Furan lignan glycoside compound in forsythia suspense leaves as well as extraction and separation method and application of furan lignan glycoside compound

Abstract

The invention discloses a furan lignan glycoside compound in forsythia suspense leaves and an extraction and separation method and application thereof. The furan lignan glycoside compound comprises a compound 1 and a compound 2, wherein the molecular formula of the compound 1 is C35H41O14; the name of the compound 1 is Phillyrigeninside B, and the structural formula of the compound 1 is shown in the description. The molecular formula of the compound 2 is C35H41O14; the name of the compound 1 is Phillyrigeninside C, and the name of the compound 2 is Phillyrigeninside C. According to the scheme, natural active substances are subjected to solvent extraction, enrichment, separation and the like by utilizing an extraction and separation method. The whole extraction process has good controllability and reproducibility, and all solvents can be recycled in the whole production process, so that in the scheme, the whole preparation process of the furan lignan glycoside compounds in the forsythia suspense leaves is simple, convenient and reasonable, and industrial production is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a furanolignan glycoside compound in forsythia leaves and an extraction and separation method and application thereof. Background Art

[0002] Forsythia suspensa (Thunb.) Vah is a shrub of the genus Forsythia in the family Oleaceae. Its dried fruit is a commonly used bulk medicinal material - Forsythia suspensa fruit (Chinese medicine Forsythia suspensa). According to the Compendium of Materia Medica, Forsythia suspensa has the effects of clearing away heat and detoxifying, reducing swelling and dispersing nodules. It is one of the traditional Chinese medicines commonly used in clinical practice in China, and is often used to treat acute wind-heat colds, lymph node tuberculosis and other diseases. In daily life, Forsythia suspensa fruit is often used in combination with honeysuckle as medicine, such as "Vitamin C Yinqiao Tablets" and "Shuanghuanglian Oral Liquid". In addition to medicinal uses, Forsythia suspensa is also used as a natural food preservative or cosmetic. In addition, Forsythia suspensa is an important oil crop, ornamental plant and soil and water conservation plant.

[0003] Forsythia leaf is the dried leaf of Forsythia, and its chemical composition is similar to that of Forsythia fruit. Compared with the traditional Chinese medicine Forsythia, Forsythia leaf is rich in resources and highly renewable. For a long time, Forsythia leaf has been used as a waste of Forsythia and has not been fully utilized, resulting in a great waste of resources. Modern pharmacological studies have shown that Forsythia leaf has the effects of preventing colds, clearing away heat and detoxifying, relieving sore throat and swelling, lowering blood pressure and anti-oxidation, and has broad application and development prospects. Forsythia leaf not only has certain therapeutic effects in preventing and treating diseases, but also has rich nutrients. Forsythia leaf has a high content of protein, dietary fiber, β-carotene and 17 amino acids, and the total amino acid content can reach 18%-20%. Therefore, in order to make full use of Forsythia leaf resources and clarify the material basis of the efficacy of Forsythia leaf, it is necessary to further study and utilize the chemical composition of Forsythia leaf. Summary of the invention

[0004] The present invention aims to provide a furanolignan glycoside compound in Forsythia suspensa leaves and an extraction and separation method and application thereof. The extraction and separation process of furanolignan glycoside compounds in Forsythia suspensa leaves of the present scheme is simple, has good controllability and reproducibility, and is convenient for industrial production.

[0005] One of the objects of the present invention is a furanolignan glycoside compound in Forsythia suspensa leaves, comprising compound 1 and compound 2; wherein the molecular formula of compound 1 is C 35 H 41 O 14 ; The name of compound 1 is: Phillyrigeninside B; The structural formula of compound 1 is as follows: Formula 1; The molecular formula of compound 2 is C 35 H 41 O 14; The name of compound 2 is: Phillyrigeninside C; The structural formula of compound 2 is as follows:

[0006]

[0007] The second object of the present invention is to provide a method for extracting and separating furanolignan glycoside compounds in Forsythia suspensa leaves, comprising the following steps:

[0008] Step 1, crushing the dried Forsythia suspensa leaves, and then refluxing and extracting them for 3-5 hours with an ethanol aqueous solution 10-15 times the mass of the Forsythia suspensa leaf raw material, and repeating the extraction several times; combining the ethanol extracts obtained by filtration, and recovering ethanol by vacuum evaporation of the combined ethanol extracts, and then obtaining a crude extract;

[0009] Step 2, suspending the crude extract obtained in step 1 in distilled water, wherein the mass ratio of the crude extract to the distilled water is 1:10-15 times; mixing well and filtering to obtain a filtrate;

[0010] Step 3, adding the filtrate obtained in step 2 to the macroporous adsorption resin and mixing thoroughly, wherein the mass ratio of the crude extract to the macroporous adsorption resin is 1:40-50; then maintaining for more than 24 hours until adsorption equilibrium is reached, after reaching the adsorption saturation state, loading the macroporous adsorption resin onto a chromatographic column, and then eluting with ethanol aqueous solutions of different concentrations at a certain flow rate; collecting each desorbed fraction segment and concentrating it under vacuum to obtain eluted fraction segments Fr.1, Fr.2, Fr.3, Fr.4, and Fr.5 of ethanol aqueous solutions of different concentrations;

[0011] Step 4: Separate and purify the eluted fraction Fr.3 obtained in step 3 to obtain compound 1 and compound 2.

[0012] As a preferred embodiment, in step 1, the dried Forsythia suspensa leaves are crushed, and then reflux extracted with a 70% ethanol aqueous solution with a volume percentage of 10-15 times the mass of the Forsythia suspensa leaf raw material for 3-5 hours, and the extraction is repeated 3 times; the ethanol extracts obtained by filtration are combined, and the ethanol is recovered by reduced pressure evaporation to obtain a crude extract.

[0013] As a preferred embodiment, in step 2, the crude extract obtained in step 1 is suspended in distilled water, wherein the mass ratio of the crude extract to distilled water is 1:10-15 times; after being fully mixed, the mixture is filtered with filter paper to obtain a filtrate.

[0014] As a preferred embodiment, in the step 3, the filtrate obtained in the step 2 is added to the macroporous adsorption resin AB-8 and mixed thoroughly, and the mass ratio of the crude extract to the macroporous adsorption resin AB-8 is 1:40-50; then the mixture is kept for more than 24 hours until adsorption equilibrium is reached, and after reaching the adsorption saturation state, the adsorption saturated macroporous adsorption resin AB-8 is loaded onto a chromatographic column with a height-to-diameter ratio of 8-10, and then eluted with ethanol aqueous solutions of different concentrations at a flow rate of 4.0 BV / h; each desorbed fraction segment is collected and concentrated under vacuum to obtain eluted fraction segments Fr.1, Fr.2, Fr.3, Fr.4, and Fr.5 of ethanol aqueous solutions of different concentrations.

[0015] As a preferred embodiment, in the step 4, the eluted fraction segment Fr.3 obtained in step 3 is subjected to medium pressure column chromatography separation and eluted with a CH3OH-H2O gradient system to obtain subcomponents Fr.3-1, Fr.3-2, Fr.3-3, and Fr.3-4. The fraction segment Fr.3-3 in the subcomponent is purified by gel column chromatography to obtain fractions Fr.3-3-1, Fr.3-3-2, and Fr.3-3-3. The obtained fraction Fr.3-3-2 is then purified by a semi-preparative HPLC column to obtain compounds 1 and 2.

[0016] As a preferred solution, in step 4, in the CH3OH-H2O gradient system, the volume percentages of methanol-water solution are 25%, 35%, 45% and 60% respectively.

