Screening preparation method of broussonetia papyrifera extract with anti-inflammatory and antioxidant activities and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a screening and preparation method of Broussonetia papyrifera branch extract with anti-inflammatory and antioxidant activities and application thereof. The anti-inflammatory and antioxidant active sites in the Broussonetia papyrifera branch are screened and separated based on the activity-oriented / spectrum-effect separation technology for the first time. The technical scheme effectively simplifies the steps of separation operation, reduces the loss of compounds in separation, and reduces the consumption of separation mobile phase and filler. At the same time, the activity-oriented separation can improve the probability of discovering active compounds, reduce the cost of determining the activity of obtained compounds, and has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a screening and preparation method of a Broussonetia papyrifera extract with anti-inflammatory and antioxidant activities and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] Broussonetia papyrifera (L.) L'Hér. ex Vent., also known as papermulberry, is a member of the genus Broussonetia in the family Moraceae, and is widely distributed in China and even the world, mainly in the Yellow River, Yangtze River and Pearl River basins in China, and also in Cambodia, Vietnam, Japan, etc. In traditional applications, the roots, bark, stems, leaves, fruits and seeds of Broussonetia papyrifera can be used as medicines. Historically, the fibers of the stems and leaves of Broussonetia papyrifera were used for papermaking and tapa cloth. The leaves of Broussonetia papyrifera have the effects of cooling blood, stopping bleeding, detumescence and treating hernia, and are often used to treat male prostate diseases. The roots and stems of Broussonetia papyrifera have the effects of dispelling wind and relieving itching and promoting urination. The fruits of Broussonetia papyrifera have the effects of tonifying kidney and improving eyesight, and can also be used as food coloring for food processing. In addition, Broussonetia papyrifera has a unique fragrance and is often used as feed for pigs, cattle and sheep. So far, researchers have extracted and identified more than 300 kinds of monomer compounds from different parts of Broussonetia papyrifera, including flavonoids, polyphenols, alkaloids, phenylpropanoids, terpenes, steroids, etc. Pharmacological studies have shown that the chemical components of Broussonetia papyrifera have anti-inflammatory, anti-tumor, antioxidant, anti-diabetic, anti-obesity, antibacterial and anti-viral activities, as well as skin whitening and anti-wrinkle activities.

[0004] Through consulting a large number of domestic and foreign literatures, it is found that the research on Broussonetia papyrifera mainly focuses on the roots, stems, leaves and fruits, and the research on the branches is very limited, which leads to ineffective utilization and great waste of resources. By separating and identifying the chemical components of the branches of Broussonetia papyrifera, screening the active components, a foundation can be laid for formulating the quality standard of the extract of the branches of Broussonetia papyrifera and comprehensively utilizing this plant resource. SUMMARY

[0005] Based on the deficiencies of the prior art in the research on the branches of Broussonetia papyrifera and the defects of the research means, the present application provides a screening and preparation method of a Broussonetia papyrifera extract with anti-inflammatory and antioxidant activities and application thereof. Specifically, based on the active-oriented / spectrum-effect separation technology, the present application has carried out systematic screening and separation of the active sites with antioxidant and anti-inflammatory activities in the branches of Broussonetia papyrifera, and finally obtained 40 compounds with the above-mentioned activities. Based on the above research results, the present application is completed.

[0006] The specific technical solutions of the present application are as follows:

[0007] In the first aspect of the present application, a screening preparation method of Broussonetia papyrifera extract with anti-inflammatory and antioxidant activities is provided, and the screening preparation method comprises the following steps:

[0008] S1, crushing Broussonetia papyrifera and adding an organic solvent to perform heating reflux extraction, concentrating and drying the medicinal liquid to obtain an extract;

[0009] S2, dispersing the extract in water and respectively using petroleum ether, ethyl acetate and n-butanol to perform extraction to obtain extracts of different parts;

[0010] S3, determining the anti-inflammatory and antioxidant activities of the extracts of different parts, and screening the ethyl acetate part with the best activity;

[0011] S4, using spectral means to determine the structures of main compounds in the ethyl acetate active part;

[0012] S5, separating the compound components of the ethyl acetate part, including using silica gel, polyamide, reverse phase ODS and dextran gel column chromatography to separate the ethyl acetate part, and purifying through preparative high performance liquid chromatography to obtain monomer components, and finally obtaining 40 active compounds.

[0013] The above separation method, a total of 40 active compounds, including: 4-methyl-5,6-dihydro-2H-pyran-2-one (1), methyl furoate (2), p-hydroxybenzaldehyde (3), vanillin (4), p-hydroxyacetophenone (5), hydroquinone (6), 3,4-dihydroxy-acetophenone (7), ω-hydroxypropioguaiacone (8), 3-hydroxy-4-methoxybenzoic acid (9), 5,4'-dihydroxydihydrostilbene-3-O-β-D-glucopyranoside (10), cinnamic acid (11), succinic acid (12), phenylacetic acid (13), protocatechuic acid (14), p-hydroxyphenylacetic acid (15), methy-5-hydroxy-2-pyridinecarboxylate (16), 3-(2-formyl-1H-pyrrol-1-yl)propanoic acid (17), thymine (18), 3-indoleacetamide (19), Indole-3-carboxamide (20), nicotinamide (21), 1H-indole-3-carboxaldehyde (22), 3-Hydroxy-5α,6α-epoxy-β-ionone (23), Geipunin (24), trans-2-(4-methoxyphenyl)-2-hexenol (25), 1,3,5-Trimethoxybenzene (26), Benzyl-β-D-glucopyranoside (27), Quercetin (28), Luteolin (29), Apigenin-7-glucoside (30), Kyohodaioside (31), Lirioresinol A (32), Fraxiresinol (33), 3-(4-hydroxy-3,5-dimethoxyphenyl)propane-1,2-diol (34), (-)-(7R,7'R,7”R,8S,8'S,8”S)-4',4”-dihydroxy-3,3',3”,5,5'-pentamethoxy-7,9':7',9-diepoxy-4,8”-oxy-8,8'-sesquineolignan-7”,9”-diol (35), Hedyotol C (36), Dehydrodiconiferyl alcohol (37), Simulanol (38), β-sitosterol (39), Daucosterol (40). The specific structural formula of compounds 1-40 is shown in Figure 7 .

