KR20220084816A - Novel compound isolated from extract of Gymnaster koraiensis - Google Patents

Abstract

The present invention relates to a novel compound isolated from honeysuckle odor and its use, and has excellent adipocyte differentiation inhibitory effect and triglyceride inhibitory effect, which can be usefully used in metabolic diseases including obesity.

Description

Novel compound isolated from extract of Gymnaster koraiensis

The present invention relates to novel compounds isolated from honey ant odor and uses thereof.

In general, obesity refers to a condition in which adipose tissue is accumulated in the body excessively. A weight at 22 is called a standard weight, a weight more than 10% more than that is called overweight, and a weight more than 20% more than that is called obesity. In recent years, obesity and its reserve group are increasing due to the lack of exercise due to the deterioration of diet and the development of transportation systems. In particular, obesity is highly related to the onset of hypertension, diabetes, fatty liver, arteriosclerosis, cerebral infarction, hyperlipidemia, peripheral vascular disorder, and ischemic heart disease, and prevention or resolution of obesity is very important in terms of prevention and treatment of these diseases. has been

Obesity, which was considered only a simple disease, has recently been classified as a disease and is reported to be associated with the development of type 2 diabetes, metabolic syndrome such as cardiovascular disease, and the development of certain cancers. Obesity is a disease that is caused by a complex action of various causes such as social, environmental, genetic, mental, and endocrine causes. According to the World Health Organization (WHO), the number of obese people worldwide has doubled since the 1980s, and in the United States, about 34% of the total population was classified as obese as of 2013. In order to reduce social and economic losses, 10% of the total national medical expenses are used for obesity-related medical services and campaigns every year. Recently, the increase in obesity rates in Asian and African countries is also remarkable. According to the British medical journal 'Lancet', in 2014, about 90 million people were obese in China. Recognizing the seriousness of obesity, which is gradually increasing, WHO set the 'World Strategy for Diet, Exercise, and Health' on the agenda in May 2004 and made efforts to solve obesity. However, so far, no country in the world has reported a decrease in obesity.

In order to prevent or eliminate obesity, improvement of lifestyles such as diet and exercise therapy is desirable, but not easily performed. In addition, there are drug therapies such as lipid absorption inhibitors and digestive absorption inhibitors as treatment for obesity, but their use is limited to severe obesity.

Drugs for the treatment of obesity include sibutramine, phentermine, diethyl-propion, phendimetrazine, benzphetamine, which are appetite suppressants that suppress appetite by stimulating the hypothalamus. (benzphetamine), metaphetamine (methamphetamine), etc. are known, and orlistat (orlistat), which inhibits absorption of ingested fat by using an inhibitor of pancreatic lipodigestive enzyme, is also known, and is an alpha-glucosidase inhibitor. Acarbose, which is used for diabetes, is also known.

Among these, various products using natural ingredients derived from ingredients that have a variety of actions and are less likely to cause side effects even when taken for a long period of time are being sold as diet food, etc. this is being done However, obesity is difficult to cure until now, and it is known that serious side effects such as insomnia, nervous anxiety, headache, increase in blood pressure, gastrointestinal disorder, hypersensitivity, bile secretion disorder, and inhibition of absorption of fat-soluble vitamins are accompanied.

On the other hand, Gymnaster koraiensis ( Gymnaster koraiensis ) is a special perennial plant in Korea. It grows in sunny and moist places, especially in Jeju-do and south of Gyeonggi-do. The height is 50 to 60 cm, and the width is 1.5 to 3 cm. The leaves are sharp at the tip, 12 to 19cm long, 1.5 to 3cm wide, and the lower part gradually narrows, hard, with few hairs on both sides, and fine serrations on the edges. It is a perennial plant with flowers blooming in October, 4-5cm in diameter, light purple, hanging on the tip of branches and main stem, and the involucre is hemispherical, about 13mm long and about 8mm in diameter. The bracts are arranged in 4 rows, and the outer part is 4 to 5mm long and about 1.5mm wide, and has a long oval shape, with hairs on the edge.

As a prior art related to honey ant odor, the liver protection use of the honey ant odor extract in Korean Patent Registration No. 1187032 (Korean Patent Registration No. 10-1187032), ACAT (cholesterol acyltransferase) inhibitory effect, and the Korean Patent Registration No. 845479, the honey ant odor extract and its fractions Pharmaceuticals for the prevention and treatment of diabetic complications, i.e., diabetic retinopathy, diabetic cataract, diabetic nephropathy, diabetic neuropathy, etc. composition, etc. However, there is no prior literature that isolates an effective substance effective for obesity.