[0017] The third object of the present invention is to use furanolignan glycoside compounds in Forsythia suspensa leaves in antibacterial and anti-inflammatory activities. Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] First, the two furanolignan glycoside compounds obtained by extraction and separation in this scheme. After activity studies, it was found that the two compounds obtained have strong antibacterial and anti-inflammatory activities. The raw material Forsythia suspensa leaves used in this scheme are rich in resources and highly renewable. This scheme avoids the waste of resources by extracting the active substances of Forsythia suspensa leaves, and the extracted compounds can be used as active ingredients of antibacterial or anti-inflammatory drugs. Based on in-depth research and development, they have potential application prospects in drug raw materials, feed additives and food.

[0019] Second, this scheme optimizes the extraction method, including: ① Drying of Forsythia suspensa leaves, solvent extraction; ② Enrichment of furanolignan glycoside compounds in Forsythia suspensa leaf extracts; ③ Separation of furanolignan glycosides and other processes. By drying the Forsythia suspensa leaves, the natural active substances are further subjected to solvent extraction, enrichment and separation by extraction and separation methods. The entire extraction process has good controllability and reproducibility, and all solvents can be recycled in the entire production process. Therefore, in this scheme, the overall preparation process of furanolignan glycoside compounds in Forsythia suspensa leaves is simple and reasonable, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the hydrogen spectrum of the compound 1Phillyrigeninside B obtained in the present invention;

[0021] Figure 2 This is the carbon spectrum of the compound 1Phillyrigeninside B obtained in the present invention;

[0022] Figure 3 is the HMBC spectrum of the compound 1Phillyrigeninside B obtained in the present invention;

[0023] Figure 4 HSQC spectrum of compound 1Phillyrigeninside B obtained in the present invention;

[0024] Figure 5 is the hydrogen spectrum of the compound 2Phillyrigeninside C obtained in the present invention;

[0025] Figure 6 is the carbon spectrum of the compound 2Phillyrigeninside C obtained in the present invention;

[0026] Figure 7 is the HMBC spectrum of the compound 2Phillyrigeninside C obtained in the present invention;

[0027] Figure 8 HSQC spectrum of compound 2Phillyrigeninside C obtained in the present invention;

[0028] Fig. 9 This is the NMR data table of Compound 1 and Compound 2 in Table 1. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and beneficial effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0030] In addition, in order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present invention can also be implemented without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0031] The embodiment of the present invention provides a method for extracting and separating furanolignan glycoside compounds in Forsythia suspensa leaves, comprising the following steps:

[0032] Step 1: crush a certain amount of dried Forsythia suspensa leaves, and then reflux extract for 3-5 hours with 70% ethanol aqueous solution with a volume percentage of 10-15 times the mass of the raw material, and repeat the extraction 3 times. Combine the ethanol extracts obtained by filtration, and evaporate under reduced pressure at 60°C to recover ethanol, and then obtain a crude extract; in this step, the Forsythia suspensa leaves are dried leaves of the medicinal plant Forsythia suspensa (Thunb.) Vah, and the leaves are harvested from June to July. Compared with the traditional Chinese medicine Forsythia (the fruit of the plant Forsythia), the Forsythia leaves are abundant in resources and highly renewable.

[0033] Step 2: suspend a certain amount of the crude extract obtained in step 1 in 10-15 times the mass of distilled water, mix thoroughly, and then filter with filter paper after reaching equilibrium.

[0034] Step 3, add the filtrate obtained in step 2 to a macroporous adsorption resin (AB-8) of 40-50 times the mass of the crude extract, mix thoroughly, and then keep for 24 hours until adsorption equilibrium is reached. After reaching the adsorption saturation state, load the macroporous adsorption resin (AB-8) onto a chromatographic column (height-to-diameter ratio 8-10), and then elute with ethanol aqueous solutions of different concentrations (20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, 95% ethanol (V / V)) at a flow rate of 4.0 BV / h. Collect each desorbed fraction and concentrate under vacuum to obtain elution fractions Fr.1, Fr.2, Fr.3, Fr.4, and Fr.5 of different ethanol concentrations.

