Screening preparation method and application of broussonetia papyrifera branch extract with anti-inflammatory and antioxidant activity

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a screening preparation method and application of a paper mulberry branch extract with anti-inflammatory and antioxidant activity. According to the invention, the antioxidant and anti-inflammatory active sites in the paper mulberry branches are systematically screened and separated for the first time on the basis of an activity-oriented / spectrum-effective separation technology. According to the technical scheme, the steps of separation operation are effectively simplified, the loss of compounds in separation is reduced, and the consumption of a separation mobile phase and filler is reduced. Meanwhile, activity-oriented separation can improve the probability of finding active compounds and reduce the cost of determining and obtaining the activity of the compounds, and the method has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a screening and preparation method of a paper mulberry tree extract with anti-inflammatory and antioxidant activities and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Broussonetia papyrifera (L.) L'Hér. ex Vent., also known as paper mulberry, is a member of the genus Broussonetia in the family Moraceae. It is widely distributed in my country and even in the world, mainly in the Yellow River, Yangtze River and Pearl River basins in China, and also found in Cambodia, Vietnam, Japan, etc. In traditional applications, the roots, bark, stems, leaves, fruits and seeds of Broussonetia can be used as medicine. Historically, the stem and leaf fibers of Broussonetia papyrifera have been used in papermaking and textiles such as tapa cloth. In traditional applications, Broussonetia papyrifera leaves have the effects of cooling blood, stopping bleeding, reducing swelling, and treating hernia, and are often used to treat male prostate diseases. The rhizomes of Broussonetia papyrifera have the effects of dispelling wind and relieving itching, and promoting urination. Broussonetia papyrifera fruit has the effects of nourishing the kidneys, improving eyesight, and relieving fatigue. Its pigment can also be used as edible pigments 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 monomer compounds from different parts of paper mulberry, including flavonoids, polyphenols, alkaloids, phenylpropanoids, terpenes, steroids, etc. Pharmacological studies have shown that paper mulberry chemical components have anti-inflammatory, anti-tumor, antioxidant, anti-diabetic, anti-obesity, antibacterial and antiviral, as well as skin whitening and anti-wrinkle activities.

[0004] By consulting a large number of domestic and foreign literature, it is found that the research on paper mulberry mainly focuses on its roots, stems, leaves and fruits, and the research on paper mulberry branches is very limited, which has led to its ineffective utilization and caused a huge waste of resources. By separating and structurally identifying the chemical components of paper mulberry branches and screening the active ingredients, it can lay a foundation for formulating the quality standards of paper mulberry branch extracts and comprehensively utilizing paper mulberry as a plant resource. Summary of the invention

[0005] Based on the above-mentioned insufficiency of the prior art in the study of paper mulberry branches and the defects of the research methods, the present invention provides a screening and preparation method of paper mulberry branch extracts with anti-inflammatory and antioxidant activities and its application. Specifically, the present invention systematically screens and separates the antioxidant and anti-inflammatory active sites in paper mulberry branches based on the activity-oriented / spectral-effect separation technology, and finally obtains 40 compounds with the above-mentioned activities. Based on the above research results, the present invention is completed.

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

[0007] The first aspect of the present invention provides a method for screening and preparing a paper mulberry tree extract having anti-inflammatory and antioxidant activities, the screening and preparation method comprising:

[0008] S1, crushing the paper mulberry branches, adding an organic solvent, heating and refluxing for extraction, concentrating the liquid, and drying to obtain an extract;

[0009] S2, after dispersing the extract with water, extracting with petroleum ether, ethyl acetate and n-butanol respectively to obtain extracts of different parts;

[0010] S3, determining the anti-inflammatory and antioxidant activities of the extracts from each part, and selecting the ethyl acetate part with the best activity;

[0011] S4. Determine the main compound structure of the active site of ethyl acetate by spectroscopy;

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

[0013] Through the above separation method, a total of 40 active compounds were obtained, 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), 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), dihydroquinone (24), scutellariae (25), 1,3,5-trimethoxybenzene (26), benzyl-β-D-glucoside (27), quercetin (28), luteolin (29) , apigenin-7-glucoside (30), edopsakura glycoside (31), tulipwood resin alcohol A (32), ash bark resin alcohol (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), dehydrodisinconyl alcohol (37), simulanol (38), β-sitosterol (39), and carotene (40). The specific structural formulas of compounds 1-40 are shown in Figure 7 .

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

[0015] The second aspect of the present invention provides the use of the paper mulberry extract or active compound obtained by the above screening and preparation method in the preparation of anti-inflammatory and / or antioxidant products.

