TWI794575B - Use of compound for preventing and/or treating of hyperlipidemia - 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 hyperlipidemia

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 also known as 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 incidence of obesity in the world has gradually increased. The World Health Organization (WHO) even described the rapidly spreading obesity as an "infectious disease" and called it "Globesity". According to the 2014 The World Health Organization estimates that about 39% of the world's population is overweight (about 1.9 billion people), and 13% of the population is obese (about 600 million people). With the change of eating habits and the improvement of quality of life, the prevalence of obesity in Taiwan is also increasing year by year. According to the National Nutrition and Health Change Survey published by the National Health Service of the Ministry of Health and Welfare, the prevalence of overweight or obesity among adults is as high as 40%. About three, while the male and female ratios are 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 of 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 hypertension, hyperlipidemia, cardiovascular disease, sleep apnea, angina, degenerative arthritis, high uric acid bone and joint diseases such as inflammation, type 2 diabetes, and cancer; Excessive fat cells in the body will secrete harmful substances that hinder the action of insulin, which will hinder the function of insulin and will not be able to effectively control the rising blood sugar, thus leading to symptoms of hyperglycemia. If the body is in a state of high blood sugar for a long time, the pancreas will be tired of secreting More insulin is used to lower blood sugar, and once the pancreas is overloaded, type 2 diabetes can develop. In addition, blood lipid refers to the fat in the blood, mainly including cholesterol and triglyceride. When the concentration of cholesterol or triglyceride circulating in the blood is higher than the normal value, it is called hyperlipidemia; while cholesterol or triglyceride When the ester value is too high, or the high-density lipoprotein concentration 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, high blood pressure, diabetes, and kidney disease.

As a result, obesity reduces quality of life and can lead to premature death, so morbidly obese patients live, on average, much less than normal-weight individuals. Although, genes may be involved in the development of obesity, the obesity epidemic is largely attributed to a high-calorie diet and a sedentary lifestyle. 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 approaches are difficult to implement and their efficacy is limited, largely due to adaptive physiological mechanisms for maintaining energy stores in the body. In addition, some drugs have been approved for long-term obesity treatment (such as orlistat, phentermine, or topiramate), but these drugs often cause serious side effects, thus limiting treatment efficacy and patient compliance; Or, bariatric surgery can significantly reduce weight, but this type of interventional surgery is not suitable for all overweight people; based on the above reasons, it is indeed necessary to study safe and effective ways to reduce body weight and fat accumulation.

To sum up, in response to the obesity and the overall health problems caused by obesity caused by the changes in life and eating habits of modern people, 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 fat decomposition. There is a real need for a composition composed of active ingredients that reduce fat formation and reduce the chance of obesity.

Therefore, one object of the present invention is to provide a composition for the preparation of a drug for the prevention and/or treatment of obesity, wherein the composition contains an effective concentration selected from the group consisting of Compounds in the group: 4-hydroxy-3-methoxycinnamic acid, also known as 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-pyranosyl] ]oxy]-2,3-dihydro-5-hydroxyl-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)benzo-4-one (5,7-Dihydroxy-2-(4-hydroxyphenyl)chroman-4-one), and any combination thereof, and a Pharmaceutically acceptable carrier.

Another object of the present invention is to provide a composition for the preparation of 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 can also be called 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O-(6-deoxy-α-L-mannopyranose Base)-β-D-pyranosyl]]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-pyranosyl]]oxy]-2 , Use of 3-dihydro-5-hydroxyl-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one for preparing medicines for preventing and/or treating obesity.

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

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

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

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

The compound 4-hydroxy-3-methoxycinnamic acid of the present invention can also be called 3-(4-hydroxyl-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)hexopyranoside, and (S)-2 , In 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 Base)-β-D-pyranosyl]]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 fat decomposition to reduce the fat content in cells. Therefore, the 4-hydroxyl-3-methoxycinnamic acid of the present invention can also be called 3-(4-hydroxyl-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 to prepare 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 an individual by oral administration, smearing, etc.

