阅读说明：本技术 一种活性化合物及其提取方法和应用、药物组合物及其应用 (Active compound, extraction method and application thereof, pharmaceutical composition and application thereof ) 是由 罗晓东 王兆杰 王欢 朱蒙 赵云丽 于 2021-08-18 设计创作，主要内容包括：本发明涉及医药技术领域,具体涉及一种活性化合物及其提取方法和应用、药物组合物及其应用。本发明提供的活性化合物,包括茋类化合物(AI、AII、AIII、AIV)、黄酮类化合物(BI、BII、BIII、BIV、BV)和Diels-Alder型加合类化合物(CI、CII、CIII、CIV、CV、CVI、CVII)中的一种或几种。本发明提供的活性化合物在小鼠体内的抗MRSA活性,与体外抗菌研究结果一致,能够有效减少脾脏、肝脏和肾脏内的细菌数量,体内抗菌效果明显优于阳性对照药Ampicillin。说明,本发明提供的活性化合物无论在体外还是在体内都具有很好的抗致病微生物活性,能够用于制备抗致病微生物药物。(The invention relates to the technical field of medicines, and particularly relates to an active compound, an extraction method and application thereof, a pharmaceutical composition and application thereof. The active compound provided by the invention comprises one or more of stilbene compounds (AI, AII, AIII and AIV), flavonoid compounds (BI, BII, BIII, BIV and BV) and Diels-Alder addition compounds (CI, CII, CIII, CIV, CV, CVI and CVII). The anti-MRSA activity of the active compound in a mouse is consistent with the in-vitro antibacterial research result, the number of bacteria in spleen, liver and kidney can be effectively reduced, and the in-vivo antibacterial effect is obviously superior to that of a positive control drug Ampicillin. The active compound provided by the invention has good anti-pathogenic microorganism activity in vitro and in vivo, and can be used for preparing anti-pathogenic microorganism medicines.)

1. An active compound, which is characterized by comprising one or more of stilbene compounds, flavonoid compounds and Diels-Alder addition compounds;

the stilbene compounds comprise one or more of a compound AI, a compound AII, a compound AIII and a compound AIV:

r in the compound AI₁Is hydroxy or methoxy, R₂Is hydrogen or geranyl, R₃Is hydrogen or isoprenyl, R₄Is hydrogen or geranyl; r in the compound AIV_5～R₁₃Independently methoxy or acetyl;

the flavonoid compound comprises one or more of compound BI, compound BII, compound BIII, compound BIV and compound BV:

r in the compound BI₁₄Is hydrogen or isoprenyl, R₁₅Is hydrogen or isoprenyl, R₁₆Is hydrogen or geranyl; r in the compound BII₁₇Is hydroxy or methoxy, R₁₈Is geranyl, R₁₉Is hydrogen; r in BV of said compound_20～R₂₇Independently methoxy or acetyl;

the Diels-Alder addition compound comprises one or more of a compound CI, a compound CII, a compound CIII, a compound CIV, a compound CV, a compound CVI and a compound CVII:

the chemical conversionR in Compound CI₂₈Is hydrogen or isoprenyl, R₂₉Is isoprenyl;

r in the compound CVII₃₀And R₃₁Independently methoxy or acetyl.

2. The active compound according to claim 1, wherein the compound AI comprises one or more of compound AI-1, compound AI-2 and compound AI-3;

r in the compound AI-1₁Is hydroxy, R₂Is hydrogen, R₃Is isoprenyl, R₄Is hydrogen;

r in the compound AI-2₁Is methoxy, R₂Is geranyl, R₃Is hydrogen, R₄Is hydrogen;

r in the compound AI-3₁Is hydroxy, R₂Is hydrogen, R₃Is hydrogen, R₄Geranyl;

the compound BI comprises a compound BI-1 and/or a compound BI-2;

r in the compound BI-1₁₄Is isoprenyl, R₁₅Is isoprenyl, R₁₆Is hydrogen;

r in the compound BI-2₁₄Is hydrogen, R₁₅Is hydrogen, R₁₆Geranyl;

the compound BII comprises a compound BII-1 and/or a compound BII-2;

r in the compound BII-1₁₇Is hydroxy, R₁₈Is geranyl, R₁₉Is hydrogen;

r in the compound BII-2₁₇Is methoxy, R₁₈Is geranyl, R₁₉Is hydrogen;

the compound CI comprises compound CI-1 and/or compound CI-2;

r in the compound CI-1₂₈Is hydrogen, R₂₉Is isoprenyl;

r in the compound CI-2₂₈Is isoprenyl, R₂₉Is isoprenyl.

3. The active compound of claim 1 or 2, further comprising a class D compound having one or more of the structures represented by formulas DI-1 to DI-10:

4. active compound according to claim 3, characterized in that it comprises the following components in parts by mass: 1-200 parts of stilbene compounds, 1-100 parts of flavonoid compounds, 1-200 parts of Diels-Alder addition compounds and 0-200 parts of D compounds.

5. The active compound of any one of claims 1, 2 or 4, wherein the active compound comprises one or more of compound AI-3, compound AII, compound BIV, compound CV and compound CVI.

6. A process for the extraction of an active compound according to any one of claims 1 to 5, comprising the steps of:

extracting Moraceae plant with alcohol solvent to obtain extract; extracting the extract with an ester solvent to obtain an ester extract;

subjecting the ester extract to first column chromatography to obtain Fr.1 component, Fr.2 component, Fr.3 component, Fr.4 component, Fr.5 component, Fr.6 component and Fr.7 component in sequence; the elution mode of the first column chromatographic separation is gradient elution, wherein an eluant used in the gradient elution is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1: 0-0: 1;

performing second column chromatographic separation on the Fr.2 component to obtain a compound AI-2, a compound AII, a compound BI-1, a compound BI-2 and a compound DI-5; an eluent adopted by the second column chromatographic separation is a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 20-100%;

subjecting the Fr.3 fraction to third column chromatography to obtain compound AI-1, compound DI-1, compound AIII, compound BIII, compound BIV, compound BII-1, compound DI-7 and compound DI-8; an eluant adopted by the third column chromatographic separation is a petroleum ether-ethyl acetate mixed solvent, and the volume ratio of petroleum ether to ethyl acetate in the mixed solvent is 8: 1-1: 1;

subjecting the Fr.4 fraction to fourth column chromatography to obtain compound DI-1, compound AI-3, compound DI-4, compound DI-6, compound BII-2, compound DI-9, compound CII and compound CVI; an eluant adopted by the fourth column chromatography is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 6: 1-1: 1;

performing fifth column chromatography on the Fr.5 component to obtain a compound CIII, a compound CIV, a compound CV, a compound CI-1, a compound CI-2 and a compound DI-10; and an eluent adopted by the fifth column chromatographic separation is a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 10-100%.

7. A process for the extraction of active compounds according to claim 5, characterized in that it comprises the following steps:

extracting Moraceae plant with alcohol solvent to obtain extract;

separating the extract by macroporous adsorption resin, and performing gradient elution on the extract by using an ethanol-water mixed solution to obtain an MA-0 component, an MA-2 component, an MA-4 component, an MA-6 component, an MA-8 component and an MA-10 component; the volume fraction of ethanol in the ethanol-water mixed solution is 0-95%;

carrying out sixth column chromatographic separation on the MA-6 component to obtain one or more of a compound AI-3, a compound AII, a compound BIV, a compound CV and a compound CVI; the elution mode of the sixth column chromatographic separation is gradient elution, an eluant adopted in the gradient elution is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1: 0-0: 1.

8. A pharmaceutical composition, which comprises the active compound of any one of claims 1 to 5 or the active compound obtained by the extraction method of any one of claims 6 to 7, a pharmaceutically acceptable adjuvant and/or a Chinese medicine.

9. Use of an active compound according to any one of claims 1 to 5, an active compound obtained by the extraction process according to any one of claims 6 to 7 or a pharmaceutical composition according to claim 8 for the preparation of an anti-pathogenic microbial agent.

10. The use according to claim 9, wherein the effective daily dose of the anti-pathogenic microbial agent is 0.01 to 1000mg/kg body weight.

Technical Field

The invention relates to the technical field of medicines, and particularly relates to an active compound, an extraction method and application thereof, a pharmaceutical composition and application thereof.

Background

Pathogenic microorganisms are a general term for bacteria, fungi, viruses and mycoplasmas that cause people to suffer from diseases, often simply referred to as pathogenic bacteria or pathogenic bacteria (pathogenic bacteria). The pathogenic bacteria have prominent characteristics of pathogenicity, and the pathogenic bacteria can obviously cause a disease from the qualitative concept, such as plague caused by plague bacteria and tuberculosis caused by tubercle bacillus; from the concept of quantity, the pathogenicity of pathogenic bacteria is divided into strong and weak pathogenicity, the pathogenicity of different bacteria is different, and the same bacteria also has strong, weak and nontoxic strains, and can be changed according to different types of hosts and different environments.

Currently, infection by multi-drug resistant pathogenic bacteria and pathogenic fungi (superbacteria and superbacteria) threatens the healthy development of human society, and more newer antibacterial drugs are urgently needed. It is known that although antibiotics can kill some pathogenic microorganisms, pathogens are affected by some drugs which are sensitive to the antibiotics for a period of time, and then the response of the pathogens to the drugs gradually weakens or even disappears, so that some pathogens can survive under normal lethal dose, namely, the antibiotics resistance is generated. Pathogenic drug resistance is currently one of the biggest threats to global health, food safety and development; however, the evolution of the drug resistance of pathogens is unprecedentedly fast due to various artificial and natural factors, the development period of novel antibiotics is too long to keep pace with the evolution pace of drug resistance, the struggle advantage of human beings against pathogenic bacteria infection is gradually weakened, and the infection and control of drug-resistant bacteria are becoming more and more difficult.

Ampicillin (ampicilin) and Berberine Hydrochloride (berberberberberberberberberberberberberyl) are commonly used antibacterial agents, however, the antibacterial activity of the above drugs against pathogenic microorganisms such as methicillin staphylococcus aureus (MRSA) is low.

Disclosure of Invention

In view of the above, the present invention aims to provide an active compound, an extraction method and application thereof, a pharmaceutical composition and application thereof, wherein the active compound provided by the present invention has excellent activity against pathogenic microorganisms.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an active compound, which comprises one or more of stilbene compounds, flavonoid compounds and Diels-Alder addition compounds;

the stilbene compounds comprise one or more of a compound AI, a compound AII, a compound AIII and a compound AIV:

r in the compound AI₁Is hydroxy or methoxy, R₂Is hydrogen or geranyl, R₃Is hydrogen or isoprenyl, R₄Is hydrogen or geranyl; r in the compound AIV_5～R₁₃Independently is aOxy or acetyl;

the flavonoid compound comprises one or more of compound BI, compound BII, compound BIII, compound BIV and compound BV:

r in the compound BI₁₄Is hydrogen or isoprenyl, R₁₅Is hydrogen or isoprenyl, R₁₆Is hydrogen or geranyl; r in the compound BII₁₇Is hydroxy or methoxy, R₁₈Is geranyl, R₁₉Is hydrogen; r in BV of said compound₂₀～R₂₇Independently methoxy or acetyl;

the Diels-Alder addition compound comprises one or more of a compound CI, a compound CII, a compound CIII, a compound CIV, a compound CV, a compound CVI and a compound CVII:

r in the compound CI₂₈Is hydrogen or isoprenyl, R₂₉Is isoprenyl;

r in the compound CVII₃₀And R₃₁Independently methoxy or acetyl.

