KR20130056940A - Compositions for preventing and treating diabetes or diabetic complications comprising extracts of acer tegmentosum maximowoca and magnolia officinalis rehd. et wils - Google Patents

Abstract

PURPOSE: A composition containing extracts of Acer tegmentosum Maxim and Magnolia officinalis Rehd. et Wils. is provided to treat diabetes or diabetes complication. CONSTITUTION: A pharmaceutical composition for preventing and treating diabetes or diabetes complication contains 0.0001-90 wt% of extracts of Acer tegmentosum Maxim and Magnolia officinalis Rehd. et Wils. as active ingredients. The extracts are prepared using water, C1-C4 alcohol, or a mixture thereof. The extracts improve antioxidation, blood glucose reduction, glucose tolerance, and insulin sensitivity. The diabetes complication is diabetes retinopathy. A food composition for preventing and treating diabetes or diabetes complication contains the extracts as an active ingredient. [Reference numerals] (AA) Normal group; (BB) Glycosuria group; (CC) Acer tegmentosum Maxim and Magnolia officinalis Rehd. et Wils mixed extract group; (DD) Single Magnolia officinalis Rehd. et Wils extract group

Description

Compositions for preventing and treating diabetes or diabetic complications comprising extracts of bee and hawthorn {Compositions for preventing and treating diabetes or diabetic complications comprising extracts of Acer tegmentosum Maximowoca and Magnolia officinalis Rehd. et Wils.}

The present invention relates to a composition for the treatment of diabetes mellitus or diabetic complications containing bee and hawthorn extract, and more specifically to a pharmaceutical composition or food for the prevention and treatment of diabetes mellitus or diabetic complications comprising a mixture of bee and hawthorn as an active ingredient. It relates to a composition.

Recently, diabetic patients are exploding due to rapid westernization, urbanization, industrialization, lifestyle and eating habits, and aging. Diabetes is emerging as the third most serious disease worldwide, and the number of patients is expected to rise to 330 million by 2025 (World Health Organization statistics). Globally, it is estimated that at least $ 153 billion in annual treatment for diabetics between the ages of 20 and 79 will cost between $ 21.3 billion and $ 396 billion by 2025. It is predicted. Considering this huge expenditure of medical expenses and the fact that a large population is exposed to diabetes, it is urgent to develop diabetes prevention, treatment, and treatment for complications.

Diabetes is a chronic metabolic disease caused by high levels of glucose in the blood. When the blood sugar level is high, pancreatic beta cells recognize this and secrete insulin, and the secreted insulin lowers blood sugar by promoting glucose absorption in target cells (muscle, liver, and fat cells). However, even after a certain period of time after meals, blood glucose levels are maintained at high levels, or even on fasting blood glucose levels at high levels are said to be diabetes. The most common symptoms of diabetes are Daum, Daurim, and Dasik, which increase the incidence of serious complications such as weight loss, eye, kidney, and nerve damage, as well as cerebral infarction, angina, and peripheral vascular disease. It is known to raise. Well-known diabetic complications include diabetic retinopathy, diabetic foot disease, diabetic nephropathy, diabetic neuropathy, various infections caused by diabetes, periodontal disease, skin disease, glucose-containing hepatomegaly, fatty liver, cirrhosis, hepatitis, acute Pancreatitis, chronic pancreatitis, diabetic diarrhea, constipation, monolithic esophagitis, gastritis, and acute gastric dilated disease (Diabetes, Korean Diabetes Association, 1992).

Diabetes can be divided into two types: insulin dependent (type 1) and insulin independent (type 2), and more than 90% of diabetics belong to type 2. Type 1 diabetes, also called pediatric diabetes, accounts for 3 to 5% of all diabetic patients in Korea and is caused by the destruction of beta-cells of interest in Isle of Langerhans due to genetic factors or autoimmune mechanisms.

