TWI685337B - Compounds for reducing fat and application thereof - Google Patents

Abstract

The present invention provides use of 3-(4-hydroxy-3-methoxyphenyl)propionic acid for manufacture of pharmaceutical composition for reducing fat. Also provided is a composition including 3-(4-hydroxy-3-methoxyphenyl)propionic acid for manufacture of pharmaceutical composition for reducing fat. The composition may further include 6,7,8,3’,4’-pentamethoxyflavone, 3,5,7,8,3’,4’-hexamethoxyflavone, or combination thereof.

Description

Fat-reducing compounds and their applications

The present invention relates to the use of a compound to reduce fat, in particular to the use of 3-(4-hydroxy-3-methoxyphenyl)propionic acid or a composition containing the compound for preparing a pharmaceutical composition for reducing fat accumulation .

The high fat and sugar diet and insufficient exercise have caused most modern people to face obesity, and because obesity has a higher chance of suffering from metabolic related diseases, such as diabetes, hyperlipidemia, hypertension, cardiovascular disease, fatty liver, etc., Serious health threats. There are also studies showing that obesity is an important carcinogen. In addition, obese people are more prone to psychological problems and social disorders. Therefore, in recent years, many medical researches have focused on finding ways to suppress obesity, trying to promote physical and mental health in this way.

Methods to suppress obesity include diet control, exercise, lifestyle modification, medication, and surgery. In addition to severely obese patients who need surgery, most people adopt diet control and exercise methods to reduce fat, because modern people are not easy to change their lifestyle due to their busy work, and they are reluctant to use non-essential drugs because of the advocacy of natural remedies. However, diet control strictly requires an individual’s balanced diet and calorie intake, which is quite difficult to implement; improper exercise may cause physical damage. In addition, these two methods have a limited effect on fat reduction, because they are not directed at fat cells, especially the fat tissue in the internal organs.

In view of this, it is necessary to develop a composition that is convenient for the general public and can effectively reduce the fat content in fat cells to prevent obesity and reduce the possibility of the occurrence of the aforementioned various metabolic diseases.

Therefore, an object of the present invention is to provide a use of 3-(4-hydroxy-3-methoxyphenyl)propionic acid for preparing a pharmaceutical composition for reducing fat accumulation.

In one embodiment of the present invention, the 3-(4-hydroxy-3-methoxyphenyl)propionic acid reduces the fat content of a fat cell.

Another object of the present invention is to provide a composition for use in preparing a pharmaceutical composition for reducing fat accumulation, wherein the composition comprises 3-(4-hydroxy-3-methoxyphenyl) propionic acid and a pharmaceutically acceptable Accepted carrier.

In one embodiment of the present invention, the aforementioned composition further comprises 6,7,8,3',4'-pentamethoxyflavonoid, 3,5,7,8,3',4'-hexamethoxyflavonoid , Or a combination thereof.

The invention discloses that several kinds of compounds with fat content reduction in fat cells can be isolated from orange peel fermentation obtained by a special fermentation process, including 3-(4-hydroxy-3-methoxyphenyl)propionic acid, 6, 7,8,3',4'-pentamethoxyflavone, and 3,5,7,8,3',4'-hexamethoxyflavone. Therefore, the present invention provides the use of 3-(4-hydroxy-3-methoxyphenyl) propionic acid or a composition containing the compound for preparing a pharmaceutical composition that reduces fat accumulation. The pharmaceutical composition may have the form of powder, granules, solution, colloid, or ointment, which can be administered to a body by oral administration.

The following will further illustrate the embodiments of the present invention with reference to the drawings. The following examples are used to illustrate the inventive features and applications of the present invention, rather than to limit the scope of the present invention. Anyone who is familiar with this skill will not deviate from it. Within the spirit and scope of the present invention, some changes and modifications can be made, so the scope of protection of the present invention shall be deemed as defined by the scope of the attached patent application.

Figure 1 shows the relative fat content of adipocytes treated with compound 1, 2 or 3.

