A flavonoid compound with lipase inhibitory activity and a preparation method and application thereof

Abstract

The application discloses a flavonoid compound with lipase inhibitory activity and a preparation method and application thereof, and is prepared from Xianmilaxinensis as raw material through pretreatment, extract immersion, silica gel column chromatography and high performance liquid chromatography separation, has a molecular formula of C 22 H 18 O5, is named as 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone, and has the following structural formula. The preparation method of the flavonoid compound with lipase inhibitory activity is to use Xianmilaxinensis as raw material, and to obtain through extract immersion, silica gel column chromatography and high pressure liquid chromatography separation. The inhibition rate of pancreatic lipase is determined by using a colorimetric method, the IC 50 value of the compound is 22.6+ / -2.6 muM, has a significant lipase inhibitory effect, and is expected to be used as a lead compound to develop a new type of lipase inhibitor drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product chemistry, and particularly relates to an isoprenyl flavone compound which is extracted and separated from the plant Artocarpus lanceolatus of Moraceae for the first time, and a use of the isoprenyl flavone compound as a lipase inhibitor. BACKGROUND

[0002] With the improvement of living standards and the reduction of physical labor, the obese population in China is increasing, and the obese population in China has exceeded 95 million, of which more than 1 million belongs to severe obesity. Long-term obesity can easily cause movement system disorders, arthritis, muscle strain or spinal nerve root compression, causing waist, leg, shoulder and back pain, and even causing joint deformation, which seriously affects limb movement. It can also cause a large accumulation of triglycerides in the liver, forming fatty liver, or increasing the load on the heart, causing blood to accumulate in the cardiovascular system, and even causing heart failure in severe cases.

[0003] The external cause of obesity is mainly due to excessive diet and insufficient activity, which increases fat intake and synthesis, and thus leads to obesity. The internal cause is mainly the disorder of fat metabolism in the body, which causes obesity. Since the lipase in the gastrointestinal tract is mainly responsible for hydrolyzing triglycerides to degrade them into diglycerides, monoglycerides and fatty acids and other products for absorption by the human body. Therefore, controlling the absorption of fat in the diet can effectively control body weight, and the lipase inhibitor existing in the diet has become an important target for the treatment of obesity drugs. There are commercialized lipase inhibitors on the market, such as orlistat, which shows good inhibitory effect, but also has a variety of gastrointestinal adverse reactions. Therefore, it is imperative to find new and efficient and safe lipase inhibitors from natural products.

[0004] Flavone is a general term for a series of compounds in which two benzene rings (A- and B-rings) with phenolic hydroxyl groups are connected to each other through a central three-carbon atom. Studies have shown that flavonoids have lipase inhibition, antiviral activity, antitumor, antioxidant free radical activity, anti-inflammatory, analgesic, lipase inhibition, and improvement of the cardiovascular system. At the same time, research has confirmed that the pharmacological effects of flavones are closely related to the chemical structure, in order to obtain more effective structure-activity relationships of these compounds, more flavonoids can be further researched and developed to find effective lead compounds and active groups, and more new flavonoids can be developed.

[0005] The origin of the sharp honey is in Malaysia and Mexico, and it is also cultivated in Hainan, Fujian, Guangxi and Yunnan of China. The mature pulp of the sharp honey is sweet and aromatic, and is rich in nutrition, which can be eaten fresh, and can also be processed into jam, preserved fruits, fruit juice, jelly and canned food, etc. In addition, modern medical research has confirmed that the sharp honey has the effects of anti-edema, lipase inhibition, anti-inflammatory, etc. A new isoprenyl flavonoid compound is separated from the sharp honey in the present application, and the compound has not been reported so far. It is worth mentioning that the compound has significant lipase inhibition effect, and can be used as a new, efficient and safe lipase inhibitor. In addition, the compound is the first isoprenyl flavonoid compound with 4-methyl furan ring found in natural products, and the structure of the compound has high novelty, and has major scientific significance for perfecting the structure-activity relationship of flavonoids. SUMMARY

[0006] The first object of the present application is to provide a flavonoid compound with lipase inhibition activity; the second object is to provide a preparation method of the flavonoid compound with lipase inhibition activity; and the third object is to provide an application of the flavonoid compound with lipase inhibition activity.

[0007] The first object of the present application is achieved by using the fine branches of the medicinal plant sharp honey (Murraya paniculata (Lour.) Spren) as raw materials, and preparing by pretreatment, extractive extraction, silica gel column chromatography and high performance liquid chromatography separation. Artocarpus champeden The compound is named as 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone, and the English name is 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone, which has the following structure:

[0008] .

