Application of urolithin B pepper acid ester in preparation of neuroprotective drugs

Abstract

The invention belongs to the technical field of medicines, and relates to application of urolithin B pepper acid ester in preparation of a neuroprotective medicine. In particular relates to application of a biphenyl compound Urolite B piperimate extracted and separated from a Zhaxun medicinal material to preparation of a neuroprotective medicine, and a preparation method of the Urolite B piperimate is reported. A biological activity test shows that the compound can remarkably improve the survival rate of primary neuronal cells damaged by glutamic acid in a glutamic acid damage model, shows a remarkable neuroprotective effect and can be used for preparing neuroprotective drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology. The present invention relates to Urolitin B piperonate, a biphenyl natural product with neuroprotective activity extracted and separated from dregs of medicinal materials, as well as a preparation method and application in neuroprotective treatment. 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 Urolitin B piperonate and a pharmaceutically acceptable salt thereof extracted and separated from slag, a preparation method thereof and uses 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 urolitin B piperonate 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 urolitin B piperonate, 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 eluted with ethanol-water for column chromatography; the eluent ethanol-water volume ratio was 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 third eluent was concentrated and further subjected to column chromatography using glycol-based silica gel, eluted with a petroleum ether:ethyl acetate gradient, and the eluent was further subjected to silica gel column chromatography. Finally, high performance liquid chromatography was used for preparative separation with acetonitrile:water = 95:5 in the volume ratio to obtain the target compound Urolitin 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 Urolitin B piperonate and its pharmaceutically acceptable salts in the preparation of neuroprotective drugs.

[0029] Beneficial technical effects

[0030] The present invention uses the MTT assay to test the protective activity of urolitin B piperonate against cortical neurons of 17-day-old Wistar rat embryos damaged by glutamate. The experiment confirmed that urolitin B piperonate exhibits significant protective activity against glutamate-induced damage in rat embryonic cortical neurons. Therefore, the urolitin B piperonate of the present invention can be used to prepare neuroprotective drugs. DETAILED DESCRIPTION

[0031] The chemical structural formula of Urolitin B piperonate referred to in the examples is as follows:

[0032]

[0033] Preparation of Urolitin B piperonate as described in Example 1

[0034] 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).

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Structural Identification: The chemical structure of Urolitin Bpiperonate was determined by conventional NMR, HRESIMS and other modern spectral techniques. Its physicochemical properties are as follows:

[0040] Colorless crystals, molecular formula is C 21 H 12 O6;

[0041] High-resolution mass spectrometry HRESIMS m / z 361.0714 [M+H] + (calculated for C 21 H 13 O6,361.0707);

[0042] NMR spectroscopy 1 H NMR (400 MHz) and 13 C NMR (100 MHz) data are shown in Table 1.

[0043] Table 1 Urolithin B piperate 1 H and 13 C NMR (ppm in CDCl3)

[0044]

[0045] Example 2 Testing of the protective activity of urolitin B piperonate on cortical neurons of rat embryos damaged by glutamate in vitro.

[0046] Experimental samples

[0047] Preparation of the test sample solution: The test sample is the pure compound Urolitin B piperonate prepared in Example 1. Accurately weigh an appropriate amount of sample and use DMSO to prepare a 10 μmol / L solution for pharmacological activity testing.

[0048] Cell line: Cortical neurons from Wistar rat embryos. Cortical neurons were harvested from Wistar rat embryos at 17 days of gestation and cultured.

[0049] Experimental methods

[0050] The MTT assay was used. Cortical neurons were harvested from 17-day-gestation Wistar rat embryos and cultured. The positive agent PHPB (10 μmol / L) and the screening compound (10 μmol / L) were pre-incubated for 1 hour. A dilution of glutamate to a final concentration of 300 μM and PHPB / compound to a final concentration of 10 μM were added to each well. After 20 hours of co-incubation, cell viability was assessed using the MTT assay.

[0051] Cell viability (%) = (mean OD value of drug administration group / mean OD value of solvent control group) × 100%.

[0052] Experimental results

[0053] Glutamate exposure for 20 hours significantly damaged rat embryonic cortical neurons, with a cell survival rate of 53.5%. Under the current experimental protocol, urolitin B piperonate at a concentration of 10 μM, combined with glutamate, significantly protected rat embryonic cortical neurons from glutamate-induced damage, demonstrating statistically significant differences compared to the model group, as shown in Table 2.

[0054] Table 2. Test of protective activity against cortical neurons of rat embryos damaged by glutamate in vitro

[0055]

[0056] Experimental Conclusion

[0057] After 20 hours of glutamate injury to primary cortical neurons, the survival rate of primary neurons in the model group decreased to 53.5%, indicating that the glutamate model was successfully established. The positive tool drug PHPB (10 μmol / L) had a neuroprotective effect with statistical significance.

[0058] The compound Urolitin B piperonate can significantly improve the survival rate of primary neuronal cells damaged by glutamate in a glutamate injury model, and has a significant neuroprotective effect in this model. Therefore, the Urolitin B piperonate of the present invention can be used to prepare neuroprotective drugs.

Claims

1. Use of a biphenyl compound having the following chemical structure and a pharmaceutically acceptable salt thereof in the preparation of neuroprotective drugs:

2. The use according to claim 1, characterized in that 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 eluted with ethanol-water for column chromatography; the eluent ethanol-water volume ratio was 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 third eluent was concentrated and further subjected to column chromatography using glycol-based silica gel, eluted with a petroleum ether:ethyl acetate gradient, and the eluent was further subjected to silica gel column chromatography. Finally, high performance liquid chromatography was used for preparative separation with acetonitrile:water = 95:5 in the volume ratio to obtain the target compound Urolitin B piperonate.

3. The use according to claim 2, characterized in that In the extraction step, the ethanol used was 95% ethanol.

4. The use according to claim 2, characterized in that In the extraction step, the extraction method is heating reflux extraction.

5. The use according to claim 2, characterized in that In the separation step, the concentrations of the first petroleum ether / ethyl acetate gradient elution were volume ratios of 1:0, 20:1, 9:1, 4:1, 1:1, and 0:1, respectively.

6. The use according to claim 2, characterized in that In the separation step, the concentrations of the second petroleum ether / ethyl acetate gradient elution were volume ratios of 1:0, 9:1, 4:1, 2:1, and 0:1, respectively.

7. The use according to claim 2, characterized in that In the separation step, the chromatographic column filler of the preparative high performance liquid HPLC is reverse phase octadecyl bonded silica gel.

8. Use of a pharmaceutical composition in the preparation of a neuroprotective drug, characterized in that: The pharmaceutical composition contains a biphenyl compound as shown in the following formula and a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier or excipient.

Images