KR101837477B1 - A composition comprising compounds isolated from Morus alba leaves for preventing or treating obesity - Google Patents

Abstract

The present invention relates to a composition comprising a compound isolated from Morus alba leaves for preventing or treating obesity. The Morus alba leaf-derived compound is excellent in the effect of inhibiting lipid accumulation in differentiation-induced adipocytes, and thus can be effectively used as a composition for preventing or treating obesity.

Description

Technical Field [0001] The present invention relates to a composition for preventing or treating obesity, which comprises a compound isolated from a mulberry leaf,

The present invention relates to a composition for preventing or treating obesity comprising a compound isolated from mulberry leaves.

The incidence of obesity has been increasing in Korea in recent years (Kim, DM et al., Obes Rev., 6 (2), 117-121, 2005.).

In general, obesity is a type of disease that is consumed among the energy consumed and accumulated in the adipocytes of the abdominal and subcutaneous adipose tissues, which is converted into triglyceride. It is a genetic, nutritional, environmental, social And a number of other causes. Obesity itself is a disease that causes disruption of daily life, but the disease itself rather than the meaning of the obesity caused by various complications are more serious problem.

Obesity can cause hyperlipemia, which leads to an increase in the amount of cholesterol or triglycerides in the blood, leading to a fatal disease such as hypertension, cardiovascular disease and stroke, and may result in peripheral tissue (Kelley, DE et al., Diabetes, 49 5), 677-683, 2000) and the increase in triglyceride accumulation in the abdominal adipose tissue (Kelley, DE et al., Am J Physiol Endocrinol Metab., 278 (5), E941-948, 2000) May cause type 2 diabetes, so long-term care and treatment is absolutely necessary. In addition, obesity management is more important for Asians because the body mass index is at least abdominal obesity higher than that of Westerners, and is highly susceptible to complications caused by arterial diseases such as hypertension, diabetes and hyperlipidemia.

To treat obesity, dietary therapy, exercise therapy, behavior modification therapy that corrects wrong eating habits and lifestyle, surgery to reduce the intestine or stomach volume in surgery, appetite suppressant, diuretic, diarrhea, or fiber to give fullness And the like are being provided. However, the methods provided so far do not exert their effects or cause many side effects. In particular, sibutramine, a type of appetite suppressant, can cause side effects such as headache, dry mouth (insatiable thirst), insomnia, and constipation, and blood pressure and pulse rate may increase slightly. You have to check. In addition, orlistat, a kind of lipolytic enzyme inhibitor, has disadvantages such as frequent bowel movements and fatty changes, and a decrease in the rate of absorption of fat-soluble vitamins in the intestines due to a decrease in fat absorption rate (Morrison, RF et al., J. Nutr., 130 (12), 3116-3121, 2000). Therefore, there is a need for a new therapeutic agent for obesity, which has fewer side effects and a certain effect.

Morus alba L.) is a common deciduous tree or shrub belonging to the genus Chrysanthemum bisporus and the mulberry of the dicotyledonous plant Nettles, and its origin is temperate and subtropical. Small branches are grayish brown or grayish white and have fine hairs but gradually disappear. Leaves are egg-shaped or long oval-shaped, round, 3 to 5 pieces long, 10 cm long, with dull serrations on the edges and pointed ends. There are hairs on the back vein with petiole.

The leaves of mulberry are called mulberry leaves and are used as a remedy for symptoms such as fever, swelling, headache, seawater, eye disease, edema, , Which scraped the outer skin of the knife and then peeled the inner skin and dried it. The above-mentioned manganese berry has been used as a therapeutic agent for fever, diuretic, shinhae, and extinction, and thus has been used as a therapeutic agent for waste heat seawater (lung cough), bronchitis, urine discomfort,

As a prior art related to mulberry leaf extract and compounds isolated therefrom, Korean Patent Laid-Open No. 10-2009-0099974 discloses a composition for preventing and treating hyperlipidemia or obesity containing an extract of Mulberry leaf as an active ingredient, Open Patent No. 10-2015-0083622 discloses an anti-obesity composition using an oak tree and mulberry. However, there is no previous report confirming the therapeutic effect of the mulberry leaf-derived compound of the present invention on obesity.

Korean Patent Laid-Open No. 10-2009-0099974, a composition for the prevention and treatment of hyperlipidemia or obesity containing an extract of Upper Lobes as an active ingredient, published on September 23, 2009. Korean Patent Laid-Open No. 10-2015-0083622, an anti-obesity composition using an oak tree and mulberry, published on July 20, 2015.