Preferably, the compound AI comprises one or more of a compound AI-1, a compound AI-2 and a compound AI-3;

r in the compound AI-1₁Is hydroxy, R₂Is hydrogen, R₃Is isoprenyl, R₄Is hydrogen;

r in the compound AI-2₁Is methoxy, R₂Is geranyl, R₃Is hydrogen, R₄Is hydrogen;

r in the compound AI-3₁Is hydroxy, R₂Is hydrogen, R₃Is hydrogen, R₄Geranyl;

the compound BI comprises a compound BI-1 and/or a compound BI-2;

r in the compound BI-1₁₄Is isoprenyl, R₁₅Is isoprenyl, R₁₆Is hydrogen;

r in the compound BI-2₁₄Is hydrogen, R₁₅Is hydrogen, R₁₆Geranyl;

the compound BII comprises a compound BII-1 and/or a compound BII-2;

r in the compound BII-1₁₇Is hydroxy, R₁₈Is geranyl, R₁₉Is hydrogen;

r in the compound BII-2₁₇Is methoxy, R₁₈Is geranyl, R₁₉Is hydrogen;

the compound CI comprises compound CI-1 and/or compound CI-2;

r in the compound CI-1₂₈Is hydrogen, R₂₉Is isoprenyl;

r in the compound CI-2₂₈Is isoprenyl, R₂₉Is isoprenyl.

Preferably, the active compound also comprises a D compound, and the D compound has one or more structures shown in formulas DI-1-DI-10:

preferably, the active compound comprises the following components in parts by mass: 1-200 parts of stilbene compounds, 1-100 parts of flavonoid compounds, 1-200 parts of Diels-Alder addition compounds and 0-200 parts of D compounds.

Preferably, the active compound comprises one or more of compound AI-3, compound AII, compound BIV, compound CV and compound CVI.

The invention provides an extraction method of an active compound in the technical scheme, which comprises the following steps:

extracting Moraceae plant with alcohol solvent to obtain extract; extracting the extract with an ester solvent to obtain an ester extract;

subjecting the ester extract to first column chromatography to obtain Fr.1 component, Fr.2 component, Fr.3 component, Fr.4 component, Fr.5 component, Fr.6 component and Fr.7 component in sequence; the elution mode of the first column chromatographic separation is gradient elution, wherein an eluant used in the gradient elution is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1: 0-0: 1;

performing second column chromatographic separation on the Fr.2 component to obtain a compound AI-2, a compound AII, a compound BI-1, a compound BI-2 and a compound DI-5; an eluent adopted by the second column chromatographic separation is a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 20-100%;

subjecting the Fr.3 fraction to third column chromatography to obtain compound AI-1, compound DI-1, compound AIII, compound BIII, compound BIV, compound BII-1, compound DI-7 and compound DI-8; an eluant adopted by the third column chromatographic separation is a petroleum ether-ethyl acetate mixed solvent, and the volume ratio of petroleum ether to ethyl acetate in the mixed solvent is 8: 1-1: 1;

subjecting the Fr.4 fraction to fourth column chromatography to obtain compound DI-1, compound AI-3, compound DI-4, compound DI-6, compound BII-2, compound DI-9, compound CII and compound CVI; an eluant adopted by the fourth column chromatography is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 6: 1-1: 1;

performing fifth column chromatography on the Fr.5 component to obtain a compound CIII, a compound CIV, a compound CV, a compound CI-1, a compound CI-2 and a compound DI-10; and an eluent adopted by the fifth column chromatographic separation is a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 10-100%.

The invention provides an extraction method of an active compound in the technical scheme, which comprises the following steps:

extracting Moraceae plant with alcohol solvent to obtain extract;

separating the extract by macroporous adsorption resin, and performing gradient elution on the extract by using an ethanol-water mixed solution to obtain an MA-0 component, an MA-2 component, an MA-4 component, an MA-6 component, an MA-8 component and an MA-10 component; the volume fraction of ethanol in the ethanol-water mixed solution is 0-95%;

carrying out sixth column chromatographic separation on the MA-6 component to obtain one or more of a compound AI-3, a compound AII, a compound BIV, a compound CV and a compound CVI; the elution mode of the sixth column chromatographic separation is gradient elution, an eluant adopted in the gradient elution is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1: 0-0: 1.

The invention provides a pharmaceutical composition, which comprises the active compound in the technical scheme or the active compound obtained by the extraction method in the technical scheme, pharmaceutically acceptable auxiliary materials and/or traditional Chinese medicines.

The invention also provides the application of the active compound in the technical scheme, the active compound obtained by the extraction method in the technical scheme or the pharmaceutical composition in the technical scheme in the preparation of anti-pathogenic microorganism drugs.

Preferably, the effective daily dose of the anti-pathogenic microorganism medicine is 0.01-1000 mg/kg body weight.

The invention provides an active compound, which comprises one or more of stilbene compounds, flavonoid compounds and Diels-Alder addition compounds;

the stilbene compounds comprise one or more of a compound AI, a compound AII, a compound AIII and a compound AIV:

r in the compound AI₁Is hydroxy or methoxy, R₂Is hydrogen or geranyl, R₃Is hydrogen or isoprenyl, R₄Is hydrogen or geranyl; r in the compound AIV_5～R₁₃Independently methoxy or acetyl;

the flavonoid compound comprises one or more of compound BI, compound BII, compound BIII, compound BIV and compound BV:

r in the compound BI₁₄Is hydrogen or isoprenyl, R₁₅Is hydrogen or isoprenyl, R₁₆Is hydrogen or geranyl; r in the compound BII₁₇Is hydroxy or methoxy, R₁₈Is geranyl, R₁₉Is hydrogen; r in BV of said compound_20～R₂₇Independently methoxy or acetyl;

the Diels-Alder addition compound comprises one or more of a compound CI, a compound CII, a compound CIII, a compound CIV, a compound CV, a compound CVI and a compound CVII:

r in the compound CI₂₈Is hydrogen or isoprenyl, R₂₉Is isoprenyl; r in the compound CVII₃₀And R₃₁Independently methoxy or acetyl.

The active compounds provided by the invention have excellent activity against pathogenic microorganisms. As shown in the example results, the results of in vivo pharmacodynamic studies show that the anti-MRSA activity of the active compound provided by the invention in mice is consistent with the results of in vitro antibacterial studies, and the active compound can effectively reduce the number of bacteria in spleen, liver and kidney, so that the mice can be recovered to be normal; the in vivo antibacterial effect of the active compound is obviously better than that of a positive control drug Ampicillin, which shows that the active compound provided by the invention has good anti-pathogenic microorganism drug effect both in vitro and in vivo. The active compound provided by the invention has the capability of treating infectious diseases and symptoms, can be used for preparing medicines for resisting diseases and symptoms caused by pathogenic microorganisms, is used for inhibiting bacterial proliferation, improving bacterial infection of skin, mucous membrane and body surface and has excellent antibacterial activity on antibiotic-resistant bacteria.

The invention provides an extraction method of the active compound in the technical scheme. The extraction method provided by the invention uses the Moraceae plants as raw materials to prepare the active compounds, has the advantages of simple operation, wide raw material source, low production cost, greenness, no pollution and suitability for industrial production.

Drawings

FIG. 1 is a graph showing the extraction scheme and scandium MRSA activity results for the active compounds of example 1;

FIG. 2 is a graph of time-kill curves for the MA-6 fraction, compound AI-3, compound AII, compound BIV, compound CV and compound CVI in test example 2;

FIG. 3 is a scanning electron micrograph of the compound CVI and Ampicillin of test example 3 after they have acted on the extracellular biofilm of pathogenic bacteria (MRSA-011), in which A1 and B1 are blank controls; a2 and A3 are compounds CVI, B1 and B2 are Ampicillin;

FIG. 4 is a graph showing the results of the test of the MA-6 fraction and berberine hydrochloride effective in inhibiting pathogenic bacteria in mice in test example 4;

FIG. 5 is a graph showing the results of measurement of the effect of the components of Compound AI-1, Compound BI-1, Compound CI-1 and MA-6 on the MRSA after contact in test example 5.

Detailed Description

The invention provides an active compound, which comprises one or more of stilbene compounds, flavonoid compounds and Diels-Alder addition compounds.

In the invention, the stilbene compounds comprise one or more of a compound AI, a compound AII, a compound AIII and a compound AIV:

in the present invention, R in the compound AI₁Is hydroxy or methylOxy radical, R₂Is hydrogen or geranyl, R₃Is hydrogen or isoprenyl, R₄Is hydrogen or geranyl; r in the compound AIV_5～R₁₃Independently methoxy or acetyl. In the present invention, the compound AI preferably includes one or more of a compound AI-1, a compound AI-2 and a compound AI-3; r in the compound AI-1₁Preferably a hydroxyl group, R₂Preferably hydrogen, R₃Preferably isoprenyl, R₄Preferably hydrogen; r in the compound AI-2₁Preferably methoxy, R₂Preferably geranyl, R₃Preferably hydrogen, R₄Preferably hydrogen; r in the compound AI-3₁Preferably a hydroxyl group, R₂Preferably hydrogen, R₃Preferably hydrogen, R₄Geranyl is preferred.

In the invention, the flavonoid compound comprises one or more of compound BI, compound BII, compound BIII, compound BIV and compound BV:

in the present invention, R in the compound BI₁₄Is hydrogen or isoprenyl, R₁₅Is hydrogen or isoprenyl, R₁₆Is hydrogen or geranyl; the compound BI preferably comprises a compound BI-1 and/or a compound BI-2; r in the compound BI-1₁₄Preferably isoprenyl, R₁₅Preferably isoprenyl, R₁₆Preferably hydrogen; r in the compound BI-2₁₄Preferably hydrogen, R₁₅Preferably hydrogen, R₁₆Geranyl is preferred. In the present invention, R in the compound BII₁₇Is hydroxy or methoxy, R₁₈Is geranyl, R₁₉Is hydrogen; the compound BII preferably comprises compound BII-1 and/or compound BII-2; r in the compound BII-1₁₇Preferably a hydroxyl group, R₁₈Preferably geranyl, R₁₉Preferably hydrogen; r in the compound BII-2₁₇Preferably methoxy, R₁₈Preference is given toIs geranyl, R₁₉Hydrogen is preferred. In the present invention, R in BV is_20～R₂₇Independently methoxy or acetyl.

In the invention, the Diels-Alder addition compound comprises one or more of a compound CI, a compound CII, a compound CIII, a compound CIV, a compound CV, a compound CVI and a compound CVII:

in the present invention, R in the compound CI₂₈Is hydrogen or isoprenyl, R₂₉Is isoprenyl; the compound CI preferably comprises compound CI-1 and/or compound CI-2; r in the compound CI-1₂₈Preferably hydrogen, R₂₉Preferably a prenyl group; r in the compound CI-2₂₈Preferably isoprenyl, R₂₉Preferably it is isoprenyl. In the present invention, R in the compound CVII₃₀And R₃₁Independently methoxy or acetyl.

In the present invention, the active compound preferably further comprises a D-type compound, and the D-type compound preferably has one or more of the structures shown in formulas DI-1-DI-10:

in the present invention, the active compound preferably comprises the following components in parts by mass: the preferable part of the stilbene compound is 1-200 parts, more preferable part is 8-150 parts, further preferable part is 8-50 parts, and most preferable part is 15-30 parts; the flavonoid compound is preferably 1-100 parts, more preferably 10-100 parts, further preferably 20-80 parts, and most preferably 35-60 parts; the Diels-Alder addition compound is preferably 1-200 parts, more preferably 20-200 parts, even more preferably 40-150 parts, and most preferably 80-120 parts; the D-type compound is preferably 0 to 200 parts, more preferably 10 to 100 parts, still more preferably 20 to 70 parts, and most preferably 35 to 55 parts.

