KR101108885B1 - Tetragonia tetragonioides extracts having antidiabetic and cholesterol-lowering activities - Google Patents

Abstract

The present invention relates to an extract obtained by extracting Tetragonia tetragonioides with an organic solvent and a functional material separated therefrom. As the extract, according to the present invention, edible without cytotoxicity is possible, α-glucosidase, It has anti-diabetic activity through α-amylase and aldose reductase inhibitory activity, and blood cholesterol inhibitory activity through HMG-CoA reductase inhibitory activity, and methyl palmitate, methyl-4- ( 2-tridecylophenyl) butanoate and methyl-3-cyclopentyl-2,3-dimethylbutanoate also exhibit antidiabetic effects through α-glucosidase inhibitory activity and inhibit HMG-CoA reductase inhibitory activity. Since the blood cholesterol inhibitory activity through, the present invention is a very useful invention in the health food industry and pharmaceutical industry.

Burnweed, antidiabetic, α-glucosidase, α-amylase, aldose reductase, HMG-CoA reductase, blood cholesterol inhibition

Description

Tetragonia tetragonioides extracts having antidiabetic and cholesterol-lowering activities}

The present invention relates to a bunchocho extract having antidiabetic and blood cholesterol inhibitory activity, and more specifically, tochogo ( Tetragonia) tetragonioides ) extracted with an organic solvent, an antidiabetic activity through α-glucosidase, α-amylase and aldose reductase inhibitory activity, and a burnt herb extract having blood cholesterol inhibitory activity through HMG-CoA reductase inhibitory activity, and It relates to a functional material separated therefrom.

Tetragonia tetragonioides is a perennial herb belonging to the Aizoaceae family called spinach or New Zealand spinach. Its height is 4060 cm, with fleshy bumps. It gradually rises up to a height of about 50 cm, and branches a little from one root to the ground, spreading out in all directions. It is distributed in New Zealand, China, Japan, South Asia, Australia, and South America, and has a strong vitality, so it grows well even in harsh and moisture-free areas such as gravel fields and rock crevices, and Korea forms colonies on the sandy beaches of southern and Jeju islands. It is growing.

Bunchocho is an edible crop that is used as a salad in foreign countries and eaten as a herb. Since ancient times, the Korean traditional medicinal herb has been considered as precious as the lettuce of the sea, and it has been known as the three herbs that are good for the stomach along with the herb from the mountain, 'Sukju Root', and the tree 'Yeok Tree'. In particular, the medicinal herb found in the Joseon Dynasty Heo-tae was looking for a treatment for Master Yoo-tae's disease (capture, rebellion, blow: today's stomach cancer). It is a plant with high treatment and prevention effect on various gastrointestinal diseases such as excessive stomach acid and indigestion.

In addition, the burnt vinegar is called the Chinese traditional medicine, so the outpost has been used for the treatment of enteritis, sepsis, carbuncle, and wind fever purpose with the efficacy of clearing, detoxification, swelling, and swelling. To date, isolated components of the plant have been reported to be β-cartene, diterpene, flavone, cerebroside, oxylic acid, sterylglucoside and polysaccharides. Anti-inflammatory and anti-ulcer activity This is being reported.

Currently, studies on the separation / analysis of the active ingredients of Korean native burned vinegar are not active, and the full efficacy and active substance structure of the burned vinegar have not been studied. There is also a lack of research on the basic ecology of plants that are the basis for cultivation.

As a conventional technique related to the burnt vinegar, Korean Patent Application No. 10-2002-0016517 discloses a bunchocho extract having apoptosis inhibitory activity and a pharmaceutical composition comprising the same, the banchocho extract is antitumor activity, preventing liver disease It shows that it can be usefully used as a prophylactic and therapeutic agent for tumors, liver diseases, etc. by showing therapeutic activity and immune enhancing activity. In addition, Korean Patent Application No. 10-2005-0092949 discloses a vinegar essential oil extract with excellent antimicrobial activity, and can be used in various fields such as purulent skin diseases such as respiratory and digestive diseases, boils and impetigo and acne preventive compositions. Teaches this outstanding burntweed essential oil extract.

