TW202112361A - Use of compound for preventing and/or treating obesity - Google Patents

Abstract

The present invention provides a use of compound for preventing and/or treating obesity, wherein the compound is selected from the group consisting of (E)-3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid)、7-[[2-O-(6-Deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]]oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one、5,7-Dihydroxy-2-(4-hydroxyphenyl) chroman-4-one, and any combination thereof. The compound can effectively promote lipolysis and/or reduce cell fat content and can effectively reduce the fat content in cells to achieve the purpose of preventing and/or treating obesity.

Description

Use of compound for preventing and/or treating obesity

The present invention relates to the use of a compound for preventing and/or treating obesity, wherein the compound is selected from the group consisting of: 4-hydroxy-3-methoxycinnamic acid can also be called 3-(4 -Hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-pyranosyl ])Oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, 5,7-dihydroxy-2-( 4-hydroxyphenyl)benzo-4-one, and any combination thereof.

In recent years, the global incidence of obesity has gradually increased. The World Health Organization (WHO) has even described the rapidly spreading obesity as "infectious disease" and called it "Global Obesity" (Globesity), according to 2014 The World Health Organization estimates that about 39% of the global population is overweight (about 1.9 billion people), and 13% of the population is obese (about 600 million people). With changes in eating habits and improved quality of life, the prevalence of obesity in Taiwan has also increased year by year. According to the survey on changes in national nutrition and health published by the National Health Administration of the Ministry of Health and Welfare, the prevalence rate of adult overweight or obesity is as high as 40%. The ratio of male to female is 49% and 40% respectively. In other words, one out of every two males in Taiwan is overweight or obese, and the average female is two to three. One is overweight or obese, and more than 200,000 of them have reached the standard for morbid obesity, and must be treated with surgery.

Obese individuals increase the risk of a variety of health problems and may lead to a variety of complications, including high blood pressure, hyperlipidemia, cardiovascular disease, sleep apnea, angina, degenerative arthritis, and hyperuricemia. Diseases such as inflammation, type 2 diabetes, and cancer; among them, due to the body Too many internal fat cells will secrete harmful substances that hinder the action of insulin, which will hinder the function of insulin, which will not be able to effectively control the rising blood sugar, which will lead to the symptoms of hyperglycemia. If the body is in hyperglycemia for a long time, the pancreas will be exhausted. More insulin to lower blood sugar, once the pancreas is overloaded, it will develop into type 2 diabetes. In addition, blood lipids refer to fats in the blood, mainly including cholesterol and triglycerides. When the concentration of cholesterol or triglycerides circulating in the blood is higher than normal, it is called hyperlipidemia; and cholesterol or triglycerides When the ester value is too high, or the concentration of high-density lipoprotein is too low, it is also called dyslipidemia. In addition to causing heart disease, hyperlipidemia is also closely related to chronic diseases such as stroke, hypertension, diabetes, and kidney disease.

Therefore, obesity reduces the quality of life and may lead to premature death, so the average life expectancy of patients with morbid obesity is much shorter than that of people with normal weight. Although genes may be involved in the development of obesity, the obesity epidemic is mainly due to high-calorie diets and sedentary lifestyles. Therefore, lifestyle changes can prevent obesity and its complications.

Although low-calorie diets and regular exercise can be used to reduce weight and treat obesity, these methods are difficult to implement and their efficacy is limited, mainly due to the adaptive physiological mechanisms that maintain energy storage in the body. In addition, some drugs have been approved for the treatment of long-term obesity (such as orlistat, phentermine, or topiramate), but these drugs often cause serious side effects, which limit the efficacy of treatment and patient compliance; and In addition, bariatric surgery can significantly cause weight loss, but this interventional surgery is not suitable for all overweight people; based on the above reasons, there is indeed a need to study safe and effective ways to reduce weight and fat accumulation.

In summary, in response to the obesity faced by modern people due to changes in life and eating habits and the overall health problems caused by obesity, and based on the improvement of modern people’s living standards and the improvement of the concept of health care, we have developed a method that can effectively promote lipolysis to It is really necessary to reduce the formation of fat and reduce the chance of obesity.