Accordingly, the present inventors confirmed that there is a preventive or therapeutic effect on obesity in honey ant lice, and by separating the active ingredient from the honey ant lichen extract having the preventive or therapeutic effect on obesity, the present invention was completed.

Republic of Korea registration number 10-1187032

Accordingly, it is an object of the present invention to provide a novel compound or a pharmaceutically acceptable salt thereof isolated from honey ant odor.

In addition, another object of the present invention is to provide a method for producing a new compound separated ring from the smell of honey ants.

In addition, the present invention is to provide an anti-obesity composition comprising a novel compound isolated from honey ant odor as an active ingredient.

Accordingly, the present invention provides a compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

In addition, the present invention comprises the steps of: 1) extracting bee ant odor with distilled water;

2) fractionating the honey ant odor water extract with n-hexane, separating the n-hexane fraction and the water fraction;

3) fractionating the water layer of step 2) with chloroform, and separating the water layer into a chloroform fraction and a water fraction;

4) fractionating the water fraction of step 3) with ethyl acetic acid to separate an ethyl acetic acid fraction and a water fraction; and

5) fractionating the water fraction of step 4) with butanol, and separating the butanol fraction and the water fraction.

In addition, the present invention provides a pharmaceutical composition for anti-obesity comprising the compound of Formula 1 as an active ingredient.

The novel compound isolated from the extract of the present invention has excellent adipocyte differentiation inhibitory effect and triglyceride inhibitory effect, which can be usefully used for metabolic diseases including obesity.

1 is a schematic diagram of separation of the novel compound of the present invention.
2 is a schematic diagram of separation of the novel compound of the present invention.
3 is a view showing LC/QTOF-MS (A) and IR spectrum (B) of the novel compound of the present invention.
4 is a diagram showing ¹ H-NMR (A) and ¹³ C-NMR (B) spectra of the novel compound of the present invention.
5 is a diagram showing HMQC (A) and 2D-NMR spectrum (B) of the novel compound of the present invention;
6 is a diagram showing the HMBC spectrum of the novel compound of the present invention.
7 is a view confirming the adipocyte induction inhibitory effect (A), the fat accumulation inhibitory effect (B), and the triglyceride inhibitory effect (C) of the novel compound of the present invention (Compound 1: Gymnasterkoreayne A (isolated from the chloroform fraction); Compound 2: 1β,6α,9β-trihydroxytrans-eudesm-3-ene-1,6-di-O-β-D-glucopyranoside (isolated from the second small fraction of the butanol fraction); Compound 3:Gymnasterkoreaside B (the butanol fraction) (isolated from the second sub-fraction); Compound 4: novel compound of the present invention; isolated from the third sub-fraction of the butanol fraction).
8 is a diagram showing the structure of the novel compound of the present invention.

The present invention may be provided in the form of a compound represented by Formula 1 or a pharmaceutically acceptable salt.

[Formula 1]

The compound of the present invention has an adipocyte differentiation inhibitory effect and has a triglyceride inhibitory effect. The compound can be isolated from the honey ant odor, and more specifically, it can be separated from the butanol fraction of the honey ant odor water extract, or the honey ant odor water extract, and the honey ant odor water extract is sequentially fractionated with n-hexane, chloroform, ethyl acetic acid and butanol. can be separated. More specifically, the compound of Formula 1 of the present invention comprises the steps of: 1) extracting the honey ant odor with distilled water;

2) fractionating the honey ant odor water extract with n-hexane, separating the n-hexane fraction and the water fraction;

3) fractionating the water layer of step 2) with chloroform, and separating the water layer into a chloroform fraction and a water fraction;

4) fractionating the water fraction of step 3) with ethyl acetic acid to separate an ethyl acetic acid fraction and a water fraction; and

5) fractionating the water fraction of step 4) with butanol, and separating the butanol fraction and the water fraction; may be prepared and finally separated from the butanol fraction.