Type 2 diabetes is caused by environmental factors such as high calories, high fat, high protein diet, lack of exercise, and stress in addition to genetic factors. It is a disease that accounts for more than 90% of diabetic patients and is particularly prone to obesity after age 40, often accompanied with hyperlipidemia, and abnormally high cholesterol or triglyceride levels (total cholesterol 220 mg / dl or more, triglyceride 150 mg / ㎗ or more) and insulin resistance. People with insulin resistance develop hyperinsulinemia because they regulate high blood sugar to normal blood sugar by releasing too much insulin. Early physiological adaptation to insulin resistance is hyperinsulinemia due to excessive proliferation of beta cells, but it does not release enough insulin to overcome the resistance of peripheral tissues to insulin, resulting in a repeated cycle of hyperglycemia and hyperinsulinemia. This results in beta cell necrosis, insulin deficiency, severe hyperglycemia and weight loss (Defronzo RA, Diabetes Rev. 5: 177-269. 1997; Polonsky KS et al., New Engl. J. Med. 334: 777-783, 1996; Reaven GM, Physiol. Rev. 75: 473-486, 1995).

All aerobic organisms, including humans, use oxygen to metabolize energy and inherently self-defense against the damage of free radicals that occur in the process, but the production of free radicals beyond the defenses of tissues is associated with aging and aging. Associated degenerative diseases and atherosclerosis, high blood pressure, diabetes, arthritis, circulatory disorders, as well as a number of diseases such as cancer. These free radicals damage factors of the immune system such as proteins, DNA, enzymes, and T-cells, causing various diseases. Particularly problematic is that free radicals attack lipid fatty acids, which are components of cellular membranes, causing lipid peroxidation. Accumulation of lipid peroxides in the body is known to degrade biological function and at the same time cause aging and geriatric diseases.

Diabetes mellitus causes high blood sugar to over-produce reactive oxygen species (ROS) by various mechanisms such as glucose autooxidation, production of advanced glycation end products, and polyol pathway activity (Nishikawa T, Kidney). Int 77, S26-S30, 2000). Indeed, many studies have reported that antioxidant defense mechanisms in the blood of diabetic patients are reduced, and that oxidative stress is increased in various tissues through the increase of oxidized DNA, proteins, and lipids (Kim Bo-hyun, Korean Journal of Endocrinology, 21) Vol. 6, 2006). Cell and tissue damage due to an imbalance in the production and processing of reactive oxygen species caused by increased oxidative stress and decreased antioxidant capacity in diabetes suggests that oxidative stress plays an important role in the development of chronic complications of diabetes (Kuroki T J Am Soc Nephtol, 14, S216-S220, 2003; Griending KK et. Al., Circulation, 108, 2034-2040, 2003; Hink U et. Al., Circ Res 88, E14-E22, 2001).

Insulin-producing pancreatic beta cells are known to be particularly vulnerable to oxidative stress, unlike other cells (Hanasaki Y et. Al., Free Radic Biol Med, 6, 845-853, 1994). This is due to the high levels of free radicals in the mitochondria of beta cells, and additional ROS production through increased beta cell NAD (P) H oxidase activation, leading to problems with antioxidant defense mechanisms (Kahn SE et. Al., Diabetologia, 46, 3-19, 2003).

In addition, insulin resistance, which is characteristic of various metabolic diseases such as obesity, metabolic syndrome and type 2 diabetes, is also closely related to oxidative stress. Oxidative stress activates stress-sensitive serine / threonine kinase signaling pathways such as JNK, NF-kB, and p38MAPK, which in turn phosphorylates several targets, including insulin receptors and IRS proteins. In other words, oxidative stress causes increased serin / threonine phosphorylation of IRS-1 and impairs insulin action including activation of Akt / PKB (Kim Sang-soo, Korea diabetes J, 32, 389 ~ 398, 2008).

