9, 10-dihydrophenanthrene compound and application thereof in liver injury treatment

Abstract

The invention provides a 9, 10-dihydrophenanthrene compound and application thereof in liver injury treatment, and particularly provides a compound shown as a formula I or a compound shown as a formula II, which shows anti-inflammatory and antioxidant activity in liver tissues, so that the 9, 10-dihydrophenanthrene compound can be used for treating liver injury diseases, such as liver injury diseases and liver injury diseases. Particularly, the liver injury diseases with inflammation and lipid peroxidation as pathological basis can be treated or prevented. # imgabs0 #

Description

[0001] This divisional application is for a Chinese patent application with the application number 202010087201.5, the filing date of February 11, 2020, and the invention title of "9,10-Dihydrophenanthrene Compounds and Their Use in the Treatment of Liver Injury". Technical Field

[0002] The present invention belongs to the field of medicinal chemistry. More specifically, it relates to a new class of 9,10-dihydrophenanthrene compounds, their preparation methods, and their use in drugs for the treatment of liver injury. Background Art

[0003] The liver is not only involved in the storage of energy and the synthesis and catabolism of various substances, including carbohydrate metabolism, protein metabolism, fat metabolism, etc., but also participates in the detoxification process of toxins and drug catabolites produced by human metabolism. As the "central hub of material metabolism", the liver is also an organ frequently invaded by various pathogenic factors or diseases. For example, drugs, viruses, alcohol, abnormal metabolism, ischemia-reperfusion, etc. can all cause damage to the liver and trigger an inflammatory response. Acute and chronic hepatitis can ultimately progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma. The high incidence of primary liver cancer globally is mainly related to the high incidence of acute and chronic hepatitis.

[0004] Currently, there are various types of common liver-protecting drugs in clinical practice. Among them, injection agents are used most frequently, such as magnesium isoglycyrrhizinate injection, reduced glutathione for injection, ademetionine 1,4-butanedisulfonate for injection, and polyene phosphatidylcholine injection. However, these drugs are relatively expensive and not suitable for long-term use. Oral dosage forms are inexpensive, convenient to use, have stable efficacy, and relatively few adverse reactions, making them suitable for long-term use by patients. However, the commonly used oral drugs in clinical practice have many defects. For example, silymarin has problems such as low bioavailability and large individual differences, while bicyclol has adverse reactions and withdrawal rebound phenomena. Therefore, the development of oral liver-protecting drugs with definite efficacy, safety, and high efficiency is of great significance for the treatment of acute and chronic liver injury in clinical practice.

[0005] Natural products are an important source for the creation of new drugs. Discovering active lead compounds from natural products and then developing innovative drugs has been proven to be an effective and important approach. 9,10-Dihydrophenanthrene compounds are a class of compounds commonly found in plants such as Orchidaceae and Juncaceae. Their structures contain more than 2 benzene rings and multiple phenolic hydroxyl groups, and they have aromaticity and certain hydrophobicity. Existing studies have shown that these compounds generally have biological activities such as anti-tumor, antispasmodic, antiplatelet aggregation, and anti-allergy, but the protective effect of such compounds on liver injury has not been reported in the literature. Summary of the Invention

[0006] The purpose of the present invention is to provide a class of 9,10-dihydrophenanthrene compounds or their derivatives that can be used for the protection of liver injury.

[0007] The first aspect of the present invention provides the use of a compound represented by the following formula I, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

[0008]

[0009] wherein,

[0010] The dotted line represents a chemical bond or none;

[0011] R a 、R b and R c are each independently selected from the group consisting of: H, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl, -CHO, -NO2;

[0012] n and p are each independently selected from the group consisting of: 0, 1, 2, 3 or 4;

[0013] m is selected from the group consisting of: 0, 1 or 2;

[0014] R d is C2-C4 alkenyl, or -(O) x -R; wherein, x is 0 or 1, and R is a structural fragment formed by removing a hydrogen atom from a structural unit selected from the group consisting of: substituted or unsubstituted substituted or unsubstituted substituted or unsubstituted substituted or unsubstituted

[0015] or R d and R a 、R b and R c together with any one of them and the carbon atoms to which they are respectively attached form a group selected from the group consisting of: substituted or unsubstituted 3-7 membered heterocyclic group, substituted or unsubstituted 3-7 membered cycloalkyl group;

[0016] Unless otherwise specified, the 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, -NO2, or a phenyl group substituted by one or more groups selected from the group consisting of: hydroxy, C1-C4 alkyl, C1-C4 alkoxy;

[0017] It is characterized by being used for preparing a pharmaceutical composition for treating or preventing liver injury.

[0018] In another preferred embodiment, the 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

[0019] In another preferred embodiment, the R d is substituted with one or more substituents selected from the group consisting of: H, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl.

[0020] In another preferred embodiment, the compound has a structure represented by the following formula I-a or I-b:

[0021]

[0022] In another preferred embodiment, the compound is selected from the group consisting of:

[0023]

[0024]

[0025] In a second aspect of the present invention, there is provided the use of a compound represented by the following formula II, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

[0026]

[0027] wherein the dashed line represents a chemical bond or none;

[0028] R d and R e each independently selected from the group consisting of: H, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl, -CHO, -NO2, or substituted or unsubstituted benzyl;

[0029] q, r each independently selected from the group consisting of: 0, 1, 2, 3 or 4;

[0030] Unless otherwise specified, the 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, -NO2, or phenyl substituted with one or more groups selected from the group consisting of: hydroxy, C1-C4 alkyl, C1-C4 alkoxy;

[0031] Characterized by being used for preparing a pharmaceutical composition for treating or preventing liver injury.

[0032] In another preferred embodiment, the compound is selected from the group consisting of:

[0033]

[0034] The third aspect of the present invention provides a use of a compound as described in the first or second aspect of the present invention, wherein the liver injury is selected from the following group: liver injury with cholestatic symptoms, or hepatitis.

[0035] In another preferred embodiment, the hepatitis is selected from the following group: toxic hepatitis (drugs, chemicals and biological toxins), alcoholic hepatitis, ischemic hepatitis.

[0036] In another preferred embodiment, the liver injury is drug-induced liver injury, preferably, the liver injury is caused by Chinese herbal medicine.

[0037] The fourth aspect of the present invention provides a compound selected from the following group:

[0038]

[0039]

[0040] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0041] Figure 1 : Protective effect of YS30 on acute CCl4-induced liver injury in mice. Levels of (A) ALT, (B) AST, and (C) LDH in mouse serum, and (D) pathological analysis of liver tissue after H&E staining. The square, arrow, and solid triangle indicate liver tissue swelling, centrilobular necrosis, and inflammatory infiltration, respectively. (E) Suzuki's score of H&E staining. ### P < 0.001, ## P < 0.01 for CCl4 group vs. Vehicle group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. CCl4 group.

[0042] Figure 2 : Effects of YS30 on levels of inflammatory factors in serum and liver lipid peroxidation. Levels of (A) TNF-α, (B) IL-6 in serum, and (C) lipid peroxidation level in liver tissue. ### P < 0.001, ## P < 0.01 vs Vehicle group, ***P < 0.001, **P < 0.01, *P < 0.01 vs CCl4 group.

[0043] Figure 3: Protective effect of YS30 on hepatic ischemia-reperfusion injury in mice. Levels of (A) ALT, (B) AST, (C) LDH in mouse serum, and (D) pathological analysis of liver tissue after H&E staining. Among them, the square, arrow, and solid triangle represent liver tissue swelling, central lobular necrosis, and inflammatory infiltration respectively. (E) Suzuki's score of H&E staining. ### P < 0.001, ## P < 0.01 I / R group vs. Sham group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. I / R group. Detailed implementation manners

[0044] Through long-term and in-depth research, the inventor of the present invention unexpectedly found that a class of 9,10-dihydrophenanthrene compounds as described in the present invention have good anti-inflammatory and antioxidant properties, and are particularly suitable for treating diseases such as liver diseases based on inflammation and lipid peroxidation as the pathological basis, including toxic hepatitis (drugs, chemicals, and biological toxins), alcoholic hepatitis, ischemic hepatitis, etc. Based on the above findings, the inventor completed the present invention.

[0045] Terms

[0046] As used herein, the term "C1-C6 alkyl" or "C1-C 10 alkyl" refers to a straight-chain or branched-chain alkyl having 1 to 6 or 1 to 10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or similar groups.

[0047] The term "C3-C6 cycloalkyl" refers to a cycloalkyl having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, methylcyclobutyl, cyclopentyl, or similar groups.

[0048] The term "halogen" refers to F, Cl, Br, and I.

[0049] In the present invention, the terms "comprising", "including" or "containing" mean that various components can be applied together to the mixtures or compositions of the present invention. Therefore, the terms "consisting essentially of..." and "consisting of..." are included in the term "comprising".

[0050] In the present invention, the term "pharmaceutically acceptable" component refers to a substance that is suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance having a reasonable benefit / risk ratio.

[0051] In the present invention, the term "effective amount" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or preventive effect. The precise effective amount for a particular subject depends on the subject's size and health status, the nature and severity of the disorder, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Accordingly, 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 judged by a clinician.

[0052] As used herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on a group are replaced by a substituent selected from the group consisting of: halogen, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C2-C6 acyl, unsubstituted or halogenated C1-C6 alkyl-hydroxy.

[0053] Unless otherwise specified, in the present invention, all compounds that appear are intended to include all possible optical isomers, such as compounds of a single chirality, or mixtures of various different chiral compounds (i.e., racemates). Among all compounds of the present invention, each chiral carbon atom can optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

[0054] As used herein, the term "compounds of the present invention" refers to the compounds represented by Formula I. This term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of the compounds of Formula I.

[0055] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention with an acid or a base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is the salts formed by the compounds of the present invention with acids. Acids suitable for forming 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, benzenesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

[0056] As used herein, the term "dimer" refers to a dimer formed by bibenzyl or stilbene, 9,10-dihydrophenanthrene or phenanthrene itself, or bibenzyl or stilbene, 9,10-dihydrophenanthrene or phenanthrene connected through C-O-C, or C-C, or simultaneously through C-O-C and C-C.

[0057] 9,10-Dihydrophenanthrene compounds or their analogs

[0058] In the early stage of this project, a series of 9,10-dihydrophenanthrene compounds were isolated from Pholidota chinensis Lindl. of the Orchidaceae family, Juncus effusus L. and Juncus setchuensis Buchen. of the Juncaceae family. Subsequently, we verified the hepatoprotective effects of these compounds in the hepatocyte injury model of endogenous toxic substances (hydrophobic bile acids), the mouse liver injury model of exogenous toxic substances (carbon tetrachloride), and the mouse liver injury model of ischemia-reperfusion.

[0059] Cholestasis is a common complication of drug-induced liver injury, accompanied by the accumulation of hydrophobic bile acids (such as deoxycholic acid (DCA) and lithocholic acid (LCA)) in hepatocytes. Hydrophobic bile acids can damage the cell basal outer membrane, organelle membranes, and specifically the outer layer of microtubule membranes, and are one of the main endogenous substances leading to hepatocyte injury. In primary rat hepatocytes and human-induced hepatic-like cells (hiHep cells), we found that these compounds could significantly counteract the hepatocyte injury caused by hydrophobic bile acids DCA and LCA in the concentration range of 0.1 - 25 μM. This suggests that these compounds may have a significant improvement effect on a type of liver injury accompanied by cholestasis symptoms, such as liver injury caused by drugs, especially Chinese herbal medicines.

[0060] The 9,10-dihydrophenanthrene compounds or their analogs isolated in the present invention specifically include:

[0061] The first category: Compounds YS1 - 37 isolated from Pholidota chinensis

[0062]

[0063]

[0064] The second category: Compounds XYW1 - 10 isolated from Juncus setchuensis Buchen.:

[0065]

[0066] The third category: Compounds XF1 - 11, 14 - 20, 24, 29, 33 isolated from Juncus effusus L.:

[0067]

[0068] Pharmaceutical compositions and administration methods

[0069] Since the compounds of the present invention have excellent activity in reversing liver injury, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used for treating, preventing, and alleviating various liver injury-related diseases, such as a type of hepatitis based on inflammation and lipid peroxidation.