For this reason, one object of the present invention is to provide a composition for the preparation of a drug for preventing and/or treating obesity, wherein the composition contains an effective concentration selected from the following Compounds in the group: 4-hydroxy-3-methoxycinnamic acid can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid)((E)-3-(4-hydroxy -3-methoxy-phenyl)prop-2-enoic acid), 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-pyranopyranosyl] ]Oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one (7-[[2-O-(6- Deoxy-α-L-mannopyranosyl)-β-D-gluco-pyranosyl]]oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), 5,7-Dihydroxy-2-(4-hydroxyphenyl)chroman-4-one (5,7-Dihydroxy-2-(4-hydroxyphenyl)chroman-4-one), and any combination thereof, and one A pharmaceutically acceptable carrier.

Another object of the present invention is to provide a composition for the preparation of a lipolysis-promoting medicine, wherein the composition contains an effective concentration of a compound selected from the group consisting of: 4-hydroxy-3 -Methoxycinnamic acid can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O-(6-deoxy-α-L-mannopyranose Yl)-β-D-pyranopyranosyl])oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4 -Ketone, 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one, and any combination thereof, and a pharmaceutically acceptable carrier.

Another object of the present invention is to provide a 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-pyranopyranosyl]]oxy]-2 The use of 3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one for the preparation of drugs for preventing and/or treating obesity.

In an embodiment of the present invention, the effective concentration of the compound is at least 10 μg/mL.

In another embodiment of the present invention, the medical composition promotes the decomposition of fat; and the medical composition promotes the decomposition of fat in adipocytes.

In another embodiment of the present invention, the 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-pyranopyranosyl]]oxy] The effective concentration of -2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one is at least 10μg/mL.

In another embodiment of the present invention, the 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-pyranopyranosyl]]oxy] -2,3-Dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one promotes the decomposition of fats.

The compound 4-hydroxy-3-methoxycinnamic acid of the present invention can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O-(6-deoxy -α-L-Mannopyranosyl)-β-D-pyranosyl]]oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H -1-benzopyran-4-one, 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one, 5-hydroxy-2-(3-hydroxy-4-methoxy Phenyl)-4-oxo-3,4-dihydro-2h-chromium-7-yl-2-o-(6-deoxyhexapyranosyl)hexapyranoside, and (S)-2 ,3-Dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, the 4-hydroxy-3- Methoxycinnamic acid can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), the 7-[[2-O-(6-deoxy-α-L-mannopyranose Yl)-β-D-pyranopyranosyl])oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4 -Ketone and the 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one can effectively promote lipolysis to reduce the fat content in cells. Therefore, the 4-hydroxy-3-methoxycinnamic acid of the present invention can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O-(6 -Deoxy-α-L-Mannopyranosyl)-β-D-pyranosyl))oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl) -4H-1-benzopyran-4-one and 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one can be used for preparation to promote lipolysis and/or reduce cell fat content The use of the composition, and the composition is a medicine, a skin care product, or a food, which can be administered to a body by oral, smearing, etc.

The following will further illustrate the implementation of the present invention in conjunction with the drawings. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone who is familiar with the art will not depart from the spirit and spirit of the present invention. Within the scope, some changes and modifications can be made. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

Fig. 1 is an HPLC fingerprint analysis chart of Daidaihua extract in one embodiment of the present invention.

Figure 2 is a hydrogen spectrum diagram of the compound TCI-CA-01 purified from the Daidaihua extract in one embodiment of the present invention.

Figure 3 is a mass spectrum of the compound TCI-CA-01 purified from the Daidaihua extract in one embodiment of the present invention.

Figure 4 is a hydrogen spectrum of the compound TCI-CA-02 purified from the Daidaihua extract in an embodiment of the present invention.

Figure 5 is a mass spectrum of the compound TCI-CA-02 purified from the Daidaihua extract in one embodiment of the present invention.

Figure 6 is a hydrogen spectrum diagram of the compound TCI-CA-03 purified from the Daidaihua extract in an embodiment of the present invention.

Figure 7 is a mass spectrum of the compound TCI-CA-03 purified from the Daidaihua extract in one embodiment of the present invention.

Figure 8 is a hydrogen spectrum diagram of the compound TCI-CA-04 purified from the Daidaihua extract in an embodiment of the present invention.

Figure 9 is a mass spectrum of the compound TCI-CA-04 purified from the Daidaihua extract in an embodiment of the present invention.

Figure 10 is the hydrogen spectrum of the compound TCI-CA-05 purified from the Daidaihua extract in one embodiment of the present invention.

