KR100436920B1 - Novel polyacetylene group compound from ginseng, process for extraction thereof and anti-obesity agent including thereof - Google Patents

Abstract

The present invention relates to a novel polyacetylene-based compound extracted from Korean ginseng, a method of extracting the same, and an anti-obesity agent containing the same, wherein the novel polyacetylene-based compound isolated and purified from ginseng according to the present invention (panaxinone A) Business Card) has an excellent effect of inhibiting obesity by inhibiting ACAT activity and reducing cholesterol from being absorbed into the body.

Description

Novel polyacetylene group compound from ginseng, process for extraction according to ginseng and anti-obesity agent, including novel novel polyacetylene-based compound extracted from ginseng

The present invention relates to a novel polyacetylene-based compound extracted from ginseng, a method for extracting the same and an anti-obesity agent containing the same. More specifically, the present invention relates to a novel polyacetylene-based compound having ACAT inhibitory activity extracted and concentrated and then purified by separating Korean ginseng with an organic solvent, an extraction method thereof, and an obesity treatment agent comprising the polyacetylene-based compound.

Acyl CoA: Cholesterol O-acyltransferase (hereinafter referred to as ACAT) is an enzyme that generally esterifies cholesterol, and its mechanism of action is largely divided into three parts of the body (intestinal, liver, and vascular wall cells). Takes place in). First, cholesterol that is absorbed from the outside or biosynthesized in the body is accumulated in a carrier called VLDL (very low density lipid) in the liver and then supplied to each organ through the blood vessel, whereby the cholesterol is converted into cholesterol ester form by ACAT. Cholesterol accumulation becomes possible in the inside. Second, in the cells that make up the arterial vessel wall, ACAT converts cholesterol into its ester form to promote the accumulation of cholesterol in the cell, which is a direct cause of atherosclerosis. Third, in the intestine, ACAT converts the ingested cholesterol into the form of esters to facilitate its absorption into the intestine. Thus, drugs that inhibit the activity of ACAT may firstly inhibit cholesterol release into the blood vessels in the liver, thereby lowering blood cholesterol levels, and secondly, prevent cholesterol from accumulating in blood vessel wall cells, thereby directly preventing arteriosclerosis. It is expected to be able. Therefore, Korean Patent Registration No. 10-027958 discloses a prophylactic and therapeutic agent for cardiovascular diseases containing ACAT activity inhibitor extract from the leaves of Takhu, and a new compound having ACAT inhibitory activity in Korean Patent Registration No. 10-254809 and The use of the compound as an agent for preventing and treating cardiovascular diseases has been disclosed.

However, drugs that inhibit the activity of ACAT can reduce the amount of cholesterol that enters the body by inhibiting the absorption of cholesterol in the intestine, and is expected to be able to treat obesity by preventing the accumulation of stored cholesterol such as the abdomen.

The cause of obesity is a symptom that is caused by excess energy. When energy intake is more than consumed or consumed less than intake, excess energy accumulates into fat, causing fat metabolism abnormally high, resulting in abnormal lipid metabolism. Obesity is known to cause high blood pressure or cardiovascular disease, type 2 diabetes, hyperlipidemia, fatty liver, osteoarthritis, infertility, gastroesophageal reflux, urinary incontinence, and increase the incidence of cancers such as breast cancer and colorectal cancer. In particular, women's obesity is caused by abnormal sex hormone metabolism and appetite centers, causing menstrual disorders and uterine weakness, causing ovarian dysfunction, causing difficulties in pregnancy. Obese women are more likely to develop endometrial cancer than postmenopausal women and have a higher incidence when they have diabetes or hypertension.In obese women, ovulation disorders and amenorrhea appear and female hormone levels decrease. Increasing body weight has been reported to increase the risk of breast cancer. Therefore, obesity is emerging as a risk factor in industrialized society. For this reason, many studies related to obesity have been conducted, but the obesity-related metabolism is so complicated that only a partial mechanism of action has been identified.

The present inventors searched for ginseng in the process of searching for an active substance that suppresses obesity from Korean native plants.

Korean ginseng is Korea's unique natural resource, and it is a representative herbal medicine of Korea, which has been widely used for thousands of years as an oriental medicine or medicine for the health of mankind. As Western medicine has shown limitations in overcoming the side effects of synthetic drugs and intractable diseases, the value of herbal medicines including ginseng is emerging.

Ginseng prevents cancer, inhibits aging, protects the liver, strengthens fatigue and improves brain function, and recently has been reported to prevent the damage of environmental hormones. Of all the herbal medicines, ginseng is a plant that grows in an extremely limited environment. It has a longer growth period than other crops and its growing method is special and difficult, and has been produced only in some selected countries around the world. Korea was a ginseng seed country by producing Korean ginseng with excellent medicinal effects due to its climate and climate as a natural growth and planting area of ginseng.