Through the above research results, oxidative stress has recently been considered as one of the important factors in the development of diabetes and complications. In addition, studies on whether antioxidant drugs can prevent or delay the onset of diabetes and diabetic complications have been attempted in diabetic animal models and diabetics. In vivo experiments have demonstrated that free radical scavengers and chemicals with antioxidant effects prevented cytotoxicity to pancreatic cells by substances such as streptozotocin and alloxanes used in diabetes-induced models (Szkudelski T, Physiol). Res, 50, 536-546, 2001). Alpha-lipoic acid, vitamins C and E, known as antioxidants, have also been reported to improve the incidence and progression of diabetes and complications in animal models (Ellis EA et. Al., Free Radic Biol Med, 28, 92-101, 2000; Cameron NE et. Al., J Clin Invest, 96, 1159-1163, 1995; Cameron NE et. Al., Diabetes Res Clin Pract, 45, 137-146, 1999).

A beetle ( Acer tegmentosum Maximowoca ) is a plant belonging to the family Mapleaceae, also called mountain cherub and mountain elm. The leaves are broad and the young stems are light green, the stems are very soft and broken well, the bark is thick and the material is white and light. Since bee has no toxicity, almost no side effect in any constitution, and the taste is light, so it is used as a blue blood agent (제) and diuretic (利 水劑). It is also used for the purpose of alcohol detoxification by restoring the liver temperature to normal and excreting moisture well. It has also been reported that ingestion of woody extract of bee trees can be used to prevent poisoning (Hur JM et al., Kor. J. Pharmacogn, 38, 67-70, 2007). In addition, detoxification, blue blood, lipolysis, diuretic, neurostable, anti-diabetic, etc. are known.

On the other hand, the hooves or hooves (厚朴, Magnolia) officinalis Rehd . et Wils.) has long been used as a health agent in Korea, Japan, and China, and leaves have been used for food in Korea and Japan (Information, Shinmingyo, Hyangjeomsa, Yeonglimsa, 469-471, 1998). The groin has been used for medicinal purposes since the bark of stems and branches has been used to treat asthma and gastrointestinal diseases. In motion therapy, it is stated that it is used for cough, diarrhea and gastrointestinal disorders when the chest and abdomen are swollen, sore, and cranky with dry stomach, digestion, convergence, urination and sputum. In `` The Ingredients and Use of Herbs '', yellow magnolia bark decoction, hubak extract sammultang, Daeseunggitang, Banhahubak extract tang, Pyungwisan, etc. are used for dysentery, habitual constipation, high blood pressure, bronchitis, gastric diseases. .

The present inventors have completed the present invention by confirming the strong antioxidant efficacy, blood sugar drop, improved glucose tolerance and insulin sensitivity in the cell and diabetic animal extracts through the cell experiments and in vivo experiments using animals. .

Therefore, the main object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of diabetes mellitus or diabetic complications containing a mixture of bee and hawthorn mixture having antidiabetic activity through a strong antioxidant effect.

Another object of the present invention is to provide a food composition for preventing and improving diabetes or diabetic complications containing the mixed extract of bee and hawthorn.

According to an aspect of the present invention, the present invention provides a pharmaceutical composition for preventing and treating diabetes mellitus or diabetic complications containing a mixture of bee and hawthorn as an active ingredient.

In the present invention, in the process of searching for plants that have antioxidative efficacy and anticipated useful activity for the prevention and treatment of diabetes and diabetic complications in various kinds of natural products, the bee and hawthorn extract in animal models through excellent antioxidant efficacy It is to be found that the anti-diabetic effect, and based on the results of the experiments showed that the mixed extract of them significantly increased compared to the single extract, it is suggested to be useful in the treatment of diabetes and diabetic complications.

In the present invention, the bee and hawthorn mixed extract can be obtained by extracting the bee and hawthorn using a conventional plant extraction method known in the art, preferably in the dried bees and hawthorn water, carbon number 1 It can obtain by adding and extracting the alcohol of -4 or these mixed solvents.

As an extraction method using the extracting solvent, conventional plant extraction methods such as hot water extraction, reflux cooling extraction, ultrasonic extraction, supercritical extraction, etc. may be used. It is appropriate to inject into the extraction solvent and to immerse for 1-15 days at 5-40 ℃ while stirring as needed or to extract by heating at 40-100 ℃ for 4-20 hours. The amount of the solvent used for extraction is preferably 5 to 20 times (weight ratio) of the raw material for extraction.