Therefore, the present inventors investigated the anti-diabetic and blood cholesterol inhibitory effects in order to obtain basic data based on the various physiological activity of the budswort, and to lay the groundwork for searching for the active substances of the wild roots. It came.

It is an object of the present invention to provide an antidiabetic and blood cholesterol inhibitory functional material isolated therefrom with an antidiabetic and blood cholesterol inhibitory activity.

The purpose of the present invention as described above is to extract the buncho vinegar with an organic solvent to obtain a bunchocho extract, to confirm their α-glucosidase, α-amylase, aldose reductase inhibitory activity and HMG-CoA reductase inhibitory activity, It was achieved by separating functional substances from the extract and confirming blood cholesterol inhibitory activity through their α-glucosidase, α-amylase inhibitory activity and HMG-CoA reductase inhibitory activity and diabetic activity in diabetic rats. .

The present invention provides a bunchocho extract having antidiabetic activity.

Bunchocho extract according to the present invention is preferably an organic solvent extract.

Methanol may be preferably used as the organic solvent, but is not limited thereto, and other organic solvents may be used.

The organic solvent extraction can be carried out at room temperature, preferably at a temperature of 15 to 25 ℃, for 4 to 8 hours.

In addition, the bunchocho extract according to the present invention is dissolved in distilled water, the organic solvent extract of the bunchocho, preferably methanol extract of the bunchocho, and then continuous in the order of n-hexane (HX), dichloromethane (DCM), and ethyl acetate (EA) N-hexane (HX) -fraction, dichloromethane (DCM) -fraction, and ethyl acetate (EA) -fraction obtained by fractionation.

Baechocho extract according to the present invention has α-glucosidase, α-amylase, aldose reductase inhibitory activity and HMG-CoA reductase inhibitory activity.

In particular, the α-glucosidase inhibitory activity of the HX fraction is the highest, and the other extracts also exhibit higher inhibitory activity than the conventional α-glucosidase activity inhibitors. In addition, Baechocho extract of the present invention exhibits α-amylase inhibitory activity at a high concentration of 10 mg / mL, shows aldose reductase inhibitory activity at a concentration of 1 mg / mL, and also shows HMG-CoA reductase inhibitory activity. In vivo experiments using an insulin-dependent diabetic mouse model showed that the administration of the bunchocho methanol extract according to the present invention showed an excellent hypoglycemic effect in diabetic rats to which streptozotocin was administered.

In another aspect, the present invention provides a composition for the prevention and treatment of diabetes mellitus and cardiovascular circulatory diseases, containing the above extracts as an active ingredient. The composition may contain a Bunchocho extract in an amount of 1 to 10 mg / mL. The composition according to the invention can be administered orally or parenterally.

In addition, the present invention provides a functional material separated from the Bunchocho extract.

The functional material according to the present invention is subjected to silica gel column chromatography of the Bunchocho extract with a solvent combination of HX / DCM = 100% HX? 100% DCM, and each fraction is separated by fractional liquid chromatography (JAI, Japan). , Stearic acid, methyl palmitate, methyl-4- (2-tridecylophenyl) butanoate, methyl-3-cyclopentyl-2,3-dimethylbutano via 1H NMR, 13C-NMR, DEPT and HMBC Eight and methyl-4-hydroxy benzoate Five materials confirmed the final structure.

<Formula 1>

Stearic acid

<Formula 2>

Methyl palmitate

<Formula 3>

Methyl-4- (2-tridecylphenyl) butanoate

<Formula 4>

Methyl-3-cyclopentyl-2,3-dimethylbutanoate

<Formula 5>

Methyl-4-hydroxy benzoate

In methyl-4- (2-tridecylphenyl) butanoate and methyl-3-cyclopentyl-2,3-dimethylbutanoate among the functional substances according to the present invention, high α-glucosidase inhibitory activity is observed. appear. In particular, methyl-3-cyclopentyl-2,3-dimethylbutanoate showed high inhibition rate.