However, many countries are paying attention to the ideal condition that ginseng has as a health food or health drug, and not only existing ginseng producing countries but also non-producing countries are actively promoting policies to encourage the research and production of ginseng. Ginseng growers include Korea, China, Japan, the United States, Canada, and parts of Europe. Most of the countries that consume ginseng are Confucian culture, including Korea, China, Japan, Taiwan, and Southeast Asia.

Korean ginseng has been used as a treatment and prevention agent for various adult diseases for a long time, and animal studies have also reported that the compounds in ginseng lower the total cholesterol in the blood. (See A.A. Qureshi, Z.Z.Din, et al., Atherosclerosis, vol. 48, 81-94 (1983).

The present invention is isolated and purified from a new polyacetylene-based compound having ACAT inhibitory activity from Panax ginseng CA Myers (Korean Panax ginseng CA Myers) to determine its structure, and confirmed that the compound has an active effect on inhibiting obesity through animal experiments It was completed by.

An object of the present invention is to provide a novel polyacetylene-based compound having ACAT inhibitory activity separated and purified from ginseng. Another object of the present invention to provide a method for producing a novel polyacetylene compound having ACAT inhibitory activity from ginseng. Still another object of the present invention is to provide an obesity treatment agent using the novel polyacetylene-based compound as an active ingredient.

The present invention extracts Korean ginseng ( Panax ginseng CA Myers) with an alcohol solvent, and purified by separation to prepare a novel polyacetylene-based compound having ACAT inhibitory activity, to determine its structure, and through the animal experiments the compound obesity It was completed by confirming that there was an effect on inhibition.

Hereinafter, the configuration and operation of the present invention.

1 shows hydrogen nuclear magnetic resonance spectra of polyacetylene-based compounds purified from ginseng.

Figure 2 shows the carbon nuclear magnetic resonance spectrum of the polyacetylene-based compound purified from ginseng.

Figure 3 shows the hydrogen-carbon nuclear magnetic resonance spectrum of the polyacetylene-based compound purified from ginseng.

Figure 4 is a graph showing the weight change that appears when the new polyacetylene-based compound according to the invention administered to the experimental animal.

The present invention relates to a novel polyacetylene compound represented by the following formula (1) having ACAT inhibitory activity isolated from ginseng.

[Formula 1]

Ginseng was washed and pulverized, and the pulverized ginseng was extracted with an alcohol solvent, preferably ethanol, and concentrated. The mixture was separated by adding ethyl acetate and water to the concentrate, and the organic layer was separated and concentrated under reduced pressure. The concentrate was purified by silica gel chromatography. To obtain the active fraction. The active fraction thus obtained was purified by medium pressure chromatography and finally purified pure active material using HPLC. As a result of structural analysis of the isolated active material through nuclear magnetic resonance (NMR) analysis, it was determined by Chemical Formula (1).

As a result of measuring the ACAT inhibitory activity of the novel polyacetylene compound of formula (1) isolated and purified from ginseng according to the present invention, IC ₅₀ is 7 µg / ml. These compounds, which inhibit the action of ACAT, can be used as therapeutic agents for obesity because they inhibit the absorption of cholesterol in the intestine and reduce the accumulation of cholesterol. And ginseng has been used as a herbal medicine and edible for a long time can be expected that the compounds of the present invention extracted from them also have no problems such as toxicity and side effects.

The polyacetylene-based compounds separated and purified according to the present invention may be formulated into a unit dosage form or a multiple dosage form such as tablets, capsules, granules, suspensions, and emulsions by conventional methods and used as therapeutic agents for obesity.

Compositions containing the compounds separated and purified according to the present invention can be parenterally or orally administered as desired, and the novel polyacetylene compounds according to the present invention can be used in an amount of 10 to 20 mg per 1 kg of body weight. Can be administered in several divided doses. Dosage levels for specific patients may vary depending on gender, age, health status, diet, time of administration, method of administration, and drug severity and disease severity.

Hereinafter, the specific configuration and operation of the present invention will be described in detail by way of examples, but the scope of the present invention is not limited only to these embodiments.

