Terpenoids in alpinia speciosa and extraction method and application thereof

Abstract

The application discloses terpenoids in Alpinia zerumbet, and an extraction method and application thereof, and belongs to the field of medicine extraction. The novel terpenoids extracted from the Alpinia zerumbet have good anti-inflammatory effects, and exhibit better nitric oxide generation inhibition activity in lipopolysaccharide-induced BV2 cells or RAW 264.7 cells, wherein, for the lipopolysaccharide-induced BV2 cells, the IC 50 value is 17.89-61.38 μ M; for the lipopolysaccharide-induced RAW 264.7 cells, the IC 50 value is 11.69-71.91 μ M. The inhibition activity of some compounds is better than that of indomethacin and minocycline. In addition, some compounds can significantly reduce the expression of iNOS and COX-2 proteins in LPS-induced BV2 cells, inhibit the expression of iNOS, IL-1β and TNF-α at the mRNA level, and obviously inhibit the nuclear translocation of NF- κ B.

Description

Technical Field

[0001] This invention relates to the field of drug extraction technology, and in particular to terpenoid compounds in Alpinia galanga, their extraction methods and applications. Background Technology

[0002] Alpinia galanga ( Alpinia zerumbet (Pers.) BLBurtt&R.M.Sm) is a genus of ginger in the family Zingiberaceae. Alpinia Alpinia galanga is a perennial plant, also known as variegated alpinia, variegated ginger, and cake leaf. It is widely distributed in subtropical and tropical regions of South America, Oceania, and Asia. In traditional medicine, many parts of Alpinia galanga can be used as medicine, and their clinical uses vary.

[0003] In recent years, although research on Alpinia galanga has increased, it has mostly focused on the pharmacological activities of essential oil active ingredients and total extracts from different parts, with very little research on its specific chemically active components, and the active ingredients remain unclear. The compounds and their activities involved in this invention have not yet been reported in any patents or literature. Summary of the Invention

[0004] In order to overcome at least one of the technical problems existing in the prior art, one of the objectives of the present invention is to provide a ginger extract.

[0005] The second objective of this invention is to provide a method for preparing Alpinia galanga extract.

[0006] A third objective of this invention is to provide a pharmaceutical composition.

[0007] A fourth objective of this invention is to provide the use of the above-mentioned pharmaceutical composition in the preparation of medicaments for the prevention or treatment of inflammatory diseases.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides an extract of Alpinia zerumbet, comprising terpenoid compounds; said terpenoid compounds are selected from... , , , , , , , , , , At least one of them.

[0009] The second aspect of the present invention provides a method for preparing the Alpinia zerumbet extract described in the first aspect of the present invention, comprising the following steps: S1: First, extract Alpinia galanga with alcohol, then extract with cyclohexane and dichloromethane in sequence to obtain dichloromethane extract; S2: The dichloromethane extract was eluted on a silica gel column using cyclohexane and ethyl acetate. The product obtained by elution with cyclohexane and ethyl acetate in a volume ratio of 2:1 to 1:2 was collected to obtain elution product A. S3: The elution product A is purified on a small-pore resin column, with methanol and water in a volume ratio of 50:50-100:0 as the eluent, to obtain elution product B. S4: The elution product B is eluted and purified on an ODS reversed-phase chromatography column using a mixture of methanol and water, followed by column chromatography purification to obtain the Alpinia galanga extract.

[0010] In some embodiments of the present invention, the Alpinia zerumbet includes at least one of Alpinia zerumbet rhizome, Alpinia zerumbet leaves, and Alpinia zerumbet fruits. In some embodiments of the present invention, the Alpinia zerumbet refers to the Alpinia zerumbet rhizome.

[0011] In some embodiments of the present invention, the column chromatography purification is performed using at least one selected from Sephadex LH-20 gel column, semi-preparative high performance liquid chromatography, and silica gel column chromatography.

[0012] In some embodiments of the present invention, the column chromatography purification includes a step of elution purification on a semi-preparative high-performance liquid chromatography column using methanol and water in a volume ratio of 50:50-95:5.

[0013] In some embodiments of the present invention, the elution product A includes mixture a, mixture b, and mixture c; The mixture a is the product obtained by elution of cyclohexane and ethyl acetate in a volume ratio of 2:1 to 1:1; The mixture b is the product obtained by elution of cyclohexane and ethyl acetate in a volume ratio of 1:1; The mixture c is the product obtained by elution of cyclohexane and ethyl acetate in a volume ratio of 1:1 to 1:2.

[0014] In some embodiments of the present invention, the elution product B includes mixture A, mixture B, mixture C, mixture D, mixture E, and mixture F; The mixture A is the elution product obtained by eluting mixture b on a small-pore resin column using a methanol aqueous solution with a volume percentage of 88-92%. The mixture B is the elution product obtained by eluting mixture a on a small-pore resin column using a methanol aqueous solution with a volume percentage of 83-87%. The mixture C is the elution product obtained by eluting mixture c on a small-pore resin column with a methanol aqueous solution of 78-82% by volume. The mixture D is the elution product obtained by eluting mixture c on a small-pore resin column with a methanol aqueous solution of 88-92% by volume. The mixture E is the elution product obtained by eluting mixture c on a small-pore resin column with a methanol aqueous solution of 68-72% by volume. The mixture F is the elution product obtained by eluting mixture c on a small-pore resin column using a methanol aqueous solution with a volume percentage of 58-62%.

[0015] In some embodiments of the present invention, step S4 specifically involves: eluting mixture A on an ODS reversed-phase chromatography column using a methanol-water solution, collecting the product obtained by elution with a methanol-water solution of 60-80% (v / v), then eluting it on a Sephadex LH-20 gel column using a methanol-water solution of 85-95% (v / v), then eluting it on a semi-preparative high-performance liquid chromatography (HPLC) column using a methanol-water solution of 70-80% (v / v), and finally eluting it on a second semi-preparative HPLC column using a methanol-water solution of 85-95% (v / v) to obtain the product with the following structural formula: The extract of Alpinia zerumbet. In some embodiments of the present invention, step S4 specifically involves: eluting mixture A on an ODS reversed-phase chromatography column with a 10-100% methanol-water solution, collecting the product obtained by elution with a 65-75% (v / v) methanol-water solution, then eluting on a Sephadex LH-20 gel column with a 85-95% (v / v) methanol-water solution, then eluting on a semi-preparative high-performance liquid chromatography (HPLC) column with a 70-75% (v / v) methanol-water solution, collecting the elution product with a retention time of 20-25 min; then eluting on a semi-preparative HPLC column with an 85-90% (v / v) methanol-water solution, collecting the elution product with a retention time of 9-10 min, to obtain the product with the structural formula […]. Alpinia galanga extract.