[0009] The second object of the present application is achieved by using the fine branches of the medicinal plant sharp honey as raw materials, and preparing by pretreatment, extractive extraction, silica gel column chromatography and high performance liquid chromatography separation, which specifically comprises the following steps:

[0010] A, pretreatment: the raw material medicinal plant sharp honey fine branches are crushed to obtain material a;

[0011] B, extractive extraction: 2-6 times of the mass of the material a of the organic extraction solvent is added into the material a, and soaked and extracted at normal temperature for 2-5 times, and the extraction time is 12-20 hours each time, the extraction liquid is combined and filtered to obtain sample extraction liquid b;

[0012] C, MCI decolorization: the sample extraction liquid is subjected to MCI column decolorization, the effluent is collected and concentrated under reduced pressure to obtain extract c;

[0013] D. Silica gel column chromatography:

[0014] 1) Add 3 to 10 times the weight of extract c to pack a column with 200 to 250 mesh silica gel, and perform gradient elution with a chloroform-methanol solution with a volume ratio of 20:1 to 1:1. Monitor the elution by TLC and combine the identical fractions.

[0015] 2) Add 3 to 10 times the weight of the extract in 200 to 250 mesh silica gel to the eluent obtained by elution with a chloroform-methanol solution in a 9:1 ratio. Then, perform gradient elution with a chloroform-acetone solution in a volume ratio of 1:0 to 1:2. Combine the identical fractions by TLC monitoring.

[0016] E. High performance liquid chromatography separation: The eluent obtained by elution with chloroform-acetone solution in an 8:2 ratio was separated and purified by high performance liquid chromatography to obtain isopentenyl flavonoids with lipase inhibitory activity.

[0017] The structures of the prepared isopentenyl flavonoids were identified by the following methods:

[0018] HRESI-MS analysis of the compound showed a quasi-molecular ion peak at 385.1049 [M+Na]. + (The calculated value is 385.1046), combined with 1 H NMR, 13 C and DEPT spectra determined its molecular formula to be C. 22 H 18 O5 has an unsaturation degree of 14.

[0019] The infrared spectrum showed carbonyl (1669) and aromatic rings (1617, 1542, 1464 cm⁻¹). -1 The resonance absorption peaks of the compound and the maximum absorption at 380, 282, and 215 nm in the ultraviolet spectrum also indicate the possible presence of an aromatic ring structure in the compound.

[0020] compound 1 H and 13 C NMR data (as shown in Table 1) Figure 1 and Figure 2 The compound contains 22 carbons and 18 hydrogens, including one 1,2,4,5-tetrasubstituted benzene ring (C-5~C-10, H-5, H-7), one 1,4-disubstituted benzene ring (C-1'~C-6', H2-2', 6 and H2-3', 5'), and one... α,β - Unsaturated carbonyl group (C-2, C-3, C-4, H-3), one (4''-methylfuran-2-yl) structural segment (C-2''~C-6'', H-3'', H-5'' and H3-6''), two methoxy groups (δ C56.0 q, δ H 3.77 s; δ C 55.7 q, δ H 3.80 s). According to typical 2 benzene rings, α,β-unsaturated carbonyl and double bond signals. Plus two benzene rings unsaturation is 8; 1 α,β-unsaturated carbonyl unsaturation is 2; pyrrole ring unsaturation is 3, there should be a ring in the compound. Further analysis of its NMR data, the presence of two oxygenated aromatic aprotic substituted carbon (C-9 and C-2) in the compound, indicating that C-9 and C-2 are connected by oxygen atom to form a pyrone ring, thus it can be inferred that compound 1 is a flavonoid compound. In addition, according to the presence of H-3 and C-4, C-10, C-1', H-5 and C-4, C-9, C-10, and H-2' and C-2 HMBC correlation (such as Figure 3 ), thus it can be further confirmed that the compound of the present application is a flavonoid structure.

[0021] Table 1. The 1 H NMR and 13 C NMR data (CDC13)

[0022]

[0023] After the parent compound is determined, the rest of the substituents such as (4"-methylfuran-2-yl) structural fragment and methoxy can be considered as substituents on the flavone. The presence of (4"-methylfuran-2-yl) structural fragment can also be confirmed by H-3" and C-2", C-4", C-5", C-6"; H-5" and C-2", C-3", C-4", C-6"; and H-6" and C-3", C-4", C-5" HMBC correlation. At the same time, through further HMBC correlation analysis, it can be confirmed that two methoxy proton signals (δ H 3.77 s and 3.80) have HMBC correlation with C-6 and C-4', confirming that the two methoxy groups are located at C-6 and C-4'. According to the HMBC correlation of H-8 and C-2", and H-3" and C-7, it can be confirmed that the (4"-methylfuran-2-yl) structural fragment is connected at C-7. Thus, the structure of compound 1 is determined. The compound is named: 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone, and the English name is: 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone.