Example 1 Isolation and Purification of Novel Polyacetylene Compounds from Ginseng

Ginseng used in the present invention was purchased from Yangnyeong-si, Geumsan, Chungcheongnam-do, as a four-year-old ginseng. The fresh 4 year old ginseng was washed well and ground using a green juicer. 7.5 L of ethanol was added to 7.5 kg of ground ginseng and immersed at room temperature for 24 hours, followed by stirring to separate the liquid and solid parts using a filter paper. After collecting the liquid phase and concentrated under reduced pressure, 2L ethyl acetate was added and stirred to separate the water layer and the organic solvent layer and extracted three times to collect the organic solvent layer and concentrated under reduced pressure. In vitro activity test during the extraction process showed that the organic solvent layer had inhibitory activity of acyl-CoA: cholesterol acyltransferase (ACAT). The organic solvent layer was concentrated to dryness under reduced pressure to obtain 50 g of a yellowish brown oily substance, dissolved in ethanol, and adsorbed and dried to an oily substance in silica gel (Merck, trade name: 9385), followed by increasing and changing the mixing ratio of ethyl acetate in normal hexane (100 : 1 to 10: 1) The material was separated by flowing. In the same manner, the active fraction was obtained using two silica gel column chromatographys, purified by medium pressure chromatography [ n -hexane: CHCl ₃ (90: 10), flow rate: 5 ml / min, UV: 254 nm], and finally Pure actives were purified using HPLC. At this time, YMC-Jsphere ODS-H80 (250 x 20 mm) was used as a separation HPLC column. Active substances were eluted at 7 ml / min with a solvent of 88% ethanol and 12% distilled water, and UV: 254 nm was detected. Finally, the pure active material isolated was obtained in a yield of 500 mg.

Example 2 Structural Analysis of the Purified Active Substance

The active material finally separated and purified by HPLC was measured at 210, 240, 253, 266 and 280 nm using an ultraviolet-visible spectrometer (Shimadzu, UV-265). Maximum IR absorbance was measured with a ratio recording infrared spectrometer (Bio-Rad Digilab Division, FTS-80). As a result, the presence of conjugated triple bonds (2235 cm ^-1 ) and carbonyl groups (1644 cm ^-1 ) was estimated.

The high resolution MS was measured using a VG70-SEQ mass spectrometer to measure (M + H) as 261.1852 and 261.1855 in the formula, so the active material was determined by molecular weight 260 and molecular formula C ₁₇ H ₂₄ O ₂ .

In nuclear magnetic resonance (NMR) analysis, 10 mg of the active substance sample was completely dried, dissolved in C6D6, placed in a 5 mm NMR tube, and subjected to NMR analysis using a Varian model. ¹ H-NMR was 300 MHz and ¹³ C-NMR was Measured at 75 MHz. The structure of the material is estimated to be 24 hydrogen and 17 carbon atoms, 2 methyl, 8 methylene, 2 oxygen-bearing metine, 4 in the DEPT spectrum The presence of two conjugated acetylenic carbons and one carbonyl carbon was estimated. In the high magnetic field (δ1.13-1.29) of the hydrogen nuclear magnetic resonance, the signals connected by methylene in the structure could not be directly determined. In the structure of the active material, δ 2.70 (1H, ddd, J = 4.0, 5.7, 6.6 Hz) and δ 2.55 (1H, m) are the presence of epoxy protons, δ 2.17 (1H, dd, J = 5.7, 18.0 Hz) and δ 1.92 (1H, dd, J = 6.6, 18.0 Hz) indicate that methylene proton is between triple bond and epoxide, δ 1.13 δ 1.29 is 12 methylene proton , Δ 2.06 (2H, q, J = 7.5 Hz) is present next to carbonyl carbon and conjugated methylene hydrogen, δ 0.79 (3H, t, J = 7.5 Hz, 1-H ) And 0.90 (3H, t, J = 6.9 Hz, 17-H) were estimated to contain terminal methyl groups. DEPT and HMQC was estimated in part by the type and position of the associated carbon and hydrogen in the spectrum (spectrum), to estimate the partial structure of the material through the ¹ H- ¹ H COSY experiment.

Hydrogen nuclear magnetic resonance of the active substance of the present invention (300 MHz, C6D6): d = 0.79 (3H, t, J = 7.5 Hz, 1-H), 0.90 (3H, t, J = 6.9 Hz, 17-H ), 1.13 1.29 (12H, bm, 11-H 16-H), 1.92 (1H, dd, J = 6.6, 18.0 Hz, 8-Ha), 2.06 (2H, q, J = 7.5 Hz, 2-H) , 2.17 (1H, dd, J = 5.7, 18.0 Hz, 8-Hb), 2.55 (1H, m, 10-H), 2.70 (1H, ddd, J = 4.0, 5.7, 6.6 Hz, 9-H) In carbon nuclear magnetic resonance (75 MHz, C6D6): d = 8.09 (q, C-1), 14.68 (q, C-17), 20.15 (t, C-8), 23.39 (t, C -16), 27.16 (t, C-11), 28.11 (t, C-12), 29.91 (t, C-13), 30.02 (t, C-14), 32.48 (t, C-15), 39.11 (t, C-2), 53.91 (d, C-9), 56.69 (d, C-10), 66.41 (s, C-6), 74.00 (s, C-5), 74.81 (s, C- 7), 85.37 (s, C-4) and 186.40 (s, C-3) were obtained, so ¹ H, ¹³ C, Cozy and HMQC spectrums were obtained. Determined by the formula (1), the novel compound isolated in the present invention was named panaxynone A. Therefore, hereinafter, the novel compound according to the present invention will be referred to as panaxynone A.