[0016] In some embodiments of the present invention, step S4 is as follows: mixture A is eluted with a methanol-water solution on an ODS reversed-phase chromatography column, and the product obtained by elution with a methanol-water solution of 50-80% (v / v) is collected. Then, the product is eluted with a methanol-water solution of 60-70% (v / v) on a semi-preparative high-performance liquid chromatography (HPLC) column to obtain the product with the following structural formula: The extract of Alpinia zerumbet. In some embodiments of the present invention, step S4 is as follows: elute mixture A on an ODS reversed-phase chromatography column with a 10-100% methanol aqueous solution, collect the product obtained by elution with a 60-70% (v / v) methanol aqueous solution, and then elute with a 62-66% (v / v) methanol aqueous solution on a semi-preparative high-performance liquid chromatography column, collect the elution product with a retention time of 22-26 min, and obtain the product with the structural formula […]. Alpinia galanga extract.

[0017] In some embodiments of the present invention, step S4 is as follows: Mixture B is eluted on an ODS reversed-phase chromatography column using a methanol-water solution, and the product obtained by elution with 50-80% methanol-water (v / v) is collected. Then, elution is performed on a silica gel column using cyclohexane and ethyl acetate (v / v) at a ratio of 100:1 to 1:1, and the product obtained by elution with cyclohexane and ethyl acetate (v / v) at a ratio of 3:1 to 1:1 is collected. Finally, elution is performed on a semi-preparative high-performance liquid chromatography system using 60-80% methanol-water (v / v) to obtain the product with the structural formula […]. and The extract of Alpinia zerumbet. In some embodiments of the present invention, step S4 is as follows: The mixture B is eluted on an ODS reversed-phase chromatography column with a 10-100% methanol-water solution, and the product obtained by elution with 60-70% methanol-water (v / v) is collected. Then, the mixture is eluted on a silica gel column with cyclohexane and ethyl acetate (v / v) at a ratio of 100:1 to 1:1, and the product obtained by elution with cyclohexane and ethyl acetate (v / v) at a ratio of 3:1 to 1:1 is collected. Finally, the mixture is eluted on a semi-preparative high-performance liquid chromatography column with a 65-70% methanol-water solution, and the eluted product with a retention time of 15-18 min is collected to obtain the product with the structural formula […]. The compound was collected, and the elution products with retention times of 19-22 min were obtained, yielding the compound with the following structural formula: Alpinia galanga extract.

[0018] In some embodiments of the present invention, step S4 is as follows: The mixture C is eluted with a methanol-water solution on an ODS reversed-phase chromatography column, and the product obtained by elution with a methanol-water solution of 60-80% (v / v) is collected. Then, the product is eluted with a methanol-water solution of 60-80% (v / v) on a semi-preparative high-performance liquid chromatography (HPLC) column, followed by elution with a methanol-water solution of 85-95% (v / v) on a semi-preparative HPLC column to obtain the product with the structural formula […]. The extract of Alpinia zerumbet. In some embodiments of the present invention, step S4 is as follows: The mixture C is eluted on an ODS reversed-phase column with a methanol-water solution of 10-100% (v / v), and the product obtained by elution with a methanol-water solution of 60-80% (v / v) is collected. Then, it is eluted in a semi-preparative high-performance liquid chromatography (HPLC) column with a methanol-water solution of 68-72% (v / v), and the eluted product with a retention time of 23-28 min is collected. Then, it is eluted in a semi-preparative HPLC column with a methanol-water solution of 90-95% (v / v), and the eluted product with a retention time of 14-17 min is collected, yielding the product with the structural formula […]. Alpinia galanga extract.

[0019] In some embodiments of the present invention, step S4 is as follows: The mixture D is eluted with an aqueous methanol solution on an ODS reversed-phase chromatography column, and the product obtained by elution with 60-80% methanol-water by volume is collected. Then, the product is eluted with 70-90% methanol-water by volume on a semi-preparative high-performance liquid chromatography column to obtain the product with the following structural formula: The extract of Alpinia zerumbet. In some embodiments of the present invention, step S4 is as follows: The mixture D is eluted on an ODS reversed-phase chromatography column using a methanol-water solution with a volume fraction of 10-100%, and the product obtained by elution with a methanol-water solution with a volume fraction of 65-75% is collected. Then, the product is eluted on a semi-preparative high-performance liquid chromatography column using a methanol-water solution with a volume fraction of 78-82%, and the eluted product with a retention time of 16-18 min is collected to obtain the product with the structural formula […]. Alpinia galanga extract.

[0020] In some embodiments of the present invention, step S4 is as follows: The mixture C is eluted with an aqueous methanol solution on an ODS reversed-phase chromatography column, and the product obtained by elution with 60-80% (v / v) of the aqueous methanol solution is collected. Then, the product is eluted with 65-75% (v / v) of the aqueous methanol solution in a semi-preparative high-performance liquid chromatography (HPLC) column, followed by elution with 85-95% (v / v) of the aqueous methanol solution in a semi-preparative HPLC column to obtain the product with the following structural formula: The extract of Alpinia zerumbet. In some embodiments of the present invention, step S4 is as follows: The mixture C is eluted on an ODS reversed-phase column with a methanol-water solution of 10-100% (v / v), and the product obtained by elution with a methanol-water solution of 65-75% (v / v) is collected. Then, the mixture is eluted on a semi-preparative high-performance liquid chromatography (HPLC) column with a methanol-water solution of 65-75% (v / v), and the eluted product obtained with a retention time of 20-22 min is collected. Then, the mixture is eluted on a semi-preparative HPLC column with a methanol-water solution of 90-95% (v / v), and the eluted product obtained with a retention time of 14-15 min is collected, yielding the product with the structural formula […]. Alpinia galanga extract.

[0021] In some embodiments of the present invention, step S4 is as follows: elute mixture B on an ODS reversed-phase chromatography column using a methanol-water solution, collect the product obtained by elution with 50-70% (v / v) methanol-water solution, and then elute with a 50-70% (v / v) methanol-water solution on a semi-preparative high-performance liquid chromatography column to obtain the product with the following structural formula: The extract of Alpinia zerumbet. In some embodiments of the present invention, step S4 is as follows: The mixture B is eluted on an ODS reversed-phase chromatography column with a methanol-water solution of 10-100% (v / v), and the product obtained by elution with methanol-water solution of 55-65% (v / v) is collected. Then, the mixture is eluted on a semi-preparative high-performance liquid chromatography column with a methanol-water solution of 60-65% (v / v), and the eluted product with a retention time of 26-27 min is collected to obtain the product with the structural formula […]. Alpinia galanga extract.

