KR20060124002A - Mixture of crude medicine extract, and method of manufacture thereof - Google Patents

Abstract

A pharmaceutical composition and health food comprising the mixture of crude medicinal herb extracts are provided to inhibit production of final saccharified products as a factor causing diabetes mellitus complication, and remove reactive oxygen species. The pharmaceutical composition comprises the mixture of crude medicinal herb extracts including ginseng, Magnolia obovata, Pueraria thunbergiana BENTH and Glycyrrhiza uralensis FISCH., wherein the mixture of crude medicinal herb extracts is prepared by mixing 5-85 wt.% of ginseng, 5-85 wt.% of Magnolia obovata, 5-85 wt.% of Pueraria thunbergiana BENTH and 5-85 wt.% of Glycyrrhiza uralensis FISCH., extracting the mixture with 10-90% alcohol, filtering and concentrating it under reduced pressure, and freeze-drying the mixture of crude medicinal herb extracts.

Description

Mixture of Crude Medicine Extract, and Method of manufacture

1 is a graph showing the inhibitory effect of the final glycation product production after 7-day culture of the mixed herbal extract according to the present invention,

Figure 2 is a graph showing the inhibitory effect of the final glycation product production after 14 days of culture of the mixed herbal extracts according to the present invention,

Figure 3 is a graph showing the final inhibitory product production inhibitory effect after 7 days of incubation of aminoguanidine as a control group,

Figure 4 is a graph showing the final inhibitory product production inhibitory effect after 14 days of incubation of aminoguanidine as a control.

The present invention relates to a mixed herbal extract and a method for manufacturing the same, and more particularly, to the final glycation product, which is one of the factors inducing diabetic complications by using the extract obtained by mixing raw ginseng, white gourd, brown root and licorice, and then extracting them. The present invention relates to a mixed herbal extract and a method for producing the same, which are effective in preventing or treating diabetic complications, preventing or delaying diabetic complications by suppressing production and removing reactive oxygen species causing oxidative stress.

The diabetic population is increasing worldwide. The WHO projected an increase in the diabetic population from about 175.52 million in 2000 to about 370 million by 2030. Similarly, Korea had less than 300,000 diabetics until the 1970s, but in 2004, the number of diabetics increased significantly to 4 million, 10% of the total population (Korea Health Industry Development Institute). 12 million people (1 in 4 people) warned of diabetes mellitus, which suffers from diabetes (2002 Diabetes Association). However, as the diabetic cure has not yet been developed worldwide, blindness due to diabetic retinopathy, diabetic cataract, nephropathy, diabetic neuropathy, etc. It is inevitable to progress to diabetes complications that can cause limb amputation.

In the past, diabetic patients died before progressing to complications, but the seriousness of diabetic complications was not recognized, but recently, the seriousness of diabetic complications is recognized due to changes in diet and life extension (average life expectancy of 74 years in Korea). Diabetes does not end with diabetes itself. It is a frightening disease defined as Metabolic syndrome, which can cause complications in one patient at the same time. In particular, chronic diabetic nephropathy is the most important cause of hemodialysis treatment and end stage renal failure, and there is no treatment other than hemodialysis treatment and organ transplantation.

These diabetic complications can be seen to progress even when blood sugar is restored normally, which is the result of irreversible advanced glycation endproducts (AGEs) produced as a result of non-enzymatic glycation of proteins due to the persistence of hyperglycemia. ) Is known as one of the main reasons.

Mechanisms that induce diabetic complications are largely explained by nonenzymatic glycation of proein, polyol pathway and oxidative stress.

Nonenzymatic glycation of protein refers to the condensation reaction of a group of amino acids such as lysine residues of a protein and reducing sugars by condensation reaction, that is, milliard reaction, without enzymatic action. endproducts, AGEs). Non-enzymatic glycosylation of protein (1) an amino acid group such as a lysine residue of a protein and an aldehyde or ketone of a reducing sugar undergo a nucleophilic addition reaction without enzymatic action to form a Schiff base, an early product. Condensing the Schiffbase and adjacent ketoamine adducts with each other to produce a reversible Amadori-type early glycosylated product; (2) It can be divided into stages in which hyperglycaemia is maintained so that the reversible amadori type of early glycosylated product is not degraded, rearranged, and cross-linked with protein to produce irreversible final glycosylated product.