[0014] For the structural identification of the above compounds, the spectral means include but are not limited to ultraviolet spectrum (UV), infrared spectrum (IR), mass spectrum (MS), nuclear magnetic resonance hydrogen spectrum (H NMR) and nuclear magnetic resonance carbon spectrum (C NMR). 1 H NMR) and nuclear magnetic resonance carbon spectrum (C NMR). 13C NMR).

[0015] In a second aspect of the present application, the Broussonetia extract or active compound obtained by the above-mentioned screening and preparation method is used for preparing an anti-inflammatory and / or antioxidant product.

[0016] The product includes, but is not limited to, food, medicine and daily chemical product.

[0017] The food can be used for human or non-human animals, and when the food is used for human, it can be health food, and when the food is used for non-human animals (including mammals and birds, especially livestock and poultry animals), it can be feed or feed additive; the medicine can also be used for human or non-human animals, and when the medicine is used for non-human animals (including mammals and birds, especially livestock and poultry animals), it can be veterinary medicine.

[0018] The daily chemical product includes cosmetic.

[0019] The above-mentioned technical solution has the following beneficial technical effects:

[0020] The above-mentioned technical solution first carries out systematic screening and separation of the anti-inflammatory and antioxidant active sites in the Broussonetia tree branch based on the activity-oriented / spectrum-effect separation technology. The above-mentioned technical solution effectively simplifies the steps of separation operation, reduces the loss of compounds in separation, and reduces the consumption of separation mobile phase and filler. At the same time, the activity-oriented separation can improve the probability of discovering active compounds, reduce the cost of determining the activity of obtained compounds, and has good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0021] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the illustrative embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application.

[0022] Figure 1 is the in-vitro ABTS free radical scavenging rate of each extraction part in Example 1 of the present application;

[0023] Figure 2 is the in-vitro DPPH free radical scavenging rate of each extraction part in Example 1 of the present application;

[0024] Figure 3 is the in-vitro total antioxidant capacity result of each extraction part in Example 1 of the present application;

[0025] Figure 4 is the in-vitro safe concentration result of each extraction part in Example 1 of the present application;

[0026] Figure 5is the determination result of the influence of each extraction part on the secretion of cytokines TNF-α, IL-1β and IL-6 of RAW264.7 in vitro in Example 1 of the present application;

[0027] Figure 6 is the result of anti-inflammatory activity of monomer components in the branch of the construction tree screened in the zebra fish inflammation model in Example 1 of the present application;

[0028] Figure 7 is the structural formula of the compound 1-40 prepared in Example 1 of the present application.

[0029] Figure 8 is the 1 H NMR and 13 C NMR spectrum of compound 1.

[0030] Figure 9 is the 1 H NMR and 13 C NMR spectrum of compound 2.

[0031] Figure 10 is the 1 H NMR and 13 C NMR spectrum of compound 3.

[0032] Figure 11 is the 1 H NMR and 13 C NMR spectrum of compound 4.

[0033] Figure 12 is the 1 H NMR and 13 C NMR spectrum of compound 5.

[0034] Figure 13 is the 1 H NMR and 13 C NMR spectrum of compound 6.

[0035] Figure 14 is the 1 H NMR and 13 C NMR spectrum of compound 7.

[0036] Figure 15 is the 1 H NMR and 13 C NMR spectrum of compound 8.

[0037] Figure 16 is the 1 H NMR and 13 C NMR spectrum of compound 9.

[0038] Figure 17is compound 10 1 H NMR and 13 C NMR spectra.

[0039] Figure 18 is compound 11 1 H NMR and 13 C NMR spectra.

[0040] Figure 19 is compound 12 1 H NMR and 13 C NMR spectra.

[0041] Figure 20 is compound 13 1 H NMR and 13 C NMR spectra.

[0042] Figure 21 is compound 14 1 H NMR and 13 C NMR spectra.

[0043] Figure 22 is compound 15 1 H NMR and 13 C NMR spectra.

[0044] Figure 23 is compound 16 1 H NMR and 13 C NMR spectra.

[0045] Figure 24 is compound 17 1 H NMR and 13 C NMR spectra.

[0046] Figure 25 is compound 18 1 H NMR and 13 C NMR spectra.

[0047] Figure 26 is compound 19 1 H NMR and 13 C NMR spectra.

[0048] Figure 27 is compound 20 1 H NMR and 13 C NMR spectra.

[0049] Figure 28 is compound 21 1 H NMR and 13 C NMR spectra.

[0050] Figure 29 is compound 22 1 H NMR and 13 C NMR spectra.

[0051] Figure 30 is compound 23 1 H NMR and 13 C NMR spectra.

[0052] Figure 31 is compound 24 1 H NMR and 13 C NMR spectra.

[0053] Figure 32 is compound 25 1 H NMR and 13 C NMR spectra.

[0054] Figure 33 is compound 26 1 H NMR and 13 C NMR spectra.

[0055] Figure 34 is compound 27 1 H NMR and 13 C NMR spectra.

[0056] Figure 35 is compound 28 1 H NMR and 13 C NMR spectra.

[0057] Figure 36 is compound 29 1 H NMR and 13 C NMR spectra.

[0058] Figure 37 is compound 30 1 H NMR and 13 C NMR spectra.

[0059] Figure 38 is compound 31 1 H NMR and 13 C NMR spectra.

[0060] Figure 39 is compound 32 1 H NMR and 13 C NMR spectra.

[0061] Figure 40 is compound 33 1H NMR and 13 C NMR spectrum.

[0062] Figure 41 It is compound 34. 1 H NMR and 13 C NMR spectrum.

[0063] Figure 42 It is compound 35. 1 H NMR and 13 C NMR spectrum.

[0064] Figure 43 It is compound 36. 1 H NMR and 13 C NMR spectrum.

[0065] Figure 44 It is compound 37. 1 H NMR and 13 C NMR spectrum.

[0066] Figure 45 It is compound 38. 1 H NMR and 13 C NMR spectrum. Detailed Implementation

[0067] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0068] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0069] As mentioned earlier, pharmacological studies have shown that paper mulberry possesses anti-inflammatory, anti-tumor, antioxidant, anti-diabetic, anti-obesity, antibacterial, and antiviral activities, as well as skin whitening and anti-wrinkle effects. Currently, most research on paper mulberry focuses on its roots, stems, leaves, and fruits, with very limited research on its branches, resulting in a significant waste of resources.