[0070] The pharmaceutical composition of the present invention contains the compound of the present invention or its pharmaceutically acceptable salt and a pharmaceutically acceptable excipient or carrier within a safe and effective amount range. The "safe and effective amount" herein refers to: an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1 - 3000 (active dose range 1 - 100 mg / kg) mg of the compound of the present invention per dose, more preferably, it contains 10 - 2000 mg of the compound of the present invention per dose. Preferably, the "per dose" is a capsule or a tablet.

[0071] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gelling substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" herein means that the components in the composition can be mixed with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers wetting agents (such as sodium dodecyl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0072] There is no particular limitation on the administration mode of the compound or pharmaceutical composition of the present invention. Representative administration modes include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0073] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier) such as sodium citrate or calcium phosphate, or with the following components: (a) fillers or bulking agents, e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, e.g., glycerol; (d) disintegrants, e.g., agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, e.g., paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, e.g., cetyl alcohol and glycerol monostearate; (h) adsorbents, e.g., kaolin; and (i) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0074] Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and release of the active compound or compounds in such compositions may be delayed and / or released in a particular part of the digestive tract. Examples of embedding components that may be used are polymeric and wax-like substances. Optionally, the active compound may also be in microcapsule form with one or more of the above excipients.

[0075] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or elixirs. Besides the active compound, the liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil or mixtures of these substances.

[0076] Besides these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, and perfumes.

[0077] Besides the active compound, the suspension may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar or mixtures of these substances.

[0078] Compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0079] Dosage forms of the compounds of the present invention for topical administration 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 that may be required, if necessary.

[0080] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0081] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage. For a person weighing 60 kg, the daily dosage is usually 1 - 2000 mg, preferably 6 - 600 mg. Of course, the specific dosage should also consider factors such as the route of administration and the health status of the patient, which are all within the scope of the skills of a skilled physician.

[0082] Compared with the prior art, the main advantages of the present invention include:

[0083] 1. This discovery first found that 9,10-dihydrophenanthrene and bibenzyl (stilbenes) have good hepatoprotective activity and can counteract the damage of primary hepatocytes or human-induced hepatic-like cells induced by hydrophobic bile acids in a relatively low concentration range. Compared with existing hepatoprotective drugs, its greatest advantage is that it can be taken orally and shows an obvious dose-dependent relationship.

[0084] 2. In addition to counteracting the liver damage induced by hydrophobic bile acids, the active compounds of the present invention also have antioxidant and anti-inflammatory activities and have potential protective effects on hepatitis with oxidative stress and inflammation as the main pathological features, such as toxic hepatitis (drugs, chemicals and biological toxins), alcoholic hepatitis, and ischemic hepatitis, which have been confirmed by animal experiments.

[0085] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0086] The instruments and materials used in the examples are as follows:

[0087] Specific rotation: Rudulph Autopol VI automatic polarimeter;

[0088] Ultraviolet spectrum: Shimadzu UV-2550 ultraviolet spectrometer;

[0089] Circular dichroism spectrum: JASCO J-810 spectropolarimeter;

[0090] Mass spectrometry: Waters 3100 SQDMS (low-resolution ESI), Waters Xevo QTof MS (high-resolution ESI);

[0091] Nuclear magnetic resonance spectrum: Varian Mercury-plus 400 nuclear magnetic resonance spectrometer, Varian MR-400 nuclear magnetic resonance spectrometer, Bruker Avance III 500 nuclear magnetic resonance spectrometer, δ (ppm), with TMS as the internal standard;

[0092] MCI resin: CHP20P (75 - 150 μm), Mitsubishi Chemical Corporation;

[0093] Column chromatography silica gel: 200 - 300 mesh, 300 - 400 mesh silica gel and silica gel H are all produced by Qingdao Marine Chemical Factory;

[0094] TLC pre-coated thin layer plate: HSGF254 is produced by Yantai Chemical Factory;

[0095] Sephadex: Sephadex LH-20: Pharmacia Biotech AB, Uppsala, Sweden;

[0096] High performance liquid chromatography and mass spectrometry combined instrument: Waters 2695 LC coupled with Waters 2998 DAD, Waters Acquity ELSD, Waters 3100 SQDMS, analytical chromatographic column model: Waters RP C-18, 3.5 μm, 4.6 mm × 100 mm;

[0097] Chiral analysis HPLC: JASCO 2000, Daicel columns, eluted with 2-propanol / hexane;

[0098] Preparative HPLC: Agilent-Varian SD1 LC coupled with Grace-Alltech 3000 ELSD, Varian M701 Fraction Collector, preparative chromatographic column Waters RP C-18, 5μm, 19mm×150mm, Waters RP C-18, 5μm, 30mm×150mm, flow rate 10.0 - 25.0 mL / min, CH3CN (Sinopharm Chemical Reagent Co., Ltd.), H2O (Milli-Q pure water system);

[0099] Color development detection method: 10% vanillin sulfate solution, UV254 and UV365 ultraviolet lamps;

[0100] Solvents for experiments: analytical grade solvents (Sinopharm Chemical Reagent Co., Ltd.), chromatographic grade solvents (Merck KGaA, Germany); Experimental animals: male ICR mice, body weight 22 - 25 g, male SD rats, body weight 200 - 250 g. All animals were purchased from Shanghai Slac Laboratory Animal Co., Ltd.;

[0101] Experimental cells: L929 cells were from the Cell Bank of the Chinese Academy of Sciences. hihep cells were transdifferentiated human hepatocytes, kindly donated by Teacher Hu Lijian from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. BMDM cells were isolated from the bone marrow of ICR mice, and primary rat hepatocytes were isolated from SD rats;

[0102] Experimental reagents: DPPH free radical was purchased from Shanghai Macklin Biochemical Co., Ltd.; olive oil, formaldehyde solution, 75% alcohol, absolute ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd.; DMSO, vitamin C, silymarin, resveratrol, dexamethasone, lipopolysaccharide (LPS) were purchased from Sigma - Aldrich; bicyclol was purchased from the National Institute for the Control of Pharmaceutical and Biological Products; 1×EBSS buffer, William's E medium, penicillin - streptomycin, fetal bovine serum (FBS), insulin - transferrin - selenium additive (ITS), 10×PBS were purchased from Gibco; high - glucose DMEM medium was purchased from Hyclone; RIPA lysis buffer, NO detection kit were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; mouse TNFα and IL - 6 ELISA kits were purchased from Hangzhou Lianke Biotechnology Co., Ltd.; ALT, AST, LDH blood biochemical detection kits were purchased from Sysmex Biotechnology Co., Ltd.; type IV collagenase was purchased from Shanghai Dingsheng Biotechnology Co., Ltd.; Percoll centrifugation solution was purchased from GE; CCK8 kit was purchased from Shanghai Yeasen Biotechnology Co., Ltd.;

[0103] Surgical instruments: ophthalmic scissors, ophthalmic forceps, 200 - mesh / 300 - mesh sieves, 1 - ml syringe, among which the scissors, forceps and sieves need to be sterilized and dried in advance;

[0104] Experimental instruments: low-temperature high-speed centrifuge (Eppendorf, 5415R), cell culture incubator (Thermo Fisher Scientific, 371), ultrapure water preparation system (Pall Cascada), inverted fluorescence microscope (Olympus CKX53), ultrasonic cell disruptor (Shanghai Biheng, SKSI), multifunctional microplate reader (Bio Tek Synergy 4), automatic blood biochemical analyzer (SYSMEX JCA-BM6010C).

[0105] Separation, purification and spectral data of the compound in Example 1

[0106] 1. Separation of 9,10-dihydrophenanthrene and bibenzyl compounds from Pholidota plants

[0107] 1.84 kg of Pholidota chinensis Lindl. collected from Xichou, Yunnan was dried in the shade and then pulverized. It was soaked in 10 L of 95% industrial ethanol at room temperature for 5 days, and this was repeated three times. The extraction solutions were combined and concentrated under reduced pressure to obtain 236 g of the total extract. The total extract was suspended in 2.0 L of water and successively extracted with petroleum ether, ethyl acetate, and n-butanol. The organic solvents were removed by distillation under reduced pressure to obtain 93 g of the brownish petroleum ether fraction, 10 g of the ethyl acetate fraction, and 45 g of the n-butanol fraction. Taking the petroleum ether fraction (93 g), after silica gel column chromatography (petroleum ether - ethyl acetate, 100:1 to 1:1), it was divided into 9 fractions. Fraction 4 was purified by silica gel column chromatography (petroleum ether - acetone 10:1 to 2:1) and then further purified by Sephadex LH-20 (MeOH) to obtain YS24 (lusianthridin, 52 mg). Fraction 5 was purified by Sephadex LH-20 (CHCl3:MeOH 1:1) to obtain YS28 (3,3',5-trihydroxybibenzyl, 63 mg) and YS32 (3,3',5-trihydroxystilbene, 21 mg). Fraction 6 was repeatedly separated and purified by silica gel column chromatography to obtain compounds YS25 (coelonin, 11 mg), YS26 (dihydropinosylvin, 140 mg), YS27 (4-hydroxy-3-methoxybibenzyl, 43 mg), YS30 (gigantol, YS30, 60 mg), YS33 (thunalbene, 45 mg), YS34 (3'-hydroxy-3,5-dimethoxystilbene, 35 mg). The small fractions obtained from fraction 7 by silica gel column chromatography were further purified by Sephadex LH-20 to obtain compounds YS23 (2-hydroxy-4,7-dimethoxy-9,10-dihydrophenanthrene, 23 mg) and YS31 (3-hydroxy-5-methoxystilbene, 11 mg). The small fractions obtained from fraction 8 by silica gel column chromatography were further purified by Sephadex LH-20 and preparative high performance liquid chromatography to obtain compounds YS1 (phochinenin A, 18 mg), YS3 - 5 (phochinenins C - E, 4, 72 and 59 mg), YS8 (phochinenin H, 27 mg), YS11 - 12 (phochinenins K and L, 7 and 4 mg), YS21 (blestrin A, 16 mg), YS29 (batatasin III, 14 mg), YS35 (shancidin, 48 mg), YS36 [1-(4-hydroxybenzyl)-4-methoxy-2,7-dihydroxy-9,10-dihydrophenanthrene, 8 mg], YS37 (isoarundinin I, 114 mg).Fraction 9 was purified by repeated silica gel column chromatography, Sephadex LH-20 column chromatography and then preparative high performance liquid chromatography to obtain compounds YS2 (phochinenin B, 23 mg), YS6-7 (phochinenins F and G, 10 and 4 mg), YS9-10 (phochinenins I and J, 63 and 11 mg), YS15-17 (phochinenins O-Q, 11, 57 and 48 mg), YS18 (gymconpin C, 60 mg), YS19 (blestrianol A, 23 mg), YS20 (flavanthrin, 115 mg), YS22 (phoyunnanin D, 24 mg). The structures of the compounds are shown in Fig. 1 for details.

[0108] 2. Physical and Chemical Properties and Spectral Data of the Compounds

[0109] YS1: Phochinenin A

[0110] Brown amorphous powder, UV (MeOH) λ 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 C 22 H 20 O4, 348.1364). 1 H NMR (300 MHz, CD3OD) δ 6.39 (1H, d, J = 2.4 Hz, H-4), 6.70 (1H, d, J = 2.4 Hz, H-6), 6.85 and 7.27 (each 1H, d, J = 15.6 Hz, H-α and α’), 6.91 (1H, dd, J = 2.1 and 2.7 Hz, H-2’), 6.84 (1H, dd, J = 2.1 and 8.1 Hz, H-4’), 7.12 (1H, dd, J = 7.6 and 8.1 Hz, H-5’), 6.87 (1H, dd, J = 2.7 and 7.6 Hz, H-6’), 3.98 (2H, s, -C H 2), 6.59 and 6.99 (each 2H, d, J = 8.7 Hz, H-2”, 3”, 5”, 6”), 3.79 (3H, s, 5-OMe).

[0111] YS2: phochinenin B

[0112] Brown amorphous powder, UV (MeOH) λ 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 C 22 H 20 O4, 348.1344). 1 H NMR (300 MHz, CD3OD) δ 6.39 (1H, d, J=2.5 Hz, H-4), 6.66 (1H, d, J=2.5 Hz, H-6), 6.80 and 7.20 (each 1H, d, J=16.0 Hz, H-α and α’), 6.83 (1H, dd, J=2.0 and 2.5 Hz, H-2’), 6.63 (1H, dd, J=2.5 and 7.6 Hz, H-4’), 7.10 (1H, dd, J=7.6 and 7.7 Hz, H-5’), 6.84 (1H, dd, J=2.0 and 7.0 Hz, H-6’), 3.96 (2H, s, -C H 2), 6.60 and 6.91 (each 2H, d, J=8.3 Hz, H-2”, 3”, 5”, 6”), 3.77 (3H, s, 3-OMe).

