KR100417287B1 - Composition for treating diabetes comprising a lower alcohol insoluble extract from hovenia dulcis thunberg. or a polysaccharide isolated therefrom - Google Patents

Abstract

PURPOSE: Provided is a therapeutic composition for diabetes which contains the lower alcohol insoluble extract fraction of Hovenia dulcis Thunberg or polysaccharides separated therefrom, as an active ingredient, and a pharmaceutically acceptable carrier. It is useful for the prevention and treatment of diabetes. CONSTITUTION: A therapeutic composition for diabetes is characterized by containing the lower alcohol insoluble extract fraction of Hovenia dulcis Thunberg or polysaccharide fractions separated therefrom and consisting of mannose, glucose, galactose, ramanose, arabinose, xylose. It further contains a pharmaceutically acceptable carrier. The lower alcohol is preferably methanol.

Description

COMPOSITION FOR TREATING DIABETES COMPRISING A LOWER ALCOHOL INSOLUBLE EXTRACT FROM HOVENIA DULCIS THUNBERG. OR A POLYSACCHARIDE ISOLATED THEREFROM}

Diabetes mellitus is a disease caused by the inability of insulin, a blood glucose control hormone, to function properly. Glucose produced by food metabolism does not move into cells and remains in the blood, resulting in high blood sugar levels.飮), multi urine (多 尿), multi-diet (多 食) three symptoms appear, accompanied by various complications. Therefore, lowering the glucose (blood sugar) content in the blood is an important factor in preventing various diseases caused by diabetic complications in diabetes.

Diabetes is the most common endocrine disorder, and the current number of patients in Korea is 2 million, representing 11% of the population aged over 30. Diabetes causes metabolic abnormalities in a variety of intracellular and external environments, including the regulation of carbohydrate metabolism, and these abnormalities cause lesions in many organs such as the visual, nervous, urinary, and pulmonary systems. Diabetes can be classified into two types. Insulin-dependent (type 1) diabetes is known to be caused by an autoimmune response to insulin-producing beta cells by genetic predisposition as an environmental factor. Insulin-independent diabetes mellitus (type 2) is not known for its etiology, but an increase in insulin resistance is known to be an important cause.

Insulin is the most ideal and effective way to treat diabetes, but its duration is short and it does not show oral action. Oral hypoglycemic agents used to date include sulfonyl-urea and biguanide-based drugs. Sulfonic acid-based drugs promote the secretion of insulin produced by the beta cells of the pancreas, thereby lowering blood glucose levels, but causing hepatotoxicity and hypoglycemia. In addition, biguanide-based drugs have side effects such as nausea, anorexia and diarrhea. Therefore, there are other mechanisms that can reduce these side effects. Development of oral diabetes treatments is required.

Diabetes is a difficult disease to treat and absolutely requires diet along with medication. In addition, the administration of insulin or oral hypoglycemic agents is limited in the fundamental treatment, and accompanied by the economic burden and the risk of side effects. Recently, attention has been paid to the hypoglycemic effect of wild edible plants for a long time as a folk medicine. There is an increasing amount of research in this area.

Lee, et al. (1988), J. Korean Soc. Food Nutr. 17, 57-61) showed that the elm extract showed hypoglycemic effect, and the ether-methanol extract of Schisandra chinensis was alloxan. It has been confirmed that diabetes mellitus has a potent hypoglycemic effect (Sheo HJ et al. (1987), J. Korean Soc. Food Nutr. 16, 262-267). In addition, it has been reported that Commelina communis L. and Yam ( Dioscorea japonica Thunb) have hypoglycemic effects in streptozotocin (STZ) -induced diabetic rats (Lim SJ and Kim MW (1992), Korean J. Nutr. 25, 511-517 ). In addition, buckwheat supplementation has been shown to reduce blood glucose and cholesterol levels in streptozotocin-induced diabetic rats (Lee JS (1994), Korean Journal of Nutrition , 27, 819-827). In addition, studies using polysaccharides isolated from natural products have been conducted. It has been reported to have activity (Tadashi K. et al. (1999), Biol. Pharm. Bull. 22: 966-970), acidic polysaccharides (TAP) and their degradation products (TAP-H) isolated from sour mushrooms Have been reported to reduce the anti-diabetic effects of diabetes mellitus, STZ-induced blood sugar, insulin, cholesterol, triglyceride and lipid peroxidation (Tadashi K. et al. (2001), Biol. Pharm. Bull 24: 1400-1403).

Therefore, the inventors of the present invention target low alcohol insoluble extract fraction of Hovenia dulcis Thunberg. And the polysaccharides isolated therefrom in the process of searching for plants that are expected to be useful for preventing and treating diabetes against various kinds of natural products. It was found to exhibit lowering activity.