The invention provides a use of 3-(4-hydroxy-3-methoxyphenyl) propionic acid for preparing a pharmaceutical composition for reducing fat accumulation, and a method comprising 3-(4-hydroxy-3-methoxyphenyl) The use of a composition of propionic acid for the preparation of a pharmaceutical composition that reduces fat accumulation. The following examples illustrate that 3-(4-hydroxy-3-methoxyphenyl)propionic acid, 6,7,8,3',4'-pentamethyl can be separated from orange peel fermented by special fermentation process Oxygen yellow Ketone, and 3,5,7,8,3’,4’-hexamethoxyflavonoids and other three compounds, each of which significantly reduces the fat content of fat cells.

definition

The numerical values used in this article are approximate values, and all experimental data are expressed within a range of 20%, preferably within a range of 10%, and most preferably within a range of 5%.

The term "pharmaceutically acceptable carrier" as used herein means that the carrier must be compatible with the active ingredient of the composition (preferably capable of stabilizing the active ingredient), and is not harmful to the individual user. Pharmaceutically acceptable carriers include water, buffer solutions, excipients, stabilizers, antioxidants, polypeptides, amino acids, carbohydrates.

Materials and Methods material

MEM-α medium (Minimum Essential Medium α medium), fetal bovine serum (FBS), penicillin/streptomycin, and phosphate buffered saline (PBS) were purchased from Thermo Fisher Scientific Corporation. Oil red O (Oil red O) was purchased from Sigma. Formaldehyde and isopropanol were purchased from Echo chemical company. Hexane (n -hexane), ethyl acetate (ethyl acetate), acetone (acetone), methanol (methanol), ethyl alcohol (ethanol), n-butanol (n -butanol), acetonitrile (acetonitrile), chloroform - d ₁ ( Deuteration degree 99.5%), methanol- d ₆ (deuteration degree 99.5%), heavy water (deuterium oxide, deuteration degree>99.8%), and dimethyl sulfoxide- d ₆ (dimethyl sulfoxide- d ₆ , deuteration (Degree>99.9%) purchased from Taiwan Merck.

microorganism

Saccharomyces cerevisiae (BCRC 20271) was purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute.

Chemical analysis instruments

The compound separation system uses column chromatography and thin layer chromatography (TLC). The medium pressure liquid chromatography (MPLC) system is Combi Flash ^® Rf+ (Teledyne ISCO); the column system is selected from Sephadex LH-20 (Amersham Biosciences), Diaion HP-20 (Mitsubishi Chemical), Merck Kieselgel 60 (40-63 μm, Art. 9385), and Merck LiChroprep® RP-18 (40-63 μm, 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; column system Choose self-analysis grade Discovery® HS C ₁₈ (250×4.6mm, 5μm; SUPELCO) and Mightysil RP-18 GP 250 (250×4.6mm, 5μm; Kanto Chemical), and semi-preparation grade Discovery® HS C ₁₈ (250× 10.0 mm, 5 μm; SUPELCO) and preparation grade Discovery® HS C ₁₈ (250×21.0 mm, 5 μm; SUPELCO). The chromatography system is equipped with UVP UVGL-25 (wavelength 254nm and 365nm). The thin-layer chromatography sheet is an aluminum sheet coated with silicone 60 F ₂₅₄ (0.25 mm; Merck) or RP-18 F _254S (0.25 mm; Merck).

The chemical structure of the compound is determined by mass spectrometry (MS) and nuclear magnetic resonance spectrometry (NMR). Specifically, two-dimensional ion trap tandem Fourier transform mass spectrometry (Bruker amaZon SL system) and electrospray ionization tandem mass spectrometry (ESI-MS/MS; Thermo Scientific Orbitrap Elite system) were used; and 400 MHz Varian 400 FT-NMR was used to obtain a For 1D and 2D NMR spectroscopy, tetramethylsilane (TMS) is used as the internal standard.

