Method for extracting novel oligomerization stilbene compounds in iris ensata thumb

Abstract

The invention discloses an extraction method of novel oligomerization stilbene compounds in iris ensata thunb. The extraction method comprises the following steps: carrying out MCI gel column elution on iris ensata thunb., and carrying out multiple preparative chromatography extraction to obtain the novel oligomerization stilbene compounds cis-upunphenylenol K, Viniferol E and Ampelopsin BF, and the novel compounds have a certain effect on non-alcoholic fatty liver.

Description

Technical Field

[0001] The present invention relates to the field of extraction and application of active substances in natural plants, and particularly to a method for extracting new oligostilbene compounds from Iris tectorum Maxim.. Background Art

[0002] With the development of modern biotechnology, people's research on active ingredients in natural products has become increasingly in-depth. Iris tectorum Maxim., as a perennial herbaceous plant of the genus Iris in the Iridaceae family, its roots and rhizomes have been widely used in traditional medicine to treat various diseases, such as inflammation, cardiovascular diseases, etc. Recent studies have shown that Iris tectorum Maxim. contains a variety of bioactive ingredients, including a special type of compound - oligostilbene compounds. These compounds have attracted the attention of scientific researchers due to their unique chemical structures and potential pharmacological effects.

[0003] Oligostilbene compounds are a class of compounds formed by connecting multiple stilbene (pronounced the same as "zhi") units through glycosidic bonds. In recent years, with the development of separation and structure identification technologies, more oligostilbene compounds with novel structures and various bioactivities have been gradually discovered. With the in-depth research, more and more oligostilbene compounds have been found, and the constituent units of their structures have also expanded from resveratrol to other various monomers and heteromonomers. The degree of polymerization of oligostilbene compounds has also developed from the original dimers and tetramers to octamers, etc. They are widely distributed in nature, especially in certain plants with relatively high contents, but their presence in Iris tectorum Maxim. has not been fully studied. Oligostilbene compounds have important application prospects due to their various bioactivities such as antioxidant, anti-inflammatory, and antibacterial. However, due to their low contents in plants and complex and diverse structures, traditional extraction and separation methods are difficult to efficiently extract these compounds from Iris tectorum Maxim.. Summary of the Invention

[0004] In order to make full use of and extract new oligostilbene compounds from Iris tectorum Maxim., the present invention intends to provide a method for extracting new oligostilbene compounds, which can obtain new compounds cis-upunaphenol K, Viniferol E and Ampelopsin BF with various activities.

[0005] The present invention provides a method for extracting oligostilbene compounds, and the structural formula of the compound is shown as Formula 10 (also referred to as Ampelopsin BF in the present invention):

[0006]

[0007] The extraction method includes the following steps:

[0008] (1) Iris lactea Pall. was eluted through an MCI gel column, successively eluted with 5-15% methanol aqueous solution, 15-25% methanol aqueous solution, 25-35% methanol aqueous solution, 35-45% methanol aqueous solution, 55-65% methanol aqueous solution, 80-90% methanol aqueous solution to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5 and Fr.6;

[0009] (2) After gel column chromatography of fraction Fr.2, fractions Fr.2-1, Fr.2-2, Fr.2-3, Fr.2-4 were obtained by preparative chromatography, and the retention times were 15.24 min, 16.63 min, 18.07 min, 19.66 min respectively. The Fr.2-1 is compound 10;

[0010] Among them, the preparative chromatography conditions include:

[0011] Chromatographic column: Preparative reversed-phase chromatographic column, preferably with a specification of 250 mm × 20 mm, 10 μm;

[0012] Mobile phase: A: Methanol / B: Water; Gradient elution was carried out using the following program: 0-25 min, 20%-80% A;

[0013] Furthermore, the concentrations of the methanol aqueous solutions are 10%, 20%, 30%, 40%, 60% and 85% in sequence.

[0014] Compound 10 was identified as a new oligostilbene compound by nuclear magnetic resonance and was named Ampelopsin BF.

