KR101290264B1 - A pharmaceutical composition and a health functional food composition for preventing, treating or improving diabetes mellitus - Google Patents

Abstract

The present invention provides a pharmaceutical composition for preventing or treating diabetes, including lotus root, plum, and spinach. The present invention also provides a composition for the prevention or improvement of dietary supplements for diabetes, including lotus root, plum, and spinach.
The composition according to the present invention includes a combination of lotus root, plum, and spinach, thereby showing a synergistic effect on the glycemic control effect compared to the case of administering each individually, so that those skilled in the art can predict the prevention, improvement or treatment of diabetes. It has a remarkable effect to the extent that it is unprecedented, and since it is composed of natural products that have been used for a long time, it is preferable because there is little risk of toxicity or side effects to the human body.

Description

A pharmaceutical composition and a health functional food composition for preventing, treating or improving diabetes mellitus}

The present invention relates to a pharmaceutical composition for the prevention, treatment, or improvement of diabetes mellitus and a composition for health functional food, and more particularly, a pharmaceutical composition for the prevention or treatment of diabetes, including a herbal compound and health for the prevention or improvement of diabetes It relates to a functional food composition.

Type 2 diabetes, which is an insulin-independent diabetes, is one of the most common metabolic disorders, characterized by insulin secretion disorders and insulin resistance, which can lead to hyperinsulin secretion from the beta cells of the pancreas, which in turn leads to hyperinsulinemia. Hyperinsulinemia already exists before it is diagnosed with diabetes, acting as an independent risk factor for many other diseases. In this process, beta-cell function eventually decreases, resulting in insulin deficiency. As a result, diabetes mellitus and fasting hyperglycemia occur after meals. Long-term diabetes eventually leads to a number of chronic complications, including microvascular complications such as retinopathy, neurodegeneration, or cardiac disease, or large vessel complications such as cardiovascular, cerebrovascular or peripheral arterial vessels. These chronic complications reduce the quality of life and significantly increase mortality in diabetics. Therefore, the management of type 2 diabetes is to prevent chronic complications, that is, to delay the onset and progression of such chronic complications.

The use of natural products, which have been underestimated as an adjuvant therapy for diabetes, is now of global interest, and research on the separation and action mechanism of bioactive substances in natural products is increasing. α-glucosidase is a brush border enzyme (maltose, sucralase, glucoamylase) of the small intestine mucosa, which has the function of breaking down disaccharides into monosaccharides. When this enzyme is inhibited, glucose absorption into the blood is extremely limited. The rise should be slow. To date, silkworms and mulberry leaf powder have been reported as a representative natural material that inhibits the action of α-glucosidase, delays the digestion and absorption of sugars, and suppresses the rise of blood sugar (Blood lowering effects of murry berry leaves.Yakak Hoegi 39: 367 -372 (1995)).

Insulin plays an important role in the maintenance of sugar homeostasis by regulating the activity of some enzymes involved in glucose metabolism and fat metabolism and gene transcription in hepatocytes through signaling pathways. Increasing the rate of glucose uptake into cells requires primarily increasing the activity of glucokinase (GK), a key regulatory enzyme in the first phase of glycolysis, and secondly glucose--6-phosphate in the presence of NADP ^+. Active in inactive form, or pyruvate dehydrogenase (PDH), which decarboxylates glucose-6-phosphate dehydrogenase or resulting pyruvate to acetyl-CoA, which acts at the initial stage of the pentose phosphate pathway oxidizing to gluconate PDH phosphatase and the like involved in the conversion of the form to PDH should have the function of promoting the use of the absorbed sugar. In addition, acetyl catalyzes the process of transporting fatty acids into the mitochondria after meals to reduce carnitine palmitoyl transferase, which aids in fatty acid oxidation and energy supply, or to transfer the resulting acetyl-CoA to malonyl-CoA, the first product of fatty acid biosynthesis. -Effects of Cordyceps militaris on key enzymes of carbohydrate metabolism.J Korean Soc Food Sci Nutr 34: has been reported to have the ability to activate CoA carboxylase (ACC) and fatty acid synthase. 1531-1535 (2005)).

In the study of Yoon (Study on mechanism involved in hypoglycemic effect on Commelina communis L. Seoul Women's Univ. Increasing the enzyme activity was increased by the hypoglycemic effect was reported. Hypoglycemic action of red ginseng components (II): investigaition of the effect of fat soluble fraction from red ginseng on enzymes related to glucose metabolism in cultured rat hepatocytes.Korean J Ginseng Sci 22: 51-59 (1998) It was reported that the GK and ACC activity lowered to were significantly increased by the red ginseng saponin component.

Hepatic GK and PDH activity is low during fasting and diabetes and decreased activity of these enzymes is a cause of hyperglycemia and has been reported to be associated with insulin resistance. In addition, when GK activity decreases, it acts to liberate the sugar synthesized throughout your life, and when GK activity increases, glycogen accumulation increases in the liver. In addition, diabetic patients with insulin resistance have been reported to be one of the factors is a decrease in the activity of ACC.