[0113] YS3: phochinenin C

[0114] Brown amorphous powder, UV (MeOH) λ 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 C 23 H 24 O5Na, 403.1521). 11H NMR (300 MHz, CD3OD) δ 6.32 (1H, d, J = 2.1 Hz, H-4), 6.26 (1H, d, J = 2.1 Hz, H-6), 2.52 and 2.69 (each 2H, m, H-α and α’), 6.51 (1H, t, J = 1.3 Hz, H-2’), 6.51 (1H, dd, J = 1.3 and 7.9 Hz, H-4’), 7.00 (1H, dd, J = 7.6 and 7.9 Hz, H-5’), 6.54 (1H, dd, J = 1.3 and 7.6 Hz, H-6’), 3.83 (2H, s, -C H 2), 6.62 (1H, d, J = 2.0 Hz, H-2”), 6.61 (1H, d, J = 8.3 Hz, H-5”), 6.45 (1H, dd, J = 2.3 and 8.3 Hz, H-6”), 3.73 and 3.69 (each 3H, s, 3 and 3”-OMe).

[0115] YS4: phochinenin D

[0116] Brown amorphous powder, [α] 20 D -0.005 (c 0.315, MeOH). UV (MeOH) λ max nm (log ε): 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 C 30 H 26 O6, 482.1729). 1 1H NMR (600 MHz, CD3OD) δ 6.57 (1H, s, H-1), 8.20 (1H, d, J = 8.3 Hz, H-5), 6.64 (1H, dd, J = 2.4 and 8.3 Hz, H-6), 6.65 (1H, d, J = 2.4 Hz, H-8), 2.75 and 2.79 (each 2H, m, H-9, 10), 6.84 and 8.15 (each 1H, s, H-1’, 4’), 6.34 and 6.38 (each 1H, d, J = 2.4 Hz, H-6’, 8’), 2.78 (4H, s, H-9’, 10’), 3.75 and 3.77 (each 3H, s, 2-OC H 3, 7’-OC H 3).

[0117] YS5: phochinenin E

[0118] Brown amorphous powder, [α] 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 C 30 H 26 O6, 482.1729). 1 H NMR (300 MHz, CD3OD) δ 6.77 and 8.03 (each 1H, s, H-1,4), 6.35 and 6.31 (each 1H, d, J=2.4 Hz, H-6,8), 2.73 (4H, s, H-9,10), 6.57 (1H, s, H-2’), 8.03 (1H, d, J=8.6 Hz, H-5’), 6.63 (1H, dd, J=2.4 and 8.6 Hz, H-6’), 6.59 (1H, d, J=2.4 Hz, H-8’), 2.50 and 2.46 (each 2H, m, H-9’,10’), 3.75 and 3.88 (each 3H, s, 7-OC H 3,3’-OC H 3).

[0119] YS6: phochinenin F

[0120] Brown amorphous powder, UV (MeOH) λ max nm (logε): 282 (4.51). EIMS m / z (rel.int): 482 [M + (100). HR-EIMS m / z: 482.1739 [M + (calcd for C 30 H 26 O6, 482.1729). 11H NMR (400 MHz, DMSO-d6) δ 6.53 (1H, s, H-1), 7.95 (1H, d, J = 8.6 Hz, H-5), 6.61 (1H, dd, J = 2.5 and 8.6 Hz, H-6), 6.65 (1H, d, J = 2.5 Hz, H-8), 2.65 (4H, s, H-9,10), 6.57 (1H, s, H-3’), 7.96 (1H, d, J = 8.6 Hz, H-5’), 6.58 (1H, dd, J = 1.6 and 8.6 Hz, H-6’), 6.56 (1H, d, J = 1.6 Hz, H-8’), 2.46 and 2.31 (each 2H, m, H-9’,10’), 3.18 and 3.81 (each 3H, s, 4-OC H 3,4’-OC H 3).

[0121] YS7: phochinenin G

[0122] Brown amorphous powder, UV (MeOH) λ max nm (log ε): 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 C 30 H 24 O6, 480.1573). 1 1H NMR (600 MHz, CD3OD) δ 6.67 (1H, s, H-2), 8.10 (1H, d, J = 8.9 Hz, H-5), 6.66 (1H, dd, J = 3.0 and 8.9 Hz, H-6), 6.58 (1H, d, J = 3.0 Hz, H-8), 2.46 and 2.22 (each 2H, m, H-9,10), 6.94 (1H, s, H-2’), 9.45 (1H, d, J = 9.4 Hz, H-5’), 7.12 (1H, dd, J = 2.7 and 9.4 Hz, H-6’), 7.11 (1H, d, J = 2.7 Hz, H-8’), 7.40 and 7.48 (each 1H, d, J = 9.0 Hz, H-9’,10’), 3.95 and 4.15 (each 3H, s, 3-OC H 3,3’-OC H 3).

[0123] YS8: phochinenin H

[0124] Brown amorphous powder, UV(MeOH) λ 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 C 30 H 26 O6, 482.1729). 1 1H NMR(600MHz, CD3COCD3) δ6.67(1H, s, H-2), 8.12(1H, d, J=8.5Hz, H-5), 6.71(1H, dd, J=2.7 and 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=2.8 and 8.8Hz, H-3’), 8.11(1H, d, J=8.8Hz, H-4’), 6.46 and 6.38(each 1H, d, J=2.3Hz, H-6’,8’), 2.46(4H, s, H-9’,10’), 3.89 and 3.83(each 3H, s, 3-OC H 3, 5’-OC H 3).

[0125] YS9: phochinenin I

[0126] Brown amorphous powder, [α] 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 C 30 H 26 O6, 482.1729). 11H NMR (300 MHz, CD3OD) δ 6.54 (1H, s, H-2), 8.22 (1H, d, J = 8.6 Hz, H-5), 6.67 (1H, dd, J = 2.8 and 8.5 Hz, H-6), 6.62 (1H, d, J = 2.8 Hz, 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 8.5 Hz, H-3’), 8.19 (1H, d, J = 8.5 Hz, H-4’), 6.33 (2H, s, H-6’,8’), 2.66 (4H, m, H-9’,10’), 3.89 and 3.83 (each 3H, s, 3-OC H 3,5’-OC H 3).

[0127] YS10: phochinenin J

[0128] Brown amorphous powder, UV (MeOH) λ max nm (log ε): 279 (4.48). EIMS m / z (rel.int): 482 [M + (37). HR-EIMS m / z: 482.1721 [M + (calcd for C 30 H 26 O6, 482.1729). 1 1H NMR (600 MHz, CD3OD) δ 6.57 (1H, s, H-2), 8.24 (1H, d, J = 8.3 Hz, H-5), 6.68 (1H, dd, J = 2.3 and 8.3 Hz, H-6), 6.64 (1H, d, J = 2.3 Hz, H-8), 2.63 (4H, s, H-9,10), 6.24 and 6.46 (each 1H, d, J = 2.6 Hz, H-1’,3’), 8.03 (1H, d, J = 9.5 Hz, H-5’), 6.64 (1H, dd, J = 2.6 and 9.5 Hz, H-6’), 6.63 (1H, d, J = 2.6 Hz, H-8’), 2.64 (4H, m, H-9’,10’), 3.77 and 3.79 (each 3H, s, 3-OC H 3,4’-OC H 3).

[0129] YS11: phochinenin K

[0130] Brown amorphous powder, UV (MeOH) λ maxnm(logε): 201(4.68), 279(4.22). EIMS m / z(rel.int): 484[M + (100). HR-EIMS m / z: 484.1894[M + (calcd for C 30 H 28 O6, 484.1786). 1 1H NMR(300 MHz, CD3OD) δ 6.81 and 8.10(each 1H, s, H-1,4), 6.31 and 6.35(each 1H, d, J=2.7 Hz, H-6,8), 2.75(4H, s, H-9,10), 6.68(1H, dd, J=2.3 and 8.3 Hz, H-6), 6.64(1H, d, J=2.3 Hz, H-8), 6.33 and 6.34(each 1H, d, J=2.4 Hz, H-4’,6’), 2.60 and 2.67(each 2H, m, H-α,α’), 6.40(1H, dd, J=2.4 and 2.6 Hz, H-2”), 6.47(1H, d, J=2.4 and 8.0 Hz, H-4”), 6.93(1H, dd, J=7.4 and 8.0 Hz, H-5”), 6.39(1H, dd, J=2.6 and 7.4 Hz, H-6”), 3.77 and 3.76(each 3H, s, 7-OC H 3, 3’-OC H 3).

[0131] YS12: phochinenin L

[0132] Brown amorphous powder, UV(MeOH) λ 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 C 30 H 28 O6, 484.1786). 11H NMR (300 MHz, CD3OD) δ 6.77 and 8.07 (each 1H, s, H-1,4), 6.31 and 6.34 (each 1H, d, J = 2.6 Hz, H-6,8), 2.71 (4H, s, H-9,10), 6.41 and 6.45 (each 1H, d, J = 1.1 Hz, H-2’,6’), 2.86 (4H, m, H-α,α’), 6.68 (1H, dd, J = 1.7 and 2.1 Hz, H-2”), 6.60 (1H, d, J = 1.7 and 7.9 Hz, H-4”), 7.09 (1H, dd, J = 7.5 and 7.9 Hz, H-5”), 6.72 (1H, dd, J = 2.1 and 7.5 Hz, H-6”), 3.74 and 3.67 (each 3H, s, 7-OC H 3,5’-OC H 3).

[0133] YS13: phochinenin M

[0134] Brown amorphous powder, UV (MeOH) λ max nm (logε): 201 (4.83), 280 (4.33). IEIMS m / z (rel.int): 484 [M + (100). HR-EIMS m / z: 484.1871 [M + (calcd for C 30 H 28 O6, 484.1786). CD data recorded on-line in hexane / ethanol 7:3 for (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). 11H NMR (300 MHz, CD3OD) δ 6.59 (1H, s, H-3), 8.03 (1H, d, J = 8.5 Hz, H-5), 6.62 (1H, dd, J = 2.7 and 8.5 Hz, H-6), 6.58 (1H, d, J = 2.7 Hz, H-8), 2.52 and 2.33 (each 2H, m, H-9, 10), 6.34 and 6.36 (each 1H, d, J = 2.5 Hz, H-4’, 6’), 2.54 and 2.56 (each 2H, m, H-α, α’), 6.38 (1H, dd, J = 2.1 and 2.1 Hz, H-2”), 6.49 (1H, d, J = 2.1 and 8.2 Hz, H-4”), 6.94 (1H, dd, J = 8.2 and 8.6 Hz, H-5”), 6.37 (1H, dd, J = 2.1 and 8.6 Hz, H-6”), 3.89 and 3.74 (each 3H, s, 2-OC H 3,3’-OC H 3).

[0135] YS14: phochinenin N

[0136] Brown amorphous powder, UV (MeOH) λ max nm (log ε): 277 (4.32), 299 (4.29). EIMS m / z (rel.int): 482 [M + (100). HR-EIMS m / z: 482.1733 [M + (calcd for C 30 H 26 O6, 484.1729). 11H NMR (600 MHz, CD3OD) δ 6.71 and 8.04 (each 1H, s, H-1,4), 6.27 and 6.32 (each 1H, d, J=2.6 Hz, H-6,8), 2.76 (4H, s, H-9,10), 6.89 and 6.35 (each 1H, d, J=2.5 Hz, H-2’,6’), 6.94 and 6.97 (each 1H, d, J=16.6 Hz, H-α,α’), 6.73 (1H, dd, J=1.7 and 2.1 Hz, H-2”), 6.55 (1H, d, J=1.7 and 8.5 Hz, H-4”), 7.02 (1H, dd, J=7.6 and 8.1 Hz, H-5”), 6.76 (1H, dd, J=2.1 and 7.6 Hz, H-6”), 3.74 and 3.86 (each 3H, s, 7-OC H 3,4’-OC H 3).