Prior arts, including the above documents, have reported the effects of treating diabetes on various wild edible plant extracts, but there has been no report on the fact that the extract of the liana has a hypoglycemic effect.

Hovenia dulcis Thunb. Is a buckthorn and deciduous broad-leaved arbor tree that is distributed at 50-800 m above sea level in Gyeonggi and south of Gangwon. It is also distributed in Japan and China. In addition to the basic species Hovenia dulcis Thunb. (Korea, Japan, China; the flowers are light green, the leaves have no hair), the varieties Hovenia dulcis var. latifolia Nakai. (Japanese specialty Northeast; leaves are very large compared to the Hovenia dulcis), Hovenia dulcis var. koreana Nakai (specialty of Korea; the flower is white) and Hovenia dulcis var. Tomentella Makino (Japanese native sake, four countries; hair on leaves) is distributed.

It is an object of the present invention to provide a lower alcohol insoluble extract of foliar leaves useful for preventing and treating diabetes.

Another object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of diabetes, comprising a low alcohol insoluble extract of the larvae and an active polysaccharide fraction isolated therefrom.

Still another object of the present invention is to provide a dietary supplement for preventing diabetes comprising a low alcohol insoluble extract of the foliar leaves as an active ingredient and a food acceptable additive.

Figure 1 shows the process of separating the methanol insoluble extraction fraction and the polymer fraction from the bark wood part,

Figure 2 shows the process of separating the methanol insoluble extraction fraction and the polymer fraction from the bark young branches,

Figure 3 shows the process of separating the methanol insoluble extraction fraction and the polymer fraction from the fruit tree fruit trees,

Figure 4 shows the process of separating the methanol insoluble extract fraction and the polymer fraction from the leaves of the bark,

5 is RI and UV spectra of methanol insoluble fractions obtained from lumber wood,

FIG. 6 is RI and UV spectra of methanol insoluble fractions obtained from larvae spp.,

FIG. 7 is RI and UV spectra of methanol insoluble fractions obtained from a ragweed fruit disease,

Figure 8 shows the weight change over time of the mice induced diabetes with streptozotocin (STZ),

Figure 9 shows the feed consumption of the streptozotocin-induced diabetic rats over time,

10 shows the plasma glucose content in the rats of the diabetic-induced control group and the extract-administered group of the larvae,

Figure 11 shows the plasma protein content in the rats of the diabetic-induced control group and the extract-administered group according to the larvae,

Figure 12 shows the plasma albumin content in the rats of the diabetic-induced control group and the extract-administered group of the larvae,

Figure 13 shows the plasma urea content in the rats of the diabetic-induced control group and the extract-administered group according to the larvae.

In accordance with the above object, the present invention provides a lower alcohol insoluble extract obtained by adding a lower alcohol to the hot water or lower alcohol extract obtained by extracting the dry leaves of the bark (Hovenia dulcis Thunb.) By hot water pressurization or lower alcohol.

"Lower alcohol" as used herein refers to any alcohol selected from methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, normal propanol, iso-propanol, normal butanol, or mixtures thereof.

Heotgae tree of the present invention, Korean (Hovenia dulcis var. Korean Nakai. ), Japanese (Hovenia dulcis var. Tomentella Makino) and Chinese (Hovenia acerba Lindl., Hovenia acerba var. Kiukiangensis CY WU, Hovenia trichocarpa Chun et Tsiang, Hovenia trichocarpa var.fulvotomemtosa) Contains all of the holly.

The lower alcohol insoluble extract of the present invention comprises the steps of 1) obtaining pressurized hot water or lower alcohol extract from the leaves of the liana; And 2) adding a lower alcohol to the extract to obtain a lower alcohol insoluble extract.

In step 1), the leaves of the barn tree can be extracted according to the pressurized hot water or lower alcohol extraction method, for example, the pressurized hot water extraction method is about 1 to 5 times, preferably 3 times the volume of the barn tree leaves. Water is added, and the pressure corresponds to about 1 to 3 atmospheres, preferably 1.5 atmospheres, for about 15 to 48 hours, preferably 30 to 12 hours at about 110 to 150 ℃, preferably 120 to 125 ℃ After extraction, the mixture is left to stand at room temperature, cooled, and the filtrate obtained by filtration is lyophilized according to a conventional freeze-drying method to obtain a pressurized hydrothermal fraction extract. In addition, the lower alcohol extraction method is added to 2 to 10 times, preferably 3 to 7 times lower alcohol relative to the volume of the bark leaves and 5 to 3 minutes while heating to 20 to 80 ℃, preferably 25 to 50 ℃ For a period of time, preferably 10 to 1 hours, ultrasonic extraction, cooling and filtration, and concentrated under reduced pressure with a rotary evaporator to obtain a lower alcohol extract from the wood of the bark tree, wherein the filtration process is repeated two or more times for the remaining residue after filtration It can be carried out. As the lower alcohol as the extraction solvent, methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, normal propanol, iso-propanol, normal butanol, or a mixture thereof may be used, with 70 to 100% methanol being most preferred.