In the present invention, the beech and hawthorn mixed extract may be included in 0.0001 to 90% by weight, preferably 0.01 to 50% by weight based on the total weight of the pharmaceutical composition.

In the present invention, the bee and hummus are considered to have no side effects such as toxicity as extracts of the present invention extracted from them as medicinal herbs that have been used for a long time and herbal medicine.

Pharmaceutical compositions containing the mixed extract of bee and hawthorn as an active ingredient, can be administered in a variety of oral or parenteral formulations, when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants And diluents such as surfactants or excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

The dosage of the mixture of bee and thick extract according to the present invention may vary depending on the sex, age, weight, and degree of disease of the patient, but it is generally appropriate to administer 10-3000 mg per adult.

In the pharmaceutical composition of the present invention, the bee and hawthorn mixture extract is characterized by having an antidiabetic effect through the antioxidant, blood sugar reduction, glucose tolerance improvement, and insulin sensitivity improving effect.

In a preferred embodiment of the present invention, it was confirmed that the bee and hawthorn mixture extract has a strong antioxidant effect in fibroblasts and rat monocytes / macrophages (RAW264.7) (see Experimental Examples 1 to 3). In addition, in the diabetes-induced mouse model using high-fat-diet (HFD), glucose tolerance test, insulin sensitivity test, and blood glucose test were performed, and the mixture of bee and hawthorn extracts for diabetes It was confirmed that there was efficacy (see Test Examples 5 to 7).

In particular, in the present invention, compared to bee alone extract or hawthorn alone extract, these mixed extracts showed a synergistic effect in the therapeutic effect on diabetes through the antioxidant effect. Therefore, it can be judged that the bee and hawthorn mixed extract according to the present invention show an elevated synergy effect than these single plant extracts.

In the pharmaceutical composition for preventing and treating diabetes mellitus or diabetic complications of the present invention, the diabetes is a chronic metabolic disease caused by high blood glucose levels and causes various diabetic complications. By treatment, the same therapeutic effect can be obtained. The diabetic complications to be treated or prevented in the present invention is a diabetic retinopathy, diabetic foot lesions, diabetic nephropathy, diabetic neuropathy, various infectious diseases caused by diabetes, periodontal disease, skin disease as a variety of conventionally known diabetic complications , Glycogen-containing hepatomegaly, fatty liver, cirrhosis, hepatitis, acute pancreatitis, chronic pancreatitis, diabetic diarrhea, constipation, monolithic esophagitis, gastritis gastritis, acute gastric dilatation, etc. (Diabetes, Korean Diabetes Association, 1992) Preferably, the diabetic complication is diabetic retinopathy.

In a preferred embodiment of the present invention, the bee and hawthorn mixture extract was tested for the effect of inhibiting neovascular production in the retina of the diabetic mouse model was confirmed that the therapeutic efficacy for diabetic complications including diabetic retinopathy (Experimental Example) 8).

According to another aspect of the present invention, the present invention provides a food composition for preventing and improving diabetes or diabetic complications containing the extract of bee and hawthorn as an active ingredient.

The bee and hawthorn mixed extract of the present invention, as described above, exhibits a prophylactic and therapeutic effect on diabetes through antioxidant efficacy. Therefore, it will be apparent that the food composition containing bee and hawthorn mixed extract as an active ingredient may exhibit a prevention and improvement effect on diabetes or diabetic complications.

The food composition of the present invention can be used as an application for prevention and improvement of the above-mentioned diabetes or diabetic complications. The food composition may further comprise a pharmaceutically acceptable food-aid additive and may be formulated in the form of a pill, powder, granule, precipitate, tablet, capsule or beverage.

In the food composition of the present invention, foods to which the bee and hawthorn mixed extract can be added include various foods, for example, beverages, gums, teas, vitamin complexes, health supplements, and the like, pills, powders, It can be used in the form of granules, acupuncture, tablets, capsules or beverages.