[0024] According to literature review, the compound of this invention is a flavonoid compound in which the isopentenyl group forms a 4-methylfuran ring, which is discovered for the first time in a natural product, and its compound structure has a high degree of novelty.

[0025] Infrared, ultraviolet, and mass spectrometry data of the compound: ultraviolet spectrum (methanol), Lambda max (log Epsilon 380 (3.74), 282 (3.91), 215 (4.22) nm; Infrared spectrum (potassium bromide tablets): Nu max 3152, 3063, 2979, 2832, 1669, 1617, 1542, 1464, 1359, 1270, 1161, 1072, 883cm -1 ; 1 H and 13 C10 NMR data (500 and 125 MHz, (C5D5N), see Table-1; positive ion mode ESIMS) m / z 385[M+Na] + HRESIMS in positive ion mode m / z 385.1049 [M+Na] + (Calculated value 385.1046, C) 22 H 18 NaO5).

[0026] The third objective of this invention, namely the lipase inhibitor activity test of the isopentenyl flavonoid compounds, is achieved as follows:

[0027] The reaction substrates 4-methylumbelliferyl oleate (4-MUO75164) and porcine pancreatic amylase (EC3.1.1.3, L3126) were purchased from Sigma-Aldrich, and phosphate-buffered saline (PBS) was purchased from Life Sciences (Thermo Fisher Scientific, China). DMSO and MeOH were analytical grade and purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; citric acid and sodium citrate were purchased from Tianjin Fuchen Chemical Reagent Factory.

[0028] Microplate reader: Tecan GENios (Tecan Group Ltd., Swizerland); 96-well plate: CorningCostar (Cambridge, MA, USA).

[0029] Lipase inhibitory activity was evaluated using a colorimetric method (Ercan et al.). Food. ChemThe inhibitory rate of the test compound on pancreatic lipase is determined by using a 96-well cell culture plate (see, for example, WO 2005, 163-169). The substrate 4-MUO generates 4-methylumbelliferone with fluorescence under the action of pancreatic lipase, and the inhibitory activity of the compound on lipase is determined by measuring the fluorescence intensity of each well after the reaction.

[0030] 0.1M citric acid-sodium citrate buffer (pH=4.2) is prepared with ultrapure water; 1.0mg / mL pancreatic lipase solution, 0.1mM 4-MUO solution, and 0.2M Na2CO3 solution are prepared with PBS (pH=7.4).

[0031] Part of the prepared lipase solution (1.0mg / ml) is heated in a 90℃ water bath for 20 minutes to inactivate the enzyme.

[0032] The compound and the positive control are respectively diluted with DMSO to an initial concentration of 10, 5, 2.5, 1.25, 0.625, and 0.3125mM.

[0033] The sample is added to the following reaction system (the concentration of DMSO is not more than 5% of the whole system): sample group: 1.0μL sample+24μL PBS+25μL enzyme; sample blank group: 1.0μL sample+24μL PBS+25μL inactivated enzyme; negative group: 1μL DMSO+24μL PBS+25μL enzyme; and negative blank group: 1.0μL DMSO+24μL PBS+25μL inactivated enzyme.

[0034] After mixing, 50μL of the reaction substrate 4-MUO (0.1mM) is added to each well, and the reaction is carried out in a 25℃ constant temperature incubator for 20 minutes. Then, 100μL of citric acid-sodium citrate solution (0.1M, pH=4.2) is added to each sample well to terminate the reaction, and the fluorescence absorbance value of each well is determined by using an enzyme marker, with an excitation wavelength of 320nm and a detection wavelength of 450nm. Each sample is repeated at least three times, and the inhibition rate is calculated according to the following formula:

[0035] Inhibition rate (%)=[1-(A sample-A sample blank) / (A negative-A negative blank)]x100

[0036] The half maximal inhibitory concentration IC 50 value of the isoprenyl flavone of the application is 22.6±2.6μM, which has a significant lipase inhibitory effect, and is expected to be used as a lead compound to develop a new type of lipase inhibitor drug. 50

[0037] The application has the following advantages:

[0038] ​(1) The compound of the present invention is the isopentenyl flavonoid of the present invention, which is isolated from the medicinal plant *Cinnamomum camphora*. *Cinnamomum camphora* has a large biological yield of twigs, the raw material source is very wide, the cost is low, and the compound is easy to isolate and prepare.

[0039] (2) The preparation method of this invention is a combination of solvent extraction, conventional column chromatography and high performance liquid chromatography. The compound preparation process is simple, the obtained compound has high purity, and subsequent industrial production is easy to achieve.

[0040] (3) The compounds of this invention are non-toxic to animals and safe to use. Their IC50 values ​​are [not specified]. 50 The value was 22.6±2.6 μM, which showed significant lipase inhibitory activity and is expected to be used as a lead compound for the development of novel lipase inhibitor drugs.