Unlike reversible Amadori type of early glycosylated products, final glycosylated products are irreversible reaction products. Once produced, they do not degrade even when blood sugar returns to normal, but accumulate in tissues during protein survival to abnormally change tissue structure and function. (Vinson, JA et al., 1996, J. Nutritinal Biochemistry 7: 559-663; Smith, PR et al., 1992, Eur. J. Biochem ., 210: 729-739).

For example, glycated albumin, one of the final glycation products produced by the reaction of glucose and various proteins, is an important factor in causing chronic diabetic nephropathy. Glycosylated albumin is more easily introduced into renal glomeruli cells than normal albumin without glycosylation, and high concentrations of glucose stimulate mesangium cells to increase extracellular matrix synthesis. Excessly introduced glycated albumin and increased extracellular matrix result in fibrosis of the glomeruli. These mechanisms continue to damage the glomerulus, leading to the stage of extreme treatment methods such as hemodialysis or organ transplantation.

In addition, chronic diabetes has been reported to accumulate collagen in the arterial wall and basal membrane protein in the renal glomeruli and to accumulate in tissues (Brownlee, M., et al., 1986, Sciences , 232, 1629-). 1632).

As described above, glycosylation occurs in proteins such as the basement membrane, plasma albumin, lens protein, fibrin, and collagen by non-enzymatic protein glycosylation reaction, and the resulting glycosylated product abnormally changes the structure and function of the diabetic retinopathy. ), Diabetes cataract, diabetic nephropathy (nephropathy), diabetic neuropathy (Neuropathy), such as chronic complications.

In addition, the final glycosylated product produced by non-enzymatic protein glycosylation reaction is known to play an important role in aging (Monnier et all., Proc. Natl. Acad. Sci. USA, 81: 583, 1984; Lee et a1., Biochem.Biophys.Res.Comm., 123: 888, l984; Diabetologia, 38: 357-394).

In addition, another mechanism that causes diabetic complications is oxidative stress, which is described as follows.

The aerobic breathing organisms constantly receive oxygen molecules (O ₂ ) into the cells, reactive oxygen species in the mitochondria or immune cells exposed to foreign antigens, and externally by reactive oxygen species (reactive oxygen species). species, ROS). These active oxygen species O ₂ ^- may be mentioned (superoxide anion), HO (hydroxyl radical), 1 O 2 (singlet oxygen), H 2 O 2 (hydrogen peroxide), HOCI (hypochlorous acid). However, there are antioxidant enzymes (for example, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase) and various antioxidant compounds (vitamin C, vitamin E, uric acid, bilirubin) that remove these reactive oxygen species in the living body. There is a balance between the generation and elimination of reactive oxygen species, maintaining cell function. However, in situations where too many reactive oxygen species are produced or the function of the antioxidant system is degraded, cells are adversely affected by reactive oxygen species, which are called "oxidative stress".

Many clinical diseases have been reported to increase oxidative stress, and the use of antioxidants in these diseases has been reported to alleviate the disease. For example, in diabetic patients, oxidative stress is higher than normal, and the administration of antioxidants reduces diabetes and its complications (Blosi, MS (1958) Antioxidant determination by the use of a stable free radical). Nature 181 : 1199-1200).

As described above, the final glycosylated product produced by the non-enzymatic protein glycosylation reaction is the main cause of the progression of diabetic complications and aging, and excessive reactive oxygen species promote the progression of complications, so to prevent the progression of diabetic complications and aging. There is a need to suppress the production of final glycation end products and reactive oxygen species.

Currently, aminoguanidine, the only synthetic agent that inhibits protein glycosylation, is a nucleophilic hydrazine, which binds with the amadori product and prevents cross-linking with the protein, thereby inhibiting the formation of the final glycation product and progressing to complications. Delay or prevent (Brownlee, M., et al., 1986, Sciences, 232, 1629-1632; Edelstein, D. et al., L992, Diabetes, 4l, 26-29). Aminoguanidine-HCl is the most promising synthetic drug for the prevention and treatment of diabetic complications, but it has been progressed up to Phase III clinical trials, but there is a problem that toxicity is induced upon long-term administration.