At this time, the amount of the bee and hawthorn mixed extract in the food or beverage can be generally added to 0.0001 to 20% by weight of the total food weight in the case of the food composition of the present invention, 0.0001 based on 100ml for the health beverage composition To 20 wt%.

The food supplementary additives defined in the present invention include food additives customary in the art, such as flavors, flavors, colorants, fillers, stabilizers and the like, and are exemplified below.

When the food composition of the present invention is in the form of a beverage, there is no particular limitation on the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and as a further beverage, various flavors or natural carbohydrate copper may be included as additional ingredients. Can be. Examples of the above-mentioned natural carbohydrate include monosaccharides such as glucose and fructose, for example, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin. Sugar and sugar alcohols such as xylitol, sorbitol and erythritol. (Such as taurine, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (such as saccharin and aspartame) are advantageously used as flavorings other than those described above . The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the food composition of the present invention.

In addition to the above, the food composition of the present invention can be used as a flavoring agent such as a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so important, but is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

As described above, according to the present invention, it was shown that the bee and hawthorn mixture extracts have a significantly increased antioxidant effect compared to the bee alone extract or the hawthorn alone extract, and elevated diabetes and diabetic complications in diabetic animal model mice. The therapeutic effect was shown. Therefore, the beech and hawthorn mixed extract according to the present invention can be used in functional foods or pharmaceutical compositions useful for the treatment and prevention of diabetes mellitus or diabetic complications.

1 is a diagram showing the results of immunostaining for VEGF in diabetic animal model retina of bee and hawthorn mixture extract,
Figure 2 is a diagram showing the immunostaining results for CD31 in the diabetic animal model retina of bee and hawthorn mixture extract.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Establishment of Extraction Method

For the production of the mixture of bee and thick leaf extract, the extraction solvent and the extraction conditions were tested as shown in Table 1 to establish the highest yield extraction method, which was used in the following examples.

As shown in Table 1, the highest yield was obtained when 70% ethanol was used as the extraction solvent, and no significant difference in yield was obtained according to the extraction conditions of 'room temperature, 3 days', '60 ° C., 6 hours'. Thus, 70% ethanol was used as the extraction solvent in the following examples.

Example 2. Preparation of Beech and Thick Mixed Extracts

Filtration obtained by filtering 9 kg of 70% ethanol (3%) at room temperature by adding 1.5 kg of wood obtained from Yeongwol-san, Gangwon-do, and dried and shredded bark trees and 1.5 kg of stem bark obtained from Gyeongdong Market. The solution was concentrated under reduced pressure with a vacuum concentrator (BUCHI, R-200, Switzerland) to obtain 312 g (yield: 10.4%) of an extract of a 1: 1 mixing ratio (weight ratio) of bee and thick pepper (Example 2-1).

In addition, in order to prepare a mixed extract of various extraction ratios (1: 2, 1: 3, 2: 1, 3: 2, 3: 1 weight ratio) of the bee and the thick gourd in the same manner, the raw materials were prepared in the following ratio (weight) The yields of the extracts obtained by the mixture extraction in%) are shown in Table 2.

Comparative example  1 and 2. Bee  And preparation of extracts of thick pepper alone

3 kg of the beech wood used in Example 2 and 3 kg of pepper bark stems were added with 9% of 70% ethanol and extracted for 3 days at room temperature, and the filtrate was filtered and concentrated under reduced pressure using a vacuum condenser (BUCHI, R-200, Switzerland). Thus, 287 g (yield: 9.6%) and 308 g (yield: 10.3%) of bee sole extract (Comparative Example 1) and hawthorn sole extract (Comparative Example 2) were obtained, respectively.

Experimental Example  One. Bee  And Thick Mixture Extract NO  Production inhibitory activity

RAW264 induced activation with a lipopolysaccharide (Lipopolysaccharide, LPS; Sigma-Aldrich, USA) for the mixed extract of Examples 2-1 to 2-6 and the extracts of Comparative Examples 1 and 2 described in Table 2 above. The NO production inhibitory activity by the sample of 7 cells was investigated.