In the present invention, the active compound preferably comprises one or more of compound AI-3(Albafurana), compound AII (Moracin G), compound BIV (Kuwanon B), compound CV (Mulberrofuran Q) and compound CVI (Kuwanon O).

In the present invention, the compound AI-3 is preferably used in the form of a pure compound AI-3 or a plant extract containing the compound AI-3, and the mass percentage content of the compound AI-3 in the plant extract containing the compound AI-3 is preferably 1 to 99%, more preferably 10 to 80%, and further preferably 50 to 60%; the plant extract of the AI-3 compound-containing plant extract is preferably Moraceae plant extract, more preferably Morus plant extract, and even more preferably cortex Mori extract. In the invention, the compound AII is preferably used in the form of pure compound AII or compound AII-containing plant extract, and the mass percentage of the compound AII in the compound AII-containing plant extract is preferably 1-99%, more preferably 10-80%, and further preferably 50-60%; the plant extract of the compound AII-containing plant extract is preferably an extract of a plant of the family moraceae, more preferably an extract of a plant of the genus morus, and even more preferably an extract of cortex mori. In the invention, the compound BIV is preferably used in the form of a pure compound BIV or a compound BIV-containing plant extract, and the mass percentage of the compound BIV in the compound BIV-containing plant extract is preferably 1-99%, more preferably 10-80%, and further preferably 50-60%; the plant extract of the BIV-containing plant extract is preferably an extract of a plant of the family moraceae, more preferably an extract of a plant of the genus morus, and even more preferably an extract of cortex mori. In the present invention, the compound CV is preferably used in the form of a pure compound CV or a compound CV-containing plant extract, and the compound CV content in the compound CV-containing plant extract is preferably 1 to 99% by mass, more preferably 10 to 80% by mass, and even more preferably 50 to 60% by mass; the plant extract of the CV-containing plant extract is preferably an extract of a plant of the family moraceae, more preferably an extract of a plant of the genus morus, and still more preferably an extract of cortex mori. In the invention, the compound CVI is preferably used in the form of a pure compound CVI or a plant extract containing the compound CVI, and the mass percentage of the compound CVI in the plant extract containing the compound CVI is preferably 1-99%, more preferably 10-80%, and further preferably 50-60%; the plant extract of the plant extract containing the compound CVI is preferably an extract of a plant of the family moraceae, more preferably an extract of a plant of the genus morus, and still more preferably an extract of cortex mori.

In the present invention, when compound AI-3 in the active compound is used in the form of a plant extract containing AI-3 compound AI-3, compound AII is used in the form of a plant extract containing compound AII, compound BIV is used in the form of a plant extract containing compound BIV, compound CV is used in the form of a plant extract containing compound CV, and compound CVI is used in the form of a plant extract containing compound CVI, the content of compound AI-3 in the active compound is preferably 0.5 to 12% by mass, more preferably 1 to 11.1% by mass, and still more preferably 1.6 to 7.5% by mass; the mass percentage content of the compound AII is preferably 0.7-4.5%, more preferably 1.2-4%, and further preferably 1.5-3.5%; the mass percentage content of the compound BIV is preferably 0.4-8.5%, more preferably 1.3-8.2%, and further preferably 2-8%; the mass percentage content of the compound CV is preferably 2-17%, more preferably 5-16%, and further preferably 10-15.8%; the mass percentage content of the compound CVI is preferably 1-15%, more preferably 1.2-14%, and even more preferably 5-13.8%.

The invention provides an extraction method of an active compound in the technical scheme, which is marked as a method I and comprises the following steps:

extracting Morus plant with alcohol solvent to obtain extract; extracting the extract with an ester solvent to obtain an ester extract;

subjecting the ester extract to first column chromatography to obtain Fr.1 component, Fr.2 component, Fr.3 component, Fr.4 component, Fr.5 component, Fr.6 component and Fr.7 component in sequence; the elution mode of the first column chromatographic separation is gradient elution, wherein an eluant used in the gradient elution is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1: 0-0: 1;

performing second column chromatographic separation on the Fr.2 component to obtain a compound AI-2, a compound AII, a compound BI-1, a compound BI-2 and a compound DI-5; an eluent adopted by the second column chromatographic separation is a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 20-100%;

subjecting the Fr.3 fraction to third column chromatography to obtain compound AI-1, compound DI-1, compound AIII, compound BIII, compound BIV, compound BII-1, compound DI-7 and compound DI-8; an eluant adopted by the third column chromatographic separation is a petroleum ether-ethyl acetate mixed solvent, and the volume ratio of petroleum ether to ethyl acetate in the mixed solvent is 8: 1-1: 1;

performing fourth column chromatography on the Fr.4 component to obtain compound DI-1, compound AI-3, compound DI-4, compound DI-6, compound BII-2, compound DI-9, compound CII, compound CVI; an eluant adopted by the fourth column chromatography is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 6: 1-1: 1;

performing fifth column chromatography on the Fr.5 component to obtain a compound CIII, a compound CIV, a compound CV, a compound CI-1, a compound CI-2 and a compound DI-10; and an eluent adopted by the fifth column chromatographic separation is a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 10-100%.

In the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.

The invention utilizes alcohol solvent to extract Moraceae plants to obtain the extract. In the present invention, the Moraceae plant is preferably a Morus plant, and more preferably one or more of leaves, roots, bark, branches and fruits of a Morus plant. In the present invention, the Moraceae plant is preferably dried and pulverized before use. The temperature and time for the drying in the present invention are not particularly limited, and drying conditions known to those skilled in the art may be used. The method of the present invention is not particularly limited, and the pulverization may be carried out in a particle size of 100 to 300 mesh. In the present invention, the volume fraction of ethanol in the ethanol solvent is preferably not less than 95%; the ratio of the mass of the Moraceae plant to the volume of the alcohol solvent is preferably 1 g: 5-300 mL, more preferably 1 g: 8 mL. In the invention, the extraction temperature is preferably 25-85 ℃, and more preferably 50-55 ℃; the extraction time is preferably 0.5-8 h, and more preferably 0.5-3 h; the extraction mode is preferably ultrasonic extraction; the conditions for the ultrasonic extraction are not particularly limited in the present invention, and the ultrasonic extraction conditions known to those skilled in the art may be used. After the extraction, the invention preferably also comprises filtering the extraction system to obtain an extracting solution and filter residue; preferably, the filter residue is repeatedly extracted for 2-3 times, and the extracting solutions are combined and then concentrated to obtain an extract; the concentration mode is preferably reduced pressure distillation; in the present invention, the concentration conditions are not particularly limited, and the concentration may be carried out to a constant weight.

After the extract is obtained, the ester solvent is used for extracting the extract to obtain the ester extract. In the present invention, the ester solvent preferably includes ethyl acetate; the amount of the ester solvent used in the present invention is not particularly limited, and may be an amount of an extractant known to those skilled in the art. After the extraction, the invention preferably further comprises concentrating the organic phase obtained by the extraction, wherein the concentration mode is preferably reduced pressure distillation; in the present invention, the concentration conditions are not particularly limited, and the concentration may be carried out to a constant weight.

After obtaining the ester extract, the invention carries out first column chromatography separation on the ester extract to sequentially obtain a Fr.1 component, a Fr.2 component, a Fr.3 component, a Fr.4 component, a Fr.5 component, a Fr.6 component and a Fr.7 component. In the invention, the elution mode of the first column chromatography separation is gradient elution, the eluent adopted by the gradient elution is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1: 0-0: 1, and more preferably 1:0, 1:3, 1:5 and 1:10 respectively. In the invention, the chromatographic column used for the first column chromatographic separation is preferably a silica gel packed column, and the specification of the chromatographic column is preferably 1 × 42 cm; the particle size of silica gel used for the first column chromatography is preferably 200-300 meshes.

After the fr.1 component, the fr.2 component, the fr.3 component, the fr.4 component, the fr.5 component, the fr.6 component and the fr.7 component are obtained, the present invention preferably further comprises performing an anti-MRSA activity test on the fr.1 component, the fr.2 component, the fr.3 component, the fr.4 component, the fr.5 component, the fr.6 component and the fr.7 component, as a result of which the fr.2 component, the fr.3 component, the fr.4 component, the fr.5 component and the fr.6 component have anti-MRSA activity. The method for testing the anti-MRSA activity is not particularly limited, and a method for testing the anti-MRSA activity, which is well known to those skilled in the art, may be used, and specifically, the method for determining MIC (minimum inhibitory concentration) or MBC (minimum bactericidal concentration) is used.

After the Fr.2 component is obtained, the invention carries out second column chromatographic separation on the Fr.2 component to obtain a compound AI-2, a compound AII, a compound BI-1, a compound BI-2 and a compound DI-5. In the invention, an eluent used for the second column chromatographic separation is a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 20-100%, and more preferably 30%; the chromatographic column used for the second column chromatographic separation is preferably an RP-C18 chromatographic column, and the specification of the RP-C18 chromatographic column is preferably 9.1 × 150mm and 5 μm.

After the Fr.3 component is obtained, the invention carries out third column chromatography separation on the Fr.3 component to obtain a compound AI-1, a compound DI-1, a compound AIII, a compound BIII, a compound BIV, a compound BII-1, a compound DI-7 and a compound DI-8. In the invention, an eluent adopted by the third column chromatography is a petroleum ether-ethyl acetate mixed solvent, and the volume ratio of petroleum ether to ethyl acetate in the mixed solvent is 8: 1-1: 1, and more preferably 5: 1; the chromatographic column adopted by the third column chromatographic separation is preferably a silica gel packed column, and the specification of the chromatographic column is preferably 4 x 60 cm; the particle size of silica gel used for the third column chromatography is preferably 100-300 meshes.

After the Fr.4 component is obtained, the Fr.4 component is subjected to fourth column chromatography separation to obtain a compound DI-1, a compound AI-3, a compound DI-4, a compound DI-6, a compound BII-2, a compound DI-9, a compound CII and a compound CVI. In the invention, an eluent adopted by the fourth column chromatography is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 6: 1-1: 1; more preferably 3: 1; the chromatographic column adopted by the fourth column chromatographic separation is preferably a silica gel packed column, and the specification of the chromatographic column is preferably 1 × 60 cm; the particle size of silica gel used for the fourth column chromatography is preferably 200-300 meshes.

After the Fr.5 component is obtained, the Fr.5 component is subjected to fifth column chromatography to obtain a compound CIII, a compound CIV, a compound CV, a compound CI-1, a compound CI-2 and a compound DI-10. In the invention, the eluent adopted by the fifth column chromatography is methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 10-100%, and more preferably 45%; the chromatographic column used for the fifth column chromatographic separation is preferably an RP-C18 chromatographic column, and the specification of the RP-C18 chromatographic column is preferably 9.1X 150mm and 5 mu m.

In the present invention, when the active compound includes one or more of compound AI-3, compound AII, compound BIV, compound CV and compound CVI, the extraction method of the active compound, denoted as method two, includes the following steps:

extracting Moraceae plant with alcohol solvent to obtain extract;

separating the extract by macroporous adsorption resin, and performing gradient elution on the extract by using an ethanol-water mixed solution to obtain an MA-0 component, an MA-2 component, an MA-4 component, an MA-6 component, an MA-8 component and an MA-10 component; the volume fraction of ethanol in the ethanol-water mixed solution is 0-95%;

carrying out sixth column chromatographic separation on the MA-6 component to obtain one or more of a compound AI-3, a compound AII, a compound BIV, a compound CV and a compound CVI; the elution mode of the sixth column chromatographic separation is gradient elution, an eluant adopted in the gradient elution is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1: 0-0: 1.

