Biphenyl compound urolithin B pepper acid ester and application thereof in anti-liver injury aspect
By extracting and isolating the biphenyl compound urolithin B piperate from dregs of the body and combining it with multiple formulation forms and administration routes, the problem of effective utilization of this compound in dregs of the body was solved, achieving significant anti-liver damage efficacy.
Patent Information
- Application Number
- CN202410253018.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-06
- Publication Date
- 2025-09-16
AI Technical Summary
The existing technology fails to effectively utilize the extraction and separation of the biphenyl compound urolithin B piperate and its pharmaceutically acceptable salts from the residue, and its application in preventing liver damage has not been fully developed.
The biphenyl compound urolithin B piperate is separated by ethanol extraction, MCI fine resin column chromatography, silica gel column chromatography and high-performance liquid chromatography, and is prepared into a pharmaceutical composition for use in various administration routes and dosage forms, including liquid, solid and semi-solid dosage forms, suitable for enteral or parenteral administration.
The effective preparation and application of the biphenyl compound urolithin B piperonate in a pharmaceutical composition was achieved, showing significant anti-liver damage effects, better than the existing positive control bicyclol, and improving cell survival rate.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medical technology and relates to a biphenyl natural product Urolitin B piperonate extracted and separated from dregs of medicinal materials, a preparation method thereof, and its use in preventing liver damage. Background Art
[0002] Zhaxun, a traditional Tibetan medicine, has been used for over 1,300 years. In Tibetan medicine, Zhaxun is classified as either earth or essence. It originates from rocky mountains, where the sun melts the rocks and condenses their essence into Zhaxun. Zhaxun is primarily produced in the Himalayas, at altitudes between 1,000 and 5,000 meters. It has a sweet and bitter taste, a cooling nature, and clears heat. Zhaxun is primarily used to treat febrile illnesses such as liver heat, kidney heat, and stomach heat, as well as phthisis, gout, eye diseases, febrile edema, and physical weakness. The ingredients involved in this invention are biphenyl compounds. Summary of the Invention
[0003] The technical problem solved by the present invention is to provide a biphenyl compound urolithin B piperate obtained by extraction and separation from slag and a pharmaceutically acceptable salt thereof, as well as a preparation method and use thereof.
[0004] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:
[0005] The first aspect of the technical solution of the present invention is to provide a biphenyl compound urolithin B piperate and a pharmaceutically acceptable salt thereof, the compound structure of which is as follows:
[0006]
[0007] The second aspect of the technical solution of the present invention is to provide a method for preparing the biphenyl compound urolithin B piperate, which is separated from slag, and the specific steps are as follows:
[0008] Extraction: The crushed residue is extracted with ethanol, and the extract is concentrated to obtain a crude extract.
[0009] Separation: The crude extract was dissolved in a small amount of ethanol, an appropriate amount of MCI fine resin filler was added and dried, the dried filler was loaded into a column, and ethanol-water was used for column chromatography elution; the eluent ethanol-water was in a volume ratio, ethanol:water in the first eluent = 0:100, ethanol:water in the second eluent = 30:70, ethanol:water in the third eluent = 60:40, and ethanol:water in the fourth eluent = 95:5; the third eluent was concentrated and further subjected to column chromatography using glycol-based silica gel, eluted with a gradient of petroleum ether:ethyl acetate, and the eluent was further subjected to silica gel column chromatography, and finally preparative separation was performed using a high performance liquid chromatograph with a volume ratio of acetonitrile:water = 95:5 to obtain the target compound urolithin B piperonate.
[0010] In the above preparation method, in the extraction step, the ethanol used is 95% ethanol.
[0011] In the above preparation method, it is characterized in that, in the extraction step, the extraction method adopted is heating reflux extraction.
[0012] In the above preparation method, it is characterized in that, in the separation step, the ratio of the eluent during the glycol-based silica gel chromatography is petroleum ether: ethyl acetate = 1:0, 9:1, 4:1, 0:1 (v / v).
