KR20180061960A - The Novel compound isolated from mulberry for enhancing anti-obesity effect - Google Patents

Abstract

The present invention relates to novel compounds, and more particularly to novel compounds isolated from mulberry leaves and antiobesity compositions containing them.

Description

[0001] The present invention relates to a compound isolated from a mulberry leaf for enhancing an anti-obesity effect,

The present invention relates to novel compounds, and more particularly to novel compounds isolated from mulberry leaves and antiobesity compositions containing them.

According to changes in diet and lifestyle, obesity related to lifestyle is increasing. Obesity through excessive fat accumulation in adipocytes is known to cause chronic diseases including diabetes, hypertension, and cardiovascular disease. Obesity is a chronic disease caused by a deficiency in the ability to control the energy balance due to leptin resistance and insulin resistance. As a result, chronic stress, excessive intake of carbohydrates, accumulation of toxic chemicals, and lack of physical activity cause obesity . Various studies have been carried out to suppress obesity. However, until now, a complete remedy without side effects has not been developed and sold.

In recent years, studies have been actively carried out to find out the mechanism of action of a natural substance in an anti-obesity material having no side effects, and it has been reported that the activity of promoting activity of pancreatic lipase and the substance having the ability of inhibiting carbohydrase, Materials that reduce area, and materials that have weight and body fat reduction capabilities have been introduced.

On the other hand, mulberry is widely distributed in tropical and temperate regions and belongs to Moracceae and Morus. Mulberry has been used as a traditional medicinal material in the private sector, such as leaves, oats, and young branches, and various physiological activities have been reported. In the mulberry leaves, rutin, quercetin, quercitrin, isoquercitrin and alkaloid have been reported. These compounds have been reported to inhibit DNA damage and cell adhesion and inflammation in human aortic endothelial cells. 1-deoxynojirimycin (1-DNJ) isolated from mulberry leaves is known to inhibit the increase of blood glucose by inhibiting α-glucosidase.

However, the anti-obesity effect of certain compounds contained in the mulberry leaves has not been disclosed to date.

Accordingly, the inventors of the present invention discovered a novel compound in studying pure isolation and assay of substances extracted from mulberry leaves, and confirmed that the compound exhibits an obesity effect by promoting lipolysis of a rat primary cell, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a novel compound isolated from mulberry leaves and an anti-obesity composition containing the same.

In one aspect, the present invention provides a novel compound.

In another aspect, the present invention provides an anti-obesity composition comprising the above compound.

The compound according to the present invention is isolated from mulberry leaves and has excellent anti-obesity effect and thus can be effectively used for prevention and treatment of obesity.

FIG. 1 shows the result of glycerol release evaluation in the primary adipose tissue cell line of the compound of Rat isolated from mulberry leaves.

The present invention provides novel compounds isolated from mulberry leaves and their pharmaceutically acceptable salts.

Such compounds may be prepared by pharmaceutically acceptable salts and solvates according to conventional methods in the art.

Salts are useful as acid addition salts formed by pharmaceutically acceptable free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, lactic acid, glycollic acid, gluconic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid and the like.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of such compounds include, unless otherwise indicated, salts of acidic or basic groups that may be present in the compounds. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, (Mesylate) and p-toluenesulfonate (tosylate) salts, which are known in the art and which are known in the art Can be manufactured through a manufacturing process.

The mulberry leaf refers to pulverized mulberry leaf powder or dry mulberry leaf produced in the country.

More specifically, the compound can be isolated by the following steps.

(a) adding ethyl acetate to the ground mulberry leaves to obtain an ethyl acetate extract; (b) adding the ethyl acetate extract to a silica gel column, sequentially eluting the solvent with hexane, dichloromethane, acetone, methanol as a mobile phase solvent with polarity increasing to obtain 141 fractions; (c) The 98th to 100th fractions (MALE 98-100, 702.5 mg) of the 141 fractions were added to a silica gel Sephadex column, and a mixed solvent of chloroform and methanol (50:50) ; And (d) adding a fifth fraction of the sixteen fractions to a silica gel column, followed by elution with an elution solvent to obtain a compound.