In addition, GK regulates glucose metabolism in the beta cells and hepatocytes of the pancreas, and acts as a glucose detector to regulate insulin secretion by recognizing blood glucose in the beta cells of the pancreas. Ko et al. (Ko BS, Kwon DY, Hong SM, Park SM. 2007. In vitro anti-diabetic effects of crude extracts of platycodi radix.Korean J Food Sci Technol 39: 701-707). It was reported that GK mRNA expression was increased because it enhanced the function of beta cells and improved insulin secretion by glucose stimulation. Kang et al. (Kang SY, Paeng JR, Seo KW, Woo JT, Kim SW, Yang IM, Kim JW, Kim YS, Kim KW, Choi YK. 1994. Regulation of glucokinase gene expression and activity in the liver of diabetic rats. J Med 47: 203-209) reported that increased GK enzyme activity and mRNA expression after administration of insulin in hepatic tissues of diabetic rats were reported to increase GK enzyme synthesis. ACC is an enzyme involved in pyruvate producing energy through the TCA cycle and converting excess glucose into fat, which is involved in the consumption of glucose into the cell.

Therefore, if the activity or expression of these GK, PDH, and / or ACC is enhanced, the potential as a substance to treat, prevent or improve diabetes can be expected.

Diabetes is a disease that must constantly manage blood sugar to a normal level, and there is a continuous need for development of drugs or functional foods for treating, preventing or improving diabetes using natural products that can effectively manage blood sugar without side effects.

 The lotus (Nelumbo nucifera) is a perennial plant that grows in ponds or marshes in Siberia, Korea. In particular, the lotus root (Nelumbo nucifera Gaertn.) Is used for food because the tip of the root stem is thicker in autumn, and it is traditionally used in oriental medicine and private house. It has been widely used because it is good for releasing blood, neuralgia, rheumatism, fatigue and recovery after fatigue, weak stomach and lungs, frequent diarrhea, stress and nervousness, hemorrhagic gastric ulcer or gastritis. Lotus root contains various physiologically active ingredients such as methylcorypalline, dimethylcocaeurine, β-sistosterol, kaempferol, quercetine and tannic acid, and studies on the efficacy of lotus root reduce plasma cholesterol, prevent heart disease and colon cancer, and prevent diabetes. This has been reported (Korean Patent Publication 2010-54287).

Prunus mume (Japanese apricot) has traditionally been used in the East, including Korea and Japan, to improve gastrointestinal and liver disease and blood circulation. MK615 compounds recently isolated from plums inhibit the proliferation of cell lines such as breast cancer, colorectal cancer and colon cancer (Kraus E, et al., Photodermatol Photoimmunol Photomed. 2003: 19: 295-302; Rang WQ, et al., Naunyn Schmiedebergs Arch Pharmacol. 2003: 367: 306-311; and Chiou WF, et al., J. Nat. Prod. 1997: 60: 708-711), in particular colon cancer cell lines It has been known to effectively induce apoptosis in cancer cells such as SW480, COLO and WiDr (Chiou WF, et al., J. Nat. Prod. 1997: 60: 708-711). In addition, the plum contains a large amount of natural phenolic phytochemicals such as flavonoids and phenolic acid to inhibit antioxidant and cancer cell proliferation (Chiou WF, et al., J. Nat. Prod). 1997: 60: 708-711 and Okayasu I, et al., Gastroenterology. 1990: 98: 694-702).

Spinach is a seed plant of the main tree, the annual herbaceous or biennial herb, a leafy stem edible vegetable. Looking at the nutritional components of spinach, there are many vitamin types, and vitamin A is the highest among vegetables. In addition, calcium, iron and oxo are a lot of good alkaline foods for children and pregnant women. In addition, saponin and good quality fiber contains spinach is effective in constipation and iron and folic acid is effective in preventing anemia. In addition, there are many beta-carotene, lutein, phenol, vitamin C, and dietary fiber in spinach. Therefore, spinach has long been known as a health food with anticancer effects.

Korean Patent Publication 2010-54287

1.Blood lowering effects of murry berry leaves. Yakhak Hoegi 39: 367-372 (1995)). 2.Effects of Cordyceps militaris on key enzymes of carbohydrate metabolism. J Korean Soc Food Sci Nutr 34: 1531-1535 (2005)). Yoon et al., Study on mechanism involved in hypoglycemic effect on Commelina communis L. Seoul Women's Univ. MSthesis 2005. Lee et al., Hypoglycemic action of red ginseng components (II): investigaition of the effect of fat soluble fraction from red ginseng on enzymes related to glucose metabolism in cultured rat hepatocytes. Korean J Ginseng Sci 22: 51-59 (1998). 5. Ko BS et al., In vitro anti-diabetic effects of crude extracts of platycodi radix. Korean J Food Sci Technol 39: 701-707 (2007). 6. Kang et al., Regulation of glucokinase gene expression and activity in the liver of diabetic rats. Korean J Med 47: 203-209 (1994). 7. Kraus E, et al., Photodermatol Photoimmunol Photomed. 2003: 19: 295-302. 8. Rang WQ, et al., Naunyn Schmiedebergs Arch Pharmacol. 2003: 367: 306-311. 9. Chiou WF, et al., J. Nat. Prod. 1997: 60: 708-711. 10. Okayasu I, et al., Gastroenterology. 1990: 98: 694-702.

Accordingly, the present inventors recognized the necessity of developing a composition that is effective in preventing, improving, or treating diabetes while minimizing side effects, and researching seasonal fruits and vegetables that are enjoyed on our table shows that a glycemic control effect The invention has been accomplished by developing a very good combination of natural composition.

