CN113244203A

CN113244203A - 9, 10-dihydrophenanthrenes and their use for the treatment of liver injury

Info

Publication number: CN113244203A
Application number: CN202010087201.5A
Authority: CN
Inventors: 叶阳; 潘国宇; 姚胜; 薛亚茹; 邓强强; 唐春萍; 彭兆亮; 柯昌强
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2020-02-11
Filing date: 2020-02-11
Publication date: 2021-08-13
Also published as: WO2021160139A1

Abstract

The invention provides a class of 9,10-dihydrophenanthrene compounds and application thereof in treatment of liver injury, and particularly provides a class of compounds shown as a formula I or a formula II, which show anti-inflammatory and antioxidant activities in liver tissues, so that the compounds can be used for treatment or prevention of liver injury diseases, particularly liver injury diseases with inflammation and lipid peroxidation as pathological bases.

Description

9, 10-dihydrophenanthrenes and their use for the treatment of liver injury

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a novel 9,10-dihydrophenanthrene compound, a preparation method thereof and application thereof in medicaments for treating liver injury.

Background

The liver not only participates in the storage of energy and the synthesis and catabolism of various substances, including glycometabolism, protein metabolism, fat metabolism, etc., but also participates in the detoxification process of toxins and drug catabolites produced by human metabolism. The liver, as the "center of mass metabolism", is also an organ that is often attacked by various pathogenic factors or diseases, such as drugs, viruses, alcohol, abnormal metabolism, ischemia-reperfusion, etc., all can cause damage to the liver and cause inflammatory reaction. Acute and chronic hepatitis eventually turns into liver fibrosis, cirrhosis and hepatocellular carcinoma. The incidence of primary liver cancer is high in the world, and is mainly related to the high incidence of acute and chronic hepatitis.

At present, the types of common liver-protecting medicines in clinic are various, wherein the use frequency of the injection is the highest, such as magnesium isoglycyrrhizinate injection, reduced glutathione for injection, adenosine methionine sulfosuccinate for injection and polyene phosphatidyl choline injection. However, the price of the medicine is high, and the medicine is not suitable for long-term administration. The oral preparation has the advantages of low price, convenient use, stable curative effect and relatively less adverse reaction, and is suitable for long-term administration by patients; however, the existing oral drugs commonly used in clinic have a plurality of defects, such as low bioavailability, large individual difference and the like of silymarin, and the bicyclol has adverse reaction and drug withdrawal and rebound phenomena. Therefore, the development of oral liver-protecting medicaments with definite curative effect, safety and high efficiency has important significance for treating clinical acute and chronic liver injury.

The natural product is an important source for creating new drugs, and the discovery of active lead compounds from the natural product and the development of the new drugs are proved to be effective important ways. The 9,10-dihydrophenanthrene compound is a compound commonly existing in plants of orchidaceae, juncaceae and the like, has more than 2 benzene rings in the structure, contains a plurality of phenolic hydroxyl groups, and has aromatic property and certain hydrophobicity. The existing research shows that the compounds generally have the biological activities of resisting tumor, relieving spasm, resisting platelet aggregation, resisting allergy and the like, but the protection effect of the compounds on liver injury is not reported in documents.

Disclosure of Invention

The invention aims to provide a 9,10-dihydrophenanthrene compound or a derivative thereof which can be used for protecting liver injury.

In a first aspect of the present invention, there is provided a use of a compound represented by the following formula I, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein the content of the first and second substances,

the dotted line represents a chemical bond or nothing;

R_a、R_band R_cEach independently selected from the group consisting of: H. hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl, -CHO, -NO₂；

n, p are each independently selected from the group consisting of: 0.1, 2, 3 or 4;

m is selected from the group consisting of: 0.1 or 2;

R_dis C2-C4 alkenyl, or- (O)_x-R; wherein x is 0 or 1, and R is a structural fragment formed by losing one hydrogen atom from a structural unit selected from the group consisting of: substituted or unsubstituted

Substituted or unsubstituted, substituted or unsubstituted

Substituted or unsubstituted

Or R_dAnd R_a、R_bAnd R_cAny of which and the carbon atoms to which each is attached together form a group selected from: a substituted or unsubstituted 3-7 membered heterocyclic group, a substituted or unsubstituted 3-7 membered cycloalkyl group;

unless otherwise specified, said substitution is by one or more substituents selected from the group consisting of: H. hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl, -CHO, -NO₂Or phenyl substituted with one or more groups selected from: hydroxy, C1-C4 alkyl, C1-C4 alkoxy;

the preparation method is characterized by being used for preparing a pharmaceutical composition for treating or preventing liver injury.

In another preferred embodiment, said R is a group selected from the group consisting of: substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

Substituted or unsubstituted

In another preferred embodiment, R is_dSubstituted with one or more substituents selected from the group consisting of: H. hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl.

In another preferred embodiment, the compound has a structure as shown in formula I-a or I-b below:

in another preferred embodiment, the compound is selected from the group consisting of:

in a second aspect of the present invention, there is provided a use of a compound represented by the following formula II, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein the dotted line represents a bond or nothing;

R_dand R_eEach independently selected from the group consisting of: H. hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl, -CHO, -NO₂Or substituted or unsubstituted benzyl;

q, r are each independently selected from the group consisting of: 0.1, 2, 3 or 4;

in a third aspect of the invention there is provided the use of a compound according to the first or second aspect of the invention, wherein the liver damage is selected from the group consisting of: liver damage with symptoms of cholestasis, or hepatitis.

In another preferred embodiment, the hepatitis is selected from the group consisting of: toxic hepatitis (drug, chemical and biotoxin), alcoholic hepatitis, and ischemic hepatitis.

In another preferred embodiment, the liver injury is drug-induced liver injury, preferably, the liver injury is Chinese herbal medicine-induced liver injury.

In a fourth aspect of the present invention, there is provided a compound selected from the group consisting of:

it is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. The description is not repeated here, but is limited to the drawings.

Drawings

FIG. 1: YS30 has protective effect on mouse CCl4 acute liver injury. Levels of (A) ALT (B) AST and C (C) LDH in mouse serum (D) liver tissue H&And E, pathological analysis after staining. Wherein the boxes, arrows and solid triangles represent liver tissue swelling, central lobular necrosis and inflammatory infiltration, respectively. (E) H&E staining Suzuki's score.^###P<0.001，^##P<0.01CCl4 group vs. vehicle group; p<0.05,**P<0.01,***P<0.001vs. CCl4 group.

FIG. 2:the effect of YS30 on the level of inflammatory factors and liver lipid peroxidation in serum. Serum (A) TNF- α (B) IL-6 levels (C) lipid peroxidation levels in liver tissue.^###P<0.001，^##P<0.01 vs. Vehicle group, P<0.001，**P<0.01，*P<0.01vs CCl4 group.

FIG. 3: YS30 protective effect on ischemia reperfusion liver injury of mice. Serum of mouse (A) ALT, (B) AST, (C) LDH level, (D) liver tissue H&And E, pathological analysis after staining. The boxes, arrows, and solid three corners indicate liver tissue swelling, central leaflet necrosis, and inflammatory infiltration, respectively. (E) H&E staining Suzuki's score.^###P<0.001，^##P<0.01I/R group vs. Sham group; p<0.05,**P<0.01,***P<I/R group at 0.001vs.

Detailed Description

The inventor has conducted extensive and intensive studies for a long time, and unexpectedly found that the 9,10 dihydrophenanthrene compounds disclosed by the invention have good anti-inflammatory and antioxidant properties, and are particularly suitable for treating diseases based on inflammation and lipid peroxidation, such as liver diseases, including toxic hepatitis (drugs, chemical substances and biotoxins), alcoholic hepatitis, ischemic hepatitis and the like. Based on the above findings, the inventors have completed the present invention.

Term(s) for

As used herein, the term "C₁-C₆Alkyl "or" C₁-C₁₀Alkyl "means a straight or branched chain alkyl group having 1 to 6 or 1 to 10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

The term "C₃-C₆Cycloalkyl "means a cycloalkyl group having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, methylcyclobutyl, cyclopentyl, or the like.

The term "halogen" refers to F, Cl, Br and I.

As used herein, the terms "comprising," "including," or "including" mean that the various ingredients may be used together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.

In the present invention, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio.

In the present invention, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the condition, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation and can be determined by a clinician.

Herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on a group are replaced with a substituent selected from the group consisting of: halogen, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C₂-C₆Acyl, unsubstituted or halogenated C1-C6 alkyl-hydroxy.

Unless otherwise specified, all occurrences of a compound in the present invention are intended to include all possible optical isomers, such as a single chiral compound, or a mixture of various chiral compounds (i.e., a racemate). In all compounds of the invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

As used herein, the term "compounds of the invention" refers to compounds of formula I. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula I.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed with acids from the compounds of the present invention. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

As used herein, the term "dimer" refers to a dimer of dibenzyl or stilbenes, 9,10-dihydrophenanthrene or phenanthrene by itself or in combination with other dibenzyl or stilbenes, 9,10-dihydrophenanthrene or phenanthrene via a C-O-C, or C-C, or both C-O-C and C-C linkage.

9, 10-dihydrophenanthrenes or their analogs

In the earlier stage of the project, a series of 9,10-dihydrophenanthrene compounds are separated from stonecrop (Pholidota chinensis Lindl.) of the genus stonecrop of the family orchidaceae, Juncus effusus L of the genus Juncus of the family juncaceae, and Juncus effusus L of the genus Juncus. Then, the liver protection effect of the compound is verified in an endogenous toxic substance (hydrophobic cholic acid) liver cell injury model, an exogenous toxic substance (carbon tetrachloride) mouse liver injury model and an ischemia-reperfusion mouse liver injury model.

In rat primary hepatocytes and human-derived induced liver-like cells (hiHep cells), we found that the compounds are capable of significantly counteracting the liver cell damage caused by the hydrophobic cholic acid (DCA) and LCA at concentrations ranging from 0.1 to 25uM, suggesting that the compounds may have a significant ameliorating effect on a type of liver damage accompanied by symptoms of cholestasis, such as liver damage caused by drugs, particularly Chinese herbal medicines.

The 9,10-dihydrophenanthrene compound or the analogue thereof obtained by separation comprises the following components in part by weight:

the first type: YS 1-37 separated from pholidota chinensis

The second type: XYW 1-10 of a compound separated from juncus effuses:

in the third category: XF 1-11, 14-20, 24, 29, 33 compounds separated from rush:

pharmaceutical compositions and methods of administration

Because the compound has excellent activity of reversing liver injury, the compound, various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof and a pharmaceutical composition containing the compound as a main active ingredient can be used for treating, preventing and relieving various diseases related to liver injury, such as hepatitis taking inflammation and lipid peroxidation as pathological bases.

The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof in a safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-3000 (active dose range 1-100 mg/kg) mg of the compound of the invention per dose, more preferably, 10-2000mg of the compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of being blended with the compounds of the present invention and with each other without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers

Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, especially cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically effective administration dose, and the daily administration dose for a human body with a weight of 60kg is usually 1-2000 mg, preferably 6-600 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Compared with the prior art, the invention has the main advantages that:

1. the discovery discovers for the first time that 9,10-dihydrophenanthrene and dibenzyl (stilbenes) have good liver protection activity, and the damage to primary hepatocytes or human-derived induced liver-like cells induced by hydrophobic cholic acid can be resisted within a lower concentration range. Compared with the existing liver-protecting drugs, the liver-protecting drug has the greatest advantages of oral administration and obvious dose dependence.