In addition, the extracts and the compounds isolated therefrom according to the present invention has a hypoglycemic function and a hypocholesterolemic effect can be applied to a variety of foods, for example, beverages, tea, candy that can be easily and easily manufactured And functional foods, such as sweets, can also be applied to prevent diabetes, lower blood cholesterol levels, therapeutics and herbal remedies.

Accordingly, the present invention provides a food composition for preventing diabetes and lowering blood cholesterol concentration, which contains an antidiabetic and blood cholesterol lowering activity, as an active ingredient, as an active ingredient. In another aspect, the present invention provides a pharmaceutical composition for preventing diabetes, lowering blood cholesterol concentration, containing as an active ingredient a budchocho extract or a compound isolated therefrom having antidiabetic activity and blood cholesterol lowering activity.

Baechocho extract according to the present invention is capable of edible without cytotoxicity, as well as anti-diabetic activity through α-glucosidase, α-amylase and aldose reductase inhibitory activity, through HMG-CoA reductase inhibitory activity Methyl palmitate, methyl-4- (2-tridecylophenyl) butanoate and methyl-3-cyclopentyl-2,3-dimethylbutanoate, which are functional substances which have cholesterol lowering activity in blood and are separated therefrom Since the anti-diabetic effect is exhibited through α-glucosidase inhibitory activity and the cholesterol lowering activity of blood is inhibited through HMG-CoA reductase inhibitory activity, the present invention is a very useful invention for the health food industry and the pharmaceutical industry.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

Example 1: Burnout  Preparation of Extract

Burnhacho used in this example was purchased as a plant native to Jeju Island and the southern part of the Korean peninsula, and used after drying.

As shown in Fig. 1, 1 kg of dried burnt vinegar was added to methanol (MeOH, 2 L), and extracted three times for 4 hours with stirring at room temperature. Filtration under reduced pressure to 5A. The filtrate was concentrated under reduced pressure with a rotary vacuum evaporator (Ratavapor R-121, Buchi, Switzerland), and 2 L of n -hexane (HX) was added to the resulting concentrate to separate the HX and H ₂ O layers. It was.

The separated H ₂ O layer was solvent fractioned in the same order as HX in 2 L of dichloromethane (DCM) and 2 L of ethyl acetate (EA). Each solvent fraction layer was concentrated under reduced pressure with a rotary vacuum evaporator to obtain MeOH fraction, HX fraction, DCM fraction and EA fraction. The concentrated fraction was dried by completely removing the solvent at room temperature and then used as a sample for activity measurement while freezing.

Example 2 Diabetes Related Enzyme Activity Analysis

Example 2-1: α- Glucosidase  Inhibitory Activity Assay

Intestinal α-glucosidase is an enzyme present in the microvilli of the small intestine mucosa. It is an essential enzyme for the digestion of carbohydrates that breaks down carbohydrates of polysaccharides into monosaccharides. Starch, broken into meals, is broken down into oligosaccharides by amylase and broken down into glucose by α-glucosidase. α-glucosidase inhibitors regulate post-prandial blood sugar by binding to α-glucosidase instead of oligosaccharides, preventing the breakdown and absorption of monosaccharides into glucose. Currently, the α-glucosidase inhibitors marketed in Korea include acarbose (acarbose) and volibose (volibose).

In this example, the degree of activity inhibition was measured in vitro using p -nitrophenyl-α-D-glucopyranoside (PNP-G) as the substrate. 10 μL of each solvent extraction sample solution was added to 40 uL of α-glucosidase (Sigma, Mo, USA) of 1 unit / mL yeast source, and reacted at 37 ° C. for 10 minutes, followed by 1 mM PNP-G solution ( 950 uL of pH 6.9) was added in potassium phosphate buffer. After reaction at 37 ° C. for 20 minutes, 1 mL of 1 M Na ₂ CO ₃ was added to terminate the reaction, and the absorbance was measured at 405 nm. MeOH was used as a negative control and 1-deoxynorjirimycin was used as a positive control.