[0013] In the above preparation method, it is characterized in that, in the separation step, the eluent for the silica gel column chromatography is diol-based silica gel chromatography, and the eluent ratio is petroleum ether:ethyl acetate = 9:1 and 4:1.
[0014] In the above preparation method, it is characterized in that, in the separation step, the chromatographic stationary phase used by the high performance liquid chromatograph is octadecyl bonded silica gel (ODS), and the mobile phase is acetonitrile-water.
[0015] The third aspect of the technical solution of the present invention is to provide a pharmaceutical composition, characterized in that the pharmaceutical composition contains the biphenyl compound and its pharmaceutically acceptable salt and pharmaceutically acceptable salt or additive described in the first aspect.
[0016] The pharmaceutical composition can be prepared according to methods known in the art. The compound of the present invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants to form any dosage form suitable for human or animal use. The content of the compound of the present invention in its pharmaceutical composition is generally 0.1-95% by weight.
[0017] The compound of the present invention or the pharmaceutical composition containing the same can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.
[0018] The dosage form can be a liquid dosage form, a solid dosage form, or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), and sprays; semisolid dosage forms can be ointments, gels, pastes, and the like.
[0019] The compound of the present invention can be prepared into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.
[0020] In order to prepare the compound of the present invention into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and solubilizers. The diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent may be water, ethanol, isopropyl alcohol, etc.; the binder may be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; the lubricant and solubilizing agent may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.
[0021] The tablets can 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.
[0022] To prepare a dosing unit as a capsule, the active ingredient compound of the present invention can be mixed with a diluent and a cosolvent, and the mixture can be placed directly in a hard or soft capsule. Alternatively, the active ingredient compound of the present invention can be first mixed with a diluent, a binder, and a disintegrant to form granules or pellets, which can then be placed in a hard or soft capsule. The same diluents, binders, wetting agents, disintegrants, and cosolvents used to prepare tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention.
[0023] To prepare the compounds of the present invention as injections, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. Examples of solubilizers or cosolvents include poloxamer, lecithin, and hydroxypropyl-β-cyclodextrin; pH adjusters include phosphates, acetates, hydrochloric acid, and sodium hydroxide; and osmotic pressure regulators include sodium chloride, mannitol, glucose, phosphates, and acetates. For lyophilized powder injections, mannitol, glucose, and the like can also be added as support agents.
[0024] Furthermore, if necessary, colorants, preservatives, perfumes, flavorings or other additives may be added to the pharmaceutical preparations.
[0025] To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known method of administration.
[0026] The dosage of the pharmaceutical compositions of the present invention can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration, and the dosage form. Generally speaking, a suitable daily dosage range of the compounds of the present invention is 0.001-5 mg / kg body weight. The above dosage can be administered as a single dosage unit or divided into several dosage units, depending on the physician's clinical experience and the dosage regimen including the use of other therapeutic means.
[0027] The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.
[0028] The fourth aspect of the technical solution of the present invention is to provide the use of the biphenyl compound urolithin B piperate and its pharmaceutically acceptable salts in the preparation of anti-liver damage drugs.
[0029] Beneficial technical effects
[0030] The compound has the ability to significantly improve cell survival rate in an in vitro liver cell injury model induced by paracetamol (APAP), which is higher than that of the positive control drug bicyclol, and can be used to prepare anti-liver injury drugs. DETAILED DESCRIPTION
[0031] Example 1 Preparation of Urolitin B piperonate
[0032]
[0033] Extraction: 25.0 kg of dried residue was crushed and extracted three times with 95% ethanol (300 L) under heating (80°C) and reflux. The resulting extract was evaporated under reduced pressure to obtain a crude extract (2000 g). The crude extract was dissolved in water (6 L), suspended uniformly, and extracted three times with petroleum ether (6 L). The resulting extract was concentrated under reduced pressure (37°C) to obtain a petroleum ether extract (200 g). The aqueous solution after the petroleum ether extraction was extracted three times with ethyl acetate (6 L). The ethyl acetate extraction fractions were combined and the ethyl acetate solvent was recovered under reduced pressure to obtain an ethyl acetate extract (900 g).