2. In addition to combating hydrophobic bile acid induced liver damage, the active compounds of the invention also have antioxidant and anti-inflammatory activity, potentially protective against hepatitis characterized by oxidative stress and inflammation, such as toxic hepatitis (drugs, chemicals and biotoxins), alcoholic hepatitis, ischemic hepatitis, and have been demonstrated in animal tests.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for which specific conditions are not noted in the following examples are generally carried out under conventional conditions or conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

The instruments and materials used in the examples are as follows:

specific optical rotation: rudilph Autopol VI autosopter;

ultraviolet spectrum: shimadzu UV-2550 type ultraviolet spectrometer;

circular dichroism spectrum: JASCO J-810 spectropolarimeter;

mass spectrum: waters 3100SQDMS (low resolution ESI), Waters Xevo QTof MS (high resolution ESI);

nuclear magnetic resonance spectroscopy: a Varian Mercury-plus 400 NMR spectrometer, a Varian MR-400 NMR spectrometer, a Bruker Avance III 500 NMR spectrometer, delta (ppm) using TMS as an internal standard;

MCI resin: CHP20P (75-150 μm), Mitsubishi Chemical Corporation;

column chromatography silica gel: 200-mesh 300-mesh silica gel, 300-mesh 400-mesh silica gel and silica gel H are produced in Qingdao ocean factories;

TLC prefabricated thin-layer plate: HSGF254 is produced in a tobacco bench chemical plant;

and (3) gelling the glucan: sephadex LH-20: pharmacia Biotech AB, Uppsala, Sweden;

high performance liquid chromatography and mass spectrometer: waters 2695LC coupling Waters 2998DAD, Waters Acquity ELSD, Waters 3100SQDMS, analytical column model: waters

RP C-18， 3.5μm，4.6mm×100mm；

Chiral analytical HPLC: JASCO 2000, Daicel columns, 2-propanol/hexane elution;

preparative HPLC: Agilent-Varian SD1 LC coupling Grace-Alltech 3000ELSD, Varian M701 Fraction Collector, preparation of chromatographic column Waters

RP C-18，5μm，19mm×150 mm，Waters

RP C-18, 5 μm, 30 mm. times.150 mm, flow rate 10.0-25.0mL/min, CH3CN (national drug group), H2O (Milli-Q pure water system);

the color development detection method comprises the following steps: 10% vanillin sulfate solution, UV254 and UV365 UV lamps;

solvents for the experiments: analytical grade solvents (national drug group), chromatographic grade solvents (Merck KGaA, Germany); experimental animals: male ICR mice, weighing 22-25g, male SD rats, weighing 200-250 g. Animals were purchased from shanghai slaike laboratory animals ltd;

experimental cells: the L929 cells were from the cell bank of the Shanghai academy of sciences of Chinese academy. The hihep cells are transdifferentiated human hepatocytes, which are gifted by health teachers who are available from biochemical cells of the Chinese academy of sciences. BMDM cells were isolated from ICR mouse bone marrow, rat primary hepatocytes from SD rats;

experimental reagent: DPPH radicals are available from shanghai mclin biochemistry science and technology limited; olive oil, formaldehyde solution, 75% alcohol, absolute ethanol from chemical agents ltd; DMSO, vitamin C, silymarin, resveratrol, dexamethasone, Lipopolysaccharide (LPS) from Sigma-Aldrich; bicyclol is purchased from China pharmaceutical biologies institute; 1 × EBSS buffer, William' E medium, streptomycin, Fetal Bovine Serum (FBS), insulin-transferrin-selenium supplement (ITS), 10 × PBS from Gibco; high-glucose DMEM medium was purchased from Hyclone; RIPA lysate and NO detection kit are purchased from Shanghai Bin Yuntian biotechnology limited company; mouse TNF alpha and IL-6ELISA kits were purchased from Hangzhou Union biotechnology, Inc.; ALT, AST and LDH blood biochemical detection kits are purchased from Hessemetkang Biotech Co., Ltd; type IV collagenase was purchased from shanghai dingsheng biotechnology limited; percoll centrifugate was purchased from GE; the CCK8 kit was purchased from Shanghai assist saint Biotech Co., Ltd;

surgical instruments: ophthalmic scissors, ophthalmic tweezers, a 200-mesh/300-mesh screen and a 1ml injector, wherein the scissors, the tweezers and the screen need to be sterilized and dried in advance;

an experimental instrument: low temperature high speed centrifuge (Eppendorf, 5415R), cell culture box (Thermo Fisher Scientific,371), ultrapure water preparation system (Pall Cascada), inverted fluorescence microscope (Olympus CKX53), ultrasonic cell disruptor (Shanghai Bianyi, SKSI), multifunctional enzyme labeling instrument (Bio Tek Synergy 4), full automatic blood biochemical analyzer (SYSMEX JCA-BM 6010C).

Isolation and purification of the Compound of example 1 and spectroscopic data

1. Separation of 9,10-dihydrophenanthrene and dibenzyl compounds from Chinese pholidota herb plant

Drying herba Bulbophylli Inconspicui 1.84kg collected from Yunnan West Domain in shade, pulverizing, soaking in 95% industrial ethanol 10L at room temperature for 5 days, repeating for three times, mixing extractive solutions, and concentrating under reduced pressure to obtain total extract 236 g. Suspending the total extract in 2.0L water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and vacuum evaporating to remove organic solvent to obtain brown petroleum ether fraction 93g, ethyl acetate fraction 10g and n-butanol fraction 45 g. A petroleum ether fraction (93g) was taken and subjected to silica gel column chromatography (petroleum ether-ethyl acetate, 100:1 to 1:1) to divide into 9 fractions. And purifying the fraction 4 by silica gel column chromatography (10: 1-2: 1 petroleum ether-acetone) and Sephadex LH-20(MeOH) to obtain YS24 (Lusithridin, 52 mg), and purifying the fraction 5 by Sephadex LH-20(CHCl3: MeOH 1:1) to obtain YS28(3,3', 5-trihydroxy dibenzyl, 63mg) and YS32(3,3', 5-trihydroxy stilbene, 21 mg). And separating and purifying the fraction 6 by repeated silica gel column chromatography to obtain compounds YS25(coelonin,11mg), YS26(dihydropinosylvin, 140mg), YS27 (4-hydroxy-3-methoxyl dibenzyl, 43mg), YS30(gigantol, YS30,60mg), YS33(thunalbene,45 mg) and YS34 (3' -hydroxy-3, 5-dimethoxystilbene, 35 mg). And purifying the fraction 7 by Sephadex LH-20 to obtain compounds YS23 (2-hydroxy-4, 7-dimethoxy-9, 10-dihydrophenanthrene, 23mg) and YS31 (3-hydroxy-5-methoxystilbene, 11 mg). The fraction 8 is subjected to silica gel column chromatography to obtain small fraction, and then subjected to Sephadex LH-20 and preparative high performance liquid purification to obtain compounds YS1 (photochinin A,18 mg), YS 3-5 (photochinins C-E,4,72 and 59mg), YS8 (photochinin H,27mg), YS11-12 (photochinins K and L,7 and 4mg), YS21 (blestrinin A,16mg), YS29(batatasin III,14 mg), YS35(shancidin,48mg), YS36[1- (4-hydroxybenzyl) -4-methoxy-2, 7-dihydroxy-9, 10 dihydrophenanthrene, 8mg ], and YS37 (isoarnidinin I,114 mg). Subjecting fraction 9 to repeated silica gel column chromatography and Sephadex LH-20 column chromatography, and purifying with preparative high performance liquid chromatography to obtain compounds YS2 (photochinin B,23 mg), YS6-7 (photochinin F and G,10 and 4mg), YS9-10 (photochinin I and J,63 and 11mg), YS 15-17 (photochinin O-Q,11,57 and 48mg), YS 18 (gymcopin C,60mg), YS19 (blesterianol A,23mg), YS20 (flavanthron, 115mg) and YS22 (photochinin D,24 mg). The structure of the compound is detailed in FIG. 1.

2. Physicochemical properties and spectral data of the Compounds

YS1:Phochinenin A

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):216(4.78)and 299(4.17).EI MS m/z(rel.int):348[M]⁺,HREIMS m/z:348.1363[M]⁺(calcd for C₂₂H₂₀O₄,348.1364).¹H NMR(300MHz,CD3OD)δ6.39(1H,d,J＝2.4Hz,H-4),6.70(1H,d,J＝2.4Hz,H-6), 6.85and 7.27(each 1H,d,J＝15.6Hz,H-αandα’),6.91(1H,dd,J＝2.1and 2.7Hz,H- 2’),6.84(1H,dd,J＝2.1and 8.1Hz,H-4’),7.12(1H,dd,J＝7.6and 8.1Hz,H-5’),6.87 (1H,dd,J＝2.7and 7.6Hz,H-6’),3.98(2H,s,-CH2),6.59and 6.99(each 2H,d,J＝8.7 Hz,H-2”,3”,5”,6”),3.79(3H,s,5-OMe).

YS2:phochinenin B

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):201(4.36),209(4.36)and 281 (4.08),EI-MS m/z(rel.int):348[M⁺].HR-EIMS m/z:348.1353[M⁺](calcd for C₂₂H₂₀O₄, 348.1344).¹H NMR(300MHz,CD3OD)δ6.39(1H,d,J＝2.5Hz,H-4),6.66(1H,d,J＝ 2.5Hz,H-6),6.80and 7.20(each 1H,d,J＝16.0Hz,H-αandα’),6.83(1H,dd,J＝2.0 and 2.5Hz,H-2’),6.63(1H,dd,J＝2.5and 7.6Hz,H-4’),7.10(1H,dd,J＝7.6and 7.7 Hz,H-5’),6.84(1H,dd,J＝2.0and 7.0Hz,H-6’),3.96(2H,s,-CH2),6.60and 6.91 (each 2H,d,J＝8.3Hz,H-2”,3”,5”,6”),3.77(3H,s,3-OMe).

YS3:phochinenin C

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):201(4.79)and 280(3.79).ESI-

MS:m/z 403.1[M+Na]⁺,379.1[M-H]^-；HR ESI-MS:m/z 403.1535(calcd for C₂₃H₂₄O₅Na, 403.1521).¹H NMR(300MHz,CD3OD)δ6.32(1H,d,J＝2.1Hz,H-4),6.26(1H,d,J＝ 2.1Hz,H-6),2.52and 2.69(each 2H,m,H-αandα’),6.51(1H,t,J＝1.3Hz,H-2’),6.51 (1H,dd,J＝1.3and 7.9Hz,H-4’),7.00(1H,dd,J＝7.6and 7.9Hz,H-5’),6.54(1H,dd, J＝1.3and 7.6Hz,H-6’),3.83(2H,s,-CH2),6.62(1H,d,J＝2.0Hz,H-2”),6.61(1H,d, J＝8.3Hz,H-5”),6.45(1H,dd,J＝2.3and 8.3Hz,H-6”),3.73and 3.69(each 3H,s,3 and 3”-OMe).

YS4:phochinenin D

Brown amorphous powder, [ alpha ]]²⁰ _D-0.005(c 0.315,MeOH).UV(MeOH)λ_max nm(logε):213 (5.04),279(4.89),296(4.77).EIMS m/z(rel.int):482[M⁺](100).HR-EIMS m/z: 482.1742[M⁺](calcd for C₃₀H₂₆O₆,482.1729).¹H NMR(600MHz,CD3OD)δ6.57(1H, s,H-1),8.20(1H,d,J＝8.3Hz,H-5),6.64(1H,dd,J＝2.4and 8.3Hz,H-6),6.65(1H,d, J＝2.4Hz,H-8),2.75and 2.79(each 2H,m,H-9,10),6.84and 8.15(each 1H,s,H- 1’,4’),6.34and 6.38(each 1H,d,J＝2.4Hz,H-6’,8’),2.78(4H,s,H-9’,10’),3.75and 3.77(each 3H,s,2-OCH ₃,7’-OCH ₃).

YS5:phochinenin E

Brown amorphous powder, [ alpha ]]²⁰ _D+0.005(c 0.325,MeOH).UV(MeOH)λ_max nm(logε):216 (4.71),279(4.52),297(4.41).EIMS m/z(rel.int):482[M⁺](100).HR-EIMS m/z: 482.1734[M⁺](calcd for C₃₀H₂₆O₆,482.1729).¹H NMR(300MHz,CD3OD)δ6.77and 8.03(each 1H,s,H-1,4),6.35and 6.31(each 1H,d,J＝2.4Hz,H-6,8),2.73(4H,s,H-9, 10),6.57(1H,s,H-2’),8.03(1H,d,J＝8.6Hz,H-5’),6.63(1H,dd,J＝2.4and 8.6Hz, H-6’),6.59(1H,d,J＝2.4Hz,H-8’),2.50and 2.46(each 2H,m,H-9’,10’),3.75and 3.88 (each 3H,s,7-OCH ₃,3’-OCH ₃).