Accordingly, it is an object of the present invention to provide an effective pharmaceutical composition for preventing or treating diabetes, including a combination of herbal medicines as an active ingredient.

Another object of the present invention to provide a dietary supplement for the prevention or improvement of effective diabetes comprising a combination of herbal medicines as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating diabetes, including lotus root, plum, and spinach.

The present invention also provides a composition for the prevention or improvement of dietary supplements for diabetes, including lotus root, plum, and spinach.

Hereinafter, the present invention will be described in more detail.

The present invention has been studied for the development of a natural product composition that is effective in diabetes but has no side effects, it is widely used as a conventional food, when combined with lotus root, plum, and spinach, which has no concern of side effects, the glycemic control effect is very excellent Revealed. Therefore, such a combination of lotus root, plum, and spinach can be used not only as an active ingredient of a pharmaceutical composition for preventing or treating diabetes, but also as an active ingredient of a nutraceutical composition for preventing or improving diabetes.

Accordingly, in one aspect, the present invention provides a pharmaceutical composition for preventing or treating diabetes, including lotus root, plum, and spinach.

In another aspect, the present invention provides a dietary supplement for the prevention or improvement of diabetes mellitus, including lotus root, plum, and spinach. Hereinafter, the pharmaceutical composition and the health functional food composition according to the present invention will be referred to as a natural product composition.

Lotus root, plum, and spinach in the natural composition is not particularly limited in the combination ratio thereof, but preferably may be included in the ratio of 10-1000 parts by weight of plum, 10-1000 parts by weight of 100 parts by weight of lotus root. have.

Natural products constituting the natural product composition of the present invention is obviously equivalent to the same speed in the art, and includes all of the same herbal medicine effective for blood sugar control.

Each of the lotus root, plum, and spinach may be contained in the composition as a solvent extract of natural products, pulverized natural products themselves, pulverized filtrate obtained by pulverizing and filtering, juice filtrate obtained by juice and filtration, and pulverized dry natural products. It does not specifically limit in the embedding form and method, such as it may contain with water. The solvent extract of the natural product may be extracted separately or together with each of the herbal medicines in water, an organic solvent or a combination thereof, and the organic solvent may be C ₁ -C ₄ alcohol, acetone, chloroform, methylene chloride, ether, ethyl Acetate, hexane, or a combination thereof may be used, but is not limited thereto. Examples of C ₁ -C ₄ alcohols include methanol, ethanol, propanol and butanol, and ethanol is most preferred.

The solvent extracts of lotus root, plum, and spinach may be prepared by any extraction method known to those skilled in the art of herbal extraction, but from 10 ° C. to 80 ° C., preferably from 10 ° C. to an extraction temperature of room temperature (about 25 ° C.). 80 hours, preferably 2 to 3 days using an extraction method such as hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction, preferably by extracting 1 to 5 consecutive times by cold extraction have. The solvent extract thus obtained can be obtained as a final extract by filtration under reduced pressure and the filtration extract is concentrated under reduced pressure at 20 to 100 ° C., preferably at room temperature (about 25 ° C.), by lyophilization with a rotary evaporator.

Each of the lotus root, plum, and spinach is a pulverized filtration obtained by being contained in the composition as a pulverized product of the natural product itself, or when it is used as a beverage as a health functional food composition, so as to feel the inherent taste of the natural product itself, or by pulverizing and filtering. It is preferably contained in the composition as a juice or juice filtrate obtained by juice and filtration. In this case, the composition for the dietary supplement is 50-70 parts by weight of plum, 100- parts by weight of lotus root, spinach 20- based on natural products in terms of appearance, flavor, texture and overall acceptability It is preferable to contain in the ratio of 40 weight part (refer Experimental Example 2 below).

The natural product composition comprising lotus root, plum, and spinach according to the present invention is effective in preventing, improving, or treating diabetes. This effect was demonstrated in the following experimental example. Specifically, α-glucosidase inhibitory activity of lotus root, plum, spinach, and mixtures thereof, and glucose metabolism related enzyme activity in the liver of Goto-Kakizaki rat and glucose metabolism related gene expression were measured at the molecular level. In addition, the effects of hypoglycemic elevation on normal and diabetic patients were examined in clinical trials, and the effects of anti-genetic toxicity on long-term intake were measured.

As a result, lotus root, plum, and spinach Each milled filtrate showed α-glucosidase inhibition and was shown to increase the enzyme activity of GK, PDH, and / or acetyl-CoA carboxylase. In addition, when dilutions of mixed ground filtrate of lotus root, plum, and spinach were treated in HepG2 cells, GK, PDH, and ACC mRNAs were significantly increased compared to the control group, increasing enzyme expression of GK, PDH, and ACC. It was shown to make (Experimental Example 1). In addition, when the dilution of the mixed grinding filtrate of lotus root, plum, and spinach according to an embodiment of the present invention to HepG2 cells, when treated with HepG2 cells with each grinding filtrate of lotus root, plum, and spinach alone In comparison, it has been shown to have a synergistic effect on the increased enzyme expression of GK, PDH, and ACC (FIGS. 1A to 1C of Experimental Example 1). In addition, as a result of performing a sugar load test on the mixed grinding filtrate of lotus root, plum and spinach according to an embodiment of the present invention, it was found that the blood glucose response area was significantly lower than that of the glucose-administered group. (glycemic load, GL) was very low as 4.7, and glyemic impact, which represents the weight of glucose with the same blood glucose response as the blood sugar response by 1 g of food, was also shown as 7 g of glucose to prevent or improve diabetes in glucose loading test. Appeared to be very effective. In addition, as a result of measuring blood glucose after actually administering a mixed pulverized filtrate of lotus root, plum and spinach according to an embodiment of the present invention to diabetic patients, fasting blood glucose was reduced to a significant level (Experimental Example below) 3).