[0015] The present invention provides a method for extracting oligostilbene compounds, and the structural formula of the compound is as shown in Formula 9 (also called Viniferol E in the present invention):

[0016]

[0017] The extraction method includes the following steps: Compound 9 was obtained by preparative chromatography of the above-mentioned fraction Fr.2-4, and the retention time was 19.66 min;

[0018] Among them, the conditions of preparative chromatography include:

[0019] Chromatographic column: Preparative reversed-phase chromatographic column, preferably with a specification of 250 mm × 20 mm, 10 μm;

[0020] Mobile phase: Acetonitrile is used as mobile phase A, water is used as mobile phase B, and isocratic elution is carried out with a mobile phase A:mobile phase B volume ratio of 10-30%:70-90% as the mobile phase.

[0021] Furthermore, the volume ratio of mobile phase A to mobile phase B is 28:72.

[0022] Compound 9 was identified as a new oligostilbene compound by its nuclear magnetic resonance and named Viniferol E.

[0023] The present invention provides a method for extracting oligostilbene compounds, and the structural formula of the compounds is shown in Formula 7 (also referred to as cis-upunaphenol K in the present invention):

[0024]

[0025] The extraction method includes the following steps: After subjecting the above-mentioned component Fr.5 to gel column chromatography, the obtained elution component is subjected to preparative chromatography to obtain the compound 7, and the retention time is 25.70 min;

[0026] Among them, the preparative chromatography conditions include:

[0027] Chromatographic column: Preparative reverse-phase chromatographic column, preferably with a specification of 250 mm × 20 mm, 10 μm;

[0028] Mobile phase: Acetonitrile is used as mobile phase A, and water is used as mobile phase B. The mobile phase is mixed in a volume ratio of mobile phase A to mobile phase B of 10-30%:70-90% and used for isocratic elution.

[0029] Furthermore, the volume ratio of mobile phase A to mobile phase B is 40:60.

[0030] Compound 7 was identified as a new oligostilbene compound by its nuclear magnetic resonance and named cis-upunaphenol K.

[0031] The preparative chromatography conditions also include: The flow rate is 5-15 mL / min;

[0032] Furthermore, the flow rate is 10 mL / min.

[0033] In the present invention, the Iris lactea Pall. is the seed coat part of Iris lactea Pall.;

[0034] Furthermore, the Iris lactea Pall. is the seed coat part of Iris lactea Pall.;

[0035] Furthermore, the preparation method of the ethyl acetate extract of the seed coat of Iris lactea Pall. includes the following steps: The seed coat of Iris lactea Pall. is extracted with a 60-80% alcohol solution at 50-80 °C and then extracted with ethyl acetate to obtain the ethyl acetate extract of the seed coat of Iris lactea Pall.

[0036] Furthermore, the alcohol solution is 75% ethanol and the extraction temperature is 60 °C.

[0037] In the present invention, the purities of Compound 10, Compound 9, and Compound 7 are not less than 90%.

[0038] The present invention also provides a method for simultaneously separating multiple novel oligostilbene compounds, comprising the following steps:

[0039] (1) Elute Iris lactea Pall. through an MCI gel column, successively eluting with 5 - 15% methanol aqueous solution, 15 - 25% methanol aqueous solution, 25 - 35% methanol aqueous solution, 35 - 45% methanol aqueous solution, 55 - 65% methanol aqueous solution, and 80 - 90% methanol aqueous solution to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, and Fr.6;

[0040] (2) After subjecting fraction Fr.2 to gel column chromatography and then preparative chromatography, fractions Fr.2 - 1, Fr.2 - 2, Fr.2 - 3, and Fr.2 - 4 are obtained, with retention times of 15.24 min, 16.63 min, 18.07 min, and 19.66 min respectively. Fraction Fr.2 - 1 is Compound 10;

[0041] Among them, the preparative chromatography conditions include: chromatographic column: preparative reversed - phase chromatographic column, preferably with a specification of 250 mm × 20 mm, 10 μm; mobile phase: A: methanol / B: water; gradient elution is carried out using the following program: 0 - 25 min, 20% - 80% A;

[0042] (3) Subject fraction Fr.2 - 4 to preparative chromatography to obtain Compound 9, with a retention time of 19.66 min;