In step 2), the pressurized hot water or lower alcohol extract obtained from the above is dried or concentrated under reduced pressure at room temperature, and the concentrate is then lowered to 30 minutes to 3 hours, preferably 10 minutes to 1 hour with lower alcohol, preferably 100% methanol. During this, the reflux extract obtained by reflux extraction 3 to 4 times was centrifuged again to collect the lower alcohol insoluble fraction to obtain a lower alcohol insoluble fraction. Alternatively, the reflux extract is filtered and the supernatant and the precipitate are separated by adding 2-8 times, preferably about 4 times the volume of lower alcohol, preferably 100% ethanol, to the unfiltered insoluble fraction and distilled water to the precipitate. After redissolution by addition, it can be lyophilized to obtain a lower alcohol insoluble fraction.

Lower alcohol insoluble extracts of other parts of the bark tree may be prepared from woody parts, young branches and fruit diseases of the bark tree according to the same method as the method of preparing the leaf extract. It is preferable to use young branches of 1 to 4 years as young branches of the barn.

In addition, polysaccharide fractions may be prepared by a method comprising the step of separating the lower alcohol insoluble extract by ion exchange resin column chromatography.

For example, after dissolving the lower alcohol insoluble extract in distilled water, 0 to 5 M aqueous sodium chloride solution was added to the ion exchange resin column in concentration step by step, followed by dialysis, concentration, and lyophilization in turn to perform each concentration. To obtain a polymer fraction consisting of a polysaccharide component of the star. At this time, the ion exchange resin may be a cation exchange resin or an anion exchange resin, a preferred cation exchange resin strong acid cations such as AG 50W-x8, Amberlite IR-120, Doex 50W-x8 Exchange resin; Weakly acidic cation exchange resins such as Amberlite IRC-50, Bio-Rex 70, Duoolite-436 and the like; Weakly basic cation exchange resins such as amberlite IRA-67, Dow's 3-x4A and the like; Strongly basic cation exchange resins such as AG 2x8, Amberlite IRA-400, Dowks 2-x8 series, and the like; Or exchange resins consisting of improved fibrous celluloses such as cation exchange resins such as CM-cellulose, SE-cellulose, and anion exchange resins such as DEAE cellulose; Or made of crosslinked dextran, eg, G-25 and G-50 beaded cation Sephadex-type resins, eg agarose, eg Sepharose CL and Biogel A Sepharose-type resins Or improved bead type ion exchange resins, such as Fractogels or Toyopearl resins, more preferred resins are Toyopearl DEAE type exchange resins, which are weakly basic anion exchange resins, the most preferred being Pearl DEAE-650C type exchange resin.

The polysaccharide is separated from the lower alcohol insoluble extract of the wood of the bark wood through the ion exchange resin, depending on the concentration of sodium chloride, in the OM sodium chloride solution, the molecular weight of 5,750 consisting of mannose, glucose, galactose, ramanose, arabinose, xylose High molecular weight polysaccharides of 21,500 to 21,600 kDa consisting of mannose, glucose, galactose, ramanose, arabinose, xylose in the polymer polysaccharide of 0.1 to 5,760 kDa, sodium chloride, mannose, glucose in the 0.2 M sodium chloride solution , 285 to 286 kDa molecular weight polysaccharide consisting of ramanose, arabinose, xylose, 0.3 M sodium chloride solution, 62.7 to 62.8 kDa and 40.6 to 40.7 consisting of mannose, glucose, galactose, arabinose, xylose Polymeric polysaccharide of kDa, mannose in 3 M sodium chloride solution, gluco , Galactose, arabinose, a polymeric polysaccharide having a molecular weight of 37.7 to 37.8 kDa composed of xylene and elute rose.

Also, it is a polysaccharide isolated from the lower alcohol insoluble extracts of the bark sprigs, and in the O M sodium chloride solution, mannose, glucose, galactose. Arabinose, a molecular weight of 68.0 to 68.1 kDa consisting of xylose and 36.4 to 36.5 kDa of a polymeric polysaccharide, a molecular weight of 22 to 23 kDa consisting of mannose, glucose, lamanose, arabinose in a 0.1 M sodium chloride solution; 233 to 234 kDa; And a polymeric polysaccharide of 200 to 201 kDa, a molecular polysaccharide having a molecular weight of 32.8 to 32.9 kDa consisting of mannose, ramanose, arabinose and xylose in a 0.2 M sodium chloride solution, mannose, glucose and arabinose in a 0.3 M sodium chloride solution. The molecular weight consisting of 88.6 to 88.7 kDa and 79.1 to 79.2 kDa of the polymer polysaccharide is eluted, and in the 3 M sodium chloride solution, the polymer polysaccharide of 216 to 217 kDa molecular weight consisting of mannose, galactose, arabinose, xylose elutes do.