Moreover, the natural product composition according to the present invention contains foods that have been proven to be safe while being used for a very long time, and thus have no toxicity and side effects. Can be.

The pharmaceutical composition according to the aspect of the present invention may include a pharmaceutically acceptable carrier or additive.

The pharmaceutical composition may be administered to a variety of mammals, including rats, mice, livestock, humans, etc., for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection. It can be administered by. Accordingly, the pharmaceutical composition may be formulated in conventional pharmaceutical formulations known in the art. The pharmaceutical composition may be formulated and administered in any dosage form including, but not limited to, oral, injectable, suppository, transdermal and non-administrative agents, but preferably liquids, suspensions, powders, granules, It may be formulated in oral dosage forms such as tablets, capsules, pills, emulsions, syrups, aerosols, or extracts.

When formulated into each of the above formulations, it may be prepared by the addition of a pharmaceutically acceptable carrier or additive necessary for the preparation of each formulation. Representatively, when formulated into a dosage form for oral administration, one or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives may be used as the carrier. More than one species can be selected and used.

The carrier and the additive may be any pharmaceutically acceptable, specifically, as a diluent, lactose, dextrose, sucrose, corn starch, soybean oil, microcrystalline cellulose, sorbitol, xylitol, or mannitol, magnesium stearate as a lubricant, As talc and a binder, polyvinylpyrrolidone or hydroxypropyl cellulose is preferable. In addition, as a disintegrant, carboxymethyl cellulose calcium, sodium starch glycolate, potassium polyacrylic acid, or crospovidone, sweetener as white sugar, fructose, sorbitol, or aspartame, stabilizer as carboxymethyl cellulose sodium, beta-cyclodextrin, As lead, or xanthan gum, and preservative, methyl paraoxybenzoate, propyl paraoxybenzoate, or potassium sorbate is preferable.

In addition to the above ingredients, in order to enhance the taste as well-known additives, natural flavors such as plum, lemon, pineapple, herbal flavor, natural pigments such as fruit juice, chlorophyllin, flavonoids, fructose, honey, sugar alcohol, sugar Sweetening ingredients such as, or may be used by mixing an acidulant such as citric acid, sodium citrate.

Among the preparations for parenteral administration, there are sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. For the preparation of non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As a base of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The pharmaceutical composition can be appropriately added to or reduced the mixing ratio of the constituent natural products as appropriate within the range that effectively maintains the therapeutic or prophylactic effect of diabetes, 0.01-80% by weight of the natural component constituting the composition according to the present invention, Preferably 1-50% by weight.

The pharmaceutical composition, in order to obtain a prophylactic or therapeutic effect of diabetes, the active ingredient of the composition based on the natural product 0.1-50 g / kg / day, preferably 1-10 g / kg / day 1 It may be administered several times or several times, and may be appropriately increased or decreased depending on the age, sex, weight, diet, excretion rate, and other drugs currently being taken. Therefore, the pharmaceutical composition according to the present invention is to be prepared in consideration of the range of effective amount, and the unit dosage form formulated in this way is specialized according to the judgment of experts and the needs of the individual to monitor or observe the administration of the drug as needed. Dosing may be done several times at regular intervals.

The health functional food composition according to another aspect of the present invention may include a food or acceptable carrier or additive.

Health functional foods defined in the present invention is a health function prescribed in the Food and Drug Administration Notice 2008-72 is sufficiently established that the newly defined functional and safety for the human body through the Act on Health Functional Foods amended in 2008 It means that it is a health functional food as listed in the regulations on the recognition of food functional ingredients.

The nutraceutical composition according to the present invention may be formulated into a conventional nutraceutical formulation known in the art. The health functional food may be prepared, for example, as a powder, granules, tablets, pills, capsules, suspensions, emulsions, syrups, dips, liquids, extracts, gums, teas, jellies, beverages, and the like. It may be formulated in the form of. As such a pharmaceutically acceptable carrier or additive, any carrier or additive known to be usable in the art for the preparation of the formulation to be prepared may be used.

The health functional food composition may comprise from 0.01 to 15% by weight, preferably 0.2 to 10% by weight of the total natural food ingredients of the composition according to the present invention, when prepared as a drink 100 mL It may be contained in a ratio of 0.1 to 30g, preferably 0.2 to 5g based on the amount, the whole drink may be composed of natural ingredients, but is not limited thereto.

The beverage may further contain other ingredients in addition to the herbal ingredients, and may further contain various flavors or natural carbohydrates, etc. that are commonly used in beverages. Examples of the natural carbohydrate include conventional sugars such as monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugars such as xylitol, sorbitol, erythritol, Of sugar alcohols may be contained. In addition, as a flavoring agent, a natural flavoring agent (e.g., tau Martin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.) may be contained. The ratio of the natural carbohydrate is preferably about 1 to 20 g, preferably about 5 to 12 g per 100 mL of beverage.

