KR101867516B1 - A novel compound isolated from Uncaria gambir and pharmaceutical composition forα-glucosidase inhibitory activity - Google Patents

Abstract

The present invention relates to a novel compound having Uncaria gambir-derived alpha-glucosidase inhibitory activities, and a pharmaceutical composition containing the same. According to the present invention, the novel compound has stronger inhibitory activities than existing alpha-glucosidase inhibitors while minimizing side effects. To this end, the phenol resorcinol derivative compound is represented by chemical formula 1.

Description

[0001] The present invention relates to a novel compound having an alpha-glucosidase inhibitory activity and to a pharmaceutical composition comprising the novel compound isolated from a non-glucosidase inhibitory activity.

The present invention relates to a novel compound having an alpha-glucosidase inhibitory activity and to a pharmaceutical composition comprising the same.

Diabetes is caused by insulin secretion problems such as decreased insulin secretion and resistance due to genetic and environmental reasons, or abnormal function of insulin, glucose in the blood can not be transferred to / stored in the cells and excessively increased in the blood, It is a serious metabolic disease with hyperglycemia which is much higher than normal people.

According to the International Diabetes Federation (IDF), the number of people with diabetes worldwide has increased from about 30 million people in 1985 to about 285 million people in 2010, and about 7 million new people will develop diabetes each year, Predicts explosive growth to about 438 million people, about 10% of all adults.

In Korea, the number has increased from about 3 million in 2007 to about 3.51 million in 2010, and the current trend is estimated to increase to about 5.4 million by 2030. Diabetes mellitus is considered the fourth most common cause of death in Korea, including complications. The increase in the number of people with diabetes is expected to be due to multiple factors such as westernization of eating habits, lack of exercise, and longevity as economic growth and income increase.

Diabetes is largely classified as type 1 diabetes (type 1 diabetes) or type 2 diabetes (type 2 diabetes).

Type 1 diabetes accounts for about 10% of total diabetes mellitus and occurs mainly in children and is caused by insulin secretion due to destructive lesions of pancreatic β-cells. Therefore, people with type 1 diabetes should be given insulin injections throughout their lifetime, and dietary controls and regular blood tests should be performed to ensure that adequate blood glucose is maintained. The cause of type 1 diabetes is known to be due to congenital factors and certain infections.

Type 2 diabetes is known to be caused by lifestyle and genetic influences, accounting for the majority of diabetic patients in Korea. Unlike type 1 diabetes, which occurs predominantly in children, it occurs mainly in adults, due to insulin resistance that does not secrete enough insulin to function properly or cells do not respond to insulin.

Existing diabetes drugs include insulin preparations, sulphone urea drugs, thiazolidinedione (TZD) drugs, biguanide drugs, α-glucosidase inhibitors, miglitinides, Incretin mimetics and DPP-IV inhibitors .

In diabetic patients, diabetic patients usually develop severe complications due to rapid rise in blood sugar levels.

α-Glucosidase inhibitor reversibly inhibits the disaccharide degradation enzyme present in the brush border of the small intestine, thereby delaying the absorption of carbohydrates in the intestine, reducing postprandial blood glucose, and improving insulin secretion delay due to hyperglycemia of non-insulin dependent diabetes mellitus .

In particular, the mechanism of α-glucosidase inhibitory action inhibits digestion and absorption of carbohydrates in the small intestine without going through insulin secretion, thereby minimizing side effects such as hypoglycemic phenomenon, hepatic toxicity, and decreased beta cell function It has the advantage of being.

However, existing products such as acarbose, voglibose, and miglitol have over-suppression of α-amylase, which is a digestive enzyme, resulting in severe side effects such as gastrointestinal disturbances such as abdominal distension and diarrhea, and hepatotoxicity.

Existing orally used diabetes medicines currently used in clinical practice may cause various side effects such as hypoglycemia, diarrhea, abdominal bloating, weight gain, lactic acid blood, cardiac toxicity, liver toxicity, In addition, the beta cells in the pancreas that are insulin secretion function are irreversibly damaged / destroyed and eventually become insulin resistant.