The invention utilizes alcohol solvent to extract Moraceae plants to obtain the extract. In the present invention, the extraction method of the extract is preferably the same as the preparation conditions of the extract in the first method, and is not repeated herein.

After the extract is obtained, the extract is segmented and separated by using macroporous adsorption resin, and the extract is subjected to gradient elution by using an ethanol-water mixed solution to obtain an MA-0 component, an MA-2 component, an MA-4 component, an MA-6 component, an MA-8 component and an MA-10 component; the ethanol in the ethanol-water mixed solution is 0-95% in volume, preferably 0%, 20%, 40%, 60%, 80% and 95% in sequence, namely MA-0 component is obtained when the ethanol is 0%, MA-2 component is obtained when the ethanol is 20%, MA-4 component is obtained when the ethanol is 40%, MA-6 component is obtained when the ethanol is 60%, MA-8 component is obtained when the ethanol is 80%, and MA-10 component is obtained when the ethanol is 95%. In the present invention, the macroporous adsorption resin is preferably macroporous adsorption resin D101.

After obtaining the MA-0 component, the MA-2 component, the MA-4 component, the MA-6 component, the MA-8 component and the MA-10 component, the invention preferably also comprises an MRSA activity test of the MA-0 component, the MA-2 component, the MA-4 component, the MA-6 component, the MA-8 component and the MA-10 component, and the test result shows that the MA-6 component has MRSA activity. In the present invention, the method for testing anti-MRSA activity is preferably the same as the method for testing anti-MRSA activity described in the aforementioned method one, and will not be described herein again.

After obtaining the MA-6 component, carrying out sixth column chromatography separation on the MA-6 component to obtain one or more of a compound AI-3, a compound AII, a compound BIV, a compound CV and a compound CVI. In the invention, the elution mode of the sixth column chromatography separation is gradient elution, an eluant adopted by the gradient elution is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 1: 0-0: 1; the chromatographic column used for the sixth column chromatographic separation is preferably a silica gel packed column, the specification of the silica gel packed column is preferably 1 x 60cm, and the particle size of the silica gel used for the sixth column chromatographic separation is preferably 200-300 meshes.

The invention also provides a pharmaceutical composition which comprises the active compound in the technical scheme or the active compound obtained by the extraction method in the technical scheme, pharmaceutically acceptable auxiliary materials and/or traditional Chinese medicines.

In the invention, the traditional Chinese medicine preferably comprises one or more of extracts of pinellia ternate, perilla fruit, bitter apricot seed, thunberg fritillary bulb, scutellaria baicalensis, coptis chinensis, gardenia and motherwort; the pharmaceutical composition preferably comprises extracts of cortex Mori, rhizoma Pinelliae, fructus Perillae, semen Armeniacae amarum, Bulbus Fritillariae Thunbergii, Scutellariae radix, Coptidis rhizoma and fructus Gardeniae, or extracts of ramulus Mori and herba Leonuri; the extract is preferably an ethanol extract.

In the invention, the pharmaceutically acceptable auxiliary materials preferably comprise one or more of a carrier, an auxiliary agent and an excipient; the auxiliary agent preferably comprises a preservative and an emulsifier; the present invention is not particularly limited with respect to the kind of the carrier, the adjuvant and the excipient, and any carrier, adjuvant and excipient known to those skilled in the art may be used. In the invention, the mass percentage of the active compound in the pharmaceutical composition is preferably 0.1-10%, and more preferably 1-5%.

In the present invention, the dosage form of the pharmaceutical composition preferably includes tablets, solutions, powders, capsules, granules, suspensions, pastes, liniments or injections.

In the present invention, the effective daily dose of the active compound and the pharmaceutical composition is independently preferably 0.01 to 1000mg/kg body weight, more preferably 0.1 to 500mg/kg body weight, and even more preferably 1 to 10mg/kg body weight.

The invention also provides the application of the active compound in the technical scheme, the active compound obtained by the extraction method in the technical scheme or the pharmaceutical composition in the technical scheme in the preparation of anti-pathogenic microorganism drugs.

In the present invention, the antipathogenic microbial drug is preferably an antipathogenic microbial drug, or an antihuman pathogenic microbial drug. In the present invention, the anti-animal pathogenic microorganism drug and the anti-human pathogenic microorganism drug are independently preferably a drug against a skin disease caused by bacterial infection, a drug for treating a disease caused by deep bacterial infection, a drug against a disease and a disorder caused by a drug-resistant microorganism, or a drug against a patient's complicated disease with low immunity against pathogenic microorganisms. In the present invention, the anti-pathogenic microorganism drug preferably includes one or more of a drug for preventing microbial infection of the skin, a drug for preventing scald, a drug for preventing microbial infection in cold injury, a drug for inhibiting bacterial proliferation, a drug for improving digestive tract, a drug for treating skin infection, and a drug for treating mucosal infection.

In the present invention, the daily effective dose of the anti-pathogenic microorganism drug is preferably 0.01 to 1000mg/kg body weight, more preferably 0.1 to 500mg/kg body weight, and still more preferably 1 to 10mg/kg body weight.

In the invention, the active compound and/or the pharmaceutical composition has the activity of resisting pathogenic microorganisms, and the anti-pathogenic microorganism medicine can be used for inhibiting the infection of pathogenic microorganisms on the surface of a biological organic body and various diseases caused by the infection of the pathogenic microorganisms, and relieving various hazards caused by the pathogenic microorganisms, such as the preparation of medicines for resisting diseases or symptoms caused by the infection of pathogenic microorganisms, the preparation of reagents for researching the pathological mechanism of the infection of the pathogenic microorganisms and the preparation of reagents for evaluating the curative effect of the infection of the pathogenic microorganisms; enough to deal with the threat of part of the drug-resistant bacteria to human health.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The extraction and separation are carried out according to the flow chart shown in figure 1, and the specific steps are as follows:

(1) crushing 10kg of dried white mulberry root-bark to the particle size of 100-300 meshes, adding 80L of 95% ethanol by volume, heating to 55 ℃, extracting for 3 hours, and filtering to obtain an extracting solution and filter residues; repeatedly extracting the residue for 3 times, mixing extractive solutions, and concentrating under reduced pressure to constant weight to obtain extract (1.58 kg); extracting the extract with ethyl acetate, and concentrating the ethyl acetate layer to constant weight to obtain ethyl acetate extract (0.56 kg);

subjecting 300g of the ethyl acetate extract to first column chromatography to obtain a Fr.1 fraction (68g), a Fr.2 fraction (16g), a Fr.3 fraction (24g), a Fr.4 fraction (42g), a Fr.5 fraction (32g), a Fr.6 fraction (61g) and a Fr.7 fraction (39g) in this order; the first column chromatography separation adopts silica gel with the particle size of 200-300 meshes, the eluent is a chloroform-methanol mixed solvent, and the volume ratio of chloroform to methanol in the eluent is 1: 0-0: 1.

(2) The fr.1 fraction, the fr.2 fraction, the fr.3 fraction, the fr.4 fraction, the fr.5 fraction, the fr.6 fraction and the fr.7 fraction were tested for anti-MRSA activity, and the test results are shown in fig. 1, according to the determination method of MIC and MBC in CLSI M100 antibacterial drug susceptibility test execution standard 2019. The test result shows that the Fr.2 component to the Fr.5 component have the MRSA resistance activity, wherein the MRSA resistance activity of the Fr.4 component and the MRSA resistance activity of the Fr.5 component are the best, and the MIC reaches 64 mu g/mL; fr.2 and Fr.3 fractions were sub-active against MRSA and had MICs of 128. mu.g/mL.

(3) Subjecting the Fr.2 fraction to second column chromatography to give AI-2(7.4mg), AII (8.5mg), Compound BI-1(6.4mg), Compound BI-2(14.3mg) and Compound DI-5(12.8 mg); wherein, the chromatographic column used for the second column chromatographic separation is an RP-C18 chromatographic column (9.1 × 150mm, 5 μm), the eluent is a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 30%.

Subjecting the Fr.3 fraction to third column chromatography to give compound AI-1(9.2mg), compound DI-1(23.4mg), compound AIII (10.7mg), compound BIII (12.9mg), compound BIV (9.8mg), BII-1(8.2mg), compound DI-7(11.6mg) and compound DI-8(16.7 mg); wherein, the third column chromatography adopts a silica gel packed column (4 multiplied by 60cm, the particle size of the silica gel is 100-300 meshes), and the eluent is a mixed solvent of petroleum ether and ethyl acetate with the volume ratio of 5: 1.

Subjecting the Fr.4 fraction to fourth column chromatography to give Compound DI-1(7.2mg), Compound AI-3(8.4mg), Compound DI-3(16.9mg), Compound DI-4(9.7mg), Compound DI-6(16.3mg), Compound BII-2(12.8mg), Compound DI-9(14.9mg), Compound CII (16.7mg) and Compound CVI (22.5 mg); wherein, the fourth column chromatography adopts silica gel packed column (1 × 60cm, silica gel particle size 200-300 mesh), and the eluent is chloroform and methanol mixed solvent with volume ratio of 3: 1.

Subjecting the Fr.5 fraction to fifth column chromatography to give Compound CIII (60.7mg), Compound CIV (14.8mg), Compound CV (19.6mg), Compound CI-1(34.8mg), Compound CI-2(26.5mg) and Compound DI-10(19.8 mg); wherein, the chromatographic column used for the fifth column chromatography is an RP-C18 chromatographic column (9.1 × 150mm, 5 μm), and the eluent is methanol water solution (the volume fraction of methanol is 45%);

the structural characterization data for each compound is as follows:

compound DI-1: c₁₄H₁₀O₄；¹H NMR(400MHz,DMSO-d₆)δ_H 9.61(3H,s),7.46(1H,d),7.10(1H,d),7.16(1H,s),6.87(3H,dd),6.42(1H,d)；13C NMR(100MHz,DMSO-d₆)δ_c:159.3,156.2,155.8,154.5,132.2,121.6,121.3,113.0,103.2,102.9,102.1,98.0；negative ESI-MS m/z 241[M-H]^-。

Compound AI-1: c₁₉H₁₈O₄；¹H NMR(400MHz,DMSO-d₆)δ_H:6.88(1H,s),7.32(1H,d),6.72(1H,dd),6.83(1H,s),6.78(2H,s),5.26(1H,d),3.31(2H,d),1.78(3H,s),1.62(3H,s)；¹³C NMR(100MHz,DMSO-d₆)δc:157.5,157.2,156.6,156.5,131.4,130.3,124.4,123.2,121.8,116.9,113.1,103.9,101.3,98.5,26.0,23.4,18.0；negative ESI-MS m/z 309[M-H]^-。

Compound AI-2: c₂₅H₂₈O₄；¹H NMR(400MHz,DMSO-d₆)δ_H:9.50(2H,s),7.40(1H,m),7.14(1H,s),6.97(1H,m),6.76(2H,d),6.30(1H,d),5.32(1H,d),4.99(1H,d),3.85(3H,s),3.57(2H,d),3.52(2H,m),3.45(3H,s),1.98(3H,s),1.77(3H,s),1.53(2H,m),1.46(2H,m)；¹³C NMR(100MHz,DMSO-d₆)δc:159.2,159.1,155.3,155.0,153.8,135.2,132.1,131.1,124.4,122.8,122.1,118.6,108.6,103.3,102.9,102.9,102.0,56.9,39.6,26.5,25.7,22.5,17.8,16.4；negative ESI-MS m/z 391[M-H]^-。