YS6：phochinenin F

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):282(4.51).EIMS m/z(rel.int):482 [M⁺](100).HR-EIMS m/z:482.1739[M⁺](calcd for C₃₀H₂₆O₆,482.1729).¹H NMR(400 MHz,DMSO-d₆)δ6.53(1H,s,H-1),7.95(1H,d,J＝8.6Hz,H-5),6.61(1H,dd,J＝2.5 and 8.6Hz,H-6),6.65(1H,d,J＝2.5Hz,H-8),2.65(4H,s,H-9,10),6.57(1H,s,H-3’), 7.96(1H,d,J＝8.6Hz,H-5’),6.58(1H,dd,J＝1.6and 8.6Hz,H-6’),6.56(1H,d,J＝ 1.6Hz,H-8’),2.46and 2.31(each 2H,m,H-9’,10’),3.18and 3.81(each 3H,s,4-OCH ₃, 4’-OCH ₃).

YS7:phochinenin G

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):211(4.60),263(4.66)357(3.48), 375(3.53).EIMS m/z(rel.int):480[M⁺](100).HR-EIMS m/z:480.1575[M⁺](calcd for C₃₀H₂₄O₆,480.1573).¹H NMR(600MHz,CD3OD)δ6.67(1H,s,H-2),8.10(1H,d,J＝ 8.9Hz,H-5),6.66(1H,dd,J＝3.0and 8.9Hz,H-6),6.58(1H,d,J＝3.0Hz,H-8),2.46 and 2.22(each 2H,m,H-9,10),6.94(1H,s,H-2’),9.45(1H,d,J＝9.4Hz,H-5’),7.12 (1H,dd,J＝2.7and 9.4Hz,H-6’),7.11(1H,d,J＝2.7Hz,H-8’),7.40and 7.48(each 1H, d,J＝9.0Hz,H-9’,10’),3.95and 4.15(each 3H,s,3-OCH ₃,3’-OCH ₃).

YS8:phochinenin H

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):211(4.70),279(4.51),295(4.40). EIMS m/z(rel.int):482[M⁺](100).HR-EIMS m/z:482.1734[M⁺](calcd for C₃₀H₂₆O₆, 482.1729).¹H NMR(600MHz,CD3COCD3)δ6.67(1H,s,H-2),8.12(1H,d,J＝8.5Hz, H-5),6.71(1H,dd,J＝2.7and 8.5Hz,H-6),6.69(1H,d,J＝2.7Hz,H-8),2.58(4H,m, H-9,10),6.67(1H,d,J＝2.8,H-1’),6.64(1H,dd,J＝2.8and 8.8Hz,H-3’),8.11(1H,d, J＝8.8Hz,H-4’),6.46and 6.38(each 1H,d,J＝2.3Hz,H-6’,8’),2.46(4H,s,H-9’,10’), 3.89and 3.83(each 3H,s,3-OCH ₃,5’-OCH ₃).

YS9:phochinenin I

Brown amorphous powder, [ alpha ]]²⁰ _D+0.008(c 0.310,MeOH).UV(MeOH)λ_max nm(logε):200 (5.61),278(5.58),297(5.44).EIMS m/z(rel.int):482[M⁺](23).HR-EIMS m/z: 482.1726[M⁺](calcd for C₃₀H₂₆O₆,482.1729).¹H NMR(300MHz,CD3OD)δ6.54(1H, s,H-2),8.22(1H,d,J＝8.6Hz,H-5),6.67(1H,dd,J＝2.8and 8.5Hz,H-6),6.62(1H,d, J＝2.8Hz,H-8),2.63(4H,s,H-9,10),6.58(1H,d,J＝2.8,H-1’),6.61(1H,dd,J＝2.8 and 8.5Hz,H-3’),8.19(1H,d,J＝8.5Hz,H-4’),6.33(2H,s,H-6’,8’),2.66(4H,m,H- 9’,10’),3.89and 3.83(each 3H,s,3-OCH ₃,5’-OCH ₃).

YS10:phochinenin J

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):279(4.48).EIMS m/z(rel.int):482 [M⁺](37).HR-EIMS m/z:482.1721[M⁺](calcd for C₃₀H₂₆O₆,482.1729).¹H NMR(600 MHz,CD3OD)δ6.57(1H,s,H-2),8.24(1H,d,J＝8.3Hz,H-5),6.68(1H,dd,J＝2.3 and 8.3Hz,H-6),6.64(1H,d,J＝2.3Hz,H-8),2.63(4H,s,H-9,10),6.24and 6.46(each 1H,d,J＝2.6Hz,H-1’,3’),8.03(1H,d,J＝9.5Hz,H-5’),6.64(1H,dd,J＝2.6and 9.5 Hz,H-6’),6.63(1H,d,J＝2.6Hz,H-8’),2.64(4H,m,H-9’,10’),3.77and 3.79(each 3H,s,3-OCH ₃,4’-OCH ₃).

YS11:phochinenin K

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):201(4.68),279(4.22).EIMS m/z (rel.int):484[M⁺](100).HR-EIMS m/z:484.1894[M⁺](calcd for C₃₀H₂₈O₆,484.1786). ¹H NMR(300MHz,CD3OD)δ6.81and 8.10(each 1H,s,H-1,4),6.31and 6.35(each 1H,d,J＝2.7Hz,H-6,8),2.75(4H,s,H-9,10),6.68(1H,dd,J＝2.3and 8.3Hz,H-6), 6.64(1H,d,J＝2.3Hz,H-8),6.33and 6.34(each 1H,d,J＝2.4Hz,H-4’,6’),2.60and 2.67(each 2H,m,H-α,α’),6.40(1H,dd,J＝2.4and 2.6Hz,H-2”),6.47(1H,d,J＝2.4 and 8.0Hz,H-4”),6.93(1H,dd,J＝7.4and 8.0Hz,H-5”),6.39(1H,dd,J＝2.6and 7.4 Hz,H-6”),3.77and 3.76(each 3H,s,7-OCH ₃,3’-OCH ₃).

YS12:phochinenin L

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):201(4.74),279(4.29,297(4.12). EIMS m/z(rel.int):484[M⁺](100).HR-EIMS m/z:484.1879[M⁺](calcd for C₃₀H₂₈O₆, 484.1786).¹H NMR(300MHz,CD3OD)δ6.77and 8.07(each 1H,s,H-1,4),6.31and 6.34(each 1H,d,J＝2.6Hz,H-6,8),2.71(4H,s,H-9,10),6.41and 6.45(each 1H,d,J＝ 1.1Hz,H-2’,6’),2.86(4H,m,H-α,α’),6.68(1H,dd,J＝1.7and 2.1Hz,H-2”),6.60 (1H,d,J＝1.7and 7.9Hz,H-4”),7.09(1H,dd,J＝7.5and 7.9Hz,H-5”),6.72(1H,dd, J＝2.1and 7.5Hz,H-6”),3.74and 3.67(each 3H,s,7-OCH ₃,5’-OCH ₃).

YS13:phochinenin M

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):201(4.83),280(4.33).I EIMS m/z (rel.int):484[M⁺](100).HR-EIMS m/z:484.1871[M⁺](calcd for C₃₀H₂₈O₆,484.1786). CD data recorded on-line in hexane/ethanol 7:3for(aS)-13:λ_max(φ):314(-2.3),299sh (-2.2),282(-2.4),268(0.9),257(-0.8),237(4.0),225(-6.9).(aR)-13:313sh(3.9),301 (4.5),282(6.0),270(-0.5),257(1.6),237(-7.8),227(6.6).¹H NMR(300MHz, CD3OD)δ6.59(1H,s,H-3),8.03(1H,d,J＝8.5Hz,H-5),6.62(1H,dd,J＝2.7and 8.5 Hz,H-6),6.58(1H,d,J＝2.7Hz,H-8),2.52and 2.33(each 2H,m,H-9,10),6.34and 6.36(each 1H,d,J＝2.5Hz,H-4’,6’),2.54and 2.56(each 2H,m,H-α,α’),6.38(1H,dd, J＝2.1and 2.1Hz,H-2”),6.49(1H,d,J＝2.1and 8.2Hz,H-4”),6.94(1H,dd,J＝8.2 and 8.6Hz,H-5”),6.37(1H,dd,J＝2.1and 8.6Hz,H-6”),3.89and 3.74(each 3H,s,2- OCH ₃,3’-OCH ₃).

YS14:phochinenin N

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):277(4.32),299(4.29).EIMS m/z (rel.int):482[M⁺](100).HR-EIMS m/z:482.1733[M⁺](calcd for C₃₀H₂₆O₆,484.1729). ¹H NMR(600MHz,CD3OD)δ6.71and 8.04(each 1H,s,H-1,4),6.27and 6.32(each 1H,d,J＝2.6Hz,H-6,8),2.76(4H,s,H-9,10),6.89and 6.35(each 1H,d,J＝2.5Hz,H- 2’,6’),6.94and 6.97(each 1H,d,J＝16.6Hz,H-α,α’),6.73(1H,dd,J＝1.7and 2.1Hz, H-2”),6.55(1H,d,J＝1.7and 8.5Hz,H-4”),7.02(1H,dd,J＝7.6and 8.1Hz,H-5”), 6.76(1H,dd,J＝2.1and 7.6Hz,H-6”),3.74and 3.86(each 3H,s,7-OCH ₃,4’-OCH ₃).

YS15:phochinenin O

Brown amorphous powder, UV (MeOH) lambda_max nm(logε):219(4.62),310(4.46).EIMS m/z (rel.int):482[M⁺](100).HR-EIMS m/z:482.1738[M⁺](calcd for C₃₀H₂₆O₆,482.1729). ¹H NMR(500MHz,CD3OD)δ6.73and 8.08(each 1H,s,H-1,4),6.29and 6.33(each 1H,d,J＝2.4Hz,H-6,8),2.71(each 2H,m,H-9,10),6.75and 6.77(each 1H,d,J＝1.3 Hz,H-2’,6’),7.09and 7.12(each 1H,d,J＝16.0Hz,H-α,α’),6.96(1H,dd,J＝1.7and 2.4Hz,H-2”),6.67(1H,d,J＝2.4and 8.0Hz,H-4”),7.14(1H,dd,J＝7.7and 8.0Hz, H-5”),7.01(1H,dd,J＝1.7and 7.7Hz,H-6”),3.72and 3.76(each 3H,s,7-OCH ₃,5’- OCH ₃).

YS16:phochinenin P

Brown amorphous powder, [ alpha ]]²⁰ _D+0.027(c 0.305,MeOH).UV(MeOH)λ_max nm(logε):212 (4.69),281(4.45),297(4.45).EIMS m/z(rel.int):482[M⁺](100).HR-EIMS m/z: 482.1736[M⁺](calcd for C₃₀H₂₆O₆,482.1729).¹H NMR(300MHz,CD3OD)δ6.63(1H, s,H-2),8.10(1H,d,J＝8.6Hz,H-5),6.66(1H,dd,J＝2.5and 8.6Hz,H-6),6.60(1H, d,J＝2.5Hz,H-8),2.47and 2.28(each 2H,m,H-9,10),6.48and 6.94(each 1H,d,J＝ 2.6Hz,H-4’,6’),6.78and 6.97(each 1H,d,J＝16.0Hz,H-α,α’),6.72(1H,dd,J＝2.1 and 2.1Hz,H-2”),6.62(1H,d,J＝2.1and 7.7Hz,H-4”),7.07(1H,dd,J＝7.7and 8.1 Hz,H-5”),6.86(1H,dd,J＝2.1and 8.1Hz,H-6”),3.89and 3.84(each 3H,s,3-OCH ₃, 5’-OCH ₃).

YS17:Rac-phochinenin Q

Brown amorphous powder, [ alpha ]]²⁰ _D 0(c 0.287,MeOH).UV(MeOH)λ_max nm(logε):205 (4.88),282(4.35).EIMS m/z(rel.int):512[M⁺](100).HR-EIMS m/z:512.1842[M⁺] (calcd for C₃₁H₂₈O₇,512.1835).¹H NMR(500MHz,CD3OD)δ6.63(1H,s,H-3),8.04 (1H,d,J＝8.6Hz,H-5),6.64(1H,dd,J＝2.4and 8.6Hz,H-6),6.60(1H,d,J＝2.4Hz, H-8),2.47(2H,m,H-9),2.38and 2.22(each 1H,m,H-10),6.22(1H,d,J＝2.1Hz,H- 2’),6.28(1H,dd,J＝1.5and 2.1Hz,H-4’),6.20(1H,d,J＝1.5Hz,H-6’),4.43and 5.37 (each 1H,d,J＝6.5Hz,H-α,α’),6.91(1H,d,J＝2.1Hz,H-2”),6.81(1H,d,J＝7.9Hz, H-5”),6.79(1H,dd,J＝1.6and 7.9Hz,H-6”),3.92,3.84and 3.71(each 3H,s,4-OCH ₃, 3’-OCH ₃,3”-OCH ₃).