A polysaccharide isolated from the lower alcohol insoluble extracts of the liana, a high molecular weight polysaccharide of 1,330 to 1,340 kDa consisting of mannose, glucose, galactose, arabinose, and xylose in 0 M sodium chloride solution, and only in 0.1 M sodium chloride solution. Polymeric polysaccharide of molecular weight 142 to 143 kDa consisting of nose, glucose, galactose, ramanose, arabinose and xylose, molecular weight consisting of mannose, glucose, galactose, lamanose and arabinose in a 0.2 M sodium chloride solution Polymeric polysaccharide of 70.6 kDa, polymeric polysaccharide of molecular weight 102-103 kDa consisting of mannose, glucose, galactose, lamanose, arabinose, xylose in 0.3 M sodium chloride solution, mannose, glucose, in 3 M sodium chloride solution A high molecular weight polysaccharide of 5.0 to 5.5 kDa composed of arabinose is eluted.

However, polysaccharides isolated from the lower alcohol extracts of the bark tree in accordance with the object of the present invention include all polysaccharides having a molecular weight of 20 kDa or more, as well as the polysaccharides eluted according to the various concentrations of sodium chloride described above.

Lower alcohol insoluble extracts obtained from the woody parts, young branches, fruits and leaves of the larvae, and the active polysaccharide fractions isolated from them according to the method, lower blood sugar levels and increase the plasma protein and albumin content in normal diabetic animals. It keeps close, and decreases the plasma urea content that increases with diabetes, it can be useful for the prevention and treatment of diabetes.

Accordingly, the present invention is for the prevention and treatment of diabetes mellitus, including a low alcohol insoluble extract of the wood, young branches, fruit and leaves of the larvae, and a polysaccharide fraction isolated therefrom as an active ingredient and a pharmaceutically acceptable carrier. It provides a pharmaceutical composition. In addition, the low alcohol insoluble extract of the leaves of the bark tree as an active ingredient and provides a dietary supplement for preventing diabetes comprising a food-acceptable additive.

The pharmaceutical composition of the present invention may further include excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc. which are commonly used, tablets, capsules, powders, granules, suspensions, emulsifiers, It may be formulated in unit dosage form or in multiple dosage pharmaceutical formulations, such as syrups, solutions or parenteral preparations.

The lower alcohol insoluble extract of the present invention or the pharmaceutical composition containing the polysaccharide fraction isolated therefrom may be parenterally or orally administered according to the desired method, and the extract of the hawthorn tree as an active ingredient per day is 0.001 per kg of body weight To 0.2 g Preferably, the amount of 0.01 to 0.05 g can be administered in one to several times. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, and degree of disease.

Lower alcohol insoluble extracts of the larvae leaves may be added to food or beverages for the purpose of preventing and treating diabetes. At this time, the lower alcohol insoluble extract is generally added to the health food composition 0.1 to 15% by weight, preferably 1 to 10% by weight of the total food weight, 1 to 30 based on 100 ml in the health beverage composition g, preferably 3 to 10 g.

The dietary supplement of the present invention does not have any particular limitation on the liquid component except for containing the lower alcohol insoluble extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colorants and neutralizers (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Examples of foods to which the lower alcohol insoluble extract and polysaccharide fraction of the present invention may be added for the development of dietary supplements include various foods, beverages, gums, vitamin complexes, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Example 1 Preparation of Lower Alcohol Insoluble Extracts of Each Part of Bare Tree

5 liters of distilled water was added to 1 kg each of woody parts, young branches, fruit and leaves of the dry bark tree, and collected from the National Arboretum, Sohol-myeon, Pocheon-gun, Gyeonggi-do, Korea. Pressurized hot water extraction was carried out with precision pore HD5-A 190 model (Korea). The extract obtained by filtration of the extract with Whatman No. 2 (USA) was dried with a freeze dryer (three-way cold-heated SHDSM24L model, Korea), followed by hydrothermal extracts of bark wood, young branches, fruits and leaves (23.4g, respectively). 12.2 g, 145.7 g and 158.3 g) were prepared (see FIGS. 1-4). Subsequently, the pressurized hot water extract obtained above was dried or concentrated under reduced pressure at room temperature, and the reflux extract obtained by refluxing the concentrate with 100% methanol for 3 to 4 times for 1 hour was again centrifuged (5,000 rpm, 15 minutes) The alcohol insoluble fractions were collected to obtain a lower alcohol insoluble fraction.