As the salt, an acid addition salt formed with a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, and organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, and the like. Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, sube Late, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like. The acid addition salt can be prepared by a conventional method, for example, by dissolving the derivative of Formula 1 in an organic solvent such as methanol, ethanol, acetone, dichloromethane, acetonitrile, etc. and adding an organic or inorganic acid to filter the resulting precipitate; It can be prepared by drying, or by distilling the solvent and an excess of acid under reduced pressure, followed by drying and crystallization in an organic solvent.

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 alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate). Furthermore, the present invention includes not only the compound represented by Formula 1 and pharmaceutically acceptable salts thereof, but also solvates, optical isomers, hydrates, and the like, which may be prepared therefrom. In addition, the present invention provides a pharmaceutical composition for preventing or treating obesity or metabolic syndrome, comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof. As in the experimental example to be described later, as confirmed by the Oil red O staining method, the compound according to the present invention selectively decomposes the excessively accumulated lipid droplets in adipocytes and the size of the accumulated fat granules is small. It was confirmed to lose weight, and the effect of inhibiting triglycerides was confirmed. These results are evidence that the compound according to the present invention can selectively reduce or eliminate only excess fat cells, that is, fat cells that can cause disease, and thus increase energy consumption through changes in metabolic processes. It can be an effective alternative to existing drugs that are concerned about the occurrence of side effects by inducing changes in the eating pattern.

Examples of the metabolic syndrome include myocardial infarction, arteriosclerosis, hyperlipidemia, hypertension, cerebral infarction, cerebral hemorrhage, fatty liver, and any one selected from the group consisting of type 2 diabetes. This metabolic syndrome is a disease that is accompanied by abnormalities in metabolic functions such as an increase in body fat, an increase in blood pressure, and abnormalities in blood lipids, and obesity due to a decrease in physical activity along with a high-fat diet intake. Therefore, treatment or prevention of obesity through the removal of excess body fat will be effective in improving the prognosis of metabolic syndrome as well. As used herein, the term “prevention” refers to any action that inhibits or delays the occurrence, spread, and recurrence of the above-described diseases by administration of the pharmaceutical composition of the present invention, and “treatment” refers to administration of the pharmaceutical composition of the present invention. This means any action that improves or beneficially changes the symptoms of the above disease.

The pharmaceutical composition according to the present invention may be formulated in an oral or parenteral dosage form according to standard pharmaceutical practice. These formulations may contain additives such as pharmaceutically acceptable carriers, adjuvants or diluents in addition to the active ingredient. Suitable carriers include, for example, physiological saline, polyethylene glycol, ethanol, vegetable oil and isopropyl myristate, and diluents include, for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or the like. or glycine, and the like, but is not limited thereto. In addition, the compounds of the present invention can be dissolved in oil, propylene glycol or other solvents commonly used for the preparation of injectable solutions. In addition, the compounds of the present invention may be formulated into ointments or creams for topical action.

A preferred dosage of the compound represented by Formula 1 according to the present invention varies depending on the condition and weight of the patient, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for desirable effects, it is preferable to administer the compound represented by Formula 1 according to the present invention at 00001 to 100 mg/kg (body weight), preferably 0001 to 100 mg/kg (body weight) per day. Administration may be administered once a day or in divided doses via oral or parenteral routes.

Depending on the administration method, the pharmaceutical composition according to the present invention may contain 0001 to 99% by weight, preferably 001 to 60% by weight of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention. .

The pharmaceutical composition of the present invention may be administered to mammals, including rats, mice, livestock, and humans, by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection.

According to another aspect of the present invention, there is provided an obesity-related metabolic disease prevention or health functional food comprising the compound of Formula 1 as an active ingredient. The health functional food means a food manufactured and processed using raw materials or ingredients useful for the human body in accordance with the Health Functional Food Act, and the functionality is to control nutrients or physiologically affect the structure and function of the human body. It may refer to ingestion for the purpose of obtaining useful effects for health purposes, such as action. The health functional food may include normal food additives, and unless otherwise specified, the standards and standards for the relevant item according to the general rules and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety. conformity can be judged by Items described in the Food Additives Codex include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; Mixtures, such as an added alkali agent, a preservative agent, and a tar dye agent, are mentioned. The health functional food can be used in various ways, such as foods and beverages for the prevention or improvement of metabolic diseases, for example, various foods, beverages, gum, tea, vitamin complexes, health functional supplements, food additives, and the like. The health functional food may be manufactured and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and pills for the purpose of preventing or improving metabolic diseases. Specifically, the health functional food in the form of tablets is granulated with a mixture of the compound, excipient, binder, disintegrant, and other additives in a conventional manner, and then a lubricant is added thereto and compression-molded, or the mixture is directly compression-molded. can be manufactured. In addition, the health functional food in the form of tablets may contain a mating agent, etc., if necessary, and may be coated with a suitable skinning agent if necessary.