[0137] YS15: phochinenin O

[0138] Brown amorphous powder, UV (MeOH) λ 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 C 30 H 26 O6, 482.1729). 1 1H NMR (500 MHz, CD3OD) δ 6.73 and 8.08 (each 1H, s, H-1,4), 6.29 and 6.33 (each 1H, d, J=2.4 Hz, H-6,8), 2.71 (each 2H, m, H-9,10), 6.75 and 6.77 (each 1H, d, J=1.3 Hz, H-2’,6’), 7.09 and 7.12 (each 1H, d, J=16.0 Hz, H-α,α’), 6.96 (1H, dd, J=1.7 and 2.4 Hz, H-2”), 6.67 (1H, d, J=2.4 and 8.0 Hz, H-4”), 7.14 (1H, dd, J=7.7 and 8.0 Hz, H-5”), 7.01 (1H, dd, J=1.7 and 7.7 Hz, H-6”), 3.72 and 3.76 (each 3H, s, 7-OC H3,5’-OC H 3).

[0139] YS16: phochinenin P

[0140] Brown amorphous powder, [α] 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 C 30 H 26 O6, 482.1729). 1 1H NMR (300 MHz, CD3OD) δ 6.63 (1H, s, H-2), 8.10 (1H, d, J=8.6 Hz, H-5), 6.66 (1H, dd, J=2.5 and 8.6 Hz, H-6), 6.60 (1H, d, J=2.5 Hz, H-8), 2.47 and 2.28 (each 2H, m, H-9,10), 6.48 and 6.94 (each 1H, d, J=2.6 Hz, H-4’,6’), 6.78 and 6.97 (each 1H, d, J=16.0 Hz, H-α,α’), 6.72 (1H, dd, J=2.1 and 2.1 Hz, H-2”), 6.62 (1H, d, J=2.1 and 7.7 Hz, H-4”), 7.07 (1H, dd, J=7.7 and 8.1 Hz, H-5”), 6.86 (1H, dd, J=2.1 and 8.1 Hz, H-6”), 3.89 and 3.84 (each 3H, s, 3-OC H 3,5’-OC H 3).

[0141] YS17: Rac-phochinenin Q

[0142] Brown amorphous powder, [α] 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 C 31 H 28 O7, 512.1835). 1 1H NMR (500 MHz, CD3OD) δ 6.63 (1H, s, H-3), 8.04 (1H, d, J = 8.6 Hz, H-5), 6.64 (1H, dd, J = 2.4 and 8.6 Hz, H-6), 6.60 (1H, d, J = 2.4 Hz, H-8), 2.47 (2H, m, H-9), 2.38 and 2.22 (each 1H, m, H-10), 6.22 (1H, d, J = 2.1 Hz, H-2’), 6.28 (1H, dd, J = 1.5 and 2.1 Hz, H-4’), 6.20 (1H, d, J = 1.5 Hz, H-6’), 4.43 and 5.37 (each 1H, d, J = 6.5 Hz, H-α, α’), 6.91 (1H, d, J = 2.1 Hz, H-2”), 6.81 (1H, d, J = 7.9 Hz, H-5”), 6.79 (1H, dd, J = 1.6 and 7.9 Hz, H-6”), 3.92, 3.84 and 3.71 (each 3H, s, 4-OC H 3,3’-OC H 3,3”-OC H 3).

[0143] YS18: gymconpin C

[0144] Brown amorphous powder, [α] 20 D +0.003 (c 0.315, MeOH). UV (MeOH) λ max nm (log ε): 216 (5.14), 280 (4.95), 296 (4.85). CD data recorded on-line in hexane / ethanol 7:3 for (aS)-18: λ max (φ): 307 (8.7), 287 sh (5.5), 271 (-9.1), 246 (8.5), 219 (-9.7). (aR)-18: 311 (-8.8), 286 sh (-5.5), 269 (10.0), 243 (-8.3), 222 (9.8).

[0145] 11H NMR (500 MHz, CD3OD) δ 6.57 (1H, s, H-3), 8.03 (1H, d, J = 8.5 Hz, H-5), 6.63 (1H, dd, J = 2.7, 8.5 Hz, H-6), 6.60 (1H, d, J = 2.7 Hz, H-8), 2.53 and 2.50 (each 2H, m, H-9, 10), 6.32 and 6.37 (each 1H, d, J = 2.5 Hz, H-1’, 3’), 7.87 and 6.76 (each 1H, s, H-5’, 7’), 27.0 (4H, s, H-9’, 10’), 3.88 and 3.74 (each 3H, s, 4-OMe, 4’-OMe).

[0146] YS19: blestrianol A

[0147] Light yellow powder, UV (MeOH) λ max nm (log ε): 276 (4.54), 285 (4.61), 200 (4.45). ESI-MS m / z 482.3 [M + H] + . 1 1H NMR (500 MHz, CD3OD) δ 2.41 - 2.57 (4H, m, H-9’, H-10’), 2.72 (4H, s, H-9, H-10), 3.26 and 3.88 (each 3H, s, 4, 4’-OMe), 6.59 and 6.62 (each 1H, s, H-3’, H-1), 6.60 (1H, d, J = 2.6 Hz, H-8’), 6.63 (1H, dd, J = 8.6, 2.6 Hz, H-6’), 6.64 (1H, dd, J = 8.4, 2.6 Hz, H-6), 6.66 (1H, d, J = 2.6 Hz, H-8), 8.05 (lH, d, J = 8.6 Hz, H-5’), 8.08 (1H, d, J = 8.4 Hz, H-5).

[0148] YS20: flavanthrin

[0149] White amorphous powder, [α] 20 D 0 (c 0.380, MeOH). CD data recorded on-line in hexane / ethanol 7:3 for (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(400 MHz, CD3OD) δ 2.36 and 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=2.3 Hz, H-1, 1’), 6.67 (2H, dd, J=8.3, 2.3 Hz, H-3, 3’), 8.07 (2H, d, J=8.3 Hz, H-4, 4’).

[0150] YS21: blestrin A

[0151] White powder, UV(MeOH) λ max nm(logε): 272sh(4.28), 281(4.36), 299(4.25). ESI-MS m / z 483.2 [M+H] + . 1 1H-NMR(400 MHz, CD3OD) δ 6.61 (1H, s, H-3), 8.06 (1H, d, J=8.6 Hz, H-5), 6.64 (1H, dd, J=2.8, 8.6 Hz, H-6), 6.61 (1H, d J=2.8 Hz, H-8), 2.55 (4H, s, H-9, 10), 6.26 and 6.51 (each 1H, d, J=2.6 Hz, H-1’, 3’), 8.01 (1H, d, J=9.2 Hz, H-5’), 6.60 - 6.64 (2H, m, H-6’, 8’), 3.86 and 3.77 (each 3H, s, 4-OMe, 4’-OMe).

[0152] YS22: phoyunnanin D

[0153] White powder, UV(MeOH) λ max nm(logε): 205(4.92), 280(4.47). ESI-MS m / z 484.2 [M+H]+ . 1 H-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”),7.0 2(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).

[0154] YS23:7-hydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene,7-hydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene

[0155] Light red oily liquid, EI-MS m / z(%):256(100). 1 H 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),6.4 5(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).

[0156] YS24: lusianthridin

[0157] Light brown powder, EI-MS m / z(%):242(100). 11H NMR (500 MHz, CD3OD), δ 7.08 (1H, t, J = 7.8 Hz, H-7), 6.84 (1H, dd, J = 7.8, 2.5 Hz, H-6), 6.81 (1H, dd, J = 7.8, 2.5 Hz, H-8), 6.57 (1H, d, J = 2.5 Hz, H-3), 6.53 (1H, d, J = 2.5 Hz, H-1), 3.95 (3H, s, 4-OCH3), 2.67 (4H, m, CH2-9, 10)

[0158] YS25: coelonin

[0159] Light brown oily liquid, EI-MS m / z (%): 242 (100). 1 1H NMR (500 MHz, acetone-d6) δ 8.07 (1H, d, J = 8.5 Hz, H-5), 6.68 (1H, d, J = 8.5 Hz, 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).

[0160] YS26: dihydropinosylvin

[0161] White needle-like solid, EI-MS m / z (%): 214 (55). 1 1H NMR (400 MHz, CDCl3) δ 2.73 - 2.90 (4H, m, H-α, α’), 6.19 (1H, t, J = 2.1 Hz, H-4), 6.24 (2H, d, J = 2.1 Hz, H-2, 6), 7.10 - 7.20 (3H, m, H-2’, 4’, 6’), 7.25 (2H, t, J = 7.5 Hz, H-3’, 5’).

[0162] YS27: 4-hydroxy-3-methoxybibenzyl

[0163] White solid, EI-MS m / z (%): 228 (69). 1H NMR (400 MHz, 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 = 8.0 Hz, H-2), 6.70 (1H, dd, J = 8.0, 1.9 Hz, H-4), 6.62 (1H, d, J = 1.9 Hz, H-6), 3.84 (3H, s, 3-OMe), 2.95–2.84 (4H, m, H-α, α').

[0164] YS28:3,3',5-trihydroxy-bibenzyl,3,3',5-trihydroxy-bibenzyl

[0165] White powder, EI-MS m / z(%):230(100). 1 H 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').

[0166] YS29:batatasin III

[0167] White solid, EI-MS m / z(%): 274(30). 1 H 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').

[0168] 6.84 (1H, d, J = 8.0 Hz, H-2), 6.68 (1H, dd, J = 8.0, 1.8 Hz, H-4), 6.63 (1H, d, J = 1.8 Hz, 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’).

[0169] YS30: gigantol YS30

[0170] Light yellow oily liquid. EI-MS m / z (%): 274 [M] + ; 1 H NMR (400 MHz; CDCl3) δ 6.84 (1H, d, J = 8.0 Hz, H-2), 6.68 (1H, dd, J = 8.0, 1.8 Hz, H-4), 6.63 (1H, d, J = 1.8 Hz, 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’).

[0171] YS31: 3-hydroxy-5-methoxystilbene 3-hydroxy-5-methoxy

[0172] White solid, EI-MS m / z (%): 226 (78); 1 H-NMR (400 MHz, CDCl3) δ 3.83 (3H, s, 5-OMe), 6.35 (1H, t, J = 2.3 Hz), 6.61 (1H, t, J = 1.8 Hz), 6.65 (1H, t, J = 1.8 Hz), 6.99 (1H, d, J = 16.5 Hz, H-α), 7.07 (1H, d, J = 16.5 Hz, H-α’), 7.26 (1H, tt, J = 7.6 and 1.4 Hz, H-4’), 7.36 (2H, t, J = 7.6 Hz, H-3’, 5’), 7.50 (2H, dd, J = 7.6 Hz, H-2’, 4’).

[0173] YS32: 3,3',5-trihydroxystilbene, 3,3',5-trihydroxystilbene

[0174] 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 2.4and0.6Hz,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’).

[0175] YS33:thunalbene

[0176] Light yellow oily colloid, EI-MS m / z(%):242(86); 1 H-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’)

[0177] YS34:3’-hydroxy-3,5-dimethoxy-stilbene, 3′-hydroxy-3,5-dimethoxystilbene

[0178] White solid, EI-MS m / z(%):256(73); 1 H 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’).

[0179] YS35:shancidin

[0180] Colorless needle-like solid, EI-MS m / z (%): 348 (100); 1 1H-NMR (500 MHz, CD3OD) δ 2.45 (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 = 2.5 Hz, H-8), 6.62 (1H, dd, J = 8.6, 2.5 Hz, H-6), 6.65 (2H, d, J = 8.5 Hz, H-3’, 5’), 6.94 (2H, d, J = 8.5 Hz, H-2’, 6’), 7.97 (1H, d, J = 8.6 Hz, H-5).

[0181] YS36: 9,10-dihydro-1-(4-hydroxybenzyl)-4-methoxy-2,7-phenanthrenediol, 1-(4-hydroxybenzyl)-4-methoxy-2,7-dihydroxy-9,10-dihydrophenanthrene

[0182] Pale yellow needle-like solid, EI-MS m / z (%): 348 (100); 1 1H-NMR (500 MHz, CD3COCD3) δ 8.02 (1H, d, J = 9.0 Hz, H-5), 6.98 (2H, d, J = 8.4 Hz, H-2’, 6’), 6.70 (2H, d, J = 8.4 Hz, H-3’, 5’), 6.68 (1H, s, H-8), 6.65 (1H, d, J = 9.0 Hz, 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).