Specifically, 8 × 10 ⁵ cells were inoculated in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) without phenol-red. After one day, the sample and 0.1 mu g / ml of lipopolysaccharide were treated and cultured for 24 hours. After that, the supernatant was recovered, and 100 μl of the supernatant was added to 96-well plate. The same amount of Griess reagent (Sigma-Aldrich, USA) was added thereto and the mixture was gently shaken at room temperature for 10 minutes and absorbance was measured at 570 nm. Sodium nitrite was used as a standard product, a calibration curve was prepared, and the amount of nitric oxide produced in the culture solution was determined for each sample. The% yield of each sample was determined assuming that the amount of produced nitric oxide in the group treated with lipopolysaccharide was 100%.

As shown in Table 3 above, as a result of confirming the inhibitory effect of NO production of Examples 2-1 to 2-6 on RAW264.7 cells, it was found that the ratio of the mixed extract of the bee and the husk of Example 2-1 was 1: 1. It showed a high NO production inhibition rate, i.e. excellent antioxidant efficacy. On the other hand, compared to the case of treatment with the bee and the thick extract of Comparative Examples 1 and 2 (approximately 74% to 89% increase) showed an increased antioxidant efficacy in all cases mixed at various mixing ratios.

Experimental Example  2. Bee  And Thick Mixture Extract ABTS Using Antioxidant ability  Measure

Antioxidant activity by the samples of fibroblasts induced oxidative stress with hydrogen peroxide (H ₂ O ₂ ) for the mixed extracts of Examples 2-1 to 2-6 and the extracts of Comparative Examples 1 and 2 described in Table 2 above Investigate. A method of indirectly measuring the antioxidant efficacy of the sample was indirectly determined by the extent to which ABTS (2,2'-Azino-di- [3-ethylbenzthiazoline sulphonate]) was oxidized to mets ⁺ by ABTS ⁺ .

Specifically, fibroblasts (1 × 10 ⁶ cells) were inoculated in a 60 mm dish using Iscove's Modified Dulbecco's Medium (IMDM, Gibco, USA) medium and incubated for 24 hours. After incubation, 200 μM of hydrogen peroxide and the sample were treated and incubated for 24 hours. Collect the cells by centrifugation and add cold buffer (5 mM potassium phosphate, pH 7.4, containing 0.9% sodium chloride and 0.1% glucose) to homogenization (60W (50%), pulse at 5 sec intervals for 1 minute). Cell lysate was prepared. 10 μl of cell lysate, 10 μl of metmyoglobin, 150 μl of chromogen, and 40 μl of hydrogen peroxide were reacted in a 96well plate for 5 minutes and the absorbance was measured at 405 nm using an ELISA reader. The% Yield of each sample was obtained by making the amount of ABTS ⁺ produced in the hydrogen peroxide treated group 100%.

As shown in Table 4, as a result of confirming the ABTS ⁺ production inhibitory effect of Examples 2-1 to 2-6 on the oxidative stress using hydrogen peroxide, as in the NO production inhibition test, the bee and the thick of Example 2-1 It showed the excellent antioxidant efficacy by showing the highest inhibition rate of ABTS ⁺ production in the 1: 1 ratio of the mixed extract. On the other hand, compared to the case of treatment with bees and thick extracts of Comparative Examples 1 and 2 alone (approximately 105.4% to 118.0% increase) showed an increased antioxidant efficacy in all cases mixed at various mixing ratios.

Experimental Example  3. Bee  And Thick Mixture Extract protein carbonyl assay Using Antioxidant ability  Measure

The most common indicator of protein oxidation is protein carbonyl content. 2,4-dinitrophenylhedrazine (DNPH) reacts with protein carbonyls to form Schiff basef, producing a product corresponding to the hydrazone. Therefore, using the DNPH reaction, the antioxidant capacity of the sample can be measured by protein carbonyl assay, which is indirectly quantified by spectrophotometric analysis of protein-hydrazone.