Figure 11 is a mass spectrum of the compound TCI-CA-05 purified from the Daidaihua extract in an embodiment of the present invention.

Figure 12 shows the compound TCI-CA-01, the compound TCI-CA-02, the compound TCI-CA-03, the compound TCI-CA-04, and the compound purified from the Daidaihua extract in one embodiment of the present invention A bar graph showing the effect of TCI-CA-05 in promoting lipolysis. ***p<0.001.

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

Use Excel software for statistical analysis. Data mean ± standard deviation (SD), said difference between the groups in a Student's t-test (student's t -test) analysis.

According to the present invention, the medicine can be manufactured into a dosage form suitable for parenterally or orally administration by using techniques well known to those skilled in the art. This includes, but not Limited to: injection (for example, sterile aqueous solution or dispersion), sterile powder, tablet, troche, lozenge Lozenge, pill, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup, elixir ), slurry and the like.

The medicine according to the present invention can be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intramuscular injection (intramuscular injection) and intravenous injection.

The medicine according to the present invention may contain a pharmaceutically acceptable carrier which is widely used in medicine manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of: solvent, emulsifier, suspending agent, decomposer ), binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, Lubricants, absorption delaying agents, liposomes and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

According to the present invention, the pharmaceutically acceptable carrier includes a solvent selected from the group consisting of water, normal saline (normal saline), phosphate buffered saline (PBS), Aqueous solution containing alcohol and combinations thereof.

According to the present invention, the drug can be administered by a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, intradermal injection Injection (intradermal injection) and intralesional injection (intralesional injection).

According to the present invention, a food product can be used as a food additive, which can be added during the preparation of raw materials by a conventional method, or added during the production process of the food, and formulated with any edible material for supply Food products consumed by humans and non-human animals.

According to the present invention, the types of food products include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

According to the present invention, column chromatography and thin layer chromatography (Thin layer chromatography, TLC) are purified from the ethyl acetate layer extract of the flower extract of the present invention. Compounds: 4-hydroxy-3-methoxycinnamic acid can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid)((E)-3-(4-hydroxy-3- methoxy-phenyl)prop-2-enoic acid), named as TCI-CA-01 in this article; 7-[[2-O-(6-deoxy-α-L-mannosyl)-β-D-pyranosyl Pyrananosyl]]oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one(7-[[2 -O-(6-Deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]]oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4 -one), named TCI-CA-02 in this article; 5,7-Dihydroxy-2-(4-hydroxyphenyl)benzo-4-one (5,7-Dihydroxy-2-(4-hydroxyphenyl)chroman -4-one), named TCI-CA-03 herein; 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2h-chromium -7-yl-2-o-(6-deoxyhexapyranosyl)hexapyranoside (5-hydroxy-2-(3-hydroxy-4-methoxy-phenyl)-4-oxo-3,4- dihydro-2h-chromen-7-yl 2-o-(6-deoxyhexopy-ranosyl)hexopyranoside), named TCI-CA-04 herein; and (S)-2,3-dihydro-5,7-dihydroxy -2-(3-Hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one((S)-2,3-Dihydro-5,7- dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one), named TCI-CA-05 in this article.

According to the present invention, the operating procedures and parameter conditions related to the chemical separation and chemical structure analysis of the mixture fall within the professional quality and routine technology of those who are familiar with the technology.

The present invention provides a method for preparing 4-hydroxy-3-methoxycinnamic acid, also called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O-(6- Deoxy-α-L-mannopyranosyl)-β-D-pyranosyl))oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)- 4H-1-benzopyran-4-one and/or 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one are used to promote lipolysis and/or reduce cell fat content The use of the composition can effectively promote the decomposition of fat to reduce the fat content in the cell.

At the same time, the composition of the present invention for promoting lipolysis and/or reducing cell fat content may also contain an effective concentration of a compound selected from the group consisting of: 4-hydroxy-3-methoxy Cinnamic acid can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O-(6-deoxy-α-L-mannosyl)-β -D-pyranopyranosyl]]oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, 5 , 7-Dihydroxy-2-(4-hydroxy-phenyl) benzo-4-one (5,7-Dihydroxy-2-(4-hydroxy-phenyl)chroman-4-one), and any combination thereof, and A pharmaceutically acceptable carrier, and the composition is a medicine, a skin care product, and a food.