[0183] YS37: isoarundinin I

[0184] White needle-like solid, MS m / z (%): 350 (100). 1H-NMR (500 MHz, CD3OD) δ 2.90 (4H, m, H-α, α’), 3.87 (2H, s, -CH2-Ph), 3.70 (3H, s, 3-OMe), 6.30 and 6.24 (each 1H, d, J = 2.4 Hz, H-4,6), 6.54 (1H, t, J = 2.3 Hz, H-2’), 6.56 (1H, dd, J = 7.9, 2.3 Hz, H-4’), 7.03 (1H, t, J = 7.9 Hz, H-5’), 6.56 (1H, dd, J = 7.9, 2.3 Hz, H-6’), 6.64 (2H, d, J = 8.5 Hz, H-3”,5”), 6.95 (2H, d, J = 8.5 Hz, H-2”,6”).

[0185] 3. Isolation of 9,10-Dihydrophenanthrene and Phenanthrene Compounds from Juncus setchuensis

[0186] 9.5 kg of dry whole herb of Juncus setchuensis was cold-extracted three times with 95% industrial ethanol (35 L) for 3 days each time. The extraction solutions were combined and the solvent was recovered under reduced pressure to obtain 445 g of total extract. The total extract was dissolved in water and partitioned with ethyl acetate and n-butanol respectively to obtain 217 g of ethyl acetate fraction, 62 g of n-butanol layer and 153 g of water layer. The ethyl acetate fraction (217 g) was subjected to silica gel column chromatography (petroleum ether - acetone 6:1 - 0:1) to obtain 8 fractions. Fraction 3 (16.7 g) was separated by silica gel column chromatography (PE - isopropyl ether 10:1 - 1:1), and the secondary fraction F3-1 was recrystallized with isopropyl ether to obtain compound XYW-1 (juncuenin A, 51 mg). The secondary fraction F3-4 was separated by Sephadex LH-20 (chloroform - methanol 1:1) to obtain compound XYW-5 (dehydrojuncuenin A, 333 mg). Fraction 4 (70.53 g) was recrystallized with chloroform to obtain compound XYW-3 (juncuenin C, 22 g). The mother liquor was subjected to MCI column chromatography (EtOH / H2O: 30% - 100%), and the secondary fraction F4-1 was purified by silica gel column chromatography (PE:EtOAc 6:1 - 1:1) and Sephadex LH-20 (chloroform - methanol 1:1) to obtain compounds 8 (829 mg), 9 (971 mg), 10 (111 mg); the secondary fraction F4-9 (735 mg) was separated by Sephadex LH-20 (chloroform - methanol: 1:1) and then by preparative TLC (PE:Actone 2:1) to obtain compound XYW-6 (dehydrojuncuenins B, 17 mg). Fraction 5 (10.146 g) was subjected to MCI column chromatography (EtOH / H2O 30% - 100%), and the secondary fraction F5-5 (314 mg) was recrystallized with chloroform to obtain compound XYW-4 (juncuenin D, 66 mg); the secondary fraction F5-10 (1.492 g) was purified by silica gel column chromatography (PE:Actone 7:2), and the obtained fraction was recrystallized (MeOH-PE) to obtain compound XYW-7 (dehydrojuncuenins C, 28 mg). Fraction 6 (12.11 g) was recrystallized with methanol, and the remaining mother liquor was subjected to MCI column chromatography (EtOH / H2O 30% - 100%). The secondary fraction F6-4 (125 mg) was further purified by SephadexLH-20 (chloroform - methanol 2:1) to obtain compound XYW-2 (juncuenin B, 32 mg).

[0187] 4. Physicochemical properties and spectral data of the compounds

[0188] XYW-1: juncuenin A (2-hydroxy-1,7-dimethyl-6-vinyl-9,10-dihydrophenanthrene)

[0189] White amorphous powder; C 18 H 18 O, EI MS m / z(%): 250 [M] + (100); HREIMS m / z 250.1358 (calcd. for C 18 H 18 O, 250.1358). 1 H NMR(300MHz, CDCl3) δ6.74 and 7.55 (each 1H, d, J=8.3Hz, H-3,4), 7.76 and 7.01 (each 1H, s, H-5,8), 2.80 (4H, s, H-9,10), 2.24 and 2.35 (each 3H, s, 1-,7-Me), 6.95 (1H, dd, J=10.9,17.4Hz, H-11), 5.69 (1H, dd, J=1.5,17.4Hz, H-12a), 5.29 (1H, dd, J=1.5,17.4Hz, H-12b), 4.84 (1H, br.s, 2-OH).

[0190] XYW-2: juncuenin B (2,6-dihydroxy-1-methyl-7-carboxy-8-vinyl-9,10-dihydrophenanthrene)

[0191] White amorphous powder; C 18 H 16 O4, EIMS m / z(%): 296 [M] + (26); HREIMS m / z 296.1050 (calcd. for C 18 H 16 O4,296.1048). 1 H NMR(300MHz, CD3OD) δ6.73 and 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).

[0192] XYW-3: juncuenin C (2,6-dihydroxy-1,7-dimethyl-8-vinyl-9,10-dihydrophenanthrene)

[0193] Colorless fine prisms (CHCl3); C 18 H 18 O2, EIMS m / z(%): 266[M] + (100); HREIMS m / z 266.1302 (calcd. for C 18 H 18 O2, 266.1307). 1 1H NMR(300MHz, CDCl3) δ 6.72 and 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), 4.67 and 4.78 (each 1H, br.s, 2-,6-OH).

[0194] XYW-4: juncuenin D (2,8a-dihydroxy-1,7-dimethyl-6-oxo-8-vinyl-9,10-dihydrophenanthrene)

[0195] Pale yellow small square prisms (CHCl3); C 18 H 18 O3, ESIMS m / z 305.1 [M+Na] + , HRESIMS m / z 305.1143 (calcd. for C 18 H 18 O3Na, 305.1154). 1 1H NMR(300MHz, DMSO-d6) δ 6.75 and 7.37 (each 1H, d, J = 8.8Hz, H-3,4), 6.32 (1H, s, H-5), 1.54 and 2.40 (each 1H, m, H-9), 2.68 and 2.86 (each 1H, m, H-10), 2.02 and 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), 5.54 and 9.69 (each 1H, br.s, 8a-,2-OH).

[0196] XYW-5: dehydrojuncuenin A (2-hydroxy-1,7-dimethyl-6-vinylphenanthrene)

[0197] White powder; C 18 H 16 O, EIMS m / z(%): 248[M] + (100), HREIMS m / z 248.1210 (calcd. for C 18 H 16 O, 248.1202). 1 H NMR(300MHz, CDCl3)δ7.15 and 8.50 (each 1H, d, J=8.9Hz, H-3,4), 8.67 and 7.62 (each 1H, s, H-5,8), 7.68 and 7.86 (each 1H, d, J=9.2Hz, H-9,10), 2.60 and 2.53 (each 3H, s, 1-,7-Me), 7.15(1H, dd, J=10.9 and 17.4Hz, H-11), 5.87(1H, dd, J=2.1 and 17.4Hz, H-12a), 5.43(1H, dd, J=2.1 and 10.9Hz, H-12b), 4.93(1H, br.s, 2-OH).

[0198] XYW-6: dehydrojuncuenin B (2,6-dihydroxy-1,7-dimethyl-8-vinylphenanthrene)

[0199] Brown powder; C 18 H 16 O2, EIMS m / z(%) 264[M] + (100), HREIMS m / z 264.1155 (calcd. C 18 H 16 O2, 264.1150). 11H NMR (300 MHz, acetone-d6) δ 7.20 and 8.20 (each 1H, d, J = 9.1 Hz, H-3,4), 8.01 (1H, s, H-5), 7.96 and 7.70 (each 1H, d, J = 9.6 Hz, H-9,10), 2.57 and 2.38 (each 3H, s, 1-,7-Me), 7.10 (1H, dd, J = 9.0 and 15.9 Hz, H-11), 5.75 (1H, dd, J = 2.5 and 15.9 Hz, H-12a), 5.70 (1H, dd, J = 2.5 and 9.0 Hz, H-12b), 8.42 and 8.66 (each 1H, br.s, 2-,6-OH).

[0200] XYW-7: dehydrojuncuenin C (2,6-dihydroxy-1-methyl-12-lactone-phenanthrene)

[0201] Yellow small cubic crystals (MeOH-PE); C 18 H 14 O4, EIMS m / z (%): 294 [M] + (69), HREIMS m / z 294.0910 (calcd. for C 18 H 14 O4, 294.0892). 1 1H NMR (300 MHz, DMSO-d6) δ 7.26 and 8.50 (each 1H, d, J = 8.8 Hz, H-3,4), 8.02 (1H, s, H-5), 7.84 and 7.76 (each 1H, d, J = 9.2 Hz, H-9,10), 2.47 (3H, s, 1-Me), 6.70 (1H, dd, J = 11.9, 18.1 Hz, H-11), 5.75 (1H, dd, J = 2.1, 18.1 Hz, H-12a), 5.70 (1H, dd, J = 2.1, 11.9 Hz, H-12b), 5.54 and 9.69 (each 1H, br.s, 8a-,2-OH).

[0202] XYW-8: 2,8-dihydroxy-1,6-dimethyl-5-vinyl-9,10-dihydrophenanthrene

[0203] White powder; C 18 H 18 O2; ESIMS m / z: 267 [M + H] +.1H-NMR(300MHz, acetone-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), 2.67 and 2.57(each 2H, m, H-9, 10), 2.25 and 2.23(each 3H, s, 1-, 6-Me).

[0204] XYW-9: Juncus phenanthrol

[0205] White powder; C 17 H 16 O2, ESI-MS m / z: 253[M + H] + , 1H NMR(300MHz, acetone-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).

[0206] XYW-10: 2,7-Dihydroxy-1-methyl-5-vinylphenanthrene

[0207] White powder, C 17 H 14 O2, ESI-MS m / z: 251[M + H] +.1H NMR (300 MHz, acetone-d6): δ 7.21 (1H, d, J = 8.8 Hz, H-3), 8.51 (1H, d, J = 8.8 Hz, H-4), 7.27 (1H, d, J = 1.5 Hz, H-6), 7.22 (1H, d, J = 1.5 Hz, H-8), 7.91 (1H, d, J = 9.0 Hz, H-9), 7.66 (1H, d, J = 9.0 Hz, H-10), 7.49 (1H, dd, J = 11.0, 17.1 Hz, H-11), 5.76 (1H, dd, J = 17.1, 1.5 Hz, H-12a), 5.44 (1H, dd, J = 11.0, 1.5 Hz, H-12b), 2.58 (3H, s, 1-Me).

[0208] 5. Separation of 9,10-Dihydrophenanthrene and Phenanthrene Compounds from Juncus effusus

[0209] The whole herb (10 kg) of Juncus effusus collected from Jinxiu County, Guangxi was crushed and soaked four times with 95% industrial ethanol for three days each time. The extraction solutions were combined and concentrated under reduced pressure to remove ethanol, obtaining a dark brown extract (605 g). The extract was transferred into a 25 L tap funnel, 10 L of water was added, and a suspension was stirred. Then it was extracted three times with petroleum ether, dichloromethane, and ethyl acetate respectively, and 75 g of the petroleum ether fraction, 165 g of the dichloromethane fraction, and 80 g of the ethyl acetate fraction were obtained by concentration under reduced pressure.

[0210] The dichloromethane fraction was treated by MCI column chromatography and eluted successively with 50, 60, 70, 80, 95% ethanol-water to obtain seven fractions. Referring to the separation and purification methods of compounds in Pholidota chinensis and Juncus setchuensis, silica gel column chromatography, Sephadex LH-20 gel column chromatography, preparative HPLC and other methods were repeatedly used for separation and purification to obtain 33 9,10-dihydrophenanthrene compounds, among which compounds XF-1 - 11, 24, 29 were 9,10-dihydrophenanthrene / phenanthrene monomers, and XF-14 - 20, 33 were 9,10-dihydrophenanthrene / phenanthrene dimers.