Therefore, various studies are being conducted to find a substitute substance having an excellent activity for inhibiting? -Glucosidase while minimizing adverse effects from natural products.

In this regard, Korean Patent No. 10-1234582 discloses a composition containing Pediococcus sp. JNU534 strain or culture thereof having alpha-glucosidase inhibitory activity, Korean Patent Registration No. 10-0468429 Korean Patent No. 10-0448447 discloses a composition containing a grape seed extract having an alpha-glucosidase inhibitory activity and a method for producing the same. .

Uncaria gambir Roxburgh is a vine plant native to Rubiaceae native to the tropics of Southeast Asia, Africa and South America, and has been widely used as a medicinal herb, hemostatic agent, and anti-ulcer agent.

Aracholine, an alkaloid, is known to have anti-inflammatory properties. Epicatechin has physiological functions such as cholesterol lowering, hypoglycemia, detoxification and aging inhibition. Polyphenols are cancer cells Inhibited the growth of the cells. However, there has been no report on the activity of inhibiting the α-glucosidase.

As a part of the development of an α-glucosidase inhibitor derived from a natural material, the present inventor has confirmed α-glucosidase inhibitory activity from various medicinal plants, discovered a novel prenyl resorcinol derivative compound from the herbal extract and fractions thereof, Glucosidase inhibitor of the present invention. The present invention has been completed based on this finding.

Korean Patent No. 10-1234582 (composition for inhibiting alpha glucosidase activity) Korean Patent No. 10-0468429 (pine extract with alpha-glucosidase inhibiting activity and its extraction method) Korean Patent No. 10-0448447 (composition containing grape seed extract having inhibitory activity of alpha glucosidase, preparation method and use thereof)

It is an object of the present invention to solve the above problems and to provide a novel compound having an α-glucosidase-inhibiting activity and a pharmaceutical composition containing the compound.

In order to solve the above-mentioned problems, the present invention provides a novel prenyl resorcinol derivative compound of the following formula (I) having an alpha-glucosidase inhibitory activity derived from an acaricide.

[Chemical Formula 1]

The compound is characterized by having an IC ₅₀ value of 15.5 ± 0.7 μM for α-glucosidase inhibitory activity.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for inhibiting alpha-glucosidase, which comprises an extract of Uncaria gambir or a fraction thereof as an active ingredient.

The acyl halide fraction is characterized by containing a novel prenyl resorcinol derivative compound of formula (1).

The ginseng fractions are mauritianin, quercetin 3- (2,6-rhamnosylgalactopyranoside), procyanidin B3, (+) - epicatechin, (+) - catechin, 5- (3 ', 4'-dihydroxyphenyl) -γ-valerolactone, protocatechuic acid Or a salt thereof.

The ginseng extract has an IC ₅₀ value of 30.3 ± 2.7 μM with respect to the α-glucosidase inhibitory activity.

The ginseng extract of the present invention may contain water or an organic solvent extract, preferably an organic solvent extract, a crude extract or a concentrate thereof.

The organic solvent is preferably at least one selected from the group consisting of lower alcohols having 1 to 4 carbon atoms, hexane, ethyl acetate, dichloromethane, ether, chloroform and acetone.

The lower alcohol having 1 to 4 carbon atoms is preferably at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, and n-butanol, and the lower alcohol having 1 to 4 carbon atoms includes anhydride or hydrated alcohol .

The alcohol, such as ethanol or isopropanol, preferably contains 1 to 100% (v / v), preferably 30 to 100%, more preferably 70 to 100%, still more preferably 90%, 95%, 100 % Alcohol. When butanol is used as an extraction solvent, it is preferable to use water-saturated butanol (75 to 85% aqueous solution of butanol).

In addition, the acaricidal fraction of the present invention can be obtained by concentrating a crude acaricidal crude extract and then fractionating it with a lower alcohol having 1 to 4 carbon atoms, an aqueous solution thereof or a non-polar solvent separately (or a combination thereof) .

The lower alcohol having 1 to 4 carbon atoms is preferably selected from the group consisting of ethanol, methanol, propanol, isopropanol, butanol and n-butanol. The non-polar solvent may be selected from the group consisting of hexane, ethyl acetate, ether, dichloromethane, chloroform and butanol, preferably n-hexane, ethyl acetate and n-butanol saturated with water. The fractions may be water fractions other than the respective organic solvent layers.