[0041] (4) The compound of the present invention is a flavonoid compound in which the isopentenyl group forms a 4-methylfuran ring for the first time in a natural product. Its compound structure has high novelty and is of great scientific significance for improving the structure-activity relationship of flavonoid compounds. Attached Figure Description

[0042] Figure 1 The carbon NMR spectrum of lipase-inhibiting flavonoids of this invention ( 13 (C NMR).

[0043] Figure 2 The proton nuclear magnetic resonance spectra of the lipase-inhibiting flavonoids of this invention (H1N) 1 H NMR);

[0044] Figure 3 The key HMBC correlation diagram of the lipase-inhibiting flavonoid compounds of this invention. Detailed Implementation

[0045] The present invention will be further described below with reference to embodiments and accompanying drawings, but this does not limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0046] It is based on the medicinal plant Spiraea japonica ( Artocarpus champeden Using (Lour.)Spren) twigs as raw material, 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone was prepared through pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography. It was named 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone and has the following structure:

[0047]

[0048] The lipase-inhibiting active flavonoid compound described in the present application is prepared from the medicinal plant Acronychia pedunculata by pretreatment, extractive extraction, silica gel column chromatography and high-performance liquid chromatography separation, and specifically comprises the following steps:

[0049] A, pretreatment: crushing the raw material Yunnan national medicinal plant Acronychia pedunculata to obtain material a;

[0050] B, extractive extraction: adding 2-6 times the mass of material a of an organic extraction solvent to material a, soaking and extracting at room temperature for 2-5 times, each time for 12-20 hours, combining the extractive extraction liquid and filtering to obtain sample extractive extraction liquid b;

[0051] C, MCI decolorization: loading the sample extractive extraction liquid onto an MCI column for decolorization, collecting the effluent and concentrating under reduced pressure to obtain extractive c;

[0052] D, silica gel column chromatography:

[0053] 1) loading 3-10 times the weight of extractive c of 200-250 mesh silica gel into a column, gradient eluting with chloroform-methanol solution with a volume ratio of 20:1-1:1, monitoring by TLC, and combining the same parts;

[0054] 2) loading 3-10 times the weight of extractive c of 200-250 mesh silica gel into a column with 9:1 chloroform-methanol solution, gradient eluting with chloroform-acetone solution with a volume ratio of 1:0-1:2, monitoring by TLC, and combining the same parts;

[0055] E, high-performance liquid chromatography separation: purifying the eluent obtained by eluting with 8:2 chloroform-acetone solution by high-pressure liquid chromatography to obtain the target lipase-inhibiting active flavonoid compound.

[0056] The organic extraction solvent in step B is 70%-100% methanol aqueous solution, 70%-100% ethanol aqueous solution or 70%-100% acetone aqueous solution.

[0057] The D step further comprises a sample mixing step of dissolving 1.5-3 times the weight of material c of an organic solvent and then adding 0.8-2.0 times the weight of 80-100 mesh silica gel to material c before column loading.

[0058] The organic solvent is pure methanol or pure acetone.

[0059] The chloroform-methanol solution in step D1) has a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4 or 1:1.

[0060] The high performance liquid chromatography separation and purification in the E step is that methanol aqueous solution with a volume concentration of 60-75% is used as a mobile phase, a flow rate is 12 mL / min, a Zorbax PrepHT GF reversed-phase preparation column with a size of 2.12*250 mm and 5 μm is used as a stationary phase, a UV detector is used for detection with a detection wavelength of 380 nm, 0.5-1.0 mL is injected each time, and the chromatographic peak of 30-42 min is collected, and the target lipase inhibition active flavonoid compound is obtained after multiple accumulation and evaporation.

[0061] The isoprenyl flavone compound provided by the application is used as a lipase inhibitor.

[0062] The preparation method specifically includes the following operations:

[0063] A, sample extraction and purification: the Xianmilazi is dried, crushed to 20-50 meshes, then extracted with a solvent for 2-5 times, each time with 2-6 times the raw material, and the extraction time is 12-20 hours; the precipitate is filtered to obtain a sample extract, and the extract is concentrated under reduced pressure to obtain a extract. The prepared ethanol extract is distributed in ethyl acetate and aqueous solution. The ethyl acetate layer is concentrated under reduced pressure to obtain a crude extract. The obtained crude extract is filtered to remove the precipitate, and then subjected to decolorization on an MCI column. The effluent is collected and concentrated under reduced pressure to obtain an extract for column chromatography separation.