(A) of the present invention is a step of obtaining an ethyl acetate extract by adding an ethyl acetate solvent to a pulverized mulberry leaf, wherein the dried mulberry leaf is pulverized using a pulverizer, and an ethyl acetate solvent is added to the pulverized mulberry leaf Sonication for 1 to 4 hours, and filtering the supernatant.

The step (b) of the present invention is primarily a step of obtaining 141 fractions. The extract of mulberry ethyl acetate obtained through the step (a) is added to a silica gel column, and then hexane, dichloromethane, acetone , Followed by sequential elution with methanol.

The type of the silica gel is not particularly limited as long as it is a silica gel commonly used in the art to which the present invention belongs, but general silica gel can be preferably used.

The step (c) of the present invention is a step of obtaining 16 secondary fractions, and the 98-100th fraction (MALE98-100, 702.5 mg) of the 141 fractions obtained through the step (b) To a packed 110 × 3 cm Sephadex column and then using a mixed solvent as the mobile phase with chloroform and methanol (50:50).

The step (d) of the present invention is a step of obtaining a pure compound. MALE5IE, which is a fifth fraction of the 16 fractions obtained through the step (c), is added to a silica gel column, and then 500 ml of hexane, ml of methanol and 100 ml of methanol.

In another aspect, the present invention provides an anti-obesity composition comprising the above compound.

The composition may be provided as a pharmaceutical composition or a food composition.

The composition may be formulated into a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient or diluent to the compound.

The composition of the pharmaceutical unit dosage form of the present invention may be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, sterile injectable solutions, suppositories and transdermal preparations according to a conventional method And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. If necessary, it is formulated using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient.

In one embodiment, the composition of the present invention may be formulated into a solid preparation for oral administration. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, Are mixed and formulated. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

In another embodiment, the composition of the present invention may be formulated and used as a liquid preparation for oral administration. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. These liquid preparations may contain various excipients in addition to commonly used inert diluents (for example, purified water, ethanol, liquid paraffin) For example, wetting agents, sweeteners, fragrances, preservatives and the like may be included.

In another embodiment, the composition of the present invention may be formulated into a formulation for parenteral, preferably intraperitoneal administration. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the sterilized aqueous solution, a suitable buffer solution such as Hank's solution, Ringer's solution or physically buffered saline may be used. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, Vegetable oil, injectable esters such as ethyl oleate, and the like can be used. If desired, preservatives, stabilizers, wetting or emulsifying agents, salts for controlling osmotic pressure and / or buffers may be used. On the other hand, in the case of suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glyceroglatin and the like may be used.

The composition formulated in the above manner may be administered through various routes including parenteral or oral (transdermal, subcutaneous, intravenous, muscular, abdominal, etc.) in an effective amount. The "effective amount" as used herein refers to an amount that shows a preventive or therapeutic effect when administered to a patient. The dose of the composition according to the present invention can be appropriately selected depending on the route of administration, subject to be administered, age, sex, weight, individual difference, and disease state. Preferably, the composition of the present invention may contain the active ingredient in an amount of 1 to 10000 μg / kg body weight / day, more preferably 10 to 1000 μg / kg body weight / day RTI ID = 0.0 > of < / RTI >

In addition, the compounds of the present invention may be used as health foods using conventional food preparation methods. The health food refers to a food added to a food material such as a beverage, a tea, a spice, a gum or a confection or the like, or a food prepared by a capsule, a powder, or a suspension, It is meaningful, unlike general medicine, there is no side effect that can occur when a food is used as a raw material for a long time, and it can be taken as an anti-obesity supplement.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to be illustrative of the present invention and not to limit the scope of the invention as defined by the appended claims. It will be obvious to those skilled in the art that the present invention is also included in the scope of the present invention.