Example 2 Characterization of Materials in Lower Alcohol Insoluble Fractions

(1) Purification and Separation of Polymer Fractions by Ion Exchange Column Chromatography

Ion-exchange resin column chromatography was carried out with methanol insoluble fractions of the lumber, young branches, fruit and leaves obtained in Example 1. 1 g of methanol insoluble fraction was dissolved in 100 ml of purified water at room temperature for 30 minutes using a stirrer. Toyo pearl (Toyo pearl ^R , Tosoh, Japan) 160 ml of DEAE-650C ion exchange resin equilibrated with demineralized distilled water was added to a 254 nm UV meter (product name, company) and RI meter (RI monitor: RI-10 model, A liquid chromatography column (35 cm long, 2.5 cm diameter, Glass Econo-column, BIO-RAD Loveborito) of a low pressure liquid chromatography system (Biologic LP, BIO-RAD Rubberatorie, USA) with EYELA, Japan Lissa, USA) and equilibrated with 500 ml of demineralized distilled water again. 100 ml of methanol insoluble fraction dissolved in demineralized distilled water was added to the DEAE-650C resin in the column and flowed to adsorb. Here, 0 M, 0.1 M, 0.2 M, 0.3 M, and 3 M NaCl solutions were eluted by a stepwise elution method in which 500 ml were flowed at an elution rate of 2 ml / hr.

As a result, the chromatogram of the methanol insoluble fraction of the elder tree was eluted with UV absorbing substances in the 0.1 M fraction, as shown in FIG. 5, and most of the eluted wood was eluted after the 0.3 M fraction. They began to elute after the 0.3 M fraction. Chromatograms of the methanol insoluble fraction of the hawthorn tree branch were eluted with UV absorbing materials in all the eluting fractions as shown in FIG. 6, and the materials absorbing RI began to elute after the 0.3M fraction. The chromatogram of the ethanol insoluble fraction of the ferns was eluted with UV absorbing material after 0.1 M fraction as shown in FIG. 7, and the material absorbing RI began to elute after 0.3 M fraction. Dialysis membranes with an average molecular weight cut-off of 1,200 Da (average flat thickness 32 mm, volume 100 ml / ft benzoyl) to remove sodium chloride contained in the elution fractions of the bark wood, young branches, fruit and leaves. Dialysis tubing, Sigma, USA) was dialyzed three times with demineralized distilled water for 4 hours and then lyophilized with a freeze dryer (Samwon cold-heat SFDSM24L, South Korea) to remove methanol insoluble in the woody parts of spruce, young branches, fruit and leaves. Polysaccharide fractions of the polymer were obtained in the fraction.

(2) Absolute molecular weight measurement by SEC-HPLC-MALLS

TSK gel 3000pw, 4000pw, 5000pw (7.8 × 300mm, Tosoh) and MALLS as pump (Spectra system p2000), guard column (TSK PWH, Tosoh), RI-detector (Shodex SE71), SEC (Size Exclusion Chromatography) column (Polygonal Laser Scattering, DawnDSP-F, Wyatt Technology) A device consisting of a detector and a developing solvent, GPC was carried out by flowing a solution made to 0.15 M NaNO ₃ in 0.02% sodium azide at a flow rate of 0.5 ml / min. Absolute molecular weight was measured by this.

As a result, as shown in Table 1, the peaks of the wood-insoluble methanol insoluble fraction are mainly five, fraction 1 has a molecular weight of 5,758,000, fraction 2 has a molecular weight of 21,520,000, fraction 3 has a molecular weight of 285,000, and fraction 4 has a molecular weight of 40,620 and 62,720 (average). Molecular weight: 51,670) and fraction 5 had a molecular weight of 37,770.

Average Molecular Weight of Ion Exchange Resin Chromatography Fractions from Wood by SEC-HPLC-MALLS NaCl concentration 0 M (fraction 1) 0.1 M (fraction 2) 0.2 M (fraction 3) 0.3 M (fraction 4) 3 M (fraction 5) Average molecular weight (Da) 5,758,000 21,520,000 285,300 51,670 37,770

In addition, as shown in Table 2, there are mainly 9 peaks of methanol insoluble fractions, and fraction 1 has 68,040 and 36,440 molecular weight (52,240 average molecular weight), fraction 2 has molecular weight of 22,500, 233,300, 200,700 (average molecular weight: 228,250), and fraction 3 The molecular weight was 32,840, fraction 4 was found to be 88,610 and 79,190 (average molecular weight: 83,900) and fraction 5 was 216,500.