The implementation of the present invention will be further described in conjunction with the drawings below. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Within the scope, some changes and modifications can be made, so the protection scope of the present invention should be defined by the scope of the appended patent application.

Fig. 1 is the HPLC fingerprint analysis spectrum of the Daidaihua extract of one embodiment of the present invention.

Fig. 2 is the hydrogen spectrum of the compound TCI-CA-01 purified from the extract of Daidaihua according to an embodiment of the present invention.

Fig. 3 is a mass spectrum of the compound TCI-CA-01 purified from the extract of Daidaihua according to an embodiment of the present invention.

Fig. 4 is the hydrogen spectrum of the compound TCI-CA-02 purified from the extract of Daidaihua according to an embodiment of the present invention.

Fig. 5 is a mass spectrum of the compound TCI-CA-02 purified from the extract of Daidaihua according to an embodiment of the present invention.

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

Fig. 7 is a mass spectrum of the compound TCI-CA-03 purified from the extract of Daidaihua according to an embodiment of the present invention.

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

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

Fig. 10 is the hydrogen spectrum of the compound TCI-CA-05 purified from the extract of Daidaihua according to an embodiment of the present invention.

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

Figure 12 is the purified compound TCI-CA-01, compound TCI-CA-02, compound TCI-CA-03, compound TCI-CA-04, and compound The bar graph of the effect of TCI-CA-05 on promoting lipolysis. ***p<0.001.

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

Statistical analysis was performed using Excel software. The data are expressed as the mean ± standard deviation (SD), and the differences among the groups were analyzed by Student's t - test.

In accordance with the present invention, pharmaceutical products may be manufactured in a dosage form suitable for parenterally or orally administered by techniques well known to those skilled in the art, including, but not Limited to: injections (eg, sterile aqueous solutions or dispersions), sterile powders, tablets, troches, lozenges (lozenge), pill, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup, elixir ), slurry (slurry) and the like.

The pharmaceutical product according to the present invention may be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intramuscular injection (intramuscular injection) and intravenous injection (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 contain one or more agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposers ), binder (binding agent), excipient (excipient), stabilizer (stabilizing agent), chelating agent (chelating agent), diluent (diluent), gelling agent (gelling agent), preservative (preservative), Lubricants, absorption delaying agents, liposomes, and the like. The selection and quantities of these reagents are within the professionalism and routine skill of those skilled in the art.

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

According to the present invention, the medicinal product may be administered by a parenteral route selected from the group consisting of subcutaneous injection, intraepidermal injection, intradermal injection, Intradermal injection and intralesional injection.

According to the present invention, the food product can be regarded as a food additive (food additive), which is added during the preparation of raw materials by known methods, or added during the production process of food, and formulated with any edible material for Food products for ingestion by humans and non-human animals.

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

According to the present invention, from the ethyl acetate layer extract of the Daidaihua extract of the present invention by column chromatography (Column chromatography) and thin layer chromatography (Thin layer chromatography, TLC), the five A compound: 4-hydroxy-3-methoxycinnamic acid, also known as 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid) ((E)-3-(4-hydroxy-3- methoxy-phenyl)prop-2-enoic acid), named as TCI-CA-01 herein; 7-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-pyranosyl pyranosyl]]oxyl]-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 here as TCI-CA-02; 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one (5,7-Dihydroxy-2-(4-hydroxyphenyl)chrom -4-one), designated herein as TCI-CA-03; 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2h-chromium -7-yl-2-o-(6-deoxyhexopyranosyl)hexapyranoside (5-hydroxy-2-(3-hydroxy-4-methoxy-phenyl)-4-oxo-3,4- dihydro-2h-chromen-7-yl 2-o-(6-deoxyhexopy-ranosyl)hexopyranoside), designated herein as TCI-CA-04; 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 in this paper TCI-CA-05. Among them, the term "Daidaihua extract" means that the Daidaihua flower is extracted with a solvent for a specific time and temperature.