Among the health functional foods in the form of capsules, hard capsules can be prepared by filling conventional hard capsules with a mixture of the compound and additives such as excipients, or their granules or coated granules, and soft capsules include the compound and excipients It can be prepared by filling a mixture with additives, such as gelatin, in a capsule base. The soft capsules may contain, if necessary, a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like. The health functional food in the form of a ring is formed by molding a mixture of the compound, excipient, binder, disintegrant, etc. in an appropriate way. It may be prepared, and if necessary, it may be coated with sucrose or other suitable skinning agent, or may be coated with starch, talc or a suitable material. The health functional food in the form of granules may be prepared in a granular form by an appropriate method of a mixture of the compound, excipients, binders, disintegrants, etc., and may contain flavoring agents, flavoring agents, etc. as necessary. In addition, the definitions of terms for the excipients, binders, disintegrants, lubricants, flavoring agents, and the like are described in documents known in the art and may include those having the same or similar functions.

Those skilled in the art to which the present invention pertains will be able to apply by changing the type, introduction ratio, etc. of each configuration based on the description of the present invention, and if equivalent technical effects are realized despite the above modifications, the present invention It will be said that it is encompassed by the technical thought of

Through the following examples, the present invention will be described in more detail, but it is obvious that the present invention is not limited by the following examples.

[Example 1] Separation of compounds of the present invention from honey ant odor extract

1. Preparation of fractions from beehive odor water extract

In 2018, the leaves grown in the test field of the Ginseng Special Crop Department of the National Academy of Horticultural and Herbal Science, Rural Development Administration, were harvested and then freeze-dried. About 10 times of distilled water was added to the honey ant odor sample, extracted three times for 24 hours at room temperature, and then filtered. The filtered extract was freeze-dried to obtain a water extract of honey ant odor. After dissolving the honey ant odor extract in 20 times distilled water, it was put into a fractionation funnel (separation funnel), the same amount of nucleic acid was added, mixed well, and separated into a water layer and a nucleic acid layer, and the above process was repeated 3 times. Chloroform was added to the water layer again to separate the water layer and the chloroform layer, and this was repeated 3 times. Thereafter, ethyl acetate and butanol layers were also obtained in the same manner as above to obtain respective fractions (see FIGS. 1 and 2 ).

2. Separation of novel compounds from butanol fraction

The butanol fraction of honey ant odor was eluted with medium-pressure liquid chromatography (packed C18 gel, Water:MeOH=1:1 to 3:7; KeyChem-Flash, YMC, Kyoto, Japan) to obtain three subfractions. , Multiple prep-HPLC (recycling, flow rate, 30.0 ml/min; UV, 210 nm; LC-Forte/R, ODS-AM column; YMC, Kyoto, Japan) was performed for small fraction 3 of these with 52% MeOH. and separated. The purely isolated product was dissolved in pyridine-d ₅ and NMR analysis was performed. Mass was measured using Hybrid LC/Q-TOF system (maXis 4G; Bruker BioSciences, Rheinstetten, Germany), and ¹ H-, ¹³ C-NMR and 2D-NMR (COSY, HMQC, HMBC) were 400 MHz FT- Measurements were made with an NMR spectrometer (AVANCE III 400 MHz (Bruker BioSciences, Rheinstetten, Germany)).

The results are shown in FIGS. 3 to 6 and Table 1 below. The isolated compound below was named 28-O-xylosyl-asterogenic acid, and has the chemical structure of Formula 1 below (C ₃₅ H ₅₇ O ₉ ).