Fibroblasts (2.5 × 10 ⁶ cells) were inoculated in a 100 mm dish using Iscove's Modified Dulbecco's Medium (IMDM, Gibco, USA) medium, and replaced with fresh medium after incubation for 24 hours. After medium replacement, hydrogen peroxide and samples were treated and incubated for 24 hours. Cells were collected by centrifugation and intracellular proteins were isolated by adding lysis buffer. After the reaction by the addition of DNPH, trichloroacetic acid (TCA), Ethanol / Ethyl acetate, guanidine hydrochloride was completed using the reaction, and the absorbance at 370 nm using an ELISA reader to quantify the carbonyl content.

As shown in Table 5, as a result of confirming the protein carbonyl production inhibitory effect of Examples 2-1 to 2-6 on the oxidative stress using hydrogen peroxide, the same as in Example 2-1 of NO and ABTS + production inhibition test It showed the highest antioxidant activity by showing the highest protein carbonyl inhibition rate in the 1: 1 ratio of the mixture of bee and hummus. On the other hand, compared to the case of treatment with bee and thick extract of Comparative Examples 1 and 2 alone (approximately 46.0% to 56.2% increase) showed an increased antioxidant efficacy in all cases mixed at various mixing ratios.

In summary, as a result of cross-checking the antioxidant capacity of the bee and the thick gourd extract through Experimental Examples 1 to 3, it was confirmed that the bee and the thick gourd extract have excellent antioxidant efficacy in in vitro test. In particular, it was confirmed that the most excellent antioxidant efficacy in the 1: 1 ratio of the bee and thick extract of the extract of Example 2-1.

In addition, in Experiments 1 to 3, an in vivo test using a diabetic animal model was performed to verify the effect on prevention and treatment of diabetes mellitus and diabetic complications of the extract of bee and hulica extract having excellent antioxidant efficacy in a 1: 1 ratio.

Experimental Example  4. Design and experiment of experimental animals

Experimental animals were housed in 4-week-old C57BL / 6 male mice (body weight 20-28g) (Korean BioLink, Korea) under constant conditions (temperature: 22 ± 2 ℃, humidity: 55 ± 10% Respectively. Ten cows were fed with water and food from a cage, and used for 1 week before the experiment.

After the end of the acclimatization period, normal diet was fed to the normal group for one week, and high fat-diet (HFD) diet was fed to the diabetic group. The bee and hulky extracts were administered 10 mg / kg of high fat diet and mixed extracts. In addition, the comparison group was administered the same extract of the bee alone and extract only.

Experimental Example  5. Blood sugar ( Blood glucose ) Inspection

The effect of the administration of the mixture of the bee and hummus extract on the blood glucose level of the diabetic animal model was investigated. As a result, the blood glucose level was 110 mg / dl in the normal group, and the blood sugar level was 178 mg / dl in the diabetic group not receiving the extract. And 137 mg / dl to some extent blood glucose reduction effect, while in the case of the bee and hulling extracts group 117 mg / dL blood sugar was reduced to a similar level as the normal group. Therefore, it was confirmed that the bee and hulked mixture extract has an excellent anti-diabetic effect.

Experimental Example  6. Glucose tolerance  ( glucose tolerance ) Inspection

The effect of the administration of the mixture of bee and hummus extract on the effect of improving glucose tolerance in the animal model of diabetes was investigated. The mixed extract was orally administered at 10 mg / kg, and after 30 minutes, glucose was administered to the mice (1 g / kg), and blood was collected from the tail vein after 45 minutes and 90 minutes to measure blood glucose levels (mg / dl). . In addition, the comparison group was administered the same extract of the bee alone and extract only.

As shown in Table 7, the bee and hulked extracts recovered blood glucose levels to normal levels 90 minutes after glucose administration in the diabetic animal model. In the group administered with the extract of the bee alone and the extract of the hoo alone, 210 mg / dl and 207 mg / dl after 90 minutes of glucose administration, respectively, showed a lower blood sugar reduction effect than the mixed extract. Therefore, the bee and hulky extract of the present invention shows a significant improvement in glucose tolerance in the diabetic animal model, it can be seen that it is effective in preventing and treating diabetes.