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 accomplishment and routine technical scope of those skilled in this technology.

The present invention provides a method for preparing 4-hydroxy-3-methoxycinnamic acid (also known as 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 is used to promote lipolysis and/or reduce cellular fat content The use of the composition can effectively promote fat decomposition to reduce the fat content in cells.

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-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 (5,7-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-hydroxyl-3-methoxycinnamic acid of the present invention can also be called 3-(4-hydroxyl-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)hexopyranoside, and (S )-2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one detailed extraction and purification method; And the five compounds were tested in promoting lipolysis in fat cells.

chemical analysis material

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

Chemical Analysis Instruments

The compounds were separated using column chromatography (Column chromatography) and thin layer chromatography (Thin layer chromatography, TLC). Specifically, the tubes for column chromatography were selected from Sephadex LH-20 (purchased from Amersham BioSciences, UK), Diaion HP-20 (purchased from Mitsubishi Chemical Co., Japan), Merck Kieselgel 60 (40-63 μm, purchased from Merck, Germany, No. Art.9385), Merck LiChroprep ^® RP-18 (40-63um, purchased from Merck, Germany, No. Art.0250). High Performance Liquid Chromatography (HPLC) system equipped with Hitachi L-2310 series pump, Hitachi L-2420 UV-VIS detector (detection wavelength is 200nm to 380nm), and D-2000 Elite data processing Software; the column system is selected from analytical grade Discovery ^® HS C18 (250×4.6mm, 5μm; SUPELCO) column and Mightysil RP-18 GP 250 (250×4.6mm, 5μm; Kanto Chemical) column, and semi-preparative grade Discovery ^® HS C18 (250×10.0mm, 5 μm; SUPELCO) column and preparative grade Discovery ^® HS C18 (250×21.0mm, 5 μm; SUPELCO) column. The chromatography system is equipped with UV lamp UVP UVGL-25 (wavelength 254nm and 365nm). The thin-layer chromatography sheet is an aluminum sheet coated with silica gel 60 F ₂₅₄ (0.25mm; purchased from Merck, Germany) or RP-18 F _254S (0.25mm; purchased from Merck, Germany).

The chemical structure of the compound was analyzed by mass spectrometry (Mass spectrometry, MS) and nuclear magnetic resonance spectrometry (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 determination, and the unit is m/z; and Use 400MHz Varian 400 FT-NMR to obtain one-dimensional and two-dimensional NMR spectra, The chemical shift (Chemical shift) is represented by δ, and the unit is ppm; tetramethylsilane (Tetramethylsilane, TMS) is used as an internal standard; the coupling constant (J) is in Hz, and the peak (Singlet) is expressed in s, d It means 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 Compounds TCI-CA-01, TCI-CA-02, TCI-CA-03, TCI-CA-04, and TCI-CA-05 of the present invention

The compound purified in this example was obtained from Citrus aurantium , which is an evergreen shrub plant of the family Rutaceae ( Rutaceae ) of the genus Citrus , also known as lime, green orange, and bitter orange. Originated from the Mediterranean Sea, it has been used by the ancient Greeks as a fungicide in aromatherapy and a sedative in phytotherapy. Daidai likes warm and humid climate and is not cold-resistant, with white or purple flowers. Daidai is known to have the effects of regulating qi and relieving depression, alleviating abdominal distension and vomiting, promoting blood circulation, breaking Qi and promoting phlegm, etc. It is also used for calming the mood and relieving tension because of the strong fragrance of the flowers of Daidai. disturbed.