[Formula 1]

[Table 1]

[Example 2] Confirmation of anti-obesity effect of the compound of the present invention

1. Confirmation of inhibition of adipocyte differentiation

(1) Preadipocytes (3T3-L1) were purchased from the American Type Culture Collection (CL-173, ATCC, VA, USA). 3T3-L1 cell growth medium, Dulbecco's modified Eagles medium (DMEM, Gibco, Canada) was used and cultured at 37°C and 5% CO2 conditions. 1×10 ⁵ cells/mL were incubated in a 12 well-plate at 37°C and 5% CO2 environment until the cell density reached 100%. Differentiation induction medium for inducing differentiation into adipocytes is 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich, St. Louis, MO, USA), 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO) , USA), 10 μg/mL insulin (Gibco, Canada), 1X P/S/G, and DMEM medium containing 10% fetal bovine serum (FBS, Gibco, Canada) were used. To induce differentiation into adipocytes, they were cultured in a differentiation induction medium for 48 hours and then replaced with a differentiation medium containing insulin 10 μg/ml. After replacing the medium every 48 hours, the differentiation induction state was confirmed. In order to check the efficacy of inhibiting differentiation by treating the compounds during the differentiation induction process, the extract was treated with the differentiation induction medium. Differentiation induced without treatment with the extract was compared as a control.

(2) Remove the adipocyte culture medium, wash twice with DPBS (Gibco, Canada), treat with 10% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and leave at room temperature for 10 minutes to fix the cells did. After that, it was washed twice with dH2O, and 60% isopropanol was added and left at room temperature for 5 minutes. After removing 60% isopropanol and staining with Oil Red O solution (Sigma, St. Louis, MO, USA) for 10 minutes, adipocyte differentiation was confirmed using a phase contrast microscope. In addition, for quantification, isopropanol (Sigma, St. Louis, MO, USA) was added to elute and recover the stained reagent, and then absorbance at 520 nm using a multi plate reader (Synergy H1, Biotek, VT, USA) was measured.

3. Measurement of triglyceride inhibitory ability

After washing with PBS in adipocyte medium, 5% NP-40/ddH2O was put and dissolved. Transfer to a new tube, heat twice at 80-100°C for 5 minutes, centrifuge at 14000×g for 2 minutes, and measure the supernatant using Triglyceride Assay Kit (bioassay system, Hayward, CA, USA).

4. Check the results

As shown in Figure 7 A to C, the compound of formula 1 of the present invention confirmed the adipocyte differentiation inhibitory ability and triglyceride inhibitory ability, so that the compound of the present invention can be used for the prevention or treatment of various metabolic diseases, in particular, obesity. confirmed that there is.

Claims (13)

A compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof;
[Formula 1]

The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound has an adipocyte differentiation inhibitory effect. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound has a neutral fat inhibitory effect. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is isolated from honey ant odor. [Claim 5] The compound or a pharmaceutically acceptable salt thereof according to claim 4, wherein the compound is isolated from a honey ant odor water extract. [Claim 6] The compound or a pharmaceutically acceptable salt thereof according to claim 5, wherein the compound is isolated from the butanol fraction of the water extract of honey ants. The compound or a pharmaceutically acceptable salt thereof according to claim 6, wherein the compound is isolated by sequential fractionation of the honey ant odor water extract with n-hexane, chloroform, ethylacetic acid and butanol. 1) extracting honey ant odor with distilled water;
2) fractionating the honey ant odor water extract with n-hexane, separating the n-hexane fraction and the water fraction;
3) fractionating the water layer of step 2) with chloroform, and separating the water layer into a chloroform fraction and a water fraction;
4) fractionating the water fraction of step 3) with ethyl acetic acid to separate an ethyl acetic acid fraction and a water fraction; and
5) fractionating the water fraction of step 4) with butanol to separate the butanol fraction and the water fraction;
[Formula 1]

Process according to claim 8, characterized in that the compound is isolated in the butanol fraction.
An anti-obesity composition comprising, as an active ingredient, the butanol fraction of the honey ant odor water extract. 11. The method of claim 10, wherein the fraction is 1) extracting honey ant odor with distilled water;
2) fractionating the honey ant odor water extract with n-hexane, separating the n-hexane fraction and the water fraction;
3) fractionating the water layer of step 2) with chloroform, and separating the water layer into a chloroform fraction and a water fraction;
4) fractionating the water fraction of step 3) with ethyl acetic acid to separate an ethyl acetic acid fraction and a water fraction; and
5) The composition for anti-obesity, which was prepared by fractionating the water fraction of step 4) with butanol. A pharmaceutical composition for anti-obesity comprising a compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

A food composition for preventing or improving obesity comprising a compound having the structure of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

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