[0211] 6. Spectral Data of Compounds

[0212] XF-1: Juncuenin H

[0213] Colorless square crystals (acetone); ESIMS m / z 249.2 [M - H] - ; HRESIMS m / z 249.1302 [M - H] - (calcd for C18H17O, 249.1279). 11H NMR (400 MHz, CDCl3) δ 6.75 and 7.48 (each 1H, d, J = 8.4 Hz, H-3,4), 7.51 and 7.13 (each 1H, d, J = 7.9 Hz, H-5,6), 6.79 (1H, dd, J = 11.4, 17.9 Hz, H-11), 5.61 (1H, dd, J = 11.4, 2.1 Hz, H-12a), 5.24 (1H, dd, J = 17.9, 2.1 Hz, H-12b), 2.91 and.276 (each 2H, m, H-9,10), 2.26 and 2.34 (each 3H, s, 1-,7-Me), 5.16 (1H, br.s, 2-OH).

[0214] XF-2: Juncuenin I

[0215] Light yellow powder; ESIMS m / z 251.1 [M-H] - ; HRESIMS m / z 251.1049 [M-H] - (calcd for C17H15O2, 251.1072). 1 1H NMR (400 MHz, acetone-d6) δ 6.86 and 7.60 (each 1H, d, J = 8.4 Hz, H-3,4), 8.12 and 7.18 (each 1H, s, H-5,8), 10.28 (1H, s, 6-CHO), 2.84 (4H, m, H-9,10), 2.21 and 2.63 (each 3H, s, 1-,7-Me), 8.42 (1H, br.s, 2-OH).

[0216] XF-3: Juncuenin J

[0217] Light yellow powder; ESIMS m / z 297.1 [M-H] - ; HRESIMS m / z 297.1501 [M-H] - (calcd for C19H21O3, 297.1491). 11H NMR (400 MHz, acetone-d6) δ 6.77 and 6.95 (each 1H, d, J = 8.3 Hz, H-3,4), 6.95 (1H, s, H-6), 2.86 and 2.40 (each 2H, m, H-9,10), 2.20 and 2.24 (each 3H, s, 1-,8-Me), 4.80 (1H, q, J = 6.3 Hz, H-11), 1.50 (3H, d, J = 6.3 Hz, H-12), 2.88 (3H, s, -OMe), 8.20 and 8.02 (each 1H, br.s, 2-,7-OH).

[0218] XF-4: Juncuenin K

[0219] Light yellow powder; ESIMS m / z 281.3 [M+H] + ; HRESIMS m / z 281.1533 [M+H] + (calcd for C19H21O2, 281.1542). 1 1H NMR (400 MHz, CDCl3) δ 6.81 and 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, 17.9 Hz, H-11), 5.61 (1H, dd, J = 14.1, 2.0 Hz, H-12a), 5.20 (1H, dd, J = 17.9, 2.0 Hz, H-12b), 2.74 and 2.79 (each 2H, m, H-9,10), 2.21 and 2.24 (each 3H, s, 1-,7-Me), 4.70 (1H, br.s, 6-OH), 3.86 (3H, s, 2-OMe).

[0220] XF-5: Juncuenin L

[0221] Light yellow powder; ESIMS m / z 310.1 [M-H] - ; HRESIMS m / z 310.1092 [M-H] - (calcd for C18H16NO4, 310.1079). 11H NMR (400 MHz, CDCl3) δ 8.46 and 6.68 (each 1H, s, H-4, 8), 6.79 (1H, dd, J = 18.5, 14.4 Hz, H-11), 5.60 (1H, dd, J = 14.4, 1.2 Hz, H-12a), 5.20 (1H, dd, J = 18.5, 1.2 Hz, H-12b), 2.67 and 2.79 (each 2H, m, H-9, 10), 2.32 and 2.28 (each 3H, s, 1-, 6-Me), 11.10 and 4.70 (each 1H, br.s, 2-, 7-OH).

[0222] XF-6: Juncuenin M

[0223] Light yellow powder; ESIMS m / z 372.1 [M-H] - ; HRESIMS m / z 372.1624 [M-H] - (calcd for C24H22NO3, 372.1600). 1 1H NMR (400 MHz, CDCl3) δ 6.64 and 7.13 (each 1H, d, J = 8.4 Hz, H-3, 4), 6.76 (1H, dd, J = 11.3, 17.8 Hz, H-11), 5.62 (1H, dd, J = 11.3, 1.9 Hz, H-12a), 5.22 (1H, dd, J = 17.8, 1.9 Hz, H-12b), 2.66 (4H, m, H-9, 10), 2.26 and 2.29 (each 3H, s, 1-, 7-Me), 9.40 (1H, s, 1’-CHO), 6.96 and 6.24 (each 1H, dd, J = 2.6, 3.6 Hz, H-2’, 3’), 4.15 (2H, s, H-5’), 5.11 and 5.23 (each 1H, br.s, 2-, 6-OH), 9.68 (1H, s, NH).

[0224] XF-7: Dehydrojuncuenin F

[0225] Light yellow powder; ESIMS m / z 246.9 [M-H] - ; HRESIMS m / z 247.1145 [M-H] - (calcd for C18H15O, 247.1123). 11H NMR (400 MHz, CDCl3) δ 7.18 and 8.44 (each 1H, d, J = 8.9 Hz, H-3,4), 8.44 and 7.46 (each 1H, d, J = 8.5 Hz, H-5,6), 8.12 and 7.88 (each 1H, d, J = 9.5 Hz, H-9,10), 7.07 (1H, dd, J = 14.4, 17.9 Hz, H-11), 5.79 (1H, dd, J = 14.4, 2.0 Hz, H-12a), 5.43 (1H, dd, J = 17.9, 2.0 Hz, H-12b), 2.59 and 2.49 (each 3H, s, 1-,7-Me), 5.48 (1H, br.s, 2-OH).

[0226] XF-8: Dehydrojuncuenin G

[0227] Light yellow powder; ESIMS m / z 249.1 [M-H] - ; HRESIMS m / z 249.0881 [M-H] - (calcd for C17H13O2, 249.0916). 1 1H NMR (400 MHz, acetone-d6) δ 7.38 and 8.60 (each 1H, d, J = 8.9 Hz, H-3,4), 9.12 and 7.74 (each 1H, s, H-6,8), 7.76 and 8.08 (each 1H, d, J = 9.2 Hz, H-9,10), 2.58 and 2.77 (each 3H, s, 1-,7-Me), 10.42 (1H, s, 6-CHO), 8.73 (1H, br.s, 2-OH).

[0228] XF-9: Dehydrojuncuenin H

[0229] Light yellow powder; ESIMS m / z 265.2 [M+H] + ; HRESIMS m / z 265.1207 [M+H] + (calcd for C18H17O2, 265.1229). 11H NMR (400 MHz, CDCl3) δ 7.25 and 8.65 (each 1H, d, J = 9.3 Hz, H-3,4), 7.19 (2H, s, H-6,8), 7.60 and 7.91 (each 1H, d, J = 9.2 Hz, H-9,10), 7.47 (1H, dd, J = 10.7, 17.3 Hz, H-11), 5.78 (1H, dd, J = 17.3, 1.6 Hz, H-12a), 5.46 (1H, dd, J = 10.7, 1.6 Hz, H-12b), 2.61 (3H, s, 1-Me), 3.97 (3H, s, 2-OMe), 5.10 (1H, br.s, 7-OH).

[0230] XF-10: Dehydrojuncuenin I

[0231] Pale yellow powder; ESIMS m / z 279.1 [M+H] + ; HRESIMS m / z 279.1035 [M+H] + (calcd for C18H15O3, 279.1021). 1 1H NMR (400 MHz, acetone-d6) δ 7.23 and 8.82 (each 1H, d, J = 9.3 Hz, H-3,4), 8.04 (1H, s, H-5), 8.18 and 8.40 (each 1H, d, J = 9.5 Hz, H-9,10), 7.14 (1H, dd, J = 11.5, 17.9 Hz, H-11), 5.85 (1H, dd, J = 11.5, 2.0 Hz, H-12a), 5.46 (1H, dd, J = 17.9, 2.0 Hz, H-12b), 2.39 (3H, s, 7-Me), 11.01 (1H, s, 1-CHO), 12.94 and 9.02 (each 1H, br.s, 2-,6-OH).

[0232] XF-11: Dehydrojuncuenin J

[0233] Pale yellow powder; ESIMS m / z 297.1 [M-H] - ; HRESIMS m / z 297.0682 [M-H] - (calcd for C18H14O2Cl, 297.0682). 11H NMR (400 MHz, CDCl3) δ 8.39 and 8.79 (1H, s, H-4,5), 7.99 and 7.68 (each 1H, d, J = 9.5 Hz, H-9,10), 7.05 (1H, dd, J = 11.4, 17.9 Hz, H-11), 5.82 (1H, dd, J = 11.4, 2.0 Hz, H-12a), 5.40 (1H, dd, J = 17.9, 2.0 Hz, H-12b), 2.63 and 2.41 (each 3H, s, 1,7-Me), 5.81 and 5.18 (each 1H, br.s, 2-,6-OH).

[0234] XF-14: Dijuncuenin C

[0235] Light yellow powder; ESIMS m / z 529.2 [M-H] - ; HRESIMS m / z 529.2370 [M-H] - (calcd for C36H33O4, 529.2379). 1 1H NMR (400 MHz, CDCl3) δ 7.45 (2H, s, H-4,4’), 7.05 (2H, s, H-5,5’), 6.76 (2H, dd, J = 17.9, 11.4 Hz, H-11,11’), 5.63 (2H, dd, J = 11.4, 1.9 Hz, H-12a,12’a), 5.22 (2H, dd, J = 17.9, 1.9 Hz, 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).

[0236] XF-15: Dijuncuenin D

[0237] Light yellow powder; ESIMS m / z 529.1 [M-H] - ; HRESIMS m / z 529.2391 [M-H] - (calcd for C36H33O4, 529.2379). 11H NMR (400 MHz, CDCl3) δ 7.45 (2H, s, H-4,4’), 6.67 (2H, s, H-8,8’), 6.86 (2H, dd, J = 17.9, 11.4 Hz, H-11,11’), 5.47 (2H, dd, J = 11.4, 1.3 Hz, H-12a,12’a), 5.14 (2H, dd, J = 17.9, 1.3 Hz, 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).

[0238] XF-16: Dijuncuenin E

[0239] Light yellow powder; ESIMS m / z 529.3 [M-H] - ; HRESIMS m / z 529.2358 [M-H] - (calcd for C36H33O4, 529.2379). 1 1H NMR (400 MHz, CDCl3) δ 7.29 and 7.68 (each 1H, s, H-4,5), 6.91 (1H, dd, J = 11.5, 17.5 Hz, H-11), 5.82 (1H, dd, J = 17.3, 0.7 Hz, H-12a), 5.40 (1H, dd, J = 11.5, 0.7 Hz, H-12b), 2.74 (4H, m, H-9,10), 2.36 and 2.26 (each 3H, s, 1-,7-Me), 7.49 and 6.68 (each 1H, s, H-4’,8’), 6.86 (1H, dd, J = 17.9, 11.3 Hz, H-11’), 5.46 (1H, dd, J = 11.3, 1.8 Hz, H-12’a), 5.14 (1H, dd, J = 17.9, 1.8 Hz, H-12’b), 2.74 (4H, m, H-9’,10’), 2.31 and 2.23 (each 3H, s, 1’,6’-Me).

[0240] XF-17: Dijuncuenin F

[0241] Light yellow powder; ESIMS m / z 527.1 [M-H] - ; HRESIMS m / z 527.2231 [M-H] -(calcd for C36H31O4, 527.2222). 1 1H NMR (400 MHz, CDCl3) δ 7.50 and 7.00 (each 1H, s, H-4,5), 6.78 (1H, dd, J=11.4, 17.9 Hz, H-11), 5.65 (1H, dd, J=11.4, 1.9 Hz, H-12a), 5.25 (1H, dd, J=17.9, 1.9 Hz, H-12b), 2.89 (4H, m, H-9,10), 2.30 and 2.22 (each 3H, s, 1-,7-Me), 4.93 and 4.85 (each 1H, s, 2-,6-OH), 6.59 and 7.66 (each 1H, d, J=9.3 Hz, H-3’,4’), 8.11 and 7.79 (each 1H, d, J=9.5 Hz, H-9’,10’), 7.14 (1H, dd, J=17.9, 11.4 Hz, H-11’), 5.88 (1H, dd, J=11.4, 1.9 Hz, H-12’a), 5.46 (1H, dd, J=17.9, 1.9 Hz, H-12’b), 2.54 and 2.45 (each 3H, s, 1’,7’-Me), 5.13 and 5.65 (each 3H, s, 2’,6’-OH).