First, prepare the Daidaihua extract, mix the Daidaihua dry material with water, alcohol, or an extraction solvent of a mixture of alcohol and water at a liquid-solid ratio of 5-20:1-5, wherein the extraction solvent is preferably water, After homogenization, extraction is carried out in a solvent at 50-100° C. for 0.5-3 hours. After cooling to room temperature after extraction, 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 Daidaihua extract of the present invention, and its high performance liquid chromatography (HPLC) fingerprint is shown in FIG. 1 .

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

Then, according to the bioassay guided fractionation method, the active components in the ethyl acetate layer extract of the Daidaihua extract of the present invention were tracked. First, 4.4 g of the ethyl acetate layer extract was separated and purified by column chromatography, in which silica gel molecular sieve tubes were used as the chromatography material, and methanol was used as the eluent. After column chromatography, a total of 7 Divide the layers (F1-F7); then, further separate and purify the divided layer F1 with medium pressure column chromatography, which is filled with Merck LiChroprep ^® RP-18 as the chromatographic material, and mixed with water and methanol It is an eluent with gradually decreasing polarity, separated and purified by column chromatography, and then combined by thin-layer chromatography to spot slices, and finally a total of 5 subdivided layers (F1-1-F1-7) were obtained.

The subdivided layer F1-3 was separated and purified by HPLC, wherein the mixed solvent system of water and acetonitrile mixed at a ratio of 4:1 was used as the mobile phase, and 6.9 mg of compounds TCI-CA-01, 8.0 mg of compound TCI-CA-02, and 12.7 mg of compound TCI-CA-03.

The subdivided layer F1-5 was separated and purified by HPLC, wherein the mixed solvent system of water and acetonitrile mixed at a ratio of 3:1 was used as the mobile phase, and 5.6 mg of compounds TCI-CA-04, and 9.2 mg of compound TCI-CA-05.

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, the five The name and chemical structure of the compound are shown in Table 1 below, the hydrogen spectrum data of TCI-CA-01 is shown in Figure 2, the mass spectrum data of TCI-CA-01 is shown in Figure 3; the hydrogen spectrum of TCI-CA-02 The spectral data is shown in Figure 4, the mass spectral data of TCI-CA-02 is shown in Figure 5; the hydrogen spectral data of TCI-CA-03 is shown in Figure 6, and the mass spectral 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, also known as 3-(4-hydroxy-3-methoxyphenyl) -2-acrylic acid) ((E)-3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid), the compound is commonly known as ferulic acid (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-mannopyranosyl]]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-ketone (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-hydroxyl-2-(3-hydroxyl-4-methoxyphenyl)-4- Oxo-3,4-dihydro-2h-chromium-7-yl-2-o-(6-deoxyhexapyranosyl)hexopyranoside (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 the compound of formula (V).

Example 3 Efficacy of the active ingredients of the Daidaihua extract of the present invention in promoting lipolysis

In this example, the compound TCI-CA-01, compound TCI-CA-02, compound TCI-CA-03, compound TCI-CA-04, and compound TCI of the present invention were carried out using mouse bone marrow stromal cells (Bone marrow stromal cells). -CA-05) in the effect test of promoting lipolysis. Wherein, the mouse bone marrow stromal cell line was purchased from the American Type Culture Collection (ATCC ^® ), with the number 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, the United States) cell culture medium, 10% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, U.S.), and added 1.0% penicillin/streptomycin (Penicillin-streptomycin, purchased from Gibco, U.S.); and using differentiation medium (Differentiation Medium) to make Mouse bone marrow stromal cells were differentiated, which contained 90% α-MEM cell culture medium, 20% fetal bovine serum, and added 1.0% penicillin/streptomycin.