The 4-hydroxy-3-methoxycinnamic acid of the present invention will also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O-( 6-deoxy-α-L-mannopyranosyl)-β-D-pyranosyl))oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl) )-4H-1-benzopyran-4-one, 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one, 5-hydroxy-2-(3-hydroxy-4 -Methoxyphenyl)-4-oxo-3,4-dihydro-2h-chromium-7-yl-2-o-(6-deoxyhexapyranosyl)hexapyranoside, and (S ) The detailed extraction and purification method of -2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one; And the five compounds were used in a test experiment to promote lipolysis in fat cells.

Chemical analysis materials

Hexane (n -hexane), ethyl acetate (ethyl acetate), acetone (acetone), methanol (methanol), ethyl alcohol (ethanol), n-butanol (n -butanol), acetonitrile (acetonitrile), chloroform -d1 (Deuterium Degree of sulfoxide 99.5%), methanol-d6 (degree of deuteration 99.5%), heavy water (deuterium oxide, degree of deuteration greater than 99.8%), and dimethyl sulfoxide- d ₆ (dimethyl sulfoxide- d ₆ , degree of deuterium> 99.9%) purchased from Merck, Taiwan.

Chemical analysis equipment

The separation of compounds utilizes column chromatography and thin layer chromatography (TLC). Specifically, the tube housing of the column chromatography is selected from Sephadex LH-20 (purchased from Amersham BioSciences, UK), Diaion HP-20 (purchased from Mitsubishi Chemical Co., Japan), Merck Kieselgel 60 (40-63μm, Purchased from Merck, Germany, code Art. 9385), Merck LiChroprep ^® RP-18 (40-63um, purchased from Merck, Germany, code Art. 0250). High Performance Liquid Chromatography (HPLC) system equipped with Hitachi L-2310 series pumps, Hitachi L-2420 UV-VIS detector (detection wavelength from 200nm to 380nm), and D-2000 Elite data processing Software; the column is selected from the analytical grade Discovery ^® HS C18 (250×4.6mm, 5μm; SUPELCO) column and Mightysil RP-18 GP 250 (250×4.6mm, 5μm; Kanto Chemical) column, as well as semi-preparative grade Discovery ^® HS C18 (250×10.0mm, 5μm; SUPELCO) column and preparative Discovery ^® HS C18 (250×21.0mm, 5μm; SUPELCO) column. Chromatography system is equipped with UV lamp UVP UVGL-25 (wavelength is 254nm and 365nm). Thin-layer chromatography sheet is coated with silicone 60 F ₂₅₄ (0.25mm; purchased from Merck, Germany) or RP-18 F _254S (0.25mm; purchased from Merck, Germany) aluminum sheet.

The chemical structure of the compound was analyzed by mass spectrometry (MS) and nuclear magnetic resonance spectrometry (NMR). Specifically, two-dimensional ion trap tandem Fourier transform mass spectrometry, electrospray ionization tandem mass spectrometry (ESI-MS/MS, Thermo Scientific Orbitrap Elite system), and Bruker amaZon SL system are used for the measurement, and the unit is m/z; and Use 400MHz Varian 400 FT-NMR to obtain 1D and 2D NMR spectra, The chemical shift is represented by δ, where the unit is ppm; Tetramethylsilane (TMS) is used as the internal standard; the coupling constant (J) is in Hz, and s is the peak (Singlet), d Table doublet (Doublet), t means triplet (Triplet), q means quartet (Quartet), p means quintet, m means multiplet (Multiplet), and brs means broad peak.

Example 1 Separation and purification of the compounds TCI-CA-01, TCI-CA-02, TCI-CA-03, TCI-CA-04, and TCI-CA-05 of the present invention

The purified compound in this example is obtained from Citrus aurantium , which is an evergreen shrub of Citrus in the Rutaceae family (Rutaceae ), also known as lime blossom, green orange, and bitter orange. Originated from the Mediterranean, it has been used by the ancient Greeks as an antiseptic for aromatherapy and a tranquilizer for phytotherapy in ancient Greece. Daidaihua likes a warm and humid climate and is not cold-resistant. The flowers are white or purplish red. Daidaihua is known to have the functions of regulating qi and relieving depression, reducing abdominal distension and pain and vomiting, promoting blood circulation, dispelling qi and promoting phlegm. It can also be used to calm the mood and relieve tension due to its strong fragrance and let you smell it. disturbed.