[0034] Separation: Dissolve the ethyl acetate extract in a small amount of ethanol, add 1800g of MCI filler and dry it. Then load the filler into a column and use ethanol-water for column chromatography elution; the volume ratio of ethanol to water in the eluent is ethanol:water = 0:100 in the first eluent, ethanol:water = 30:70 in the second eluent, ethanol:water = 60:40 in the third eluent, and ethanol:water = 95:5 in the fourth eluent; the volume of the eluent for each eluent is 12L.
[0035] The third elution fraction was evaporated to dryness and then subjected to column chromatography on glycol-based silica gel using a gradient elution of petroleum ether:ethyl acetate in a volume ratio of 1:0, 9:1, 4:1, and 0:1, respectively. The volume of each gradient elution was 4000 mL. Similar fractions were combined based on TLC color development to yield 15 components A through O.
[0036] The extract of component E (45.0 g) and the extract of F (58.5 g), i.e., petroleum ether / ethyl acetate volume ratios of 9:1 and 4:1, were combined to obtain 103.5 g of extract (named EF), which was further subjected to silica gel H column chromatography, eluted with petroleum ether / ethyl acetate volume ratios of 9:1, 2:1 and 0:1, respectively, to obtain 9 components EF1 to EF9 by combining similar fractions according to TLC color development (anisaldehyde-sulfuric acid-ethanol color developer), which were evaporated to dryness for use.
[0037] Finally, EF2 (1.30 g) was dissolved in acetonitrile and separated using high-performance liquid chromatography (HPLC) with detection at 260 nm to yield the target compound, Urolitin B piperonate. The column was a 250 mm × 20 mm / 5 μm RP-C18 column, the flow rate was 10 ml / min, and the mobile phase was a 95 / 5 (v / v) ratio of CH₃CN / H₂O.
[0038] Structural Identification: The chemical structure of Urolitin B piperonate was determined by X-ray diffraction and nuclear magnetic resonance spectroscopy. Its physicochemical properties are as follows:
[0039] Colorless crystals, molecular formula is C 21 H 12 O6;
[0040] High-resolution mass spectrometry HRESIMS m / z 361.0714 [M+H] + (calculated for C 21 H 13 O6,361.0707);
[0041] NMR spectroscopy 1 H NMR (400 MHz) and 13C NMR (100 MHz) data are shown in Table 1.
[0042] Table 1 Urolitin B piperonate 1 H and 13 C NMR (ppm in CDCl3)
[0043]
[0044]
[0045] Pharmacological experiments
[0046] Experimental sample: Urolitin B piperonate prepared in Example 1
[0047] Preparation of the test sample solution: The test sample is the pure compound Urolitin Bpiperonate prepared in Example 1. Accurately weigh an appropriate amount of sample and prepare a 10 μmol / L solution using DMSO for pharmacological activity testing.
[0048] Cell line: Human hepatocellular carcinoma HepG2 cells, which retain the characteristics of normal human hepatocytes. They were grown in DMEM (100 U / ml penicillin and 100 μg / ml streptomycin) supplemented with 10% fetal bovine serum at 37°C, 5% CO2, and saturated humidity. They were digested and passaged using a solution containing 0.25% trypsin and 0.02% EDTA.
[0049] Experimental methods
[0050] (1) Effects of compounds on HepG2 cell proliferation
[0051] The MTT method was used. HepG2 cells were seeded in a 96-well cell culture plate and cultured for 24 hours. Different concentrations of the test compound were then added. A solvent control group was also established, with three parallel wells for each drug concentration. After 48 hours of drug exposure, the culture medium was discarded, and 100 μl of MTT (0.5 mg / ml) solution was added to each well. The cells were cultured for another 4 hours. The MTT solution was discarded, and 150 μl of DMSO was added to each well. The cells were mixed and shaken, and the absorbance was measured at 570 nm using a microplate reader. Cell viability (%) = (mean OD value of the drug-treated cells / mean OD value of the solvent control cells) × 100%.