[0022] In some embodiments of the present invention, step S4 is as follows: The mixture E is eluted with a methanol-water solution on an ODS reversed-phase chromatography column, and the product obtained by elution with 50-80% (v / v) methanol-water solution is collected. Then, the product is eluted with a 60-70% (v / v) methanol-water solution on a semi-preparative high-performance liquid chromatography column to obtain the product with the following structural formula: The extract of Alpinia zerumbet. In some embodiments of the present invention, step S4 is as follows: The mixture E is eluted on an ODS reversed-phase chromatography column with a methanol-water solution of 10-100% (v / v), and the product obtained by elution with a methanol-water solution of 60-70% (v / v) is collected. Then, the mixture is eluted on a semi-preparative high-performance liquid chromatography column with a methanol-water solution of 65-70% (v / v), and the eluted product with a retention time of 24-25 min is collected to obtain the product with the structural formula […]. Alpinia galanga extract.

[0023] In some embodiments of the present invention, step S4 is as follows: Mixture B is eluted on an ODS reversed-phase chromatography column using a methanol-water solution, and the product obtained by elution with 40-70% methanol-water solution (v / v) is collected. Then, the mixture is eluted on a silica gel column using a mixture of cyclohexane and ethyl acetate, and the product obtained by elution with cyclohexane and ethyl acetate in a v / v ratio of 50:1 to 5:1 is collected. Finally, the mixture is eluted on a semi-preparative high-performance liquid chromatography column using 60-70% methanol-water solution (v / v) to obtain the product with the following structural formula: The extract of Alpinia zerumbet. In some embodiments of the present invention, step S4 is as follows: The mixture B is eluted on an ODS reversed-phase chromatography column with a methanol-water solution of 10-100% (v / v), and the product obtained by elution with methanol-water solution of 50-60% (v / v) is collected. Then, the mixture is eluted on a silica gel column with a mixture of cyclohexane and ethyl acetate at a v / v ratio of 100:1-1:3, and the product obtained by elution with cyclohexane and ethyl acetate at a v / v ratio of 50:1-5:1 is collected. Finally, the mixture is eluted on a semi-preparative high-performance liquid chromatography column with a methanol-water solution of 60-65% (v / v), and the eluted product with a retention time of 16-18 min is collected to obtain the product with the structural formula […]. Alpinia galanga extract.

[0024] In some embodiments of the present invention, step S4 is as follows: The mixture F is eluted on an ODS reversed-phase chromatography column using a methanol-water solution, and the product obtained by elution with a methanol-water solution of 40-70% (v / v) is collected. Then, the product is eluted on a semi-preparative high-performance liquid chromatography column using a methanol-water solution of 60-70% (v / v), followed by elution on a semi-preparative high-performance liquid chromatography column using a methanol-water solution of 50-60% (v / v) to obtain the product with the structural formula […]. The extract of Alpinia zerumbet. In some embodiments of the present invention, step S4 is as follows: The mixture F is eluted on an ODS reversed-phase chromatography column with a methanol-water solution of 10-100% (v / v), and the product obtained by elution with a methanol-water solution of 50-60% (v / v) is collected. Then, the mixture is eluted on a semi-preparative high-performance liquid chromatography column with a methanol-water solution of 63-65% (v / v), and the eluted product with a retention time of 18-19 min is collected. Then, the mixture is eluted on a semi-preparative high-performance liquid chromatography column with a methanol-water solution of 50-55% (v / v), and the eluted product with a retention time of 40-45 min is collected, yielding the product with the structural formula […]. Alpinia galanga extract.

[0025] All terpenoid compounds provided in this invention are novel compounds.

[0026] In some embodiments of the present invention, the semi-preparative high-performance liquid chromatography column includes, but is not limited to, the following columns: COSMOSIL 5C18-AR-Ⅱ 10 × 250 mm, COSMOSIL 5C18-MS-Ⅱ 10 × 250 mm, COSMOSILPBr 10 × 250 mm, HPLCONE 5C18A 10 × 250 mm, HPLCONE 5C18A 10 × 250 mm, HPLCONE 5C18C 10 × 250 mm, and HPLCONE 5C18D 10 × 250 mm, etc.

[0027] A third aspect of the invention provides a pharmaceutical composition comprising the Alpinia galanga extract described in the first aspect of the invention and pharmaceutically acceptable excipients.

[0028] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is tablets, capsules, pills, granules, oral solutions, oral suspensions, syrups, injections, sterile powders for injection, aerosols, powder sprays, or sprays.

[0029] In some embodiments of the present invention, the pharmaceutical compositions of the present invention are suitable for multiple routes of administration and can thus be formulated into any pharmaceutically acceptable dosage form. For example, the above-described pharmaceutical compositions can be administered to patients or subjects requiring such treatment via oral, parenteral, rectal, or pulmonary administration. When used for oral administration, the pharmaceutical compositions can be formulated into oral preparations, such as conventional oral solid preparations, like tablets, capsules, pills, granules, etc.; or into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When formulating oral preparations, suitable fillers, binders, disintegrants, lubricants, etc., can be added. When used for parenteral administration, the above-described pharmaceutical compositions can also be formulated into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When formulating injections, conventional methods in the existing pharmaceutical field can be used for production; when preparing injections, excipients may not be added, or suitable excipients may be added depending on the properties of the drug. When used for rectal administration, the pharmaceutical compositions can be formulated into suppositories, etc. When used for pulmonary administration, the pharmaceutical compositions can be formulated into inhaled preparations, aerosols, powder inhalers, or sprays, etc.

[0030] In some embodiments of the invention, pharmaceutically acceptable excipients are substances that are non-toxic, compatible with the active ingredient, and otherwise biologically suitable for use in organisms. The selection of a specific excipient will depend on the route of administration or the type and state of disease for treating a particular patient. Examples of pharmaceutically acceptable excipients include, but are not limited to, solvents, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and / or oils conventional in the pharmaceutical field. The composition may also contain excipients such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavoring agents, and aromatizers.

[0031] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0032] Exemplary granulating agents and / or dispersants include potato starch, corn starch, cassava starch, sodium starch glycolate, clay, alginate, guar gum, citrus pomace, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (cross-linked polyvinylpyrrolidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (cross-linked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0033] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth gum, carrageenan, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, glyceryl triacetate monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxylated polymethylene, polyacrylic acid, acrylic polymers, and carboxyethylene polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (20), poly... ethylene-oxygenated sorbitol (60), polyoxyethylene-oxygenated sorbitol monooleate (80), sorbitol monopalmitate (40), sorbitol monostearate (60), sorbitol tristearate (65), glyceryl monooleate, sorbitol monooleate (80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate), sucrose fatty acid esters, polyethylene glycol fatty acids Esters (e.g.), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether (30)), poly(ethylene-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, F-68, poloxamer P-188, cetrimonium bromide, cetylpyridine chloride, benzalkonium chloride, docusatesodium and / or mixtures thereof.