The pharmaceutical composition and the composition for health functional food according to the present invention may be administered at the same time lotus root, plum, and spinach contained as an active ingredient, but the extent to show the effect of preventing, improving or treating diabetes of the composition It may be administered sequentially at predetermined intervals within the range. If the combination of lotus root, plum and spinach is to be administered at predetermined intervals, each active ingredient may be formulated in a separate, separate formulation. In the case where the lotus root, plum and spinach are to be administered simultaneously, they may be formulated in a uniformly mixed form in one formulation, or may be formulated in separate formulations and then simultaneously administered.

As described above, the composition according to the present invention comprises a combination of lotus root, plum, and spinach, thereby showing a synergistic effect in the glycemic control effect than when administered separately, preventing, ameliorating or treating diabetes. It is preferable to have a remarkable effect to those skilled in the art unpredictable, and since it consists of natural ingredients that have been used for a long time, there is little risk of toxicity or side effects to the human body.

FIG. 1A shows that GK mRNA was treated with HepG2 cells after 24 hours of dilution of 50 to 1000-fold dilutions of the grinding filtrate of lotus root, plum and spinach, and the mixed grinding filtrate of lotus root, plum and spinach of Example 1. It is a graph showing the result of measuring the expression level.
FIG. 1B shows PDH mRNA after treatment with HepG2 cells for 24 hours with dilution of 50 to 1000-fold dilutions of the grinding filtrate of lotus root, plum and spinach and the mixed grinding filtrate of Example 1 of lotus root, plum and spinach. It is a graph showing the result of measuring the expression level.
FIG. 1C shows ACC mRNA after 24 hours of dilution of 50-1000-fold dilutions of lotus root, plum and spinach, and 50-1000 times the mixed grinding filtrate of lotus root, plum and spinach. It is a graph showing the result of measuring the expression level.
P <0.05 vs control, Mix, mixture; S, spinach; JA, plum; LR, lotus root and a, ab and b on the graph mean that they are significantly different as p> 0.05 in Duncan's multiple range test. The mixed grinding filtrate of lotus root, plum, and spinach was prepared and diluted 50 times. Mix 50, 100-fold dilution was Mix 100, and 1000-fold dilution was labeled Mix 1000. μg of the grinding filtrate of lotus root, plum, and spinach, respectively. It is expressed in units of / mL. That is, S100 means 100 μg / mL of spinach, JA10 means 10 μg / mL of plum, and LR1 means 1 μg / mL of lotus root. The control is indicated as c.
Figure 2 is a graph showing the average of the results of measuring the tail DNA content before and after ingestion to determine whether lymphocyte DNA damage recovery after ingesting a functional drink according to an embodiment of the present invention to six diabetic patients 3 weeks .

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

1-3 in comparison: grinding filtrate preparation of lotus root, plum, and spinach respectively

5g of lotus root (Comparative Example 1), Plum (Comparative Example 2), and Spinach (Comparative Example 3), which were purchased from the market and washed cleanly, were individually crushed using a grinder (HMF-3000S, EL Nissey Electric Co., Ltd., Korea). After grinding, the resultant was filtrated with whatman paper (No. 1) to prepare a ground filtrate of lotus root, plum, and spinach.

Example 1 Preparation of Mixed Grinding Filtrate of Lotus Root, Plum, and Spinach

5 g of lotus root, plum and spinach, which were purchased in the market and washed cleanly, were mixed and ground together using a grinding machine (HMF-3000S, Elnische Electric Co., Korea), and then filtered using whatman paper (No. 1). Thereby, the mixed grinding filtrate of lotus root, plum, and spinach was produced.

Example 2: Preparation of Ethanol Extracts of Lotus Root, Plum, and Spinach

Lotus root, plums, and spinach purchased from the market and washed clean were used for the preparation of the extract.

1 kg each of lotus root, plum and spinach in a well-pulverized state was added to an extractor, and 2 L of an 80% ethanol aqueous solution was added thereto, followed by cold washing for 3 days at room temperature to obtain respective liquid extracts. After filtering each of the liquid extracts and concentrated under reduced pressure at 50 ℃ with a rotary evaporator (rotary evaporator) and completely remove the solvent in a vacuum oven to dry and then powderized to finally obtain each ethanol dry extract.

Experimental Example 1: Measurement of activity and gene expression of enzymes related to glucose metabolism

1-1. Experimental Method

Liver extraction from Goto-kakizaki rats to determine GK, PDH, ACC enzyme activity, followed by washing with physiological saline, homogenization with buffer (0.02 M EDTA, 0.002 M dithioerythritol (DTT), 0.1 M triethanolamine) and 3,000 at 4 The supernatant (cytosol and mitochondria) was obtained by centrifugation for 15 minutes at rpm. A buffer solution was added to the supernatant and centrifuged at 24,650 x g for 15 minutes at 4 ° C to separate cytosol and mitochondria pellets. 1 mL of buffer was added to 1 mL of mitochondrial pellets, and centrifuged at 24,650 × g for 30 minutes at 4 ° C. The filtrates of Example 1 and Comparative Examples 1-3 were used as samples.