Therefore, in recent years, due to the limitations of the development of disease treatments by conventional synthetic compounds, and problems related to side effects and toxicity during treatment, development of disease treatments based on herbal medicines has been actively progressed.

Therefore, the present inventors have the effect of inhibiting the production of the final glycosylated raw ginseng, bakbak, brown root, licorice, in particular, the mixed herbal extract consisting of 5 to 85% by weight each of the excellent glycemic acid production inhibitory effect of diabetic complications The present invention has been found to be useful for preventing and aging, as well as for preventing and aging, as well as for preventing and treating.

In order to solve the above problems, the present applicant has continuously studied the causes, prescriptions, and treatment methods of diabetic complications, and the present invention has the effect of inhibiting the production of the final glycated product, one of the causes of diabetic complications, and active oxygen. It is an object of the present invention to provide a mixed herbal extract obtained by extracting a mixture of raw ginseng, pakbak, brown root and licorice having an antioxidant effect of eliminating species.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetic complications and anti-aging or delaying containing the mixed herbal extract as an active ingredient.

Still another object of the present invention is to provide a functional food for preventing or treating diabetic complications and for preventing or delaying aging, containing the mixed herbal extract as an active ingredient.

The mixed herbal extract according to the present invention for achieving the object of the present invention is obtained by mixing with 5 ~ 85% by weight each of raw ginseng, bakbak, brown root and licorice, extracted with an alcohol or an aqueous alcohol solution and then concentrated under reduced pressure and then freeze-dried. It features.

In addition, the pharmaceutical composition and functional food for preventing or treating diabetic complications according to the present invention is characterized in that it contains the mixed herbal extract as an active ingredient.

In addition, the anti-aging or delaying pharmaceutical composition and functional food according to the present invention is characterized in that it contains the mixed herbal extract as an active ingredient.

First, the herbal medicines used in the present invention will be described.

Wonsam is the root of the flower of Somyangji Flower, and it is a perennial plant of rose and wild flowers that grows on sunny lawns in high mountains at the foot of high mountains in northern Korea, Gangwon-do, central Gyeonggi-do and Gyeongsangbuk-do.

Wonsam has been loved as a guanggu food for folk remedies, and it is known to be excellent in bone formation and osteomyelitis because it contains ingredients that exceed ginseng. It is used as a substitute for ginseng for people who have a fever due to ginseng. Ingredients include Flavonoid, Tannin, and Protocatechuic Acid. In traditional Chinese medicine, swelling, dysphagia, clear fever, detoxification, fish, haemostasis, carbuncle, diarrhea, and bohe ( 효능), serpentine 毒死 咬傷 (등에) is excellent in efficacy.

Magnolia Bark is a dried bark of Magnolia obovata, M. officinalis, M. officinalis var. Biloba belonging to the Magnolia family . Efficacy in the lower part of the body, and treats wet weight, complete earth and sand, food gas, abdominal constipation, and dam- ing swelling. Pharmacopoeia Commission, Republic of China Pharmacopoeia, Part I, 204, Chemical Industry Press, Beijing), essential oils such as alpha, beta, gamma-oidesmol (magnolol) and honokiol (honokiol), alkaloids, and saponins. Pharmacological effects include anti-allergic efficacy (Shin, TY, et al., 2001, Arch. Pharm., Res. , 24: 249-255), apoptosis effect (Park, HJ, et al., 2001, Arch. Pharm , Res. , 24: 342-348), NO synthesis inhibitory effect, TNF-α expression inhibitory effect (Son, HJ, et al., 2000, Planata med ., 66: 467-471), antifungal effect (Bang, KH, et al., 2000, Arch. Pharm, Res ., 23: 46-49), mental stability effects (Kuribara, H., et al, 1999, J. Pharm. Pharmacol ., 51: 97-103) and It has been reported to have various effects such as skin cancer inhibitory effect (Komoshima, T. et al., 1991, J. Nat. Prod. , 54: 816-822).