YS18:gymconpin C

Brown amorphous powder, [ alpha ]]²⁰ _D+0.003(c 0.315,MeOH).UV(MeOH)λ_max nm(logε):216 (5.14),280(4.95),296(4.85).CD data recorded on-line in hexane/ethanol 7:3for(aS)-18: λ_max(φ):307(8.7),287sh(5.5),271(-9.1),246(8.5),219(-9.7).(aR)-18:311(-8.8), 286sh(-5.5),269(10.0),243(-8.3),222(9.8).

¹H NMR(500MHz,CD3OD)δ6.57(1H,s,H-3),8.03(1H,d,J＝8.5Hz,H-5),6.63 (1H,dd,J＝2.7,8.5Hz,H-6),6.60(1H,d,J＝2.7Hz,H-8),2.53and 2.50(each 2H,m, H-9,10),6.32and 6.37(each 1H,d,J＝2.5Hz,H-1’,3’),7.87and 6.76(each 1H,s,H- 5’,7’),27.0(4H,s,H-9’,10’),3.88and 3.74(each 3H,s,4-OMe,4’-OMe).

YS19:blestrianol A

Pale yellow powder, UV (MeOH) lambda_max nm(logε):276(4.54),285(4.61),200(4.45).ESI- MS m/z 482.3[M+H]⁺.¹H NMR(500MHz,CD3OD)δ2.41-2.57(4H,m,H-9’,H-10’), 2.72(4H,s,H-9,H-10),3.26and 3.88(each 3H,s,4,4’-OMe),6.59and 6.62(each 1H,s, H-3’,H-1),6.60(1H,d,J＝2.6Hz,H-8’),6.63(1H,dd,J＝8.6,2.6Hz,H-6’),6.64(1H, dd,J＝8.4,2.6Hz,H-6),6.66(1H,d,J＝2.6Hz,H-8),8.05(lH,d,J＝8.6Hz,H-5’), 8.08(1H,d,J＝8.4Hz,H-5).

YS20:flavanthrin

White amorphous powder, [ alpha ]]²⁰ _D 0(c 0.380,MeOH).CD data recorded on-line in hexane/ethanol 7:3for(aS)-20:λ_max(φ):315(22.0),303sh(13.6),286sh(7.0),268 (-18.9),251sh(5.6),236(20.4),221(-25.1),202(-30.9).(aR)-20:315(-39.1),305sh (-26.8),285sh(-13.6),269(-34.6),250sh(-12.8),236(-36.2),220(32.8),204 (21.9).ESI-MS m/z.483.2[M+H]⁺.¹H NMR(400MHz,CD3OD)δ2.36and 2.50(each 4H,m,H-9,10,9’,10’),3.92(6H,s,7-OMe,7’-OMe),6.59(each 1H,s,H-6,6’),6.66(2H, d,J＝2.3Hz,H-1,1’),6.67(2H,dd,J＝8.3,2.3Hz,H-3,3’),8.07(2H,d,J＝8.3Hz,H- 4,4’).

YS21:blestrin A

White powder, UV (MeOH) lambda_max nm(logε):272sh(4.28),281(4.36),299(4.25).ESI- MS m/z 483.2[M+H]⁺.¹H-NMR(400MHz,CD3OD)δ6.61(1H,s,H-3),8.06(1H,d,J＝ 8.6Hz,H-5),6.64(1H,dd,J＝2.8,8.6Hz,H-6),6.61(1H,d J＝2.8Hz,H-8),2.55(4H, s,H-9,10),6.26and 6.51(each 1H,d,J＝2.6Hz,H-1’,3’),8.01(1H,d,J＝9.2Hz,H-5’), 6.60-6.64(2H,m,H-6’,8’),3.86and 3.77(each 3H,s,4-OMe,4’-OMe).

YS22:phoyunnanin D

White powder, UV (MeOH) lambda_max nm(logε):205(4.92),280(4.47).ESI-MS m/z 484.2 [M+H]⁺.¹H-NMR(400MHz,Acetone-d₆)δ6.38and 6.43(1H,d,J＝2.5Hz,H-1,3),8.27 (1H,d,J＝8.7Hz,H-5),6.64(1H,dd,J＝2.8,8.7Hz,H-6),6.69(1H,d J＝2.8Hz,H-8), 2.69(4H,m,H-9,10),6.41and 6.50(each 1H,d,J＝2.7Hz,H-1’,3’),6.63(1H,m,H- 2”),6.61(1H,ddd,J＝7.8,2.5,0.9Hz,H-4”),7.02(1H,t,J＝7.8Hz,H-5”),6.59(1H, dd,J＝7.8,0.9Hz,H-6”),2.73(4H,m,H-α’,α”),3.80and 3.70(each 3H,s,2-OMe,6’- OMe).

YS23 [ 7-hydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene ], 7-hydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene ]

A light red oily liquid, EI-MS m/z (%): 256(100).¹H NMR(500MHz,CDCl3):δ8.10 (1H,d,J＝8.5Hz,H-5),6.73(1H,d,J＝8.5Hz,H-6),6.68(1H,s,H-8),6.45(1H,s,H- 3),6.41(1H,s,H-1),3.85(3H,s,4-OCH3),3.82(3H,s,2-OCH3),2.73(4H,m,CH2-9, 10).

YS24:lusianthridin

Light brown powder, EI-MS m/z (%): 242(100).¹H NMR(500MHz,CD3OD),δ7.08(1H, t,J＝7.8Hz,H-7),6.84(1H,dd,J＝7.8,2.5Hz,H-6),6.81(1H,dd,J＝7.8,2.5Hz,H- 8),6.57(1H,d,J＝2.5Hz,H-3),6.53(1H,d,J＝2.5Hz,H-1),3.95(3H,s,4-OCH₃), 2.67(4H,m,CH₂-9,10)

YS25:coelonin

A light brown oily liquid, EI-MS m/z (%): 242(100).¹H NMR(500MHz,acetone-d₆)δ 8.07(1H,d,J＝8.5Hz,H-5),6.68(1H,d,J＝8.5Hz,H-6),6.66(1H,s,H-8),6.45(1H, s,H-3),6.37(1H,s,H-1),3.86(3H,s,4-OCH3),2.70(4H,m,CH₂-9,10).

YS26 Dihydroppinosylvin

White acicular solid, EI-MS m/z (%): 214(55).¹H NMR(400MHz,CDCl3)δ2.73-2.90 (4H,m,H-α,α’),6.19(1H,t,J＝2.1Hz,H-4),6.24(2H,d,J＝2.1Hz,H-2,6),7.10-7.20 (3H,m,H-2’,4’,6’),7.25(2H,t,J＝7.5Hz,H-3’,5’).

YS27 4-hydroxy-3-methoxybibenzyl

White solid, EI-MS m/z (%): 228(69), 1H NMR (400MHz, CDCl3) δ 7.32-7.27(2H, m, H-3 ', 5'), 7.24-7.17(3H, m, H-2 ', 4', 6 '), 6.85(1H, d, J ═ 8.0Hz, H-2),6.70(1H, dd, J ═ 8.0,1.9Hz, H-4),6.62(1H, d, J ═ 1.9Hz, H-6),3.84(3H, s,3-OMe), 2.95-2.84 (4H, m, H- α, α').

YS28:3,3',5-trihydroxy-bibenzyl,3,3', 5-trihydroxy-dibenzyl

White powder, EI-MS m/z (%):230(100).¹H NMR(500MHz,CDCl₃)δ7.08(1H,t,J＝ 7.5Hz,H-5’),6.72(1H,m,H-2’),6.69(1H,m,H-6’),6.65(1H,dd,J＝7.5,2.0,H-4’), 6.23(1H,s,H-6),6.23(1H,s,H-2),6.19(1H,s,H-4),2.77(4H,m,H-α,α’).

YS29:batatasin III

White solid, EI-MS m/z (%) 274(30).¹H NMR(400MHz,CDCl₃)δ7.09(1H,t,J＝ 7.8,H-5’),6.65(1H,d,J＝7.8,H-6’),6.57(1H,d,J＝7.4,H-4’),6.60(1H,s,H-2’),6.82 (1H,d,J＝8.2Hz,H-2),6.73(1H,d,J＝2.0Hz,H-6),6.63(1H,dd,J＝8.1,2.0Hz,H- 4),3.85(3H,s,4-OCH3),2.75(4H,m,H-α,α’).

6.84(1H,d,J＝8.0Hz,H-2),6.68(1H,dd,J＝8.0,1.8Hz,H-4),6.63(1H,d,J＝ 1.8Hz,H-6),6.35–6.30(1H,m,H-4’),6.27-6.23(2H,m,H-2’,6’),3.84(3H,s,3- OMe),3.75(3H,s,4’-OMe),2.97-2.58(4H,m,H-α,α’).

YS30:gigantolYS30

A yellowish oily liquid. EI-MS M/z (%) 274[ M ]]⁺；¹H NMR(400MHz；CDCl₃)δ6.84 (1H,d,J＝8.0Hz,H-2),6.68(1H,dd,J＝8.0,1.8Hz,H-4),6.63(1H,d,J＝1.8Hz,H- 6),6.35–6.30(1H,m,H-4’),6.27–6.23(2H,m,H-2’,6’),3.84(3H,s,3-OMe),3.75 (3H,s,4’-OMe),2.97-2.58(4H,m,H-α,α’).

YS31 3-hydroxy-5-methoxystilbene 3-hydroxy-5-methoxy

White solid, EI-MS m/z (%): 226 (78);¹H-NMR(400MHz,CDCl₃)δ3.83(3H,s,5- OMe),6.35(1H,t,J＝2.3Hz),6.61(1H,t,J＝1.8Hz),6.65(1H,t,J＝1.8Hz),6.99 (1H,d,J＝16.5Hz,H-α),7.07(1H,d,J＝16.5Hz,H-α’),7.26(1H,tt,J＝7.6and 1.4 Hz,H-4’),7.36(2H,t,J＝7.6Hz,H-3’,5’),7.50(2H,dd,J＝7.6Hz,H-2’,4’).

YS32(3,3', 5-trihydroxystilbene,3,3', 5-trihydroxystilbene)

Light yellow oily colloid, EI-MS m/z (%): 228 (100);¹H-NMR(400MHz,CD₃OD)δ6.19 (1H,t,J＝2.1Hz,H-2),6.47(2H,d,J＝2.1Hz,H-4),6.68(1H,ddd,J＝7.8and 2.4and 0.6Hz,H-5’),6.92(1H,d,J＝16.2Hz,H-α),6.93(1H,t,J＝2.4Hz,H-2’),6.96(1H,d, J＝16.2Hz,H-α’),6.97(1H,d,J＝7.8Hz,H-6’),7.15(1H,t,J＝7.8Hz,H-4’).

YS33:thunalbene

light yellow oily colloid, EI-MS m/z (%) (242) (86);¹H-NMR(500MHz,CD₃OD)δ6.95 (1H,brs,H-2),6.68(2H,dd,J＝7.8,1.9Hz,H-4),7.15(1H,t,J＝7.8Hz,H-5),7.00 (1H,d,J＝7.2Hz,H-6),7.01(1H,d,J＝16.2Hz,H-α),6.98(1H,d,J＝16.4Hz,H-α’), 6.59(1H,brs,H-2’),6.57(1H,brs,H-6’),6.28(1H,t,J＝2.1Hz,H-4’),3.78(3H,s, OCH₃-5’)

YS34 3 '-hydroxy-3, 5-dimethoxy-stilbene, 3' -hydroxy-3,5-dimethoxy-stilbene

A white solid, EI-MS m/z (%): 256 (73);¹H NMR(400MHz,CDCl₃)δ7.21(1H,t, J＝7.7Hz,H-5),7.05(1H,d,J＝7.7Hz,H-6),7.00(1H,d,J＝16.7Hz,H-α),6.98(1H, d,J＝16.7Hz,H-α’),6.96(1H,d,J＝2.3Hz,H-2),6.74(dd,1H,J＝7.7,2.3Hz,H-4), 6.64(2H,d,J＝2.2Hz,H-2’,6’),6.39(1H,t,J＝2.2Hz,H-4’),3.81(s,6H,OCH₃-3’,5’).