TABLE 1

Α-Glucosidase Inhibitory Activity of Bunchocho extract

 Values are the average of three experiments.

The degree of inhibition of the activity of the organic solvent extract of Burnacho was expressed as IC ₅₀ (mg / mL). Α-glucosidase inhibitory activity of HX fraction was determined to be the highest at 0.723 mg / mL, EA fraction was 1.356 mg / mL, and DCM fraction was inhibited to 1.536 mg / mL. Was slightly higher at 5.527 mg / mL. The 1-deoxynorzirimycin used as a positive control was 3.230 mg / mL, and it was determined that the separation and purification of HX fractions of Baecho was able to develop an excellent inhibitor of α-glucosidase activity.

Example 2-2 α-amylase Inhibitory Activity

Α-amylase from human saliva (Sigma, Mo, USA) and pig pancreas Inhibitory activity against α-amylase (Sigma, Mo, USA) was measured using starch as a substrate. 1 unit / mL of α-amylase and 1.0% starch were mixed and reacted at 20 ° C. for 3 minutes, and a DNS coloring reagent was added. Color development was carried out by heating at 80 ° C. and 100 ° C. for 15 minutes, and absorbance was measured at 540 nm after cooling. Acarbose (Sigma, Mo, USA) was used as a positive control.

TABLE 2

Α-amylase Inhibitory Activity of Bunchocho extract

TABLE 3

Inhibitory Activity of Hexane Fraction of Baechoencho and Acarbose on α-amylase

Values are the average of three experiments.

In human saliva-derived α-amylase inhibitory activity, HX fraction showed high activity of 71.73% at a concentration of 5 mg / mL, and DCM and EA fractions of 54.96% and 74.44% at a high concentration of 10 mg / mL, respectively. Activity appeared. In the pancreatic-derived pancreatic-derived α-amylase inhibitory activity, the DCM and EA fractions of the vinegar showed high inhibitory activity of 92.83% and 86.74% at 5 mg / mL, respectively, and the HX fraction also showed high inhibitory activity of 66.58% at the same concentration. Inhibitory activity was shown. In addition, the 50% inhibition was measured for the HX fraction and the acarbose used as the hypoglycemic agent. As a result, α-amylase derived from human saliva showed 2882.19 ug / mL and 163.81 ug / mL of acarbose. In the case of pancreas-derived α-amylase, HX fraction showed 2043.30 ug / mL and acarbose 150.04 ug / mL.

Example 2-3: Aldoz  Reductase inhibitory activity

Aldose reductase was measured using DL-glyceraldehyde as substrate. 10 uL of each sample prepared by concentration, 500 uL of 0.1 M SPB buffer, 0.1 M DL-glyceraldehyde, and 1.5 mM β-NADPH were added and pre-reacted at 25 ° C. for 3 minutes. 0.4 units of aldose reductase of human muscle cell origin were added and immediately measured for absorbance at 340 nm for 3 minutes.

TABLE 4

Aldose Reductase Inhibitory Activity of Bunchocho extract

Values are the average of three experiments.

An aldose reductase inhibitor is an enzyme inhibitor that inhibits aldose reductase, which converts glucose into sorbitol, and aims to reduce the production of sorbitol.

Many studies have shown that polyol metabolic abnormalities cause diabetic complications. As a result, the MeOH extract of Baechocho was 44.44% at 1 mg / mL concentration, and similar activity was observed in other fractions. In particular, the HX fraction was relatively high at 51.85%.