Kelley, D. E. et al., Fuel selection in human skeletal muscle in insulin resistance: a reexamination, Diabetes, 49 (5), 677-683, 2000. Kelley, D. E. et al., Subdivisions of subcutaneous abdominal adipose tissue and insulin resistance, Am J Physiol Endocrinol Metab., 278 (5), E941-948, 2000. Kim, D. M. et al., Prevalence of obesity in Korea, Obes Rev., 6 (2), 117-121, 2005. Morrison, R. F. et al., Hormonal signaling and transcriptional control of adipocyte differentiation, J. Nutr., 130 (12), 3116-3121, 2000.

It is an object of the present invention to provide a composition for preventing or treating obesity comprising a compound isolated from mulberry leaves.

The invention mulberry leaf (Morus alba D-glucopyranoside (Compound 1), morazine J (Compound 2), 2,5-anhydro-3-O-β-D- Galactopyranosyl-D-mannitol (Compound 3) and (6R, 9R) -3-oxo-α-ionone 9-0-β-glucopyranoside Which comprises a compound as an active ingredient, to a pharmaceutical composition for preventing or treating obesity.

[Chemical Formula 1]

Compounds isolated from the mulberry leaves can be obtained from extracts of mulberry leaves extracted with water, C1 to C4 lower alcohols or a mixed solvent thereof. The C1 to C4 lower alcohols include methanol, ethanol, propanol, isopropanol, Butanol and the like can be used. The mulberry leaf extract may be a fraction obtained by re-fractionation with an organic solvent. The organic solvent may be further fractionated using hexane, ethyl acetate, n-butanol, or the like, or a combination of these solvents may be sequentially fractionated, And fractionating the above fractions by chromatography.

The chromatography was performed by silica gel column chromatography, flash column chromatography, sephadex LH-20 column chromatography, RP-18 column chromatography (RP -18 column chromatography, thin layer chromatography (TLC), medium pressure liquid chromatography and high performance liquid chromatography (HPLC).

Meanwhile, the compound of the present invention can be synthesized according to a conventional method in the art, and can also be prepared as a pharmaceutically acceptable salt.

The pharmaceutical composition according to the present invention can be formulated into a suitable form together with a commonly used pharmaceutically acceptable carrier. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and, when administered to humans, does not normally cause an allergic reaction such as gastrointestinal disorders, dizziness, or the like.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterilized injection solutions according to a conventional method . Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, But are not limited to, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraoxybenzoate, propylparaxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, stabilizers, binders, disintegrants, surfactants and the like which are usually used. Solid formulations 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 and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

The pharmaceutical composition comprising the compound of formula (I) as an active ingredient of the present invention can be administered to mammals such as rats, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. The dosage will depend on the age, sex, body weight, the particular disease or condition being treated, the severity of the disease or condition, the time of administration, the route of administration, the absorption, distribution and excretion of the drug, It depends on judgment. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several doses. The dose is not intended to limit the scope of the invention in any way.

The present invention also provides a health functional food for preventing or ameliorating obesity comprising at least one compound selected from the group of the compounds of the formula (1) isolated from mulberry leaves and a pharmaceutically acceptable food-aid additive. The compound may be added in an amount of 0.001 to 100% by weight to the health functional food of the present invention. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids, and examples of foods to which the compound of the present invention can be added include various foods, beverages, gums, tea, vitamins .

The present invention relates to a composition for preventing or treating obesity, which comprises a compound isolated from a mulberry leaf, wherein the mulberry leaf-derived compound is excellent in the effect of inhibiting fat accumulation in differentiation induced fat cells, May be useful as a composition.

Fig. 1 is a graph showing the cell survival rate for adipocytes when the compounds 1 to 4 of the present invention are treated. Fig.
Fig. 2 shows the results of confirming that the compounds 1 to 4 of the present invention have an effect of inhibiting fat accumulation in 3T3-L1 cells induced to differentiate into adipocytes.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

< Example  1. Isolation of mulberry-derived compounds>

2.9 kg of mulberry leaves were refluxed with methanol to obtain 380 g of mulberry extract. The extract was fractionated with hexane, ethyl acetate and butanol. The ethyl acetate fraction (15.0 g) and the butanol fraction (68.0 g) containing a large amount of the active ingredient were purified by reverse phase column chromatography and preparative HPLC to obtain morazine M 6-β-D-glucopyranoside (compound 1 D-mannitol (Compound 3, 90 mg) and (6R, 5-dihydroxy-D-galactopyranosyl- 9R) -3-oxo-α-ionone 9-O- β-glucopyranoside (Compound 4, 22.0 mg).