[0016] The products include but are not limited to food, medicine and daily chemical products.

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

[0018] The daily chemical products include cosmetics.

[0019] Beneficial technical effects of the above technical solution:

[0020] The above technical solution is the first to systematically screen and separate the antioxidant and anti-inflammatory active sites in paper mulberry branches based on activity-guided / spectral separation technology. The above technical solution effectively simplifies the steps of separation operation, reduces the loss of compounds during separation, and reduces the consumption of separation mobile phase and filler. At the same time, activity-guided separation can increase the probability of discovering active compounds and reduce the cost of determining the activity of compounds, which has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

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

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

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

[0025] Figure 4 It is the result of the safe concentration of RAW264.7 in vitro for each extraction part in Example 1 of the present invention;

[0026] Figure 5The results of the determination of the effects of the extraction parts in Example 1 of the present invention on the secretion of cytokines TNF-α, IL-1β, and IL-6 by RAW264.7 in vitro;

[0027] Figure 6 This is the result of screening the anti-inflammatory activity of monomer components in the dendrobium of the zebrafish inflammation model in Example 1 of the present invention;

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

[0029] Figure 8 is compound 1 1 H NMR and 13 C NMR spectrum;

[0030] Fig. 9 is compound 2 1 H NMR and 13 C NMR spectrum;

[0031] Fig.10 is compound 3 1 H NMR and 13 C NMR spectrum;

[0032] Fig.11 is compound 4 1 H NMR and 13 C NMR spectrum;

[0033] Fig.12 is compound 5 1 H NMR and 13 C NMR spectrum;

[0034] Fig.13 is compound 6 1 H NMR and 13 C NMR spectrum;

[0035] Fig.14 is compound 7 1 H NMR and 13 C NMR spectrum;

[0036] Fig.15 is compound 8 1 H NMR and 13 C NMR spectrum;

[0037] Fig.16 is compound 9 1 H NMR and 13 C NMR spectrum;

[0038] Fig.17is compound 10 1 H NMR and 13 C NMR spectrum;

[0039] Fig.18 is compound 11 1 H NMR and 13 C NMR spectrum.

[0040] Fig.19 is compound 12 1 H NMR and 13 C NMR spectrum.

[0041] Fig. 20 is compound 13 1 H NMR and 13 C NMR spectrum.

[0042] Fig.21 is compound 14 1 H NMR and 13 C NMR spectrum.

[0043] Fig. 22 is compound 15 1 H NMR and 13 C NMR spectrum.

[0044] Fig.23 is compound 16 1 H NMR and 13 C NMR spectrum.

[0045] Fig.24 is compound 17 1 H NMR and 13 C NMR spectrum.

[0046] Fig.25 is compound 18 1 H NMR and 13 C NMR spectrum.

[0047] Fig.26 is compound 19 1 H NMR and 13 C NMR spectrum.

[0048] Fig. 27 is compound 20 1 H NMR and 13 C NMR spectrum.

[0049] Fig.28 is compound 21 1 H NMR and 13 C NMR spectrum.

[0050] Fig.29 It is compound 22 1 H NMR and 13 C NMR spectrum.

[0051] Fig.30 It is compound 23 1 H NMR and 13 C NMR spectrum.

[0052] Fig.31 is compound 24 1 H NMR and 13 C NMR spectrum.

[0053] Fig.32 is compound 25 1 H NMR and 13 C NMR spectrum.

[0054] Fig.33 is compound 26 1 H NMR and 13 C NMR spectrum.

[0055] Fig.34 is compound 27 1 H NMR and 13 C NMR spectrum.

[0056] Fig.35 It is compound 28 1 H NMR and 13 C NMR spectrum.

[0057] Fig.36 It is compound 29 1 H NMR and 13 C NMR spectrum.

[0058] Fig.37 is compound 30 1 H NMR and 13 C NMR spectrum.

[0059] Fig.38 is compound 31 1 H NMR and 13 C NMR spectrum.

[0060] Fig.39 It is compound 32 1 H NMR and 13 C NMR spectrum.

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

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

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

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

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

[0066] Fig.45 It is compound 38 1 H NMR and 13 C NMR spectrum. DETAILED DESCRIPTION

[0067] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

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

[0069] As mentioned above, pharmacological studies have shown that paper mulberry has anti-inflammatory, anti-tumor, antioxidant, anti-diabetic, anti-obesity, antibacterial and anti-viral, as well as skin whitening and anti-wrinkle activities. Currently, most of the research on paper mulberry focuses on its roots, stems, leaves and fruits, while research on its branches is very limited, resulting in a huge waste of resources.