YS35:shancidin

colorless needle-like solid, EI-MS m/z (%): 348 (100);¹H-NMR(500MHz,CD₃OD)δ2.45 (2H,m,H-9),2.57(2H,m,H-10),3.88(3H,s,4-OMe),3.93(2H,s,H-11),6.52(1H,s, H-3),6.58(1H,d,J＝2.5Hz,H-8),6.62(1H,dd,J＝8.6,2.5Hz,H-6),6.65(2H,d,J＝8.5Hz,H-3’,5’),6.94(2H,d,J＝8.5Hz,H-2’,6’),7.97(1H,d,J＝8.6Hz,H-5).

YS36 (9, 10-dihydro-1- (4-hydroxybenzyl) -4-methoxy-2,7-phenanthrenediol, 1- (4-hydroxybenzyl) -4-methoxy-2, 7-dihydroxy-9, 10-dihydrophenanthrene)

Pale yellow needle-like solid, EI-MS m/z (%): 348 (100);¹H-NMR(500MHz,CD₃COCD₃)δ 8.02(1H,d,J＝9.0Hz,H-5),6.98(2H,d,J＝8.4Hz,H-2’,6’),6.70(2H,d,J＝8.4Hz, H-3’,5’),6.68(1H,s,H-8),6.65(1H,d,J＝9.0Hz,H-6),6.62(1H,s,H-3),4.05(2H,s, CH₂-11),3.83(3H,s,4-OCH3),2.62(4H,m,CH2-9,10).

YS37:isoarundinin I

white needle-like solid, MS m/z (%) 350(100).¹H-NMR(500MHz,CD₃OD)δ2.90(4H, m,H-α,α’),3.87(2H,s,-CH₂-Ph),3.70(3H,s,3-OMe),6.30and 6.24(each 1H,d,J＝ 2.4Hz,H-4,6),6.54(1H,t,J＝2.3Hz,H-2’),6.56(1H,dd,J＝7.9.2.3Hz,H-4’),7.03 (1H,t,J＝7.9Hz,H-5’),6.56(1H,dd,J＝7.9,2.3Hz,H-6’),6.64(2H,d,J＝8.5Hz,H- 3”,5”),6.95(2H,d,J＝8.5Hz,H-2”,6”).

3. Separation of 9,10-dihydrophenanthrene and phenanthrene compounds in wild rush

Drying medulla Junci (9.5 kg), extracting with 95% industrial ethanol (35L) for 3 times (3 days each time), mixing extractive solutions, and recovering solvent under reduced pressure to obtain total extract 445 g. Dissolving the total extract in water, and respectively extracting with ethyl acetate and n-butanol to obtain ethyl acetate part 217g, n-butanol layer 62g and water layer 153 g. The ethyl acetate fraction (217g) was passed through a silica gel column (petroleum ether-acetone 6:1 to 0:1) to obtain 8 fractions. And (3) separating the fraction 3(16.7g) by silica gel column chromatography (PE-isopropyl ether 10: 1-1: 1), recrystallizing the obtained secondary fraction F3-1 by isopropyl ether to obtain a compound XYW-1 (juncunin A,51mg), and carrying out Sephadex LH-20 (chloroform-methanol 1:1) on the secondary fraction F3-4 to obtain a compound XYW-5 (dehydrojuncunin A,333 mg). Recrystallizing fraction 4(70.53g) with chloroform to obtain compound XYW-3(juncuenin C,22g), and subjecting the mother liquor to MCI column chromatography (EtOH/H)₂30-100% of O), purifying the obtained secondary fraction F4-1 by silica gel column chromatography (PE: EtOAc 6: 1-1: 1) and Sephadex LH-20 (chloroform-methanol 1:1) to obtain compounds 8(829mg), 9(971mg) and 10(111 mg); secondary F4-9(735mg) was separated by Sephadex LH-20 (chloroform-methanol: 1:1) and subjected to preparative TLC (PE: Actone 2:1) to give compound XYW-6 (dehydrojuncumins B,17 mg). Subjecting fraction 5(10.146g) to MCI column chromatography (EtOH/H2O 30% -100%) to obtain secondary fraction F5-5(314mg), and recrystallizing with chloroform to obtain compound XYW-4 (juncunin D,66 mg); the secondary fraction F5-10(1.492g) was purified by silica gel column chromatography (PE: Actone 7:2) and the resulting fraction was recrystallized (MeOH-PE) to give compound XYW-7 (dehydrojuncumens C, 28 mg). Recrystallizing fraction 6(12.11g) with methanol, subjecting the residual mother liquor to MCI column chromatography (EtOH/H2O 30% -100%) to obtain secondary fraction F6-4(125mg), and purifying with Sephadex LH-20 (chloroform-methanol 2:1)Compound XYW-2(juncuenin B,32mg) was obtained.

4. Physicochemical Properties and spectral data of the Compounds

XYW-1 juncuenin A (2-hydroxy-1, 7-dimethyl-6-vinyl-9, 10-dihydrophenanthrene)

A white amorphous powder; c₁₈H₁₈O,EI MS m/z(％):250[M]⁺(100)；HREIMS m/z 250.1358 (calcd.for C₁₈H₁₈O,250.1358).¹H NMR(300MHz,CDCl₃)δ6.74and 7.55(each 1H,d,J ＝8.3Hz,H-3,4),7.76and 7.01(each 1H,s,H-5,8),2.80(4H,s,H-9,10),2.24and 2.35 (each 3H,s,1-,7-Me),6.95(1H,dd,J＝10.9,17.4Hz,H-11),5.69(1H,dd,J＝1.5,17.4 Hz,H-12a),5.29(1H,dd,J＝1.5,17.4Hz,H-12b),4.84(1H,br.s,2-OH).

XYW-2 juncuenin B (2, 6-dihydroxy-1-methyl-7 carboxy-8-vinyl-9, 10-dihydrophenanthrene)

A white amorphous powder; c₁₈H₁₆O₄,EIMS m/z(％):296[M]⁺(26)；HREIMS m/z 296.1050 (calcd.for C₁₈H₁₆O₄，296.1048).¹H NMR(300MHz,CD₃OD)δ6.73and 7.41(each 1H, d,J＝8.3Hz,H-3,4),7.13(1H,s,H-5),2.73(4H,m,H-9,10),2.17(3H,s,1-Me),7.03 (1H,dd,J＝11.0,17.8Hz,H-11),5.04(1H,dd,J＝1.4,17.8Hz,H-12a),5.42(1H,dd,J＝ 1.4,17.8Hz,H-12b).

XYW-3: juncunein C (2, 6-dihydroxy-1, 7-dimethyl-8 vinyl-9, 10-dihydrophenanthrene)

Colorless fine needle crystals (CHCl)₃)；C₁₈H₁₈O₂,EIMS m/z(％):266[M]⁺(100)；HREIMS m/z 266.1302(calcd.for C₁₈H₁₈O₂,266.1307).¹H NMR(300MHz,CDCl₃)δ6.72and 7.40 (each 1H,d,J＝8.4Hz,H-3,4),7.06(1H,s,H-5),2.77(4H,m,H-9,10),2.01(6H,s,1-,7- Me),6.75(1H,dd,J＝11.3,16.7Hz,H-11),5.61(1H,dd,J＝2.1,16.7Hz,H-12a),5.29 (1H,dd,J＝2.1,16.7Hz,H-12b),4.67and 4.78(each 1H,br.s,2-,6-OH).

XYW-4 juncuenin D (2,8 a-dihydroxy-1, 7-dimethyl-6-carbonyl-8-vinyl-9, 10-dihydrophenanthrene)

Pale yellow, small cubic crystals (CHCl 3); c₁₈H₁₈O₃，ESI MS m/z 305.1[M+Na]⁺,HRESIMS m/z 305.1143(calcd.for C₁₈H₁₈O₃Na,305.1154).¹H NMR(300MHz,DMSO-d₆)δ6.75and 7.37(each 1H,d,J＝8.8Hz,H-3,4),6.32(1H,s,H-5),1.54and 2.40(each 1H,m,H-9), 2.68and 2.86(each 1H,m,H-10),2.02and 1.93(each 3H,s,1-,7-Me),6.70(1H,dd,J＝ 11.9,18.1Hz,H-11),5.75(1H,dd,J＝2.1,18.1Hz,H-12a),5.70(1H,dd,J＝2.1,11.9Hz, H-12b),5.54and 9.69(each 1H,br.s,8a-,2-OH).

XYW-5: dehydrojuncunin A (2-hydroxy-1, 7-dimethyl-6-vinyl phenanthrene)

A white powder; c₁₈H₁₆O,EIMS m/z(％):248[M]⁺(100),HREIMS m/z 248.1210(calcd. for C₁₈H₁₆O,248.1202).¹H NMR(300MHz,CDCl3)δ7.15and 8.50(each 1H,d,J＝8.9 Hz,H-3,4),8.67and 7.62(each 1H,s,H-5,8),7.68and 7.86(each 1H,d,J＝9.2Hz,H- 9,10),2.60and 2.53(each 3H,s,1-,7-Me),7.15(1H,dd,J＝10.9and 17.4Hz,H-11), 5.87(1H,dd,J＝2.1and 17.4Hz,H-12a),5.43(1H,dd,J＝2.1and 10.9Hz,H-12b),4.93 (1H,br.s,2-OH).

XYW-6: dehydrojuncunin B (2, 6-dihydroxy-1, 7-dimethyl-8-vinyl phenanthrene)

A brown powder; c₁₈H₁₆O₂,EIMS m/z(％)264[M]⁺(100),HREIMS m/z 264.1155(calcd. C₁₈H₁₆O₂,264.1150).¹H NMR(300MHz,acetone-d₆)δ7.20and 8.20(each 1H,d,J＝9.1 Hz,H-3,4),8.01(1H,s,H-5),7.96and 7.70(each 1H,d,J＝9.6Hz,H-9,10),2.57and 2.38(each 3H,s,1-,7-Me),7.10(1H,dd,J＝9.0and 15.9Hz,H-11),5.75(1H,dd,J＝2.5 and 15.9Hz,H-12a),5.70(1H,dd,J＝2.5and 9.0Hz,H-12b),8.42and 8.66(each 1H, br.s,2-,6-OH).

XYW-7: dehydrojuncunin C (2, 6-dihydroxy-1-methyl-12-lactone-phenanthrene)

Yellow cristobalite (MeOH-PE); c₁₈H₁₄O₄,EIMS m/z(％):294[M]⁺(69),HREIMS m/z 294.0910(calcd.for C₁₈H₁₄O₄,294.0892).¹H NMR(300MHz,DMSO-d₆)δ7.26and 8.50 (each 1H,d,J＝8.8Hz,H-3,4),8.02(1H,s,H-5),7.84and 7.76(each 1H,d,J＝9.2Hz, H-9,10),2.47(3H,s,1-Me),6.70(1H,dd,J＝11.9,18.1Hz,H-11),5.75(1H,dd,J＝2.1, 18.1Hz,H-12a),5.70(1H,dd,J＝2.1,11.9Hz,H-12b),5.54and 9.69(each 1H,br.s,8a-, 2-OH).

XYW-8: 2, 8-dihydroxy-1, 6-dimethyl-5-vinyl-9, 10-dihydrophenanthrene

A white powder; c₁₈H₁₈O₂；ESI MS m/z:267[M+H]⁺.¹H-NMR(300MHz,actone-d₆):δ 6.69(1H,d,J＝8.5Hz,H-3)，7.44(1H,d,J＝8.5Hz,H-4),6.75(1H,s,H-7),6.81(1H, dd,J＝11.3,18.5Hz,H-11),5.47(1H,dd,J＝11.3,2.1Hz,H-12a),5.19(1H,dd,J＝18.5, 2.1Hz,H-12b),2.67and 2.57(each 2H,m,H-9,10),2.25and 2.23(each 3H,s,1-,6-Me).

XYW-9: rush phenanthrol

A white powder; c₁₇H₁₆O₂,ESI-MS m/z:253[M+H]⁺,¹H NMR(300MHz,actone-d₆)：δ 6.85(1H,d,J＝8.4Hz,H-3),7.21(1H,d,J＝8.4Hz,H-4)；6.89(1H,d,J＝2.7Hz,H- 6),6.91(1H,d,J＝2.7Hz,H-8),6.94(1H,dd,J＝10.9,17.9Hz,H-11)，5.62(1H,dd,J ＝17.9,1.5Hz,H-12a),5.20(1H,dd,J＝10.9,1.5Hz,H-12b),2.67(4H,m,H-9,10),2.22 (3H,s,1-Me).