[0043] Among them, the preparative chromatography conditions include: chromatographic column: preparative reversed - phase chromatographic column, preferably with a specification of 250 mm × 20 mm, 10 μm; mobile phase: acetonitrile as mobile phase A, water as mobile phase B, and the mobile phase is mixed in a volume ratio of mobile phase A: mobile phase B of 10 - 30%: 70 - 90% for isocratic elution;

[0044] (4) After subjecting fraction Fr.5 to gel column chromatography, the eluted fraction is subjected to preparative chromatography to obtain Compound 7, with a retention time of 25.70 min;

[0045] Among them, the preparative chromatography conditions include: chromatographic column: preparative reversed - phase chromatographic column, preferably with a specification of 250 mm × 20 mm, 10 μm; mobile phase: acetonitrile as mobile phase A, water as mobile phase B, and the mobile phase is mixed in a volume ratio of mobile phase A: mobile phase B of 10 - 30%: 70 - 90% for isocratic elution.

[0046] In the present invention, the percentages before aqueous methanol solution all refer to volume fractions. For example, "40% aqueous methanol solution" means an aqueous methanol solution with a methanol volume fraction of 40%, and the same applies to other cases.

[0047] The beneficial effects of the present invention are as follows:

[0048] (1) The present invention uses alcohol extraction, column chromatography, and combined with preparative liquid chromatography to extract and prepare three new oligostilbene compounds, cis-upunaphenol K, Viniferol E, and Ampelopsin BF, from Iris lactea Pall., and more comprehensively develops the active substances of Iris lactea Pall.

[0049] (2) The present invention more comprehensively explores the medicinal value of Iris lactea Pall. and expands its clinical application, providing more reference bases for developing potential plant-derived drugs for the treatment of non-alcoholic fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is the extraction flow chart;

[0051] Figure 2 is the purity analysis of Compound 1;

[0052] Figure 3 is the mass spectrum of Compound 1;

[0053] Figure 4 is the purity analysis of Compound 2;

[0054] Figure 5 is the mass spectrum of Compound 2;

[0055] Figure 6 is the purity analysis of Compound 3;

[0056] Figure 7 is the mass spectrum of Compound 3;

[0057] Figure 8 is the purity analysis of Compound 4;

[0058] Figure 9 is the mass spectrum of Compound 4;

[0059] Figure 10 is the purity analysis of Compound 5;

[0060] Figure 11 is the mass spectrum of Compound 5;

[0061] Figure 12 is the purity analysis of Compound 6;

[0062] Figure 13 is the mass spectrum of Compound 6;

[0063] Figure 14 For the purity analysis of Compound 7;

[0064] Figure 15 For the mass spectrum of Compound 7;

[0065] Figure 16 For the purity analysis of Compound 8;

[0066] Figure 17 For the mass spectrum of Compound 8;

[0067] Figure 18 For the purity analysis of Compound 9;

[0068] Figure 19 For the mass spectrum of Compound 9;

[0069] Figure 20 For the purity analysis of Compound 10;

[0070] Figure 21 For the mass spectrum of Compound 10;

[0071] Figure 22 For the changes in the viability of HepG2 cells by Compound cis-upunaphenol K, Viniferol E and Ampelopsin BF;

[0072] Figure 23 For the effects of Compound cis-upunaphenol K, Viniferol E and Ampelopsin BF on lipid accumulation;

[0073] Figure 24 For the effects of Compound cis-upunaphenol K, Viniferol E and Ampelopsin BF on the TG content;

[0074] Figure 25 For the effects of Compound cis-upunaphenol K, Viniferol E and Ampelopsin BF on the TC content;

[0075] Figure 26 For the effects of Compound cis-upunaphenol K, Viniferol E and Ampelopsin BF on the SREBP-1c protein; Figure 27 For the effects of Compound cis-upunaphenol K, Viniferol E and Ampelopsin BF on the expression of FAS protein;

[0076] Figure 28Effects of compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF on ACC protein expression;

[0077] Figure 29 Effects of compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF on SCD-1 protein expression. Detailed implementation manners

[0078] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0079] Example 1 Extraction of oligostilbenoid compounds from Iris lactea Pall.