Puerariae Radix is a dried root of Pueraria thunbergiana ( P. lobata ), a perennial mutant belonging to the legumes. Its weakness is persimmon, sour, flat, and rain. 들어가) Enter the stomach (해) (解 表), Sujin (透疹), Saengjin (生 津), Governor (효능) shows the efficacy. Pharmacological actions include antipyretic, blood pressure lowering, memory enhancement, increased cerebral blood flow, coronary artery dilation, cardiac function, and antiarrhythmic effects (Kim, Ho-Cheol, Herbal Pharmacology, Moon-Dang, 92-94, 2001).

Licorice (Glycyrrhizae Raidx) is the dried root and rhizome of perennial herb, Glycyrrhiza glabra , G. uralensis and other related plants. Entering into the rain, stomach, stomach, heart, lungs, middle-aged, middle-aged, detoxification, lubrication branch, salary It has the effect of Jitong (통) and Harmonic Pharmaceutical (調和 諸 藥). The main components include glycyrrhizin, a triterpen saponin, and flavonoid compounds such as liquiritin. Pharmacological actions include adrenal cortex hormone-like action, anti-ulcer efficacy, smooth group relaxation, hepatoprotective action, anti-inflammatory, anti-allergic action, and antiviral action (Kim Ho-cheol, Herbal Pharmacology, Mumun-dang, 434-436, 2001).

Hereinafter, the present invention will be described in detail.

I. Mixed Herbal Extracts

The mixed herbal extract according to the present invention is obtained by mixing raw ginseng, groin, brown root and licorice at 5 to 85% by weight, respectively, extracting with an alcohol or an aqueous alcohol solution and then concentrating under reduced pressure and freeze drying.

Method for producing a mixed herbal extract of the mixing step of mixing 5 to 85% by weight of each of the raw ginseng, bakbak, brown root and licorice; An extract preparation step of extracting the mixture mixed in the mixing step using 10-90% alcohol, filtering, and then concentrating under reduced pressure to prepare an extract; The extract obtained in the extract preparation step consists of a drying step of drying in a freeze dryer.

The preparation method of the mixed herbal extract is described in more detail as follows.

1. Herbal Medicine Mixing Stage

Grind the raw ginseng, white gourd, brown root and licorice, respectively, and then mix them at 5 to 85% by weight.

2. Extract Preparation Step

The mixture mixed in the mixing step is extracted at room temperature using 10 ~ 90% alcohol. At this time, the alcohol can be used 10 to 90% C ₁ to C ₄ alcohol, it is preferable to use 80% alcohol, the amount is preferably added 5 to 10 times (w / v) of the herbal medicine. .

The extract extracted as described above is concentrated under reduced pressure.

3. Drying Step

The extract prepared in the extract manufacturing step is lyophilized in a freeze dryer.

The mixed herbal extract thus obtained was confirmed in vitro experiments to inhibit glycation of bovine serum albumin (BSA), and the final glycosylated product produced 50% inhibitory concentration, ie, IC ₅₀ is a positive control aminoguanidine [ It showed a much lower concentration (14.39 μg / ml) than 86.91 μg / ml (15-day culture), indicating an excellent inhibitory effect on the production of the final glycosylated product.

II. Pharmaceutical composition containing a mixed herbal extract as an active ingredient

The present invention relates to a pharmaceutical composition for preventing or treating diabetic complications and an anti-aging or delaying pharmaceutical composition comprising a mixed herbal extract obtained by mixing and extracting raw ginseng, groin, brown root and licorice as an active ingredient. Mixed herbal extracts exhibit therapeutic activity in diabetic complications or aging, such as inhibiting the production of the final glycation end products, the main cause of diabetes complications or aging, and can be administered orally or parenterally during clinical administration. Can be used as

The mixed herbal extract of the present invention may be administered in an effective amount through various routes of administration. The use together contains a pharmaceutically acceptable carrier. More specifically, pharmaceutically acceptable carriers can be any standard pharmaceutical carrier that can be used in known formulations such as sterile solutions, tablets, coated tablets and capsules.