(1) α-glucosidase measurement

5 mg rat intestinal acetone powder (Sigma Aldrich, USA) is mixed with 1 mL of maleic acid buffer at 4 ° C., sonicated for 30 minutes on ice and centrifuged at 4 ° C. 11,000 rpm for 30 minutes. The supernatant was then used as the enzyme solution of α-glucosidase. 50 μL enzyme solution, 50 μL sample, 100 μL 0.1 M maleic acid buffer was preincubated at 37 ° C. for 10 minutes and then reacted at 37 ° C. for 30 minutes by adding 50 μL 0.4% maltose substrate solution. After boiling for 5 minutes at 100 ℃ left in an ice bath and measured using a glucose kit (glucose kit). The positive control group was compared with acarbose (acarbose).

(2) GK enzyme activity

85 mM Hepes-NaOH (pH 7.4), 1000 mM KCl, 25 mM DTT, 75 mM MgCl ₂ , 10 mg / mL BSA, 50 mM NAD ⁺ , 4 units glucose-6-phosphate dehydrogenase, 1 M glucose After adding the cytosol and the sample and reacting for 10 minutes at 37 ° C, absorbance values were measured for 10 minutes at 37 nm and 340 nm by adding 500 mM ATP. GK activity measurement units are expressed as nmole of NADH generated for 1 min per mg of cytosolic protein.

(3) PDH enzyme activity

The reaction buffer (50 mM KH ₂ PO ₄ , 1 mM MgCl ₂ , 2.5 mM NAD ⁺ , 0.2 mM TPP, 0.13 mM CoA, 0.32 M DTT) and 2 mM pyruvate were added and reacted at 30 ° C. for 3 minutes, followed by sample and mitochondrial tissue solution. The initial absorbance at 340 nm was measured and the amount of NADH generated was measured for 3 minutes.

(4) ACC enzyme activity

Reaction buffer (50 mM Tris-HCl, pH 7.5), 3.75 mM glutathione, 0.75 mg fetal bovine serum, 0.125 mM NADH, 0.125 mM acetyl-CoA, 0.5 mM potassium phosphoenolpyruvate, 6 μg lactate dehydrogena First, 10 mM MgCl ₂ , 15 μg pyruvate kinase, 3.75 mM ATP) were added and reacted at 37 ° C. for 2 minutes, and then 25 mM KHCO ₃ was added thereto and incubated for 1 minute. And the mitochondria and the sample was added and reacted for 3 minutes at 37 ℃ and the amount of NAD ⁺ was measured for 3 minutes at 340 nm.

(5) Measurement of HepG2-cell glucose metabolism related gene (GK, PHD, ACC) expression change using real-time PCR

After diluting the sample with HepG2-cells 1x10 ⁶ cells incubated for 24 hours and incubating for 24 hours, cells were collected using 1 mL of TRIzol Reagent to extract total RNA and the concentration of the extracted RNA was nanodrops (nano drop). RNA extracted from HepG2-cell was used for real-time PCR using GK, PHD and ACC, and GAPDH was used as housekeeping gene. Reverse transcription was performed by mixing 1 μg total RNA, 30 pmole oligo dT19, dH ₂ O, and reacting at 70 ° C. for 5 minutes, and using 5X RT buffer (250 mM Tris-HCl; pH 8.3, 375 mM KCl, 15 mM MgCl). ₂ , 50 mM DTT) and 1 mM dNTPs were added and reacted at 37 ° C for 5 minutes, and then 200 U M-MLV reverse transcriptase was added to synthesize cDNA for 1 hour at 37 ° C and 15 minutes at 70 ° C. Using the synthesized cDNA as a template, 2x QuantiTect SYBR Green PCR Master Mix, 0.5 μM primers, and RNase-free water were mixed to 20 μL and amplified by SuperBio. The reaction conditions were incubated for 10 minutes at 95 ℃, 50 ℃ denature (denaturation) at 94 ℃ 15 seconds, annealing (annealing) at 55 ℃ 30 seconds for PDH and ACC, 59 ℃ 20 seconds for GK, Extension was carried out at 72 ° C. for 30 seconds. The amplification results were obtained by Delta CT analysis, and the melting curve of the PCR reaction was confirmed, and the purity of the amplification product was determined based on the normal gene expression. concentration). The sequence of the used primer is as follows.

1-2. Experiment result

The inhibitory activity of α-glycosidase of lotus root, plum, and spinach prepared in Comparative Example 1-3 was confirmed, and the inhibitory power thereof was compared with that of acarbose, a representative hypoglycemic agent, used for the treatment of type 2 diabetes. Indicated. According to Table 2, α-glucosidase inhibitory activity was found to be 30.4% lotus root, 20.0% plum, 5.2% spinach, α-glycosidase inhibitory activity of akaboose, a positive control was 84.8%.

Table 3 shows the results of measuring the effect of grinding filtrate of each of the lotus root, plum, and spinach of Comparative Example 1-3 on the GK, PDH, ACC enzyme activity of HepG2 cells. According to Table 3, GK activity of glucose metabolism-associated enzyme was not shown in lotus root, and only PDH (5.57-fold) and ACC (5.27-fold) activity were significantly increased compared to the control group. GK, PDH, and ACC activities of plum were significantly increased by 7.91 times, 2.45 times, and 3.95 times, respectively. Spinach significantly increased PDH and ACC activity by 2.16 and 5.50 fold, respectively, compared to the control.

After diluting the lotus root, plum and spinach of Comparative Example 1-3 and the diluted solution of the lotus root, plum and spinach mixed grinding filtrate of Example 1 by 50-1000 times for 24 hours in HepG2 cells, Results of determining the expression level of GK, PDH, ACC mRNA are shown in Figures 1a to 1c.