[0064] B, primary silica gel column chromatography: the obtained extract is diluted with 1.5-3 times of acetone or methanol, then mixed with silica gel in an amount of 0.8-2.0 times the weight of the extract, and the mixed silica gel is 100-150 mesh. The mixed sample is subjected to column chromatography on silica gel, the silica gel for column chromatography is 200-250 mesh, and the weight of the silica gel used is 3-10 times the weight of the extract; gradient elution is performed with a mixed organic solvent of chloroform and methanol in a volume ratio of 20:1-1:1, the gradient eluate of each gradient is collected and concentrated, and TLC monitoring is performed to combine the same parts to obtain 6 components (A-F).

[0065] C, secondary silica gel column chromatography: the component b (the chloroform-methanol 9:1 part) in the B step is further subjected to silica gel column chromatography: the silica gel for column chromatography is 200-250 mesh, the weight of the silica gel used is 3-10 times the weight of the extract, gradient elution is performed with a mixed organic solvent of chloroform and acetone in a volume ratio of 1:0-1:2, and the gradient eluate of each gradient is collected and concentrated.

[0066] D, high performance liquid chromatography separation: the component C (the part eluted with the mixed organic solvent of chloroform and acetone 8:2) in the C step is subjected to high performance liquid chromatography separation and purification to obtain the flavonoid compound.

[0067] Further, preferably, the solvent of the A step is an aqueous acetone solution with a volume concentration of 70-100%, an aqueous ethanol solution with a volume concentration of 70-100%, or an aqueous methanol solution with a volume concentration of 70-100%.

[0068] Further, preferably, before the silica gel column chromatography in the C step, the extract is diluted with acetone or methanol in an amount of 1.5-3 times the weight of the extract, and then the sample is mixed with 80-100 mesh silica gel in an amount of 0.8-2.0 times the weight of the extract, and then loaded.

[0069] Further, preferably, in the C step, the volume ratio of chloroform and methanol mixed organic solvent used in gradient elution is 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1 in turn (each time elution is performed until no component flows out, i.e., no component remains in the distillation flask after the solvent is evaporated, and then the next gradient is changed).

[0070] Further, preferably, the high-performance liquid chromatography separation and purification in the E step is performed using an aqueous methanol solution with a volume concentration of 60-75% as the mobile phase, a flow rate of 12 mL / min, a Zorbax PrepHT GF reversed-phase preparative column with a size of 2.12*250 mm and a particle size of 5 μm as the stationary phase, a UV detector with a detection wavelength of 380 nm, an injection amount of 0.5-1.0 mL each time, and the chromatographic peak collected at 30-42 min, and the pure compound is obtained after multiple accumulations and evaporation.

[0071] The raw medicinal plant Jiumila used in the present application is not limited by region and variety, and can achieve the content of the present application,

[0072] The present application is further described below using Jiumila raw materials from different regions in Yunnan:

[0073] Example 1

[0074] The present application provides a preparation method of the lipase-inhibiting active flavonoid compound, which comprises the steps of extract preparation, silica gel column chromatography and high-performance liquid chromatography separation, and uses Jiumila as the raw medicinal plant, and the specific operation is as follows:

[0075] The medicinal plant Jinyingla used is produced in Honghehekou, Yunnan. The raw material is the sample of the medicinal plant Jinyingla after being crushed or cut into segments. The sample is soaked in 70% methanol water solution for extraction for 4 times, each time for 15 hours. The combined extract is decolorized by MCI column, and the eluent is concentrated under reduced pressure to obtain extract. The extract is dissolved in 2 times the mass of methanol, then 90 mesh silica gel is added for mixing, and the mixture is loaded on a column. The column is eluted with chloroform-methanol eluent with a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1, respectively, by gradient elution. The gradient eluent is collected, concentrated, and monitored by TLC. The same parts are combined to obtain 6 parts A-F. The collected sample B (9:1) part 235 g is loaded on a 220 mesh silica gel column with 8 times the mass of extract. The column is eluted with chloroform-acetone eluent with a volume ratio of 1:0, 1:1 and 1:2, respectively, by gradient elution. The gradient eluent is collected, concentrated, and monitored by TLC. The same parts are combined to obtain 3 parts, wherein the 8:2 part 28.5 g is further purified by HPLC. The mobile phase is 66% methanol, the flow rate is 12 ml / min, the column is 2.12 x 250 mm, and the column temperature is 5 µ The Zorbax PrepHT GF reversed-phase preparation column with a mobile phase of methanol is used as the stationary phase, and the ultraviolet detector is used for detection with a wavelength of 380 nm. Each time, 0.8 mL is injected, and the chromatographic peak of 35.2 min is collected. After multiple accumulations, the sample is evaporated to dryness to obtain the lipase-inhibiting active prenyl flavonoid compound.