Compound AI-3: c₂₄H₂₆O₄；¹H NMR(400MHz,CD₃OD)δ_H:7.17(1H,d),6.89(1H,s),6.79(2H,s),6.73(1H,d),6.24(1H,s),5.41(1H,m),5.01(1H,m),3.60(2H,d),2.06(2H,m),1.99(2H,m),1.89(3H,m),1.52(3H,m),1.48(3H,s)；¹³C NMR(100MHz,CD₃OD)δc:159.6,155.6,155.5,153.5,135.6,133.8,131.8,125.0,123.2,122.6,118.5,112.8,112.4,103.6,103.4,103.0,102.2,40.5,27.3,25.4,23.2,17.3,16.2；negative ESI-MS m/z 377[M-H]^-。

Compound DI-2: c₁₉H₁₈O₅；¹H NMR(400MHz,DMSO-d₆)δ_H:9.44(2H,br s),7.30(1H,s),7.06(1H,s),6.95(1H,s),6.70(2H,d),6.23(1H,d),5.17(1H,s),3.68(1H,dd),3.02(1H,dd),2.72(2H,dd),1.31(3H,s),1.22(3H,m)；¹³C NMR(100MHz,DMSO-d₆)δc:159.2,159.2,154.9,154.2,151.5,132.0,122.4,121.3,103.2,102.8,102.6,117.5,101.5,98.8,77.7,68.5,31.7,26.1,20.8；ESI-MS m/z325[M-H]^-。

Compound AII: c₁₉H₁₆O₄；¹H NMR(400MHz,DMSO-d₆)δ_H:7.86(1H,s),7.51(1H,s),7.10(1H,d),7.01(1H,s),6.40(1H,s),5.99(1H,d),5.86(1H,m),3.35(1H,m),3.31(1H,m),3.13(1H,dd),2.78(1H,dd),2.34(3H,s)；¹³C NMR(100MHz,DMSO-d₆)δc:160.5,158.0,157.9,153.9,136.6,134.0,127.1,121.4,120.6,120.0,118.3,104.6,104.4,103.0,75.97,24.25,21.0；negative ESI-MS m/z307[M-H]^-。

Compound AIII: c₁₉H₂₀O₄；¹H NMR(400MHz,DMSO-d₆)δ_H:9.55(1H,s),9.36(1H,s),9.02(2H,s),7.35(1H,d),7.10(1H,d),6.71(1H,d),6.42(2H,s),6.32(1H,d),6.24(1H,d),5.17(1H,m),3.15(2H,m),1.70(3H,s),1.61(3H,s)；¹³C NMR(100MHz,DMSO-d₆)δc:158.7,156.7,156.7,156.2,137.9,130.3,127.8,126,124.2,123.1,117.2,114.5,108.2,103.5,103.2,102.7,25.7,22.9,17.7；negative ESI-MS m/z 311[M-H]^-。

Compound BI-1: c₂₅H₂₆O₆；¹H NMR(400MHz,DMSO-d₆)δ_H:13.27(1H,s),9.77(1H,s),8.83(1H,s),6.41(1H,d),6.10(1H,dd),5.96(1H,s),4.97(1H,m),3.02(2H,d),2.51(2H,d),1.63,1.60(6H,br s),1.56(3H,s),1.43(3H,s)；¹³C NMR(100MHz,DMSO-d₆)δc:181.8,164.0,162.1,161.7,161.0,160.4,159.2,156.0,132.9,131.3,130.5,123.1,121.9,119.7,113.5,107.0,106.8,103.8,102.7,97.6,25.7,25.6,23.6,22.6,17.8,17.6；negative ESI-MS m/z421[M-H]^-。

Compound BI-2: c₂₅H₂₆O₆；¹H NMR(400MHz,DMSO-d₆)δ_H:13.06(1H,s),7.56(1H,s),6.97(1H,s),6.55(1H,m),6.38(1H,d),6.17(1H,d),5.29(1H,m),5.08(1H,s),3.19(2H,d),2.08(1H,s),2.02(2H,m),1.70(3H,s),1.55(3H,s),1.44(3H,s)；¹³C NMR(100MHz,DMSO-d₆)δc:182.2,164.4,162.4,161.8,160.6,159.8,157.7,135.6,131.2,128.9,126.9,124.4,123.1,110.3,109.0,107.1,103.9,99.0,94.1,41.0,27.1,26.7,25.8,18.0,16.3；negative ESI-MS m/z 421[M-H]^-。

Compound BIII: c₂₅H₂₄O₆；¹H NMR(400MHz,DMSO-d₆)δ_H:13.20(1H,s),9.90(2H,m),7.16(1H,d),6.53(1H,d),6.48(1H,d),6.39(1H,dd),6.22(1H,s),5.68(1H,d),3.04(2H,d),1.57(3H,s),1.42(6H,s),1.40(3H,s)；¹³C NMR(100MHz,DMSO-d₆)δc:182.2,162.3,161.4,161.1,158.9,157.0,152.2,131.7,131.4,128.0,121.9,120.4,114.3,111.2,107.3,104.7,103.2,100.9,99.2,78.4,28.1,27.7,25.8,24.1,17.7；negative ESI-MS m/z419[M-H]^-。

Compound DI-3: c₂₅H₂₆O₇；¹H NMR(400MHz,DMSO-d₆)δ_H:13.26(1H,s),9.87(1H,s),9.80(1H,s),7.17(1H,d),6.51(1H,d),6.44(1H,dd),6.36(1H,d),6.21(1H,s),5.68(1H,d),2.34(2H,m),2.09(2H,m),1.41(6H,s),0.97(6H,m)；¹³C NMR(100MHz,DMSO-d₆)δc:182.0,161.8,160.9,160.4,158.4,156.4,151.7,131.1,127.5,121.2,114.1,110.7,106.7,104.1,102.7,100.3,98.7,78.0,68.6,41.9,28.8,28.8,27.6,27.6,20.0；negative ESI-MS m/z437[M-H]^-。

Compound DI-4: c₂₅H₂₄O₆；¹H NMR(400MHz,DMSO-d₆)δ_H:9.87(1H,s),9.80(1H,s),7.17(1H,d),6.51(1H,d),6.44(1H),6.37(1H),6.21(1H,s),5.68(1H,d),2.34(2H,m),1.46(2H,m),1.42(6H,s),0.97(6H,s)；¹³C NMR(100MHz,DMSO-d₆)δc:182.2,162.0,161.1,160.6,158.6,156.0,151.9,131.3,127.7,121.4,114.3,110.9,106.9,104.4,102.9,100.5,98.9,78.2,68.8,42.1,29.0,29.0,27.8,27.8,20.2；negative ESI-MS m/z419[M-H]^-。

Compound BIV: c₂₅H₂₄O₆；¹H NMR(400MHz,DMSO-d₆)δ_H:13.04(1H,s),7.03(1H,d),6.71(1H,d′),6.41(1H,d),6.29(1H,s),6.20(1H,s),5.74(1H,d),5.03(1H,d),2.98(2H,d),1.54(3H,s),1.40(6H,s),1.33(3H,s)；¹³C NMR(100MHz,DMSO-d₆)δc:182.1,164.5)161.9,161.2,158.3,155.5,151.0,131.1,130.6,129.8,121.7,120.7,116.9,113.9,110.6,108.3,104.1,98.8,93.9,75.7,27.8,27.8,25.9,23.9,17.6；negative ESI-MS m/z419[M-H]^-。

Compound DI-5: c₁₅H₁₂O₇；¹H NMR(400MHz,DMSO-d₆)δ_H:11.94(1H,s),7.18(1H,dd),6.37(1H,d),6.30(1H,dd),5.94(1H,d),5.87(1H,d),5.37(1H,d),4.37(1H,d)；¹³C NMR(100MHz,DMSO-d₆)δc:198.5,167.1,163.7,163.4,159.0,157.5,130.2,114.0,106.8,102.8,100.8,96.2,95.2,78.2,70.5；negative ESI-MS m/z 303[M-H]^-。

Compound DI-6: c₁₅H₁₂O₆；¹H NMR(400MHz,DMSO-d₆)δ_H:12.17(s),7.20(1H,d),6.35(1H,d),6.27(1H,dd),5.87(2H,br s),5.59(1H,dd),2.51(2H,dd)；¹³C NMR(100MHz,DMSO-d₆)δc:197.3,167.1,164.0,163.9,159.0,156.2,128.7,115.8,106.9,102.8,102.1,96.1,95.3,74.3,41.5；negative ESI-MS m/z287[M-H]^-。

Compound BII-1: c₂₅H₂₈O₆；¹H NMR(400MHz,DMSO-d₆)δ_H:12.16(1H,s),9.39(1H,s),7.02(1H,s),6.40(1H,s),5.86(2H,d),5.57(1H,dd),5.24(1H,d),5.07(1H,m),3.12(2H,d),3.23(1H,dd),2.60(1H,dd),2.03(2H,m),1.97(2H,m),1.64(3H,s),1.60(3H,s),1.54(3H,s)；¹³C NMR(100MHz,DMSO-d₆)δc:197.0,166.8,163.7,163.6,156.0,153.7,134.6,130.9,128.1,124.3,123.3,118.3,115.0,102.5,101.8,95.8,95.0,74.2,41.3,41.3,27.4,26.4,25.6,17.7,16.0；negative ESI-MS m/z423[M-H]^-。

Compound BII-2: c₂₆H₃₀O₆；¹H NMR(400MHz,DMSO-d₆)δ_H:12.15(1H,s,OH-5),10.77(1H,s,OH-7),9.66(1H,s,OH-2′),7.12(1H,s,H-6′),6.47(1H,s,H-3′),5.88(1H,dd,J＝4.5,2.1Hz,H-6),5.87(1H,dd,J＝4.5,2.1Hz,H-8),5.62(1H,dd,J＝12.7,3.0Hz,H-2),5.21(1H,m,H-2″),5.07(1H,d,J＝6.9Hz,H-7″),3.74(3H,s,OCH₃-4′),3.23(1H,dd,J＝17.1,12.8Hz,H-3a),3.15(2H,d,J＝7.1Hz,H-1″),2.63(1H,dd,J＝17.1,3.1Hz,H-3b),2.03(2H,m,H-6″),1.97(2H,m,H-5″),1.64(3H,s,H-4″),1.60(3H,s,H-10″),1.53(3H,s,H-9″)；¹³C NMR(100MHz,DMSO-d₆)δc:197.1(s,C-4),167.0(s,C-7),164.0(s,C-5),163.8(s,C-4a),158.1(s,C-4'),154.3(s,C-2'),135.2(s,C-3"),131.1(s,C-8"),128.1(d,C-6'),124.5(d,C-7"),123.2(d,C-2"),120.0(s,C-5'),116.2(s,C-1'),102.1(s,C-8a),99.3(d,C-3'),96.1(d,C-6),95.3(d,C-8),74.2(d,C-2),55.3(q,OCH₃-4'),41.5(t,C-3),39.6(t,C-5"),27.8(t,C-1"),26.6(t,C-6"),25.9(q,C-10"),17.9(q,C-9"),16.2(q,C-4")；negative ESI-MS m/z437[M-H]^-。