First, prepare the flower extract, mix the dried flower material and the extraction solvent of water, alcohol, or alcohol-water mixture at a liquid-to-solid ratio of 5-20:1-5. The extraction solvent is preferably water. After homogenization, extract in a solvent at 50-100°C for 0.5-3 hours. After extraction, it was cooled to room temperature, and the crude extract was filtered through a 400 mesh filter to obtain a filtrate. Finally, the filtrate was concentrated under reduced pressure at 45-70° C. to obtain the flower extract of the present invention. The high performance liquid chromatography (HPLC) fingerprint spectrum is shown in FIG. 1.

Next, take 10 liters of the flower extract of the present invention for separation and purification, mix ethyl acetate and water at a ratio of 1:1, and extract the flower extract three times by liquid partition extraction. The extracts of the ethyl acetate layer are combined and concentrated and dried under reduced pressure to obtain 4.4 g of the ethyl acetate layer extract of the flower extract of the present invention.

Next, according to the Bioassay guided fractionation method, the active ingredients in the ethyl acetate layer extract of the Daidaihua extract of the present invention were tracked. Firstly, 4.4 g of ethyl acetate layer extract was separated and purified by column chromatography. Among them, a silica gel molecular sieve tube was used as the chromatographic material and methanol was used as the eluent. After column chromatography, a total of 7 were obtained. Divided layers (F1-F7); then, the divided layer F1 is further separated and purified by medium pressure column chromatography, filled with Merck LiChroprep ^® RP-18 as the chromatographic material, and mixed with water and methanol In order to extract the liquid and gradually reduce its polarity, it is separated and purified by column chromatography, then spotted and combined by thin layer chromatography, and finally a total of 5 sub-division layers (F1-1-F1-7) are obtained.

Then the sub-division layer F1-3 was separated and purified by HPLC, in which a mixed solvent system of water and acetonitrile mixed in a ratio of 4:1 was used as the mobile phase, and 6.9 mg of compound TCI-CA-01 and acetonitrile were obtained after separation and purification. 8.0 mg of compound TCI-CA-02, and 12.7 mg of compound TCI-CA-03.

The sub-division layer F1-5 was separated and purified by HPLC, in which a mixed solvent system of water and acetonitrile mixed in a ratio of 3:1 was used as the mobile phase, and after separation and purification, 5.6mg of compound TCI-CA-04, And 9.2 mg of compound TCI-CA-05.

The compounds TCI-CA-01, TCI-CA-02, TCI-CA-03, TCI-CA-04, and TCI-CA-05 were analyzed by nuclear magnetic resonance spectrometer and mass spectrometer and compared with the literature to determine the five The name and chemical structure of the compound are shown in Table 1. The hydrogen spectrum data of TCI-CA-01 is shown in Figure 2, and the mass spectrum data of TCI-CA-01 is shown in Figure 3; the hydrogen of TCI-CA-02 The spectrum data is shown in Figure 4, the mass spectrum data of TCI-CA-02 is shown in Figure 5; the hydrogen spectrum data of TCI-CA-03 is shown in Figure 6, and the mass spectrum data of TCI-CA-03 is shown in Figure 7. As shown; the hydrogen spectrum data of TCI-CA-04 is shown in Figure 8, the mass spectrum data of TCI-CA-04 is shown in Figure 9; the hydrogen spectrum data of TCI-CA-05 is shown in Figure 10, TCI- The mass spectrum data of CA-05 is shown in Figure 11; among them, the compound TCI-CA-01 is 4-hydroxy-3-methoxycinnamic acid, which can also be called 3-(4-hydroxy-3-methoxyphenyl) -2-acrylic acid)((E)-3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid), this compound is commonly known as Ferulic acid and its structural formula is formula (I) Compound; Compound TCI-CA-02 is 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-pyranopyranosyl]]oxy]-2 ,3-Dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one (7-[[2-O-(6-Deoxy-α-L- mannopyranosyl)-β-D- glucopyranosyl]]oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), commonly known as Naringin (Naringin), its structural formula is formula (II ) Compound; compound TCI-CA-03 is 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one (5,7-Dihydroxy-2-(4-hydroxyphenyl)chroman-4- one), commonly known as Naringenin (Naringenin), its structural formula is the compound of formula (III); compound TCI-CA-04 is 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4- Oxo-3,4-dihydro-2h-chromium-7-yl-2-o-(6-deoxyhexapyranosyl)hexapyranoside (5-hydroxy-2-(3-hydroxy-4- methoxyphenyl)-4-oxo-3,4-dihydro-2h-chromen-7-yl 2-o-(6-deoxyhexopy-ranosyl)hexopyranoside), commonly known as neohesperidin (Neohesperidin), its structural formula is formula (IV ) Compound; and compound TCI-CA-05 is (S)-2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzene Pyran-4-one ((S)-2,3-Dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one), commonly known as orange The structural formula of Hesperetin is a compound of formula (V).

