to a conventional tablet manufacturing method

To manufacture.

Formulation Example 3. Preparation of Capsule

Equestrian Equestrian Equestrian Equestrian Fraction 50 mg of Example 1

100 mg corn starch

100 mg lactose

2 mg of magnesium stearate

Mixing the above ingredients according to a conventional capsule preparation method and filling it into a gelatin capsule

To prepare a capsule.

Formulation Example 4. Preparation of injection

Equestrian Equestrian Equestrian Equestrian Fraction 50 mg of Example 1

Suitable amount of sterile distilled water for injection

proper amount of pH adjuster

Manufactured with the above ingredients per ampoule (2 ml) according to a conventional injection preparation method

do.

Formulation Example 5. Preparation of liquid formulation

Equestrian Equestrian Equestrian Equestrian Fraction 100 mg of Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

Dissolve each component by adding each component to purified water according to the conventional method for preparing liquid

After adding lemon scent in an appropriate amount, mix the above ingredients and add purified water to

Is adjusted to 100 ml by adding purified water, then filled in a brown bottle and sterilized.

To manufacture the agent.

Formulation Example 6. Preparation of health food composition

1000 mg of ethyl acetate fraction of Equestrian Equestrian horse of Example 1

Vitamin mixture right amount

Vitamin A acetate 70 ㎍

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 ㎍

Vitamin C 10 mg

Biotin 10 ㎍

Nicotinic acid amide 1.7 mg

Folic acid 50 ㎍

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is relatively suitable for healthy food.

Powder was mixed and formulated in a preferred embodiment, but the mixing ratio was arbitrarily modified.

It is okay to do so, and the above ingredients are mixed according to the usual health food manufacturing method.

Well, granules can be prepared and used in the preparation of health food compositions according to conventional methods.

have.

Formulation Example 7. Preparation of healthy beverage

1000 mg of ethyl acetate fraction of Equestrian Equestrian horse of Example 1

Citric acid 1000 mg

100 g oligosaccharides

2 g of plum concentrate

1 g taurine

After adding purified water to mix the above ingredients according to the general health drink manufacturing method of 900 ml, the resulting solution is stirred and heated at 85°C for about 1 hour, and the resulting solution is filtered and obtained in a sterilized 2 liter container and sealed and sterilized. After refrigerated storage, it is used to prepare the health beverage composition of the present application.

The composition ratio is a mixture of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.

Claims (2)

A cognitive improvement functional food composition containing both phenolic compounds dicaffeoyl glucose isomer Ⅰ and Ⅱ. The cognitive improvement functional food composition of claim 1, wherein the food composition has any one formulation of powder, tablet, capsule, injection, and liquid.

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