1a to 1c, when the mixed grinding filtrate of Example 1 was diluted 1000-fold in HepG2 cells, the mRNAs of GK, PDH, and ACC were 3.4 compared to the control treated only with growth media. Pear, 2.2-fold, and 2.3-fold expressions were significantly increased (p <0.05). The GK gene was significantly increased in the 1000-fold dilution of the mixed milled filtrate, and PDH and ACC were also mixed in the mixed milled filtrate of Example 1 by 1000 than the group treated with lotus root, plum and spinach milled filtrate alone. Gene expression was significantly increased in pear-diluted samples.

In Goto-kakizaki rats, treatment of lotus root (except GK activity), plum and spinach, respectively, increased GK, PDH, and ACC activity of glucose metabolism-regulating enzymes. Comparison of GK, PDH, and ACC gene expression by treatment of lotus root, plum and spinach, respectively, showed a 3.4-fold increase in GK gene expression only in Mix 1000 (1000-fold dilution of lotus root, plum and spinach mixed grinding filtrate). In the other treatment groups except Mix 1000, there was no significant effect on the expression level of the control gene. Therefore, it can be said that the mixed pulverized filtrate of lotus root, plum and spinach has a synergistic effect in the expression of glycometabolase gene of human-derived hepatocytes compared with lotus root, plum and spinach alone.

Experimental Example  2: Sensory evaluation

2-1. Experimental Method

Ten members of graduate school and undergraduate students of the Department of Food and Nutrition, Kyungnam University, were selected and trained enough for the sensory test. The sensory characteristics used in the palatability test were 9-point item scales, with 9 points being very liking from 1 point dislike for appearance, flavor, texture, and overall palatability.

To prepare a variety of mixed drinks according to an embodiment of the present invention by mixing the grinding filtrate of lotus root, plum, and spinach prepared in Comparative Examples 1 to 3 in various ratios based on natural products and then sensory test Was carried out.

2-2 results

As a result of the sensory test, the mixed functional beverage of Example 3 (Example 3) having a blending ratio of Table 4 based on natural products was found to be excellent in overall preference.

Experimental Example 3

3-1. Experimental Method

1) Sugar load test after eating functional drink in normal adult

Ten normal adults were taken at the fingertips on the morning of the experiment, and fasting blood glucose was measured. Blood glucose levels were measured at 15, 30, 45, 60, 90 and 120 minutes after sample ingestion at the fingertips, respectively. The blood glucose measurement was repeated twice with a blood glucose meter. In order to compare the results of the blood glucose response with the control group of 50 g of glucose, the blood glucose response area after ingesting 75 mL of the mixed functional drink of Example 3 and the glucose concentration area for 2 hours after the ingestion of glucose was measured. Comparing the results calculated as a percentage of the glycemic load.

Glycemic load (GL) is a quantitative measure of how fast carbohydrates in a particular food are broken down and absorbed into glucose by taking into account the actual intake of the food, based on the GI. Since quantitative aspects such as the amount of intake per person are not considered, GL was calculated considering the actual food intake.

Blood glucose level (GL) = weight of carbohydrate per serving (g) X GI / 100

In addition, the glycemic impact, which is the weight of glucose (glycemic glucose equivalent, GGE / g) showing the same glycemic response by 1 g of a given food was measured.

GGE / g = (IAUC _food / IAUC _glucose ) x (Wt _glucose / Wt _food ) x 1g

(IAUC: incremental area under the curve)

2) Long-term blood glucose control and anti-genetic toxicity evaluation of diabetic patients

(1) Blood collection before and after eating mixed functional drinks

Six diabetic patients were given the mixed functional drink once a day (75 mL) for 3 weeks at 3-5 o'clock, which was the experimental group, and after one week wash out again, the placebo beverage (the same amount as the mixed functional drink) Commercial pear juice containing glucose) was ingested for 3 weeks to compare as a control.

Blood glucose was measured after fasting for 8 hours or more before and after ingestion. Blood was collected for Comet assay, and whole blood was placed in a heparinized sterile test tube.

(2) comet assay

a. Leukocyte DNA Damage Measurement

Fresh blood was collected from Histopaque 1077 to isolate only white blood cells and used in this experiment. The isolated white blood cells were mixed with 75 µl of 0.7% low melting agarose gel (LMA), and the suspension of whole blood and LMA was evenly dispersed over a normal slide coated with 1% normal melting agarose (NMA), and then covered with a cover glass. Stored in the refrigerator at 4 ℃. Once the gel has hardened, remove the cover glass and cover one more layer with 75 μl of 0.7% LMA solution on top of it and place it in cold alkaline lysis buffer (2.5M NaCl, 100mM EDTA, 10mM tris) prepared for cell lysis. Immediately before use, 1% Triton X-100 was mixed and the slide was immersed in low temperature, dark room for 1 hour to release the double screw of DNA. After dissolving the slides were arranged in an electrophoresis tank and unwinding with 4 ℃ cold buffer (300mM NaOH, 10mM Na2EDTA). After 20 minutes, electrophoresis was performed for 20 minutes at 25V / 300 ± 3 mA. After electrophoresis, wash thoroughly with 0.4M Tris buffer solution (pH 7.4), stain the nuclei with 20 μl / ml concentration of ethidium bromide, observe on a fluorescence microscope (Leica, Germany), and send each through a CCD camera. Nucleus images were analyzed on a computer equipped with a comet image analyzing system (Komet 4, Kinetics, UK).