[0080] 1. Experimental methods

[0081] 1.1 Instruments and materials

[0082] Bruker DRX-500MHz nuclear magnetic resonance spectrometer (Bruker, Germany); LC-IT-TOF mass spectrometer (Shimadzu, Japan); Waters 2535 semi-preparative liquid chromatograph (Waters, USA); Agilent 1260 high performance liquid chromatograph (Agilent, USA); Büchi rotary evaporator (Büchi, Switzerland); SHB-3 circulating multi-purpose vacuum pump (Yuhua Instrument Co., Ltd., Gongyi); ZF-1 triple-purpose ultraviolet instrument (Shanghai Jingke Industrial Co., Ltd.); column chromatography silica gel (80 - 100, 200 - 300 mesh, Qingdao Kangyexin Medicinal Silica Gel Desiccant Co., Ltd.); GF254 silica gel plate (50 - 100nm, Linyi Haixiang Chemical Co., Ltd.); Sephadex LH-20 (Amersham Biosciences, Sweden), preparative reversed-phase chromatographic column (250mm×20mm, 10μm, Daisogel, Japan); RP-C18 (40 - 63μm, Merck, Germany); microporous resin MCI (Mitsubishi Chemical, Japan); chromatographic grade methanol (Shanghai Xingke High Purity Solvents Co., Ltd.).

[0083] 1.2 Extraction of compounds

[0084] (1) Extraction of ethyl acetate fraction from the seed coat of Iris lactea Pall.

[0085] Extraction of Iris lactea Pall. var. chinensis (Fisch.) Koidz. seed coat with ethyl acetate: The seed coat was extracted with 75% ethanol at 60 °C for 3 times, 3 h each time. The extracts of the 3 times were combined and concentrated under reduced pressure to obtain an extract. Then the extract was dispersed in water and extracted with ethyl acetate to finally obtain the ethyl acetate extract.

[0086] (2) Extraction of compounds

[0087] 20 g of the Iris lactea Pall. var. chinensis (Fisch.) Koidz. component (ethyl acetate fraction of the seed coat) was mixed with microporous resin (MCI) and preliminarily separated with 10%, 20%, 30%, 40%, 60% and 85% methanol / water solutions to obtain 6 fractions (Fr.1 - Fr.6). Fraction Fr.2 (20% methanol / water solution) was subjected to gel column chromatography with chloroform:methanol (1:1) as the mobile phase. The obtained fraction was then separated by preparative liquid chromatography (preparative chromatographic conditions: preparative reversed-phase chromatographic column (250 mm × 20 mm, 10 μm, Daisogel Co., Ltd., Japan); mobile phase: A: methanol / B: water; gradient elution was carried out using the following program: 0 - 25 min, 20% - 80% A, flow rate: 10 mL / min) to obtain Fr.2 - 1 (compound 10), Fr.2 - 2 (compound 5), Fr.2 - 3 (compound 6) and Fr.2 - 4, with retention times of 15.24 min, 16.63 min, 18.07 min and 19.66 min respectively. Among them, Fr.2 - 4 was further separated by preparative liquid chromatography (preparative reversed-phase chromatographic column (250 mm × 20 mm, 10 μm, Daisogel Co., Ltd., Japan); isocratic elution, mobile phase: acetonitrile:water = 28:72, flow rate: 10 mL / min) to obtain compound 9, with a retention time of 19.66 min. Fraction Fr.3 (30% methanol / water solution) was subjected to gel column chromatography with methanol, and the eluted fraction was then separated by preparative liquid chromatography (methanol:water = 34:66) to obtain compounds 2 and 3 respectively. Fraction Fr.4 (40% methanol / water solution) was subjected to silica gel column chromatography with chloroform:methanol = 15:1, and the obtained fraction was then subjected to gel column chromatography with methanol as the mobile phase to obtain compound 1. After gel column chromatography of Fr.5 (60% methanol / water solution) (mobile phase: chloroform:methanol = 1:1), the eluted fraction was then separated by preparative liquid chromatography (preparative reversed-phase chromatographic column (250 mm × 20 mm, 10 μm, Daisogel Co., Ltd., Japan); isocratic elution, mobile phase: acetonitrile:water = 40:60, flow rate: 10 mL / min) to obtain compounds 4 and 7 respectively, and the retention time of compound 7 was 25.70 min. Fr.6 (85% methanol / water solution) was subjected to gel column chromatography with methanol as the mobile phase, and the eluted fraction was first separated by preparative liquid chromatography (acetonitrile:water = 40:60) and then by preparative liquid chromatography (methanol:water = 52:48) to obtain compound 8. The detailed separation flow chart is as Figure 1 shown.