Compound DI-7: c₂₅H₂₄O₇(ii) a A yellow powder;¹H NMR(400MHz,DMSO-d₆)δ_H:7.32(1H,s,H-6'),6.98(1H,s,H-14),6.92(1H,d,J＝8.0Hz,H-11),6.80(1H,d,J＝8.0Hz,H-12),6.27(1H,s,H-3'),5.72(1H,d,J＝2.0Hz,H-8),4.59(1H,d,J＝2.0Hz,H-6),4.46(1H,dd,J＝13.0,2.5Hz,H-2),3.72(2H,s,H-3),3.31(6H,s,H-16,17),1.60(3H,s,H-18)；¹³C NMR(100MHz,DMSO-d₆)δc:196.8(s,C-4),167.0(s,C-7),164.1(s,C-5),163.8(s,C-8a),155.4(s,C-2'),153.8(s,C-4'),137.0(s,C-13),135.3(s,C-10),128.2(s,C-9),127.3(d,C-12),122.8(d,C-11),121.4(d,C-6'),121.1(d,C-14),119.2(s,C-1'),114.5(s,C-5'),103.8(d,C-3'),101.9(s,C-4a),95.1(d,C-8),94.9(d,C-6),77.6(s,C-15),74.6(d,C-2),41.8(t,C-3),26.6(q,C-16),26.6(q,C-17),19.9(q,C-18)；negative ESI-MS m/z435[M-H]^-。

compound DI-8: c₂₅H₂₆O₆(ii) a A yellow powder;¹H NMR(400MHz,DMSO-d₆)δ_H:12.19(1H,s,OH-5),9.59(1H,s,OH-2'),7.33(1H,s,H-6'),6.23(1H,s,H-3'),5.96(1H,d,J＝5.5Hz,H-10),5.89(1H,s,H-6),5.80(1H,s,H-8),5.59(1H,dd,J＝13.5,2.5Hz,H-2),3.44(1H,d,J＝6.4,5.5Hz,H-9),2.62(2H,dd,J＝17.0,13.5Hz,H-3),1.90(2H,m,H-13),1.85(1H,m,H-15),1.68(3H,m,H-12),1.36(3H,s,H-17),1.21(2H,m,H-14),1.08(3H,m,H-18)；¹³C NMR(100MHz,DMSO-d₆)δc:197.3(s,C-4),167.0(s,C-7),164.0(s,C-5),163.8(s,C-8a),154.1(s,C-2'),153.0(s,C-4'),134.1(s,C-11),127.9(d,C-6'),123.0(d,C-10),117.4(s,C-1'),115.8(s,C-5'),103.4(d,C-3'),102.1(s,C-4a),96.2(d,C-6),95.3(d,C-8),76.3(s,C-16),74.8(d,C-2),41.0(t,C-3),38.9(d,C-15),31.5(d,C-9),30.2(t,C-13),26.5(q,C-17),25.6(q,C-18),23.7(q,C-12),19.7(t,C-14)；negative ESI-MS m/z421[M-H]^-。

compound DI-9: c₂₅H₂₂O₆；¹H NMR(400MHz,DMSO-d₆)δ_H:12.20(1H,s,OH-5),7.94(1H,s,H-6'),7.62(1H,s,H-6”),7.19(1H,d,J＝8.0Hz,H-3”),7.06(1H,m,H-4”),6.43(1H,s,H-3'),5.93(1H,d,J＝1.0Hz,H-8),5.90(1H,d,J＝1.0H_Z,H-6),5.69(1H,dd,J＝12.0,5.0H_Z,H-2a),3.54(1H,d,J＝5.0H_Z,H-3),2.65(1H,dd,J＝12.0,5.0H_Z,H-2b),2.33(1H,s,H-10”),1.54(2H,s,H-8”,9”).¹³C NMR(100MHz,DMSO-d₆)δc:196.8(s,C-4),166.7(s,C-7),163.5(s,C-5),163.3(s,C-8a),156.1(s,C-4'),153.5(s,C-2'),136.8(s,C-5”),134.9(s,C-2”),127.7(s,C-1”),127.6(d,C-4”),123.3(d,C-3”),122.6(d,C-6'),121.7(d,C-6”),118.4(s,C-5'),113.5(s,C-1'),104.0(d,C-3'),101.6(s,C-4a),95.8(d,C-6),95.0(d,C-8),77.6(s,C-7”),73.6(t,C-2),41.0(d,C-3),27.5(q,C-8”),27.4(q,C-9”),20.8(q,C-10”)；negative ESI-MS m/z417[M-H]^-。

Compound CII: c₃₄H₂₆O₈；¹H NMR(400MHz,DMSO-d₆)δ_H:9.76(1H,s,OH-5′),9.62(1H,s,OH-10″),9.60(1H,s,OH-12″),9.45(1H,s,OH-18″),9.33(1H,s,OH-6),7.42(1H,s,H-4),7.12(1H,s,H-14″),7.08(1H,d,J＝8.4Hz,H-20″),7.04(1H,s,H-3),6.95(1H,d,J＝1.6Hz,H-6′),6.92(1H,d,J＝2.0Hz,H-7),6.84(1H,d,J＝1.6Hz,H-2′),6.76(1H,dd,J＝8.4,2.4Hz,H-5),6.43(1H,dd,J＝8.4,2.4Hz,H-19″),6.35(1H,m,H-2″),6.33(1H,d,J＝2.4Hz,H-17″),6.26(1H,m,H-11″),6.09(1H,m,H-13″),3.30(1H,overlapped,H-5″),3.20(1H,overlapped,H-3″),2.81(1H,dd,J＝17.2,5.2Hz,H-4″),2.67(1H,dd,J＝17.2,5.2Hz,H-6″a),1.93(1H,dd,J＝17.2,5.2Hz,H-6″b),1.75(3H,s,H-7″)；¹³C NMR(100MHz,DMSO-d₆)δc:159.0(s,C-10″),157.3(s,C-18″),157.0(s,C-12″),156.7(s,C-6),156.0(s,C-2),155.5(s,C-5′),153.7(s,C-3′),153.6(s,C-16″),152.3(s,C-7a),133.0(s,C-1″),129.7(s,C-1′),129.4(d,C-14″),127.3(d,C-20″),122.0(d,C-2″),121.4(s,C-3a),121.0(d,C-4),116.2(s,C-4′),115.3(s,C-15″),112.8(d,C-5),112.3(s,C-9″),109.2(d,C-19″),106.1(d,C-13″),104.4(d,C-6′),103.7(d,C-2′),103.6(d,C-17″),102.9(d,C-11″),101.9(s,C-8″),101.3(d,C-3),97.7(d,C-7),35.9(d,C-3″),35.4(t,C-6″),33.9(d,C-5″),27.4(d,C-4″),23.7(q,C-7″)；negative ESI-MS m/z 561[M-H]^-。

Compound CIII: c₃₉H₃₂O₈；¹H NMR(400MHz,DMSO-d₆)δ_H:9.84(1H,s,OH-5′),9.78(1H,s,OH-12″),9.61(1H,s,OH-18″),9.31(1H,s,OH-6),7.39(1H,d,J＝8.4Hz,H-4),7.15(1H,d,J＝8.4Hz,H-20″),7.10(1H,d,J＝8.7Hz,H-14″),7.07(1H,s,H-3),6.98(1H,d,J＝2.2Hz,H-7),6.92(2H,d,J＝1.7Hz,H-2′,6′),6.84(1H,dd,J＝8.4,2.2Hz,H-5),6.74(1H,d,J＝10.0H_Z,H-21″),6.57(1H,dd,J＝8.4,2.5Hz,H-19″),6.41(1H,d,J＝5.4H_Z,H-2″),6.31(1H,d,J＝2.5Hz,H-17″),6.24(1H,d,J＝8.7H_Z,H-13″),3.23(1H,d,J＝5.4H_Z,H-3″),3.18(1H,s,H-4″),2.78(1H,d,J＝5.4Hz,H-5″),2.68(1H,dd,J＝17.0,5.4Hz,H-6″a),1.92(1H,dd,J＝17.0,5.4Hz,H-6″b),1.78(3H,s,H-7″)；¹³C NMR(100MHz,DMSO-d₆)δc:157.5(s,C-5′),157.1(s,C-16″),156.2(s,C-7a),155.7(s,C-6),154.2(s,C-10″),153.8(s,C-3′),153.7(s,C-2),152.2(s,C-18″),151.6(s,C-12″),133.3(s,C-1″),130.0(s,C-1′),129.0(t,C-22″),128.3(d,C-14″),127.5(d,C-20″),121.8(d,C-2″),121.6(d,C-4),121.2(s,C-3a),117.4(s,C-9″),117.2(t,C-21″),116.3(s,C-15″),113.0(d,C-5),112.1(s,C-4′),110.2(s,C-11″),109.4(d,C-19″),107.4(d,C-13″),104.6(d,C-2′),103.7(d,C-6′),103.0(d,C-17″),102.2(d,C-3),101.0(s,C-8″),97.9(d,C-7),75.9(s,C-23″),36.7(d,C-4″),35.8(t,C-6″),33.9(d,C-3″),27.7(q,C-25″),27.5(d,C-5″),27.0(q,C-24″),24.0(q,C-7″)；negative ESI-MS m/z 627[M-H]^-。

Compound CIV: c₃₄H₂₂O₈；¹H NMR(400MHz,DMSO-d₆)δ_H:7.40(1H,d,J＝8.0H_Z,H-20″),7.23(1H,d,J＝8.8H_Z,H-6′),7.20(1H,d,J＝8.0H_Z,H-2″),7.07(1H,d,J＝8.0H_Z,H-4),6.93(2H,d,J＝8.8H_Z,H-6″,3),6.75(1H,d,J＝8.0H_Z,H-2′),6.47(1H,s,H-7),6.43(1H,s,H-5),6.40(3H,d,J＝2.0H_Z,H-11″,17″,19″),6.12(1H,dd,J＝8.0,2.0H_Z,H-14″),6.08(1H,m,H-13″),1.50(3H,s,H-7″)；¹³C NMR(100MHz,DMSO-d₆)δc:160.1(s,C-12″),160.0(s,C-18″),157.5(s,C-10″),157.4(s,C-6),156.5(s,C-7a),155.9(s,C-5′),154.1(s,C-2),153.9(s,C-3′),152.9(s,C-16″),139.7(s,C-1″),133.4(s,C-1′),130.5(d,C-14″),130.0(s,C-3″),129.9(s,C-5″),127.1(d,C-2″),125.5(d,C-20″),122.8(s,C-3a),121.9(d,C-4),121.8(d,C-6″),121.6(s,C-4″),116.7(s,C-9″),115.0(s,C-15″),113.2(d,C-5),112.2(s,C-4′),109.6(d,C-19″),108.7(d,C-6′),108.1(s,C-8″),106.3(d,C-13″),106.2(d,C-2′),103.4(d,C-17″),103.3(d,C-11″),102.7(d,C-3),97.9(d,C-7),24.6(q,C-7″)；negative ESI-MS m/z 557[M-H]^-。

Compound CV: c₃₄H₂₄O₁₀；¹H NMR(400MHz,DMSO-d₆)δ_H:9.23(1H,s,OH-6),7.48(1H,d,J＝8.4H_Z,H-4),7.39(1H,d,J＝8.4H_Z,H-14”),7.12(1H,s,H-3),6.91(1H,s,H-7),6.84(1H,s,H-6'),6.80(1H,dd,J＝8.4,1.5H_Z,H-5),6.75(1H,d,J＝8.7H_Z,H-20”),6.57(1H,s,H-2'),6.53(1H,dd,J＝8.4,1.5Hz,H-13”),6.27(1H,d,J＝2.4H_Z,H-6),6.26(1H,m,H-19”),6.11(1H,d,J＝1.5H_Z,H-11”),3.25(1H,s,H-3”),2.71(1H,s,H-5”),2.56(2H,dd,J＝14.1,3.6H_Z,H-6”),1.66(3H,s,H-7”)；¹³C NMR(100MHz,DMSO-d₆)δc:195.3(s,C-2”),172.1(s,C-12”),166.7(s,C-10”),160.0(s,C-2),159.8(s,C-3′),156.1(s,C-18”),155.6(s,C-7a),155.0(s,C-16”),154.2(s,C-6),154.1(s,C-5'),133.8(d,C-14”),131.3(s,C-1'),126.2(d,C-20”),121.5(s,C-3a),121.2(d,C-4),113.2(s,C-9”),112.9(s,C-15”),112.7(d,C-5),112.0(d,C-19”),109.9(s,C-8”),109.8(s,C-4'),108.3(d,C-6′),104.3(d,C-2'),102.4(d,C-13”),102.0(d,C-3),98.0(d,C-17'′),97.9(d,C-7),97.8(d,C-11”),91.7(s,C-4”),74.8(s,C-1”),48.4(d,C-3”),37.0(d,C-5”),30.56(t,C-6”),22.5(q,C-7”)；negative ESI-MS m/z 591[M-H]^-。