[0076] The structure of the prepared lipase-inhibiting active prenyl flavonoid compound is identified by the following method:

[0077] The HRESI-MS of the compound shows that the quasi-molecular ion peak is 385.1049 [M+Na] + (calculated value is 385.1046), combined with 1 H NMR, 13 C and DEPT spectrum determines that the molecular formula of the compound is C 22 H 18 O5, and the unsaturation degree is 14.

[0078] The infrared spectrum shows the resonance absorption peaks of carbonyl (1669) and aromatic ring (1617, 1542, 1464 cm -1 ). The ultraviolet spectrum has maximum absorption at 380, 282, 215 nm, which also indicates that there may be an aromatic ring structure in the compound.

[0079] The HRESI-MS of the compound shows that the quasi-molecular ion peak is 385.1049 [M+Na] 1 H and 13 C NMR data (as shown in Table 1, Figure 1 and Figure 2) shows that the compound contains 22 carbons and 18 hydrogens, including 1 1,2,4,5-tetrasubstituted benzene ring (C-5~C-10, H-5, H-7), 1 1,4-disubstituted benzene ring (C-1 '~C-6', H2-2',6 and H2-3',5'), and 1 α,β - unsaturated carbonyl (C-2, C-3, C-4, H-3), one (4"-methylfuran-2-yl) moiety (C-2"~C-6", H-3", H-5" and H3-6"), two methoxy groups (δ C 56.0 q, δ H 3.77 s; δ C 55.7 q, δ H 3.80 s). According to the typical signals of 2 benzene rings, α,β-unsaturated carbonyl and double bond. In addition to the unsaturation of two benzene rings, 8; the unsaturation of 1 α,β-unsaturated carbonyl is 2; the unsaturation of pyrrole ring is 3, there should be one more ring in the compound. Further analysis of the NMR data of the compound, the presence of two oxidized aromatic aprotic substituted carbons (C-9 and C-2) in the compound, indicating that C-9 and C-2 are connected by oxygen atom to form a pyrone ring, thus it can be inferred that compound 1 is a flavonoid compound. In addition, according to the HMBC correlation of H-3 and C-4, C-10, C-1 ', H-5 and C-4, C-9, C-10, and H-2' and C-2 in the compound (such as Figure 3 ), it can be further confirmed that the compound of the present application is a flavonoid structure.

[0080] After the parent compound is determined, the remaining substituents such as (4"-methylfuran-2-yl) moiety and methoxy group can be regarded as substituents on the flavone. The presence of (4"-methylfuran-2-yl) moiety can also be confirmed by HMBC correlation of H-3" and C-2", C-4", C-5", C-6"; H-5" and C-2", C-3", C-4", C-6"; and H-6" and C-3", C-4", C-5". At the same time, through further HMBC correlation analysis, it can be known that the two methoxy proton signals (δ H3.77 s and 3.80) and C-6 and C-4', it can be confirmed that two methoxyl groups are located at C-6 and C-4'. According to the HMBC correlation of H-8 and C-2" and H-3" and C-7, it can be confirmed that the (4"-methylfuran-2-yl) moiety is connected at C-7. Thus far, the structure of compound 1 is determined. The compound is named as: 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone. Through literature search, the compound of the present application is the first time to be found in natural products as a flavone compound with isopentenyl group forming a 4-methylfuran ring, and the compound structure has high novelty.

[0081] Example 2

[0082] The present example provides a preparation method of the lipase inhibitory activity flavone compound according to the present application, which comprises the steps of extract immersion, silica gel column chromatography and high performance liquid chromatography separation, and uses medicinal plant Jiumilai as raw material, and the specific operation is as follows:

[0083] The medicinal plant Jiumilai used is produced in Yuanjiang, Yuxi, Yunnan, and the raw material is a sample of crushed or segmented Jiumilai fine branches. The sample is soaked and extracted for 5 times with 80% ethanol aqueous solution, each time for 12 h. The extract liquid is combined and subjected to decolorization on an MCI column, and the eluent is concentrated under reduced pressure to obtain an extract. The extract is dissolved in acetone with a mass of 3 times of the extract, then 100 mesh silica gel is added for mixing, and the mixed sample is subjected to column packing with 250 mesh silica gel. Gradient elution is performed with chloroform-methanol eluent with a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1, respectively. The gradient eluent is collected, concentrated, monitored by TLC, and the same parts are combined to obtain 6 parts A-F. Among them, the sample B (9:1) part 271 g is collected, 10 times of 250 mesh silica gel is added to the extract column, and gradient elution is performed with chloroform-acetone eluent with a volume ratio of 1:0, 1:1 and 1:2, respectively. The gradient eluent is collected, concentrated, monitored by TLC, and the same parts are combined to obtain 3 parts, among which the 8:2 part 26.7 g is further subjected to high performance liquid chromatography separation with 66% methanol as mobile phase, a flow rate of 12 ml / min, a column of 2.12 x 250 mm, 5 µ Zorbax PrepHT GF reversed phase preparation column with a mobile phase of methanol and 0.1% formic acid in water, a flow rate of 12 ml / min, a column of 2.12 x 250 mm, 5 22 H 18 O5。