Average Molecular Weight of Ion Exchange Resin Chromatography Fractions of Young Branches by SEC-HPLC-MALLS NaCl concentration 0 M (fraction 1) 0.1 M (fraction 2) 0.2 M (fraction 3) 0.3 M (fraction 4) 3 M (fraction 5) Average molecular weight (Da) 52,240 228,250 32,840 83,900 216,500

As shown in Table 3, the peaks of the most insoluble methanol insoluble fraction were four, and fraction 1 was 1,330,000, fraction 2 was 142,800, fraction 3 was 70,540, fraction 4 was 102,400, and fraction 5 was 5,300.

Molecular Weight for Each Peak of Methanol Insoluble Fraction NaCl concentration 0 M (fraction 1) 0.1 M (fraction 2) 0.2 M (fraction 3) 0.3 M (fraction 4) 3 M (fraction 5) Average molecular weight (Da) 1,330,000 142,800 70,540 102,400 5,300

From the above results, it was proved that the fractions eluted by ion exchange chromatography were polymeric substances.

(3) analysis per composition

For analysis and confirmation of the composition of each fraction, the Hakomori method [Hakomori, S, J. Biochem. Tokyo , 55 , p205-209. 1964) and the Wege method [Waeghe, T. J et al., Carbohydr. Res. , 123 , p281-304. (1983) methylation analysis. That is, after methylation of the sample (500 µg), the methylated sample was collected by a Sep-Pak C ₁₈ cartridge (Supelco, USA) in which ethanol was adsorbed. Acidic sugars in methylated polysaccharides are reduced to LiB (C ₂ H ₅ ) ₃ D (super-dupride, 1 mL, room temperature, 1 hour, Aldrich) dissolved in tetrahydrofuran (THF) Recovered with a Pack C ₁₈ cartridge. This was hydrolyzed with 1.0 M trifluoroacetic acid (TFA) at 121 ° C. for 2 hours and then reduced to NaBD ₄ to carry out an acetylation reaction. Partially methylated alditol acetate was analyzed by GC. Methylated alditol acetates were identified by ion fragmentation and relative elution time, and the peaks were determined by the peak area and reaction factor of the flame ionization detector (FID).

In addition, GC analysis for the compositional analysis of each polysaccharide fraction was carried out using a Varian CP-3800 gas chromatography device (Varian Co. USA) under the following conditions:

Detector: FID

Column: 0.25 mm ID Fused Silica Capillary GC Column (30 cm × 0.25 mm × 0.2 μm; Supelco SP-2380)

Column temperature: 230 ° C, injector temperature: 250 ° C, detector temperature: 250 ° C

Mobile phase: helium (1.0 mL / min).

As a result of the experiment, the composition (mannose 1 basis) of each fraction separated from the methanol insoluble fraction of the bark, young branches and fruit of the larvae was as shown in Tables 4 to 6, respectively.

Composition Per Composition of Methanol Insoluble Fraction of Wood Mannose Glucose Galactose Ramanoz Arabinose Xylose Fraction 1 One 3 6.1 0.21 4.58 3.56 Fraction 2 One 1.29 5.04 1.32 5.93 2.26 Fraction 3 One 7.54 - 8.76 3.23 0.31 Fraction 4 One 6.14 2.21 - 3.46 2.62 Fraction 5 One 4.79 2.66 - 1.93 0.34

Compositional Composition of Young Methanol Insoluble Fraction Mannose Glucose Galactose Ramanoz Arabinose Xylose Fraction 1 One 3.14 0.88 - 0.57 0.57 Fraction 2 One 0.92 - 1.4 2.14 - Fraction 3 One - - 0.69 1.23 0.61 Fraction 4 One 0.31 - - 2.9 - Fraction 5 One - 6.84 - 1.5 1.45

Composition Per Composition of Fruit Methanol Insoluble Fraction Mannose Glucose Galactose Ramanoz Arabinose Xylose Fraction 1 One 6 3.1 - 1.58 - Fraction 2 One 3.29 2.04 0.32 1.93 0.26 Fraction 3 One 2.51 12.53 187 13.43 - Fraction 4 One 1.13 7.81 3.64 5.86 0.75 Fraction 5 One 3.95 - - 1.93 -

Example 3: Diabetes Inhibitory Effect of Low Alcohol Insoluble Extracts

(Step 1) Raising Laboratory Animals and Inducing Diabetes

Streptozotocin (STZ) is a compound that selectively produces cytotoxicity against beta-cells in the pancreas, and studies have been conducted on hypoglycemic effects using STZ-induced diabetes as a diabetic model (Konno C). et al., Planta medica 51: 162-163 (1985); Hikino H. et al., Planta Medica 54: 1-3 (1988)).