In order to confirm 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, the mouse bone marrow stroma was first Cells were differentiated into adipocytes, and ^8x104 mouse bone marrow stromal cells were cultured in a 24-well culture dish containing 0.5mL of the above-mentioned preadipocyte expansion medium, cultured at 37°C for 7 days, and replaced every 3 days during the period After 7 days, observe the formation of lipid droplets with a microscope to ensure that the cells are completely differentiated, and then divide the cells into the following seven groups: (1) the control group (Mock) containing only cell culture medium, (2) Add 10 μg/mL of the compound TCI-CA-01 of the present invention to the experimental group, (3) add 10 μg/mL of the compound TCI-CA-02 of the present invention to the experimental group, (4) add 10 μg/mL of the compound TCI-CA-02 of the present invention The experimental group of CA-03, (5) the experimental group adding 10 μg/mL of the compound TCI-CA-04 of the present invention, (6) the experimental group adding 10 μg/mL of the compound TCI-CA-05 of the present invention, and (7 ) was added 10 μg/mL of the compound TCI-CA-02 of the present invention plus 10 μg/mL of the compound TCI-CA-03 of the present invention to the experimental group, and cultured at 37°C for 7-10 days, and also replaced with a fresh one every 3 days Differentiation medium.

Then, use a glycerol detection kit (purchased from Cayman Chemical, USA) to detect the value of extracellular glycerol, where triglycerides stored in cells are decomposed to produce free glycerol and fatty acids, so by measuring extracellular The glycerol content in the cell can be inferred the value of fat breakdown in the cell. This kit is used to measure the product content of the coupled enzyme reaction system that produces bright purple products to quantify the value of extracellular glycerol, and use the standard to establish a standard curve to calculate the value of extracellular glycerol in the sample. The cell culture supernatant of each well was collected, and 25 μL of the cell culture supernatant was transferred to a new 96-well culture plate, and then 100 μL of reconstituted free glycerol assay reagent (Free Glycerol Assay Reagent) was added to each well React at room temperature for 15 minutes, then read the value at OD _540nm . And use Excel software to conduct 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 of the present invention in promoting lipolysis are shown in FIG. 12 . It can be seen from Figure 12 that after the action of the compound TCI-CA-01 of the present invention, compared with the control group, it can effectively promote the decomposition of fat; and the compound TCI-CA-02 of the present invention and the compound TCI-CA-03 After that, compared with the control group, it can significantly promote 8% and 15% lipolysis respectively; after the compound TCI-CA-04 and compound TCI-CA-05 of the present invention act, they are similar to the values of the control group. The results show that the active ingredients compound TCI-CA-01, compound TCI-CA-02, and compound TCI-CA-03 in the Daidaihua extract of the present invention can effectively promote lipolysis to reduce the fat content in cells. However, after compound TCI-CA-02 and compound TCI-CA-03, which have excellent ability to promote lipolysis, are mixed, the effect of promoting lipolysis after the action of fat cells is similar to that of the control group. This result shows that it is not arbitrary Combination of compounds that can promote fat breakdown can effectively increase their effect on promoting fat breakdown.

In summary, the compound 4-hydroxyl-3-methoxycinnamic acid of the present invention can also be called 3-(4-hydroxyl-3-methoxyphenyl)-2-acrylic acid), 7-[[2-O -(6-deoxy-α-L-mannopyranosyl)-β-D-pyranosyl]]oxy]-2,3-dihydro-5-hydroxy-2-(4-hydroxy Phenyl)-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 In (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 The pyran-4-one and the 5,7-dihydroxy-2-(4-hydroxyphenyl)benzo-4-one can effectively promote fat decomposition to reduce the fat content in cells. Therefore, the 4-hydroxyl-3-methoxycinnamic acid of the present invention can also be called 3-(4-hydroxyl-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 to prepare 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, and can be administered to an individual by oral administration, smearing, etc.

Claims (3)

A composition for the preparation of a drug for preventing and/or treating hyperlipidemia, wherein the composition comprises 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-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, any combination thereof, and a pharmaceutically acceptable carrier. The use as claimed in claim 1, wherein the effective concentration of the compound is at least 10 μg/mL. The use as claimed in claim 1, wherein the composition promotes the decomposition of fat in adipocytes.

Images