Fat-reducing efficacy test

The following examples used adipocytes differentiated from a mouse interstitial cell line OP9 (ATCC CRL-2749) purchased from the American Type Culture Collection (ATCC). OP9 cells were seeded at 8×10 ⁴ cells/well in a 24-well culture plate containing 500 μL of preadipocyte expansion medium (90% MEM-α medium, 20% FBS, 1% penicillin/streptomycin) at 37°C Incubate for 7 days. During the cell culture period, the medium was changed to adipocyte differentiation medium (90% MEM-α medium, 20% FBS, 1% penicillin/streptomycin) every 3 days. After 7 days, the formation of oil droplets in the cells was observed under a microscope (ZEISS; magnification 400x) to confirm their complete differentiation into adipocytes. Thereafter, the sample to be tested is added to the cells and cultured at 37°C for 7 to 10 days, during which the adipocyte differentiation medium is changed every 3 days. Finally, the medium was removed and the cells were washed with PBS solution for oil red O staining and determination of the relative fat content of each group of cells. The relative fat content is the ratio (expressed as a percentage) of the fat content of the cells in the experimental group to the cells in the control group. The control group cell lines were treated with adipocyte differentiation medium without test samples in a similar manner.

Preparation of Oil Red O dye liquor

Dissolve the dye oil Red O completely in 100% isopropyl alcohol to prepare a 3mg/mL oil red O stock solution. In order to obtain the oil red O dye solution available, the stock solution was diluted with secondary water to a concentration of 1.8 mg/mL immediately before use and filtered through a 0.22 μm filter membrane.

Oil red O staining

The fat content in the cells was determined by oil red O staining. Before staining, wash the cells with PBS solution and fix the cells with 10% formaldehyde for 30 minutes. After the fixed cells were washed once with PBS solution, they were rinsed with 60% isopropyl alcohol for 1 minute. Thereafter, the cells were stained with Oil Red O staining solution for 1 hour, and after removing the staining solution, the cells were stained with 60% isopropyl alcohol for 5 seconds. After staining, the cells were washed with PBS solution and observed with a microscope (ZEISS Axio Vert. A1), or 100% isopropyl alcohol was used to dissolve the intracellular stain for 10 minutes for quantification. For quantification, 100 μL of the aforementioned dissolving agent-isopropanol solution was transferred to a 96-well plate, and the absorbance at 510 nm was measured using an ELISA (enzyme-linked immunosorbent assay, BioTek) disk reader (BioTek). Quantitative results are statistically analyzed using Student's t test of Excel software.

Example 1 Preparation of orange peel ferment

First, wash, dry, and chop the orange peel ( Citrus reticulata ) (about 0.5 to 1 cm in length). In addition, a yeast culture solution is prepared, which is an aqueous solution containing 1% to 5% (w/v) yeast protein hydrolysate (yeast peptone) and 10% to 15% (w/v) glucose. The yeast culture solution can be heated at 50°C to 100°C for 0.5 to 2 hours, and then cooled to room temperature. Adding 0.01% to 0.5% (w/v) Saccharomyces cerevisiae ( Saccharomyces cerevisiae ; BCRC 20271) to the yeast culture broth, and standing culture at 25°C to 35°C for 3 to 5 days to obtain a yeast culture (yeast culture) . Thereafter, the orange peel and the yeast culture are mixed in a weight ratio of 1:15 to 3:10, and left to ferment at 25°C to 35°C for 3 to 10 days to obtain a fermented orange peel.

Example 2 Analysis of Fat-Reducing Active Components of Fermented Orange Peel

To confirm the fat-reducing active ingredient in the orange peel ferment prepared by the method of Example 1, 10 liters of orange peel ferment was prepared according to the method described in Example 1, and analyzed according to the following steps. First, extract the orange peel fermented by ethyl acetate (water) and water in equal proportions. Times. The obtained ethyl acetate layer extracts were combined, and then concentrated and dried under reduced pressure to obtain a total of about 3.6 g of ethyl acetate layer extracts. After that, the remaining aqueous layer extract was extracted three times in the manner of n-butanol and water in equal liquid phase distribution. The obtained n-butanol layer extract and the aqueous layer extract were combined separately, and then concentrated and dried under reduced pressure to obtain a total of about 10.8 g of n-butanol layer extract and a total of about 199 g of water layer extract.

The fermented orange peel and the extracts of the above three fermented orange peels (ethyl acetate layer extract, n-butanol layer extract and water layer extract) were tested for fat reduction efficacy, and the results showed that the orange peel fermented product and the All three extracts can significantly reduce fat in fat cells, especially the ethyl acetate layer extract shows the best effect of inhibiting fat accumulation. Therefore, the fat-reducing component is further separated from the ethyl acetate layer extract.