[0052] (2) Protective effect of the compound on paracetamol-induced hepatocyte damage in vitro
[0053] The MTT assay was used. HepG2 cells were seeded in a 96-well cell culture plate and cultured for 24 hours. Non-toxic concentrations of the test compound and paracetamol (APAP, final concentration 8 mM) were then added. A positive drug control group (bicyclol), a solvent blank control group, and a model group were also established. The cells were treated for a further 48 hours. The culture medium was discarded, and 100 μl of MTT (0.5 mg / ml) solution was added to each well. The cells were cultured for another 4 hours. The MTT solution was discarded, and 150 μl of DMSO was added to each well. The cells were mixed and shaken, and the absorbance was measured at 570 nm using a microplate reader. Cell viability (%) = (mean OD value of the drug group / mean OD value of the solvent control group) × 100%.
[0054] Experimental results
[0055] 1. Cytotoxicity: When treated with HepG2 cells at a concentration of 10 μM for 48 h, under the current experimental protocol, Urolitin Bpiperonate had no significant toxicity to HepG2 cells, and the cell survival rate was greater than 90% (data not shown).
[0056] 2. Protective Effect against APAP-Induced Hepatocellular Damage: Results are shown in Table 2. Treatment of HepG2 cells with 8 mM APAP for 48 hours significantly damaged the cells, with cell survival significantly reduced (47.53%) compared to the blank control group. Under the current experimental protocol, Urolitin B piperonate demonstrated significant protective effects against APAP-induced HepG2 cell damage, demonstrating statistically significant differences compared to the model group.
[0057] Table 2: Effects on cell survival of HepG2 cells damaged by APAP
[0058]
[0059] Experimental Conclusion
[0060] The compound Urolitin B piperonate has the ability to significantly improve cell survival rate in an in vitro hepatocyte injury model induced by paracetamol (APAP), which is higher than that of the positive control drug bicyclol, and can be used to prepare anti-liver injury drugs.
Claims
1. Biphenyl compound urolithin B piperate or a pharmaceutically acceptable salt thereof; 2. The method for preparing the biphenyl compound urolithin B piperate according to claim 1, wherein The method comprises the following steps: Extraction: The crushed medicinal materials are extracted with ethanol, and the extract is concentrated to obtain a crude extract; Separation: The crude extract was dissolved in a small amount of ethanol, an appropriate amount of MCI fine resin filler was added and dried, the dried filler was loaded into a column, and ethanol-water was used for column chromatography elution; the eluent ethanol-water was in a volume ratio, ethanol:water in the first eluent = 0:100, ethanol:water in the second eluent = 30:70, ethanol:water in the third eluent = 60:40, and ethanol:water in the fourth eluent = 95:5; the third eluent was concentrated and further subjected to column chromatography using glycol-based silica gel, eluted with a gradient of petroleum ether:ethyl acetate, and the eluent was further subjected to silica gel column chromatography, and finally preparative separation was performed using a high performance liquid chromatograph with a volume ratio of acetonitrile:water = 95:5 to obtain the target compound urolithin B piperonate.
3. The preparation method according to claim 2, characterized in that In the extraction step, the ethanol used was 95% ethanol.
4. The preparation method according to claim 2, characterized in that In the extraction step, the extraction method adopted is heating reflux extraction.
5. The preparation method according to claim 2, characterized in that In the separation step, the eluent for diol-based silica gel chromatography was petroleum ether:ethyl acetate, with a volume ratio of petroleum ether:ethyl acetate = 1:0, 9:1, 4:1, and 0:
1.
6. The preparation method according to claim 2, characterized in that In the separation step, the eluent for silica gel column chromatography is diol-based silica gel chromatography, and the eluent ratio is petroleum ether:ethyl acetate = 9:1 and 4:1 by volume.
7. The preparation method according to claim 2, characterized in that In the separation step, the chromatographic stationary phase used in the high performance liquid chromatography is octadecyl bonded silica gel ODS.
8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the biphenyl compound urolithin B piperate or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable additive according to claim 1.
9. Use of the biphenyl compound urolithin B piperate or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-liver injury drug.