XYW-10: 2, 7-dihydroxy-1-methyl-5-vinylphenanthrene

White powder, C₁₇H₁₄O₂,ESI-MS m/z:251[M+H]⁺.¹H NMR(300MHz,actone-d₆):δ 7.21(1H,d,J＝8.8Hz,H-3),8.51(1H,d,J＝8.8Hz,H-4),7.27(1H,d,J＝1.5Hz,H-6), 7.22(1H,d,J＝1.5Hz,H-8),7.91(1H,d,J＝9.0Hz,H-9),7.66(1H,d,J＝9.0Hz,H- 10),7.49(1H,dd,J＝11.0,17.1Hz,H-11),5.76(1H,dd,J＝17.1,1.5Hz,H-12a),5.44 (1H,dd,J＝11.0,1.5Hz,H-12b),2.58(3H,s,1-Me).

5. Separation of 9,10-dihydrophenanthrene and phenanthrene compounds in rush

Crushing rush (J.effusus) whole plant (10kg) collected from Jinxiu county, Guangxi, soaking in 95% industrial ethanol four times for three days each time, combining the extractive solutions, and concentrating under reduced pressure to remove ethanol to obtain dark brown extract (605 g). Transferring the extract into a 25L faucet bottle, adding 10L water, stirring to obtain a suspension, sequentially extracting with petroleum ether, dichloromethane and ethyl acetate for three times, and concentrating under reduced pressure to obtain 75g of petroleum ether fraction, 165g of dichloromethane fraction and 80g of ethyl acetate fraction.

The dichloromethane fraction is processed by MCI column chromatography, and eluted sequentially with 50, 60, 70, 80, and 95% ethanol-water to obtain seven fractions. According to the separation and purification method of the compounds in the pholidota chinensis and the physalis pubescens, the separation and purification are carried out by repeatedly using the means of silica gel column chromatography, Sephadex LH-20 gel column chromatography, preparative HPLC and the like to obtain 33 9,10-dihydrophenanthrene compounds, wherein the compounds XF-1-11, 24 and 29 are 9, 10-dihydrophenanthrene/phenanthrene monomers, and the compounds XF-14-20 and 33 are 9, 10-dihydrophenanthrene/phenanthrene dimers.

6. Spectral data of the compound

XF-1:Juncuenin H

Colorless square crystals (acetone); ESIMS M/z 249.2[ M-H ]]^-；HRESIMS m/z 249.1302[M-H]^-(calcd for C18H17O,249.1279).¹H NMR(400MHz,CDCl₃)δ6.75and 7.48(each 1H,d, J＝8.4Hz,H-3,4),7.51and 7.13(each 1H,d,J＝7.9Hz,H-5,6),6.79(1H,dd,J＝11.4, 17.9Hz,H-11),5.61(1H,dd,J＝11.4,2.1Hz,H-12a),5.24(1H,dd,J＝17.9,2.1Hz,H- 12b),2.91and.276(each 2H,m,H-9,10),2.26and 2.34(each 3H,s,1-,7-Me),5.16(1H, br.s,2-OH).

XF-2:Juncuenin I

Light yellow powder; ESIMS M/z 251.1[ M-H ]]^-；HRESIMS m/z 251.1049[M-H]^-(calcd for C17H15O2,251.1072).¹H NMR(400MHz,acetone-d₆)δ6.86and 7.60(each 1H,d,J＝8.4 Hz,H-3,4),8.12and 7.18(each 1H,s,H-5,8),10.28(1H,s,6-CHO),2.84(4H,m,H-9,10), 2.21and 2.63(each 3H,s,1-,7-Me),8.42(1H,br.s,2-OH).

XF-3:Juncuenin J

Light yellow powder; ESIMS M/z 297.1[ M-H ]]^-；HRESIMS m/z 297.1501[M-H]^-(calcd for C19H21O3,297.1491).¹H NMR(400MHz,acetone-d₆)δ6.77and 6.95(each 1H,d,J＝ 8.3Hz,H-3,4),6.95(1H,s,H-6),2.86and 2.40(each 2H,m,H-9,10),2.20and 2.24 (each 3H,s,1-,8-Me),4.80(1H,q,J＝6.3Hz,H-11),1.50(3H,d,J＝6.3Hz,H-12), 2.88(3H,s,-OMe),8.20and 8.02(each 1H,br.s,2-,7-OH).

XF-4:Juncuenin K

Light yellow powder; ESIMS M/z 281.3[ M + H ]]⁺；HRESIMS m/z 281.1533[M+H]⁺(calcd for C19H21O2,281.1542).¹H NMR(400MHz,CDCl₃)δ6.81and 7.49(each 1H,d,J＝8.6 Hz,H-3,4),7.09(1H,s,H-5),6.75(1H,dd,J＝14.1,17.9Hz,H-11),5.61(1H,dd,J＝ 14.1,2.0Hz,H-12a),5.20(1H,dd,J＝17.9,2.0Hz,H-12b),2.74and 2.79(each 2H,m, H-9,10),2.21and 2.24(each 3H,s,1-,7-Me),4.70(1H,br.s,6-OH),3.86(3H,s,2-OMe).

XF-5:Juncuenin L

Light yellow powder; ESIMS M/z 310.1[ M-H ]]^-；HRESIMS m/z 310.1092[M-H]^-(calcd for C18H16NO4,310.1079).¹H NMR(400MHz,CDCl₃)δ8.46and 6.68(each 1H,s,H-4,8), 6.79(1H,dd,J＝18.5,14.4Hz,H-11),5.60(1H,dd,J＝14.4,1.2Hz,H-12a),5.20(1H,dd,J＝ 18.5,1.2Hz,H-12b),2.67and 2.79(each 2H,m,H-9,10),2.32and 2.28(each 3H,s,1-,6-Me), 11.10and 4.70(each 1H,br.s,2-,7-OH).

XF-6:Juncuenin M

Light yellow powder; ESIMS M/z 372.1[ M-H ]]^-；HRESIMS m/z 372.1624[M-H]^-(calcd for C24H22NO3,372.1600).¹H NMR(400MHz,CDCl₃)δ6.64and 7.13(each 1H,d,J＝8.4 Hz,H-3,4),6.76(1H,dd,J＝11.3,17.8Hz,H-11),5.62(1H,dd,J＝11.3,1.9Hz,H- 12a),5.22(1H,dd,J＝17.8,1.9Hz,H-12b),2.66(4H,m,H-9,10),2.26and 2.29(each 3H,s,1-,7-Me),9.40(1H,s,1’-CHO),6.96and 6.24(each 1H,dd,J＝2.6,3.6Hz,H-2’, 3’),4.15(2H,s,H-5’),5.11and 5.23(each 1H,br.s,2-,6-OH),9.68(1H,s,NH).

XF-7:Dehydrojuncuenin F

Light yellow powder; ESIMS M/z 246.9[ M-H ]]^-；HRESIMS m/z 247.1145[M-H]^-(calcd for C18H15O,247.1123).¹H NMR(400MHz,CDCl₃)δ7.18and 8.44(each 1H,d,J＝8.9 Hz,H-3,4),8.44and 7.46(each 1H,d,J＝8.5Hz,H-5,6),8.12and 7.88(each 1H,d,J＝ 9.5Hz,H-9,10),7.07(1H,dd,J＝14.4,17.9Hz,H-11),5.79(1H,dd,J＝14.4,2.0Hz, H-12a),5.43(1H,dd,J＝17.9,2.0Hz,H-12b),2.59and 2.49(each 3H,s,1-,7-Me),5.48 (1H,br.s,2-OH).

XF-8:Dehydrojuncuenin G

Light yellow powder; ESIMS M/z 249.1[ M-H ]]^-；HRESIMS m/z 249.0881[M-H]^-(calcd for C17H13O2,249.0916).¹H NMR(400MHz,acetone-d₆)δ7.38and 8.60(each 1H,d,J＝ 8.9Hz,H-3,4),9.12and 7.74(each 1H,s,H-6,8),7.76and 8.08(each 1H,d,J＝9.2Hz, H-9,10),2.58and 2.77(each 3H,s,1-,7-Me),10.42(1H,s,6-CHO),8.73(1H,br.s,2- OH).

XF-9:Dehydrojuncuenin H

Light yellow powder; ESIMS M/z 265.2[ M + H ]]⁺；HRESIMS m/z 265.1207[M+H]⁺(calcd for C18H17O2,265.1229).¹H NMR(400MHz,CDCl₃)δ7.25and 8.65(each 1H,d,J＝9.3 Hz,H-3,4),7.19(2H,s,H-6,8),7.60and 7.91(each 1H,d,J＝9.2Hz,H-9,10),7.47 (1H,dd,J＝10.7,17.3Hz,H-11),5.78(1H,dd,J＝17.3,1.6Hz,H-12a),5.46(1H,dd,J ＝10.7,1.6Hz,H-12b),2.61(3H,s,1-Me),3.97(3H,s,2-OMe),5.10(1H,br.s,7-OH).

XF-10:Dehydrojuncuenin I

Light yellow powder; ESIMS M/z 279.1[ M + H ]]⁺；HRESIMS m/z 279.1035[M+H]⁺(calcd for C18H15O3,279.1021).¹H NMR(400MHz,acetone-d₆)δ7.23and 8.82(each 1H,d,J＝ 9.3Hz,H-3,4),8.04(1H,s,H-5),8.18and 8.40(each 1H,d,J＝9.5Hz,H-9,10),7.14 (1H,dd,J＝11.5,17.9Hz,H-11),5.85(1H,dd,J＝11.5,2.0Hz,H-12a),5.46(1H,dd,J ＝17.9,2.0Hz,H-12b),2.39(3H,s,7-Me),11.01(1H,s,1-CHO),12.94and 9.02(each 1H,br.s,2-,6-OH).

XF-11:Dehydrojuncuenin J

Light yellow powder; ESIMS M/z 297.1[ M-H ]]^-；HRESIMS m/z 297.0682[M-H]^-(calcd for C18H14O2Cl,297.0682).¹H NMR(400MHz,CDCl₃)δ8.39and 8.79(1H,s,H-4,5), 7.99and 7.68(each 1H,d,J＝9.5Hz,H-9,10),7.05(1H,dd,J＝11.4,17.9Hz,H-11), 5.82(1H,dd,J＝11.4,2.0Hz,H-12a),5.40(1H,dd,J＝17.9,2.0Hz,H-12b),2.63and 2.41(each 3H,s,1,7-Me),5.81and 5.18(each 1H,br.s,2-,6-OH).

XF-14:Dijuncuenin C

Light yellow powder; ESIMS M/z 529.2[ M-H ]]^-；HRESIMS m/z 529.2370[M-H]^-(calcd for C36H33O4,529.2379).¹H NMR(400MHz,CDCl₃)δ7.45(2H,s,H-4,4’),7.05(2H,s, H-5,5’),6.76(2H,dd,J＝17.9,11.4Hz,H-11,11’),5.63(2H,dd,J＝11.4,1.9Hz,H-12a, 12’a),5.22(2H,dd,J＝17.9,1.9Hz,H-12,12’a),2.84(4H,m,H-9,9’),2.80(4H,m,H- 10,10’),2.32(6H,s,1-,1’-Me),2.23(6H,s,7-,7’-Me),5.36(2H,s,3-,3’-OH),4.73(2H, br.s,6-,6’-OH).

XF-15:Dijuncuenin D

Light yellow powder; ESIMS M/z 529.1[ M-H ]]^-；HRESIMS m/z 529.2391[M-H]^-(calcd for C36H33O4,529.2379).¹H NMR(400MHz,CDCl₃)δ7.45(2H,s,H-4,4’),6.67(2H,s, H-8,8’),6.86(2H,dd,J＝17.9,11.4Hz,H-11,11’),5.47(2H,dd,J＝11.4,1.3Hz,H-12a, 12’a),5.14(2H,dd,J＝17.9,1.3Hz,H-12b,12’b),2.70(4H,m,H-9,9’),2.76(4H,m,H- 10,10’),2.33(6H,s,1-,1’-Me),2.23(6H,s,6-,6’-Me),5.43(2H,s,2-,2’-OH),5.99(2H, br.s,7-,7’-OH).