[0070] Therefore, the application provides a method for separating and preparing and screening anti-oxidation and anti-inflammation active ingredients in Broussonetia papyrifera, which comprises the following steps: crushing Broussonetia papyrifera into coarse powder, extracting the coarse powder by using a solvent, recovering the solvent, and freeze-drying or drying under reduced pressure to prepare dry extract; suspending the extract in water, and sequentially extracting the suspension by using different organic solvents, and recovering the solvents to obtain different extract parts. The radical scavenging capacity of the extract parts is determined by using DPPH method and ABTS method, and the T-AOC (total antioxidant capacity) is determined by using the diphenyl picrylhydrazyl method to determine the anti-oxidation active part; the ability of the extract parts to release cytokines from RAW264.7 cells is determined by using ELISA method to determine the anti-inflammation active part. 1 H-NMR, 13 C-NMR and other spectrum methods are used to analyze the active parts, the structure type of the active compounds is deduced by combining with the literature, and the optimal separation scheme is selected and formulated from the separation methods such as silica gel column chromatography, gel column chromatography, macroporous resin column chromatography, reverse phase column chromatography and preparative high performance liquid chromatography according to the structure characteristics of the active compounds, so that the active parts are rapidly enriched and accurately separated to obtain the target compounds. Meanwhile, a CuSO4-induced zebrafish inflammation model is established, ibuprofen is used as a positive control drug, the number of zebrafish macrophage migrations is determined by using a stereoscopic fluorescence microscope, and the anti-inflammatory activities of the obtained partial active compounds are further studied.

[0071] Specifically, in one typical embodiment of the application, a screening and preparation method of Broussonetia papyrifera extract with anti-inflammatory and anti-oxidation activities is provided, and the screening and preparation method comprises the following steps:

[0072] S1, crushing Broussonetia papyrifera and adding an organic solvent to perform heating reflux extraction, and concentrating and drying the medicinal liquid to obtain an extract;

[0073] S2, dispersing the extract in water, and extracting the dispersion by using petroleum ether, ethyl acetate and n-butanol respectively to obtain extract parts of different parts;

[0074] S3, determining the anti-inflammatory and anti-oxidation activities of the extract parts, and screening the ethyl acetate part with the best activity;

[0075] S4, determining the main compound structure of the ethyl acetate active part by using spectrum methods;

[0076] S5, separating the compound components of the ethyl acetate part, including separating the ethyl acetate part by using silica gel, polyamide, reverse phase ODS and dextran gel column chromatography, and purifying the monomer components by using preparative high performance liquid chromatography, and finally obtaining 40 active compounds.

[0077] In the step S1, the organic solvent can be ethanol, and more further, 80% ethanol.

[0078] The heating and reflux extraction can be performed 2-4 times. In one specific embodiment of the invention, the heating and reflux extraction method is as follows: heating and refluxing with 80% ethanol 3 times, 2 hours each time, with the ethanol volume being 10, 8, and 8 times the volume of the medicinal material, respectively. The extracts obtained from each reflux extraction are combined and concentrated under reduced pressure for subsequent processing.

[0079] In step S2, the volume ratio of water to organic solvent (petroleum ether, ethyl acetate, and n-butanol) is 1:0.5-5, preferably 1:1. The extraction is performed 2-6 times for each extraction solvent, preferably 4 times.

[0080] In step S3, the specific methods for determining the antioxidant and anti-inflammatory activities in the extracts of each part include, but are not limited to, using the DPPH method, ABTS method and diphenylpicrylhydrazine method to determine the T-AOC (total antioxidant capacity) antioxidant activity of extracts from different parts of paper mulberry tree branches; and using cell inflammation models and / or animal inflammation models to determine the anti-inflammatory activity of extracts from different parts of paper mulberry tree branches;

[0081] More specifically, the cell inflammation model used was RAW246.7 cells. By measuring the effects of each extraction fraction on the release of cytokines (TNF-α, IL-1β, IL-6) from RAW246.7 cells, active extraction fractions with anti-inflammatory effects were screened.

[0082] The animal inflammation model used is a CuSO4-induced zebrafish inflammation model. By measuring the effect of each extraction fraction on the migration of zebrafish macrophages, active extraction fractions with anti-inflammatory effects are screened. In one specific embodiment of the invention, the anti-inflammatory activity of some isolated active compounds is detected using the above-mentioned animal inflammation model.

[0083] The results showed that the antioxidant active sites of the paper mulberry tree were the ethyl acetate and n-butanol sites; the anti-inflammatory active sites were also the ethyl acetate and n-butanol sites. Considering both antioxidant and anti-inflammatory activities, the ethyl acetate site exhibited the best activity; therefore, further research on the chemical composition of the ethyl acetate site is recommended.

[0084] In step S4, the spectroscopic methods used for structural identification of the above compounds include, but are not limited to, ultraviolet (UV) spectroscopy, infrared (IR) spectroscopy, mass spectrometry (MS), and proton nuclear magnetic resonance (NMR) spectroscopy. 1 H NMR) and carbon nuclear magnetic resonance (NMR) 13 (C NMR).

[0085] In another embodiment of the present application, the separation method of the ethyl acetate fraction in step S5 comprises the following steps: taking the ethyl acetate fraction extract, 100-200 mesh silica gel, silica gel column, first gradient elution with petroleum ether: ethyl acetate (79:1, 49:1, 29:1, 19:1, 14:1, 9:1, 5:1, 3:1, 1:1, 1:4, 1:9, 0:1), and then with ethyl acetate: methanol (79:1, 39:1, 19:1, 9:1, 4:1, 2:1, 0:1), thin layer chromatography detection, and combining the same fractions to obtain E1-E12.

[0086] E5 and E8 were repeatedly recrystallized to obtain compound 39 and compound 40.

[0087] E7 was subjected to gel column chromatography, methanol elution purification, and then preparative high performance liquid chromatography purification (3 mL / min, MeOH:H2O = 32:68) to obtain compound 3, compound 4, compound 5, and compound 11.

[0088] E8 was subjected to ODS column chromatography, gradient elution with methanol: water (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0) to obtain 6 fractions of E8a-E8f, and E8a was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 15:85) to obtain compound 6 and compound 12.

[0089] E8b was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 20:80) to obtain compound 1 and compound 7, and E8d was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 36:65) to obtain compound 23 and compound 24.