The extract of the present invention may include not only extracts or fractions obtained by the above-mentioned extraction solvent but also extracts obtained through ordinary purification processes. For example, various additional purification methods, such as separation using an ultrafiltration membrane with a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity) The fractions obtained through this step are also included in the fractions of the present invention.

The extract or fraction of the present invention can be prepared in powder form by an additional process such as vacuum distillation and freeze-drying or spray-drying.

The composition of the present invention may be prepared into a pharmaceutical composition through a conventional method and administered to a human body, and the following specific examples are illustrative, but the present invention is not limited thereto.

The pharmaceutical composition of the present invention may be prepared by using physiologically acceptable adjuvants. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants, have.

In addition, one or more pharmaceutically acceptable carriers may be optionally formulated into pharmaceutical compositions.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.

Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added.

In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The dosage of the pharmaceutical composition according to the present invention should be a pharmaceutically effective amount. A " pharmaceutically effective amount " means an amount sufficient to prevent or treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level will vary depending on factors such as the formulation method, the condition and weight of the patient, The severity of the disease, the mode of administration, the route and time of administration, the rate of excretion, the responsiveness, and the like. Effective amounts may vary depending on the route of treatment, the use of excipients, and the likelihood of use with other agents, as will be appreciated by those skilled in the art.

In another aspect of the present invention, there is provided a composition for treating type 2 diabetes and an alpha glucosidase inhibiting food comprising an ash extract or fraction as an active ingredient.

Since the food composition may be a functional food according to the purpose of the present invention, the present invention provides a composition for treating type 2 diabetes and an alpha glucosidase inhibitory food, wherein the food is a health functional food.

The functional food is a food emphasizing the biological control function of food, and is added with added value so as to function and manifest to a specific purpose using physical, biochemical and biotechnological methods. These functional food ingredients are designed and manufactured so that the body control functions related to the regulation of the body defense and the body rhythm, the prevention and the recovery of the disease are sufficiently exhibited to the living body, and the food can be supplemented with food, a sweetener or a functional ingredient ≪ / RTI >

Examples of the food composition according to the present invention include various foods such as beverages, gums, tea, vitamin complexes, and health supplement foods. Examples of the food composition include natural fruit juice, fruit juice drink, . The food composition of the present invention can be prepared in the form of tablets, tablets, granules, powders, capsules, liquid solutions, rings and the like according to known production methods.

In addition, the food composition of the present invention may further contain various conventional flavors, natural carbohydrates and the like. Flavors include natural flavors such as tau Martin and stevia extract, and synthetic flavors such as saccharin and aspartame. The above-mentioned natural carbohydrates may include polysaccharides such as disaccharides such as glucose, monosaccharide such as fructose, maltose, sucrose, dextrin, cyclodextrin, sugar alcohols such as xylitol, sorbitol, erythritol, and the like . In addition, the food composition of the present invention may further contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring agents, heavy stabilizers (cheese, chocolate etc.), pectic acid and its salts, Salt, organic acids such as anhydrous citric acid, protective colloid thickener, pH adjusting agent, stabilizer, preservative, glycerin, alcohol, carbonating agent used in carbonated drinks, natural fruit juice, fruit juice etc. Lt; RTI ID = 0.0 > additive < / RTI > These additives may be used independently or in combination.

As described above, the novel compounds having an α-glucosidase inhibitory activity according to the present invention and a pharmaceutical composition containing the same have stronger inhibitory activity than conventional α-glucosidase inhibitors At the same time, side effects can be minimized.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made in detail to the preferred embodiments of the present invention and the accompanying drawings. The detailed description of the functions and configurations of the present invention will be omitted if it is determined that the gist of the present invention may be unnecessarily blurred.

Materials and methods

UV spectra were confirmed using a Hitachi U-2000 spectrophotometer (Hitachi, Tokyo, Japan). ¹ H and ¹³ C NMR spectra were measured on a Varian VNS 600 instrument (600 MHz, 150 MHz) (2D analysis). Chemical shifts were recorded in delta (ppm), and TMS was used as an internal standard. FABMS spectra were obtained using a Micro Mass Auto Spec OA-TOF spectrometer (Micromass, Manchester, UK).