[0070] In view of this, the present invention provides a method for separating, preparing and screening antioxidant and anti-inflammatory active ingredients in paper mulberry branches, comprising: crushing paper mulberry branches into coarse powder, extracting with solvents, recovering the solvents, freeze-drying or drying under reduced pressure to make dry extracts, suspending the extracts in water, extracting with different organic solvents in turn, recovering the solvents, and obtaining different extraction parts. The free radical scavenging ability of the above-mentioned extraction parts was determined by the DPPH method and the ABTS method, and the T-AOC (total antioxidant capacity) was determined by the diphenylpicrylhydrazine method to identify the antioxidant active parts; the ability of the above-mentioned extraction parts to release cytokines from RAW264.7 cells was determined by the ELISA method to determine the anti-inflammatory active parts. 1 H-NMR, 13 C-NMR and other spectral methods were used to analyze the above active sites, and the structural types of active compounds were deduced in combination with literature. Then, according to the structural characteristics of the active compounds, the best separation scheme was screened and formulated from silica gel column chromatography, gel column chromatography, macroporous resin column chromatography, reversed phase column chromatography and preparative high performance liquid chromatography separation methods, and the active sites were quickly enriched and accurately separated to obtain the target compounds. At the same time, a CuSO4-induced zebrafish inflammation model was established, ibuprofen was used as a positive control drug, and the number of zebrafish macrophage migration was determined by stereo fluorescence microscopy, and the anti-inflammatory activity of some of the active compounds obtained was further studied.

[0071] Specifically, in a typical embodiment of the present invention, a method for screening and preparing a paper mulberry tree extract having anti-inflammatory and antioxidant activities is provided, the screening and preparation method comprising:

[0072] S1, crushing the paper mulberry branches, adding an organic solvent, heating and refluxing for extraction, concentrating the liquid, and drying to obtain an extract;

[0073] S2, after dispersing the extract with water, extracting with petroleum ether, ethyl acetate and n-butanol respectively to obtain extracts of different parts;

[0074] S3, determining the anti-inflammatory and antioxidant activities of the extracts from each part, and selecting the ethyl acetate part with the best activity;

[0075] S4. Determine the main compound structure of the active site of ethyl acetate by spectroscopy;

[0076] S5. Separating the compound components in the ethyl acetate part, including using silica gel, polyamide, reversed phase ODS, dextran gel column chromatography to separate the ethyl acetate part, and purifying it through preparative high performance liquid chromatography to obtain monomer components, and finally obtaining 40 active compounds.

[0077] Wherein, in step S1, the organic solvent may be ethanol, and further may be 80% ethanol;

[0078] The heating reflux extraction can be performed 2-4 times. In a specific embodiment of the present invention, the specific method of the heating reflux extraction is: heating reflux extraction with 80% ethanol for 3 times, each time for 2 hours, and the amount of ethanol used is 10, 8, and 8 times the volume of the medicinal material, respectively. The medicinal solution obtained from each reflux extraction is combined, and concentrated under reduced pressure for subsequent treatment.

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

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

[0081] More specifically, the cell inflammation model uses RAW246.7 cells, and the active extraction parts with anti-inflammatory effects are screened by measuring the effects of various extraction parts on the release of cytokines (TNF-α, IL-1β, IL-6) from RAW246.7 cells;

[0082] The animal inflammation model used is a CuSO4-induced zebrafish inflammation model, and the active extraction parts with anti-inflammatory effects are screened by measuring the effects of various extraction parts on the migration of zebrafish macrophages. In a specific embodiment of the present invention, the above-mentioned animal inflammation model is used to detect the anti-inflammatory activity of some active compounds isolated.

[0083] The results showed that the antioxidant activity sites of Broussonetia papyrifera branches were ethyl acetate and n-butanol, and the anti-inflammatory activity sites of Broussonetia papyrifera branches were ethyl acetate and n-butanol. Combining the antioxidant and anti-inflammatory activity results, the ethyl acetate site had the best activity, so further research on the chemical composition of the ethyl acetate site was considered.

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

[0085] In another specific embodiment of the present invention, in step S5, in another specific embodiment of the present invention, the method for separating the ethyl acetate part comprises the following steps: taking the ethyl acetate part extract, mixing the sample with 100-200 mesh silica gel, loading the silica gel column, 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), and then with ethyl acetate: methanol (79:1, 39:1, 19:1, 9:1, 4:1, 2:1, 0:1) gradient elution, 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 and purified by eluting with methanol, and then purified by preparative high performance liquid chromatography (3 mL / min, MeOH:H2O=32:68) to obtain Compound 3, Compound 4, Compound 5, and Compound 11.