Example 3 The effect of the active ingredient of the flower extract of the present invention in promoting lipolysis

In this example, mouse bone marrow stromal cells (Bone marrow stromal cells) were used to carry out the compound TCI-CA-01, compound TCI-CA-02, compound TCI-CA-03, compound TCI-CA-04, and compound TCI of the present invention. -CA-05) Test for the effect of promoting fat decomposition. Among them, the mouse bone marrow stromal cell line was purchased from the American Type Culture Collection (ATCC ^® ), numbered CRL-2749 ^TM . The cells were cultured in pre-adipocyte expansion medium (Pre-adipocyte Expansion Medium) before differentiation, which contained 90% α-MEM (Minimum Essential Medium Eagle-Alpha Modification, purchased from Gibco, USA) cell culture medium, 10% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, USA), and 1.0% of penicillin/streptomycin (Penicillin-streptomycin, purchased from Gibco, USA); and using the differentiation medium (Differentiation Medium) to make Mouse bone marrow stromal cells are differentiated, which contains 90% α-MEM cell culture medium, 20% fetal bovine serum, and 1.0% penicillin/streptomycin.

In order to prove that compound TCI-CA-01, compound TCI-CA-02, compound TCI-CA-03, compound TCI-CA-04, and compound TCI-CA-05 have the effect of promoting lipolysis, firstly, the mouse bone marrow stroma Cells are differentiated into adipocytes, 8x10 ⁴ mouse bone marrow stromal cells are cultured in a 24-well culture dish containing 0.5 mL of the above-mentioned pre-adipocyte expansion medium, cultured at 37°C for 7 days, and replaced every 3 days during this period After 7 days of fresh differentiation culture medium, observe the formation of lipid droplets under a microscope to ensure that the cells are fully differentiated. Then divide the cells into the following seven groups: (1) Control group containing only cell culture medium (Mock), (2) The experimental group added 10 μg/mL compound TCI-CA-01 of the present invention, (3) the experimental group added 10 μg/mL compound TCI-CA-02 of the present invention, (4) added 10 μg/mL compound TCI- of the present invention The experimental group of CA-03, (5) the experimental group added 10 μg/mL of the compound TCI-CA-04 of the present invention, (6) the experimental group added 10 μg/mL of the compound TCI-CA-05 of the present invention, and (7) ) Add 10μg/mL of the compound TCI-CA-02 of the present invention plus 10μg/mL of the compound TCI-CA-03 of the present invention to the experimental group, and incubate at 37°C for 7-10 days, and also change fresh every 3 days The differentiation medium.

Next, use a glycerol test kit (purchased from Cayman Chemical, USA) to detect the value of extracellular glycerol. After the triglycerides stored in the cells are broken down, free glycerol and fatty acids are produced, so by measuring the extracellular The content of glycerol can infer the value of fat breakdown in the cell. This kit uses the measurement of the product content of the coupled enzyme reaction system that produces a bright purple product to quantify the value of extracellular glycerol, and establishes a standard curve with standards to calculate the value of extracellular glycerol in the sample. Collect the cell culture supernatant of each well, and transfer 25μL of the cell culture supernatant to a new 96-well culture plate, and then add 100μL of reconstituted free glycerol assay reagent (Free Glycerol Assay Reagent) to each well React for 15 minutes at room temperature, and then read the value at _{OD 540nm.} And use Excel software to perform student t-test to determine whether there is a statistically significant difference between the two sample groups (*p value<0.05; **p value<0.01; ***p value<0.001).