According to the bioassay guided fractionation method, using Sephadex LH-20 column and methanol eluent, the ethyl acetate layer extract (about 3.6g) was subjected to column chromatography to separate 5 divided layers (Marked as F1 to F5, respectively). After that, using a silica gel column Merck Kieselgel 60 (40-63 μm, Art. 9385) and an eluent mixed with dichloromethane and methanol at a volume ratio of 19:1, column chromatography was performed on the F2 divided layer, and the resulting effluent After separation by thin layer chromatography, 11 divided layers (marked as F2-1 to F2-11, respectively) can be obtained. Next, using a silica gel column and an eluent mixed with n-hexane and ethyl acetate in a volume ratio of 2:3, column chromatography was performed on the F2-4 divided layers, and the resulting effluent was separated by thin-layer chromatography. Thirteen divided layers are obtained (labeled as F2-4-1 to F2-4-13, respectively).

The layers divided into F2-4-11 and F2-4-13 were further separated by high performance liquid chromatography (HPLC). When the mobile phase is a mixture of methanol and water in a volume ratio of 3:7, compound 1 (approximately 16.2 mg) can be obtained from the F2-4-11 divided layer, and compound 2 (approximately 9.3) can be obtained from the F2-4-13 divided layer mg).

In addition, using a silica gel column and an eluent mixed with n-hexane and ethyl acetate in a volume ratio of 1:1, the column division of F3 was subjected to column chromatography, and the resulting effluent was separated by thin layer chromatography to obtain 8 Divided layers (labeled as F3-1 to F3-8 respectively). After that, using a silica gel column and an eluent mixed with dichloromethane and methanol in a volume ratio of 9:1, column chromatography was performed on the F3-5 divided layers, and the resulting effluent was separated by thin-layer chromatography and then separated by HPLC After separation (the mobile phase is a mixture of methanol and water in a volume ratio of 3:7), compound 3 (about 4.7 mg) can be obtained.

The chemical structures of compounds 1, 2, and 3 were determined by mass spectrometer and nuclear magnetic resonance spectrometer analysis. Their names and structural formulas are shown in Table 1 below.

Example 3 Evaluation of compound 1-3 for reducing fat

In this example, the fat reduction effect of compounds 1, 2, and 3 (with a concentration of 4 μg/mL) was evaluated using the fat reduction efficacy test. The results are shown in Figure 1 and Table 2 below; * and ** in the figure represent p< 0.05 and p<0.01. According to Table 2, compared with the control group, Compounds 1, 2, and 3 all significantly inhibited fat accumulation in adipocytes, which reduced fat content by approximately 20%, 17%, and 24%, respectively.

In summary, the above experiments show that the orange peel fermentation process of the present invention can produce several compounds that can reduce fat accumulation. Therefore, the present invention provides a 3-(4-hydroxy-3-methoxyphenyl) propionic acid (ie Use of the compound 3) or the composition containing the compound for preparing a pharmaceutical composition for reducing fat accumulation. The pharmaceutical composition may have the form of powder, granules, solution, colloid, or ointment, which can be administered to a body by oral administration.

Claims (6)

A use of 3-(4-hydroxy-3-methoxyphenyl) propionic acid for preparing a pharmaceutical composition for reducing fat accumulation. The use as described in item 1 of the patent application scope, wherein the 3-(4-hydroxy-3-methoxyphenyl) propionic acid reduces the fat content of a fat cell. A composition is used for preparing a pharmaceutical composition for reducing fat accumulation, wherein the composition comprises 3-(4-hydroxy-3-methoxyphenyl) propionic acid and a pharmaceutically acceptable carrier. The use as described in item 3 of the patent application scope, wherein the composition further comprises 6,7,8,3',4'-pentamethoxyflavone, 3,5,7,8,3',4'- Hexamethoxyflavone, or a combination thereof. The use as described in item 3 of the patent application scope, wherein the pharmaceutical composition is administered orally. The use as described in item 3 of the patent application scope, wherein the pharmaceutical composition has a dosage form of powder, granules, solution, colloid, or paste.

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