Compound CI-1: c₄₀H₃₆O₁₁；¹H NMR(400MHz,DMSO-d₆)δ_H:13.00(1H,s,OH-5),12.88(1H,s,OH-23),10.50(1H,s,OH-7),10.44(1H,s,OH-25),9.99(1H,s,OH-31),9.77(1H,s,OH-29),8.98(1H,s,OH-2'),8.87(1H,s,OH-4'),7.27(1H,s,H-27),6.56(1H,d,J＝8.4Hz,H-33),6.52(1H,s,H-3’),6.41(1H,dd,J＝8.4,2.0H_Z,H-5’),6.12(1H,d,J＝8.4H_Z,H-32),5.98(1H,s,H-24),5.96(1H,s,H-6),5.92(1H,d,J＝8.8H_Z,H-26),5.10(1H,d,J＝7.2H_Z,H-10),4.23(1H,s,H-14),3.19(1H,s,H-19),3.07(2H,m,H-9),1.83(1H,m,H-18),1.64(3H,s,H-12),1.39(6H,m,H-13,17)；¹³C NMR(100MHz,DMSO-d₆)δc:208.1(s,C-21),181.7(s,C-4),164.6(s,C-23),164.4(s,C-25),161.8(s,C-7),161.2(s,C-4′),160.6(s,C-8a),159.5(s,C-2),156.7(s,C-2′),156.2(s,C-29),155.6(s,C-31),155.1(s,C-5),133.2(s,C-16),132.8(d,C-33),131.6(s,C-11),131.5(d,C-6′),131.4(d,C-27),123.6(d,C-15),122.2(d,C-10),120.0(s,C-28),119.5(d,C-3),114.4(s,C-22),111.7(s,C-1′),107.6(d,C-26),107.1(d,C-27),106.7(d,C-32),104.1(s,C-4a),103.2(d,C-24),102.9(d,C-3′),101.9(d,C-30),97.8(d,C-6),46.2(d,C-20),37.6(t,C-18),37.4(d,C-19),25.9(q,C-12),23.9(t,C-9),23.4(d,C-14),22.9(q,C-17),17.8(q,C-13)；negative ESI-MS m/z 691[M-H]^-。

Compound CI-2: c₄₅H₄₄O₁₁；¹H NMR(400MHz,DMSO-d₆)δ_H:13.37(1H,s,OH-5),12.87(1H,s,OH-23),7.11(1H,d,J＝8.0H_Z,H-6'),7.10(1H,d,J＝8.0H_Z,H-27),6.57(1H,d,J＝8.0H_Z,H-33),6.56(1H,d,J＝2.0H_Z,H-3'),6.52(1H,d,J＝2.8H_Z,H-5'),6.40(1H,d,J＝2.0H_Z,H-30),6.13(1H,d,J＝8.0H_Z,H-26),5.98(1H,d,J＝2.8Hz,H-32),5.95(1H,s,H-6),5.11(1H,m,H-10),5.10(1H,m,H-15),5.02(1H,m,H-35),4.23(1H,m,H-14),4.17(1H,m,H-20),3.19(2H,d,J＝7.0H_Z,H-9),3.14(2H,d,J＝2.0H_Z,H-34),1.84(2H,s,H-18),1.63(6H,s,H-37,38),1.57(3H,s,H-17),1.45(6H,s,H-12,13)；¹³C NMR(100MHz,DMSO-d₆)δc:208.6(s,C-21),182.1(s,C-4),162.4(s,C-23),162.0(s,C-7),161.2(s,C-25),160.8(s,C-4′),160.6(s,C-8a),159.0(q,C-2),156.8(s,C-2′),156.3(s,C-5),155.5(s,C-29),155.0(s,C-31),132.2(s,C-16),131.6(s,C-11),130.8(d,C-33),130.7(s,C-36),129.7(d,C-6'),123.6(d,C-15),122.6(d,C-27),122.2(d,C-35),122.0(s,C-28),120.0(d,C-10),119.5(d,C-3),114.1(s,C-22),113.8(s,C-24),111.8(s,C-1′),107.2(s,C-8),107.0(d,C-26),106.9(d,C-32),106.8(d,C-5'),104.1(s,C-4a),103.2(d,C-30),103.0(d,C-3'),97.8(d,C-6),45.9(d,C-20),37.3(t,C-18),37.2(d,C-19),25.9(q,C-12),25.8(q,C-37),23.9(t,C-9),23.0(d,C-14),21.7(q,C-17),21.6(t,C-34),18.0(q,C-13),17.8(q,C-38)；negative ESI-MS m/z 759[M-H]^-。

Compound DI-10: c₃₅H₃₀O₁₁；¹H NMR(400MHz,DMSO-d₆)δ_H:13.03(1H,s,OH-5),12.18(1H,s,OH-10″),9.06(1H,s,OH-7),8.86(1H,s,OH-12″),8.27(1H,s,OH-4″),7.58(1H,d,J＝9.0H_Z,H-14″),6.95(1H,d,J＝9.0H_Z,H-6′),6.72(1H,d,J＝9.0H_Z,H-20″),6.13(3H,m,H-6,8,5′),5.90(4H,m,H-11″,13″,17″,19″),5.61(1H,dd,J＝13.0,3.0H_Z,H-2),5.26(1H,s,H-2″),4.32(1H,m,H-4″),3.44(3H,m,H-3″,5″,3),3.08(1H,m,H-6″a),2.09(1H,m,H-6″b),1.69(3H,m,H-7″)；¹³C NMR(100MHz,DMSO-d₆)δc:208.6(s,C-8″),197.0(s,C-4),166.5(s,C-7),164.2(s,C-5),164.1(s,C-8a),163.6(s,C-10″),163.5(s,C-12″),156.0(s,C-18″),155.6(s,C-16″),153.4(s,C-4′),152.3(s,C-2′),136.4(d,C-20″),132.7(s,C-1″),132.4(d,C-14″),130.9(d,C-6′),125.5(d,C-2″),125.1(d,C-4″),124.7(s,C-15″),117.1(s,C-1′),116.9(s,C-9″),108.0(s,C-3′),107.3(d,C-13″),106.9(d,C-19″),106.9(d,C-5′),105.9(d,C-11″),102.6(s,C-4a),101.7(d,C-17″),95.8(d,C-6),95.1(d,C-8),73.8(d,C-2),42.2(t,C-3),41.9(t,C-6″),37.7(d,C-5″),37.6(d,C-3″),23.1(q,C-7″)；negative ESI-MS m/z625[M-H]^-。

Compound CVI: c₄₀H₃₈O₁₁；¹H NMR(400MHz,DMSO-d₆)δ_H:13.09(1H,s,OH-5),12.23(1H,s,OH-10″),9.37(1H,s,OH-7),9.12(3H,s,OH-2′,4′,12″),8.88(1H,s,OH-18″),7.32(1H,d,J＝9.0H_Z,H-14″),7.02(1H,d,J＝9.0H_Z,H-6′),6.73(1H,d,J＝9.0H_Z,H-20″),6.13(2H,dd,J＝9.0,2.5H_Z,H-17″,13″),6.04(1H,d,J＝9.0Hz,H-5′),5.96(1H,dd,J＝9.0,2.5H_Z,H-19″),5.93(3H,d,J＝2.0H_Z,H-2,6,8),5.55(1H,s,H-2″),5.22(1H,d,J＝6.0H_Z,H-2″′),5.15(2H,d,J＝10.0H_Z,H-4″,3″),4.15(1H,m,H-5″),3.19(2H,m,H-6″),2.10(1H,d,J＝3.18H_Z,H-3),2.02(3H,s,H-7″),1.63(3H,s,H-4″′),1.56(3H,s,H-5″′)；¹³C NMR(100MHz,DMSO-d₆)δc:208.7(s,C-8″),196.9(s,C-4),166.9(s,C-7),164.5(s,C-8a),164.3(s,C-5),163.9(s,C-12″),163.8(s,C-10″),156.2(s,C-18″),155.9(s,C-16″),155.5(s,C-2′),153.3(s,C-4′),136.5(s,C-1″),133.7(d,C-20″),131.1(d,C-14″),125.4(s,C-3″′),125.3(d,C-6′),124.4(d,C-2″),121.1(s,C-15″),120.7(d,C-2″′),115.7(s,C-1′),115.5(s,C-9″),114.3(s,C-11″),107.4(s,C-3′),107.4(d,C-19″),106.2(d,C-13″),106.1(d,C-5′),102.2(s,C-4a),102.1(d,C-17″),96.0(d,C-6),95.3(d,C-8),73.8(d,C-2),48.9(d,C-4″),41.9(t,C-3),37.2(d,C-3″),37.1(d,C-5″),37.0(t,C-6″),26.2(t,C-1″′),25.8(q,C-7″),18.8(q,C-5″′),17.9(q,C-4″′)；negative ESI-MS m/z 693[M-H]^-。

Example 2

Active composition derived from ramulus mori

(1) Crushing 10g of branches of a dry plant mulberry Morus alba into particles with the particle size of 100-300 meshes, adding 80mL of 95% ethanol by volume, heating to 50 ℃, performing ultrasonic extraction for 0.5h, and filtering to obtain an extracting solution and filter residues; repeatedly extracting the filter residue with ultrasound for 3 times, mixing extractive solutions, and concentrating under reduced pressure to constant weight to obtain extract (1.05 g); and (3) sectionally separating the extract by adopting macroporous adsorption resin D101, eluting by using ethanol-water mixed solutions with the volume fractions of 1L of ethanol being 0%, 20%, 40%, 60%, 80% and 95% in sequence respectively to obtain an MA-0 component, an MA-2 component, an MA-4 component, an MA-6 component, an MA-8 component and an MA-10 component in sequence respectively.

(2) The MA-0 fraction, MA-2 fraction, MA-4 fraction, MA-6 fraction, MA-8 fraction and MA-10 fraction were subjected to an anti-MRSA activity test in accordance with the procedure of step (2) of example 1, and the result of the test was that the MA-6 fraction had an anti-MRSA activity.

(3) Carrying out sixth column chromatographic separation on the MA-6 component to obtain a compound AI-3, a compound AII, a compound BIV, a compound CV and a compound CVI; the elution mode of the sixth column chromatographic separation is gradient elution, an eluant adopted by the gradient elution is a chloroform-methanol mixed solvent, the volume ratio of chloroform to methanol in the mixed solvent is 1: 0-0: 1, and the sixth column chromatographic separation adopts a silica gel packed column (1 multiplied by 60cm, the particle size of silica gel is 200-300 meshes).

The mass percentage of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV in the MA-6 component are respectively 5.2 percent, 7.8 percent, 12.4 percent, 1.2 percent and 14.5 percent.

Example 3

Active composition derived from mulberry leaves

The active composition was extracted according to the method of example 2, except that the branches of the plant Morus alba were replaced with the leaves of the plant Morus alba, and the mass percentages of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV in the MA-6 fraction were 7.5%, 8.2%, 12.0%, 3.4% and 15.7%, respectively.