Example 3: Burnout  Separation of Functional Substances from Extracts

The Bunchocho extract prepared in Example 1 was separated for each solvent to obtain 70 g of MeOH extract, 14,35 g of HX fraction, 8.67 g of DCM fraction, and 6.99 g of EA fraction (see FIG. 1). Among them, the HX fractions were subjected to silica gel column chromatography with a solvent combination of HX / DCM = 100% HX? 100% DCM, and each fraction was subjected to fractional liquid chromatography (JAI, Japan). As an analytical column, a multi-column GS-310 (particle 13 μm) was used and separation was performed at 254 nm using a UV detector. 100% MeOH was used as the developing solvent, and the flow rate was adjusted to 5-7 mL / min according to the nature of each fraction. For the structural analysis of the separated material, high performance liquid chromatography (HPLC; CBM-20A, Shimadzu, Japan) and gas chromatography mass spectrometer (GC / MS; HP6890, Agilent, USA) were used, respectively, ODS-C18 column (250). 4.6 mm, 5 μm, Japan) and HP-5 column (Agilent, USA) were used.

Final structure was confirmed by 1H NMR, 13C-NMR, DEPT, HMBC. The result is stearic acid, methyl palmitate, methyl-4- (2-tridecylphenyl) butanoate, methyl-3-cyclopentyl-2,3-dimethylbutanoate and methyl-4-hydroxy benzoate 5 Materials were separated.

<Formula 1>

Stearic acid

<Formula 2>

Methyl palmitate

<Formula 3>

Methyl-4- (2-tridecylphenyl) butanoate

<Formula 4>

Methyl-3-cyclopentyl-2,3-dimethylbutanoate

<Formula 5>

Methyl-4-hydroxy benzoate

Example 4 Antidiabetic Effect of Separation Substances

The inhibitory activity against α-glucosidase was examined for the isolated material. Kinetic assayed inhibitory activity using various concentrations of PNPG as a substrate for different concentrations of compounds.

TABLE 5

Investigation of α-glucosidase and α-amylase inhibitory activity and α-glucosidase kinetic on the isolated substance showed no activity in stearic acid and methyl palmitate and methyl-4- (2-tridecylphenyl Butanoate and methyl-3-cyclopentyl-2,3-dimethylbutanoate showed high α-glucosidase inhibitory activity. In particular, methyl-3-cyclopentyl-2,3-dimethylbutanoate exhibited a high inhibition rate of 63.7% at 2.52 mM, thereby confirming anti-diabetic function, and burned vinegar was an edible plant with no cytotoxicity as a health food. Also showed the possibility.

Example 5: Burnout  Antidiabetic Effect of Extracts

In the case of diabetic hyperglycemia, pancreatic beta cells are damaged by autoimmune reactions or chemicals and cause abnormalities in insulin synthesis and secretion, resulting in insulin deficiency, and thus type 1 diabetes mellitus. In addition, the blood insulin concentration is normal or hyperinsulinemia, and can be classified as a type 2 diabetes in which hyperglycemia occurs due to abnormal glucose metabolism due to high insulin resistance in peripheral tissues.

The clinical symptoms of type 1 diabetes are mainly acute in young patients. Osmotic symptoms include: Daum, Dasik, polyuria, nocturnal enuresis, weight loss, fatigue and boredom. Additional symptoms include muscle spasms, sunken vision, filamentous fungal infections of the mouth and genital area, or skin bacterial infections. It was confirmed through an in vivo experiment using an insulin-dependent diabetic mouse model as an extract of the burnt vinegar (see FIGS. 3 and 4).

1. Preparation of experimental animals

In this example, 6 weeks old male ICR mice were supplied and used by Samtaco Bio Korea. The animal was bred in the environment of temperature 21 ± 2 ℃ and humidity 55 ± 10%, and the feed was used solid food for sterilization by experimental irradiation sterilized by Samtaco Bio Korea Co., Ltd. Feeding, pre-breeding was controlled by 12 hours each day and night to avoid possible stress.