3-2. Experiment result

1) Results of glucose load test according to functional drink intake in normal persons

The results of measuring the blood glucose response area are shown in Table 5 below.

According to the results, the total area of the blood glucose response in the intake of the functional beverage was significantly lower than in the 50 g intake of glucose. GL is included in low GL when it is 20 or less, and the functional beverage glucose index value is 4.7. In addition, the glycemic impact of functional drinks is 7, which is the same as the glycemic response after 7 g of glucose.

2) Effect on long-term blood sugar control and antigenetic toxicity after 3 weeks of functional drink

Fasting blood glucose decreased significantly from 186.7 ± 21.2 to 148.3 ± 9.1 after 3 weeks of intake of functional drinks, but after 3 weeks of placebo drink (commercial pear juice), blood glucose returned to its original level, but there was no significant difference. .

3) Changes in DNA Damage in Human Lymphocytes

Lymphocyte DNA damage in the body was measured by the length of the destroyed fragment of the cell after the COMET assay using an electrophoresis of alkaline environment, expressed in tail DNA (%). The results are shown in Fig.

According to Figure 2, DNA damage was significantly inhibited after ingesting the functional beverage for three weeks. Therefore, long-term intake of functional drinks is believed to be involved in repairing damaged lymphocyte DNA by scavenging the free radicals produced by diabetes.

Formulation Example 1 Preparation of Tablet

Using the herbal composition prepared in Example 2, tablets for oral administration using the wet granulation method and the dry granulation method with the following composition are prepared by tableting after granules are prepared.

Formulation Example 2: Preparation of Capsule

Using the herbal composition prepared in Example 2 to fill a gelatin capsule in the following composition to prepare a capsule.

Formulation Example 3: Preparation of Ointment

Using the herbal composition prepared in Example 2 to prepare an ointment with the following composition.

Formulation Example 4 Preparation of Injection

Using the herbal composition prepared in Example 2 to prepare an injection with the following composition.

Formulation Example 5 Preparation of Liquid

Using the herbal composition prepared in Example 2 to prepare a liquid formulation with the following composition.

Formulation Example 6 Preparation of Powder

Using the herbal composition prepared in Example 2 was mixed in the following composition and filled in an airtight fabric to prepare a powder.

Formulation Example 7: Preparation of Pills

Heat 100 mL of honey, filter it, heat it again, and filter again three or four times, then rub it with your hands to heat it until it becomes viscous, yellow dust, and not sticky in your hands after cooling. . Water, walnut oil and vinegar may be used instead of honey. The hot honey prepared as described above is kneaded by adding 5 g of the herbal composition prepared in Example 2 to form a pill by molding the dough into a ring of equal size by using an artificial or a machine. The prepared pills are dried in a cool and dry place and then stored in a cool and airtight place.

Formulation Example 8 Preparation of Suspending Agent

0.1 g of the herbal composition prepared in Example 2 was stirred and mixed in 100 mL of water with polyethylene glycol, polyvinylpyrrolidone, carboxymethylcellulose, bentonite or polysorbate 80 as a suspending agent, and saccharin sodium, syrup as a sweetener. , Sucrose, honey, or D-mannitol is mixed to prepare a suspending agent according to the conventional method for preparing a suspending agent.

Formulation Example 9 Preparation of Transdermal Agent

Using the herbal composition prepared in Example 2, a transdermal agent is prepared according to a conventional method with the following composition.

Formulation Example 10 Preparation of Chewing Gum

Chewing gum is prepared using conventional methods using the herbal composition prepared in Example 2 in the following composition and content.

Formulation Example 11 Preparation of Beverage

Using the herbal composition prepared in Example 2 to prepare a beverage using conventional methods in the following composition and content.

Formulation Example 12 Preparation of Candy

Using the herbal composition prepared in Example 2, candy is prepared using conventional methods in the following composition and content.

A mixture of 20% herbal composition and 80% crystalline lactitol is diluted with purified water and placed in a sauce pen. The batch is first heated on a hot plate until all material is solubilized, then the batch is transferred to a vacuum cooker and further heated. The formed blend forms a viscous mass even at relatively low temperatures. The syrup is then transferred from the cooker to the thick plate and cured until a suitable tissue is formed for drop rolling. The cured mass is fed through a drop roller. Once imprinted, the candy has an acceptable quality.

Claims (6)

A pharmaceutical composition for preventing or treating diabetes, including lotus root, plum, and spinach. A health functional food composition for preventing or improving diabetes, including lotus root, plum, and spinach. The composition according to claim 1 or 2, wherein the composition comprises 10 to 1000 parts by weight of plum and 10 to 1000 parts by weight of spinach, based on 100 parts by weight of the lotus root. The solvent according to claim 1 or 2, wherein the lotus root, plum and spinach are each selected from the group consisting of its own pulverized product, pulverized filtrate, dry pulverized product, or water, C ₁ -C ₄ alcohol and combinations thereof. A composition characterized in that the crude extract of. The composition according to claim 1, which is in the form of a powder, granules, tablets, capsules, suspensions, emulsions, syrups, solutions, aerosols, extracts, injections, transdermal or suppositories. 3. A composition according to claim 2 in the form of a liquid, suspension, powder, granule, tablet, capsule, pill, extract, tea, jelly, or beverage.

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