Freshly weaned male Sprague-Dawley rats were fed with free feed and water for 2 to 3 weeks and then weighed 200 to 300 g per 10 rats per group in the normal control, diabetes-induced control, Example 1 The lower alcohol insoluble extracts prepared for each part of the barn wood prepared (neck, young branches, fruit and leaves) were divided into six groups of test groups to be treated. Diabetes-induced control and test groups of rats were induced by intraperitoneal injection of STZ dissolved in 5 mM citrate buffer (pH 4.5) at a dose of 60 mg / kg body weight. Normal controls received only the same volume of citrate buffer. In order to determine the presence of diabetic animals from the day after STZ administration using a test stick glucoside (Glucotest stick, gel Pharmaceuticals (share), Uriscan ^TM) for diabetic counties it had nyodang inspection. One week after the STZ administration, rats in the test group were dissolved in water at a concentration of 0.5% instead of water, and then administered 100 ml per horse for 10 days.

(Step 2) Check the antidiabetic effect

(2-1) Analysis of body weight and feed intake

The amount of feed consumed by the test animals was measured at a certain time every day, and when the first day of administration of the low alcohol insoluble extract for each part of the bark tree was 0 days, the weight was measured with an animal balance at a certain time every day for 8 days. The effects of low alcohol insoluble extract on the body weight and feed intake of diabetic rats were compared.

As a result, in the case of body weight, as shown in FIG. 8, the normal control group increased 43.3% compared to the original body weight, and the diabetes-induced control group decreased 13.9% on day 8, and lower alcohols of woody parts, young branches, fruits and leaves of larvae The test groups administered with each insoluble extract showed a slight decrease, but showed no significant difference. The reason that the weight loss was not shown in the test group administered with the bark extract is thought to be that the symptoms of diabetes are lighter.

On the other hand, in the feed intake, the diabetes-induced control group consumed about 13.8% more of the feed than the normal control group, but there was no significant difference between the control group and the test group administered with the extract of the bark tree (FIG. 9).

As a result, the diabetic control group showed a typical phenomenon of diabetes, which consumed a lot of feed and reduced weight, whereas all the test groups administered with extracts of the locusts maintained their normal weight, indicating that the symptoms of diabetes improved. have.

(2-3) Blood Chemistry Analysis

(1) Collection of Analytical Samples

After 10 days of administration of STZ-induced diabetic rats with low alcohol insoluble extracts, the blood was drawn from the heart with a capillary tube containing heparin under ether anesthesia, and then centrifuged at 4 ° C and 2,000 rpm for 15 minutes. Separately, only plasma was taken and placed in a cap tube and stored frozen at -20 ° C until analysis.

(2) analysis

To determine the effect of lower alcoholic extracts of bark wood, young branches, fruits and leaves on diabetes by changing the carbohydrate, lipid and protein metabolism caused by diabetes, thawed frozen plasma and glucose and protein in plasma , Albumin and urea (BUN) were measured using a blood biochemical analyzer (Chiron / Diagnostics Express Plus (USA)). The results are as follows.

Plasma glucose

As can be seen in Figure 10, the diabetes-induced control group showed a higher plasma glucose level of 169% compared to the normal control group. In addition, the test group administered low alcohol insoluble extracts of woody, young branches, fruits and leaves of the larvae showed higher levels of 70.8%, 36.7%, 125% and 67%, respectively, than the control group. Were 36.6%, 49.3%, 16.4% and 38.0%, respectively. The hypoglycemic effect was observed in the extract-administered groups of all the larvae, and it is estimated that components exhibiting the hypoglycemic effect existed in each site of the larvae. STZ causes hyperglycemia by impairing the rapid insulin secretion response to glucose in the early stages rather than causing absolute insulin deficiency, particularly in β-cells. Therefore, such a significant hypoglycemic effect may be due to the improvement of insulin sensitivity to glucose or the impaired function of β-cells rapidly by administration of the extracts of the locusts.

Plasma Proteins and Albumin

Plasma protein and albumin concentrations were significantly increased in the test group administered the low alcohol insoluble extract of each part of the bark tree compared to the diabetes-induced control group (FIGS. 11 and 12). This result means that the lower alcohol insoluble extract of each part of the bark tree promotes the production of albumin and protein necessary for physiological physiological activity, which is decreased during diabetes-induced diabetes.

Urea Content

As can be seen in Figure 13, plasma urea concentrations were 122.3%, 45.35%, 68.3%, 75.5%, and 23.5 in the diabetic-induced control group and the rat bark, young branches, fruit and leaf extract administration test groups, respectively, compared to the normal control group. However, in the test administration group of the extracts of the larvae, the percentage of urea increased by 35.3%, 24.3%, 21.0% and 44.5%, respectively, compared to the diabetes-induced control group. It was confirmed to decrease by. From this result, it can be seen that the liana extracts resolve the symptoms of increased urea caused by diabetes.

The pharmaceutical composition of the present invention comprising a lower alcohol insoluble extract and / or a polysaccharide fraction isolated therefrom as a active ingredient and a pharmaceutically acceptable carrier, and a lower alcohol insoluble extract as a active ingredient The dietary supplement of the present invention comprising an acceptable additive as is very useful for the prevention and treatment of diabetes.