[0034] Exemplary binders include starch (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., gum arabic, sodium alginate, Irish moss extract, panwar gum, ghatti gum, mucilage of isapola husk, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate, and larch arabinogalactan), alginate, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethyl methacrylate, waxes, water, ethanol, and / or mixtures thereof.

[0035] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoal preservatives, alcohol preservatives, acid preservatives, and other preservatives. In some embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0036] Exemplary antioxidants include alpha-tocopherol, ascorbic acid, ascorbate palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0037] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridine chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerol, hexetidine, imidureurium, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0038] Exemplary buffers include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium fructate, valeric acid, calcium hydrogen phosphate, phosphoric acid, trivalent calcium phosphate, calcium hydroxide, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginate, pyrogen-free raw water, isotonic saline, Ringer's solution, ethanol, and mixtures thereof.

[0039] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0040] Exemplary natural oils include bitter almond oil, almond oil, avocado oil, babassu coconut oil, bergamot oil, blackcurrant seed oil, borage oil, juniper oil, chamomile oil, canola oil, caraway oil, Brazilian wax palm oil, castor oil, cinnamon oil, cocoa butter, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, flaxseed oil, geraniol oil, gourd oil, grapeseed oil, hazelnut oil, hyssop oil, isopropyl myristate, jojoba oil, kukui argan oil, and mixed lavender oil. Lavender oil, lemon oil, litsea cubeba oil, macadamia nut oil, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange oil, orange sea bream oil, palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, camellia oil, peppermint oil, sea buckthorn oil, sesame oil, shea butter, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, ailanthus oil, vetiver oil, walnut oil, and wheat germ oil. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, caprylic triglyceride, cyclomethicone, diethyl sebate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0041] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may also contain inert diluents (such as water or other solvents), solubilizers, and emulsifiers commonly used in the art, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (e.g., cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitol, and mixtures thereof. In addition to inert diluents, oral compositions may include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers. In some embodiments for parenteral administration, the conjugates described herein are mixed with solubilizers (such as alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof).

[0042] A fourth aspect of the present invention provides the use of the Alpinia galanga extract described in the first aspect of the present invention in the preparation of a medicament for the prevention or treatment of inflammatory diseases.

[0043] In some embodiments of the present invention, the inflammatory diseases include dermatitis, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, frozen shoulder, tendinitis, tenosynovitis, tendinitis, myositis, hepatitis, cystitis, nephritis, Sjögren's syndrome, multiple sclerosis, or acute and chronic inflammatory diseases.

[0044] The beneficial effects of this invention are: the extract of Alpinia zerumbet in this invention has a good anti-inflammatory effect, exhibiting good nitric oxide production inhibition activity in lipopolysaccharide-induced BV2 cells or RAW 264.7 cells. Specifically, for lipopolysaccharide-induced BV2 cells, its IC50 value is significantly higher than that of other lipopolysaccharide-induced BV2 cells. 50 The value is 17.89-61.38 μ M; For lipopolysaccharide-induced RAW 264.7 cells, its IC50... 50 The value is 11.69-71.91 μ M. Some compounds showed superior inhibitory activity compared to indomethacin and minocycline. Furthermore, some compounds significantly reduced LPS-induced expression of iNOS and COX-2 proteins in BV2 cells, inhibited the expression of iNOS, IL-1β, and TNF-α at the mRNA level, and significantly suppressed NF-κB. κ The nuclear translocation of B has potential for further development.

[0045] The extraction method in this invention is to extract terpenoid compounds from Alpinia galanga. The extraction method is simple, the extraction reagents are readily available, the extraction steps are simple and easy to operate, and it can achieve large-scale extraction. Attached Figure Description

[0046] Figure 1 This is an X-ray single-crystal diffraction pattern of compound 1 in Example 1.

[0047] Figure 2 The above are the ECD spectra of compounds 2-5 and 7-8 from Example 1.

[0048] Figure 3 The above are the ECD spectra of compounds 9-11 from Example 1.

[0049] Figure 4 This is a graph showing the expression levels of iNOS and COX-2 proteins in LPS-induced BV2 cells of compounds 3-4 from Example 1.

[0050] Figure 5 This is a graph showing the mRNA translation levels of compounds 3-4 from Example 1 in LPS-induced BV2 cells, including iNOS and IL-1β.

[0051] Figure 6 Compound 3 in Example 1 inhibits LPS-induced NF-κB in BV2 cells. κ Test image of B-core translocation.

[0052] Figure 7 Compound 4 in Example 1 inhibits LPS-induced NF-κB in BV2 cells. κ Test image of B-core translocation. Detailed Implementation

[0053] The specific implementation of the present invention will be further described in detail below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased commercially.

[0054] Example 1 This example provides a series of terpenoid compounds from Alpinia galanga, denoted as compounds 1-11. The structural formulas of compounds 1-11 are as follows: .

[0055] This example also provides a method for extracting the above-mentioned terpenoid compounds. The specific extraction steps are as follows: 20 kg of dried rhizomes of Alpinia zerumbet were chopped and extracted by reflux with a 70% (v / v) ethanol aqueous solution for 4 hours each time. The extracts were combined and concentrated until alcohol-free, yielding approximately 13 L of suspension. The suspension was then extracted sequentially with cyclohexane and dichloromethane. The dichloromethane extract was purified by silica gel column chromatography (column size: 90 × 1200 mm) using cyclohexane and ethyl acetate as eluents (sequentially using v / v ratios of 100:1, 25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 1:2). The resulting compounds 1-11 were designated as D1, D2, and D3. The D1, D2, and D3 fractions were obtained by combining similar fractions based on thin-layer chromatography colorimetric analysis. The specific eluent formulations for fractions D1-D3 are shown in Table 1 below.

[0056] Table 1. Eluent ratios for eluted components D1, D2, and D3.

[0057] Fraction D1 was eluted using a small-pore resin column chromatography system with a methanol:water eluent ratio of 50:50-100:0 (column size: 60.8 × 190 mm). The product obtained by elution with an eluent ratio of 85:15 was collected and designated as D11. Fraction D2 was eluted using a methanol:water eluent ratio of 50:50-100:0 (column size: 60.8 × 190 mm). The product obtained by elution with an eluent ratio of 90:10 was collected and designated as D21. Fraction D3 was eluted using a small-pore resin column chromatography system with a methanol:water eluent ratio of 50:50-100:0 (column size: 60.8 × 190 mm). The products obtained by elution with eluents at volume ratios of 60:40, 70:30, 80:20, and 90:10 were collected and denoted as D31, D32, D33, and D34, respectively. The eluent formulations corresponding to D11, D21, D31, D32, D33, and D34 are shown in Table 2 below.