< Example  2. Measurement of cell viability>

To determine the cell viability of the mulberry-derived compound prepared in Example 1 of the present invention, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed.

The microplate was treated with 3T3-L1 preadipocyte and 40 μM of each compound 1 to 4, followed by incubation in a CO ₂ cell incubator (Heracell 150i, thermo) at 37 ° C. under 5% CO ₂ for 72 hours . Thereafter, 200 쨉 l of 5 ㎎ / ml MTT solution was added to each well, followed by reaction for 60 minutes. Then, the formazan crystal produced through the above procedure was dissolved in DMSO and the absorbance at 565 nm was measured , Is shown in Fig.

Referring to FIG. 1, the compounds 1 to 4 of the present invention showed cell viability of 95% or more, indicating that they do not show cytotoxicity to adipocytes, unlike the control group, digitonin.

< Example  3. Estimation of fat accumulation inhibitory effect>

3T3-L1 adipocytes were cultured in DMEM (with 4.5 g / L D-Glucose, Dulbecco's modified Eagle's medium, GIBCO) containing 10% BS (bovine serum, GIBCO) and 1% PS (penicillin / streptomycin) Cells were cultured in a CO ₂ cell incubator (Heracell 150i, thermo) at 37 ° C and 5% CO ₂ , and subcultured at intervals of 2 to 3 days.

When 3T3-L1 adipocytes are filled with about 80-90% of the cell culture dish, the cells are separated using 0.25% trypsin-EDTA (GIBCO), then the cells are resuspended in a centrifugal separator and resuspended in 10% And diluted to 60,000 cells per ml. 200 μl of the solution was added to each well of a 96-well plate, and the cells were cultured until the wells were filled. Then, 1 μg / ml insulin, 0.5 mM IBMX (3-Isobutyl-1-Methylxanthine) and 1 μM dexamethasone The differentiation induction medium contained was mixed with each of the compounds 1 to 4 of the present invention at a concentration of 40 μM and the cell culture medium was replaced. After 3 days, 10% FBS medium was replaced with medium containing 10 μg / ml insulin and compounds 1 to 4 of the present invention at a concentration of 40 μM respectively, followed by further incubation for 2 days. In the 10% FBS medium, And then cultured for another 2 days.

The cells were then washed with PBS, fixed with 10% formaldehyde solution for at least 1 hour, washed with 60% isopropanol, and 100 μl of Oil Red O solution was added for 10 minutes Lt; / RTI &gt; After 10 minutes, the cells were washed 4 times with sterilized water, and the oil red dye was dissolved in 100% isopropanol. The fat accumulation rate was measured at an absorbance of 520 nm, and the results are shown in Table 1 and FIG.

compound Fat accumulation rate (%) Control group 100 ± 1.7 Compound 1 40 [mu] M (16.2 [mu] g / ml) 78.8 ± 5.4 * Compound 2 40 [mu] M (10.9 [mu] g / ml) 63.4 ± 8.9 *** Compound 3 40 [mu] M (13.1 [mu] g / ml) 70.6 ± 10.6 *** Compound 4 40 [mu] M (14.8 [mu] g / ml) 65.3 ± 4.0 EGCG 100 [mu] M (45.8 [mu] g / ml) 69.7 ± 5.4 *** Ethyl acetate fraction of mulberry leaves 500 [mu] g / ml 79.5 ± 2.3 * Butanol fraction of mulberry leaves 1000 mu g / ml 81.2 ± 1.5 *

Referring to Table 1 and FIG. 2, the compound of the present invention showed a fat accumulation inhibitory effect 3 times or more higher than that of a positive control group, EGCG (Epigallocatechin Gallate), and showed a fat accumulation inhibitory effect 30 times or more higher than that of a mulberry leaf fraction, It can be effectively used as a composition for preventing or treating cancer.

< Example  4. Assessment of neutral fat accumulation inhibition efficacy>

The procedure of Example 3 was repeated except that Nile Red solution was used instead of Oil Red O solution and allowed to stand for 10 minutes. Then, fluorescence (emission: 485 nm, excitation: 540 nm) was measured and shown in Table 2 below.

compound Triglyceride accumulation rate (%) Control group 100.2 ± 0.95 Compound 2 40 μM 81.5 ± 5.8 EGCG 100 μM 57.3 ± 2.0

As shown in Table 2, Compound 2 of the present invention showed an effect of inhibiting the accumulation of triglycerides, and thus it could be effectively used as a composition for preventing or treating obesity.