Claims (16)

A pharmaceutical composition for preventing and treating diabetes, comprising a polysaccharide selected from the group consisting of the following polysaccharides isolated from methanol insoluble extract of Hovenia dulcis Thunb., And comprising a pharmaceutically acceptable carrier: A polysaccharide having a molecular weight of 5,750 to 5,760 kDa, isolated from methanol insoluble extracts of the bark wood, and consisting of mannose, glucose, galactose, ramanose, arabinose, xylose, A polysaccharide having a molecular weight of 21,500 to 21,600 kDa, isolated from methanol insoluble extracts of the bark wood, and consisting of mannose, glucose, galactose, ramanose, arabinose, xylose, A polysaccharide having a molecular weight of 285 to 286 kDa, isolated from the methanol insoluble extract of the lumber wood, and consisting of mannose, glucose, ramanose, arabinose, xylose, A polysaccharide of 62.7-62.8 kDa or 40.6-40.7 kDa, molecular weight 62.7-62.8 kDa, isolated from methanol insoluble extracts of the bark wood, and composed of mannose, glucose, galactose, arabinose, xylose, A polysaccharide having a molecular weight of 37.7 to 37.8 kDa, isolated from the methanol insoluble extract of the lumber wood, and consisting of mannose, glucose, galactose, arabinose, xylose, Mannose, glucose, galactose, isolated from methanol insoluble extracts of eggplants 1-4 years old. Arabinose, a polysaccharide having a molecular weight of 68.0 to 68.1 kDa or 36.4 to 36.5 kDa consisting of xylose, A polysaccharide of 22-23 kDa, 233-234 kDa, or 200-201 kDa, isolated from methanol insoluble extracts of 1-4 years old branches, consisting of mannose, glucose, lamanose, arabinose, A polysaccharide having a molecular weight of 32.8 to 32.9 kDa, isolated from methanol insoluble extracts of eggplants of 1 to 4 years old, consisting of mannose, ramanose, arabinose, xylose, Polysaccharide isolated from methanol insoluble extracts of eggplants of 1 to 4 years old, molecular weight 88.6 to 88.7 kDa or 79.1 to 79.2 kDa, consisting of mannose, glucose, arabinose, A polysaccharide having a molecular weight of 216 to 217 kDa, isolated from methanol insoluble extract of eggplants 1-4 years old, consisting of mannose, galactose, arabinose, xylose, A polysaccharide having a molecular weight of 1,330 to 1,340 kDa, isolated from methanol insoluble extracts of the liana, and consisting of mannose, glucose, galactose, arabinose, xylose, A polysaccharide having a molecular weight of 142 to 143 kDa, isolated from the methanol insoluble extract of the liana fruit tree, consisting of mannose, glucose, galactose, ramanose, arabinose, xylose, A polysaccharide having a molecular weight of 70.5 to 70.6 kDa, isolated from methanol insoluble extracts of the liana, and consisting of mannose, glucose, galactose, ramanose, arabinose, A polysaccharide having a molecular weight of 102 to 103 kDa, isolated from methanol insoluble extracts of the liana, and consisting of mannose, glucose, galactose, ramanose, arabinose, xylose, A polysaccharide having a molecular weight of 5.0 to 5.5 kDa, isolated from methanol insoluble extracts of the liana fruit tree, consisting of mannose, glucose and arabinose. The method of claim 1, Hovenia dulcis var.Koreana Nakai, Hovenia dulcis var. Tomentella Makino and Hovenia acerba Lindl., Hovenia acerba var. Kiukiangensis CY WU, Hovenia trichocarpa Chun et Tsiang, Hovenia trichocarpa Chun et Tsiang var. fulvotomemtosa). The method of claim 1, The polysaccharide is 1) extracting pressurized hot water or methanol with a woody part, 1-4 year-old eggplant or fruit of the bark; 2) adding methanol to the extract to obtain a methanol insoluble extract; And 3) A composition comprising the step of separating the methanol insoluble extract by ion exchange resin chromatography using 0-5 M sodium chloride eluate. The method of claim 3, wherein Pressurized hot water extraction method of step 1) is added 1 to 5 times the volume of water to the wood parts, 1-4 year-old branches or fruit disease of the dry bark tree, and 15 minutes to 48 ℃ at 110-150 ℃ at a pressure of 1-3 atmospheres Composition for extracting for time. The method of claim 3, wherein The methanol extraction method of step 1) is characterized by extracting for 10 minutes to 1 hour while adding 3 to 7 times the volume of methanol to the wood parts, 1-4 year old branches or fruit bottles of the dry bark; Composition. delete delete delete delete delete delete delete delete delete delete delete