[0058] Table 2. Eluent ratios corresponding to components D11, D21, D31, D32, D33, and D34

[0059] Component D21 was eluted using an ODS reversed-phase column with methanol and water at a volume ratio of 10:90-100:0. The product obtained by elution with methanol and water at a volume ratio of 70:30 was collected. Then, the product was eluted with methanol and water at a volume ratio of 90:10 on a Sephadex LH-20 gel column, followed by elution with methanol and water at a volume ratio of 71:29 on a semi-preparative high-performance liquid chromatography (HPLC) column (HPLCONE 5C8A 10 × 250 mm), and the elution product with a retention time of 23.1 min was collected. Finally, the product was eluted with methanol and water at a volume ratio of 89:11 on a semi-preparative HPLC column (COSMOSIL 5C18-AR-Ⅱ 10 × 250 mm), and the elution product with a retention time of 9.3 min was collected to obtain compound 1.

[0060] Component D21 was eluted on an ODS reversed-phase column using methanol and water at a volume ratio of 10:90-100:0. Products eluted with methanol and water at volume ratios of 60:40 and 70:30 were collected. Then, the eluent was eluted with methanol and water at a volume ratio of 64:36 on a semi-preparative high-performance liquid chromatography column (HPLCONE 5C8A 10 × 250 mm). The eluent with a retention time of 24.7 min was collected to obtain compound 2.

[0061] Component D11 was eluted on an ODS reversed-phase column using methanol and water at a volume ratio of 10:90–100:0. Products eluted with methanol and water at volume ratios of 60:40 and 70:30 were collected. Then, the product was eluted on a silica gel column using cyclohexane and ethyl acetate at a volume ratio of 100:1–1:1. Products eluted with cyclohexane and ethyl acetate at volume ratios of 3:1 and 1:1 were collected. Finally, the product was eluted in a semi-preparative high-performance liquid chromatography (HPLC) column (COSMOSIL 5C18-AR-Ⅱ 10 × 250 mm) using methanol and water at a volume ratio of 68:32. The elution product with a retention time of 16.7 min was collected to give compound 3; the elution product with a retention time of 20.3 min was collected to give compound 4.

[0062] Component D33 was eluted on an ODS reversed-phase column using methanol and water at a volume ratio of 10:90-100:0. The product obtained by elution with methanol and water at a volume ratio of 70:30 was collected. Then, the product was eluted again in a semi-preparative high-performance liquid chromatography (HPLC) column using methanol and water at a volume ratio of 69:31 (column: COSMOSIL 5C18-AR-Ⅱ 10 × 250 mm), and the eluent with a retention time of 25.1 min was collected. Finally, the product was eluted again in a semi-preparative HPLC column using methanol and water at a volume ratio of 92:8 (column: COSMOSIL PBr 10 × 250 mm), and the eluent with a retention time of 15.5 min was collected to obtain compound 5.

[0063] Component D34 was eluted on an ODS reversed-phase column using methanol and water at a volume ratio of 10:90-100:0. The product obtained by elution with methanol and water at a volume ratio of 70:30 was collected. Then, the product was eluted with methanol and water at a volume ratio of 80:20 on a semi-preparative high-performance liquid chromatography column (column: COSMOSIL 5C18-AR-Ⅱ 10 × 250 mm). The eluted product with a retention time of 17.4 min was collected to obtain compound 6.

[0064] Component D33 was eluted on an ODS reversed-phase column using methanol and water at a volume ratio of 10:90-100:0. The product obtained by elution with methanol and water at a volume ratio of 70:30 was collected. Then, the product was eluted again in a semi-preparative high-performance liquid chromatography (HPLC) column using methanol and water at a volume ratio of 69:31 (column: COSMOSIL 5C18-AR-Ⅱ 10 × 250 mm), and the eluent with a retention time of 21.5 min was collected. Finally, the product was eluted again in a semi-preparative HPLC column using methanol and water at a volume ratio of 92:8 (column: COSMOSIL PBr 10 × 250 mm), and the eluent with a retention time of 14.6 min was collected to obtain compound 7.

[0065] Component D11 was eluted on an ODS reversed-phase column using methanol and water at a volume ratio of 10:90-100:0. The product obtained by eluting with methanol and water at a volume ratio of 60:40 was collected. Then, the product was eluted with methanol and water at a volume ratio of 62:38 on a semi-preparative high-performance liquid chromatography column (column: COSMOSIL 5C18-AR-Ⅱ 10 × 250 mm). The eluted product with a retention time of 26.4 min was collected to obtain compound 8.

[0066] Component D32 was eluted on an ODS reversed-phase column using methanol and water at a volume ratio of 10:90-100:0. Products eluted with methanol and water at volume ratios of 60:40 and 70:30 were collected. Then, the product was eluted with methanol and water at a volume ratio of 66:34 on a semi-preparative high-performance liquid chromatography column (column: COSMOSIL 5C18-AR-Ⅱ 10× 250 mm). The eluted product with a retention time of 24.8 min was collected to obtain compound 9.

[0067] Component D11 was eluted on an ODS reversed-phase column using methanol and water at a volume ratio of 10:90–100:0. Products eluted with methanol and water at volume ratios of 50:50 and 60:40 were collected. Then, the product was eluted on a silica gel column using cyclohexane and ethyl acetate at a volume ratio of 100:1–1:3. Products eluted with cyclohexane and ethyl acetate at volume ratios of 50:1, 10:1, and 5:1 were collected. Finally, the product was eluted on a semi-preparative high-performance liquid chromatography column using methanol and water at a volume ratio of 64:36 (column: COSMOSIL 5C18-AR-Ⅱ 10 × 250 mm). The eluted product with a retention time of 17.3 min was collected to give compound 10.

[0068] Component D31 was eluted on an ODS reversed-phase column using methanol and water at a volume ratio of 10:90-100:0. Products eluted with methanol and water at volume ratios of 50:50 and 60:40 were collected. Then, the product was eluted on a semi-preparative high-performance liquid chromatography column using methanol and water at a volume ratio of 64:36 (column: COSMOSIL 5C18-AR-Ⅱ 10 × 250 mm), and the eluent with a retention time of 18.2 min was collected. Finally, the product was eluted on a semi-preparative high-performance liquid chromatography column using methanol and water at a volume ratio of 53:47 (column: HPLCONE 5C8A 10 × 250 mm), and the eluent with a retention time of 42.1 min was collected to obtain compound 11.