Example 4

Active composition derived from mulberry

The active composition was extracted according to the method of example 2, which differs from example 3 in that the branches of the plant Morus alba were replaced with mature ears of the plant Morus alba, and the mass percentage contents of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV in the MA-6 fraction were 4.1%, 2.3%, 6.3%, 1.2% and 10.1%, respectively.

Example 5

Active composition derived from cortex Mori

The active composition was extracted according to the method of example 2, which differs from example 3 in that the branches of the plant Morus alba were replaced with mature ears of the plant Morus alba, and the mass percentage contents of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV in the MA-6 fraction were respectively 11.1%, 13.0%, 7.9%, 17.5% and 13.9%, respectively.

Example 6

Cortex Mori traditional Chinese medicine compound-cortex Mori soup

Weighing 20g of white mulberry root-bark, 5g of pinellia ternate, 5g of perilla fruit, 5g of bitter almond, 5g of thunberg fritillary bulb, 5g of scutellaria baicalensis, 5g of coptis chinensis and 5g of gardenia, crushing the materials into particles with the particle size of 100-300 meshes, adding 500mL of ethanol with the volume fraction of 95%, heating the mixture to 50 ℃, performing ultrasonic extraction for 0.5 hour, and filtering the mixture to obtain an extracting solution and filter residues; carrying out ultrasonic extraction on the filter residue for 3 times, combining extracting solutions, and concentrating under reduced pressure to constant weight to obtain a pharmaceutical composition (7.39 g); wherein, the mass percentage of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV in the active composition are respectively 0.5 percent, 0.4 percent, 1.2 percent, 0.7 percent and 2.0 percent in sequence.

Example 7

Mulberry twig Chinese medicine compound-mulberry twig decoction

Weighing 20g of white mulberry root-bark and 6g of motherwort, crushing the white mulberry root-bark and the motherwort into particles with the particle size of 100-300 meshes, adding 300mL of 95% ethanol by volume fraction, heating the mixture to 50 ℃, performing ultrasonic extraction for 0.5h, and filtering the mixture to obtain an extracting solution and filter residues; carrying out ultrasonic extraction on the filter residue for 3 times, combining the extracting solutions, and concentrating under reduced pressure to constant weight to obtain a pharmaceutical composition (2.9 g); wherein, the mass percentage of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV in the pharmaceutical composition are respectively 5.7 percent, 6.7 percent, 13.8 percent, 4.5 percent and 15.8 percent in sequence.

Example 8

Mulberry twig Chinese medicine compound-mulberry twig decoction

Weighing 20g of white mulberry root-bark and 6g of motherwort, crushing the white mulberry root-bark and the motherwort into particles with the particle size of 100-300 meshes, adding 300mL of 95% ethanol by volume fraction, heating the mixture to 50 ℃, performing ultrasonic extraction for 0.5h, and filtering the mixture to obtain an extracting solution and filter residues; carrying out ultrasonic extraction on the filter residue for 3 times, combining the extracting solutions, and concentrating under reduced pressure to constant weight to obtain a pharmaceutical composition (2.9 g); wherein, the mass percentage of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV in the pharmaceutical composition is respectively 0.5 percent, 0.4 percent, 1.2 percent, 0.7 percent and 2.0 percent.

Example 9

Preparation of medicinal composition powder

The active compound compositions of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV prepared in the examples 1 to 4 were mixed with an excipient at a mass ratio of 9:1, respectively, to obtain a pharmaceutical composition powder.

Example 10

Preparation of pharmaceutical composition tablets

The active compound compositions of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV prepared in the examples 1 to 4 and the excipient are respectively mixed according to the mass ratio of 1: 5-1: 10, and the mixture is granulated and tableted to obtain the pharmaceutical composition tablet.

Example 11

Preparation of pharmaceutical composition capsule

The active compound compositions of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV prepared in the examples 1 to 4 and the excipient are mixed according to the mass ratio of 5:1 and then are encapsulated to obtain the pharmaceutical composition capsule.

Example 12

Preparation of pharmaceutical composition granules

The active compound compositions of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV prepared in the examples 1 to 4 and the excipient are mixed according to the mass ratio of 5:1 and then granulated to obtain the pharmaceutical composition granules.

Example 13

Preparation of medicinal composition granule

The active compound compositions of the compound AI-3, the compound BIV, the compound CVI, the compound AII and the compound CV prepared in the examples 1 to 4 and the excipient are mixed according to the mass ratio of 5:1 and then granulated to obtain the medicinal composition granules.

Test example 1

The effect of the active compounds and the positive control drugs Kanamycin (Kanamycin), Ampicillin (ampicilin), Methicillin (Methicillin) and Berberine (Berberine) on pathogenic microorganisms.

The test method comprises the following steps: CLSI M100 antibacterial drug susceptibility test execution standard 2019, which comprises the following steps:

pathogenic microorganisms: MRSA-031, MRSA-011, MRSA-003, and S.aureusATCC4330.

1g of the MA-6 fraction, compound AI-1, compound AI-2, compound AI-3, compound AII, compound AIII, compound BI-1, compound BI-2, compound BIII, compound BIV, compound BII-1, compound BII-2, compound CII, compound CIII, compound CIV, compound CV, compound CI-1, compound CI-2, compound CVI, Kanamycin, Ampicillin, Methicillin and Berberine in step (1) of example 2 was dissolved in Phosphate Buffered Saline (PBS) at pH 7.4, respectively, to obtain drug solutions to be tested.

In a 96-well cell culture plate, the drug solution to be tested was added in different volumes to a final concentration of 0.1. mu.L, TSB medium was added to 49. mu.L per well, and then 50. mu.L of pathogenic microorganism suspension (1X 10)⁷CFU/mL), final volume 100 μ L, same concentration of DMSO added as a control group, and three wells repeated per group. Place 96-well plates in 5% CO₂After culturing for 24h at 37 ℃ in a constant temperature incubator, the absorbance is measured by an enzyme-labeling instrument OD600nm, and the test is repeated three times. Operating according to CLSI 2019 standard. The MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) test results of the drugs to be tested are shown in table 1.

TABLE 1 Effect of active Compounds and Positive control drugs on pathogenic microorganisms

As can be seen from Table 1, the anti-MRSA activity of the compound AI-2, the compound AI-3, the compound AII, the compound AIII, the compound BI-2, the compound BIII, the compound BIV, the compound BII-1, the compound BII-2, the compound CII, the compound CIII, the compound CIV, the compound CV, the compound CI-2 and the compound CVI is better than that of Methicillin and Berberine, and the active compounds provided by the invention have good anti-pathogenic bacteria activity.

Test example 2

Effect of Compound AI-3(Albafurana), Compound AII (Moracin G), Compound BIV (Kuwanon B), Compound CV (Mulberrofuran Q) and Compound CVI (Kuwanon O) on MRSA-011

The test method comprises the following steps: CLSI M100 antibacterial drug susceptibility test execution standard 2019, which comprises the following steps:

the MA-6 fraction, albafuran a, Moracin G, Kuwanon b, Mulberrofuran Q, and Kuwanon O in step (1) of example 2 were dissolved in Phosphate Buffered Saline (PBS) at pH 7.4, respectively, to obtain drug solutions to be tested. Adding different volumes of the mixture into a 24-hole cell culture plate respectivelyThe final concentrations of the drug solutions to be tested were 1 × MIC (minimum inhibitory concentration), 2 × MIC, and 4 × MIC, respectively, TSB medium was added to each well to 0.9mL, and then 100 μ L of pathogenic microorganism suspension (1 × 10)⁷CFU/mL), final volume 1mL, DMSO at the same concentration was added as a control group, and each group was repeated three wells. After culturing the 24-well plate in a constant temperature incubator at 37 ℃, respectively culturing the plate in 0, 1, 2, 3, 4, 6, 12, 16, 20 and 24 hours, taking out 20 mu L of culture solution from each well, diluting the culture solution to different times by using sterile PBS solution, coating the diluted culture solution on TSA solid culture medium, culturing for a certain time, recording the number of growing colonies under the appropriate dilution times, wherein the unit is log10(CFU/mL), and repeating the test for three times, wherein the time-sterilization curve results of the MA-6 component, the compound AI-3, the compound AII, the compound BIV, the compound CV and the compound CVI are shown in figure 2. As can be seen from FIG. 2, compound AI-3, compound BIV and compound CVI inhibit the growth of MRSA-011, and are dose-dependent, the higher the compound concentration is, the better the sterilization effect is, the sterilization rate is rapidly increased, the rapid sterilization effect is realized, the action effect is in dose-effect relationship, and the compound belongs to the sterilization type compound; the compound AII and the compound CV have the fastest sterilization rate within 6h, the sterilization rate is gradually reduced after 6h, and the sterilization effect is continuously achieved, but when the concentration is increased to 4 XMIC, the sterilization rate is increased, and the bacterial number is rapidly reduced after 8h, so that the compound AII and the compound CV can be judged to have time dependence on the inhibition of the growth of MRSA-011, the slow sterilization effect is achieved, and the compound is a bacteriostatic compound.

Test example 3

Effect of Compound CVI on the extracellular biofilm of pathogenic bacteria

Respectively incubating the MRSA-011 extracellular biomembrane structure with compound CVI with different densities (1 × MIC and 2 × MIC) and positive control drug Ampicillin, observing the MRSA extracellular structure by a scanning electron microscope, and observing the anti-biomembrane structure potential of the compound CVI in visualization. The results of the effect of compound CVI on MRSA-011 are shown in FIG. 3, in which A1-A3 are respectively the control, compound CVI with 1 × MIC concentration, and scanning electron microscope image after the effect of compound CVI with 2 × MIC concentration, and B1-B3 are respectively the control, Ampicillin with 1 × MIC concentration, and scanning electron microscope image after the effect of Ampicillin with 2 × MIC concentration. As can be seen from FIG. 3, the biomembrane treated by the compound CVI shows only a few small and sparse colonies under a scanning electron microscope and has no recognizable complete biomembrane structure, while the treatment of the positive drug Ampicillin reduces the aggregation of some bacteria but still has a clear complete structure, which indicates that the active compound provided by the invention has better anti-pathogenic bacteria activity.

Test example 4

EXAMPLE 5 Effect of MA-6 fraction obtained in step (1) on pathogenic bacteria infection model mice

Establishing an MRSA-011 infection model of Kunming mouse abdominal cavity, performing gastric lavage treatment on the mouse by using PBS solution (40mg/kg) of active MA-6 component and PBS solution (50mg/kg) of Berberine Hydrochloride (Berberine Hydrochloride), testing colony counts in Spleen (Spleen), Liver (Liver) and Kidney (Kidney) after continuously using the medicine for 5 days, and testing the result that the MA-6 component and the Berberine Hydrochloride effectively inhibit pathogenic bacteria in the mouse body as shown in figure 4, wherein P is less than 0.05. As can be seen from FIG. 4, the active composition derived from cortex Mori has antibacterial activity against MRSA-011 infection model mice, reduces lethality (Mortality) of mice infected with acute drug-resistant bacteria, and has stronger in vivo antibacterial effect than the positive control drug, berberine hydrochloride (P < 0.05).

Test example 5

Determination of post-contact Effect on MRSA

The test was carried out in accordance with the method of test example 2, and the drugs to be tested were compound AI-1(Moracin), compound BI-1(Kuwanon C), compound CI-1(Kuwanon G) and the MA-6 fraction obtained in step (1) of example 5.

The results of the determination of the effects of the AI-1 compound, the BI-1 compound, the CI-1 compound and the MA-6 compound on the exposure to MRSA-011 are shown in FIG. 5. As can be seen from FIG. 5, compound AI-1 is effective in killing bacteria for a long period of time, and the antibacterial activity of compound AI-1 has mild persistence.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.