For HPLC analysis, a YMC-Pack ODS A-302 column (4.6 mm i.d. × 150 mm; YMC Co., Kyoto, Japan) was used.

Gradient elution was carried out using 1% HCOOH and Acetonitrile as mobile phase solvents.

The column temperature was maintained at 40 占 폚, the flow rate of the mobile phase was maintained at 1.0 ml / min, and the compound was detected at 280 nm.

Column chromatography was performed using Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan) and YMC GEL ODS AQ 120-50S (YMC Co., Kyoto, Japan).

Thin-layer chromatography (TLC) was performed on Kieselgel 60 F ₂₅₄ plates (0.25 mm layer thickness, Merck, Darmstadt, Germany). A 10% sulfuric acid solution was used as a color development reagent. The UV irradiation wavelength was 254 and 365 nm when TLC spot was detected.

The culls were collected in Hunan Province, China in September 2011 and purchased from Humanherb Inc. (Daegu, Korea).

α-glucosidase was purchased from Sigma-Aldrich Co., USA, and all reagents used in the experiment were sterilized before use.

Analysis of the inhibitory activity of α-glucosidase was carried out by slightly modifying the previous method. (0.02 unit of α-glucosidase, 50 μL) and 1 mM p- nitrophenyl-α-D-glucopyranoside (50 μL) were mixed in 50 mM phosphate buffer (pH 7.0) and the test sample was dissolved in 5% DMSO (10 μL). After 20 minutes of reaction at 37.5 ° C, 2 M NaOH was added to quench the reaction. The released p-nitrophenol was measured at 410 nm using a UV microplate reader (Infinite F200; Tecan Austria GmBH, Grodig, Austria). IC ₅₀ was calculated by linear regression analysis. Acarbose was used as a positive control, and all analyzes were performed three times.

Extraction and separation

The dried crude extract was pulverized and then extracted with 3 L of 80% methyl alcohol (MeOH) at room temperature for 24 hours. After extraction, MeOH was removed by concentration under reduced pressure.

The collected methanol extract (435 g) was subjected to column chromatography using a Diaion HP-20 column (2.8 cm id 9 58 cm) using an H ₂ O-MeOH mixed solvent (0.2 L) as an elution solvent.

UG01-UG06 was obtained by increasing the methanol content. The activity of UG01-UG06 was tested for each fraction. As a result, UG01 (H ₂ O) and UG04 (60% MeOH) Respectively. (UG01: 63.9 ± 3.5 μg / mL, UG04: 30.3 ± 2.7 μg / mL)

Compounds were separated using reverse phase HPLC for the most active eluents UG01 and UG04.

Compound 4 (t _R 8.4 min, 26.0 mg), Compound 5 (t _R 7.7 min, 11.6 mg) was subjected to column chromatography using a YMC GEL ODS AQ 120-50S column (1.1 cm id x 38 cm) and the compound _{6 (t R 5.9 min, 56.3} mg) was isolated.

Compound 1 (t _R 11.3 min, 16.5 mg), Compound 2 (t _R 9.7 min, 20.8 mg) was subjected to column chromatography using a YMC GEL ODS AQ 120-50S column (1.1 cm id x 42 cm) for eluent UG04. a, compound _{3 (t R 10.1min, 6.2mg)} , compound 7 _(R t 8.9min, 2.8mg) and compound 8 _(R t 5.1min, 2.7mg) was isolated.

¹ H, ¹³ C NMR and FABMS were used to identify the structure of eight compounds.

The following formulas (1) to (8) show the structures of the eight separated compounds.

Compound 2 (m-2) is mauritianin, Compound 3 is quercetin 3- (2,6-rhamnosylgalactopyranoside), Compound 4 is procyanidin B3 and Compound 5 is (+) - epicatechin, Compound (6) was identified as (+) - catechin, compound 7 (7) was identified as 5- (3 ', 4'-dihydroxyphenyl) -γ-valerolactone and compound 8 (8) as protocatechuic acid.