[0035] Step 4. The fraction Fr.3 obtained in step 3 is separated by medium pressure column chromatography, gel column chromatography, semi-preparative HPLC and the like. In the following embodiments, the filler of the medium pressure column chromatography of the fraction Fr.3 is C18 reverse phase filler, the filler of the gel column chromatography is dextran gel LH-20, and the semi-preparative HPLC column is a reverse phase ODS-A column. Compounds 1 and 2 are finally separated and their structures are identified by organic compound spectral analysis methods such as NMR, HR-MS, IR, and UV.

[0036] Example 1

[0037] 5.6 kg of dried Forsythia suspensa leaves were crushed, and then refluxed and extracted for 3 hours with 70% ethanol aqueous solution with a volume percentage of 15 times the mass of the raw material, and the extraction was repeated 3 times until the color of the extract became lighter. The obtained ethanol extracts were combined and evaporated under reduced pressure at 60°C, and then 190 g of crude extract was obtained.

[0038] The crude extract (180 g) was suspended in 15 times the mass of distilled water, mixed thoroughly, and then filtered with filter paper after reaching equilibrium. The filtrate was added to a macroporous adsorption resin (AB-8) 40 times the mass of the crude extract, mixed thoroughly, and then kept for 24 hours until adsorption equilibrium was reached. After reaching the adsorption saturation state, the macroporous adsorption resin (AB-8) was loaded onto a glass chromatographic column (height diameter 8), and then eluted with ethanol aqueous solutions of different concentrations (20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, 95% ethanol, V / V) at a flow rate of 4.0 BV / h, and 3 BV was eluted at each concentration. Each desorbed fraction was collected and concentrated under vacuum to obtain elution fractions Fr.1 (40.5 g), Fr.2 (54.3 g), Fr.3 (28.5 g), Fr.4 (15.6 g), and Fr.5 (34.5 g) of ethanol aqueous solutions of different concentrations.

[0039] The fraction Fr.3 (28.5 g) was separated by stainless steel medium pressure column chromatography (inner diameter: 5 cm, column length: 60 cm, ODS-A, 75 μm), and eluted by CH3OH-H2O gradient system according to different volume percentages of CH3OH and H2O (25% methanol, 35% methanol, 45% methanol, 60% methanol, V / V), and the elution volume for each concentration was 2.0 L. Four subcomponents Fr.3-1 (10.2 g), Fr.3-2 (9.5 g), Fr.3-3 (1.5 g), and Fr.3-4 (6.8 g) were obtained. Fraction Fr.3-3 (1.5 g) was purified by LH-20 column chromatography (glass column, inner diameter: 3 cm, column length: 100 cm, eluted with MeOH) to obtain fractions Fr.3-3-1 (0.2 g), Fr.3-3-2 (0.4 g), and Fr.3-3-3 (0.5 g), and then fraction Fr.3-3-2 (0.4 g) was purified by semi-preparative HPLC ODS-A column [stainless steel column, inner diameter: 3 cm, column length: 30 cm, H2O-MeOH (volume ratio of H2O: MeOH is 4:6)] to obtain compound 1 (20.8 mg, t R =15.3min) and compound 2 (12.3mg, t R =19.2min).

[0040] Example 2

[0041] 5 kg of dried Forsythia suspensa leaves were crushed, and then refluxed and extracted for 5 hours with 70% ethanol aqueous solution with a volume fraction of 10 times the mass of the raw material, and the extraction was repeated 3 times until the color of the extract became lighter. The ethanol extracts were combined and evaporated under reduced pressure at 60°C, and then 180 g of crude extract was obtained.

[0042] The crude extract (160 g) was suspended in 10 times the mass of distilled water, mixed thoroughly, and then filtered with filter paper after reaching equilibrium. The filtrate was added to a macroporous adsorption resin (AB-8) 50 times the mass of the crude extract, mixed thoroughly, and then kept for 24 hours until adsorption equilibrium was reached. After reaching the adsorption saturation state, the macroporous adsorption resin (AB-8) was loaded onto a glass chromatographic column (height-to-diameter ratio 10), and then eluted with ethanol aqueous solutions of different concentrations (20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, 95% ethanol, V / V) at a flow rate of 4.0 BV / h, and 3 BV was eluted at each concentration. Each desorbed fraction was collected and concentrated under vacuum to obtain elution fractions of different ethanol concentrations Fr.1 (36.4 g), Fr.2 (47.6 g), Fr.3 (26.8 g), Fr.4 (13.5 g), and Fr.5 (30.8 g).