[0088] E8 was purified by ODS column chromatography with a gradient elution of 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 E8a-E8f. E8a was further purified by preparative HPLC (3 mL / min, MeOH:H2O=15:85) to obtain compounds 6 and 12.

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

[0090] The column was packed with 200-300 mesh silica gel wet method, E9 was loaded, and 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). The fractions were combined by thin layer chromatography to obtain 11 fractions E9a-E9k. E9h was purified again by preparative HPLC (3 mL / min, MeOH:H2O=45:55) to obtain compound 25 and compound 26; E9k was purified again by preparative HPLC (3 mL / min, MeOH:H2O=8:92, 17:83, 50:50) to obtain 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) as gradient elution to obtain 4 fractions (E9i1-E9i4). E9i2 was purified by preparative HPLC (3 mL / min, MeOH:H2O=85:15, 70:30) to obtain compound 2, compound 13, and compound 14; E9i3 was purified by preparative HPLC (3 mL / min, MeOH:H2O=65:35, 55:45, 40:60) to obtain compound 8, compound 16, and compound 32.

[0091] E10 was separated by polyamide chromatography 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) as gradient elution and detected by thin layer chromatography. The fractions were combined to obtain 10 fractions, E10a-E10j. E10c was purified by preparative HPLC (3 mL / min, MeOH:H2O=82:18) to obtain compounds 34, 35 and 36; E10d was purified by preparative HPLC (3 mL / min, MeOH:H2O=60:40, 48:52) to obtain compounds 19 and 38; E10e was purified by preparative HPLC (3 mL / min, MeOH:H2O=55:45) to obtain compound 27; E10f was purified by preparative HPLC (3 mL / min, MeOH:H2O=17:83, 35:65) to obtain compound 15 and compound 20; E10i was purified by preparative HPLC (3 mL / min, MeOH:H2O=70:30) to obtain compound 29.

[0092] E11 was separated by polyamide column chromatography and gradient 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) and detected by thin layer chromatography. The fractions were combined to obtain 10 fractions, E11a-E11j. E11b was re-purified by preparative HPLC (3 mL / min, MeOH:H2O=10:90) to obtain compound 21; E11g was re-purified by preparative HPLC (3 mL / min, MeOH:H2O=50:50) to obtain compounds 28, 30 and 31; E11h was re-purified by preparative HPLC (3 mL / min, MeOH:H2O=58:42, 45:55) to obtain compound 10; E11i was re-purified by preparative HPLC (3 mL / min, MeOH:H2O=50:50) to obtain compound 22.

[0093] Through the above separation method, a total of 40 active compounds were obtained, 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), 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), dihydroquinone (24), scutellariae (25), 1,3,5-trimethoxybenzene (26), benzyl-β-D-glucoside (27), quercetin (28), luteolin (29) , apigenin-7-glucoside (30), edopsakura glycoside (31), tulipwood resin alcohol A (32), ash bark resin alcohol (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), dehydrodisinconyl alcohol (37), simulanol (38), β-sitosterol (39), and carotene (40). The specific structural formulas of compounds 1-40 are shown in Figure 7 .

[0094] In another specific embodiment of the present invention, there is provided the use of the paper mulberry extract or active compound obtained by the above screening and preparation method in the preparation of anti-inflammatory and antioxidant products.

[0095] The paper mulberry branch extract includes the extracts of the ethyl acetate part and the n-butanol part, and preferably the extract of the ethyl acetate part.

[0096] The products include but are not limited to food, medicine and daily chemical products.

[0097] Among them, the food can be used for humans or non-human animals. When the food is used for humans, it can be a health food. When the food is used for non-human animals (including mammals and birds, especially livestock and poultry), it can be a feed or feed additive; the medicine can also be used for humans or non-human animals. When the medicine is used for non-human animals (including mammals and birds, especially livestock and poultry), it can be a veterinary drug.

[0098] The daily chemical products include cosmetics.

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

[0100] Example 1: Preparation of Extraction Parts of Paper Basil Branches

[0101] 5 kg of dried material of Broussonetia papyrifera branches was crushed into coarse powder, and extracted with 80% ethanol under heating and reflux for 3 times, each time for 2 hours, and the amount of ethanol used was 10, 8, and 8 times the volume of the material, respectively. The liquid was filtered, combined, concentrated under reduced pressure, and dried under reduced pressure to obtain 776 g of extract. After the extract was dispersed with appropriate amount of water (1:1-1:5, g:mL), it was extracted with petroleum ether, ethyl acetate, and n-butanol in sequence (extractant: mother liquor (v / v) = 1:1, 4 times). The extracts of each part were concentrated and dried under reduced pressure to obtain petroleum ether part (2.4 g), ethyl acetate part (23 g), and n-butanol part (57 g), respectively, and stored in a refrigerator.