Example 3: ACAT Activity Assay of Panaxinone A

The activity measurement of the ACAT (acyl-CoA: cholesteryl acyltransferase) inhibitor was used with a slight modification of the Ericson method. As the ACAT activating enzyme source, microsomes partially purified from livers of experimental rats not contaminated with specific microorganisms were used in the laboratory of the Korea Research Institute of Bioscience and Biotechnology. The substrates were cholesterol and radiolabeled oleoyl. ) Co-A was reacted to measure the degree of reaction by the amount of radioactivity contained in the reaction product, cholesterol ester (cholesterol ester). Simply stated, a mixture of cholesterol dissolved in acetone and Triton WR-1339 dissolved in acetone is completely suspended in water, the acetone is removed with nitrogen gas, and then potassium-phosphate buffer (K-phosphate buffer, pH). 7.4, the final concentration of 0.1 M) to stabilize the enzymatic reaction, add 30 μM of bovine serum albumin to the final concentration, 10 μl of sample dissolved in DMSO and shake for 30 minutes at 37 ℃ After the reaction, the reaction was carried out with a substrate of [1- ¹⁴ C] oleoyl-Co A (0.04 μCi) and shaking at 37 ° C. for 30 minutes, followed by adding 1 ml of isopropanol-heptane. Stop the mixture, add 0.6 ml of n -heptane and 0.4 ml of KPB buffer, mix well, and let stand for 2 minutes. After separating, take 200 µl of the supernatant into a scintillation vial and scintillation cock. 4 ml of tail, Lipoluma, Lumac Co.) was added to measure the amount of cholesteryl oleate produced at the scintillation counter (Packard Delta-200). ACAT inhibitory activity was calculated according to the following equation.

Inhibitory activity (%) = [1- (T-B / C1-B)] x 100

T: Put the sample in the enzyme reaction solution, cpm value of the test

C: cpm value of the control without the sample in the enzyme reaction solution

B: cpm value of the control in which the sample was added without the enzyme source

Wave naksi non this acyl co A cholesterol acyl-trans spread a result of measuring a cyclase inhibition rate of the concentration that inhibits the enzyme activity to 50% that is was IC ₅₀ is measured by 7 ㎍ / ml, because it was the Active Compounds molecular weight 260. The IC ₅₀ Calculated to 26.7 μM.

Example 4 Animal Activity Test of Panaxinone A

In order to detect the obesity-related in vivo activity of the active substance, the test animal was a free feeding laboratory animal feed (first feed) with a free feeding laboratory animal feed (first feed). ZDF (fa / fa, Hanlan, USA) three-week-old rats that were systematically maintained for obesity experiments were subjected to pure quarantine for one week. The average weight of four-week-old rats was 123 ± 2 g. The feed water was freely fed with autoclaved tab water for 20 minutes at 121 ° C., and the feed was sterilized with custom cholesterol-added experimental animal feed (Oriental. Co. Ltd. Japan).

The control group received only cholesterol-added experimental animal feed without feeding panaxinone A. After 21 days, the control group fed freely by changing to ordered experimental animal feed (first feed). Except for the 10 mg / kg / day, the same conditions as the control group were tested. The results of measuring the body weight of the experimental animals for 56 days are shown in Table 1 and FIG. 4.

Weight Comparison of Rats Fed with Panaxinone A According to the Present Invention Time (week) Panaxinone A salary group (g) Control (g) 0 123 122 One 188 201 2 225 246 3 262 286 4 291 318 5 316 346 6 334 373 7 342 391 8 375 407

In the comparison of control and panaxinone A-weighted groups at weekly intervals, 6.5%, 8.6%, 8.4%, 8.5%, 8.7%, 10.5%, 13%, 7.9 after the first week compared to the control. Body weight loss was observed by about 8%, and body weight was reduced in the 8-week animal test, but no signs of toxicity were observed in the experimental animals.

As described above, the present invention provides a novel polyacetylene-based compound that inhibits ACAT activity from ginseng to prevent cholesterol from being absorbed into the body, thereby reducing body weight and reducing obesity. Since it is effective, it is a very useful invention for the industrial field of medicine.

Claims (4)

Polyacetylene type compound represented by following General formula (1). [Formula 1] The method of preparing a compound according to claim 1, which comprises pulverizing ginseng, extracting with an alcohol solvent, concentrating the extract, and separating the layers by adding ethyl acetate and water to the concentrate, and then separating and purifying the organic layer by vacuum concentration. . delete An obesity treatment agent comprising the compound of claim 1 as an active ingredient.