[0090] E9 was loaded on a column of silica gel (200-300 mesh) and eluted with petroleum ether: ethyl acetate (50:1, 25:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1) gradient. The fractions were combined by thin layer chromatography to give 11 fractions, E9a-E9k. E9h was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 45:55) to give compound 25 and compound 26; E9k was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 8:92, 17:83, 50:50) to give compound 9 (2 mg), compound 17, compound 18 and compound 37; E9i (500 mg) was purified by ODS column chromatography with methanol:water (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 10:0) gradient to give 4 fractions (E9i1-E9i4). E9i2 was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 85:15, 70:30) to give compound 2, compound 13 and compound 14; E9i3 was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 65:35, 55:45, 40:60) to give compound 8, compound 16 and compound 32.

[0091] E10 was separated by polyamide column chromatography and eluted with petroleum ether:dichloromethane (9:1, 4:1, 3:1, 2:1, 1:1, 1:5, 1:8, 0:1) and dichloromethane:methanol (10:1, 9:1, 7:1, 5:1, 2:1, 1:1, 0:1) gradient. The fractions were combined by thin layer chromatography to give 10 fractions, E10a-E10j. E10c was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 82:18) to give compound 34, compound 35 and compound 36; E10d was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 60:40, 48:52) to give compound 19 and 38; E10e was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 55:45) to give compound 27; E10f was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 17:83, 35:65) to give compound 15 and compound 20; E10i was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 70:30) to give compound 29.

[0092] E11 was separated by polyamide column chromatography, eluted with petroleum ether: dichloromethane (1 :0, 9:1, 3:1, 2:1, 1:1, 0:1), dichloromethane:methanol (90:1, 70:1, 50:1, 25:1, 20:1, 15:1, 10:1, 9:1, 5:1, 2:1, 1:1, 0:1) gradient, detected by thin layer chromatography, and the fractions were combined to give 10 fractions of E11a-E11j. E11b was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 10:90) to give compound 21; E11g was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 50:50) to give compound 28, compound 30, compound 31; E11h was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 58:42, 45:55) to give compound 10; E11i was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 50:50) to give compound 22.

[0093] The 40 active compounds obtained by the above separation method include: 4-methyl-5,6-dihydro-2H-pyran-2-one (1), methyl furfurate (2), p-hydroxybenzaldehyde (3), vanillin (4), p-hydroxyacetophenone (5), hydroquinone (6), 3,4-dihydroxy-acetophenone (7), ω-hydroxypropioguaiacone (8), 3-hydroxy-4-methoxybenzoic acid (9), 5,4'-dihydroxydihydrostilbene-3-O-β-D-glucopyranoside (10), cinnamic acid (11), succinic acid (12), phenylacetic acid (13), protocatechuic acid (14), p-hydroxyphenylacetic acid (15), methy-5-hydroxy-2-pyridinecarboxylate (16), 3-(2-formyl-1H-pyrrol-1-yl)propanoic acid (17), thymine (18), 3-indoleacetamide (19), Indole-3-carboxamide (20), nicotinamide (21), 1H-indole-3-carboxaldehyde (22), 3-Hydroxy-5α,6α-epoxy-β-ionone (23), Geipunin (24), trans-2-(4-methoxyphenyl)-chromen-8-ol (25), 1,3,5-trimethoxybenzene (26), benzyl-β-D-glucopyranoside (27), quercetin (28), luteolin (29), apigenin-7-glucoside (30), kisshikuloside (31), Lirioresinol A (32), Fraxiresinol (33), 3-(4-hydroxy-3,5-dimethoxyphenyl)propane-1,2-diol (34), (-)-(7R,7'R,7”R,8S,8'S,8”S)-4',4”-dihydroxy-3,3',3”,5,5'-pentamethoxy-7,9':7',9-diepoxy-4,8”-oxy-8,8'-sesquineolignan-7”,9”-diol (35), Hedyotol C (36), dehydrodiconiferyl alcohol (37), simulanol (38), β-sitosterol (39), daucosterol (40). The specific structural formula of compounds 1-40 is shown in Table 1. Figure 7 .

[0094] In another specific embodiment of the present application, the Broussonetia koidzumii extract or active compound obtained by the above screening preparation method is used for preparing anti-inflammatory and antioxidant products.

[0095] The Broussonetia papyrifera extract includes the extract of the ethyl acetate fraction and the n-butanol fraction, and is preferably the extract of the ethyl acetate fraction.

[0096] The product includes, but is not limited to, food, medicine and daily chemical product.

[0097] The food can be used by human or non-human animals, and when the food is used by human, it can be health food, and when the food is used by non-human animals (including mammals and birds, especially livestock and poultry animals), it can be feed or feed additive; the medicine can also be used by human or non-human animals, and when the medicine is used by non-human animals (including mammals and birds, especially livestock and poultry animals), it can be veterinary drug.

[0098] The daily chemical product includes cosmetic.

[0099] The application is further explained by the following examples, but the application is not limited by the examples. It should be understood that the examples are only used to illustrate the application and not used to limit the scope of the application. In the examples of the application, the preparation of high performance liquid chromatography purification is carried out by using FL-H080G type semi-preparative liquid chromatograph (Welch Xtimate C18 chromatographic column).

[0100] Example 1: Preparation of each extraction fraction of Broussonetia papyrifera

[0101] 5 kg of dried Broussonetia papyrifera medicinal material is crushed into coarse powder, and extracted by 80% ethanol by heating reflux for 3 times, 2 h each time, and the ethanol is 10, 8 and 8 times the volume of the medicinal material, respectively. The medicinal liquid is filtered, combined, concentrated under reduced pressure, and dried under reduced pressure to obtain 776 g of extract. The extract is dispersed with an appropriate amount of water (1:1-1:5, g:mL), and then extracted with petroleum ether, ethyl acetate and n-butanol (extraction agent: mother liquor (v / v) = 1:1, 4 times), respectively. Each extraction liquid is concentrated and dried under reduced pressure to obtain 2.4 g of petroleum ether fraction, 23 g of ethyl acetate fraction and 57 g of n-butanol fraction, respectively, which are stored in a refrigerator.