XF-16:Dijuncuenin E

Light yellow powder; ESIMS M/z 529.3[ M-H ]]^-；HRESIMS m/z 529.2358[M-H]^-(calcd for C36H33O4,529.2379).¹H NMR(400MHz,CDCl₃)δ7.29and 7.68(each 1H,s,H-4,5), 6.91(1H,dd,J＝11.5,17.5Hz,H-11),5.82(1H,dd,J＝17.3,0.7Hz,H-12a),5.40(1H, dd,J＝11.5,0.7Hz,H-12b),2.74(4H,m,H-9,10),2.36and 2.26(each 3H,s,1-,7-Me), 7.49and 6.68(each 1H,s,H-4’,8’),6.86(1H,dd,J＝17.9,11.3Hz,H-11’),5.46(1H,dd, J＝11.3,1.8Hz,H-12’a),5.14(1H,dd,J＝17.9,1.8Hz,H-12’b),2.74(4H,m,H-9’,10’), 2.31and 2.23(each 3H,s,1’,6’-Me).

XF-17:Dijuncuenin F

Light yellow powder; ESIMS M/z 527.1[ M-H ]]^-；HRESIMS m/z 527.2231[M-H]^-(calcd for C36H31O4,527.2222).¹H NMR(400MHz,CDCl₃)δ7.50and 7.00(each 1H,s,H-4,5), 6.78(1H,dd,J＝11.4,17.9Hz,H-11),5.65(1H,dd,J＝11.4,1.9Hz,H-12a),5.25(1H, dd,J＝17.9,1.9Hz,H-12b),2.89(4H,m,H-9,10),2.30and 2.22(each 3H,s,1-,7-Me), 4.93and 4.85(each 1H,s,2-,6-OH),6.59and 7.66(each 1H,d,J＝9.3Hz,H-3’,4’),8.11 and 7.79(each 1H,d,J＝9.5Hz,H-9’,10’),7.14(1H,dd,J＝17.9,11.4Hz,H-11’),5.88 (1H,dd,J＝11.4,1.9Hz,H-12’a),5.46(1H,dd,J＝17.9,1.9Hz,H-12’b),2.54and 2.45 (each 3H,s,1’,7’-Me),5.13and 5.65(each 3H,s,2’,6’-OH).

XF-18:Dijuncuenin G

Light yellow powder; ESIMS M/z 527.2[ M-H ]]^-；HRESIMS m/z 527.2234[M-H]^-(calcd for C36H31O3,527.2222).¹H NMR(400MHz,CDCl₃)δ7.58and 6.71(each 1H,s,H-4,8), 6.71(1H,dd,J＝11.3,17.9Hz,H-11),5.13(1H,dd,J＝11.3,1.9Hz,H-12a),5.05(1H, dd,J＝17.9,1.9Hz,H-12b),2.80(4H,m,H-9,10),2.28and 2.19(each 3H,s,1-,6-Me), 4.78and 4.90(each 1H,s,2-,7-OH),6.59and 7.70(each 1H,d,J＝9.3Hz,H-3’,4’),8.10 and 7.78(each 1H,d,J＝9.5Hz,H-9’,10’),7.13(1H,dd,J＝17.9,11.4Hz,H-11’),5.87 (1H,dd,J＝11.4,1.9Hz,H-12’a),5.46(1H,dd,J＝17.9,1.9Hz,H-12’b),2.55and 2.45 (each 3H,s,1’-,7’-Me),4.96and 5.70(each 3H,s,2’-,6’-OH).

XF-19:Dijuncuenin H

Light yellow powder; ESIMS M/z 527.3[ M-H ]]^-；HRESIMS m/z 527.2233[M-H]^-(calcd for C36H31O4,527.2222).¹H NMR(400MHz,CDCl₃)δ7.32and 6.81(each 1H,s,H-3,6), 6.72(1H,dd,J＝10.9,15.1Hz,H-11),5.42(1H,dd,J＝10.9,1.1Hz,H-12a),4.83(1H, dd,J＝15.1,1.1Hz,H-12b),2.82(4H,m,H-9,10),2.33and 2.28(each 3H,s,1-,8-Me), 6.62and 7.70(each 1H,d,J＝9.3Hz,H-3’,4’),8.10and 7.79(each 1H,d,J＝9.5Hz,H- 9’,10’),7.12(1H,dd,J＝15.9,11.4Hz,H-11’),5.87(1H,dd,J＝11.4,1.9Hz,H-12’a), 5.46(1H,dd,J＝15.9,1.9Hz,H-12’b),2.55and 2.44(each 3H,s,1’-,7’-Me).

XF-20:Dijuncuenin I

Light yellow powder; ESIMS M/z 525.3[ M-H ]]^-；HRESIMS m/z 525.2084[M-H]^-(calcd for C36H29O4,525.2066).¹H NMR(400MHz,CDCl₃)δ7.07and 9.04(each 1H,d,J＝9.3 Hz,H-3,4),7.37and 7.72(each 1H,d,J＝9.4Hz,H-9,10),7.43(1H,dd,J＝11.3,18.0 Hz,H-11),5.82(1H,dd,J＝11.3,1.6Hz,H-12a),5.44(1H,dd,J＝18.0,1.6Hz,H-12b), 2.49(6H,s,1-,8-Me),4.99and 5.13(each 1H,br.s,2-,7-OH),6.36and 7.37(each 1H,d, J＝9.4Hz,H-3’,4’),8.18and 7.82(each 1H,d,J＝9.4Hz,H-9’,10’),7.19(1H,dd,J＝ 17.9,11.4Hz,H-11’),5.92(1H,dd,J＝11.4,1.8Hz,H-12’a),5.52(1H,dd,J＝17.9,1.8 Hz,H-12’b),2.52and 2.46(each 3H,s,1’-,7’-Me),4.77and 5.30(each 1H,br.s,2’-,6’- OH).

XF-24:2, 7-dihydroxy-1, 8-dimethyl-5-vinyl-9, 10-dihydrophenanthrene

White amorphous powder, C₁₈H₁₈O₂,ESIMS m/z：289.3[M+Na]⁺；¹H NMR(400MHz, CDCl₃):δ6.66(1H,d,J＝8.5Hz,H-3),7.25(1H,d,J＝8.5Hz,H-4),6.87(1H,s,H-6), 2.69(4H,br.s,H-9,10),6.90(1H,dd,J＝10.8,17.4Hz,H-11),5.16(1H,dd,J＝10.8, 1.2Hz,H-12a),5.58(1H,dd,J＝17.4,1.2Hz,H-12b),2.26and 2.30(3H,s,1-,8-Me).

XF-29 Dehydrojuncunin B (2, 7-dihydroxy-1, 6-dimethyl-5-vinyl-phenanthrene)

White amorphous powder, C₁₈H₁₆O₂,ESIMS m/z：287.3[M+Na]⁺；¹H NMR(400MHz, DMSO-d₆)δ7.22and 8.18(each 1H,d,J＝8.6Hz,H-3,4),7.90(1H,s,H-5),7.85and 7.63(each 1H,d,J＝9.2Hz,H-9,10),7.09(1H,dd,J＝18.4,12.0Hz,H-11),5.79(1H, dd,J＝12.8,1.6Hz,H-12a),5.35(1H,dd,J＝12.8,1.6Hz,H-12b),2.44and 2.28(each 3H,s,1-,6-Me).

XF-33:8- [4- (2, 7-dihydroxy-1, 6-dimethyl-5-vinyl-9, 10-dihydrophenanthrene) yl ] -2, 7-dihydroxy-1, 6-dimethyl-5-vinyl-phenanthrene

White amorphous powder, C₃₆H₃₂O₄,ESIMS m/z:551.3[M+H]⁺,¹H NMR(400MHz, CDCl₃)δ7.02and 8.87(each 1H,d,J＝9.2Hz,H-3,4),7.37and 7.78(each 1H,d,J＝ 9.6Hz,H-9,10),7.32(1H,dd,J＝18.0,11.2Hz,H-11),5.76(1H,dd,J＝18.0,1.6Hz, H-12a),5.38(1H,dd,J＝11.2,1.6Hz,H-12b),2.56and 2.52(each 3H,s,1-,6-Me),7.50 and 6.70(each 1H,s,H-4’,8’),2.85and 2.76(each 2H,m,H-9’,10’),6.83(1H,dd,J＝ 18.0,11.2Hz,H-11’),5.36(1H,dd,J＝2.0,18.0Hz,H-12’a),5.11(1H,dd,J＝2.0,11.2 Hz,H-12’b),2.53and 2.20(each 3H,s,1’-,6’-Me).

EXAMPLE 2 Effect of Compounds on combating hydrophobic cholic acid hepatotoxicity

Cholestasis is a common complication of drug-induced liver injury, accompanied by accumulation of hydrophobic cholic acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA), in hepatocytes, which are capable of disrupting the outer membrane of the cell basement, the membrane of organelles, and specifically the outer layer of the microtubule membrane, one of the major endogenous substances responsible for hepatocyte injury. The hydrophobic cholic acid-induced hepatocyte injury model can be used for primarily evaluating the effect of the compound on cholic acid toxicity resistance, so as to speculate the protective effect of the compound on a type of hepatic injury accompanied with cholestasis symptoms, such as hepatic injury caused by medicines, especially Chinese herbal medicines.

1. Experimental protocol

1.1 Compounds against DCA-induced injury of rat Primary hepatocytes

Separating rat primary hepatic cells by two-step perfusion method according to 3 × 10⁵The cells were inoculated at a density of one/ml in a 96-well plate and administered 4h after adherence. After incubating rat primary hepatocytes with 200. mu.M DCA and the corresponding compounds for 24h, the cell viability was determined by the CCK8 method. The absorbance at 450nm is measured by a multifunctional microplate reader, and the absorbance is in direct proportion to the cell activity. The protection effect of the compound is judged according to the activity of the cells.

1.2 Compounds against DCA-induced Hihep cell injury

Culturing the hihep cells in HMM (heparin) medium,when the cells grow to 90% confluence, the cells are grown at 1X 10⁵The cells were inoculated in 96-well plates at a density of one/ml, and administered after 3 days of culture. The hhep cells were incubated with 250 μ M DCA and the corresponding compound for 24 h. Cell viability was then determined by the CCK8 method, which was performed as described above.

1.3 Compounds against LCA-induced Hihep cell injury

The hihep was cultured as described above. The hhep cells were incubated with 30 μ M LCA and the corresponding compounds for 24 h. Cell viability was then determined by the CCK8 method, which was performed as described above.

The above experiment was repeated three times, and the results of the administration group showing high cell viability and a statistical difference (P <0.05) between the two compared with the cholic acid-administered group alone (DCA and LCA) were considered to be active; any of the three models, active, were considered to be capable of antagonizing the hydrophobic bile acid hepatotoxicity.

2. Results of the experiment

The experimental result shows that the tested 9,10-dihydrophenanthrene compound shows a relatively obvious effect of resisting the hepatotoxicity of hydrophobic cholic acid in the concentration range of 0.1-25 uM, has a potential liver protection effect, and especially has a potential protection effect on liver injury accompanied with cholestasis symptoms, such as liver injury caused by medicines, especially Chinese herbal medicines. The results of the specific experiments are shown in table 1.

EXAMPLE 3 protective Effect of Compound YS30 on liver injury of mouse CCl4

CCl₄The induced mouse acute liver injury model is one of the common models for evaluating the drug to resist the acute liver injury before clinic. The mechanism of injury is CCl₄The liver is metabolized into free radicals by metabolic enzymes such as CYP2E1 and CYP2B, and the free radicals are bound with nucleic acids, proteins and lipid nodules in liverSynthesis results in denaturation of nucleic acids, hindered protein synthesis, and lipotropy. The pathological changes can lead to cell death, release damage mode factors (DAMPs), cause activation of immune cells, induce inflammatory responses and cause downstream liver injury. Oxidative stress and inflammation are two important pathological processes of liver injury, and the model can be used for evaluating the protective effect of the compound on hepatitis with inflammation and oxidative stress as main pathological bases. Including toxic hepatitis (drugs, chemicals and biotoxins), alcoholic hepatitis, and liver damage caused by ischemic hepatitis.