Typically, the carrier may include excipients such as starch, milk, sugar, certain types of clays, gelatin, stearic acid, talc, vegetable oils or oils, gums, glycols, or other known excipients, and also flavors, pigments Additives and other ingredients may be included.

The preparation for administering the composition containing the herbal extract of the present invention as an active ingredient can be administered by oral, intravenous, intramuscular, or transdermal administration in a conventional manner, but is not limited thereto.

In actual clinical administration, it can be administered in a variety of oral and parenteral formulations, which are formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like that are commonly used.

Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like form at least one excipient such as starch, calcium carbonate, sucrose or lactose. (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Oral liquid preparations include suspensions, liquid solutions, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin.

In addition, the pharmaceutical compositions of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection or intramuscular injection. In order to formulate into a parenteral formulation, the herbal extracts are mixed with water or stabilizers or buffers to prepare a solution or suspension, which is prepared in unit dosage form of ampoules or vials.

The dosage of the pharmaceutical composition is appropriately selected depending on the absorbency, inactivation rate, rate of excretion, the age, sex and condition of the patient, and the severity of the disease to be treated in the body. Can be administered in divided doses.

In the pharmaceutical composition of the present invention, when the herbal extract is contained as an active ingredient, the effective dose is 500 to 2,000 mg / kg / day, preferably 500 to 1,000 mg / kg / day, 1 to 3 times a day. It may be administered separately. The exact amount, route of administration, and frequency of the agent can be readily determined according to the nature of the agent, the weight and condition of the subject to be administered, and the nature of the particular derivative to be used.

III. Functional food containing mixed herbal extract as an active ingredient

The present invention relates to a functional food for preventing or treating diabetic complications and an anti-aging or delaying functional food containing a mixed herbal extract obtained by mixing and extracting raw ginseng, groin, brown root and licorice as an active ingredient. Other food ingredients may be added depending on the merchandise and consumer preference of the final product.

When the mixed herbal extract is used as a food additive, the mixed herbal 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 may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of food or beverage, the mixed herbal medicine of the present invention is added in an amount of 15 wt% or less, preferably 10 wt% or less with respect to the raw material. However, in the case of long-term intake for health and hygiene or for health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. have.

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, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages, vitamin complexes, and the like, and includes all of the functional foods in a conventional sense.

The functional food of the present invention may contain various flavors, natural carbohydrates, and the like as additional ingredients, like ordinary beverages. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and sugar alcohols such as polysaccharides, xylitol, sorbitol and erythritol, and destrin, cyclodextrin and liver. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents and the like used in beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of from 0.1 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail by the following examples and experimental examples. However, the following Examples and Experimental Examples are merely illustrative of the contents of the present invention and the scope of the invention is not limited by the Examples and Experimental Examples.

Example 1 Preparation of Mixed Herbal Extract

After grinding ground ginseng, licorice, groceries, and brown roots into powder and mixing the same amount, 1 liter of 80% ethyl alcohol aqueous solution (ethyl alcohol: distilled water = 80: 20) was added at room temperature (20 to 30) and extracted for 24 hours. The ethyl alcohol extract was filtered with filter paper, and extracted five times in the same manner. All the extracts thus obtained were collected, concentrated under reduced pressure, and dried in a lyophilizer.

[Experimental Example 1] Analysis of inhibitory efficacy of the final glycation product production of mixed herbal extracts according to the present invention

Bovine serum albumin (hereinafter referred to as BSA: US Sigma) was selected as a protein source, and the BSA was added to 50 mM phosphate buffer (pH 7.4) to a concentration of 10 mg / ml. Prepared by addition. As a sugar source, a mixture of 25 mM fructose and 25 mM glucose was used. A fructose and glucose mixture was added to the BSA solution prepared above. After mixing the mixed herbal extract prepared in Example 1 in 15% Tween 80, it was added to the mixed solution of the BSA and sugar and incubated at 37 ℃ for 7 days, 14 days. At this time 0.02% sodium azide was added as an antibacterial agent.