[0102] The antioxidant activity of each extraction part was determined by the DPPH method. The specific steps are as follows: 10 mg of extracts of different polar extraction parts of the arborvitae were taken respectively, and the test solutions with mass concentrations of 0.1, 0.5, 1.0, 1.5, and 2.0 mg / mL were prepared with anhydrous ethanol, and 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL vitamin C solutions were prepared with water. 2 mL of each test solution was taken in a 10 mL test tube, and 2 mL of the fully dissolved DPPH ethanol solution (0.1 mmol / L) was added in portions, stoppered and shaken, and tin foil was used to block light. It was kept away from light at room temperature for 30 minutes, placed in a 1 cm colorimetric dish, put into a spectrophotometer, and the absorbance was measured at 517 nm. The absorbance of the test solution was measured as Ai Purified water was used as the blank control group, and the absorbance was A0. Anhydrous ethanol was used instead of DPPH ethanol solution as the background group, and the absorbance was A j According to formula (1) (2), the scavenging rate of DPPH free radicals by different mass concentrations of Vc and sample solutions was calculated and the IC 50 .

[0103] The calculation formula for the scavenging rate of DPPH free radicals by each extraction part is:

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

[0105] The scavenging rate of the sample on DPPH free radical (%) = [A0-(A i -A j )] / A0×100%(2)

[0106] The antioxidant activity of each extraction part was determined by the ABTS method. The specific steps are as follows: 10 mg of extracts of different polar extraction parts of the arborvitae were taken respectively, and prepared into mass concentrations of 0.05, 0.1, 0.15, 0.2, and 0.25 mg / mL with anhydrous ethanol, and 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL vitamin C solutions were prepared with water. The ABTS reagent in the kit was taken into a test tube, the tube cap was screwed, shaken, and placed at room temperature for 12 to 16 hours to obtain ABTS concentrated solution. ABTS solution (7.4 mmol / L) and potassium persulfate solution (2.6 mmol / L) were mixed in a volume ratio of 1:1, and placed at room temperature in the dark for 12 hours to obtain ABTS mother liquor, which was diluted 45 times with purified water as ABTS free radical working solution. Accurately measure 2 mL of ABTS free radical working solution and 1 mL of each test solution, shake well, and measure the absorbance at 734 nm after 30 minutes at room temperature in the dark. The absorbance of the test solution was measured as A i Purified water was used as the blank control group, with an absorbance of A0, and ABTS working solution was used as the background group, with an absorbance of A j The scavenging rate of DPPH free radicals by sample solutions of different mass concentrations was calculated according to formula (3) and the IC 50 .

[0107] The study found that the antioxidant activity of paper mulberry is in the ethyl acetate part, ethanol part and n-butanol part.

[0108] The calculation formula for the ABTS free radical scavenging rate of each extraction part is:

[0109] ABTS free radical scavenging rate (%) = [A0-(Ai -A j )] / A0×100% (3)

[0110] RAW246.7 cells were selected as target cells, and the active extracts with anti-inflammatory effects were screened by measuring the effects of each extraction fraction on the release of cytokines from target cells. Some of the isolated compounds were selected to establish a CuSO4-induced zebrafish inflammation model, and ibuprofen was used as a positive control drug. The number of zebrafish macrophage migration was determined by stereofluorescence microscopy to determine its 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 15mL centrifuge tube was taken, 3mL of serum-containing culture medium was added, the liquid in the frozen tube was added to the above-mentioned centrifuge tube containing culture medium, and the mixture was gently blown to mix, centrifuged at 1000r / min~1500r / min at room temperature for 5min~10min, the supernatant was removed, 3mL~5mL of serum-containing culture medium was added, and the cell suspension was gently blown to mix, and added to the culture bottle. (2) Culture: Place the cells in a culture flask containing complete culture medium and allow them to grow adherently at 38°C and 5% CO2. When the cells have grown to more than 80% of the culture flask, the cells can be subcultured. (3) Subculture: Discard the culture medium in the culture flask, add 1mL to 3mL of trypsin, and incubate at 37°C for 2 to 5 minutes. If the cytoplasm shrinks and the intercellular space increases, immediately add an equal volume of culture medium containing serum to terminate digestion. Gently blow the cells at the bottom of the flask repeatedly to detach the adherent cells from the bottom of the flask and suspend them to form a cell suspension. Inoculate into new culture flasks according to the three-pass method and place them in the incubator for culture.