[0102] The antioxidant activity of each extraction fraction is determined by DPPH method, and the specific steps are as follows: 10 mg of extract of each extraction fraction of Broussonetia papyrifera is prepared into test solution with a mass concentration of 0.1, 0.5, 1.0, 1.5 and 2.0 mg / mL by using anhydrous ethanol, and a vitamin C solution with a concentration of 0.02, 0.04, 0.06, 0.08 and 0.1 mg / mL is prepared by using water. 2 mL of each test solution is taken in a 10 mL test tube, 2 mL of dissolved and fully dissolved DPPH ethanol solution (0.1 mmol / L) is added, a tin foil paper is used to shield and avoid light, and the test tube is shaken, sealed and shaken uniformly, placed in a tin foil paper for 30 min at room temperature, placed in a 1 cm cuvette, and placed in a spectrophotometer to measure the absorbance at 517 nm. The absorbance of the test solution is Ai Purified water was used as the blank control group with absorbance A0, and anhydrous ethanol was used instead of DPPH ethanol solution as the background group with absorbance A0. j The scavenging rates of DPPH free radicals by different mass concentrations of Vc and sample solutions were calculated according to formulas (1) and (2), and the IC50 was obtained through SPSS statistical analysis. 50 .

[0103] The formula for calculating the DPPH free radical scavenging rate of each extraction fraction is as follows:

[0104] Vitamin C DPPH free radical scavenging rate (%) = (1-A) Vc / A i )×100% (1);

[0105] Sample DPPH radical scavenging rate (%) = [A0 - (A i -A j )] / A0×100%(2)

[0106] The antioxidant activity of each extracted fraction was determined using the ABTS method. The specific steps are as follows: 10 mg of extracts from different polarity fractions of *Broussonetia papyrifera* branches were prepared with anhydrous ethanol to obtain vitamin C solutions with concentrations of 0.05, 0.1, 0.15, 0.2, and 0.25 mg / mL, and with water to obtain solutions with concentrations of 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL. The ABTS reagent from the kit was placed in a test tube, capped, and shaken well. The solution was incubated at room temperature for 12–16 h to obtain a concentrated ABTS solution. The ABTS solution (7.4 mmol / L) and potassium persulfate solution (2.6 mmol / L) were mixed at a 1:1 volume ratio and incubated at room temperature in the dark for 12 h to obtain the ABTS stock solution. The stock solution was diluted 45 times with purified water to obtain the ABTS free radical working solution. 2 mL of the ABTS free radical working solution and 1 mL of each test sample solution were accurately measured, shaken thoroughly, and incubated at room temperature in the dark for 30 min. The absorbance was then measured at 734 nm. The absorbance of the test solution was measured to be A. i Purified water was used as the blank control group with absorbance A0, and ABTS working solution was used as the background group with absorbance A0. j The scavenging rate of DPPH free radicals by sample solutions of different mass concentrations was calculated according to formula (3), and the IC50 was obtained by SPSS statistical analysis. 50 .

[0107] Studies have found that the antioxidant activity of paper mulberry is concentrated in the ethyl acetate fraction, the ethanol fraction, and the n-butanol fraction.

[0108] The formulas for calculating the ABTS free radical scavenging rate of each extraction fraction are as follows:

[0109] ABTS free radical scavenging rate (%) = [A0 - (Ai -A j )] / A0x100% (3)

[0110] RAW246.7 cells were selected as target cells, and the anti-inflammatory active extraction fractions were screened by determining the effects of each extraction fraction on the release of cytokines from target cells. Partially isolated compounds were selected, and a CuSO4-induced zebrafish inflammation model was established. Ibuprofen was used as a positive control drug, and the number of zebrafish macrophage migrations was determined by stereoscopic fluorescence microscopy to determine the anti-inflammatory activity. The culture method of RAW246.7 cells is as follows: (1) Cell recovery: after the frozen cells were taken out, they were quickly dissolved at 37°C. A 15 mL centrifuge tube was taken, 3 mL of serum-containing medium was added, the liquid in the frozen tube was added to the centrifuge tube containing the medium, and it was mixed gently. After centrifugation at 1000 r / min-1500 r / min at room temperature for 5 min-10 min, the supernatant was removed, 3 mL-5 mL of serum-containing medium was added, and the cell suspension was obtained by gently mixing. It was added to the culture bottle. (2) Culture: the cells were placed in a culture bottle containing complete medium for adherent growth, and the culture conditions were 38°C and 5% CO2. When the cells grew more than 80% of the culture bottle, the cells could be passaged. (3) Passage: the culture solution in the culture bottle was discarded, 1 mL-3 mL of trypsin was added, and it was incubated at 37°C for 2-5 min. If the cytoplasm retracts and the intercellular space increases, an equal volume of serum-containing culture solution is immediately added to terminate the digestion. The cells on the bottom of the bottle were gently blown and beaten in sequence to make the adherent cells detach from the bottom of the bottle and form a cell suspension. According to one transmission, it was inoculated into a new culture bottle and placed in a culture box for culture.

[0111] The safety concentration of each extraction fraction on RAW246.7 cells was determined by CCK-8 method, and the specific method was as follows: an appropriate amount of ethyl acetate extraction fraction was prepared into sample solution with a concentration of 0.15625, 0.3125, 0.625, 0.125, 0.25, 0.5, 1, 2, 3, 5 mg / mL using DMEM culture solution. The sample solution of n-butanol sample was prepared in the same way. RAW246.7 cells were adjusted to a concentration of 5x10 5 6 / mL and added to a 96-well cell culture plate, with 6 replicate wells per well. 200 μL of sample solution was added to each well, with 4 wells per concentration. A blank control group (containing only DEME medium without cells) and a normal group (200 μL of DMEM culture solution was added) were set up at the same time. They were incubated in a 37°C, 5% CO2 incubator. After 24 h, 10 μL of CCK-8 solution was added to each well, and the absorbance values of the drug group (A), the normal group (B), and the control wells (C) were determined at 450 nm after incubation in the incubator for 2 h. The cell viability was calculated according to formula (4). The safety concentration of the drug was determined according to the calculation results.

[0112] Cell viability (%) = (A-C) / (B-C) x 100% (4)

[0113] The content of cytokines released by RAW246.7 was determined by ELISA method, and the specific method is as follows: set the RAW264.7 cells treated only with growth medium as the blank group, the RAW264.7 cells treated with LPS (1 μg / mL) as the model group, and the cells treated with LPS (1 μg / mL) and different extraction samples (0.25 mg / mL) as the drug groups. The cell density was 5 x 10 5 The cells were seeded in 12-well plates at a density of 5 x 10

[0114] The data processing method of the above anti-inflammatory experiment is: the data is represented by `X±SD, the experimental data is analyzed by one-way ANOVA using GraphPad prism 9.0 software, and the differences between the experimental data are analyzed by multiple comparison Turkey test. P<0.05 is judged as having significant difference.