[0088] 1.3 Identification of Compounds

[0089] Ten monomeric compounds were tested and analyzed by a Bruker DRX-500MHz nuclear magnetic resonance spectrometer.

[0090] 2 Experimental Results

[0091] 2. Purity Analysis and Structure Identification of Compounds

[0092] Based on nuclear magnetic resonance data and relevant literature analysis, ten compounds were identified as oligostilbene compounds. Compound 1: cis-vitisin B (20 mg, purity 96%); Compound 2: Viniferin (20 mg, purity 96%); Compound 3: Vitisin A (79 mg, purity 96%); Compound 4: Vitisin B (20 mg, purity 90%); Compound 5: Hopeaphenol (20 mg, purity 96%); Compound 6: Isohopeaphenol (20 mg, purity 96%); Compound 7: cis-upunaphenol K (10 mg, purity 90%); Compound 8: Vitisin C (20 mg, purity 96%); Compound 9: Viniferol E (20 mg, purity 96%); Compound 10: Ampelopsin BF (1 mg, purity 96%). The purities and mass spectra of the above ten compounds are as Figures 2 to 21 shown. The structural formulas of Compounds 1-10 are as follows:

[0093]

[0094] The beneficial effects of the novel compounds cis-upunaphenol K, Viniferol E and Ampelopsin BF of the present invention are demonstrated by the following test examples:

[0095] Test Example 1 Effect of Oligostilbene Compounds on Non-alcoholic Fatty Liver

[0096] 1 Experimental Method

[0097] 1.1. Chemicals and Reagents

[0098] Compound 7 (cis-upunaphenol K), Compound 9 (Viniferol E) and Compound 10 (Ampelopsin BF) were isolated from Iris lactea Pall. Sodium oleate (OA) was purchased from Xi'an KunChuang Technology Development Co., Ltd. Oil Red O and MTT were purchased from Beijing Solarbio. DMEM medium was purchased from Corning. Fetal bovine serum (FBS) was from Zhejiang Sijiqing. Triglyceride (TG) and total cholesterol (TC) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). BCA assay kit and protein lysis buffer were purchased from Beyotime (Shanghai, China). Antibodies FAS, SCD-1, SREBP-1c and ACC were purchased from Cell Signaling Technology.

[0099] 1.2. HepG2 cell culture

[0100] HepG2 is a human hepatocellular carcinoma cell line, purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM containing 10% FBS at 37 °C and 5% CO2.

[0101] 1.3. Cell viability assay

[0102] Cell viability was detected by the MTT method. HepG2 cells in the logarithmic growth phase were adjusted to a concentration of 1×10 5 cells / well, and 150 μL per well was inoculated into a 96-well plate and cultured for 24 h. The original medium was replaced with a compound solution (cis-upunaphenol K, Viniferol E, Ampelopsin BF) containing different concentrations, and a blank control (Con) was set. After continuous culture for 48 h, the absorbance value (A) at a wavelength of 490 nm was detected by the MTT method. The cell survival rate was calculated according to the formula: cell survival rate / % = A treatment group / A blank group × 100%.

[0103] 1.4. Oil Red O staining

[0104] Oil Red O staining was used to determine the lipid droplet content in HepG2 cells. HepG2 cells were cultured in a 6-well plate at a density of 5×10 4 cells / mL, and 0.5 mM OA was added to the medium. Cells were treated with 10 μM cis-upunaphenol K, Viniferol E, Ampelopsin BF for 48 hours. After treatment, the cells were washed twice with PBS and then fixed with 4% paraformaldehyde for 30 minutes. Subsequently, the treated cells were stained with Oil Red O solution for 1 hour. Finally, the cells were observed and photographed under an inverted optical microscope.