[0111] The CCK-8 method was used to determine the safe concentration of each extraction part for RAW246.7 cells. The specific method is as follows: Take an appropriate amount of the ethyl acetate extraction part and use DMEM culture medium to prepare sample solutions with concentrations of 0.15625, 0.3125, 0.625, 0.125, 0.25, 0.5, 1, 2, 3, and 5 mg / mL. The sample solution of the n-butanol sample was prepared in the same way. The RAW246.7 cells were adjusted to a concentration of 5×10 5 / ml, add to 96-well cell culture plates, set up 6 replicates in each well. Add 200μL of sample solution to each well, repeat 4 wells for each concentration, set up a blank control group (containing only DEME culture medium, no cells) and a normal group (add 200μL DMEM culture medium), and place them back in a 37°C, 5% CO2 incubator for incubation. After 24h, add 10μL CCK-8 solution to each well, incubate in the incubator for 2h, and measure the absorbance value A of the drug group, the absorbance value (B) of the normal group, and the absorbance value (C) of the control well at 450nm. Calculate cell viability according to formula (4). Determine the safe concentration of the drug based on the calculation results.

[0112] Cell viability (%) = (AC) / (BC) × 100% (4)

[0113] The ELISA method was used to determine the content of cytokines released by RAW246.7. The specific method was as follows: RAW264.7 cells treated with growth medium only were set as the blank group, RAW264.7 cells treated with LPS (1 μg / mL) were set as the model group, and cells treated with LPS (1 μg / mL) and different extraction samples (0.25 mg / mL) were set as the drug group. The cell density was 5×10 5 Cells / well were seeded in a 12-well plate, with 6 replicate wells in each group, and placed in a cell culture incubator for 24 hours. Afterwards, the cell supernatant was collected and the test was performed according to the following steps: (1) The reagent kit was kept at room temperature, and the standard and working solutions were prepared in advance. (2) Blank wells, standard wells, and sample wells were set up. The wells with only sample diluent were used as blank wells, and 100 μL / well of sample, standard, or sample diluent was added respectively. 3 wells were set up in each group, and the ELISA plate was sealed with a sealing film and incubated at 37°C for 90 minutes. (3) After the incubation, the liquid in the wells was removed, and the wells were dried. 100 μL of antibody working solution was added to each well, and the plate was sealed with a film, and incubated at 37°C for 60 minutes. (4) After the incubation, the liquid in the wells was removed, and an equal volume of washing buffer solution was added. The wells were soaked for 1 minute, and the waste liquid was discarded. The plate was washed 3 times and then dried. (5) 100 μL / well of ABC working solution was added, and the plate was sealed with a film, and incubated at 37°C for 30 minutes. (6) After the incubation, remove the liquid in the wells, add an equal volume of washing buffer solution, soak for 1 min, remove the waste liquid, repeat the wash 3 times and spin dry. (7) Add 90 μL TMB colorimetric solution / well, seal the plate with a sealing film, protect from light, and react at 37°C for 20 min. (8) After the reaction, add 100 μL TMB stop solution to each well and detect its absorbance at 450 nm.

[0114] The data processing method of the above anti-inflammatory experiment is as follows: the data are expressed as `X±SD, and the experimental data are analyzed by one-way ANOVA using GraphPad prism 9.0 software, and the differences between experimental data are analyzed using multiple comparison Turkey test. P<0.05 is considered to be significantly different.

[0115] The results showed that the antioxidant activity sites of Broussonetia papyrifera branches were ethyl acetate and n-butanol, and the anti-inflammatory activity sites of Broussonetia papyrifera branches were ethyl acetate and n-butanol. Combining the antioxidant and anti-inflammatory activity results, the ethyl acetate site had the best activity, so further research on the chemical composition of the ethyl acetate site was considered.

[0116] pass 1 H NMR and 13The structures of the main compounds 1-40 in the ethyl acetate site were determined by C NMR and other spectral methods and by comparison with literature reports: 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 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), dihydroquinone(24), scutellariae(25), 1,3,5-trimethoxybenzene(26), benzyl-β-D-glucoside(27), quercetin(28), luteolin(29), apigenin-7-glucoside(30), edopsin(31), liriodendron resinol A(32), ash resinol(33), 3-(4-hydroxy-3,5-dimetho xyphenyl)propane-1,2-diol(34), (-)-(7R,7'R,7"R,8S,8'S,8"S)-4',4"-dihydroxy-3,3',3",5,5'-pe ntamethoxy-7,9':7',9-diepoxy-4,8"-oxy-8,8'-sesquineolignan-7",9"-diol(35), Hedyotol C(36), dehydrodisinconiferyl alcohol(37), simulanol(38), β-sitosterol(39), and carotene(40).

