Alkaloid compounds isolated from kadsura coccinea and preparation method and application thereof

Abstract

The application relates to the technical field of medicines, in particular to an alkaloid compound separated from white earth and a preparation method and application thereof. The alkaloid compound or a pharmaceutically acceptable salt thereof has a structure as shown in formula I or formula II: wherein R is H or methoxy. The novel alkaloid compound is separated from white earth, and is verified by experiments to have good anti-hepatic fibrosis activity and has a good application prospect in the field of anti-hepatic fibrosis drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an alkaloid compound isolated from Smilax glabra, its preparation method, and its application. Background Technology

[0002] Liver fibrosis is an essential stage in the progression of chronic liver disease to cirrhosis and liver cancer, and it is the core pathological change in chronic liver disease caused by various factors such as viral infection, excessive alcohol consumption, and hereditary diseases. Currently, liver fibrosis and early cirrhosis are considered reversible, and timely treatment of liver fibrosis can inhibit its progression to cirrhosis and liver cancer. Current clinical treatments for liver fibrosis or cirrhosis include etiological treatment targeting the primary disease, reducing inflammation and immune responses, and liver transplantation; however, these methods have limitations such as significant side effects, high medical costs, and a lack of donors. Therefore, identifying key anti-liver fibrosis candidate drugs and elucidating their mechanisms of action has significant social and economic value.

[0003] Hepatic stellate cell activation is a key trigger for the development and progression of liver fibrosis, and inhibiting hepatic stellate cell activation is an important intervention strategy to prevent the progression of liver disease to fibrosis. Under normal circumstances, hepatic stellate cells are in a resting state. In chronic liver injury, damaged hepatocytes, sinusoidal endothelial cells, and Kupffer cells release pro-fibrotic cytokines, such as TGF-β1, activating hepatic stellate cells. Activated hepatic stellate cells express large amounts of the cytoskeletal protein α-SMA, migrate to the damaged site, and transform into a myofibroblast-like phenotype, synthesizing extracellular matrix proteins such as Collagen I and secreting them extracellularly, participating in the progression of liver fibrosis. Therefore, inhibiting hepatic stellate cell migration has the potential to treat liver fibrosis. Evaluating the expression of α-SMA and cell contraction in hepatic stellate cells can comprehensively assess their migration capacity. Previous studies have suggested that the low molecular weight G protein RhoA is a key protein regulating cell migration, and its active form, RhoA-GTP, plays an important role in cytoskeletal remodeling-mediated cell movement.

[0004] Traditional Chinese medicine and herbal formulas have extensive clinical experience in the treatment of liver fibrosis, characterized by good efficacy, strong activity, and minimal toxic side effects. Natural compounds can serve as important sources of lead compounds in the development of drugs for treating liver fibrosis, such as glycyrrhizic acid preparations, with representative drugs being magnesium isoglycyrrhizate injection and diammonium glycyrrhizate enteric-coated capsules. In addition, silymarin (a flavonoid extracted from silymarin) also has anti-fibrotic effects, with representative drugs being silymarin meglumine tablets and silymarin capsules, which are widely used clinically.

[0005] White sarsaparilla is the dried rhizome of *Heterosmilax yunnanensis* Gagnep., a plant belonging to the genus *Heterosmilax* in the family Liliaceae. It is mainly distributed in Shaanxi, Gansu, Anhui, Sichuan, and Yunnan provinces. It possesses properties such as clearing heat and detoxifying, removing dampness, and promoting joint mobility. It is the only auxiliary ingredient in the anti-tumor traditional Chinese medicine compound *Kushen* injection, and its highly polar aqueous fraction is used medicinally. Chemical composition and pharmacological activity studies have shown that the aqueous fraction of white sarsaparilla contains phenolic glycosides, flavonoid glycosides, sterol glycosides, azo glycosides, as well as some monophenolic acids, amino acids, and polysaccharides, exhibiting diuretic, anti-inflammatory, joint-promoting, antibacterial, detoxifying, and analgesic activities. However, current research on white sarsaparilla is limited, and the types and quantities of compounds are not comprehensive enough. The highly polar fraction mainly consists of glycosides, and pharmacological activity studies primarily focus on the crude extract.

[0006] Currently, there are no reports of alkaloid compounds being isolated from the traditional Chinese medicine Smilax glabra, nor are there any reports of corresponding isolation and preparation methods or anti-liver fibrosis activity.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] One object of the present invention is to provide alkaloid compounds isolated from Smilax glabra.

[0009] Another object of the present invention is to provide a method for preparing alkaloid compounds.

[0010] Another object of the present invention is to provide the use of alkaloid compounds isolated from Smilax glabra and their pharmaceutically acceptable salts, pharmaceutical compositions, etc., in the preparation of drugs for treating liver fibrosis.

[0011] To achieve the above-mentioned objectives of the present invention, one aspect of the present invention provides alkaloid compounds or pharmaceutically acceptable salts thereof, said compounds having a structure as shown in Formula I or Formula II:

[0012]

[0013] Wherein, R is H or methoxy.

[0014] Another aspect of the present invention provides a method for preparing any of the above-described alkaloid compounds, comprising the following steps:

[0015] (a) The white sclerotium was extracted by reflux with 92-98% ethanol aqueous solution, 82-88% ethanol aqueous solution, 72-78% ethanol aqueous solution and water in sequence, and the extracts were combined and concentrated to obtain the first extract.

[0016] (b) The first extract is extracted with an organic solvent, and the remaining aqueous phase is precipitated with alcohol. The supernatant is collected and concentrated to obtain the second extract. The second extract is purified by macroporous resin column chromatography, gel column chromatography and liquid chromatography to obtain the alkaloid compound.

[0017] In a specific embodiment of the present invention, the organic solvent includes at least one of ethyl acetate, petroleum ether, dichloromethane, and n-butanol.

[0018] In a specific embodiment of the present invention, in the alcohol precipitation, ethanol is used to adjust the aqueous phase to an ethanol aqueous solution with a volume fraction of 82% to 88%.

[0019] In a specific embodiment of the present invention, the macroporous resin column chromatography includes: eluting with a water-ethanol gradient and collecting the eluent in a 30% ethanol aqueous solution. Further, elution is performed sequentially with ethanol aqueous solutions of 0%, 5%, 15%, 30%, 50%, 70%, and 95% (v / v), and the eluent in a 30% ethanol aqueous solution is collected.

[0020] In a specific embodiment of the present invention, the gel column chromatography includes: eluting with a water-ethanol gradient and collecting the eluent in a 0% ethanol aqueous solution. Further, elution is performed sequentially with ethanol aqueous solutions of 0%, 5%, 15%, 30%, 50%, 70%, and 95% by volume.

[0021] In a specific embodiment of the present invention, the liquid chromatography column purification includes: purifying compounds I and II using a C18 reversed-phase semi-preparative liquid chromatography column.

[0022] In another aspect, the present invention provides the use of any of the aforementioned alkaloid compounds or their pharmaceutically acceptable salts in the preparation of drugs for treating liver fibrosis.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned alkaloid compounds or their pharmaceutically acceptable salts and pharmaceutically acceptable excipients.

[0024] In a specific embodiment of the present invention, the mass fraction of the alkaloid compound or its pharmaceutically acceptable salt in the pharmaceutical composition is 0.1% to 95%.

[0025] In specific embodiments of the present invention, the dosage form of the pharmaceutical composition includes at least one of tablets, capsules, pills, granules, and suspensions.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention isolates novel alkaloid compounds from Smilax glabra, which, as verified by experiments, exhibit good anti-liver fibrosis activity and have promising application prospects in the field of anti-liver fibrosis drugs. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0029] This invention provides, in one aspect, alkaloid compounds or pharmaceutically acceptable salts thereof, wherein the alkaloid compounds have a structure as shown in Formula I or Formula II:

[0030]

[0031] Wherein, R is H or methoxy.

[0032] This invention, through the discovery of the pharmacodynamic material basis and active lead compounds of commonly used Chinese medicines, combined with clinical applications, literature review, and laboratory screening for anti-liver fibrosis activity, found that several compounds isolated from the Chinese medicine Smilax glabra have good anti-liver fibrosis activity.

[0033] Formula I is a novel tryptophan-CC-coupled catechin spirocyclic carbon glycoside compound, and Formula II is a 2,3-diketopyrazine compound. These compounds can significantly inhibit the RhoA-GTP activity of blastocysts and limit their motility and migration ability. Among them, compound I has the most obvious effect, indicating that this series of compounds is expected to become a novel candidate drug for anti-liver fibrosis.

[0034] Another aspect of the present invention provides a method for preparing any of the above-mentioned alkaloid compounds, comprising the following steps:

[0035] (a) The white sclerotium was extracted by reflux with 92-98% ethanol aqueous solution, 82-88% ethanol aqueous solution, 72-78% ethanol aqueous solution and water in sequence, and the extracts were combined and concentrated to obtain the first extract.

[0036] (b) The first extract was extracted with an organic solvent, and the remaining aqueous phase was precipitated with alcohol. The supernatant was collected and concentrated to obtain the second extract. The second extract was purified by macroporous resin column chromatography, gel column chromatography and liquid chromatography to obtain alkaloid compounds.

[0037] In the reflux extraction of *Smilax glabra*, four extraction solvents were used sequentially. First, a 92-98% (v / v) ethanol aqueous solution was used for reflux extraction, and the supernatant was collected by filtration. Then, an 82-88% (v / v) ethanol aqueous solution was used for reflux extraction of the *Smilax glabra* residue obtained from the previous filtration, and the supernatant was collected by filtration. Next, a 72-78% (v / v) ethanol aqueous solution was used for reflux extraction of the *Smilax glabra* residue obtained from the previous filtration, and the supernatant was collected by filtration. Finally, water was used for reflux extraction of the *Smilax glabra* residue obtained from the previous filtration, and the supernatant was collected by filtration. The extracts were combined, and the solvent was recovered and removed by vacuum distillation to obtain the first extract.

[0038] In different embodiments, the volume fraction of the ethanol aqueous solution with a volume fraction of 92% to 98% can be 92%, 93%, 94%, 95%, 96%, 97%, 98%, or any combination thereof; the volume fraction of the ethanol aqueous solution with a volume fraction of 82% to 88% can be 82%, 83%, 84%, 85%, 86%, 87%, 88%, or any combination thereof; and the volume fraction of the ethanol aqueous solution with a volume fraction of 72% to 78% can be 72%, 73%, 74%, 75%, 76%, 77%, 78%, or any combination thereof.

[0039] In a specific embodiment of the present invention, in reflux extraction, each extraction solvent is extracted 2 to 3 times, and the extraction time for each extraction is 1.5 to 2.5 hours.

[0040] In different implementation methods, each extraction solvent can be used for extraction two or three times, and the extraction time for a single extraction can be 1.5 hours, 2 hours, 2.5 hours, etc. After each extraction, the supernatant is collected by filtration, and then the extraction solvent is added again for extraction.

[0041] In each extraction, the amount of extraction solvent can be adjusted according to actual needs. The target compound of the present invention can be obtained from the extract by using conventional material-liquid ratio extraction. Optimizing the material-liquid ratio can appropriately improve the yield of the target compound.

[0042] In a specific embodiment of the present invention, the organic solvent includes at least one selected from ethyl acetate, petroleum ether, dichloromethane, and n-butanol. The first extract is mixed with the organic solvent and extracted using the organic solvent, such as three times, to remove the organic phase. The remaining aqueous phase is then collected for subsequent alcohol precipitation.

[0043] In a specific embodiment of the present invention, during alcohol precipitation, ethanol is used to adjust the aqueous phase to an aqueous solution with an ethanol volume fraction of 82% to 88%.

[0044] In different embodiments, during alcohol precipitation, the aqueous phase can be adjusted to an aqueous ethanol solution with an ethanol volume fraction of 82%, 83%, 84%, 85%, 86%, 87%, 88%, or any combination thereof. The aqueous phase remaining after organic solvent extraction is subjected to alcohol precipitation, filtered to remove large molecules of sugars, proteins, etc., and then concentrated to obtain a second extract.

[0045] In a specific embodiment of the present invention, macroporous resin column chromatography includes: eluting with a water-ethanol gradient and collecting the eluent in a 30% ethanol aqueous solution. In the water-ethanol gradient elution, the volume fraction of ethanol is 0% to 95%. Further, elution is performed sequentially with ethanol aqueous solutions of 0%, 5%, 15%, 30%, 50%, 70%, and 95% volume fractions, and the eluent in the 30% ethanol aqueous solution is collected.

[0046] In practice, the second extract is dissolved in water before being loaded for chromatography separation. HP20 macroporous resin can be used, but it is not limited to this. For macroporous resin column chromatography, the column specifications can be a column diameter d = 14.5 cm, a column height h = 82 cm, and a column volume of 13.5 L, but these are not limited to these specifications.

[0047] In a specific embodiment of the present invention, gel column chromatography includes: eluting with a water-ethanol gradient and collecting the eluent in a 0% ethanol aqueous solution. In the water-ethanol gradient elution, the volume fraction of ethanol is 0% to 95%. Further, elution is performed sequentially with ethanol aqueous solutions of 0%, 5%, 15%, 30%, 50%, 70%, and 95% volume fractions.

[0048] In a specific embodiment of the present invention, the gel column used in gel column chromatography is a Sephadex LH-20. In actual operation, the target eluent collected by macroporous resin column chromatography is concentrated, dissolved in water, and then loaded for further chromatographic separation. The column specifications in gel column chromatography can be a column diameter d = 11.7 cm and a column height h = 67.0 cm, but are not limited to these.

[0049] In a specific embodiment of the present invention, the liquid chromatography column purification includes: purifying compounds I and II using a C18 reversed-phase semi-preparative liquid chromatography column.

[0050] Compound II of the present invention comprises two structures, II1 and II2, as detailed below:

[0051]

[0052] In another aspect, the present invention provides the use of any of the above-mentioned alkaloid compounds or their pharmaceutically acceptable salts in the preparation of drugs for treating liver fibrosis.

[0053] The alkaloid compounds isolated from Smilax glabra in this invention have shown significant inhibitory effects on the motility of rat hepatic stellate cells in in vitro anti-hepatic fibrosis activity evaluation, thus exhibiting anti-hepatic fibrosis efficacy.

[0054] In another aspect, the present invention provides a pharmaceutical composition comprising any of the above-mentioned alkaloid compounds or their pharmaceutically acceptable salts and pharmaceutically acceptable excipients.

[0055] The pharmaceutical compositions of the present invention may include one or more of the above-mentioned alkaloid compounds or their pharmaceutically acceptable salts.

[0056] Among them, pharmaceutically acceptable salts include, but are not limited to, any one or more of sodium salts, potassium salts, and magnesium salts.

[0057] In a specific embodiment of the present invention, the mass fraction of alkaloids or their pharmaceutically acceptable salts in the pharmaceutical composition is 0.1% to 95%.

[0058] In different embodiments, the mass fraction of the alkaloid compound or its pharmaceutically acceptable salt in the composition may be 0.1%, 0.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0059] The amount of alkaloids or their pharmaceutically acceptable salts in a pharmaceutical composition can be adjusted according to the actual type of excipients, dosage form, and dosage requirements.

[0060] For example, in a unit dosage form, the amount of the alkaloid compound of the present invention or its pharmaceutically acceptable salt in the pharmaceutical composition may be 0.1 to 100 mg, such as 4 to 50 mg.

[0061] The dosage of the pharmaceutical composition of the present invention depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the frequency of administration and the purpose of treatment. Therefore, the therapeutic dosage of the present invention can be adjusted according to the actual situation to achieve the required effective therapeutic amount and complete the purpose of prevention or treatment.

[0062] In specific embodiments of the present invention, the appropriate daily dose range of the alkaloid compound or its pharmaceutically acceptable salt is 0.001 to 100 mg / kg, preferably 0.1 to 60 mg / kg, and more preferably 1 to 30 mg / kg.

[0063] For example, the daily dose of the alkaloid compound of the present invention or its pharmaceutically acceptable salt taken by adult patients may be 10-500 mg, preferably 20-100 mg, which may be taken once or in 2-3 divided doses; the daily dose taken by children may be 5-30 mg, preferably 10-20 mg, which may be taken once or in 2-3 divided doses.

[0064] In specific embodiments of the present invention, the dosage form of the pharmaceutical composition includes liquid dosage forms or solid dosage forms. Conventional liquid dosage forms include, but are not limited to, true solutions, colloids, microparticles, emulsions, and suspensions; conventional solid dosage forms include, but are not limited to, tablets, capsules, pills, and granules. It can also be in the form of drops, aerosols, powders, suppositories, lyophilized powder injections, etc., and can be adjusted according to actual drug administration needs.

[0065] The pharmaceutical composition of the present invention can be a conventional formulation, or any one of a sustained-release formulation, a controlled-release formulation, a targeted formulation, and various microparticle delivery systems.

[0066] The dosage forms and corresponding preparation methods of the pharmaceutical compositions of the present invention can be prepared according to conventional methods. For example, the alkaloid compounds of the present invention or their pharmaceutically acceptable salts can be compounded with one or more pharmaceutically acceptable excipients to prepare a composition in the corresponding dosage form or dosage for human or animal use.

[0067] The drug compositions of the present invention can be administered via the enteric or non-enteric route, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum, or rectum. For example, the route of administration can be by injection, including but not limited to intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and acupoint injection.

[0068] In specific embodiments of the present invention, pharmaceutically acceptable excipients include, but are not limited to, any one or more of solvents, emulsifiers, thickeners, suspending agents, diluents, absorbents, excipients, fillers, disintegrants, disintegration inhibitors, binders, humectants, and lubricants.

[0069] For tablets, excipients that can be used include, but are not limited to, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone. Disintegrants include: dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium lauryl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors include: sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters include: quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants include: talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets, etc.

[0070] For pill formulations, excipients that can be used include, but are not limited to, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, glyceryl monostearate, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfonate, methylcellulose, ethylcellulose, etc.

[0071] For example, for capsules, the alkaloid compounds of the present invention or their pharmaceutically acceptable salts can be mixed with various excipients, and the resulting mixture can be placed in hard gelatin capsules or soft capsules. Alternatively, the alkaloid compounds of the present invention or their pharmaceutically acceptable salts can be formulated into microcapsules, suspended in an aqueous medium to form a suspension, or filled into hard capsules or formulated as injectables for application.

[0072] When the alkaloid compounds of the present invention or their pharmaceutically acceptable salts are formulated into injectable preparations, such as solutions, suspension solutions, emulsions, or lyophilized powders for injection, these preparations may be aqueous or non-aqueous and may contain one or more pharmacodynamically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants, or dispersants. For example, diluents may be selected from water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan esters, fatty acid esters, etc. Furthermore, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol may be added to the injectable formulations. In addition, conventional solubilizers, buffers, pH adjusters, etc., may also be added.

[0073] In practical applications, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical composition according to actual needs.

[0074] The source of the white sclerotium in the following specific embodiments of the present invention is: purchased from Shanxi Zhendong Pharmaceutical Co., Ltd., place of origin: Anshun, Guizhou, identified by Guo Baolin, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

[0075] Example 1

[0076] This embodiment provides a method for isolating compounds I, II1, and II2 from *Smilax glabra*, comprising the following steps:

[0077] (1) Take 100 kg of dried tuber of Smilax glabra, crush it and extract it by reflux with 95% ethanol aqueous solution for 2 hours each time, and filter and collect the supernatant. Then extract it by reflux with 85% ethanol aqueous solution for 2 hours each time, and filter and collect the supernatant. Then extract it by reflux with 75% ethanol aqueous solution for 2 hours each time, and filter and collect the supernatant. Then extract it by reflux with water for 2 hours each time, and filter and collect the supernatant. Combine all the supernatants, remove the ethanol by depressurization and recover the solvent to obtain about 24.5 kg of extract.

[0078] (2) The extract was extracted with an equal volume of ethyl acetate three times. The ethyl acetate phase and the aqueous phase were collected separately. The aqueous phase was then adjusted to an ethanol aqueous solution with a volume fraction of 85%. The extract was then filtered to remove large molecules of sugar, protein and other substances. The extract was concentrated to obtain a second extract of about 6142g.

[0079] (3) Dissolve the second extract obtained in step (2) in 3L of water and perform HP20 macroporous resin column chromatography separation twice (d = 14.5cm, h = 82cm, column volume 13.5L). Elute with 0%, 5%, 15%, 30%, 50%, 70%, and 95% ethanol aqueous solution in volume fractions, and collect the 30% ethanol aqueous solution eluent.

[0080] (4) Dissolve the 30% ethanol aqueous solution eluent (about 110g) collected in step (3) in 250mL of water, load the sample by wet method, and perform Sephadex LH-20 gel column chromatography (d=11.7cm, h=67.0cm). Elute sequentially with ethanol aqueous solutions of volume fractions of 0%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, and 95%, and collect the 0% ethanol aqueous solution eluent.

[0081] (5) The 0% ethanol aqueous solution eluent collected in step (4) was purified by C18 reversed-phase semi-preparative liquid chromatography column. The eluent was a mixed solution of acetonitrile, water and acetic acid with a volume ratio of 16:84:0.1, to obtain compound I (5.3 mg), compound II1 (5.3 mg) and compound II2 (9.1 mg).

[0082] The structures were identified by spectroscopic methods such as UV, IR, NMR, and MS. Compound I is a novel tryptophan C-C coupled catechin spirocyclic carbon glycoside, while compounds II1 and II2 are two 2,3-diketopyrazine compounds.

[0083] The specific spectral information and NMR signal assignments for the three compounds are as follows:

[0084] The structural formula of compound I is as follows:

[0085]

[0086] Compound I: Light purple amorphous powder; specific rotation UV(MeOH)λ max (logε)283(3.25),292sh(3.11),208(3.90)nm; ECD(c 0.2mg / mL,MeOH)λ max (Δε)213.0(-48.99),232(+35.88),283(-13.06)nm; IRν max 3367,2930,1722,1627,1530,1493,1462,1440,1340,1289,1245,1124,1060,998,923,820,749cm -1 HRESIMS m / z 707.2082 [M+H] + (calcd for C 35 H 35 O 14 N2,707.2083).

[0087] 1 H NMR (500MHz, CD3OD), δ H:4.97(1H,overlap,H-2),4.07(1H,dd,J=4.5,8.5Hz,H-3),2.63(1H,dd,J=4.5,20.0Hz,H-4α),2.37(1H,m,H-4β),6.06(1H,s,H-6),6.58(1H,d,J=2.5Hz,H-10),6.37(1H,d,J=8.5Hz,H-13),6.12(1H,dd,J=2.0,8.5Hz,H-14),5.13(1H,s,H-1'),2.51(2H,m,H-3'),3.97(1H,m,H-4'),4.13(1H,m,H-5'),3.11(1H,dd,J=5.0,11.5Hz,H-6'α),2.95(1H,dd,J=7.0,11.5Hz,H-6'β),7.54(1H,d,J=8.0Hz,H-4"),6.90(1H,dt,J=1.0,8.0Hz,H-5”),6.96(1H,dt,J=1.0,8.0Hz,H-6”),7.23(1H,d,J=8.0Hz,H-7"),3.53(1H,dd,J=4.0,15.0Hz,H-9"α),3.27(1H,dd,J=5.5,15.0Hz,H-9"β),4.90(1H,m,H-10"),H-12"(unshowed).

[0088] 13 C NMR(125MHz,CD3OD),δ C :80.8(C-2),67.6(C-3),25.0(C-4),101.6(C-4a),157.9(C-5),91.7(C-6),159.8(C-7),105.9(C-8),151.4(C-8a),131.9(C-9),113.5(C-10),145.5(C-11),145.1(C-12),116.3(C-13),118.0(C-14),47.1(C-1'),120.2(C-2'),44.7(C-3'),72.3(C-4'),89.3(C-5'),63.4(C-6'),133.3(C-1"),108.9(C-2"),129.3(C-3"),119.6(C-4"),119.3(C-5"),122.1(C-6"),111.7(C-7"),137.5(C-8"),27.9(C-9"),54.5(C-10"),168.9(C-11"),171.3(C-13"),174.1(C-14"),C-12"(unshowed).

[0089] The structural formula of compound II1 is as follows:

[0090]

[0091] Compound II1: White amorphous powder; specific rotation [α]20D-46.6 (c 0.10, MeOH); UV(MeOH)λ max (logε)303(4.29),245(4.14),225(4.23)nm; IRν max 3337,2926,1698,1673,1607,1533,1508,1456,1385,1278,1238,1178,1071,1023,841,770,699cm -1 HRESIMS m / z 560.1877 [M+H] + (calcd for C 26 H 30 O 11 N3,560.1875).

[0092] 1 H NMR (500MHz, DMSO-d6), δ H :8.50(1H,d,J=5.5Hz,H-4),3.29(1H,m,H-5a),3.69(1H,overlap,H-5b),3.68(1H,overlap,H-6),3.40(2H,m,H-7),6.88( 1H,t,J=6.0Hz,H-8),2.99(3H,s,H-9),6.68(2H,d,J=8.5Hz,H-2',6'),7.73(2H,d,J=8.5Hz,H-3',5'),5.05(1H,d,J=8.0Hz ,H-1"),3.32(1H,t,J=8.0Hz,H-2"),3.34(1H,m,H-3"),3.25(1H,t,J=9.0Hz,H-4"),3.78(1H,m,H-5"),4.12(1H,dd,J=8.0 ,12.0Hz,H-6"a),4.53(1H,dd,J=2.0,12.0Hz,H-6"b),7.08(2H,d,J=8.5Hz,H-2"',6"'),7.81(2H,d,J=8.5Hz,H-3"',5"').

[0093] 13 C NMR (125MHz, DMSO-d6), δ C:157.4(C-2),157.3(C-3),39.0(C-5),55.3(C-6),41.9(C-7),34.0(C-9),152. 4(C-1'),111.0(C-2',6'),131.3(C-3',5'),116.4(C-4'),165.6(C-7'),99.5( C-1”),73.1(C-2”),76.4(C-3”),70.2(C-4”),74.0(C-5”),63.4(C-6”),160.4( C-1"'),115.7(C-2"',6"'),131.1(C-3"',5"'),124.9(C-4"'),167.2(C-7"').

[0094] The structural formula of compound II2 is as follows:

[0095]

[0096] Compound II 2: White amorphous powder; specific rotation [α]20D-41.8 (c 0.10, MeOH); UV(MeOH)λ max (logε)296(4.45),254(4.42),215(4.69)nm; IRν max 3357,2925,1699,1674,1606,1532,1511,1464,1417,1337,1276,1218,1179,1070,1025,768,701cm -1 HRESIMS m / z 590.1981 [M+H] + (calcd for C 27 H 32 O 12 N3,590.1981).

[0097] 1 H NMR (500MHz, DMSO-d6), δ H:8.48(1H,d,J=5.5Hz,H-4),3.29(1H,dd,J=5.5,12.0Hz,H-5a),3.67(1H,t,J=5.5Hz,H-5b),3.66(1H,m,H-6),3.40(2H,m,H-7),6.87(1H ,t,J=6.0Hz,H-8),2.99(3H,s,H-9),6.67(2H,d,J=8.5Hz,H-2',6'),7.71(2H,d,J=8.5Hz,H-3',5'),5.10(1H,d,J=8.0Hz,H-1"),3.34(1H ,overlap,H-2"),3.33(1H,overlap,H-3"),3.25(1H,m,H-4"),3.76(1H,m,H-5"),4.10(1H,dd,J=7.5,12.0Hz,H-6"a),4.51(1H,dd,J=2. 0,12.0Hz,H-6"b),7.46(1H,d,J=2.0Hz,H-3"'),7.42(1H,dd,J=2.0,8.0Hz,H-5"'),7.17(1H,d,J=8.0Hz,H-6"'),3.79(3H,s,2"'-OCH3).

[0098] 13 C NMR (125MHz, DMSO-d6), δ C :157.4(C-2),157.3(C-3),39.2(C-5),55.2(C-6),41.9(C-7),34.0(C-9),152.4(C-1'), 111.0(C-2',6'),131.3(C-3',5'),116.4(C-4'),165.6(C-7'),99.2(C-1”),73.0(C-2”) ,76.7(C-3”),70.1(C-4”),74.0(C-5”),63.4(C-6"),149.9(C-1"'),148.4(C-2"'),112. 6(C-3"'),124.4(C-4"'),122.6(C-5"'),114.2(C-6"'),167.2(C-7"'),55.5(2"'-OCH3).

[0099] Experimental Example 1

[0100] Immunofluorescence detection of primary hepatic stellate cell activation levels

[0101] Primary hepatic stellate cells were isolated from male BALB / c 6-month-old rats, anesthetized with sodium pentobarbital (45 mg / kg, ip) after overnight fasting. The rats were fixed dorsally, and the upper abdomen was dissected along the midline to expose and fix the portal vein and inferior vena cava. The inferior vena cava was perfused with EGTA buffer preheated to 37°C. After successful perfusion, retrograde perfusion of the liver was performed. Three minutes later, protease and collagenase solutions were perfused at the same flow rate and temperature for 5–7 minutes. After perfusion, liver tissue was carefully removed, collagenase was added, and the cells were placed in a suspension cell culture incubator for digestion for 10 minutes. The cells were then filtered through a 75 μm filter, and the cell suspension was collected into centrifuge tubes. The resulting cell suspension was centrifuged at 100 g for 3 minutes, the precipitate being hepatocytes, and the supernatant containing non-parenchymal liver cells. Cells in the supernatant were centrifuged at 1700g for 22 min in 12% Nycodenz separation medium using a density gradient. The intermediate hazy layer was carefully aspirated to obtain high-purity hepatic stellate cells. Culture conditions were DMEM medium (containing 10% FBS and 100U penicillin-streptomycin), 37℃, 5% CO2. After cell adhesion, cells in the test group were treated with compounds I, II1, and II2 (final concentration 1 μM), respectively. After 12 h of treatment, the DMEM medium was replaced simultaneously with that of the TGF-β1 group, and TGF-β1 (1 ng / mL) was added. The control group was treated with DMEM medium replaced with an equal volume of PBS. The activation level of primary hepatic stellate cells was detected by immunofluorescence staining after 24 h. The main steps of immunofluorescence staining include: fixation with 4% paraformaldehyde; permeabilization with 0.5% Triton; blocking with 3% BSA; incubation with primary antibodies (anti-α-SMA: Boster, diluted 1:100 with PBS; anti-Collagen I: Abcam, diluted 1:100 with PBS) at 4°C overnight; and addition of fluorescently labeled secondary antibodies (goat anti-rabbit Alexa Fluor). TM 488: Invitrogen, diluted 1:100 with PBS; Goat anti-mouse Alexa Fluor TM 647: Invitrogen (diluted 1:100 with PBS), mounted with mounting medium containing DAPI.

[0102] Immunofluorescence stained slides were observed under an inverted microscope, and high-resolution immunofluorescence images of Collagen I and α-SMA protein expression were obtained using an OLYMPUS FV31S-SW system. The mean fluorescence intensity of each field of view was measured using Image-J. Three fields of view were randomly selected for each specimen, and the mean value represented the mean fluorescence intensity of Collagen I and α-SMA within a single sample. The expression differences of Collagen I and α-SMA among the groups were compared using a one-way ANOVA. The results showed that the fluorescence intensity of Collagen I and α-SMA proteins in cells treated with compounds I, II1, and II2 was significantly lower than that in the TGF-β1 group. Experimental results are expressed as mean ± standard deviation, where ### represents P < 0.001 compared to the control group, and *** represents P < 0.001 compared to the TGF-β1 group. The experimental results are shown in Tables 1 and 2.

[0103] Table 1. Mean fluorescence intensity of Collagen I in primary hepatic stellate cells as detected by immunofluorescence.

[0104]

[0105]

[0106] Table 2. Mean fluorescence intensity of α-SMA in primary hepatic stellate cells as determined by immunofluorescence.

[0107] Group Mean fluorescence intensity of α-SMA (AU) p-value control group 21.46±5.50 TGF-β1 group 67.12±17.42 <0.001### Group 1 of TGF-β1+ compounds 20.93±6.39 <0.001*** Group 2 of TGF-β1+ compounds 23.70±9.27 <0.001*** Group 3 of TGF-β1+ compounds 23.73±8.11 <0.001***

[0108] Experiment Example 2

[0109] Collagen contraction assay to detect the migration level of primary hepatic stellate cells

[0110] 0.795 mL of type I rat tail collagen was added to 0.205 mL of PBS, followed by 22.6 μL of 0.1 mol / L NaOH aqueous solution. The mixture was quickly mixed to prepare a 1 mL rat tail collagen solution. The isolated primary hepatic stellate cells were counted and diluted to a concentration of 7.5 × 10⁻⁶. 6 After the cell count reached 1 mL, 1 mL of cell suspension was mixed with 5 mL of rat tail collagen solution and seeded into 24-well plates, 400 μL per well. The plates were incubated at 37°C with 5% CO2 for 1 h. After the solution solidified, the gel was gently scraped along the edge of the gel with a 10 μL pipette tip to detach it from the culture plate. After separation, 500 μL of DMEM medium was added to each well for further culture. Cell grouping and treatment were the same as in Experiment 1.

[0111] After the collagen gel is released, the contraction of cells on the gel plate causes the gel plate to contract centripetally, forming a near-circular shape with a halo. After 24 hours, the contracted area of ​​each group stabilized. Photos were taken at a fixed height, and the ratio of the contracted gel area to the area of ​​each well in the 24-well plate (i.e., gel area percentage) was measured to compare the contraction effects of each group. The results showed that the gel area treated with compounds II1 and II2 was higher than that of the TGF-β1 group, but there was no significant difference compared to the TGF-β1 group; the gel area treated with compound I was significantly higher than that of the TGF-β1 group. Experimental results are expressed as mean ± standard deviation, and a one-way ANOVA was used to compare the differences between the control group and the TGF-β1 group, and between the TGF-β1 group and the tested compound groups. ### indicates P < 0.001 compared to the control group, and *** indicates P < 0.001 compared to the TGF-β1 group. The experimental results are shown in Table 3.

[0112] Table 3. Relative gel area after contraction of primary hepatic stellate cells as determined by the collagen contraction method.

[0113] Group Gel area percentage (%) p-value control group 93.73±3.21 TGF-β1 group 79.63±3.60 <0.001### Group 1 of TGF-β1+ compounds 91.23±2.13 <0.001*** Group 2 of TGF-β1+ compounds 81.82±1.41 0.6030ns Group 3 of TGF-β1+ compounds 83.99±2.31 0.1052ns

[0114] Experimental Example 3

[0115] Detection of RhoA protein expression by immunoblotting

[0116] When the hepatic stellate cell line LX2 reached the logarithmic growth phase and had a confluence of over 80%, it was treated using the same grouping and treatment methods as in Experiment 1. After treatment, the culture medium was discarded, and RIPA lysis buffer was added for lysis. The cells were centrifuged at 12000g for 30 min to extract total protein. After protein quantification, SDS-PAGE electrophoresis was performed, and proteins of different molecular weights were separated. Subsequently, the electrophoretically separated bands were transferred from the gel to a PVDF membrane using a wet transfer method, and the proteins adsorbed onto the membrane surface. After blocking the PVDF membrane with 5% skim milk for 2 hours, it was incubated with primary antibodies (anti-Collagen I: Abcam; anti-α-SMA: BOSTER; anti-RhoA: Cell Signaling; anti-GAPDH: Zhongshan Jinqiao, diluted 1:2000 with 3% BSA) overnight at 4°C. After washing with TBST, it was labeled with horseradish peroxidase-labeled secondary antibodies (goat anti-rabbit / goat anti-mouse: Zhongshan Jinqiao, diluted 1:2000 with TBST) and incubated at room temperature for 2 hours. Imaging was performed using the Tanon AIIDOC imaging system to detect the expression of Collagen I, α-SMA, and RhoA proteins in LX2 cells of each group. The immunoblotting results were quantitatively analyzed using Image-J. The results showed that the expression of Collagen I and α-SMA proteins in cells treated with compounds I, II1, and II2 was significantly lower than that in the TGF-β1 group; the expression of RhoA protein in cells treated with compounds II1 and II2 was significantly lower than that in the TGF-β1 group. Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used to compare the differences between the control group and the TGF-β1 group, and between the TGF-β1 group and the test compound group. Where ### indicates P < 0.001 compared to the control group, and **, **, ** indicate P < 0.001, P < 0.01, and P < 0.05 compared to the TGF-β1 group. The experimental results are shown in Tables 4, 5, and 6.

[0117] Table 4. Differences in Collagen I protein expression in LX2 cell line as detected by Western blotting.

[0118] Group Collagen I relative expression level p-value control group 1.00±0.04 TGF-β1 group 4.25±0.56 <0.001### Group 1 of TGF-β1+ compounds 1.71±0.10 <0.001*** Group 2 of TGF-β1+ compounds 2.38±0.41 <0.001*** Group 3 of TGF-β1+ compounds 1.86±0.40 <0.001***

[0119] Table 5. Detection of α-SMA protein expression in LX2 cell line by Western blotting.

[0120] Group α-SMA relative expression level p-value control group 1.00±0.20 TGF-β1 group 3.94±0.81 <0.001### Group 1 of TGF-β1+ compounds 1.77±0.17 <0.001*** Group 2 of TGF-β1+ compounds 1.88±0.15 <0.001*** Group 3 of TGF-β1+ compounds 2.10±0.42 0.0014**

[0121] Table 6. Detection of RhoA protein expression differences in LX2 cell line by Western blotting.

[0122] Group RhoA relative expression level p-value control group 1.00±0.11 TGF-β1 group 2.09±0.17 <0.001### Group 1 of TGF-β1+ compounds 1.55±0.19 0.0122* Group 2 of TGF-β1+ compounds 1.86±0.14 0.3489ns Group 3 of TGF-β1+ compounds 1.51±0.24 0.0082**

[0123] Experiment Example 4

[0124] Detection of RhoA protein activity by G-LISA

[0125] The RhoA downstream effector, the Rhotekin gene (RhoA effector), possesses a RhoA binding domain (RBD), which specifically binds to activated RhoA-GTP. Based on this principle, the GPT enzyme activity of cellular RhoA protein can be detected to evaluate the migration level of hepatic stellate cells. When the LX2 hepatic stellate cell line reached the logarithmic growth phase and had a confluence of over 80%, it was treated, with the same grouping and treatment methods as in Experiment 1. After treatment, the RhoA-GTP activity level was detected using the G-LISA RhoA activation detection kit (Cytoskeleton). After discarding the culture medium, lysis buffer was added, and the mixture was centrifuged at 10000g for 1 min to obtain the stock protein solution, and the protein concentration was quantified. Equal amounts of protein were added three times to 96-well plates coated with the Rhotekin RhoA binding domain and incubated at 4°C for 1 h. Wells containing only lysis buffer served as blank samples. In the samples, the GTP-bound form of RhoA protein molecules (activated state) could bind to the plate, while the GDP-bound form of RhoA protein did not bind. After washing away the unbound protein, the RhoA-GTP bound in each well was labeled with anti-RhoA antibody, and then incubated with HRP-labeled secondary antibody for 15 min. After incubation, the active protein was quantified at 490 nm absorbance using a microplate spectrophotometer. The results showed that the RhoA-GTP activity after treatment with compounds I, II1, and II2 was significantly lower than that in the TGF-β1 group. The experimental results are expressed as mean ± standard deviation, and the differences between the control group and the TGF-β1 group, and between the TGF-β1 group and the test compound group were compared using a one-way ANOVA. ### indicates P < 0.001 compared to the control group, and *** indicates P < 0.001 compared to the TGF-β1 group. The experimental results are shown in Table 7.

[0126] Table 7. Differences in RhoA protein activity in LX2 cell lines as detected by G-LISA.

[0127] Group RhoA activity (fold change compared to control group) p-value control group 1.00±0.17 TGF-β1 group 5.02±0.45 <0.001### Group 1 of TGF-β1+ compounds 2.04±0.21 <0.001*** Group 2 of TGF-β1+ compounds 3.32±0.32 <0.001*** Group 3 of TGF-β1+ compounds 1.97±0.47 <0.001***

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An alkaloid compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound has the structure shown in Formula I: 。 2. The method for preparing the alkaloid compound according to claim 1, characterized in that, Includes the following steps: (a) The white sclerotium was extracted by reflux with 92-98% ethanol aqueous solution, 82-88% ethanol aqueous solution, 72-78% ethanol aqueous solution and water in sequence, and the extracts were combined and concentrated to obtain the first extract. (b) The first extract is extracted with an organic solvent, and the remaining aqueous phase is precipitated with alcohol. The supernatant is collected and concentrated to obtain the second extract. The second extract is purified by macroporous resin column chromatography, gel column chromatography and C18 reversed-phase semi-preparative liquid chromatography to obtain the alkaloid compound. The organic solvent is selected from at least one of ethyl acetate, petroleum ether, dichloromethane, and n-butanol; The macroporous resin column chromatography includes: elution with a water-ethanol gradient, and collection of the eluent in a 30% ethanol aqueous solution; The gel column chromatography includes: elution using a water-ethanol gradient and collection of the eluent in a 0% ethanol aqueous solution.

3. The method for preparing alkaloid compounds according to claim 2, characterized in that, In the alcohol precipitation process, the aqueous phase is adjusted to an aqueous solution with an ethanol volume fraction of 82% to 88% using ethanol.

4. The method for preparing alkaloid compounds according to claim 2, characterized in that, In the macroporous resin column chromatography, elution was carried out sequentially with ethanol aqueous solutions of volume fractions of 0%, 5%, 15%, 30%, 50%, 70%, and 95%, and the eluent of 30% ethanol aqueous solution was collected.

5. The method for preparing alkaloid compounds according to claim 2, characterized in that, In the gel column chromatography, elution was performed sequentially using ethanol-water solutions with volume fractions of 0%, 5%, 15%, 30%, 50%, 70%, and 95%.

6. The use of the alkaloid compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating liver fibrosis.

7. A pharmaceutical composition, characterized in that, This includes the alkaloid compound of claim 1 or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, In the pharmaceutical composition, the alkaloid compound or its pharmaceutically acceptable salt has a mass fraction of 0.1% to 95%.

9. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition includes at least one of tablets, capsules, pills, granules, and suspensions.

Images