[0069] The characterization data of compounds 1-11 are as follows: Compound 1 is a colorless, bulky crystal; high-resolution mass spectrometry indicates a quasi-molecular ion peak. m / z 371.2204, [M+Na] + (calcd. for C 21 H 32 O4Na, 371.2198), the molecular formula is presumed to be C 21 H 32 O4. = + 10.75 (c 0.09, MeOH). By analyzing the proton and carbon spectra of compound 1, 1 H- 1 The structure of compound 1 was determined using HCl COSY, HSQC, DEPT-135, HMBC, NOESY, and single-crystal diffraction data. The NMR spectral data for compound 1 are shown in Table 3 below, and the X-ray single-crystal diffraction pattern is shown below. Figure 1 As shown.

[0070] Compound 2 is a colorless oily substance; high-resolution mass spectrometry indicates a quasi-molecular ion peak. m / z 291.1967, [M + H] + (calcd. for C 18 H 27 O3, 291.1960), the predicted molecular formula is C. 18 H 26 O3. = + 10.00 ( c 0.21, MeOH). By analyzing the proton and carbon spectra of compound 2, 1 H- 1 The structure of compound 2 was determined by ¹H COSY, HSQC, DEPT-135, HMBC, NOESY, and ECD calculations. The ¹H and ¹³C NMR spectra of compound 2 are shown in Table 3 below, and the ECD plot is shown in [Figure 1]. Figure 2 As shown.

[0071] Compound 3 is a colorless oily substance; high-resolution mass spectrometry indicates a quasi-molecular ion peak. m / z 355.1869, [M + Na] + (calcd. for C 20 H 28 O4Na, 355.1885), the molecular formula is presumed to be C 20 H 28 O4. = + 20.07 ( c 0.29, MeOH). By analyzing the proton and carbon spectra of compound 3, 1 H- 1 The structure of compound 3 was determined by ¹H COSY, HSQC, DEPT-135, HMBC, NOESY, and ECD calculations. The ¹H and ¹³C NMR spectra of compound 3 are shown in Table 3 below, and the ECD plot is shown in Figure 4. Figure 2 As shown.

[0072] Compound 4 is a colorless oily substance; high-resolution mass spectrometry indicates a quasi-molecular ion peak.m / z 341.2080, [M + Na] + (calcd. for C 20 H 30 O3Na, 341.2093), the predicted molecular formula is C 20 H 30 O3. = + 25.42 ( c 0.48, MeOH). By analyzing the proton and carbon spectra of compound 4, 1 H- 1 The structure of compound 4 was determined by ¹H COSY, HSQC, DEPT-135, HMBC, NOESY, and ECD calculations. The ¹H and ¹³C NMR spectra of compound 4 are shown in Table 4 below, and the ECD plot is shown in [Figure 1]. Figure 2 As shown.

[0073] Compound 5 is a pale yellow oily substance; high-resolution mass spectrometry indicates a quasi-molecular ion peak. m / z 389.2306, [M+Na] + (calcd. for C 21 H 34 O5Na, 389.23O4), the molecular formula is presumed to be C 21 H 34 O5. = + 32.92 ( c 0.05, MeOH). By analyzing the proton and carbon spectra of compound 5, 1 H- 1 The structure of compound 5 was determined by using HCl COSY, HSQC, DEPT-135, HMBC, NOESY, NMR calculations, and ECD calculations. The NMR spectral data for compound 5 are shown in Table 4 below, and the ECD plot is shown in... Figure 2 As shown.

[0074] Compound 6 is a pale yellow oily substance; high-resolution mass spectrometry indicates a quasi-molecular ion peak. m / z 375.2508, [M+Na] + (calcd. for C 21 H 36 O4Na, 375.2511), the molecular formula is presumed to be C 21 H 36 O4. = + 23.91 ( c 0.41, MeOH). By analyzing the proton and carbon spectra of compound 6,1 H- 1 The structure of compound 6 was determined by ¹H COSY, HSQC, DEPT-135, HMBC, and NOESY spectra. The ¹H and ¹³C NMR spectra of compound 6 are shown in Table 4 below.

[0075] Compound 7 is a white powder; high-resolution mass spectrometry indicates a quasi-molecular ion peak. m / z 343.2253, [M+Na] + (calcd. for C 20 H 32 O3Na, 343.2249), the predicted molecular formula is C 20 H 32 O3. = + 46.27 ( c 0.10, MeOH). By analyzing the proton and carbon spectra of compound 7, 1 H- 1 The structure of compound 7 was determined by ¹H COSY, HSQC, DEPT-135, HMBC, NOESY, and ECD calculations. The ¹H and ¹³C NMR spectra of compound 7 are shown in Table 5 below, and the ECD plot is shown in Figure 6. Figure 2 As shown.

[0076] Compound 8 is a colorless oily substance; high-resolution mass spectrometry indicates a quasi-molecular ion peak. m / z 267.1956, [M+H] + (calcd. for C 16 H 27 O3, 267.1960), the predicted molecular formula is C. 16 H 26 O3. = -7.90 ( c 0.37, MeOH). By analyzing the proton and carbon spectra of compound 8, 1 H- 1 The structure of compound 8 was determined by using HCl COSY, HSQC, DEPT-135, HMBC, NOESY spectra, NMR calculations, and ECD calculations. The NMR spectral data for compound 8 are shown in Table 5 below, and the ECD plot is shown in... Figure 2 As shown.

[0077] Compound 9 is a pale yellow oily substance; high-resolution mass spectrometry indicates a quasi-molecular ion peak. m / z 345.2037, [M+Na] + (calcd. for C19 H 30 O4Na, 345.2042), the predicted molecular formula is C 19 H 30 O4. = + 12.24 ( c 0.34, MeOH). By analyzing the proton and carbon spectra of compound 9, 1 H- 1 The structure of compound 9 was determined by ¹H COSY, HSQC, DEPT-135, HMBC, NOESY, and ECD calculations. The ¹H and ¹³C NMR spectra of compound 9 are shown in Table 5 below, and the ECD plot is shown in Figure 6. Figure 3 As shown.

[0078] Compound 10 is a colorless oily substance; high-resolution mass spectrometry indicates a quasi-molecular ion peak. m / z 251.1657, [MH] - (calcd. for C 15 H 23 O3, 251.1647), the predicted molecular formula is C. 15 H 24 O3. = -31.11 ( c 0.09, MeOH). By analyzing the proton and carbon spectra of compound 10, 1 H- 1 The structure of compound 10 was determined by ¹H COSY, HSQC, DEPT-135, HMBC, NOESY, and ECD calculations. The ¹H and ¹³C NMR spectra of compound 10 are shown in Table 6 below, and the ECD plot is shown in [Figure 1]. Figure 3 As shown.