[0117] The method for separating compound monomers in the ethyl acetate part comprises the following steps: taking 23 g of the ethyl acetate part extract, mixing the sample with 100-200 mesh silica gel in a ratio of 1:1, loading the sample on a silica gel column (213 g, 100-200 mesh), firstly eluting 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), and then eluting with ethyl acetate:methanol (79:1, 39:1, 19:1, 9:1, 4:1, 2:1, 0:1, v:v), each gradient elution having 3 column volumes, a flow rate of 10 mL / min, performing thin layer chromatography detection, combining the same fractions to obtain E1-E12.

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

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

[0120] E8 was subjected to ODS column chromatography with a gradient elution of 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), 5 column volumes per gradient elution, and a flow rate of 3 mL / min to obtain 6 fractions E8a-E8f. E8a was further purified by preparative HPLC (3 mL / min, MeOH:H2O=15:85) to obtain compounds 6 and 12;

[0121] E8b was purified again by preparative HPLC (3 mL / min, MeOH:H2O=20:80) to obtain compounds 1 and 7; E8d was purified again by preparative HPLC (3 mL / min, MeOH:H2O=36:65, v:v), with each gradient elution taking 45 min, to obtain compounds 23 and 24.

[0122] A 200-300 mesh silica gel wet-packed column was used, and E9 (3.5 g) was loaded. The column was eluted with a gradient of 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). Each gradient elution was 3 column volumes. The flow rate was 5 mL / min. The fractions were combined by thin layer chromatography to obtain 11 fractions, E9a-E9k. E9h was re-purified by preparative HPLC (3 mL / min, MeOH:H2O=45:55) to obtain compounds 25 and 26; E9k was re-purified by preparative HPLC (3 mL / min, MeOH:H2O=8:92, 17:83, 50:50, v:v), with each gradient elution taking 45 min to obtain compounds 9 (2 mg), 17, 18 and 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 elution with 3 column volumes for each gradient elution, and a flow rate of 5 mL / min to obtain 4 fractions (E9i1-E9i4). E9i2 was purified again by preparative HPLC (FL-H080G semi-preparative liquid chromatograph) (3 mL / min, MeOH:H2O=85:15, 70:30, v:v), with each gradient elution taking 45 min to obtain compound 2, compound 13, and compound 14; E9i3 was purified again by preparative HPLC (3 mL / min, MeOH:H2O=65:35, 55:45, 40:60, v:v), with each gradient elution taking 45 min to obtain compound 8, compound 16, and compound 32.

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

[0124] E11 (5 g) was separated by silica gel column chromatography (48 g column), firstly with petroleum ether: dichloromethane (1:0, 9:1, 3:1, 2:1, 1:1, 0:1, v:v) gradient elution, each gradient elution was 3 column volumes, flow rate 10 mL / min, then with 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 elution, each gradient elution was 3 column volumes, flow rate 10 mL / min. Thin layer chromatography was used for detection, and the fractions were combined to obtain 10 fractions E11a-E11j. E11b was re-purified by preparative HPLC (3 mL / min, MeOH:H2O=10:90) to obtain compound 21; E11g was re-purified by preparative HPLC (3 mL / min, MeOH:H2O=50:50) to obtain compounds 28, 30 and 31; E11h was re-purified by preparative HPLC (3 mL / min, MeOH:H2O=58:42, 45:55, v:v) for 45 min to obtain compound 10; E11i was re-purified by preparative HPLC (3 mL / min, MeOH:H2O=50:50) to obtain compound 22.

[0125] The anti-inflammatory experiment of CuSO4-induced zebrafish inflammation model was established, ibuprofen was used as a positive control drug, and the number of zebrafish macrophage migration was determined by stereo fluorescence microscopy. The specific method is as follows: JS7 mature male and female zebrafish of the transgenic zebrafish strain Tg (zlyz: EGFP) with green fluorescent labeled inflammatory cells were selected, and placed in a bathtub with baffles containing system water at a ratio of 1:2 for male and female, and cultured overnight, and the water temperature was set to 28.5℃. The baffles were removed the next day for breeding, and the embryos were collected and placed in the fish tank and sterilized with methylene blue solution, and then moved into the water for zebrafish embryo culture, and cultured in a constant temperature incubator at 28℃ for 72h for use. After 72 hours of embryonic development, normal zebrafish fry were selected and transferred into 24-well cell culture plates, with 10 fry in each well. The experiment was divided into a blank group, a model group, and a positive 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 solutions. After 24 hours of cultivation, except for the blank group, the culture medium was replaced with a solution of 4 μL CuSO4 solution (20 mmol / mL) plus 1996 μL fish water for modeling in the dark for 1 hour. After the modeling was completed, the zebrafish were washed with culture water, anesthetized with 0.1% tricaine solution, and the migration of macrophages near the lateral line of zebrafish was observed and photographed using a fluorescence microscope, and the number of macrophages that migrated above the lateral line was counted. The anti-inflammatory activity of the compounds isolated above was verified, and the results are as follows Figure 6 shown.