Experimental Example  7. Insulin sensitivity ( Insulin sensitivity ) Inspection

We investigated whether the administration of the mixture of bee and hummus extracts is effective in improving insulin resistance in diabetic animal models. 30 minutes after the oral administration of the mixture of the bee and the vulgar extract was administered insulin (0.5unit / kg), blood was collected from the tail vein after 45 minutes, 90 minutes to measure the blood glucose concentration (mg / dl). In addition, the comparison group was administered the same extract of the bee alone and extract only.

As shown in Table 8, the mixture of bee and thick extract extract the blood glucose level to the normal level 90 minutes after the insulin administration in the diabetic animal model. In the group administered with the extract of bee sole alone and the extract of beetle alone, the blood glucose reduction effect was lower than that of the mixed extracts as 137 mg / dl and 130 mg / dl, respectively, 90 minutes after the administration of insulin. By showing a marked improvement in insulin sensitivity in the animal model of diabetes, it can be seen that it is effective in preventing and treating diabetes.

Experimental Example  8. Retinopathy  Research (diabetic complication effect)

In order to evaluate whether the administration of the mixture of the bee and the thick leaf extract is effective in diabetic complications, in particular diabetic retinopathy, the effects on renal vessel formation using the retina of the diabetic animal model were investigated. In the present experiment, the expression of vascular endothelial growth factor (VEGF), which is a protein involved in angiogenesis through immunostaining, in the retinal tissues of diabetic animal models was administered orally every 10 mg / kg of the mixture of bee and hulica extract daily. The expression level of the vascular marker cluster of differentiation 31 (CD31) was measured by chemical staining. In addition, the comparison group was administered the same extract of the bee alone and extract only.

The measurement results are shown in FIGS. 1 and 2. 1 is a diagram showing the immunostaining results for VEGF in the diabetic animal model retina of the bee and hawthorn mixed extract, Figure 2 is a diagram showing the immunostaining results for CD31 in the diabetic animal model retina of the bee and hawthorn mixed extract to be. The results of FIGS. 1 and 2 are summarized in Table 9 below.

As shown in Table 9 above, as a result of confirming the inhibitory effect of the renal blood vessel generation of the mixture of bee and thick leaf extract, the expression of VEGF and CD31 was significantly increased in the diabetic group compared to the normal group, but the group treated with the bee and thick leaf extract group In VEGF and CD31 expression decreased in almost the same effect as the normal group. However, in the group administered with the extract of bee alone, 1.21 (VEGF), 1.27 (CD31), respectively, and the group administered with the extract alone, 1.24 (VEGF) and 1.29 (CD31), respectively, lower the expression of VEGF and CD31 than the mixed extract. Indicated. Therefore, it can be seen that the mixture of bee and thick extracts has an effect on the prevention and treatment of diabetic complications, especially diabetic retinopathy by inhibiting neovascularization.

Claims (6)

A pharmaceutical composition for preventing and treating diabetes mellitus or diabetic complications comprising a mixture of bee and hawthorn as an active ingredient.
The method of claim 1, wherein the bee and hawthorn mixed extract is extracted by adding water, C1-C4 alcohol, or a mixed solvent thereof to the bee and hawthorn dry matters, for preventing and treating diabetes or diabetic complications. Composition.
The pharmaceutical composition for preventing or treating diabetes or diabetic complications of claim 1, comprising 0.0001 to 90% by weight of the bee and hawthorn mixture extract based on the total weight of the composition.
[Claim 2] The pharmaceutical composition for preventing or treating diabetic or diabetic complications of claim 1, wherein the bee and hawthorn mixed extract has an antidiabetic effect through antioxidant, blood sugar reduction, glucose tolerance improvement, and insulin sensitivity improvement.
The method of claim 1, wherein the diabetic complication is diabetic retinopathy, characterized in that for preventing or treating diabetes or diabetic complications.
Food composition for the prevention and improvement of diabetes mellitus or diabetic complications containing a mixture of bee and hawthorn as an active ingredient.

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