The control group was cultured with a mixture of BSA and sugar, and the blanks of the test group and the control group (blank) were prepared, respectively. All cultures were prepared by four to avoid errors as much as possible, immediately before the culture was filled with nitrogen gas (purity: 99.999%) to prevent contamination. After 7 days and 14 days, the content of the final glycated product produced in the culture was analyzed. The final glycosylated product is fluorescence, brown, not only has a physicochemical property that can cross-link, but also has a ligand that can be recognized by cell membrane receptors. The amount of the final glycated product having these characteristics was measured by a spectrofluorometer (Excitation: 350 nm, Emission: 450 nm) and analyzed for the degree of inhibition of production thereof.

The production inhibition rate is calculated by the following formula.

Production Inhibition Rate (%) = 100- (Fluorescence Intensity of Sample-Fluorescence Intensity of Sample Blank) / (Fluorescence Intensity of Control-Fluorescence Intensity of Control Blank) × 100

Comparative Example 1 Analysis of Inhibitory Effect of Aminoguanidine on Production of Final Glycosylated Products

As a positive control of the production inhibitory effect of the final glycosylated product, aminoguanidine was dissolved in distilled water at a constant concentration, and then cultured for 7 days and 14 days in the same manner as in Experiment 1, and the results are shown in Tables 1 and 3. 4 is shown.

 division  Herbal Mixture Composition  Aminoguanidine  Incubation period  Concentration (µg / ml)  Generation Inhibition Rate (%)  Concentration (µg / ml)  Generation Inhibition Rate (%)  7 days 10 48.79 ± 3.5 18.5 14.42 ± 2.16 15 68.41 ± 5.31 37 37.12 ± 3.19 25 84.51 ± 5.51 74 55.47 ± 1.34  14 days 10 14.83 ± 2.23 18.5 23.52 ± 1.78 15 38.08 ± 1.77 37 40.95 ± 3.34 25 61.84 ± 0.83 74 63.19 ± 1.79

As shown in Table 1, the inhibitory effect of the production of the final glycosylated product was 7 and 4 times higher than that of the amino guanidine, which was positive control, for 7 days and 14 days of cultivation of the extract of raw ginseng, black root and licorice. It can be seen that the final glycation product inhibition effect is very excellent.

[Experimental Example 2] Analysis of active acid control inhibition effect of mixed herbal extracts according to the present invention

Cell culture was carried out by the Chinese hamster lung fibroblast V79-4 cells (ATCC CCL-93) from the American Type Culture Collection (ATCC), DMEM (10% fetal calf serum, 1% antibiotics) medium Suspension was incubated in 37 ℃, 5% CO ₂ incubator. At this time, the cells growing in the log phase was mainly used for the experiment.

Sample preparation was used by dissolving in Dimethysulfoxide (DMSO) solution. At this time, in order to exclude the effect of DMSO when the cells were treated, the final concentration was within 0.1%. The concentration of the sample was 10 μg / ml.

Experimental method used DCHF-DA (dichlorodihydrofluorescin discetate) measurement method, DCHF-DA method is a fluorometric method for measuring H ₂ O ₂ in the cell is a non-fluorescent substance dichlorodihydrofluorescin diacetate (DCHF-DA) into the cell It is converted to dichlorodihydrofluorescin (DCHF) by the esterase enzyme and reacted with H ₂ O _{2 in the} cell to measure the degree of fluorescence developed by oxidation with dichlorodfluorescin (DCF). (Rosenkranz, AR, Schmaldienst, S., Stuhlmeier, KM , Chen, W., Knapp, W., and Z labinger, GJ (1992) A microplate assay for the detection of oxidative products using 2 ', 7'-dichlorofluorescin diacetate.J. Immunol.Meth . 156: 39-47).