[0115] The results show that the antioxidant active fraction of B. papyrifera is the ethyl acetate fraction and the n-butanol fraction; the anti-inflammatory active fraction of B. papyrifera is the ethyl acetate fraction and the n-butanol fraction. According to the results of antioxidant activity and anti-inflammatory activity, the ethyl acetate fraction has the best activity, so further study on the chemical composition of the ethyl acetate fraction is considered.

[0116] By 1 H NMR and 13CNMR spectroscopic methods and comparison with literature reports, the structures of the major compounds 1-40 in the ethyl acetate fraction were determined as follows: 4-methyl-5,6-dihydro-2H-pyran-2-one (1), methyl furoate (2), p-hydroxybenzaldehyde (3), vanillin (4), p-hydroxyacetophenone (5), hydroquinone (6), 3,4-dihydroxy-acetophenone (7), ω-hydroxypropioguaiacone (8), 3-hydroxy-4-methoxybenzoic acid (9), 5,4'-dihydroxydihydrostilbene-3-O-β-D-glucopyranoside (10), cinnamic acid (11), succinic acid (12), phenylacetic acid (13), protocatechuic acid (14), p-hydroxyphenylacetic acid (15), methy-5-hydroxy-2-pyridinecarboxylate (16), 3-(2-formyl-lH-pyrrol-l-yl)propanoic acid (17), thymine (18), 3-indoleacetamide (19), Indole-3-carboxamide (20), nicotinamide (21), lH-indole-3-carboxaldehyde (22), 3-Hydroxy-5α,6α-epoxy-β-ionone (23), Geiporin (24), trans-2-(4-methoxyphenyl)-l-ethoxy-3-buten-2-ol (25), 1,3,5-trimethoxybenzene (26), benzyl-β-D-glucopyranoside (27), quercetin (28), luteolin (29), apigenin-7-glucoside (30), Kyohodaioside (31), Lirioresinol A (32), Fraxiresinol (33), 3-(4-hydroxy-3,5-dimethoxyphenyl)propane-l,2-diol (34), (-)-(7R,7'R,7"R,8S,8'S,8"S)-4',4"-dihydroxy-3,3',3",5,5'-pentamethoxy-7,9':7',9-diepoxy-4,8"-oxy-8,8'-sesquineolignan-7",9"-diol (35), Hedyotol C (36), dehydrodiconiferyl alcohol (37), simulanol (38), β-sitosterol (39), daucosterol (40).

[0117] The compound monomer separation method of the ethyl acetate part includes the following steps: taking ethyl acetate part extract 23 g, 100-200 mesh silica gel 1:1, silica gel column (213 g, 100-200 mesh) is loaded, first with petroleum ether: ethyl acetate (79:1, 49:1, 29:1, 19:1, 14:1, 9:1, 5:1, 3:1, 1:1, 1:4, 1:9, 0:1, v:v), then with ethyl acetate: methanol (79:1, 39:1, 19:1, 9:1, 4:1, 2:1, 0:1, v:v) gradient elution, 3 column volumes for each gradient elution, the flow rate is 10 mL / min, thin layer chromatography detection, and the same flow fraction is combined to obtain E1-E12.

[0118] E5 and E8 are repeatedly recrystallized to obtain compound 39 and compound 40.

[0119] E7 (0.5 g) is subjected to Sephadex LH-20 gel column chromatography, methanol elution purification, and then subjected to preparative high performance liquid chromatography purification (3 mL / min, MeOH:H2O=32:68) to obtain compound 3, compound 4, compound 5 and compound 11.

[0120] E8 is subjected to ODS column chromatography, with methanol:water (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0, v:v) gradient elution, 5 column volumes for each gradient elution, the flow rate is 3 mL / min, to obtain 6 flow fractions of E8a-E8f, and E8a is subjected to preparative high performance liquid chromatography purification (3 mL / min, MeOH:H2O=15:85) to obtain compound 6 and compound 12;

[0121] E8b is subjected to preparative high performance liquid chromatography purification (3 mL / min, MeOH:H2O=20:80) to obtain compound 1 and compound 7; E8d is subjected to preparative high performance liquid chromatography purification (3 mL / min, MeOH:H2O=36:65, v:v), 45 min for each gradient elution, to obtain compound 23 and compound 24.

[0122] Silica gel (200-300 mesh) was wet packed into a column and E9 (3.5 g) was loaded. Elution was performed with petroleum ether: ethyl acetate (50:1, 25:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1, v:v) gradient, 3 column volumes for each gradient, at a flow rate of 5 mL / min. Fractions were combined by thin layer chromatography to give 11 fractions, E9a-E9k. E9h was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 45:55) to give compound 25 and compound 26; E9k was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 8:92, 17:83, 50:50, v:v), 45 min for each gradient, to give compound 9 (2 mg), compound 17, compound 18 and compound 37; E9i (500 mg) was purified by ODS column chromatography with methanol:water (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 10:0, v:v) gradient, 3 column volumes for each gradient, at a flow rate of 5 mL / min, to give 4 fractions (E9i1-E9i4). E9i2 was further purified by preparative high performance liquid chromatography (FL-H080G semi-preparative liquid chromatograph) (3 mL / min, MeOH:H2O = 85:15, 70:30, v:v), 45 min for each gradient, to give compound 2, compound 13 and compound 14; E9i3 was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 65:35, 55:45, 40:60, v:v), 45 min for each gradient, to give compound 8, compound 16 and compound 32.

[0123] E10 (3.5 g) was separated by silica gel column chromatography (360 g packed column), eluted with petroleum ether: dichloromethane (9:1, 4:1, 3:1, 2:1, 1:1, 1:5, 1:8, 0:1, v:v) first, 3 column volumes for each, flow rate 10 mL / min, then dichloromethane: methanol (10:1, 9:1, 7:1, 5:1, 2:1, 1:1, 0:1, v:v) gradient, 3 column volumes for each, flow rate 10 mL / min. Fractions were combined based on thin layer chromatography detection, and 10 fractions, E10a-E10j, were obtained. E10c was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 82:18, v:v), eluted for 30 min, to obtain compound 34, compound 35, compound 36; E10d was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 60:40, 48:52, v:v), eluted for 30 min, to obtain compound 19, 38; E10e was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 55:45, v:v), eluted for 30 min, to obtain compound 27; E10f was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 17:83, 35:65, v:v), eluted for 35 min, to obtain compound 15, compound 20; E10i was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 70:30), to obtain compound 29.

