Alkaloid compound separated from white poria as well as preparation method and application of alkaloid compound

Abstract

The invention relates to the technical field of medicines, in particular to an alkaloid compound separated from white poria as well as a preparation method and application of the alkaloid compound. The alkaloid compound or the pharmaceutically acceptable salt thereof has a structure as shown in a formula I or a formula II: # imgabs0 #, and R is H or methoxyl. According to the invention, the novel alkaloid compound is separated from argil poria, and experiments prove that the novel alkaloid compound has good anti-hepatic fibrosis activity and has a good application prospect in the field of anti-hepatic fibrosis drugs.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to an alkaloid compound separated from white turmeric and a preparation method and application thereof. Background Art

[0002] Liver fibrosis is an inevitable stage for chronic liver disease to develop into cirrhosis and liver cancer. It is the core pathological change of chronic liver disease caused by various reasons such as viral infection, excessive drinking, and genetic diseases. It is currently believed that liver fibrosis and early cirrhosis are 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 causal treatment of the primary disease, reduction of inflammation and immune response, liver transplantation, etc., but these methods have limitations such as large side effects, high medical costs, and lack of donors. Therefore, finding key anti-liver fibrosis candidate drugs and clarifying their mechanisms of action have important social significance and economic value.

[0003] Hepatic stellate cell activation is a key trigger for the development of liver fibrosis, and inhibiting hepatic stellate cell activation is an important intervention strategy to prevent liver disease from progressing to fibrosis. Under normal circumstances, hepatic stellate cells are in a quiescent state. In chronic liver injury, damaged hepatic parenchymal cells, hepatic sinusoidal endothelial cells, and Kupffer cells release profibrotic cytokines, such as TGF-β1, to activate hepatic stellate cells. Activated hepatic stellate cells express a large amount of cytoskeletal protein α-SMA, migrate to damaged sites and transform into myofibroblast-like cell phenotypes, synthesize extracellular matrix proteins such as Collagen I and secrete them into the extracellular space, and participate in the progression of liver fibrosis. Therefore, inhibiting the migration of hepatic stellate cells has the potential to treat liver fibrosis. Evaluation of the expression of α-SMA in hepatic stellate cells and cell contraction can comprehensively evaluate the migration ability of hepatic stellate cells. Previous studies have suggested that the small molecular weight G protein RhoA is a key protein regulating cell migration and movement, and its active form RhoA-GTP plays an important role in cell movement mediated by cytoskeletal reorganization.

[0004] Traditional Chinese medicine and Chinese herbal compound have a lot of clinical experience in the treatment of liver fibrosis, which has the characteristics of good efficacy, strong activity and low toxicity and side effects. Natural compounds can be used as an important source of lead compounds in the research and development of drugs for the treatment of liver fibrosis, such as glycyrrhizic acid preparations, represented by magnesium isoglycyrrhizinate injection and diammonium glycyrrhizinate enteric-coated capsules; in addition, silymarin (a flavonoid extracted from milk thistle) also has anti-fibrosis effects, represented by silybin meglumine tablets and silybin capsules, and has been widely used in clinical practice.

[0005] Bai Tuling is the dried rhizome of Heterosmilax yunnanensis Gagnep., a plant of the genus Smilax in the Liliaceae family. It is mainly distributed in Shaanxi, Gansu, Anhui, Sichuan, and Yunnan provinces. It has the effects of clearing away heat and detoxifying, removing dampness, and promoting joint function. It is the only ministerial drug in the anti-tumor Chinese medicine compound Kushen injection, and its highly polar water part is used as medicine. Studies on chemical components and pharmacological activities have shown that the water part of Bai Tuling contains chemical components such as phenolic acid glycosides, flavonoid glycosides, sterol glycosides, azoglycosides, and some monophenolic acids, amino acids, and polysaccharides, and has pharmacological activities such as diuresis, anti-inflammatory, promoting joint function, antibacterial, detoxification, and analgesia. However, there are few studies on Bai Tuling at present, and the types and quantities of compounds are not comprehensive enough. The highly polar part is mainly composed of glycoside compounds, and the pharmacological activity research is also mainly based on crude extracts.

[0006] At present, there is no report on the isolation of alkaloid compounds from the traditional Chinese medicine Bai Tuling, nor is there any report on the corresponding separation and preparation method and anti-liver fibrosis activity.

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

[0008] One object of the present invention is to provide alkaloid compounds separated from Psoralea corylifolia.

[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 Psoralea corylifolia and pharmaceutically acceptable salts, pharmaceutical compositions, etc. thereof in the preparation of drugs against liver fibrosis.

[0011] In order to achieve the above-mentioned object of the present invention, the present invention provides an alkaloid compound or a pharmaceutically acceptable salt thereof, wherein the compound has 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 one of the above-mentioned alkaloid compounds, comprising the following steps:

[0015] (a) sequentially extracting the white earthworm with 92% to 98% ethanol aqueous solution, 82% to 88% ethanol aqueous solution, 72% to 78% ethanol aqueous solution and water by volume, combining and concentrating the extracts to obtain a first extract;

[0016] (b) extracting the first extract with an organic solvent, precipitating the remaining aqueous phase with alcohol, collecting and concentrating the supernatant to obtain a second extract; purifying the second extract with macroporous resin column chromatography, gel column chromatography and liquid chromatography column 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: using water-ethanol gradient elution, collecting 30% ethanol aqueous solution eluate. Further, using 0%, 5%, 15%, 30%, 50%, 70%, 95% ethanol aqueous solution volume fractions for elution, collecting 30% ethanol aqueous solution eluate.

[0020] In a specific embodiment of the present invention, the gel column chromatography comprises: using water-ethanol gradient elution, collecting 0% ethanol aqueous solution eluate. Further, using 0%, 5%, 15%, 30%, 50%, 70%, 95% ethanol aqueous solution for elution in sequence.

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

[0022] Another aspect of the present invention provides use of any one of the above-mentioned alkaloid compounds or pharmaceutically acceptable salts thereof in the preparation of drugs for resisting liver fibrosis.

[0023] Another aspect of the present invention provides a pharmaceutical composition comprising any one of the above-mentioned alkaloid compounds or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

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

[0025] In a specific embodiment 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 present invention has the following beneficial effects:

[0027] The novel alkaloid compound separated from the white turmeric powder is verified by experiments to have good anti-liver fibrosis activity and has good application prospects in the field of anti-liver fibrosis drugs. DETAILED DESCRIPTION

[0028] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0029] In one aspect, the present invention provides an alkaloid compound or a pharmaceutically acceptable salt thereof. The alkaloid compound has a structure as shown in Formula I or Formula II:

[0030]

[0031] Wherein, R is H or methoxy.

[0032] The present invention discovers the material basis of the efficacy of commonly used traditional Chinese medicines and active precursors, combines clinical application, literature research and laboratory anti-liver fibrosis activity screening, and finds that several compounds separated from the traditional Chinese medicine Bai Tuling have good anti-liver fibrosis activity.

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

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

[0035] (a) sequentially extracting the white earthworm with 92% to 98% ethanol aqueous solution, 82% to 88% ethanol aqueous solution, 72% to 78% ethanol aqueous solution and water by volume, combining and concentrating the extracts to obtain a first extract;

[0036] (b) extracting the first extract with an organic solvent, precipitating the remaining aqueous phase with alcohol, collecting and concentrating the supernatant to obtain a second extract; purifying the second extract with macroporous resin column chromatography, gel column chromatography and liquid chromatography column to obtain an alkaloid compound.

[0037] Among them, when reflux extraction is performed on the white earth ling, four kinds of extraction solvents are used in sequence to extract the white earth ling, firstly, an ethanol aqueous solution with a volume fraction of 92% to 98% is used to reflux extraction on the white earth ling, and the supernatant extract is filtered and collected; then, an ethanol aqueous solution with a volume fraction of 82% to 88% is used to reflux extraction on the white earth ling filter residue obtained by filtering in the previous step, and the supernatant extract is filtered and collected; then, an ethanol aqueous solution with a volume fraction of 72% to 78% is used to reflux extraction on the white earth ling filter residue obtained by filtering in the previous step, and the supernatant extract is filtered and collected; then, water is used to reflux extraction on the white earth ling filter residue obtained by filtering in the previous step, and the supernatant extract is filtered and collected, and each extract is combined, and the solvent is recovered by reduced pressure distillation, and the solvent is removed to obtain a first extract.

[0038] For example, in different embodiments, the volume fraction of the ethanol aqueous solution with a volume fraction of 92% to 98% may be 92%, 93%, 94%, 95%, 96%, 97%, 98% or a range consisting of any two of them; the volume fraction of the ethanol aqueous solution with a volume fraction of 82% to 88% may be 82%, 83%, 84%, 85%, 86%, 87%, 88% or a range consisting of any two of them; the volume fraction of the ethanol aqueous solution with a volume fraction of 72% to 78% may be 72%, 73%, 74%, 75%, 76%, 77%, 78% or a range consisting of any two of them.

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

[0040] For example, in different embodiments, the extraction times of each extraction solvent can be 2 or 3 times, and the time of a single extraction can be 1.5 h, 2 h, 2.5 h, etc. After each extraction, the supernatant is collected by filtration, and the extraction solvent is added again for re-extraction.

[0041] In each extraction, the amount of the extraction solvent can be adjusted according to actual needs. The target compound of the present invention can be obtained in the extract by extraction with a conventional solid-liquid ratio. By optimizing the solid-liquid ratio, the yield of the target compound can be appropriately improved.

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

[0043] 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 an ethanol volume fraction of 82% to 88%.

[0044] For example, in different embodiments, during the alcohol precipitation, ethanol can be used to adjust the aqueous phase to an ethanol aqueous solution with an ethanol volume fraction of 82%, 83%, 84%, 85%, 86%, 87%, 88% or any two of them. The remaining aqueous phase is extracted with an organic solvent and precipitated with alcohol, and macromolecular sugars, proteins and other substances are filtered out, and then concentrated to obtain a second extract.

[0045] In a specific embodiment of the present invention, the macroporous resin column chromatography includes: using water-ethanol gradient elution, collecting 30% ethanol aqueous solution eluate. Wherein, the volume fraction of ethanol in the water-ethanol gradient elution is 0% to 95%. Further, elution is carried out in sequence using 0%, 5%, 15%, 30%, 50%, 70%, 95% ethanol aqueous solution by volume fraction, and collecting 30% ethanol aqueous solution eluate.

[0046] In actual operation, water is used to dissolve the second extract, and then the sample is loaded for chromatography separation. Among them, the macroporous resin can be HP20, but is not limited to this. In the macroporous resin column chromatography, the column specifications can be a column diameter of d=14.5cm, a column height of h=82cm, and a column volume of 13.5L, but are not limited to this.

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

[0048] In a specific embodiment of the present invention, in the gel column chromatography, the gel column is Sephadex LH-20. In actual operation, the target eluate collected by the macroporous resin column chromatography is concentrated, then dissolved in water, and then chromatographically separated. In the gel column chromatography, the column specifications can be column diameter d = 11.7 cm, column height h = 67.0 cm, but are not limited thereto.

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

[0050] Among them, the compound II of the present invention includes two structures, II1 and II2, which are specifically as follows:

[0051]

[0052] Another aspect of the present invention provides use of any one of the above alkaloid compounds or a pharmaceutically acceptable salt thereof in the preparation of an anti-liver fibrosis drug.

[0053] The alkaloid compounds separated from the white radix turmeric of the present invention have a significant inhibitory effect on the movement of rat hepatic stellate cells in an in vitro anti-hepatic fibrosis activity evaluation and have an anti-hepatic fibrosis effect.

[0054] Another aspect of the present invention provides a pharmaceutical composition comprising any one of the above-mentioned alkaloid compounds or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

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

[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, in the pharmaceutical composition, the mass fraction of the alkaloid compound or the pharmaceutically acceptable salt thereof is 0.1% to 95%.

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

[0059] In the pharmaceutical composition, the amount of the alkaloid compound or its pharmaceutically acceptable salt can be adjusted according to the actual type of excipients, dosage form and dosage requirements.

[0060] For example, in a unit dosage form, in a pharmaceutical composition, the amount of the alkaloid compound of the present invention or a pharmaceutically acceptable salt thereof can 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 gender, age, weight, personality and individual response of the patient or animal, the route of administration, the number of administrations, and the purpose of treatment. Therefore, the therapeutic dose of the present invention can be adjusted according to actual conditions to achieve the requirement of its therapeutically effective amount and achieve the purpose of prevention or treatment.

[0062] In a specific embodiment of the present invention, the suitable daily dosage range of the alkaloid compound of the present invention or a pharmaceutically acceptable salt thereof 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 dosage of the alkaloid compound of the present invention or its pharmaceutically acceptable salt taken by adult patients can be 10 to 500 mg, preferably 20 to 100 mg, which can be taken once or in 2 to 3 times; the daily dosage for children can be 5 to 30 mg, preferably 10 to 20 mg, which can be taken once or in 2 to 3 times.

[0064] In a specific embodiment of the present invention, the dosage form of the pharmaceutical composition includes a liquid dosage form or a solid dosage form, etc. Among them, conventional liquid dosage forms include but are not limited to true solutions, colloids, microparticle dosage forms, emulsion dosage forms, suspension dosage forms, etc.; conventional solid dosage forms include but are not limited to tablets, capsules, pills, granules, etc. It can also be a dripping pill, aerosol, powder, suppository, freeze-dried powder injection, etc., which can be adjusted according to actual administration needs.

[0065] The pharmaceutical composition of the present invention may be a common preparation, or any one of a sustained-release preparation, a controlled-release preparation, a targeted preparation, and various microparticle drug delivery systems.

[0066] The dosage form of the pharmaceutical composition of the present invention and the corresponding preparation method can be prepared according to conventional methods, such as the alkaloid compound of the present invention or its pharmaceutically acceptable salt can be compounded with one or more pharmaceutically acceptable excipients to prepare a composition in the form of a corresponding dosage form or dosage for human or animal use.

[0067] The administration route of the pharmaceutical composition of the present invention can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneum or rectum, etc. For example, the administration route can be injection, including but not limited to intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, acupoint injection, etc.

[0068] In a specific embodiment 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, wetting agents and lubricants.

[0069] For example, for tablets, the 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, white clay, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and adhesives, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone, etc. ; disintegrants, such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets, etc.

[0070] For pills, the excipients that can be used include but are not limited to diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, glyceryl monostearate, kaolin, talc, etc.; binders, such as gum arabic, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.

[0071] 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. The alkaloid compounds of the present invention or their pharmaceutically acceptable salts can also be made into microcapsules, suspended in an aqueous medium to form a suspension, or loaded into hard capsules or made into injections for use.

[0072] For example, the alkaloid compound of the present invention or its pharmaceutically acceptable salt is made into an injection preparation, such as a solution, a suspension solution, an emulsion, or a freeze-dried powder injection. Such a preparation may be aqueous or non-aqueous, and may contain one or more pharmacologically acceptable carriers, diluents, adhesives, lubricants, preservatives, surfactants, or dispersants. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, fatty acid esters, and the like. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, or glycerol may be added to the injection preparation. In addition, conventional cosolvents, buffers, pH regulators, and the like may also be added.

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

[0074] The source of the white earth ling in the following specific embodiments of the present invention is: purchased from Shanxi Zhendong Pharmaceutical Co., Ltd., produced in Anshun, Guizhou, and appraised by Guo Baolin from the 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 white earthworm, comprising the following steps:

[0077] (1) Take 100 kg of dried tubers of Psoralea corylifolia, crush them, and then use 95% by volume ethanol aqueous solution for reflux extraction for 3 times, each time for 2 hours, and filter and collect the supernatant; then use 85% by volume ethanol aqueous solution for reflux extraction for 3 times, each time for 2 hours, and filter and collect the supernatant; then use 75% by volume ethanol aqueous solution for reflux extraction for 3 times, each time for 2 hours, and filter and collect the supernatant; then use water for reflux extraction for 3 times, each time for 2 hours, and filter and collect the supernatant; combine all the supernatants, reduce the pressure to recover the solvent and remove the ethanol, and obtain about 24.5 kg of extract.

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

[0079] (3) The second extract obtained in step (2) was dissolved in 3 L of water, and separated by HP20 macroporous resin column chromatography (d = 14.5 cm, h = 82 cm, column volume 13.5 L) twice, and eluted with ethanol aqueous solutions with volume fractions of 0%, 5%, 15%, 30%, 50%, 70%, and 95%, respectively, and the eluate with 30% ethanol aqueous solution was collected.

[0080] (4) The 30% ethanol aqueous solution eluate (about 110 g in total) collected in step (3) was dissolved in 250 mL of water, wet loaded, and subjected to Sephadex LH-20 gel column chromatography (d=11.7 cm, h=67.0 cm), and eluted with ethanol aqueous solutions with volume fractions of 0%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, and 95%, respectively, and the 0% ethanol aqueous solution eluate was collected.

[0081] (5) The 0% ethanol aqueous solution eluate collected in step (4) was purified using a C18 reverse phase semi-preparative liquid chromatography column, with the elution phase being a mixed solution of acetonitrile, water and acetic acid in 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 analyzed and identified by spectroscopic methods such as UV, IR, NMR, and MS. Compound Ⅰ is a tryptophan CC-coupled catechin spirocyclic carbon glycoside compound with a novel skeleton, and compounds Ⅱ1 and Ⅱ2 are two 2,3-diketopyrazine compounds.

[0083] The spectroscopic information and NMR signal attribution of the three specific 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 (500 MHz, 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 (500 MHz, 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 (125 MHz, 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 II2: 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 (500 MHz, 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 (125 MHz, 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 activation level of primary hepatic stellate cells

[0101] Primary hepatic stellate cells were isolated from male BALB / c 6-month-old mice, which were fasted overnight and anesthetized with sodium pentobarbital (45 mg / kg, ip). The back was fixed, the upper abdomen was opened along the midline of the abdomen, the portal vein and the inferior vena cava were exposed and fixed, and the EGTA buffer preheated to 37°C was used to perfuse from the inferior vena cava. After success, the liver was reversely perfused. After 3 minutes, the protease and collagenase solutions were perfused at the same flow rate and temperature for 5 to 7 minutes. After the perfusion was completed, the liver tissue was carefully removed, collagenase was added, and it was placed in a suspension cell culture incubator for digestion for 10 minutes. Then, the cell suspension was filtered with a 75μm filter and collected in a centrifuge tube. The resulting cell suspension was centrifuged at 100g for 3 minutes, and the precipitate was hepatocytes, and the supernatant was liver non-parenchymal cells. The supernatant cells were placed in 12% Nycodenz separation medium for density gradient centrifugation at 1700g for 22min, and the middle mist layer was carefully aspirated to obtain high-purity hepatic stellate cells. The culture conditions were DMEM medium (containing 10% FBS and 100U penicillin-streptomycin), 37°C, 5% CO2. After culturing until the cells adhered to the wall, the test group cells were added with compounds Ⅰ, Ⅱ1 and Ⅱ2 (final concentration was 1μM), and after 12h of treatment, the DMEM medium was replaced synchronously with the TGF-β1 group cells and the cytokine TGF-β1 (1ng / mL), and the control group replaced the DMEM medium with an equal volume of PBS. After 24h, the activation level of primary hepatic stellate cells was detected by immunofluorescence staining. The main steps of immunofluorescence staining include: 4% paraformaldehyde fixation; 0.5% Triton permeabilization; 3% BSA blocking; primary antibody incubation (anti-α-SMA: Boster, diluted 1:100 with PBS; anti-Collagen I: Abcam, diluted 1:100 with PBS) at 4°C overnight; addition of fluorescently labeled secondary antibody (goat anti-rabbit Alexa Fluor TM 488: Invitrogen, diluted 1:100 in PBS; goat anti-mouse AlexaFluor TM 647: Invitrogen, diluted 1:100 with PBS), and mounted with mounting medium containing DAPI.

[0102] The slides after immunofluorescence staining were placed under an inverted microscope for observation, and the OLYMPUS FV31S-SW system was used to obtain high-definition immunofluorescence images of Collagen I and α-SMA protein expression. Image-J was used to measure the average fluorescence intensity of each field of view. Three fields of view were randomly selected for each specimen, and the mean value represented the average fluorescence intensity of Collagen I and α-SMA in one sample. The expression differences of Collagen I and α-SMA in each group were compared by one-way variance test. The results showed that the fluorescence intensity of Collagen I and α-SMA proteins in cells treated with compounds Ⅰ, Ⅱ1 and Ⅱ2 were significantly lower than that in the TGF-β1 group. The experimental results are expressed as mean ± standard deviation, where ### is P<0.001 compared with the control group, and *** is P<0.001 compared with the TGF-β1 group. The experimental results are shown in Tables 1 and 2.

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

[0104]

[0105]

[0106] Table 2 Average fluorescence intensity of α-SMA in primary hepatic stellate cells detected by immunofluorescence

[0107] Group α-SMA mean fluorescence intensity (AU) P-value Control group 21.46±5.50 TGF-β1 group 67.12±17.42 <0.001### TGF-β1+compound 1 group 20.93±6.39 <0.001*** TGF-β1+compound 2 group 23.70±9.27 <0.001*** TGF-β1+compound 3 group 23.73±8.11 <0.001***

[0108] Experimental Example 2

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

[0110] Take 0.795 mL of rat tail collagen type I and add it to 0.205 mL of PBS, then add 22.6 μL of 0.1 mol / L NaOH aqueous solution, mix quickly, and prepare 1 mL of rat tail collagen solution. Count the isolated primary hepatic stellate cells and dilute to 7.5×10 6 After the cells were concentrated to 500 μL / mL, 1 mL of cell suspension was mixed with 5 mL of rat tail collagen solution and inoculated into a 24-well plate, adding 400 μL to each well. Place in a 37°C, 5% CO2 incubator for 1 hour. After the solution solidifies, use a 10 μL pipette tip to gently scrape along the edge of the gel to separate the gel from the culture plate. After the two are separated, 500 μL of DMEM culture medium is added to each well for culture. Cell grouping and treatment methods are the same as those in Experimental Example 1.

[0111] When the collagen gel is free, the contraction of the cells on the gel plate will drive the gel plate to contract centripetally, forming a quasi-circular shape with a halo. After 24 hours, the contraction area of ​​each group was stable. The photos were taken at a fixed height and the ratio of the gel area after contraction in each group to the area of ​​each well of the 24-well plate (i.e., the gel area ratio) was measured to compare the contraction effects of each group. The results showed that the gel area after treatment with compounds Ⅱ1 and Ⅱ2 was higher than that of the TGF-β1 group, but there was no significant difference compared with the TGF-β1 group; the gel area after treatment with compound Ⅰ was significantly higher than that of 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 by a one-way ANOVA test. Among them, ### is P<0.001 compared with the control group, and *** is P<0.001 compared with 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 detected by collagen contraction method

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

[0114] Experimental Example 3

[0115] Immunoblotting to detect RhoA protein expression

[0116] When the hepatic stellate cell line LX2 was cultured to the logarithmic growth phase and the confluence was above 80%, it was treated, and the grouping and treatment methods were the same as those in Experimental Example 1. After the treatment, the medium was discarded, RIPA lysis buffer was added for lysis, and the total cell protein was extracted by centrifugation at 12000g for 30 minutes. After protein quantification, SDS-PAGE electrophoresis was performed to separate proteins of different molecular weights. Subsequently, the bands separated by electrophoresis were transferred from the gel to the PVDF membrane by wet transfer, and the proteins were adsorbed on the membrane surface. After the PVDF membrane was blocked 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) at 4°C overnight, rinsed with TBST, and 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. The expression of Collagen I, α-SMA, and RhoA proteins in LX2 cells of each group was detected using the Tanon AIIDOC imaging system, and 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. 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 test. Among them, ### means P<0.001 compared with the control group, **, *, * means P<0.001, P<0.01, P<0.05 compared with the TGF-β1 group. The experimental results are shown in Tables 4, 5, and 6.

[0117] Table 4 Differences in the expression of Collagen I protein in LX2 cell lines detected by immunoblotting

[0118] Group Relative expression of Collagen I P-value Control group 1.00±0.04 TGF-β1 group 4.25±0.56 <0.001### TGF-β1+compound 1 group 1.71±0.10 <0.001*** TGF-β1+compound 2 group 2.38±0.41 <0.001*** TGF-β1+compound 3 group 1.86±0.40 <0.001***

[0119] Table 5 Immunoblotting to detect differences in α-SMA protein expression in LX2 cell lines

[0120] Group Relative expression of α-SMA P-value Control group 1.00±0.20 TGF-β1 group 3.94±0.81 <0.001### TGF-β1+compound 1 group 1.77±0.17 <0.001*** TGF-β1+compound 2 group 1.88±0.15 <0.001*** TGF-β1+compound 3 group 2.10±0.42 0.0014**

[0121] Table 6 Immunoblotting to detect the difference in RhoA protein expression in LX2 cell lines

[0122] Group Relative expression of RhoA P-value Control group 1.00±0.11 TGF-β1 group 2.09±0.17 <0.001### TGF-β1+compound 1 group 1.55±0.19 0.0122* TGF-β1+compound 2 group 1.86±0.14 0.3489ns TGF-β1+compound 3 group 1.51±0.24 0.0082**

[0123] Experimental Example 4

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

[0125] The RhoA downstream effector Rhotekin gene (RhoA effector) has a RhoA binding domain (RBD), which can specifically bind to the activated RhoA-GTP. Based on the above principle, the GPTase activity of the cell RhoA protein can be detected to evaluate the migration level of hepatic stellate cells. When the hepatic stellate cell line LX2 is cultured to the logarithmic growth phase and the confluence is more than 80%, it is treated, and the grouping and treatment methods are the same as those in Experimental Example 1. After the treatment is completed, the G-LISA RhoA Activation Detection Kit (Cytoskeleton) is used to detect the activity level of RhoA-GTP. After discarding the culture medium, the lysis buffer is added, 10000g, and the protein stock solution is obtained after centrifugation for 1min, and the protein concentration is quantified. The same amount of protein is added three times to a 96-well plate coated with the Rhotekin RhoA binding domain and incubated at 4°C for 1h. The wells containing only the lysis buffer are used as blank samples. The GTP-bound RhoA protein molecules (activated state) in the sample can bind to the plate, while the GDP-bound RhoA protein does not bind. After washing away the unbound proteins, the bound RhoA-GTP in each well was labeled with anti-RhoA antibody, and then incubated with HRP-labeled secondary antibody for 15 minutes. After the incubation, the active protein was quantified at 490nm 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 of 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 the TGF-β1 group and the test compound group were compared using a one-way ANOVA test. Among them, ### is P<0.001 compared with the control group, and *** is P<0.001 compared with the TGF-β1 group. The experimental results are shown in Table 7.

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

[0127] Group RhoA activity (fold of control group) P-value Control group 1.00±0.17 TGF-β1 group 5.02±0.45 <0.001### TGF-β1+compound 1 group 2.04±0.21 <0.001*** TGF-β1+compound 2 group 3.32±0.32 <0.001*** TGF-β1+compound 3 group 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 a structure as shown in Formula I or Formula II: Wherein, R is H or methoxy.

2. The method for preparing the alkaloid compound according to claim 1, characterized in that: The steps include: (a) sequentially extracting the white earthworm with 92% to 98% ethanol aqueous solution, 82% to 88% ethanol aqueous solution, 72% to 78% ethanol aqueous solution and water by volume, combining and concentrating the extracts to obtain a first extract; (b) extracting the first extract with an organic solvent, precipitating the remaining aqueous phase with alcohol, and collecting and concentrating the supernatant to obtain a second extract; The second extract is subjected to macroporous resin column chromatography, gel column chromatography and liquid chromatography column purification to obtain the alkaloid compound.

3. The method for preparing an alkaloid compound according to claim 2, characterized in that: The organic solvent comprises at least one of ethyl acetate, petroleum ether, dichloromethane and n-butanol; Preferably, in the alcohol precipitation, ethanol is used to adjust the aqueous phase to an ethanol aqueous solution with an ethanol volume fraction of 82% to 88%.

4. The method for preparing an alkaloid compound according to claim 2, characterized in that: The macroporous resin column chromatography comprises: using water-ethanol gradient elution, collecting 30% ethanol aqueous solution eluate; Preferably, elution is performed using ethanol aqueous solutions with volume fractions of 0%, 5%, 15%, 30%, 50%, 70% and 95% in sequence, and the eluate with 30% ethanol aqueous solution is collected.

5. The method for preparing an alkaloid compound according to claim 2, characterized in that: The gel column chromatography comprises: using water-ethanol gradient elution, collecting 0% ethanol aqueous solution eluate; Preferably, in the gel column chromatography, elution is performed using ethanol aqueous solutions with volume fractions of 0%, 5%, 15%, 30%, 50%, 70% and 95% in sequence.

6. The method for preparing an alkaloid compound according to claim 2, characterized in that: Compounds Ⅰ and Ⅱ were purified by C18 reverse phase semi-preparative liquid chromatography column.

7. Use of the alkaloid compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for resisting liver fibrosis.

8. A pharmaceutical composition, characterized in that It comprises the alkaloid compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 and a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 8, characterized in that In the pharmaceutical composition, the mass fraction of the alkaloid compound or the pharmaceutically acceptable salt thereof is 0.1% to 95%.

10. The pharmaceutical composition according to claim 8, characterized in that The dosage form of the pharmaceutical composition includes at least one of tablets, capsules, pills, granules and suspensions.

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