[0079] Compound 11 is a colorless oily substance; high-resolution mass spectrometry indicates a quasi-molecular ion peak. m / z 261.1826, [M + Na] + (calcd. for C 15 H 26 O2Na, 261.1830), the predicted molecular formula is C 15 H 26 O2. = -10.80 ( c 0.45, MeOH). By analyzing the proton and carbon spectra of compound 11, 1 H- 1The structure of compound 11 was determined by ¹H COSY, HSQC, DEPT-135, HMBC, NOESY, and ECD calculations. The ¹H and ¹³C NMR spectra of compound 11 are shown in Table 6 below, and the ECD plot is shown in [Figure 1]. Figure 3 As shown.

[0080] Using CDCl3 as a solvent, 1 The H-NMR data were obtained at 600 MHz. 13 The C-NMR data were obtained at 150 MHz. The 1H NMR and 1C NMR spectra of compounds 1-3 are shown in Table 3 below.

[0081] Table 3. 1H NMR and 1C NMR data

[0082] Using CDCl3 as a solvent, compounds 4 and 6 1 The H-NMR data were obtained at 400 MHz. 13 The C-NMR data were obtained at 100 MHz for compound 5. 1 The H-NMR data were obtained at 600 MHz. 13 The C-NMR data were obtained at 150 MHz. The 1H NMR and 1C NMR spectra of compounds 4-6 are shown in Table 4 below.

[0083] Table 4. 1H NMR and 1C NMR data

[0084] Using CDCl3 as a solvent, compounds 7 and 9 1 The H-NMR data were obtained at 600 MHz. 13 The C-NMR data were obtained at 150 MHz for compound 8. 1 The H-NMR data were obtained at 400 MHz. 13 The C-NMR data were obtained at 100 MHz. The 1H NMR and 1C NMR spectra of compounds 7-9 are shown in Table 5 below.

[0085] Table 5. 1H NMR and 1C NMR data

[0086] Using CDCl3 as a solvent, compounds 10 and 11 1 The H-NMR data were obtained at 600 MHz. 13The C-NMR data were obtained at 150 MHz. The 1H NMR and 1C NMR spectra of compound 10-11 are shown in Table 6 below.

[0087] Table 6. 1H NMR and 1C NMR data

[0088] Performance testing The in vitro anti-inflammatory activities of 11 compounds extracted from Alpinia zerumbet in Example 1 were tested. Specifically, lipopolysaccharide (LPS)-induced inflammation models of BV2 and RAW 264.7 cells were used. The accumulation of nitrite in the culture medium was measured using the Griess reagent method to assess nitric oxide (NO) production levels and evaluate the in vitro anti-inflammatory activities of the compounds. Furthermore, the toxicity of the compounds to BV2 and RAW 264.7 cells was detected using the MTT assay, with minocycline and indomethacin used as positive controls.

[0089] RAW 264.7 cells were grown at 3 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 hours in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C and 5% CO2. Cells were stimulated with 100 ng / mL LPS, followed by treatment with different concentrations of compounds. After 24 hours of culture at 37°C, the cell-free supernatant was transferred to another 96-well plate and mixed with Griess reagent (containing 1% p-aminobenzenesulfonic acid, 0.1% N-1-naphthylethylenediamine, and 2.5% phosphate solution) at a 1:1 volume ratio. Absorbance was measured at 540 nm using a microplate reader, and the NO concentration in the supernatant was calculated based on the sodium nitrite standard curve. After measuring the NO concentration in the cell culture supernatant, MTT solution (0.5 mg / mL) was added to the original 96-well plate, and the plates were cultured for another 4 hours. Absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated by comparing live cells in the untreated group to determine the cytotoxicity of the test compound.

[0090] BV2 cells were used at a rate of 3 × 10⁻⁶ per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates and cultured for 24 hours in DMEM medium containing 10% FBS and 1% penicillin-streptomycin solution at 37 °C and 5% CO2. Cells were stimulated with 500 ng / mL LPS, followed by treatment with different concentrations of compounds. After 24 hours of culture at 37 °C, the cell-free supernatant was transferred to another 96-well plate and mixed with Griess reagent (containing 1% p-aminobenzenesulfonic acid, 0.1% N-1-naphthylethylenediamine, and 2.5% phosphate solution) at a 1:1 volume ratio. Absorbance was measured at 540 nm using a microplate reader, and the NO concentration in the supernatant was calculated based on the sodium nitrite standard curve. After measuring the NO concentration in the cell culture supernatant, MTT solution (0.5 mg / mL) was added to the original 96-well plate, and the plates were cultured for another 4 hours. Absorbance was measured at 490 nm using a microplate reader. Cell viability was calculated by comparing the viable cells in the untreated group to determine the cytotoxicity of the test compounds. All data were obtained through triplicate experiments. Specific test results are shown in Tables 7 and 8.

[0091] Table 7. Inhibitory effects of compounds 1-11 on NO production in RAW 264.7 cells.

[0092] Table 8. Inhibitory effects of compounds 1-11 on NO production in BV2 cells.

[0093] As shown in Tables 7 and 8, compounds 1-11 in this invention all possess certain anti-inflammatory activities. Among them, they exhibit anti-inflammatory activity against the IC50 generated by NO in RAW264.7 cells. 50 The range is 11.69-71.91. μ M, IC50 for NO production in BV2 cells 50 The range is 17.89-61.38. μ M; Compared with indomethacin, compounds 4, 6, and 7 exhibited superior inhibitory effects on NO production in RAW 264.7 cells. Compared with minocycline, compound 4 showed superior inhibitory effects on NO production in BV2 cells.

[0094] BV2 cells were seeded into 6-well plates and cultured overnight in a CO2 incubator. The cells were then treated with LPS (500 ng / mL) alone or with different concentrations (10, 20, 40 ng / mL). μSamples containing compounds 3 and 4 (M) were treated together for 24 h. Total cellular protein was extracted using RIPA cell lysis buffer. Protein concentration was determined using a BCA protein assay kit. Protein samples were boiled in loading buffer for 5 min, separated by 10% SDS (polyacrylamide gel) electrophoresis, and transferred to a PVDF membrane for electroporation at 100 V for 100 min. The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, and then incubated overnight at 4 °C with the primary antibody iNOS (ab178945) / COX-2 (pro-12375-1-AP). After three washes, the membrane was incubated with the secondary antibody anti-Rabbit (CST-7074p2) for 2 h at room temperature and developed with ECL fluorescence. Specific test results are as follows: Figure 4 As shown. By Figure 4 It was found that compounds 3 and 4 reduced the expression levels of iNOS and COX-2 proteins in LPS-induced BV2 cells in a dose-dependent manner.