[0124] E11 (5 g) was separated by silica gel column chromatography (48 g packed column), eluted with petroleum ether: dichloromethane (1:0, 9:1, 3:1, 2:1, 1:1, 0:1, v:v) first, 3 column volumes for each, flow rate 10 mL / min, then dichloromethane: methanol (90:1, 70:1, 50:1, 25:1, 20:1, 15:1, 10:1, 9:1, 5:1, 2:1, 1:1, 0:1, v:v) gradient, 3 column volumes for each, flow rate 10 mL / min. Fractions were combined based on thin layer chromatography detection, and 10 fractions, E11a-E11j, were obtained. E11b was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 10:90), to obtain compound 21; E11g was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 50:50), to obtain compound 28, compound 30, compound 31; E11h was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 58:42, 45:55, v:v) eluted for 45 min, to obtain compound 10; E11i was further purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O = 50:50), to obtain compound 22.

[0125] The CuSO4-induced zebrafish inflammation model was established, and ibuprofen was used as a positive control drug. The number of macrophage cell migration was determined by stereoscopic fluorescence microscopy. The specific method of the anti-inflammatory experiment was as follows: the green fluorescently labeled inflammatory cell transgenic zebrafish strain Tg(zlyz:EGFP) JS7 mature male and female zebrafish were selected, and placed in a water tank with a partition according to the ratio of 1:2, and cultured overnight, and the water temperature was set to 28.5°C. The next day, the partition was removed for mating, the embryos were collected and placed in a fish tank, and methylene blue solution was added for sterilization, and then transferred to zebrafish embryo culture water, and incubated in a 28°C constant temperature incubator for 72h for standby. After the embryos developed for 72h, the normal zebrafish larvae were selected and transferred to a 24-well cell culture plate, 10 tails per well. The experiment was divided into a blank group, a model group, a positive drug group, and an experimental drug group. The positive drug group was added with 10 μmol / mL ibuprofen, and the experimental drug group was added with different doses of drug solution. After 24h of incubation, 4 μL of CuSO4 solution (20 mmol / mL) was added to the culture solution of the blank group, and 1996 μL of fish water was added to the solution to avoid light modeling for 1h. After modeling, the zebrafish were washed with culture water, 0.1% tricaine solution was added for anesthesia, and the number of macrophage cells migrating above the lateral line was counted by using a fluorescence microscope to observe and take pictures of the zebrafish. Figure 6 The anti-inflammatory activity of the compound separated in the above part was verified, and the results are shown in

[0126] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can modify the technical solutions described in the foregoing embodiments or make equivalent replacements of some parts. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application. Although the specific embodiments of the present application have been described above, the present application is not limited to the above. Those skilled in the art should understand that various modifications or changes made to the technical solutions of the present application without creative labor are still within the protection scope of the present application.

Claims

1. A method for screening preparation of Broussonetia kaempferi extract having anti-inflammatory and antioxidant activities, characterized by, The screening preparation method comprises: S1, crushing the broussonetia papyrifera and adding 80% ethanol for heating reflux extraction, concentrating and drying the medicinal liquid to obtain an extract; S2, dispersing the extract in water and respectively using petroleum ether, ethyl acetate and n-butanol for extraction to obtain extractives of different parts; S3, determining the anti-inflammatory and antioxidant activities of the extractives of different parts, and screening the ethyl acetate part with the best activity; S4, determining the structures of main compounds in the ethyl acetate active part by using spectral means; S5, separating the compound components of the ethyl acetate part, including using silica gel, polyamide, reverse phase ODS and dextran gel column chromatography to separate the ethyl acetate part, and purifying by preparative high performance liquid chromatography to obtain monomer components, and finally obtaining 39 active compounds; The 39 compounds are respectively: 4-methyl-5,6-dihydro-2H-pyran-2-one (1), furfuryl methyl ester (2), p-hydroxybenzaldehyde (3), vanillin (4), p-hydroxyacetophenone (5), hydroquinone (6), 3,4-dihydroxy-acetophenone (7), ω-hydroxypropioguaiacone (8), 3-hydroxy-4-methoxybenzoic acid (9), 5,4'-dihydroxydihydrostilbene-3-O-β-D-glucopyranoside (10), cinnamic acid (11), succinic acid (12), phenylacetic acid (13), protocatechuic acid (14), p-hydroxyphenylacetic acid (15), methy-5-hydroxy-2-pyridinecarboxylate (16), 3-(2-formyl-1H-pyrrol-1-yl)propanoic acid (17), thymine (18), 3-indoleacetamide (19), Indole-3-carboxamide (20), nicotinamide (21), 1H-indole-3-carboxaldehyde (22), 3-Hydroxy-5α,6α-epoxy-β-ionone (23), digipronin (24), trans-2-hydroxycinnamic acid (25), 1,3,5-trimethoxybenzene (26), benzyl-β-D-glucopyranoside (27), quercetin (28), luteolin (29), apigenin-7-glucoside (30), kisshikuloside (31), Lirioresinol A (32), 3-(4-hydroxy-3,5-dimethoxyphenyl) propane-1,2-diol (34), (-)-(7R,7'R,7''R,8S,8'S,8''S)-4',4''-dihydroxy-3,3',3'',5,5'-pentamethoxy-7,9':7',9-diepoxy-4,8''-oxy-8,8'-sesquineolignan-7'',9''-diol (35), Hedyotol C (36), dehydrodiconiferyl alcohol (37), simulanol (38), β-sitosterol (39), daucosterol (40); The separation method of the ethyl acetate part in step S5 includes the following steps: taking the ethyl acetate part extract, 100-200 mesh silica gel, silica gel column packing, gradient elution with petroleum ether: ethyl acetate (79:1, 49:1, 29:1, 19:1, 14:1, 9:1, 5:1, 3:1, 1:1, 1:4, 1:9, 0:1), and then with ethyl acetate: methanol (79:1, 39:1, 19:1, 9:1, 4:1, 2:1, 0:1), thin layer chromatography detection, and combining the same fractions to obtain E1-E12; E5 and E8 were repeatedly recrystallized to obtain compound 39 and compound 40; E7 was subjected to gel column chromatography and methanol elution purification, and then was subjected to preparative high performance liquid chromatography purification under the conditions of 3 mL / min, MeOH:H2O=32:68 to obtain compound 3, compound 4, compound 5 and compound 11; E8 was subjected to ODS column chromatography and gradient elution with methanol: water (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0) to obtain 6 fractions of E8a-E8f, and E8a was subjected to preparative high performance liquid chromatography purification under the conditions of 3 mL / min, MeOH:H2O=15:85 to obtain compound 6 and compound 12; E8b was subjected to preparative high performance liquid chromatography purification under the conditions of 3 mL / min, MeOH:H2O=20:80 to obtain compound 1 and compound 7; and E8d was subjected to preparative high performance liquid chromatography purification under the conditions of 3 mL / min, MeOH:H2O=36:65 to obtain compound 23 and compound 24; Silica gel (200-300 mesh) was wet packed in a column and E9 was loaded. Gradient elution was performed with petroleum ether: ethyl acetate (50:1, 25:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1). Fractions were combined by thin layer chromatography to obtain 11 fractions, E9a-E9k. Compound 25 and compound 26 were obtained by preparative high performance liquid chromatography purification of E9h under the conditions of 3 mL / min, MeOH:H2O = 45:

55. Compound 9, compound 17, compound 18 and compound 37 were obtained by preparative high performance liquid chromatography purification of E9k under the conditions of 3 mL / min, MeOH:H2O = 8:92, 17:83, 50:

50. Four fractions, E9i1-E9i4, were obtained by ODS column chromatography purification of E9i with gradient elution of methanol:water (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 10:0). Compound 2, compound 13 and compound 14 were obtained by preparative high performance liquid chromatography purification of E9i2 under the conditions of 3 mL / min, MeOH:H2O = 85:15, 70:

30. Compound 8, compound 16 and compound 32 were obtained by preparative high performance liquid chromatography purification of E9i3 under the conditions of 3 mL / min, MeOH:H2O = 65:35, 55:45, 40:

60. E10 was separated by polyamide column chromatography, eluted with petroleum ether: dichloromethane (9:1, 4:1, 3:1, 2:1, 1:1, 1:5, 1:8, 0:1), dichloromethane:methanol (10:1, 9:1, 7:1, 5:1, 2:1, 1:1, 0:1) as a gradient, detected by thin layer chromatography, and combined fractions to obtain 10 fractions of E10a-E10j; E10c was further purified by preparative high performance liquid chromatography, the conditions of which were 3 mL / min, MeOH:H2O=82:18, to obtain compound 34, compound 35, compound 36; E10d was further purified by preparative high performance liquid chromatography, the conditions of which were 3 mL / min, MeOH:H2O=60:40, 48:52, to obtain compound 19, 38; E10e was further purified by preparative high performance liquid chromatography, the conditions of which were 3 mL / min, MeOH:H2O=55:45, to obtain compound 27; E10f was further purified by preparative high performance liquid chromatography, the conditions of which were 3 mL / min, MeOH:H2O=17:83, 35:65, to obtain compound 15, compound 20; E10i was further purified by preparative high performance liquid chromatography, the conditions of which were 3 mL / min, MeOH:H2O=70:30, to obtain compound 29; E11 was separated by polyamide column chromatography, eluted with petroleum ether: dichloromethane (1:0, 9:1, 3:1, 2:1, 1:1, 0:1), dichloromethane:methanol (90:1, 70:1, 50:1, 25:1, 20:1, 15:1, 10:1, 9:1, 5:1, 2:1, 1:1, 0:1) as a gradient, detected by thin layer chromatography, and combined fractions to obtain 10 fractions of E11a-E11j; E11b was further purified by preparative high performance liquid chromatography, the conditions of which were 3 mL / min, MeOH:H2O=10:90, to obtain compound 21; E11g was further purified by preparative high performance liquid chromatography, the conditions of which were 3 mL / min, MeOH:H2O=50:50, to obtain compound 28, compound 30, compound 31; E11h was further purified by preparative high performance liquid chromatography, the conditions of which were 3 mL / min, MeOH:H2O=58:42, 45:55, to obtain compound 10; E11i was further purified by preparative high performance liquid chromatography, the conditions of which were 3 mL / min, MeOH:H2O=50:50, to obtain compound 22.

2. The screening preparation method of claim 1, wherein In the step S1, the heating reflux extraction is 2-4 times.

3. The method of claim 2, wherein the screening is performed by a method comprising: The specific method of the heating reflux extraction is: heating reflux extraction for 3 times with 80% ethanol, 2 hours each time, and the ethanol dosage is 10, 8 and 8 times the volume of the medicinal material respectively; the medicinal liquid obtained by each reflux extraction is combined, concentrated under reduced pressure and subjected to subsequent treatment.

4. The screening preparation method of claim 1, wherein In the step S2, the volume ratio of water to each organic solvent is 1:0.5-5; the extraction times are 2-6 times for each extraction solvent; and the organic solvent is petroleum ether, ethyl acetate and n-butanol.

5. The method of claim 4, wherein the screening is performed by a method comprising: In the step S2, the volume ratio of water to each organic solvent is 1:1; and the extraction times are 4 times for each extraction solvent.

6. The screening preparation method of claim 1, wherein In the step S3, the specific method for determining the antioxidant and anti-inflammatory activities in each part of the extract includes: using DPPH method, ABTS method and diphenyl picryl hydrazine method to determine the antioxidant activity of T-AOC on the extract of different parts of the broussonetia papyrifera; and using a cell inflammation model and / or an animal inflammation model to determine the anti-inflammatory activity of the extract of different parts of the broussonetia papyrifera.

7. The method of claim 6, wherein the screening is performed by a method comprising: The cell inflammation model uses RAW246.7 cells, and the animal inflammation model uses a CuSO4-induced zebrafish inflammation model.

8. The screening preparation process of claim 1 wherein, In the step S4, for the structural identification of the compounds, the spectral means used include ultraviolet spectrum, infrared spectrum, mass spectrum, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum.

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