Example  1. Separation of novel compounds of the present invention from mulberry leaves

First, dried mulberry leaves collected from Sangju (purchased in September 2015 by hot air drying in resident silkworms) were purchased. 1.93 kg of the crushed leaves were placed in a 5 L Erlenmeyer flask, filled with 5 L of hexane, sonicated for 2 hours, and then the supernatant was transferred to Whatman no. 2 filter paper. The hexane extract was extracted three times by the above method to obtain 30.18 g of a nucleic acid layer. Next, to the powder remaining after the extraction with hexane, ethyl acetate was extracted in the same manner as in hexane to obtain 39.06 g. The thus-obtained ethyl acetate extract (33.94 g) was placed in an 80 × 6 cm column packed with 850 g of silica gel, and then sequentially eluted with a polar solvent increasing in hexane, dichloromethane, acetone, and methanol to obtain 141 fractions . The obtained 141 fractions 98-100 fractions (MALE98-100 fractions, 702.5 mg) were added to a 100 x 3 cm column packed with Sephadex, and a mixed solvent of 500 ml of chloroform and 500 ml of methanol was flowed, and 16 Subsequent fractions were obtained. Sixteenth fractions (MALE5IE, 265.8 m 2) thus obtained were added to a column packed with silica gel again, and a mixed solvent of 500 ml of hexane, 400 ml of chloroform and 100 ml of methanol was flowed to obtain 68.8 mg of a pure substance .

Example  2. Of MALE98 Test solution  pharmacy

2.0 mg of pure MALE98 was dissolved in DMSO and used as the glycerol release data.

Example  3. Glycerol release measurement

After 9-10 weeks of age SD rats were euthanized, adipose tissue was collected and washed with 1% (BSA content) Krebs-Ringer solution. The washed adipose tissue was transferred to collagenase solution, cut into small pieces with scissors, and cultured in a shaking incubator (150 rpm, 37 ° C) for 1 hour. After culturing, the cells were filtered using a cell strain of 100 uM (Falcon, 352360). Transfer 10 ml to a 15 ml tube and centrifuge at 1000 rpm for 1 min. The supernatant was collected and shaken by adding 1% (BSA content) Krebs-Ringer solution to the same volume, centrifuged at 1000 rpm for 20 seconds, And repeatedly washed with adipocyte. After washing, the supernatant was dispensed into 24 wells using 3% (BSA content) Krebs-Ringer solution. After 1 hour incubation, samples were treated and free glycerol assay was performed 24 hours later.

The free glycerol assay was carried out by transferring 24 well samples into microtube, incubating in a 65 ° C water bath for 15 minutes, centrifuging at 1,000 rpm for 3 minutes, and collecting the supernatant. A sample of 10 λ accumulated in a 96-well plate was added and Free Glycerol Reagent 200 λ was added. After reacting at 200 rpm for 15 minutes in an agitator, the absorbance was measured at a wavelength of 540 nm. The concentration was calculated using a standard curve.

× glycerol standard 농도* Glycerol content =

× glycerol standard concentration

= 시료 처리한 well의 흡광도

= Absorbance of the well treated sample

= 배지만 처리한(Negative control) well의 흡광도

= Absorbance of negative control wells

= Glycerol standard 처리한 well의 흡광도

= Absorbance of Glycerol standard treated wells

As a result, the glycerol release increased in a concentration-dependent manner when MALE98 was treated, and increased about 1.6 times when treated with 200 mg / ml of the compound purely isolated from mulberry leaves (see FIG. These results suggest that the compound of the present invention increases the glycerol release content in the primary adipose tissue of Rat and accelerates the degradation of adipocytes, and that the increase in the glycerol release content causes the weight loss by decomposing mast cells.

Claims (1)

A composition for anti-obesity comprising a compound isolated from mulberry leaf extract.

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