[0105] 1.5. Measurement of TG and TC contents

[0106] Commercially available kits were used to determine the intracellular TG and TC contents. In the presence of 0.5 mM OA, HepG2 cells were cultured at a density of 5×10 4 cells per milliliter in 6-well plates. The cells were incubated for 48 h in the presence or absence of 10 μM cis-upunaphenol K, Viniferol E, and Ampelopsin BF. The treated cells were digested with trypsin. The digested and centrifuged cells were lysed by sonication on ice for 5 minutes. Subsequently, the TG and TC contents were determined according to the kit instructions.

[0107] 1.6. Western blot analysis

[0108] Cells were washed with cold PBS and lysed in RIPA buffer containing protease inhibitors. The collected lysates were centrifuged at 12,000 g for 15 minutes at 4 °C, and the supernatants were collected. 20 μg of protein samples were separated on 10% SDS polyacrylamide gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked with skim milk solution for 1 hour at room temperature and then incubated with primary antibodies overnight at 4 °C. The membranes were washed 3 times with TBST buffer and incubated with secondary antibodies for 1 hour at room temperature. β-actin was used as an internal reference protein. The blots were visualized using a 5200 Multi Luminescent imaging system (Tanon, Shanghai, China), and the band intensities were analyzed using ImageJ.

[0109] 1.7. Statistical analysis

[0110] Data analysis was performed using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA, USA). The results were expressed as the mean standard deviation (SD) of three independent experiments. Unpaired t-tests (for two groups) or one-way ANOVA (for three groups or more) were used for intergroup comparisons. P < 0.05 was considered statistically significant.

[0111] 2 Results analysis

[0112] 1.1 Effects of compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF on cell viability

[0113] The MTT assay was initially used to determine the cytotoxicity of compounds 7, 9, and 10, namely compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF, against HepG2 cells. AsFigure 22 As shown, compared with the control group, the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF (1 - 100 μM) had no cytotoxic effect on the viability of HepG2 cells. Therefore, the optimal treatment concentration of cis-upunaphenol K, Viniferol E, and Ampelopsin BF was 10 μM.

[0114] 1.2 Effects of the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF on lipid accumulation

[0115] To investigate whether the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF affect intracellular lipid accumulation, Oil Red O staining was performed. Compared with the control group (Con), 0.5 mM OA led to a significant increase in lipid droplet accumulation in HepG2 cells ( Figure 23 ). Compared with the OA group (Mod), the intracellular lipid droplets were significantly reduced after treatment with the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF.

[0116] 1.3 Effects of the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF on lipid content

[0117] By measuring the effects of the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF on the contents of TG and TC. The experimental results showed that compared with the control group (Con), 0.5 mM OA led to a significant increase in the contents of TG and TC in HepG2 cells ( Figure 24 and Figure 25 ). Compared with the OA group (Mod), the contents of TG and TC in cells were significantly reduced after treatment with the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF ( Figure 24 and Figure 25 ).

[0118] 1.4 Effects of the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF on protein expression

[0119] To further determine the potential mechanisms of the inhibitory effects of the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF on lipid accumulation, the expressions of adipogenesis-related proteins SREBP-1c, FAS, ACC, and SCD-1 were analyzed by Western blotting. As follows Figure 26 , Figure 27 , Figure 28 and Figure 29 As shown, compared with the control group (Con), 0.5 mM OA led to a significant increase in the expression levels of SREBP-1c, FAS, ACC, and SCD-1 in HepG2 cells. Compared with the OA group (Mod), the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF could reduce the expression levels of SREBP-1c, FAS, ACC, and SCD-1 in HepG2 cells. The results indicate that the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF can reduce lipid accumulation in OA-treated HepG2 cells by decreasing the expression of adipogenic proteins.