Compound 1 (Formula 1) was obtained as an amorphous yellow powder [α] _D ²⁵ -153.0 ° ( c 0.1, MeOH).

The molecular formula was identified as C ₁₂ H ₁₄ O ₃ using positive HRFABMS and showed a pseudomolecular ion peak at m / z 206.0938 [M] ⁺ .

The absorbance at 286 nm showed the presence of aromatic groups.

Table 1 shows the ¹ H and ¹³ C NMR data of Compound 1.

¹ H NMR spectrum of the compound 1 is a _{δ H 6.63 (1H, s,} H-8) and 6.50 (1H, s, H- 5) an aromatic signal _{in, δ H 1.55 (3H, s} , H-13) aryl in methyl single line showed a resorcinol moiety.

In addition, the spectrum _{δ H 4.57 (1H, m,} H-3) a peroxide in the (oxygenated) methine proton and _{δ H 3.08 (1H, dd,} J = 15.6, 4.8 Hz, H-4a) and 2.31 (1H, (1H, m, H-11b), 1.70 (1H, m, H-11b), benzylic methylene protons and 隆_H 2.11 And 1.62 (1H, m, H-12b), respectively.

In ¹³ C NMR and HSQC results for Compound 1, δ _C The presence of spirocyclopropane moieties in chroman signals and δ _C 43.6 (ⓒ-11) and 30.8 (ⓒ-12) was shown in 80.6 (ⓒ-2), 74.8 (ⓒ-3), and 37.6

These spectroscopic properties and ¹ H- ¹ H COZY correlations show that Compound 1 is a prenylated resorcinol derived from an extremely rare oxidized spirocyclopropane moiety, as shown below.

This is expected to be derived from rearrangement and oxidation of isopropene units.

The connection points between cyclopropane and methyl residues were clearly confirmed by HMBC and NOESY experiments. According to the above results, the planar structure of Compound 1 has been identified as a novel prenyl resorcinol derivative with peroxidized spirocyclopropane, which is named gambicarin.

α- glucosidase  Inhibitory activity

Table 2 below shows the inhibitory activities of? -Glucosidase of the isolated compounds 1 to 8. All compounds were measured three times and validated at the p < 0.05 level. Acarbose was used as a positive control.

Among these compounds, the new compound 1 derived from the acaricide exhibited a superior α-glucosidase inhibitory activity (15.5 ± 0.7 μM) when compared with other compounds and Acarbose (312.6 ± 3.8 μM) currently marketed as an α-glucosidase inhibitor.

From the above results, it has been shown that the novel compound derived from catecholamines has potential as an inhibitor of alpha-glucosidase and a therapeutic agent for diabetes mellitus, and it is expected that the adverse effects on human body derived from natural products can be minimized.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken as limiting the scope of the present invention. The present invention can be variously modified or modified. The scope of the invention should, therefore, be construed in light of the claims set forth to cover many of such variations.

Claims (9)

A novel prenyl resorcinol derivative compound represented by the following formula (1).
[Chemical Formula 1]

The method according to claim 1,
The compound
Characterized by being derived from catechesis
A novel prenyl resorcinol derivative compound.
The method according to claim 1,
The compound
Characterized in that it has an alpha-glucosidase inhibitory activity
A novel prenyl resorcinol derivative compound.
The method of claim 3,
The compound
Characterized by having an IC ₅₀ value of 15.5 ± 0.7 μM for α-glucosidase inhibitory activity
A novel prenyl resorcinol derivative compound.
A novel prenyl resorcinol derivative compound represented by the following formula (1), mauritianin, quercetin 3- (2,6-rhamnosylgalactopyranoside), 5- (3 ', 4'- dihydroxyphenyl) As including
A pharmaceutical composition for the treatment of diabetes through alpha-glucosidase inhibition.
[Chemical Formula 1]

delete 6. The method of claim 5,
The compound of formula (1)
Alpha-glucosidase, characterized in that with a IC ₅₀ value of 15.5 ± 0.7 μM for the inhibitory activity
A pharmaceutical composition for the treatment of diabetes through alpha-glucosidase inhibition.


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