[0043] The fraction Fr.3 (26.8 g) was separated by stainless steel medium pressure column chromatography (inner diameter: 5 cm, column length: 60 cm, ODS-A, 75 μm), and eluted by a CH3OH-H2O gradient system with different volume percentages of CH3OH and H2O mixed solution (25% methanol, 35% methanol, 45% methanol, 60% methanol, V / V), and the elution volume for each concentration was 1.8 L. Four subcomponents Fr.3-1 (8.5 g), Fr.3-2 (9.0 g), Fr.3-3 (1.3 g), and Fr.3-4 (6.0 g) were obtained. Fraction Fr.3-3 was purified by LH-20 column chromatography (glass column, inner diameter: 3 cm, column length: 100 cm, eluted with MeOH) to obtain fractions Fr.3-3-1 (0.1 g), Fr.3-3-2 (0.3 g), and Fr.3-3-3 (0.6 g), and then fraction Fr.3-3-2 (0.3 g) was purified by semi-preparative HPLC ODS-A column [stainless steel column, inner diameter: 3 cm, column length: 30 cm, H2O-MeOH (H2O: MeOH volume ratio is 4:6)] to obtain compound 1 (15 mg, t R =15.3min) and compound 2 (10 mg, t R =19.2min).

[0044] Example 3

[0045] 6 kg of dried Forsythia suspensa leaves were crushed, and then refluxed and extracted for 4 hours with 70% ethanol aqueous solution 13 times the mass of the raw material, and the extraction was repeated 3 times. The ethanol extracts were combined, filtered and evaporated under reduced pressure at 60° C., and then 200 g of crude extract was obtained.

[0046] The crude extract (190 g) was suspended in 13 times the mass of distilled water, mixed thoroughly, and then filtered with filter paper after reaching equilibrium. The filtrate was added to a macroporous adsorption resin (AB-8) 45 times the mass of the crude extract, mixed thoroughly, and then kept for 24 hours until adsorption equilibrium was reached. After reaching the adsorption saturation state, the macroporous adsorption resin (AB-8) was loaded onto a chromatographic column (height-diameter ratio 9), and then eluted with ethanol aqueous solutions of different volume percentages (20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, 95% ethanol, V / V) at a flow rate of 4.0 BV / h, and 3 BV was eluted at each concentration. Each desorbed fraction was collected and concentrated under vacuum to obtain elution fractions Fr.1 (38.5 g), Fr.2 (50.2 g), Fr.3 (30.6 g), Fr.4 (15.6 g), and Fr.5 (33.8 g) of different ethanol concentrations.

[0047] The fraction Fr.3 (30.6 g) was separated by stainless steel medium pressure column chromatography (inner diameter: 5 cm, column length: 60 cm, ODS-A, 75 μm), and eluted by a CH3OH-H2O gradient system with a mixed solution of CH3OH and H2O at different volume percentages (25% methanol, 35% methanol, 45% methanol, 60% methanol, V / V), and the elution volume for each concentration was 2.2 L. Four subcomponents Fr.3-1 (9.2 g), Fr.3-2 (10.5 g), Fr.3-3 (1.7 g), and Fr.3-4 (7.5 g) were obtained. Fraction Fr.3-3 was purified by LH-20 column chromatography (glass column, inner diameter: 3 cm, column length: 100 cm, MeOH) to obtain fractions Fr.3-3-1 (0.1 g), Fr.3-3-2 (0.4 g), and Fr.3-3-3 (0.5 g), and then fraction Fr.3-3-2 (0.4 g) was purified by semi-preparative HPLC ODS-A column [stainless steel column, inner diameter: 3 cm, column length: 30 cm, H2O-MeOH (H2O: MeOH volume ratio is 4:6)] to obtain compound 1 (20 mg, t R =15.6min) and compound 2 (11 mg, t R =19.4min).