[0120] Based on the above experimental results, it was found that the compounds cis-upunaphenol K, Viniferol E, and Ampelopsin BF have the effects of reducing lipid accumulation and increasing the redox level, and thus have the effect of anti-nonalcoholic fatty liver.

[0121] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting oligostilbene compounds, characterized in that: The structural formula of the compound is shown in Formula 10: The extraction method comprises the following steps: (1) passing the iris through an MCI gel column for elution, and eluting with 5-15% methanol aqueous solution, 15-25% methanol aqueous solution, 25-35% methanol aqueous solution, 35-45% methanol aqueous solution, 55-65% methanol aqueous solution, and 80-90% methanol aqueous solution in sequence to obtain components Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, and Fr.6; (2) After subjecting component Fr.2 to gel column chromatography, components Fr.2-1, Fr.2-2, Fr.2-3, and Fr.2-4 were obtained by preparative chromatography, with retention times of 15.24 min, 16.63 min, 18.07 min, and 19.66 min, respectively. Fr.2-1 is compound 10; Wherein, the preparative chromatography conditions include: Chromatographic column: Preparative reverse phase chromatographic column, preferably with specifications of 250 mm × 20 mm, 10 μm; Mobile phase: A: methanol / B: water; gradient elution was performed using the following program: 0-25 min, 20%-80% A; Furthermore, the concentrations of the methanol aqueous solution are 10%, 20%, 30%, 40%, 60% and 85% respectively.

2. A method for extracting oligostilbene compounds, characterized in that: The structural formula of the compound is shown in Formula 9: The extraction method comprises the following steps: subjecting the component Fr.2-4 of claim 1 to preparative chromatography to obtain compound 9 with a retention time of 19.66 min; The conditions for preparative chromatography include: Chromatographic column: Preparative reverse phase chromatographic column, preferably with specifications of 250 mm × 20 mm, 10 μm; Mobile phase: acetonitrile is mobile phase A, water is mobile phase B, and a mixture of mobile phase A and mobile phase B in a volume ratio of 10-30%:70-90% is used as the mobile phase for isocratic elution.

3. A method for extracting oligostilbene compounds, characterized in that: The structural formula of the compound is as shown in Formula 7 The extraction method comprises the following steps: subjecting the component Fr.5 of claim 1 to gel column chromatography, and subjecting the obtained eluted component to preparative chromatography to obtain the compound 7, with a retention time of 25.70 min; The preparative chromatography conditions include: Chromatographic column: Preparative reverse phase chromatographic column, preferably with specifications of 250 mm × 20 mm, 10 μm; Mobile phase: acetonitrile is mobile phase A, water is mobile phase B, and a mixture of mobile phase A and mobile phase B in a volume ratio of 35-45%:55-65% is used as the mobile phase for isocratic elution.

4. The extraction method according to claim 2, characterized in that The volume ratio of mobile phase A:mobile phase B was 28:

72.

5. The extraction method according to claim 3, characterized in that The volume ratio of mobile phase A:mobile phase B is 40:

60.

6. The extraction method according to claim 1, characterized in that The component Fr.2 was subjected to gel column chromatography using chloroform:methanol in a volume ratio of 0.5-1.5:0.5-1.5 as the mobile phase; further, the chloroform:methanol=1:

1.

7. The extraction method according to any one of claims 1 to 3, characterized in that The preparative chromatography conditions also include: a flow rate of 5 to 15 mL / min; further, the flow rate is 10 mL / min.

8. The extraction method according to any one of claims 1 to 3, characterized in that The Iris is the Iris seed coat part; further, the Iris is the Iris seed coat ethyl acetate extract.

9. The preparation method according to claim 8, characterized in that: The preparation method of the ethyl acetate extract of Iris irritans seed coat comprises the following steps: extracting the Iris irritans seed coat with a 60-80% alcohol solution at 50-80°C, and then extracting with ethyl acetate to obtain the ethyl acetate extract of Iris irritans seed coat; further, the alcohol solution is 75% ethanol, and the extraction temperature is 60°C.

10. The extraction method according to any one of claims 1 to 3, characterized in that The purity of the compound 10, compound 9 and compound 7 is not less than 90%.

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