2. Control

Streptozotocin (Sigma Co., USA) dissolved in citrate buffer (4 ° C, pH 4.2) in the abdominal cavity of mice fasted for at least 14 hours before the start of the experiment was administered at a level of 65 mg / kg body weight after 48 hours. Only mice with a blood glucose content of 200 mg / dL or more were selected to cause diabetes.

Blood glucose was measured by a blood glucose sensor (Roche Diagnostics GmbH., Germany) with blood collected from the tail vein at a certain time. The mice used in the experiment were placed in a polycarbonate breeding box (260 × 410 × 20 mm) in the experimental animal breeding room by arranging 5 animals per experiment group based on blood sugar and body weight.

3. Experimental group

As described above, streptozotocin (Sigma Co., USA) was administered at a level of 65 mg / kg body weight, and 48 hours later, only mice having a blood glucose content of at least 200 mg / dL were selected as having diabetes. Burnchocho MeOH extract was dissolved in PBS at 500 mg / kg body weight and orally administered 400 uL daily. Body weight and blood glucose were measured after fasting 12 hours every 2 days.

Example 6: HMG - CoA  Reductase enzyme activity measurement

HMG-CoA reductase inhibitory activity, a rate limiting enzyme in relation to cholesterol biosynthesis inhibitory activity, was measured using a kit (HMG-CoA Reductase Assay Kit; sigma, Mo, U.S.A). In other words, 184 μL of 1X assay buffer, 5 μL of extracted sample solution, 4 μL of NADPH, 12 μL of HMG-CoA, and 2 μL of HMGR were added to the 96 well plate, and the mixture was mixed well for 10 seconds, and then measured for 10 minutes at 340 nm using an ELISA meter. It was. The results are shown in Table 6 below.

TABLE 6

Example 7 Separation of Materials HMG - CoA  Reductase inhibitory effect

HMG-CoA reductase inhibitory effect on the separation material was measured in the same manner as in Example 6. The results are shown in Table 7 below.

<Table 7>

Excessive increase in blood cholesterol is known as a risk factor for coronary artery disease, and research on blood cholesterol elevation inhibitors has been conducted in various fields. Blood cholesterol levels are regulated in vivo by two processes, biosynthesis and degradation, which act as rate limiting enzymes during HMG-CoA reductase biosynthesis. When intracellular cholesterol content is increased, HMG-CoA reductase activity is inhibited by the mediation of SREBP. Therefore, it can be determined that the decrease in hepatic HMG-CoA reductase activity has an effect of lowering blood cholesterol concentration.

1 is a view schematically showing a burntweed extraction process according to the present invention.

Figure 2 shows (a) methyl-4- (2-tridecyl) butanoate (■, 13.87 mM; ◆, 1.387 mM; ▲, no extract) and (b) methyl-3-cyclopentyl-2,3- Lineweaver-Burk plot analysis for inhibition kinetics of α-glucosidase inhibition effect by dimethylbutanoate (■, 25.2 mM; ◆, 2.52 mM; ▲, no extract). .

3 and 4 is a diagram showing the anti-diabetic effect of Bunchocho extract according to the present invention.

Claims (6)

delete delete Anti-diabetic activity, characterized in that the methanol extract was dissolved in distilled water and then the fraction was continuously fractionated in the order of n-hexane (HX), dichloromethane (DCM) and ethyl acetate (EA). Buncho vinegar extract with blood cholesterol lowering activity. Antidiabetic activity and blood content containing methyl-4- (2-tridecylphenyl) butanoate or methyl-3-cyclopentyl-2,3-dimethylbutanoate compound isolated from the extract of Pat. 3 as an active ingredient Cholesterol-lowering active composition. delete Diabetes prevention and blood cholesterol which contain methyl-4- (2-tridecylphenyl) butanoate or methyl-3-cyclopentyl-2,3-dimethyl butanoate compound isolated from the extract of Pat. 3 Pharmaceutical composition for reducing the concentration.

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