[0048] The molecular formula of compound 1 of Example 1-3 is C 35 H 41 O 14; The name of compound 1 is: Phillyrigeninside B; The molecular formula of compound 2 is C 35 H 41 O 14 ; The name of compound 2 is: Phillyrigeninside C; The structural formulas of compound 1 and compound 2 are:

[0049]

[0050] The two compounds were named Phillyrigeninside B (1) and Phillyrigeninside C (2), and the spectral data of the compounds were:

[0051] Phillyrigeninside B(1), light yellow jelly. [α] 2 D 5 =-45.4 (c 0.21, MeOH). UVλ max (MeOH)(logε):224(4.02),258(3.54). IRν max (KBr)cm -1 :3371,2944,1733,1596,1518 and 1034. H NMR spectra of compounds ( 1 H-NMR, DMSO, 600 MHz)) and carbon spectra ( 13 C-NMR, DMSO, 150 MHz) see Table 1. HRESI-MS: m / z 685.2475 [M+H] + (Structural Formula C 35 H 41 O 14 The calculated value is 685.2496).

[0052] Phillyrigeninside C(2), light yellow jelly. [α] 2 D 5 =-66.3 (c 0.14, MeOH).Uvλ max (MeOH)(logε):226(3.86),262(3.47).IRν max (KBr)cm -1 :3368,2945,1735,1598,1517and1032. H NMR spectrum of the compound ( 1 H-NMR, DMSO, 600 MHz)) and carbon spectra ( 13C-NMR, DMSO, 150 MHz) see Table 1. HRESI-MS: m / z 685.2480 [M+H] + (Structural Formula C 35 H 41 O 14 Calculated 685.2496). Table 1. NMR data of compound 1 and compound 2 ( 1 H:600MHz; 13 C:150MHz; in ppm, J in Hz, in DMSO).

[0053]

[0054]

[0055] The biological activities of Compound 1 and Compound 2 extracted and separated in Examples 1-3 were evaluated below.

[0056] Evaluation of antimicrobial activity:

[0057] The antibacterial activity of the isolated compounds against Staphylococcus aureus, Escherichia coli, Streptococcus viridans, Bacillus subtilis and Candida albicans was detected using the standard broth (nutrient solution, purchased) microdilution method, and the MIC (minimum inhibitory concentration) of compounds 1-2 was determined, and gentamicin was used as a positive control.

[0058] The standard broth microdilution method includes the following steps: select pure culture colonies on a Brucella agar plate, prepare a bacterial suspension with a 0.5 McFarland turbidity unit of the strain to be tested, and use it as the initial inoculum bacterial suspension. Prepare a bacterial suspension with a concentration equivalent to the 0.5 McFarland turbidity standard, dilute it with nutrient solution (standard broth) at a ratio of 1:200 (bacterial suspension: nutrient solution = 1:200) to obtain a suspension, and add 100 μL of the suspension (concentration of about 5×10 4CFU / mL), sealed and placed in a 35°C ordinary air incubator, incubated for 48 hours to observe the results. The test compound (64.0 mg) was diluted in Me2SO (500 μL) and mixed with the bacterial strain cultured in nutrient solution (9.5 mL). The initial concentration of the test compound was 6.4 mg / mL, and concentrations of 2.5, 5.0, 10, 20, 40, 80, 160, 320 and 640 μg / mL of the isolated compounds were obtained by serial dilution. The antibacterial test was performed by transferring each test compound of different concentrations (1 mL) to a new test tube containing only bacterial culture (1 mL). The lowest drug concentration that completely inhibits bacterial growth in the small hole is the MIC. The test is established when bacteria in the positive control well (ie, without antibiotics) grow significantly. Record the MIC value of the highest drug concentration that inhibits bacterial growth.