[0084] Example 3

[0085] The present embodiment provides a preparation method of the lipase-inhibiting active flavonoid compound according to the present application, which comprises the steps of extract immersion, silica gel column chromatography and high performance liquid chromatography separation, and uses medicinal plant Xianmilas as raw material, and the specific operation is as follows:

[0086] The medicinal plant Xianmilas used is produced in Menghai, Xishuangbanna, Yunnan, and the raw material is medicinal plant Xianmilas (after the sample of fine branches is crushed or cut into sections, it is soaked and extracted twice with 80% acetone aqueous solution, each time for 12 h. The extract immersion is obtained by combining the extract liquid, decolorizing on an MCI column, and concentrating the eluent under reduced pressure; the extract immersion is dissolved in 1.5 times the mass of methanol, then 80-mesh silica gel is added for mixing, and the sample is packed on a column after mixing; gradient elution is performed with chloroform-methanol eluent with a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1, respectively, the gradient eluent is collected, concentrated, monitored by TLC, and the same parts are combined to obtain six parts A-F; among them, 229 g of the collected sample B (9:1) part is packed on a 200-mesh silica gel column with 3 times the amount of extract immersion, gradient elution is performed with chloroform-acetone eluent with a volume ratio of 1:0, 1:1 and 1:2, respectively, the gradient eluent is collected, concentrated, monitored by TLC, and the same parts are combined to obtain three parts, among which 23.6 g of the 8:2 part is packed on a 2.12 x 250 mm column with 66% methanol as the mobile phase, a flow rate of 12 ml / min, and 5 µ m Zorbax PrepHT GF reversed-phase preparation column as the stationary phase, and a UV detector with a detection wavelength of 380 nm, 0.5-1.0 mL of sample each time, collection of the chromatographic peak at 35.2 min, and multiple accumulations and evaporation to dryness to obtain the lipase-inhibiting active flavonoid compound according to the present application. Identification: the identification method is the same as in Example 1, and the result is that the molecular formula is C 22 H 18 O5。

[0087] Example 4

[0088] Any of the compounds prepared in Examples 1-3 is a yellow gum. The determination method is the same as in Example 1, and it is confirmed that the compound prepared in Examples 1-3 is the 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone described.

[0089] Example 5

[0090] Any of the benzopyran lactone compounds prepared in Examples 1-3 is subjected to safety evaluation, and the test conditions are as follows: the compound according to the present application is proved to be low in animal toxicity and safe in use by mouse bone marrow micronucleus test, Ames test and TK gene mutation test.

[0091] Example 6

[0092] The isoprenyl flavonoid compounds prepared in Examples 1-3 were subjected to lipase inhibition activity test, and the test conditions and results are as follows:

[0093] The reaction substrate 4-methylumbelliferyl oleate (4-MUO 75164) and porcine pancreatic amylase (EC 3.1.1.3, L3126) were purchased from Sigma Company, and the phosphate buffer PBS was purchased from Life Company (Thermo Fisher Scientific, China). DMSO and MeOH were both analytical pure and purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; citric acid and sodium citrate were purchased from Tianjin Fumeng Chemical Reagent Factory.

[0094] The enzyme reader Tecan GENios (Tecan Group Ltd., Swizerland); 96-well plates (Corning Costar, Cambridge, MA, USA).

[0095] The lipase inhibition activity evaluation was carried out by colorimetry (Ercan et al, Food. Chem ., 205, 163-169), and the determination of the inhibition rate of the test compound on pancreatic lipase was completed by 96-well cell culture plates. The substrate 4-MUO generates 4-methylumbelliferone with fluorescence under the action of pancreatic lipase, and the inhibition activity of the compound on lipase is determined by measuring the fluorescence intensity of each well after reaction.

[0096] 0.1M citric acid-sodium citrate buffer (pH=4.2) was prepared with ultrapure water; 1.0mg / mL pancreatic lipase solution, 0.1mM 4-MUO solution, and 0.2M Na2CO3 solution were prepared with PBS (pH=7.4).

[0097] Part of the prepared lipase solution (1.0mg / ml) was heated in a 90°C water bath for 20 minutes to inactivate the enzyme.

[0098] The compound and positive control were respectively diluted with DMSO to an initial concentration of 10, 5, 2.5, 1.25, 0.625, 0.3125mM of the test solution.