[0126] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. A method for screening and preparing a paper mulberry branch extract with anti-inflammatory and antioxidant activities, characterized in that: The screening preparation method comprises: S1, crushing the paper mulberry branches, adding an organic solvent, heating and refluxing for extraction, concentrating the liquid, and drying to obtain an extract; S2, after dispersing the extract with water, extracting with petroleum ether, ethyl acetate and n-butanol respectively to obtain extracts of different parts; S3, determining the anti-inflammatory and antioxidant activities of the extracts from each part, and selecting the ethyl acetate part with the best activity; S4. Determine the main compound structure of the active site of ethyl acetate by spectroscopy; S5. Separating the compound components in the ethyl acetate part, including using silica gel, polyamide, reversed phase ODS, dextran gel column chromatography to separate the ethyl acetate part, and purifying it through preparative high performance liquid chromatography to obtain monomer components, and finally obtaining 40 active compounds.

2. The screening and preparation method according to claim 1, characterized in that: In the step S1, the organic solvent is ethanol, and further is 80% ethanol.

3. The screening and preparation method according to claim 1, characterized in that: The heating reflux extraction is performed 2-4 times. Furthermore, the specific method of the heating reflux extraction is: heating reflux extraction with 80% ethanol for 3 times, each time for 2 hours, and the amount of ethanol used is 10, 8, and 8 times the volume of the medicinal material respectively; the medicinal liquid obtained from each reflux extraction is combined, and concentrated under reduced pressure for subsequent processing.

4. The screening and preparation method according to claim 1, characterized in that: In step S2, the volume ratio of water to the organic solvent (petroleum ether, ethyl acetate and n-butanol) is 1:0.5-5, preferably 1:1; the number of extractions is 2 to 6 times for each extraction solvent, preferably 4 times.

5. The screening and preparation method according to claim 1, characterized in that: In step S3, the specific method for determining the antioxidant and anti-inflammatory activities of the extracts from each part includes using the DPPH method, ABTS method and diphenylpicrylhydrazine method to determine the T-AOC (total antioxidant capacity) of the extracts from different parts of the paper mulberry branches; and using a cell inflammation model and / or an animal inflammation model to determine the anti-inflammatory activity of the extracts from different parts of the paper mulberry branches.

6. The screening and preparation method according to claim 5, characterized in that: The cell inflammation model uses RAW246.7 cells, and the active extraction parts with anti-inflammatory effects are screened by measuring the effects of various extraction parts on the release of cytokines (TNF-α, IL-1β, IL-6) from RAW246.7 cells; The animal inflammation model used is a CuSO4-induced zebrafish inflammation model. By measuring the effects of various extraction parts on the migration of zebrafish macrophages, active extraction parts with anti-inflammatory effects are screened; further, the above-mentioned animal inflammation model is used to detect the anti-inflammatory activity of some active compounds isolated.

7. The screening and preparation method according to claim 1, characterized in that: In step S4, for the structural identification of the compound, the spectral means adopted include ultraviolet spectroscopy, infrared spectroscopy, mass spectroscopy, nuclear magnetic resonance hydrogen spectrum and nuclear magnetic resonance carbon spectrum.

8. The screening and preparation method according to claim 1, characterized in that: In the step S5, 40 active compounds are obtained, 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), dihydrochloride (24), scutellariae (25), 1,3,5-trimethoxybenzene (26), benzyl-β-D-glucoside (27), quercetin (28), luteolin (29), apigenin-7-glucoside (30), edoprosin (31 )、Liriodendron resin A(32), Ash resin alcohol(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), dehydrodisinconiferyl alcohol(37), simulanol(38), β-sitosterol(39), Carotene(40).

9. Use of the paper mulberry extract or active compound obtained by the screening and preparation method according to any one of claims 1 to 8 in the preparation of anti-inflammatory and / or antioxidant products.

10. The use according to claim 9, characterized in that: The products include food, medicine and daily chemical products.

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