< Example  5. Toxicity test>

Example  5-1. Acute toxicity

This experiment was conducted to investigate the toxicity of the compound 1 or 4 of the present invention to an animal body acutely (within 24 hours) when an excessive amount of the compound 1 or 4 of the present invention was consumed in a short period of time and determine the mortality rate. Twenty ICR mice were prepared, and 10 mice were assigned to each group. In the control group, only 30% PEG-400 alone was administered, and the test group was orally administered with Compound 1 or 4 at a concentration of 1.0 g / kg, respectively. After 24 hours of administration, the respective mortality rates were examined. As a result, both the control group and the test group to which Compound 1 or 4 was administered survived.

Example  5-2. Experimental group  And control organ organs and tissue toxicity experiments

To evaluate the effect of the compound 1 or 4 of the present invention at a concentration of 1.0 g / kg on the long-term toxicity of the C57BL / 6J mice, Blood samples were taken from the animals 8 weeks later and the concentrations of GPT (glutamate-pyruvate transferase) and BUN (blood urea nitrogen) in the blood were measured using Select E (vital scientific NV, Netherland) instrument. As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were cut from each animal and histological observation was performed with an optical microscope through a conventional tissue section production process, but no abnormal abnormalities were observed.

< Formulation example  1. Pharmaceutical preparations>

Formulation example  1-1. Sanje  Produce

2 g of the compound 1 or 4 of the present invention and 1 g of lactose were mixed and filled in an airtight container to prepare a powder.

Formulation example  1-2. Manufacture of tablets

100 mg of Compound 1 or 4 of the present invention, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropylcellulose and 2 mg of magnesium stearate were mixed and then tableted according to a conventional preparation method Tablets were prepared.

Formulation example  1-3. Preparation of capsules

100 mg of the compound 1 or 4 of the present invention, 100 mg of microcrystalline cellulose, 60 mg of lactose hydrate, 20 mg of low-substituted hydroxypropylcellulose and 2 mg of magnesium stearate were mixed, The ingredients were mixed and filled into gelatin capsules to prepare capsules.

Formulation example  1-4. Injection preparation

10 mg of the compound 1 or 4 of the present invention was mixed with the sterilized distilled water suitable amount and the pH adjuster in the amount of the above-mentioned ingredients in accordance with the usual preparation method of injection.

< Formulation example  2. Food Manufacturing>

Formulation example  2-1. Manufacture of cooking seasonings

Compound 1 or 4 of the present invention was added to the cooking sauce in an amount of 1 wt% to prepare a cooking sauce for health promotion.

Formulation example  2-2. Manufacture of flour food products

A food for health promotion was prepared by adding 0.1% by weight of Compound 1 or 4 of the present invention to wheat flour and preparing bread, cake, cookies, crackers and noodles using the mixture.

Formulation example  2-3. soup  And gravies

Compound 1 or 4 of the present invention was added to soup and juice at 0.1 wt% to prepare health promotion soup and juice.

Formulation example  2-4. Manufacture of dairy products

Compound 1 or 4 of the present invention was added to milk in an amount of 0.1 wt%, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation example  2-5. Vegetable juice manufacturing

0.5 g of the compound of the present invention 1 or 4 was added to 1,000 ml of tomato juice or carrot juice to prepare vegetable juice for health promotion.

Formulation example  2-6. Manufacture of fruit juice

0.1 g of the compound of the present invention 1 or 4 was added to 1,000 ml of apple juice or grape juice to prepare fruit juice for health promotion.

Claims (5)

Mulberry leaf (Morus alba leaves) to a new Mora M 6-β-D- glucopyranoside (Compound 1), Mora new J (compound 2) and (6R, 9R) of the formula (1) separated from the 3-oxo- wherein the active ingredient is at least one compound selected from the group consisting of? -ionyl 9-O-? - glucopyranoside (Compound 4).
[Chemical Formula 1]

The method according to claim 1,
The pharmaceutical composition for preventing or treating obesity is characterized in that the compound inhibits fat accumulation. 3. The method according to claim 1 or 2,
Wherein the composition is formulated into a pharmaceutical dosage form by addition of a pharmaceutically acceptable carrier, excipient or diluent. Mulberry leaf (Morus alba leaves) to a new Mora M 6-β-D- glucopyranoside (Compound 1), Mora new J (compound 2) and (6R, 9R) of the formula (1) separated from the 3-oxo- and at least one compound selected from the group consisting of? -Ionyl 9-O-? - glucopyranoside (Compound 4) as an active ingredient.
[Chemical Formula 1]

5. The method of claim 4,
Wherein said compound inhibits fat accumulation. 2. A health functional food for preventing or ameliorating obesity.

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