Detailed description of the experimental method, V79-4 cells were inoculated in 96 wells so as to be about 3 × 105 cells per well, and then incubated in a 37%, 5% CO ₂ incubator for 16 hours to allow the cells to adhere well. After the sample was treated with 10μg / ml cells were incubated for 30 minutes in 37 ℃, 5% CO ₂ incubator. 10 μl of H ₂ O ₂ (stock 20 mM) was added (final concentration 1 mM), and then incubated for 30 minutes at 37 ° C. in a 5% CO ₂ incubator. 20 μl of DCHF-DA (stock 500 M) was added thereto, followed by measurement with a spectrofluorometer (excitation 485 nm, emission 535 nm). The active oxygen species inhibition rate was determined by comparing with the sample treatment group in which only active oxygen species formation was measured without adding a sample, and the sample was added to eliminate the active oxygen species. All experiments were repeated three times.

Reactive oxygen species inhibition rate is calculated by the following formula.

Statistical test results were tested for significance using ANOVA analysis, and p value <0.05 was determined to be significant.

Experimental Example 3 The ability to induce oxygen radicals by activating cells was measured by DCF-DA by treatment of H ₂ O ₂ with V79-4 cells, resulting in 65% scavenging at 10 μg / ml of the mixed herbal composition. Action was shown. This means that the 65% scavenging rate of the mixed herbal composition is very good, compared with 85% scavenging rate at 50mg / ml.

Experimental Example 4 Toxicity Test of Mixed Herbal Extracts of the Present Invention

As a result of the acute toxicity test in the experimental mice of the mixed herbal extract according to the present invention, the minimum lethal dose (LD50) upon oral administration or intraperitoneal injection is 6,000 mg / kg or more, which shows no toxic effect at all. As can be seen, therefore, the mixed herbal extract of the present invention can be safely administered to a living body.

Preparation Example 1 Preparation of Tablet

The herbal extract prepared according to the embodiment 100.0mg, corn starch 90.0mg, lactose 175.0 mg, 15.0 mg L-hydroxypropyl cellulose, polyvinylpyrrolidone 905.0 mg and ethanol appropriately mixed with a wet granule method After granulation, 1.8 mg of magnesium stearate was added and mixed, and one tablet was compressed to 400 mg.

Preparation Example 2 Preparation of Capsule

The herbal extract prepared according to the Example 100.0 mg, corn starch 80.0 mg, lactose 175.0mg and magnesium stearate 1.8mg were uniformly mixed and filled in 360mg each one capsule.

As described above, the mixed herbal extract according to the present invention inhibits the production of the final glycated product, which is one of the causes of diabetic complications at low concentrations, significantly lower than the positive control group aminoguanidine, and also removes the active oxygen species in the positive control group. Compared to the excellent efficacy is excellent.

Thus, the mixed herbal extract according to the present invention is a diabetic complication, namely diabetic retinopathy, diabetic cataract, diabetic nephropathy caused by the production of the final glycation end products and weakening of the oxidative defense system in the human body. It can be applied as a pharmaceutical composition and functional food for the prevention and treatment of diabetes mellitus (Neuropathy) and the like.

In addition, the inhibition rate of oxidative stress is reduced when inhibiting the production of the end glycated product, it can be applied to the pharmaceutical composition and functional food for preventing and delaying aging caused by oxidative stress.

Claims (6)

Mixed herbal extract, characterized in that obtained by mixing with 5 ~ 85% by weight of raw ginseng, bakbak, brown root and licorice each time, extracted with an alcohol or an aqueous alcohol solution and then concentrated under reduced pressure. A mixing step of mixing 5 to 85% by weight of raw ginseng, white gourd, brown root and licorice; An extract preparation step of extracting the mixture mixed in the mixing step using 10-90% alcohol, filtering, and then concentrating under reduced pressure to prepare an extract; Mixed herbal extract production method comprising the drying step of drying the extract obtained in the extract manufacturing step in a freeze dryer. A pharmaceutical composition for preventing or treating diabetic complications containing the mixed herbal extract obtained by claim 1 as an active ingredient. Functional food for preventing or treating diabetic complications containing the mixed herbal extract obtained by claim 1 as an active ingredient. Anti-aging or delayed pharmaceutical composition containing the mixed herbal extract obtained by claim 1 as an active ingredient. Anti-aging or delayed functional food containing the mixed herbal extract obtained by claim 1 as an active ingredient.

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