[0059] References: (Liu, P., Wang, YR, Gao, JY, Lu, ZY, Yin, WP, Deng, RX, 2014. Resveratrol Trimers from Seed Cake of Paeonia rockii, Molecules 19, 19549-19556; Liu, P., Li,

[0060] Table 2 Antibacterial activity of compounds (MIC, μg / mL).

[0061]

[0062] Evaluation of anti-inflammatory activity

[0063] The anti-inflammatory activity of the isolated compounds was evaluated by LPS-induced mouse macrophage RAW264.7 cell detection model, and dexamethasone was used as a positive control drug.

[0064] Experimental grouping: The concentration of RAW264.7 cell suspension was adjusted to 1×10 4Each well was added to a 96-well plate, 80 μL per well, 4 replicates per group, and cultured in a 37°C, 5% CO2 incubator. The experiment was divided into 9 groups, and the treatment methods of each group were as follows: (1) Normal group, 80 μL of culture medium; (2) Model group: 80 μL of LPS (lipopolysaccharide, 10 μg / mL); (3) Positive control group: 80 μL of LPS aqueous solution (lipopolysaccharide, 10 μg / mL) and 80 μL of dexamethasone aqueous solution (10 μmol / mL) were added to this group. (4) 6 groups of samples with different concentrations: 80 μL of 10 μg / mL LPS was added to each group, and 5, 10, 20, 40, 80, 160 μmol / mL of the test compound 1 aqueous solution and 5, 10, 20, 40, 80, 160 μmol / mL of the test compound 2 aqueous solution were added to each group (the test objects included compound 1 and compound 2, where the amount of compound 1 aqueous solution added to each group was 80 μL, and the amount of compound 2 aqueous solution added to each group was 80 μL. The concentrations of the aqueous solution of compound 1 and the aqueous solution of compound 2 are the same, for example, 80 μL of 10 μg / mL LPS aqueous solution, 80 μL of 5 μmol / mL aqueous solution of compound 1 to be tested and 80 μL of 5 μmol / mL aqueous solution of compound 2 to be tested are added to the first single group, and 80 μL of 10 μg / mL LPS aqueous solution, 80 μL of 10 μmol / mL aqueous solution of compound 1 to be tested and 80 μL of 10 μmol / mL aqueous solution of compound 2 to be tested are added to the second single group, and so on).

[0065] Nitric oxide content: After 24 hours of culture, 50 μL of cell supernatant from each group was taken to the ELISA plate, and 50 μL of Griess I and Griess II reagents of equal volume were added, and the absorbance at 540 nm was measured by UV-visible spectrophotometer. A standard curve was drawn with NaNO2 at concentrations of 1, 2, 5, 10, 20, 40, 60, and 100 μmol / mL, and the NO concentration in the cell culture supernatant and the inhibitory effect of the test substance on NO release were calculated based on the standard curve.

[0066] The inhibitory effects of compounds 1 and 2 on LPS-induced NO production in macrophages were tested and their IC 50 (50% inhibition concentration, μM) values ​​were 46.53±5.24 and 51.33±6.17, respectively. Compared with the positive control dexamethasone (IC 50 35.64±4.12 μM), compounds 1 and 2 had moderate anti-inflammatory activity.

[0067] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A furanolignan glycoside compound from Forsythia suspensa leaves, characterized in that: Including compound 1 and compound 2; wherein the molecular formula of compound 1 is C 35 H 41 O 14 ; The name of compound 1 is: Phillyrigeninside B; The structural formula of compound 1 is as follows: The molecular formula of compound 2 is C 35 H 41 O 14 ; The name of compound 2 is: Phillyrigeninside C; The structural formula of compound 2 is as follows:

2. The method for extracting and separating furanolignan glycoside compounds from Forsythia suspensa leaves according to claim 1, characterized in that: The steps include: Step 1, crushing the dried Forsythia suspensa leaves, and then refluxing and extracting them for 3-5 hours with an ethanol aqueous solution 10-15 times the mass of the Forsythia suspensa leaf raw material, and repeating the extraction several times; combining the ethanol extracts obtained by filtration, and recovering ethanol by vacuum evaporation of the combined ethanol extracts, and then obtaining a crude extract; Step 2, suspending the crude extract obtained in step 1 in distilled water, wherein the mass ratio of the crude extract to the distilled water is 1:10-15 times; mixing well and filtering to obtain a filtrate; Step 3, adding the filtrate obtained in step 2 to the macroporous adsorption resin and mixing thoroughly, wherein the mass ratio of the crude extract to the macroporous adsorption resin is 1:40-50; then maintaining for more than 24 hours until adsorption equilibrium is reached, after reaching the adsorption saturation state, loading the macroporous adsorption resin onto a chromatographic column, and then eluting with ethanol aqueous solutions of different concentrations at a certain flow rate; collecting each desorbed fraction segment and concentrating it under vacuum to obtain eluted fraction segments Fr.1, Fr.2, Fr.3, Fr.4, and Fr.5 of ethanol aqueous solutions of different concentrations; Step 4: Separate and purify the eluted fraction Fr.3 obtained in step 3 to obtain compound 1 and compound 2.

3. The method for extracting and separating furanolignan glycoside compounds from Forsythia suspensa leaves according to claim 2, characterized in that: In the step 1, the dried Forsythia suspensa leaves are crushed, and then reflux extracted with a 70% ethanol aqueous solution with a volume percentage of 10-15 times the mass of the Forsythia suspensa leaves raw material for 3-5 hours, and the extraction is repeated 3 times; the ethanol extracts obtained by filtration are combined, and ethanol is recovered by reduced pressure evaporation to obtain a crude extract.

4. The method for extracting and separating furanolignan glycoside compounds from Forsythia suspensa leaves according to claim 2, characterized in that: In the step 2, the crude extract obtained in the step 1 is suspended in distilled water, wherein the mass ratio of the crude extract to distilled water is 1:10-15 times; after being fully mixed, the mixture is filtered with filter paper to obtain a filtrate.

5. The method for extracting and separating furanolignan glycoside compounds from Forsythia suspensa leaves according to claim 2, characterized in that: In the step 3, the filtrate obtained in the step 2 is added to the macroporous adsorption resin AB-8 and mixed thoroughly, and the mass ratio of the crude extract to the macroporous adsorption resin AB-8 is 1:40-50; then the mixture is kept for more than 24 hours until adsorption equilibrium is reached. After the adsorption saturation state is reached, the adsorption saturated macroporous adsorption resin AB-8 is loaded onto a chromatographic column with a height-to-diameter ratio of 8-10, and then eluted with ethanol aqueous solutions of different concentrations at a flow rate of 4.0 BV / h; each desorbed fraction segment is collected and concentrated under vacuum to obtain eluted fraction segments Fr.1, Fr.2, Fr.3, Fr.4, and Fr.5 of ethanol aqueous solutions of different concentrations.

6. The method for extracting and separating furanolignan glycoside compounds from Forsythia suspensa leaves according to claim 2, characterized in that: In the step 4, the eluted fraction segment Fr.3 obtained in step 3 is separated by medium pressure column chromatography and eluted with a CH3OH-H2O gradient system to obtain subcomponents Fr.3-1, Fr.3-2, Fr.3-3, and Fr.3-4. The fraction segment Fr.3-3 in the subcomponent is purified by gel column chromatography to obtain fractions Fr.3-3-1, Fr.3-3-2, and Fr.3-3-3. The obtained fraction Fr.3-3-2 is then purified by semi-preparative HPLC column to obtain compounds 1 and 2.

7. The method for extracting and separating furanolignan glycoside compounds from Forsythia suspensa leaves according to claim 6, characterized in that: In step 4, in the CH3OH-H2O gradient system, the volume percentages of methanol-water solution are 25%, 35%, 45% and 60% respectively.

8. Use of the furanolignan glycoside compound in Forsythia suspensa leaves in antibacterial and anti-inflammatory activities according to claim 1.

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