[0242] XF-18: Dijuncuenin G

[0243] Light yellow powder; ESIMS m / z 527.2 [M-H] - ; HRESIMS m / z 527.2234 [M-H] - (calcd for C36H31O3, 527.2222). 11H NMR (400 MHz, CDCl3) δ 7.58 and 6.71 (each 1H, s, H-4,8), 6.71 (1H, dd, J = 11.3, 17.9 Hz, H-11), 5.13 (1H, dd, J = 11.3, 1.9 Hz, H-12a), 5.05 (1H, dd, J = 17.9, 1.9 Hz, H-12b), 2.80 (4H, m, H-9,10), 2.28 and 2.19 (each 3H, s, 1-,6-Me), 4.78 and 4.90 (each 1H, s, 2-,7-OH), 6.59 and 7.70 (each 1H, d, J = 9.3 Hz, H-3’,4’), 8.10 and 7.78 (each 1H, d, J = 9.5 Hz, H-9’,10’), 7.13 (1H, dd, J = 17.9, 11.4 Hz, H-11’), 5.87 (1H, dd, J = 11.4, 1.9 Hz, H-12’a), 5.46 (1H, dd, J = 17.9, 1.9 Hz, H-12’b), 2.55 and 2.45 (each 3H, s, 1’-,7’-Me), 4.96 and 5.70 (each 3H, s, 2’-,6’-OH).

[0244] XF-19: Dijuncuenin H

[0245] Light yellow powder; ESIMS m / z 527.3 [M-H] - ; HRESIMS m / z 527.2233 [M-H] - (calcd for C36H31O4, 527.2222). 11H NMR (400 MHz, CDCl3) δ 7.32 and 6.81 (each 1H, s, H-3,6), 6.72 (1H, dd, J = 10.9, 15.1 Hz, H-11), 5.42 (1H, dd, J = 10.9, 1.1 Hz, H-12a), 4.83 (1H, dd, J = 15.1, 1.1 Hz, H-12b), 2.82 (4H, m, H-9,10), 2.33 and 2.28 (each 3H, s, 1-,8-Me), 6.62 and 7.70 (each 1H, d, J = 9.3 Hz, H-3’,4’), 8.10 and 7.79 (each 1H, d, J = 9.5 Hz, H-9’,10’), 7.12 (1H, dd, J = 15.9, 11.4 Hz, H-11’), 5.87 (1H, dd, J = 11.4, 1.9 Hz, H-12’a), 5.46 (1H, dd, J = 15.9, 1.9 Hz, H-12’b), 2.55 and 2.44 (each 3H, s, 1’-,7’-Me).

[0246] XF-20: Dijuncuenin I

[0247] Light yellow powder; ESIMS m / z 525.3 [M-H] - ; HRESIMS m / z 525.2084 [M-H] - (calcd for C36H29O4, 525.2066). 11H NMR (400 MHz, CDCl3) δ 7.07 and 9.04 (each 1H, d, J = 9.3 Hz, H-3,4), 7.37 and 7.72 (each 1H, d, J = 9.4 Hz, H-9,10), 7.43 (1H, dd, J = 11.3, 18.0 Hz, H-11), 5.82 (1H, dd, J = 11.3, 1.6 Hz, H-12a), 5.44 (1H, dd, J = 18.0, 1.6 Hz, H-12b), 2.49 (6H, s, 1-,8-Me), 4.99 and 5.13 (each 1H, br.s, 2-,7-OH), 6.36 and 7.37 (each 1H, d, J = 9.4 Hz, H-3’,4’), 8.18 and 7.82 (each 1H, d, J = 9.4 Hz, H-9’,10’), 7.19 (1H, dd, J = 17.9, 11.4 Hz, H-11’), 5.92 (1H, dd, J = 11.4, 1.8 Hz, H-12’a), 5.52 (1H, dd, J = 17.9, 1.8 Hz, H-12’b), 2.52 and 2.46 (each 3H, s, 1’-,7’-Me), 4.77 and 5.30 (each 1H, br.s, 2’-,6’-OH).

[0248] XF-24: 2,7-Dihydroxy-1,8-dimethyl-5-vinyl-9,10-dihydrophenanthrene

[0249] White amorphous powder, C 18 H 18 O2, ESIMS m / z: 289.3 [M+Na] + ; 1 1H NMR (400 MHz, CDCl3): δ 6.66 (1H, d, J = 8.5 Hz, H-3), 7.25 (1H, d, J = 8.5 Hz, H-4), 6.87 (1H, s, H-6), 2.69 (4H, br.s, H-9,10), 6.90 (1H, dd, J = 10.8, 17.4 Hz, H-11), 5.16 (1H, dd, J = 10.8, 1.2 Hz, H-12a), 5.58 (1H, dd, J = 17.4, 1.2 Hz, H-12b), 2.26 and 2.30 (3H, s, 1-,8-Me).

[0250] XF-29: dehydrojuncuenin B (2,7-Dihydroxy-1,6-dimethyl-5-vinyl-phenanthrene)

[0251] White amorphous powder, C18 H 16 O2, ESIMS m / z: 287.3 [M+Na] + ; 1 H NMR (400 MHz, DMSO-d6) δ 7.22 and 8.18 (each 1H, d, J = 8.6 Hz, H-3,4), 7.90 (1H, s, H-5), 7.85 and 7.63 (each 1H, d, J = 9.2 Hz, H-9,10), 7.09 (1H, dd, J = 18.4, 12.0 Hz, H-11), 5.79 (1H, dd, J = 12.8, 1.6 Hz, H-12a), 5.35 (1H, dd, J = 12.8, 1.6 Hz, H-12b), 2.44 and 2.28 (each 3H, s, 1-,6-Me).

[0252] XF-33: 8-[4-(2,7-dihydroxy-1,6-dimethyl-5-ethenyl-9,10-dihydrophenanthrene)yl]-2,7-dihydroxy-1,6-dimethyl-5-ethenyl-phenanthrene

[0253] White amorphous powder, C 36 H 32 O4, ESIMS m / z: 551.3 [M+H] + , 1 H NMR (400 MHz, CDCl3) δ 7.02 and 8.87 (each 1H, d, J = 9.2 Hz, H-3,4), 7.37 and 7.78 (each 1H, d, J = 9.6 Hz, H-9,10), 7.32 (1H, dd, J = 18.0, 11.2 Hz, H-11), 5.76 (1H, dd, J = 18.0, 1.6 Hz, H-12a), 5.38 (1H, dd, J = 11.2, 1.6 Hz, H-12b), 2.56 and 2.52 (each 3H, s, 1-,6-Me), 7.50 and 6.70 (each 1H, s, H-4’,8’), 2.85 and 2.76 (each 2H, m, H-9’,10’), 6.83 (1H, dd, J = 18.0, 11.2 Hz, H-11’), 5.36 (1H, dd, J = 2.0, 18.0 Hz, H-12’a), 5.11 (1H, dd, J = 2.0, 11.2 Hz, H-12’b), 2.53 and 2.20 (each 3H, s, 1’-,6’-Me).

[0254] Effect of the compound in Example 2 against the hepatotoxicity of hydrophobic bile acids

[0255] Cholestasis is a common complication of drug-induced liver injury, accompanied by the accumulation of hydrophobic bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA), in hepatocytes. Hydrophobic bile acids can damage the outer membrane of the cell basement, organelle membranes, and specifically the outer layer of the microtubule membrane, and are one of the main endogenous substances leading to hepatocyte injury. The hepatocyte injury model induced by hydrophobic bile acids can be initially used to evaluate the effect of compounds against the toxicity of bile acids, thereby inferring the protective effect of compounds on a type of liver injury accompanied by cholestasis symptoms, such as liver injury caused by drugs, especially traditional Chinese herbs.

[0256] 1. Experimental protocol

[0257] 1.1 Protection of compounds against DCA-induced primary rat hepatocyte injury

[0258] Primary rat hepatocytes were isolated by a two-step perfusion method and seeded in 96-well plates at a density of 3×10 5 cells / ml. After 4 h of attachment, drugs were administered. After co-incubating primary rat hepatocytes with 200 μM DCA and the corresponding compounds for 24 h, the cell viability was measured by the CCK8 method. The absorbance at 450 nm was measured using a multifunctional microplate reader, and the absorbance is proportional to the cell viability. The protective effect of the compound was judged according to the cell viability.

[0259] 1.2 Protection of compounds against DCA-induced Hihep cell injury

[0260] Hihep cells were cultured in HMM (Hepatocytes Maintaining Medium) medium. When the cells reached a confluence rate of 90%, the cells were seeded in 96-well plates at a density of 1×10 5 cells / ml. After 3 days of culture, drugs were administered. Hihep cells were co-incubated with 250 μM DCA and the corresponding compounds for 24 h. Subsequently, the cell viability was measured by the CCK8 method, and the operation method was the same as above.

[0261] 1.3 Protection of compounds against LCA-induced Hihep cell injury

[0262] Hihep cells were cultured according to the above method. Hihep cells were co-incubated with 30 μM LCA and the corresponding compounds for 24 h. Subsequently, the cell viability was measured by the CCK8 method, and the operation was the same as above.

[0263] The above experiments were repeated three times. Results showing that the cell survival rate in the drug administration group was higher than that in the bile acid alone administration group (DCA and LCA) and there was a statistically significant difference between the two (P<0.05) were considered to be active; any compound that was active in one of the three models was considered to be able to counteract the hepatotoxicity of hydrophobic bile acids.

[0264] 2. Experimental results

[0265] The experimental results showed that the tested 9,10-dihydrophenanthrene compounds exhibited obvious effects against the hepatotoxicity of hydrophobic bile acids in the concentration range of 0.1 - 25 μM, with potential hepatoprotective effects. Especially for liver injury accompanied by cholestasis symptoms, such as liver injury caused by drugs, especially traditional Chinese medicines, it has potential protective effects. The specific experimental results are shown in Table 1.

[0266]

[0267]

[0268] Protective effect of compound YS30 in Example 3 against CCl4-induced liver injury in mice

[0269] The CCl4-induced acute liver injury model in mice is one of the commonly used models for preclinical evaluation of drugs against acute liver injury. Its injury mechanism is that CCl4 is metabolized by metabolic enzymes such as CYP2E1 and CYP2B in the liver into free radicals, which combine with nucleic acids, proteins, and lipids in the liver, resulting in nucleic acid denaturation, protein synthesis inhibition, and fatty degeneration. The above pathological changes will lead to cell death, release damage-associated molecular patterns (DAMPs), activate immune cells, induce inflammatory responses, and thus lead to downstream fulminant liver injury. Oxidative stress and inflammation are two important pathological processes of liver injury, and this model can be used to evaluate the protective effects of compounds against a type of hepatitis with inflammation and oxidative stress as the main pathological basis. It includes liver injury caused by toxic hepatitis (drugs, chemicals, and biological toxins), alcoholic hepatitis, and ischemic hepatitis.

[0270] 1. Experimental protocol

[0271] 1.1 CCl4 acute liver injury model

[0272] Sixty ICR mice were randomly divided into 7 groups, with 8 mice in each group: solvent group, YS30 high-dose (40 mg / kg·day) single-dose administration group, CCl4 acute model group, CCl4 model plus positive drug bicyclol group (200 mg / kg·day), CCl4 model plus YS30 low-dose (10 mg / kg·day), medium-dose (20 mg / kg·day), and high-dose (40 mg / kg·day) groups. YS30 was continuously administered by gavage at the corresponding dose for 7 days, once a day. Bicyclol was continuously administered by gavage at the corresponding dose for 3 days. The solvent group and the CCl4 acute model group were continuously administered by gavage with 0.5% Tween 80 in solvent for 7 days. 1 h after the last administration, 0.5% CCl4 olive oil solution (10 ml / kg) was subcutaneously injected. The solvent group and the YS30 single-dose administration group were subcutaneously injected with the corresponding volume of olive oil. All mice were fasted and allowed free access to water, and samples were collected 16 h after modeling. After anesthetizing the mice with sodium pentobarbital, blood was collected by eye removal. The mice were dissected, and the liver was taken. A part was washed with normal saline and then quickly frozen in liquid nitrogen and stored at -80 °C for subsequent detection of biochemical indexes. The remaining part was soaked in formalin for hematoxylin-eosin staining.