The test results of compound TCI-CA-01, compound TCI-CA-02, compound TCI-CA-03, compound TCI-CA-04, and compound TCI-CA-05 in promoting lipolysis of the present invention are shown in FIG. 12. It can be seen from Figure 12 that the compound TCI-CA-01 of the present invention can effectively promote the decomposition of fat compared with the control group after the action of the compound TCI-CA-01 of the present invention; and the compound TCI-CA-02 and the compound TCI-CA-03 of the present invention can effectively promote the decomposition of fat. Later, compared with the control group, it can significantly promote lipolysis by 8% and 15%, respectively; after the compound TCI-CA-04 of the present invention and the compound TCI-CA-05 are used, the values are similar to those of the control group. This result shows that the active ingredient compound TCI-CA-01, compound TCI-CA-02, and compound TCI-CA-03 in the flower extract of the present invention can effectively promote lipolysis and reduce the fat content in cells. However, after mixing the compound TCI-CA-02 and the compound TCI-CA-03, which have excellent lipolysis-promoting ability, after the action of adipocytes, their lipolysis-promoting effect is similar to that of the control group. This result shows that it is not arbitrary The combination of compounds that promote lipolysis can effectively add to the effect of promoting lipolysis.

In summary, the compound 4-hydroxy-3-methoxycinnamic acid of the present invention can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O -(6-Deoxy-α-L-mannopyranosyl)-β-D-pyranosyl))oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyl Phenyl)-4H-1-benzopyran-4-one, 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one, 5-hydroxy-2-(3-hydroxyl -4-methoxyphenyl)-4-oxo-3,4-dihydro-2h-chromium-7-yl-2-o-(6-deoxyhexapyranosyl)hexapyranoside, and (S)-2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, the 4 -Hydroxy-3-methoxycinnamic acid can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), the 7-[[2-O-(6-deoxy-α-L -Mannopyranosyl)-β-D-pyranosyl]]oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzene Pyran-4-one and the 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one can effectively promote lipolysis to reduce the fat content in cells. Therefore, the 4-hydroxy-3-methoxycinnamic acid of the present invention can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O-(6 -Deoxy-α-L-Mannopyranosyl)-β-D-pyranosyl))oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl) -4H-1-benzopyran-4-one and 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one can be used for preparation to promote lipolysis and/or reduce cell fat content The purpose of the composition, And the composition is a medicine, a skin care product, or a food, which can be administered to a body by oral, smearing or other methods.

Claims (10)

Use of a composition for preparing a medicine for preventing and/or treating obesity, wherein the composition contains an effective concentration of a compound selected from the group consisting of: 3-(4-hydroxy-3-methyl Oxyphenyl)-2-acrylic acid)((E)-3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid), 7-[[2-O-(6-deoxy-α- L-Mannopyranosyl)-β-D-pyranosyl]]oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1- Benzopyran-4-one (7-[[2-O-(6-Deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]]oxy]-2,3-dihydro-5-hydroxy-2 -(4-hydroxyphenyl)-4H-1-benzopyran-4-one), 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one(5,7-Dihydroxy-2-( 4-hydroxyphenyl)chroman-4-one), any combination thereof, and a pharmaceutically acceptable carrier. The use as described in item 1 of the scope of the patent application, wherein the effective concentration of the compound is at least 10 μg/mL. The use described in item 1 of the scope of patent application, wherein the composition promotes the decomposition of fat. The use described in item 3 of the scope of patent application, wherein the composition promotes the decomposition of fat in adipocytes. The use of a composition for preparing a drug for promoting lipolysis, wherein the composition contains an effective concentration of a compound selected from the group consisting of: 4-hydroxy-3-methoxycinnamic acid may also be It is called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O-(6-deoxy-α-L-mannosyl)-β-D-pyranosyl Pyrananosyl])oxy)-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, 5,7-di Hydroxy-2-(4-hydroxyphenyl)benzo-4-one, any combination thereof, and a pharmaceutically acceptable carrier. The use as described in item 5 of the scope of patent application, wherein the effective concentration of the compound is at least 10 μg/mL. The use as described in item 5 of the scope of patent application, wherein the composition promotes the decomposition of fat in adipocytes. A kind of 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-pyranosyl]]oxy]-2,3-dihydro-5- Hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one is used for the preparation of drugs for preventing and/or treating obesity. The use described in item 8 of the scope of patent application, wherein the 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-pyranosyl]] The effective concentration of oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one is at least 10 μg/mL. The use described in item 8 of the scope of patent application, wherein the 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-pyranosyl]] Oxyoxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one promotes fat decomposition.

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