[0099] The sample was added according to the following reaction system (the concentration of DMSO was not more than 5% of the whole system): sample group: 1.0μL sample+24μL PBS+25μL enzyme; sample blank group: 1.0μL sample+24μL PBS+25μL inactivated enzyme; negative group: 1μL DMSO+24μL PBS+25μL enzyme; negative blank group: 1.0μL DMSO+24μL PBS+25μL inactivated enzyme.

[0100] After mixing the samples, add 50 μL of 4-MUO (0.1 mM) to each well. Incubate at 25°C for 20 min. Then, stop the reaction by adding 100 μL of citrate-sodium citrate solution (0.1 M pH=4.2) to each well. Measure the absorbance of each well using a microplate reader. The excitation wavelength is 320 nm, and the detection wavelength is 450 nm. Each sample should be repeated at least three times. Calculate the inhibition rate using the following formula:

[0101] Inhibition rate (%) = [1 - (Sample A – Sample A Blank) / (Negative A – Negative A Blank)] × 100

[0102] The half-maximal inhibitory concentration (IC50) was calculated using SPSS 19.0 statistical analysis software. 50 The values ​​indicate that the IC50 of the isopentenyl flavonoids of this invention... 50 With a concentration of 22.6 ± 2.6 μM, it exhibits significant lipase inhibitory activity and holds promise as a lead compound for the development of novel lipase inhibitor drugs.

Claims

1. A flavonoid compound with lipase-inhibiting activity, characterized in that, It was prepared from *Hylocereus undatus* twigs through pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography. It was named 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone, and its English name is 6,4'-dimethoxy-7-(4-methylfuran-2-yl)-flavone. It has the following structure: 。 2. A method for preparing the flavonoid compound with lipase-inhibiting activity as described in claim 1, characterized in that, It is prepared from the Yunnan ethnic minority medicinal plant *Trifolium repens* branches through pretreatment, extract extraction, silica gel column chromatography, and high-performance liquid chromatography separation, specifically including the following steps: A. Pre-treatment: The raw material, Yunnan ethnic medicinal honey, is finely crushed or cut into sections to obtain material a; B. Extraction of extract: Add 2 to 6 times the mass of organic extraction solvent to material a, and extract by soaking at room temperature 2 to 5 times, with each extraction time being 12 to 20 hours. Combine the extracts and filter to obtain sample extract b. C. MCI decolorization: The sample extract was decolorized by MCI column, the eluent was collected and concentrated under reduced pressure to obtain extract c; D. Silica gel column chromatography: 1) Add 3 to 10 times the weight of extract c to pack a column with 200 to 250 mesh silica gel, and perform gradient elution with a chloroform-methanol solution with a volume ratio of 20:1 to 1:

1. Monitor the elution by TLC and combine the identical fractions. 2) The eluent obtained by eluting with a 9:1 chloroform-methanol solution was concentrated under reduced pressure to obtain component d. 3 to 10 times the weight of the obtained component d was packed into a column with 200 to 250 mesh silica gel and gradient eluted with a chloroform-acetone solution with a volume ratio of 1:0 to 1:

2. The same fractions were combined by TLC monitoring. E. High performance liquid chromatography separation: The eluent obtained by elution with chloroform-acetone solution in a ratio of 8:2 was separated and purified by high performance liquid chromatography to obtain flavonoids with lipase inhibitory activity as the target analyte. High-performance liquid chromatography (HPLC) separation and purification used a 60-75% (v / v) methanol-water solution as the mobile phase, a flow rate of 12 mL / min, and a chromatography wavelength of 2.12 × 250 mm. μ A Zorbax PrepHT GF reversed-phase preparative column was used as the stationary phase, and the UV detector was set at a wavelength of 380 nm. Each injection was 0.5 to 1.0 mL, and the chromatographic peaks were collected for 30 to 42 min. After multiple injections, the peaks were evaporated to dryness to obtain the target flavonoid compounds.

3. The preparation method according to claim 2, characterized in that, The organic extraction solvent mentioned in step B is a 70%~100% methanol aqueous solution, a 70%~100% ethanol aqueous solution, or a 70%~100% acetone aqueous solution.

4. The preparation method according to claim 2, characterized in that, Step D, before loading the column, also includes a sample mixing step where the sample is dissolved in an organic solvent at 1.5 to 3 times its weight of material c, and then mixed with 80 to 100 mesh silica gel at 0.8 to 2.0 times its weight of material c.

5. The preparation method according to claim 4, characterized in that, The organic solvent is pure methanol or pure acetone.

6. The preparation method according to claim 2, characterized in that, The volume ratios of the chloroform-methanol solution mentioned in step 1) are 20:1, 9:1, 8:2, 7:3, 6:4 and 1:

1.

7. The application of the flavonoid compound with lipase-inhibiting activity as described in claim 1, characterized in that, The application of the lipase-inhibiting flavonoids in the preparation of lipase inhibitors.

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