[0273] 1.2 Detection of blood biochemical indexes

[0274] After the collected mouse blood samples were allowed to stand for 30 min, they were centrifuged at 8000 rpm for 10 min to obtain serum. The contents of ALT, AST, and LDH in the blood were detected using an automatic blood biochemical analyzer (SYSMEX JCA-BM6010C).

[0275] 1.3 H&E staining

[0276] Mouse liver lobes of the same lobe were soaked in formalin for histopathological analysis. Liver tissue of the same part was paraffin-embedded, 3-μm sections were obtained, and H&E staining was performed. The pathological changes of the liver were observed under a microscope, and the congestion, vacuoles, necrosis, and inflammation of the liver tissue were comprehensively evaluated using Suzuki's scoring method.

[0277] 1.4 Detection of inflammatory factors

[0278] ELISA kits for inflammatory factors (TNF-α and IL-6) from Biocolor were used to detect the contents of inflammatory factors in mouse serum.

[0279] 1.5 Detection of MDA level

[0280] The MDA level in tissue samples was detected using a MDA kit from Beyotime. 20-30 mg of liver tissue samples were taken, pre-cooled PBS was added at a ratio of 1:9 for homogenization, and the supernatant was taken after centrifugation at 12000 rpm / min at 4 °C for 10 min. The corresponding protein concentration was detected using a BCA kit, and then the MDA content in the tissue was detected according to the instructions of the MDA kit.

[0281] 2. Experimental Results

[0282] The experimental results showed that oral administration of YS30 (10, 20, 40 mg / kg) dose-dependently reduced the levels of ALT, AST, and LDH in mice with liver injury ( Figure 1 A-C), and simultaneously improved the pathological changes in liver tissues ( Figure 1 D-1E). This indicated that YS30 could significantly improve CCl4-induced liver injury.

[0283] Further detection of the effects of YS30 on the levels of inflammatory factors in serum and lipid peroxidation levels in the liver showed that ( Figure 2 A-C) YS30 had a significant improvement effect on the levels of inflammatory factors TNF-α, IL-6, and liver lipid peroxidation (MDA) in acute liver injury, suggesting that YS30 had potential protective effects on a type of liver injury with inflammation and oxidative stress as the main pathological basis, such as toxic hepatitis (drugs, chemicals, and biological toxins), alcoholic hepatitis, and ischemic hepatitis.

[0284] Example 4 Protective Effect of Compound YS30 on Ischemia-Reperfusion Liver Injury in Mice

[0285] Clinically, liver ischemia-reperfusion injury occurs in situations such as liver transplantation, hepatectomy, trauma, hemorrhagic shock, and other systemic low blood flow diseases such as sepsis, respiratory failure, and congestive heart failure. The mechanism of this injury is related to the oxidative stress response during the ischemic period and the explosive inflammatory response during the reperfusion period. Using a 70% warm ischemia-reperfusion model in mice can simulate perioperative liver injury in clinical practice and evaluate the protective effect of compounds on this type of liver injury.

[0286] The compounds described in the present invention all have antioxidant and / or anti-inflammatory activities similar to those of YS30 in vitro,

[0287] 1. Experimental Protocol

[0288] 1.1 Small Ischemia-Reperfusion Liver Injury Model

[0289] C57 / BL mice were divided into 3 groups: sham operation group (Sham group), ischemia-reperfusion group (I / R), and YS30 administration group (I / R + YS30). YS30 at 40 mg / kg was dissolved in normal saline and administered via the tail vein at 24 h, 12 h, and 1 h before surgery. According to the literature reports, a 70% warm ischemia-reperfusion model was constructed. After anesthetizing the mice with 4% chloral hydrate (intraperitoneal injection), an incision was made along the midline of the abdomen, and the hepatic artery, portal vein, and bile duct were isolated. Non-invasive micro hemostatic clips were used to clamp the hepatic artery, portal vein, and bile duct of the left and middle lobes of the liver to cause 70% hepatic ischemia. After 60 min of ischemia, the non-invasive hemostatic clips were removed, blood supply was restored, the abdomen was sutured, and blood and tissue samples of each group were collected after 6 h of reperfusion.

[0290] 1.2 Detection of blood biochemical indexes

[0291] The method was the same as above

[0292] 1.3 H&E staining

[0293] The method was the same as above

[0294] 2. Experimental results

[0295] The results were as Figure 3 shown. The experimental results showed that YS30 could significantly improve liver injury in mice caused by ischemia-reperfusion, significantly inhibit the levels of ALT, AST, and LDH in the serum of mice, and at the same time could significantly improve the pathological changes of liver tissue, indicating that YS30 has the application prospect of counteracting perioperative liver injury clinically.

[0296] Example 5 Effects of 9,10-dihydrophenanthrene active compounds on oxidative stress and inflammation in the pathological process of liver injury

[0297] 1. Evaluation of antioxidant activity of 9,10-dihydrophenanthrene compounds

[0298] 1.1 Experimental protocol

[0299] DPPH free radical scavenging experiment: Weigh a certain amount of DPPH reagent, dissolve it with absolute ethanol to prepare a 0.2 mM DPPH absolute ethanol solution, and place it in a refrigerator at 4 °C for later use. Dissolve the compound to be tested in DMSO to form a 100 mM stock solution, and then dilute it 1:1000 in absolute ethanol solution to form a 100 μM test solution. Take 100 μl of the compound to be tested (100 μM) or the positive drug vitamin C (100 μM) and add 100 μl of DPPH free radical ethanol solution (0.2 mM) to a 96-well plate. At the same time, set up a DPPH free radical group without adding drugs (replaced by 100 μl of absolute ethanol). For both the positive drug and the compound to be tested, set up corresponding negative control wells without DPPH free radicals (replaced by 100 μl of absolute ethanol) to deduct the influence of the color of the test sample itself on the reading. After mixing, let it stand at room temperature for 30 min, and then measure the absorbance at 517 nm.

[0300] DPPH free radical scavenging rate = [OD DPPH-control -(OD DPPH-sample -OD sample-control )] / OD DPPH-control *100%

[0301] Among them, OD DPPH-control represents the average value of the OD values of the group without adding the compound but adding DPPH

[0302] OD DPPH-sample represents the OD value of the compound + DPPH group

[0303] OD sample-control represents the OD value of the group without adding DPPH but adding the compound

[0304] 1.2 Experimental results

[0305] The DPPH free radical scavenging rate of silymarin (100 μM) is about 35%. We stipulate that a compound with a DPPH free radical scavenging rate better than that of silymarin and with statistical significance (p < 0.05) is a strong antioxidant. The results show that among the 46 compounds tested, 39 compounds (100 μM) have a DPPH free radical scavenging rate greater than 35%, which is defined as having strong antioxidant activity. The results are shown in Table 2.

[0306] 2. Evaluation of the anti-inflammatory activity of 9,10-dihydrophenanthrene compounds

[0307] 2.1 Experimental protocol

[0308] Isolate mouse bone marrow macrophages (BMDM), and induce monocyte colonies for one week with high-glucose DMEM medium containing 15% L929 cell supernatant. Seed the cells into a 96-well plate at a seeding density of 5×10 5cells / ml. After culturing for 24 h, the corresponding drugs were added for incubation. The 10 mM solution of the positive drug or the compound to be tested was dissolved in high-glucose DMEM medium containing 10% FBS at a ratio of 1:1000. The drug to be tested (10 μM) and 1 μg / ml of LPS were incubated with BMDM simultaneously, and the supernatant was collected after 24 h for the detection of NO. The level of NO was used to reflect the strength of the inflammatory response in the reaction system, thereby judging the anti-inflammatory effect of the compound.

[0309] 2.2 Experimental results

[0310] The positive drug dexamethasone (10 μM) could significantly inhibit the release of NO by BMDM. Comparing the drug administration group with the LPS-induced group, the NO level after drug administration was lower than that of the LPS-induced group, and the compounds with a statistically significant difference (p < 0.05) between the two were defined as having anti-inflammatory effects. The results showed that among the 46 compounds tested, 21 compounds had significant anti-inflammatory effects. The results are shown in Table 2.

[0311] Table 2: Antioxidant and anti-inflammatory effects of 9,10-dihydrophenanthrene compounds

[0312]

[0313]

[0314] The above test results indicate that the 9,10-dihydrophenanthrene compounds with hepatoprotective activity have antioxidant and / or anti-inflammatory properties similar to those of YS30, and also have potential protective effects on a type of liver injury mainly based on inflammation and oxidative stress, such as toxic hepatitis (drugs, chemicals, and biological toxins), alcoholic hepatitis, and ischemic hepatitis.

[0315] Conclusion:

[0316] The CCl4-induced acute liver injury model in mice is one of the commonly used models for preclinical evaluation of the hepatotoxicity of drugs against xenobiotics (such as alcohol, drugs, and chemical poisons). Its injury mechanism is that CCl4 is metabolized by metabolic enzymes such as CYP2E1 and CYP2B in the liver into free radicals, which combine with nucleic acids, proteins, and lipids in the liver, resulting in nucleic acid denaturation, protein synthesis inhibition, and fatty degeneration. The above pathological changes will lead to cell death, release damage-associated molecular patterns (DAMPs), activate immune cells, induce inflammatory responses, and thus lead to downstream fulminant liver injury. After in vivo pharmacodynamic verification, we found that the representative compound YS30 (gigantol) has a good protective effect on CCl4-induced acute liver injury at the animal level, and can significantly reduce the elevation of ALT, AST, and LDH caused by liver injury ( Figure 1 A), and histopathological examination found that YS30 could improve pathological changes such as vacuoles, necrosis, and inflammation in liver tissue (Figure 1 B). In addition, the results of animal experiments also showed that YS30 could reduce the levels of inflammatory factors and lipid peroxidation in liver tissues ( Figure 2 ), suggesting its potential therapeutic effects on a type of hepatitis with inflammation and lipid peroxidation as the pathological basis, including toxic hepatitis (drugs, chemicals, and biological toxins), alcoholic hepatitis, ischemic hepatitis, etc.

[0317] To verify this conjecture, we further investigated the in vivo liver protection efficacy of YS30 in a mouse model of ischemia-reperfusion injury. Clinically, liver ischemia-reperfusion injury occurs in situations such as liver transplantation, hepatectomy, trauma, hemorrhagic shock, and other systemic low blood flow diseases such as sepsis, respiratory failure, and congestive heart failure. The mechanism of this injury is related to the oxidative stress response during the ischemia period and the explosive inflammatory response during the reperfusion period. The experimental results showed that YS30 significantly improved the elevation of serum ALT, AST, and LDH and the pathological changes in liver tissues caused by ischemia-reperfusion. It can be seen that YS30 has potential therapeutic effects in clinical perioperative liver injury.

[0318] In summary, the 9,10-dihydrophenanthrene compounds of the present invention have good liver protection effects and have potential application values in the treatment of drug-induced liver injury, toxic hepatitis, alcoholic hepatitis, and perioperative liver injury accompanied by cholestasis symptoms.

[0319] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. Use of a compound represented by the following formula II, or a pharmaceutically acceptable salt, hydrate or solvate thereof: Among them, The dotted line represents a chemical bond or none; R d and R e each independently selected from the group consisting of: H, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl, -CHO, -NO2, or substituted or unsubstituted benzyl; R e ' is a substituted or unsubstituted benzyl group; q is selected from the group consisting of: 0, 1, 2 or 3; r is selected from the group consisting of: 0, 1, 2, 3 or 4; Unless otherwise specified, the substitution is substituted by one or more substituents selected from the group consisting of: H, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, C2-C4 alkenyl, -CHO, -NO2, or phenyl substituted by one or more groups selected from the group consisting of: hydroxy, C1-C4 alkyl, C1-C4 alkoxy; It is characterized in that it is used for preparing a pharmaceutical composition for treating or preventing liver injury.

2. The use according to claim 1, characterized in that, The compound is selected from the group consisting of:

3. Use of a compound represented by the following formula 30, or a pharmaceutically acceptable salt, hydrate or solvate thereof, characterized in that, The compound is selected from the group consisting of:

4. The use according to claim 1 or 3, characterized in that, The liver injury is selected from the group consisting of: liver injury with cholestatic symptoms, or hepatitis.

5. The use according to claim 4, characterized in that, The hepatitis is selected from the group consisting of: toxic hepatitis (drugs, chemicals and biotoxins), alcoholic hepatitis, ischemic hepatitis.

6. The use according to claim 4, wherein, The liver injury is drug-induced liver injury, preferably, the liver injury is herbal medicine-induced liver injury.

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