1. Experimental protocol

1.1CCl4 model of acute liver injury

ICR mice were 60, randomized into 7 groups of 8: the vehicle group, YS30 high dose (40 mg/kg. day) single administration group, CCl4 acute model group, CCl4 model group and positive drug bicyclol group (200 mg/kg. day), CCl4 model group and YS30 low dose (10 mg/kg. day), medium dose (20 mg/kg. day) and high dose (40 mg/kg. day). YS30 was gavaged continuously for 7 days at the corresponding dose, once a day. The corresponding doses of bicyclol were administered by gavage for 3 consecutive days. The vehicle group and CCl4 acute modeling group were perfused with vehicle 0.5% Tween 80 for 7 days. 1h after the last administration, 0.5% CCl4 olive oil solution (10ml/kg) was injected subcutaneously. The vehicle group and the YS 30-alone group were injected subcutaneously with the corresponding volume of olive oil. All mice were fasted, had free access to water and samples were taken 16h after molding. Anesthetizing a mouse with sodium pentobarbital, collecting blood from eyeballs, dissecting the mouse, collecting liver, cleaning a part of the liver with normal saline, quickly freezing the part in liquid nitrogen, and storing the part at-80 ℃ for subsequent detection of biochemical indexes. The remaining part was soaked in formalin for hematoxylin-eosin staining.

1.2 detection of blood Biochemical indicators

Standing the collected mouse blood sample for 30min, centrifuging at 8000rpm for 10min, and collecting serum. The ALT, AST and LDH contents in blood were measured by a fully automatic hemocytometer (SYSMEX JCA-BM 6010C).

1.3H & E staining

The same liver lobe of the mouse was taken and soaked in formalin for histopathological analysis. The same part of liver tissue is taken for paraffin embedding, 3um sections are obtained for H & E staining, pathological changes of the liver are observed by a microscope, and congestion, vacuole, necrosis and inflammation of the liver tissue are comprehensively evaluated by a Suzuki's scoring method.

1.4 detection of inflammatory factors

Detection of inflammatory factor content in mouse serum by ELISA kit of inflammatory factors (TNF-alpha and IL-6) of syndromic organisms

1.5MDA level detection

The level of MDA in the tissue sample was detected using the pecan MDA kit. Taking 20-30mg liver tissue samples, adding precooled PBS (phosphate buffer solution) for homogenate according to the ratio of 1:9, centrifuging at 12000rpm/min at 4 ℃ for 10min, taking supernatant, detecting the concentration of corresponding protein by using a BCA (burst cutting edge) kit, and then detecting the MDA content in the tissues according to the MDA kit instruction.

2. Results of the experiment

The experimental results show that YS30 administered orally (10,20,40mg/kg) can reduce the levels of ALT, AST and LDH in liver injury mice dose-dependently (FIGS. 1A-C) and improve the pathological changes of liver tissues (FIGS. 1D-1E). It is shown that YS30 can obviously improve liver injury induced by CCl 4.

The influence of YS30 on the level of inflammatory factors in serum and the level of lipid peroxidation in liver is further detected, and the result shows that (figures 2A-C), YS30 has obvious improvement effect on the levels of inflammatory factors TNF-alpha and IL-6 in acute liver injury and liver lipid peroxidation (MDA), which indicates that YS30 has potential protection effect on liver injury of a type taking inflammation and oxidative stress as main pathological bases, such as toxic hepatitis (drugs, chemical substances and biological toxins), alcoholic hepatitis and ischemic hepatitis.

EXAMPLE 4 protective Effect of Compound YS30 on ischemia-reperfusion liver injury in mice

Clinically, ischemia reperfusion injury of the liver occurs in the case of liver transplantation, hepatectomy, trauma, hemorrhagic shock, and other systemic hypovolemic diseases such as sepsis, respiratory failure, congestive heart failure, and the like. The mechanism of this injury is associated with oxidative stress during ischemia and explosive inflammatory responses during reperfusion. A mouse 70% thermal ischemia reperfusion model is adopted, so that the clinical perioperative liver injury can be simulated, and the protective effect of the compound on the liver injury can be evaluated.

The compounds of the invention all have antioxidant or/and anti-inflammatory activity similar to that of YS30 in vitro,

1. experimental protocol

1.1 Small ischemia reperfusion liver injury model

C57/BL mice were divided into 3 groups: sham group (Sham group), ischemia reperfusion group (I/R), YS30 administration group (I/R + YS 30). YS 3040 mg/kg was dissolved with physiological saline, administered via caudal vein 24h, 12h, 1h before operation, respectively, a 70% ischemia reperfusion model was constructed according to literature reports, after a mouse was anesthetized with 4% chloral hydrate (intraperitoneal injection), the mouse was opened along the midline of the abdomen, the hepatic artery, hepatic portal vein, bile duct were isolated, the hepatic left lobe and hepatic middle lobe hepatic artery, hepatic portal vein, bile duct were clamped with a noninvasive microscopic hemostatic clamp to cause 70% ischemia of the liver, after 60min of ischemia, the noninvasive hemostatic clamp was removed, blood supply was restored, abdominal cavity was sutured, and each group of blood samples and tissue samples were collected after reperfusion for 6 h.

1.2 blood Biochemical index detection

The method is as above

1.3H & E staining

The method is as above

2. Results of the experiment

The result is shown in fig. 3, and the experimental result shows that YS30 can significantly improve the liver injury of the mouse caused by ischemia-reperfusion, has significant inhibitory effect on ALT, AST and LDH levels in the serum of the mouse, and can significantly improve the pathological change of liver tissue, which indicates that YS30 has an application prospect in resisting the liver injury in the clinical perioperative period.

Example 59 Effect of 10-dihydrophenanthrenes on oxidative stress and inflammation in the pathological course of liver injury

1. Evaluation of antioxidant Activity of 9,10-dihydrophenanthrene Compound

1.1 protocol

DPPH free radical scavenging experiments: weighing a certain amount of DPPH reagent, dissolving with absolute ethanol to prepare 0.2mM DPPH absolute ethanol solution, and placing in a refrigerator at 4 ℃ for later use. The test compound was dissolved in DMSO to form a 100 mM stock solution, which was diluted in absolute ethanol at a ratio of 1:1000 to form a 100uM test solution. 100ul of the test compound (100uM) or the positive drug vitamin C (100uM) and 100ul of DPPH free radical ethanol solution (0.2mM) were added to a 96-well plate, and a DPPH free radical group without drug (100 ul of absolute ethanol was used instead) was set at the same time. The positive drug and the compound to be tested are provided with corresponding negative control holes without adding DPPH free radical (100 ul of absolute ethyl alcohol is used for replacing DPPH free radical), so that the influence of the self color of the test sample on the reading is deducted. After mixing, the mixture was allowed to stand at room temperature for 30min, and then the absorbance at 517nm was measured.

DPPH radical scavenging Rate ═ OD_DPPH-control-(OD_DPPH-sample-OD_{sample-control})]/OD_DPPH-control*100％

Wherein, OD_DPPH-controlMean OD values of the groups without compound and DPPH

OD_DPPH-sampleOD values of the Compound + DPPH group

OD_{sample-control}OD value of the group without DPPH addition

1.2 results of the experiment

Silymarin (100 μ M) has a DPPH radical clearance of about 35%, we specify that DPPH radical clearance is superior to silymarin, and compounds with statistical differences (p <0.05) are strong antioxidants. The results show that of the 46 compounds tested, 39 (100. mu.M) had DPPH radical clearance greater than 35%, defined as a strong antioxidant effect. The results are shown in Table 2.

2. Evaluation of anti-inflammatory Activity of 9,10-dihydrophenanthrene Compound

2.1 protocol

Mouse bone marrow macrophages (BMDM) were isolated and monocyte colonies were induced for one week with high glucose DMEM medium containing 15% L929 cell supernatant. Cells were seeded into 96-well plates at a density of 5X 10⁵One per ml. And adding corresponding medicines for incubation after 24 hours. The positive drug or the test compound 10mM solution was dissolved in 10% FBS-containing high-glucose DMEM medium at a ratio of 1: 1000. The test drug (10. mu.M) andBMDM was incubated at 1ug/ml LPS and after 24h the supernatant was collected for NO detection. The level of NO reflects the intensity of the inflammatory response in the system. Thereby judging the anti-inflammatory effect of the compound.

2.2 results of the experiment

The positive drug dexamethasone (10 μ M) can significantly inhibit the release of NO from BMDM, the group administered is compared with the LPS-induced group, the NO level after administration is lower than that of the LPS-induced group, and the compound with the statistical difference (p <0.05) between the two is defined as having anti-inflammatory effect. The results show that 21 of the 46 compounds tested had significant anti-inflammatory effects. The results are shown in Table 2.

Table 2: antioxidant and anti-inflammatory effects of 9,10-dihydrophenanthrene compounds

The test results show that the 9,10-dihydrophenanthrene compound with the liver protection activity has similar anti-oxidation or/and anti-inflammatory properties to YS30, and has potential protection effects on liver injuries of the type which take inflammation and oxidative stress as main pathological bases, such as toxic hepatitis (drugs, chemical substances and biological toxins), alcoholic hepatitis and ischemic hepatitis.

And (4) conclusion:

the mouse acute liver injury model induced by CCl4 is one of the common models for pre-clinical evaluation of hepatotoxicity of drugs against heterologous substances (such as alcohol, drugs, chemical poisons). The damage mechanism is that CCl4 is metabolized into free radicals in liver by metabolic enzymes such as CYP2E1 and CYP2B, and the free radicals are combined with nucleic acid, protein and lipid in liver to cause nucleic acid denaturation, protein synthesis obstruction and fat degeneration. Such pathological changes lead to cell death, release of damage mode factors (DAMPs), activation of immune cells, induction of inflammatory responses and subsequent outbreak of liver damage downstream. Through in vivo efficacy verification, we find that a representative compound YS30(gigantol) has a good protective effect on acute liver injury induced by CCl4 at an animal level, can obviously reduce the elevation of ALT, AST and LDH caused by liver injury (figure 1A), and find that YS30 can improve pathological changes such as vacuole, necrosis and inflammation in liver tissues by histopathological detection (figure 1B). In addition, animal experiment results show that YS30 can reduce the level of inflammatory factors and lipid peroxidation in liver tissues (figure 2), and the potential therapeutic effect is suggested to the hepatitis of a type based on inflammation and lipid peroxidation as pathology, including toxic hepatitis (drugs, chemical substances and biological toxins), alcoholic hepatitis, ischemic hepatitis and the like.

To verify this hypothesis, we further examined the in vivo hepatoprotective efficacy of YS30 in a mouse ischemia reperfusion injury model. Clinically, ischemia reperfusion injury of the liver occurs in the cases of liver transplantation, hepatectomy, trauma, hemorrhagic shock, and other systemic low blood flow diseases such as sepsis, respiratory failure, congestive heart failure, etc. The mechanism of this injury is associated with oxidative stress during ischemia and explosive inflammatory responses during reperfusion. The experimental result shows that YS30 obviously improves the elevation of ALT, AST and LDH in the serum of the mouse caused by ischemia-reperfusion and the histopathological change of the liver. YS30 can be seen to have a potential therapeutic effect on liver injury in the clinical liver perioperative period.

In conclusion, the 9,10-dihydrophenanthrene compound has a good liver protection effect and has potential application values in treatment of drug-induced liver injury, toxic hepatitis, alcoholic hepatitis and liver injury in a perioperative period of the liver accompanied by bile stasis symptoms.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it will be appreciated that various changes or modifications may be made by those skilled in the art after reading the above teachings of the present invention, and such equivalents are intended to fall within the scope of the appended claims.

Claims

1. Use of a compound of formula I, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein the content of the first and second substances,

the dotted line represents a chemical bond or nothing;

m is selected from the group consisting of: 0.1 or 2;

Substituted or unsubstituted, substituted or unsubstituted

Substituted or unsubstituted

2. The use according to claim 1, wherein R is_dSubstituted with one or more substituents selected from the group consisting of: H. hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl.

3. The use according to claim 1, wherein the compound has the structure shown in formula I-a or I-b:

4. the use according to claim 1, wherein said compound is selected from the group consisting of:

5. use of a compound of formula II, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

wherein the dotted line represents a bond or nothing;

6. The use according to claim 5, wherein the compound is selected from the group consisting of:

7. the use of claim 1 or 5, wherein the liver injury is selected from the group consisting of: liver damage with symptoms of cholestasis, or hepatitis.

8. The use of claim 7, wherein the hepatitis is selected from the group consisting of: toxic hepatitis (drug, chemical and biological toxin), alcoholic hepatitis, and ischemic hepatitis.

9. The use of claim 7, wherein the liver injury is drug induced liver injury, preferably, liver injury is chinese herbal induced liver injury.

10. A compound selected from the group consisting of: