A method for isolating anti-hepatitis active substances from Lycium ruthenicum Murray

Abstract

The present invention discloses a method for separating anti-hepatitis active substances from Lycium ruthenicum Murr., which relates to the technical field of compound separation. The Lycium ruthenicum Murr. is made into an extract paste with methanol extract, and then evenly mixed with polyamide powder and sieved to obtain sieved powder. The sieved powder is loaded into a chromatographic column, and eluted with a water / methanol / dichloromethane three-phase system to separate the active substances. Then, the active substances are further separated with water and methanol as the elution items, and finally, compound A is separated and purified. The present invention for the first time separates and purifies compound A from Lycium ruthenicum Murr., and verifies that compound A has the effect of treating hepatitis.

Description

Technical Field

[0001] The present invention relates to the technical field of compound separation, and particularly to a method for separating anti-hepatitis active substances from Lycium ruthenicum Murr. Background Art

[0002] Lycium ruthenicum Murr. is a newly discovered wild medicinal and edible plant in places such as the Qaidam Basin in Qinghai in recent years. The nutritional value of Lycium ruthenicum Murr. is higher than that of ordinary wolfberries, containing various nutritional components such as proteins, vitamins, and minerals, with relatively high edible value. Its fruits also contain rich anthocyanin components, having antioxidant and anti-allergic functions, enhancing human immunity and improving sleep.

[0003] Research shows that Lycium ruthenicum Murr. contains a variety of bioactive components. Its mature berries are rich in polyphenols such as anthocyanins, having effects such as antioxidant, potential prevention and treatment of cardiovascular system diseases, etc. Among them, polyphenols including anthocyanins and flavonoid compounds show various bioactivities such as anti-inflammatory, cardiovascular protection, and anti-tumor through their strong antioxidant free radical scavenging effects.

[0004] Currently, the research on the active substances in Lycium ruthenicum Murr. is still relatively weak, and the exploration, separation, and extraction of the active substances in Lycium ruthenicum Murr. still await further research. Summary of the Invention

[0005] The main technical problem solved by the present invention is to provide a method for separating anti-hepatitis active substances from Lycium ruthenicum Murr. By extracting Lycium ruthenicum Murr. with methanol, and then further separating and purifying the extracted active components, a compound A with anti-hepatitis activity is finally obtained.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for separating anti-hepatitis active substances from Lycium ruthenicum Murr., comprising the following steps:

[0008] (1) Soak and extract the dried fruits of Lycium ruthenicum Murr. with methanol under room temperature and dark conditions. Extraction conditions: liquid-solid ratio 20 mL / g, extract 3 times in total, 4 - 5 days each time. Filter the extract, and perform dark-light reduced pressure concentration and combination to obtain the methanol extract paste of Lycium ruthenicum Murr. fruits;

[0009] (2) Mix the methanol extract paste prepared in step (1) with dried polyamide powder in a mass ratio of 1:1, dry it in an oven at 40 °C and then grind it, and pass through a 20-mesh sieve to obtain the sieved powder;

[0010] (3) Load the sieved powder obtained in step (2) into a small medium-pressure chromatography column, connect a medium-pressure chromatography column filled with MCI to a preparative liquid chromatography, and perform dry loading; use a water / methanol / dichloromethane three-phase system for elution, and a total of three components are obtained: Fr1 is obtained with a retention time of 12 - 39 min, Fr2 is obtained with a retention time of 39 - 139 min, and Fr3 is obtained with a retention time of 139 - 230 min;

[0011] (4) Further separate Fr2 obtained in step (3), use methanol and / or water as the mobile phase, and perform gradient elution to obtain Fr2-5 with a retention time of 76 - 130 min;

[0012] (5) Separate Fr2-5 obtained in step (4), load it into a C18 preparative chromatography column, and perform gradient elution with water / methanol as the mobile phase: Fr2-5-4 is obtained with a retention time of 21 - 27 min;

[0013] (6) Separate Fr2-5-4 obtained in step (5), use a C18 preparative chromatography column, use water / methanol as the mobile phase, and perform gradient elution to obtain Fr2-5-4-1 as a yellow powdery substance, which is the target compound A;

[0014] The structural formula of compound A is as follows:

[0015] Furthermore, the elution conditions in step (3) are:

[0016] 0 - 120 min, 100% water - 100% methanol;

[0017] 120 - 180 min, 100% methanol - 100% dichloromethane;

[0018] 180 - 210 min, 100% methanol;

[0019] 210 - 240 min, 100% water;

[0020] Flow rate: 50 mL / min, detection wavelength: 210 nm.

[0021] Furthermore, the elution conditions in step (4) are:

[0022] 0 - 120 min, 0 - 100% methanol;

[0023] 120 - 140 min, 100% methanol;

[0024] Flow rate: 50 mL / min;

[0025] Detection wavelength: 254 nm;

[0026] Filler: CHP20P gel chromatography;

[0027] Column specification: 49×460 mm.

[0028] Furthermore, the elution conditions in step (5):

[0029] 0–60–65–90 min, 30%–42%–70%–95% methanol,

[0030] Flow rate: 19 mL / min;

[0031] Detection wavelength: 210 nm.

[0032] Furthermore, the elution conditions in step (6):

[0033] 0–60 min, 30%–35% methanol;

[0034] Flow rate: 18 mL / min;

[0035] Detection wavelength: 210 nm.

[0036] Furthermore, the specification of the C18 preparative chromatographic column is 21.2×250 mm, 5 μm.

[0037] The present invention also provides the use of compound A in the preparation of a drug for preventing or treating liver injury.

[0038] Among them, the application of the drug is in the drug for preventing or treating hepatitis.

[0039] Among them, the liver injury is chemical liver injury.

[0040] Furthermore, the chemical liver injury is liver injury caused by CCl4.

[0041] According to the separation and purification path of the present invention, compound A is present in the extraction parts of Fr2, Fr2-5 or Fr2-5-4, and the contents in the above parts increase in sequence. Based on the preventive or therapeutic effect of compound A on liver injury, Fr2, Fr2-5 or Fr2-5-4 containing compound A also have the above activities.

[0042] The "hepatitis" in the present invention refers to the general term for liver inflammation caused by chemical substances. Chemical liver injury generally refers to the damage of the liver caused by various chemical factors, including drug-induced hepatitis, and also includes hepatitis caused by chemical poisons, which can also be called toxic hepatitis.

[0043] In the pharmaceutical composition of compound A or its stereoisomers, solvates, hydrates, pharmaceutically acceptable salts or co-crystals of the present invention, pharmaceutically acceptable excipients may be contained.

[0044] As used herein, "pharmaceutically acceptable" refers to a substance that includes any substance that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host to which it is administered.

[0045] The pharmaceutically acceptable excipients described in the present invention are the general term for all additional materials in a drug other than the main drug. The excipients should have the following properties: (1) They are non-toxic to the human body and have few side effects; (2) They are chemically stable and are not easily affected by temperature, pH, storage time, etc.; (3) They have no incompatibility with the main drug and do not affect the efficacy and quality inspection of the main drug; (4) They do not interact with the packaging material. The excipients in the present invention include but are not limited to fillers (diluents), lubricants (glidants or anti-adhesives), dispersants, wetting agents, binders, regulators, solubilizers, antioxidants, bacteriostatic agents, emulsifiers, disintegrants, etc. Binders include syrup, gum arabic, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methyl cellulose, etc.), gelatin paste, syrup, starch paste or polyvinylpyrrolidone, etc.; Fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; Lubricants include colloidal silicon dioxide, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc.; Disintegrants include starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinylpyrrolidone or microcrystalline cellulose, etc.; Wetting agents include sodium lauryl sulfate, water or alcohol, etc.; Antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutylhydroxyanisole, etc.; Bacteriostatic agents include 0.5% phenol, 0.3% cresol, 0.5% chlorobutanol, etc.; Regulators include hydrochloric acid, citric acid, potassium (sodium) hydroxide, sodium citrate and buffers (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; Emulsifiers include polysorbate-80, sorbitan monooleate, pluronic F-68, lecithin, soya lecithin, etc.; Solubilizers include tween-80, bile, glycerol, etc. The term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or a base that is suitable for use as a drug. The above acids and bases are generalized Lewis acids and bases. Acids suitable for forming salts include but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.

[0046] There is no particular limitation on the mode of administration of the compounds or pharmaceutical compositions of the present invention. Representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0047] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier) such as sodium citrate or calcium phosphate, or with the following components: (a) fillers or bulking agents, e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, e.g., glycerol; (d) disintegrating agents, e.g., agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retarders, e.g., paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, e.g., cetyl alcohol and glycerol monostearate; (h) adsorbents, e.g., kaolin; and (i) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0048] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents, and release of the active compound or compounds in such compositions can be delayed and released in a part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric and wax-like substances. If necessary, the active compound can also be in the form of microcapsules with one or more of the above excipients.

[0049] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents conventionally used in the art such as water or other solvents, solubilizing agents, and emulsifying agents, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 - butanediol, dimethylformamide, and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances and the like.

[0050] In addition to these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, and perfumes.

[0051] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar, or mixtures of these substances and the like.

[0052] Compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0053] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required, if necessary.

[0054] The compounds of the present invention can also be used in injection preparations. Among them, the injections are selected from liquid injections (aqueous injections), sterile powders for injection (powder injections) or tablets for injection (referring to molded tablets or machine-pressed tablets prepared by aseptic operation of drugs, dissolved in injection water for subcutaneous or intramuscular injection when in use).

[0055] Among them, in addition to the above compounds, the injection powder contains at least an excipient. The excipient in the present invention is an ingredient intentionally added to the drug, and its amount used should not have pharmacological properties. However, the excipient can contribute to the processing, dissolution or dissolution, drug delivery through the targeted delivery route or contribute to stability.

[0056] The "C18 preparative chromatographic column" is a commonly used reverse-phase chromatographic column. Its principle is to use a C18 alkyl chain immobilized on a silica gel matrix as the stationary phase to separate polar substances from non-polar substances. The alkyl chain of the C18 chromatographic column can interact with the non-polar substances in the sample, making them stay on the stationary phase for a longer time and unable to pass through the column quickly, thus achieving separation. On the contrary, polar substances cannot interact with the alkyl chain, so they can pass through the column quickly. The C18 chromatographic column is suitable for separating mixtures of compounds with different polarities, such as drugs, natural products and organic compounds, etc. Its separation effect is affected by multiple factors such as the nature of the stationary phase, the nature of the sample and the nature of the mobile phase.

[0057] The DMEM is a culture medium containing various amino acids and glucose, which is developed on the basis of the MEM culture medium. Compared with MEM, the amounts of various components are increased, and it is also divided into high-glucose type (higher than 4500 mg / L) and low-glucose type (lower than 1000 mg / L). The high-glucose type is beneficial for cells to anchor and grow at a position, and is suitable for tumor cells with faster growth and more difficult attachment, etc.

[0058] The beneficial effects of the present invention are:

[0059] The present invention first isolated and purified Compound A from Lycium ruthenicum Murr., and according to research findings, Compound A has certain activity in preventing or treating liver injury, laying a solid foundation for the basic research on the activity of Lycium ruthenicum Murr. Description of the Drawings

[0060] Figure 1 MCI separation and preparation map of the fruit extract of Lycium ruthenicum Murr.;

[0061] Figure 2 MCI separation and preparation map of Fr2;

[0062] Figure 3 Separation and preparation map of Fr2-5;

[0063] Figure 4 Separation and preparation map of Fr2-5-4;

[0064] Figure 5 Separation and preparation map of Fr2-5-4-1;

[0065] Figure 6 Mass spectrum of Compound Fr2-5-4-1;

[0066] Figure 7 Compound Fr2-5-4-1's 1 HNMR spectrum;

[0067] Figure 8 Compound Fr2-5-4-1's 13 CNMR spectrum;

[0068] Figure 9 Bar graph of the effect of monomeric compounds on the proliferation of HepG2 cells;

[0069] Figure 10 Bar graph of the protective effect of monomeric compounds on CCl4-induced HepG2 cell injury;

[0070] Figure 11 Bar graph of the effect of monomeric compounds on the AST activity in HepG2 cells;

[0071] Figure 12 Bar graph of the effect of monomeric compounds on the ALT activity in HepG2 cells. Detailed Embodiments

[0072] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described herein are only a part of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the protection scope of the present invention.

[0073] DMSO: Dimethyl sulfoxide.

[0074] Example 1

[0075] A method for separating anti-hepatitis active substances from Lycium ruthenicum Murr., comprising the following steps:

[0076] (1) Weigh 10.0 kg of dried Lycium ruthenicum Murr. fruits, add 200 L of methanol and soak them under dark conditions at room temperature for 3 extractions, with each extraction lasting for 4 days. After filtration, the extracts are concentrated under reduced pressure in the dark and combined to obtain 3.643 kg of the methanol extract paste of Lycium ruthenicum Murr. fruits.

[0077] (2) Mix the methanol extract paste prepared in step (1) with dry polyamide powder at a mass ratio of 1:1, dry it in an oven at 40 °C, grind it, and sieve it through a 20-mesh sieve to obtain the sieved powder.

[0078] (3) Weigh 50.00 g of the sieved powder obtained in step (2) and load it into a small medium-pressure chromatography column (26×100 mm), and connect it to a medium-pressure chromatography column (49×460 mm) filled with MCI for dry loading. Use a three-phase system of water / methanol / dichloromethane for elution: 0 - 120 min, 100% water - 100% methanol; 120 - 180 min, 100% methanol - 100% dichloromethane; 180 - 210 min, 100% methanol; 210 - 240 min, 100% water; flow rate: 50 mL / min, detection wavelength: 210 nm. A total of three fractions, Fr1, Fr2, and Fr3, are obtained. As shown, Fr2 with a retention time of 39 - 139 min is 511.3 g, and the yield is 15.1%. Figure 1 As shown, Fr2 with a retention time of 39 - 139 min is 511.3 g, and the yield is 15.1%.

[0079] (4) Further separate the Fr2 obtained in step (3) using methanol and / or water as the mobile phase for gradient elution. The elution conditions are: 0 - 120 min, 0 - 100% methanol; 120 - 140 min, 100% methanol; flow rate: 50 mL / min; detection wavelength: 254 nm; packing material: MCI; column specification: 49×460 mm. As shown, Fr2-5 with a retention time of 76 - 130 min is 175.5 g, and the yield is 34.3%. Figure 2 As shown, Fr2-5 with a retention time of 76 - 130 min is 175.5 g, and the yield is 34.3%.

[0080] (5) Separate the Fr2-5 obtained in step (4) and load it into a C18 preparative chromatography column with a specification of 21.2×250 mm and 5 μm. Use water / methanol as the mobile phase for gradient elution: 0 - 60 - 65 - 90 min, 30% - 42% - 70% - 95% methanol, flow rate: 19 mL / min; detection wavelength: 210 nm. Figure 3As shown, Fr2-5-4 was obtained at a retention time of 21 - 27 min, weighed 6.3 g, and the yield was 3.6%.

[0081] (6) Separate Fr2-5-4 in step (5) using a C18 preparative chromatographic column with a specification of 21.2.×250 mm and 5 μm.

[0082] Using water / methanol as the mobile phase, perform gradient elution: 0% for 60 min, 30 - 35% methanol; flow rate: 18 mL / min; detection wavelength: 210 nm; as Figure 4 and Figure 5 shown, Fr2-5-4-1 was obtained at a retention time of 25 - 26 min, which is the yellow powdery target compound A, weighed 270 mg, and the yield was 4.3%.

[0083] As Figure 6 , Figure 7 and Figure 8 shown, the MS, 1 1H-NMR, 13 13C-NMR data of compound A are as follows:

[0084] ESI-MS m / z: 197 [M-H] - , chemical formula C9H 10 O5;

[0085] 1 1H-NMR (DMSO, 800 MHz): δ: 7.54 (2H, s, H-2, 6), 5.38 (1H, s, 3,5-OH), 3.83 (3H, s, -OCH3);

[0086] 13 13C-NMR (DMSO, 800 MHz): δ: 119.23 (C1), 110.51 (C-2), 151.81 (C-3), 139.39 (C-4), 151.81 (C-5), 104.58 (C-6), 166.20 (C-7), 51.73 (C-8), 56.15 (-OCH3).

[0087] The above data are all consistent with the MS, 1 1H-NMR, 13 13C-NMR standard spectral data reported in the literature for the known compound methyl 3-methoxygallate, so it was identified as Methyl 3,4-dihydroxy-5-methoxy-benzoate.

[0088] Structural formula of methyl 3-methoxygallate (compound A)

[0089] Evaluation of the in vitro liver-protecting effect of Methyl 3,4-dihydroxy-5-methoxy-benzoate

[0090] 1. Experimental materials and reagents

[0091] 1.1 Experimental materials

[0092] The monomeric compound Methyl 3,4-dihydroxy-5-methoxy-benzoate.

[0093] Human hepatocellular carcinoma HepG2 cell line: The Collection of Type Cultures of the Chinese Academy of Sciences (Catalog number: SCSP-510).

[0094] 1.2 Preparation methods of main reagents

[0095] (1) Cell cryopreservation solution: In a laminar flow hood, mix DMEM medium: fetal bovine serum: DMSO in a ratio of 7:2:1, and prepare it freshly before use.

[0096] (2) Complete DMEM medium: First add 5 mL of penicillin-streptomycin double antibody to 500 mL of fresh DMEM medium and mix well, then add 50 mL of fetal bovine serum to prepare a DMEM medium containing 10% serum.

[0097] (3) MTT solution: Weigh 50.00 mg of MTT powder, dissolve it in PBS buffer and make up the volume to 10 mL. Sonicate it for dissolution under light protection, filter and sterilize it with a 0.22 μm microporous membrane, and store it frozen at -20°C in the dark after aliquoting.

[0098] (4) Preparation of CCl4 injury solution: Mix CCl4 and absolute ethanol at a ratio of 1:1 and add it to DMEM culture medium (final concentration: 0.1% v / v) to obtain CCl4 injury solution and store it in a 4°C refrigerator. Shake it well before use.

[0099] (5) Preparation of monomeric compound solution: Weigh 4.925 mg of Methyl 3,4-Dihydroxy-5-Methoxy-benzoate monomeric compound, dissolve it in 5 mL of DMSO to prepare a stock solution with a concentration of 5 mmol / L. Store the stock solution of the test sample in a -20°C refrigerator and dilute it as needed for the experiment.

[0100] 2. Experimental methods

[0101] 2.1 Determination of cell viability by MTT method

[0102] The MTT colorimetric method (tetramethylthiazolyl tetrazolium micro - enzyme reaction colorimetric method) was used to determine cell viability. The specific method was as follows: HepG2 cells in the logarithmic growth phase were digested with trypsin for 2 - 3 min, then a certain amount of complete DMEM medium was added, and the cell density was adjusted to 1×10 4 cells / well and seeded in a 96 - well cell plate. After culturing for 24 h, the culture medium was discarded. Then, 200 μL of DMEM medium containing different concentrations of drugs and 1% serum was added respectively. Six replicates were set in each group. After culturing for a period of time, the medium was discarded, and fresh DMEM medium containing 1% serum was added. Then, 20 μL of MTT solution (final concentration 0.5 mg / mL) was added to each well and cultured for another 4 h. After reaching the preset time, the medium in the 96 - well plate was discarded, 200 μL of DMSO was added to each well, and finally the absorbance value of each well at a wavelength of 490 nm was measured using an enzyme - linked immunosorbent assay (ELISA) reader.

[0103] 2.2 Extraction of total cellular proteins

[0104] The culture medium in the culture dish was discarded, and the cells were washed twice with PBS. Then, according to the required protein concentration, 40 - 100 μL of cell lysis buffer (RIPA:PMSF = 100:1 v / v) was added, and the cells were lysed on ice at 4℃ for 5 min. After lysis, the cells were completely scraped off using a cell scraper, transferred to a centrifuge tube, centrifuged at 12000 rpm at 4℃ for 15 min, and the supernatant was aspirated into another centrifuge tube and stored at - 80℃.

[0105] 2.3 Determination of hepatic enzyme activity in cells

[0106] HepG2 cells in the logarithmic growth phase were taken, digested with trypsin, and the concentration of the cell suspension was adjusted with complete DMEM medium containing 10% serum. Then the cells were seeded in a 6 - well plate for culture, 3 mL per well. Then the 6 - well plate was placed in a CO2 incubator for 24 h until the cells were completely adherent. The 6 - well plate was taken out, and the cells were set up with a blank control group, a model group, and a combined treatment group of compound A and 0.1% CCl4. After culturing the cells in each group for 6 h, the supernatant was discarded, and the cell proteins were extracted for the determination of ALT and AST indexes.

[0107] 3. Experimental results

[0108] 3.1 Effects of monomeric compounds on the proliferation of HepG2 cells

[0109] As Figure 9 can be seen, when the monomeric compound was applied to HepG2 cells at three concentration ranges of 5 - 20 μM for 24 h, no obvious cytotoxicity was observed at all three concentrations, and there was no significant difference compared with the blank control group. Subsequently, 5, 10, and 20 μM were continued to be selected for subsequent experiments.

[0110] 3.2 Protective effect of monomeric compounds on CCl4-induced HepG2 cell injury

[0111] As Figure 10 shown, after CCl4 injury, compared with the normal group, the survival rate of cells in the model group decreased extremely significantly, indicating that CCl4 can induce injury to HepG2 cells and inhibit their activity (p<0.01). Compared with the model group, after simultaneous administration of monomeric compounds to cells, the cell viability can be increased to varying degrees at three action concentrations, showing extremely significant differences compared with the model group (p<0.01).

[0112] Note: Figure 10 Compared with the blank control group, ##p<0.01; compared with the CCl4 model group, **p<0.01.

[0113] 3.3 HepG2 cell liver enzyme activities

[0114] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are present in various human cells and have the highest content in liver cells. When the body is stimulated by the outside world, such as drug-induced liver poisoning or chemical-induced liver poisoning, the corresponding liver cells are damaged, resulting in an increase in cell membrane permeability and their release, increasing the concentration of liver enzymes in the extracellular environment. Therefore, AST and ALT are usually used as important indicators for the clinical diagnosis of viral hepatitis and toxic hepatitis, and are also sensitive markers for acute liver cell injury.

[0115] As Figures 11 to 12 shown, compared with the blank control group, the enzyme activities of AST and ALT in the model group cells increased extremely significantly, and the difference between the two was statistically significant (p<0.01), indicating that CCl4 significantly damaged the cells, leading to abnormal cell

[0116] structure and function. Compared with the model group, pre-incubation with different concentrations of monomeric compounds and 0.1% CCl4 injury solution medium can reduce the levels of ALT and AST in cells, showing extremely significant differences (p<0.01), and presenting a dose-dependent effect, indicating that although monomeric compounds cannot completely resist the cell damage caused by CCl4, they can reduce the degree of cell damage to a certain extent. Observing the changes of various indicators can comprehensively determine the regulatory effect on liver injury. After the monomeric compound acts on the cells, the enzyme activities of ALT and AST in the cells decrease, indicating that under the intervention of the monomeric compound, the liver cell injury decreases and the liver enzyme activity decreases.

[0117] Note: Figure 11 and Figure 12, compared with the blank control group, ##p < 0.01; compared with the CCl4 model group, **p < 0.01.

Claims

1. A method for separating anti-hepatitis active substances from Lycium ruthenicum Murr., characterized in that, It includes the following contents: (1) Take the methanol extract paste of Lycium ruthenicum Murr. fruits; (2) Mix the methanol extract paste of Lycium ruthenicum Murr. fruits with dry polyamide powder at a mass ratio of 1:1, dry and then grind, and pass through a 20-mesh sieve to obtain the sieved powder; (3) Take the sieved powder in step (2) and load it into a small medium-pressure chromatographic column, connect a medium-pressure chromatographic column filled with MCI and a preparative liquid chromatograph, and perform dry sample loading; use a water / methanol / dichloromethane three-phase system for elution: 0 - 120 min, 100% water - 100% methanol; 120 - 180 min, 100% methanol - 100% dichloromethane; 180 - 210 min, 100% methanol; 210 - 240 min, 100% water; flow rate: 50 mL / min, detection wavelength: 210 nm; a total of three components are obtained: Fr1 is obtained with a retention time of 12 - 39 min, Fr2 is obtained with a retention time of 39 - 139 min, and Fr3 is obtained with a retention time of 139 - 230 min; (4) For Fr2 obtained in step (3), use methanol and / or water as the mobile phase for gradient elution: 0 - 120 min, 0 - 100% methanol; 120 - 140 min, 100% methanol; flow rate: 50 mL / min; detection wavelength: 254 nm; packing: CHP20P gel chromatography; Fr2-5 is obtained with a retention time of 76 - 130 min; (5) For Fr2-5 in step (4), connect a C18 preparative chromatographic column and perform gradient elution with water / methanol as the mobile phase: 0 - 60 - 65 - 90 min, 30% - 42% - 70% - 95% methanol, flow rate: 19 mL / min; detection wavelength: 210 nm; Fr2-5-4 is obtained with a retention time of 21 - 27 min; (6) For Fr2-5-4 in step (5), use a C18 preparative chromatographic column and perform gradient elution with water / methanol as the mobile phase: 0% - 60 min, 30 - 35% methanol; flow rate: 18 mL / min; detection wavelength: 210 nm; Fr2-5-4-1, which is the yellow powdery target compound A, is obtained with a retention time of 25 - 26 min; The structural formula of compound A is as follows:

2. The method according to claim 1, wherein The elution conditions in step (4): Column specification: 49×460 mm.

3. The method according to claim 1, wherein The specification of the C18 preparative chromatographic column is 21.2×250 mm, 5 μm.

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