[0095] BV2 cells were seeded in 12-well plates and cultured overnight to allow cell adhesion. After adhesion, compound 3 (10, 20, 40 g) was used respectively. μ M) and compound 4 samples (10, 20, 40) μ BV2 cells were treated with M and LPS (500 ng / mL) and cultured for 24 h. Total RNA was then extracted from the samples. After RNA quantification, the RNA was reverse transcribed into cDNA and the cDNA sequence was amplified. Quantitative analysis and detection were performed using a PCR device. Primer information: iNOS sequence: 5'-AGCCAAGCCCTCACCTACTT-3' (F) (SEQ ID NO:1); 5'-GCCT CCAATCTCTGCCTATC-3' (R) (SEQ ID NO:2); COX-2 sequence: 5'-TGAGTACCGCAAACGCTTCTC-3' (F) (SEQ ID NO:3); 5'-TGGACGAGGTTTTTCCACCAG-3' (R) (SEQ ID NO:4); IL-1β sequence: 5'-GTTGACGGACCCCAAAAGAT-3' (F) (SEQ ID NO:5); 5'-CCTCATCCTGGAAGGTCCAC-3' (R) (SEQ ID NO:6); TNF-α sequence: 5'-TATGGCTCAGGGTCCAACTC-3' (F) (SEQ ID NO:1). ID NO:7); 5'-GGAAAGCCCATTTGAGTCCT-3' (R) (SEQ ID NO:8), specific test results are as follows Figure 5 As shown. By Figure 5It was found that in LPS-induced BV2 cells, compound 3 significantly reduced the translation levels of iNOS, IL-1β and TNF-α mRNA, while compound 4 significantly reduced the translation levels of iNOS, IL-1β and COX-2 mRNA.

[0096] BV2 cells were seeded in 48-well plates and treated with LPS (500 ng / mL) alone or with compound 3 (10, 20, 40). μ M) and compound 4 samples (10, 20, 40) μ After 4 h of combined treatment with M), the supernatant was discarded, the mixture was washed with PBS, fixed with 4% paraformaldehyde for 15 min, washed with PBS, treated with 0.1% Triton X-100 solution for 20 min, washed with PBS, blocked with 5% BSA solution for 30 min, washed with PBS, and then incubated with the first antibody p-NF-κB at 4 ℃. κ Incubate overnight with B p65 (sc-166748). After washing three times, incubate with the secondary antibody anti-mouse (Az 1200) for 3 h at room temperature. Nuclear morphology changes (blue fluorescence) and NF-κB staining were observed using Hoechst 33258 staining. κ The distribution of B (green fluorescence), and the specific test results are as follows: Figure 6 and 7 As shown. By Figure 6 and Figure 7 It is known that compound 3 can inhibit LPS-induced NF-κB in BV2 cells. κ Nuclear translocation of B, compound 4 can inhibit LPS-induced NF-κB in BV2 cells. κ nuclear translocation of B.

[0097] In summary, compounds 1-11 in this invention are novel compounds extracted from Alpinia zerumbet, possessing novel structures and good anti-inflammatory activity. Compound 4, in particular, exhibits excellent in vitro anti-inflammatory activity. Compounds 3 and 4 significantly reduce the expression of iNOS and COX-2 proteins in LPS-induced BV2 cells. Compound 3 inhibits the expression of iNOS, IL-1β, and TNF-α at the mRNA level, and compound 4 inhibits the expression of iNOS, IL-1β, and COX-2 at the mRNA level in a dose-dependent manner. Immunofluorescence staining observation shows that compounds 3 and 4 can significantly inhibit NF-α expression. κ The nuclear translocation of B has potential for further development.

[0098] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A kind of ginger extract, characterized in that: Includes terpenoids; said terpenoids are selected from... , , , , , , , , , , At least one of them.

2. The method for preparing the Alpinia zerumbet extract according to claim 1, characterized in that: Includes the following steps: S1: First, extract Alpinia galanga with alcohol, then extract with cyclohexane and dichloromethane in sequence to obtain dichloromethane extract; S2: The dichloromethane extract was eluted on a silica gel column using cyclohexane and ethyl acetate, and the product obtained by elution with cyclohexane and ethyl acetate in a volume ratio of 2:1 to 1:2 was collected to obtain elution product A. S3: The elution product A is purified on a small-pore resin column, with methanol and water in a volume ratio of 50:50-100:0 as the eluent, to obtain elution product B. S4: The elution product B is eluted and purified on an ODS reversed-phase chromatography column using a mixture of methanol and water, followed by column chromatography purification to obtain the Alpinia galanga extract.

3. The method for preparing the extract of Alpinia galanga according to claim 2, characterized in that: The column chromatography purification is performed using at least one of Sephadex LH-20 gel column, semi-preparative high performance liquid chromatography, and silica gel column chromatography. And / or, the column chromatography purification includes the step of elution purification on a semi-preparative high-performance liquid chromatography column using methanol and water in a volume ratio of 50:50-95:

5.

4. The method for preparing the extract of Alpinia galanga according to claim 2, characterized in that: The elution product A includes mixture a, mixture b, and mixture c; The mixture a is the product obtained by elution of cyclohexane and ethyl acetate in a volume ratio of 2:1 to 1:1; The mixture b is the product obtained by elution of cyclohexane and ethyl acetate in a volume ratio of 1:1; The mixture c is the product obtained by elution of cyclohexane and ethyl acetate in a volume ratio of 1:1 to 1:

2.

5. The method for preparing the extract of Alpinia galanga according to claim 4, characterized in that: The elution product B includes mixture A, mixture B, mixture C, mixture D, mixture E, and mixture F; The mixture A is the elution product obtained by eluting mixture b on a small-pore resin column using a methanol aqueous solution with a volume percentage of 88-92%. The mixture B is the elution product obtained by eluting mixture a on a small-pore resin column using a methanol aqueous solution with a volume percentage of 83-87%. The mixture C is the elution product obtained by eluting mixture c on a small-pore resin column with a methanol aqueous solution of 78-82% by volume. The mixture D is the elution product obtained by eluting mixture c on a small-pore resin column with a methanol aqueous solution of 88-92% by volume. The mixture E is the elution product obtained by eluting mixture c on a small-pore resin column with a methanol aqueous solution of 68-72% by volume. The mixture F is the elution product obtained by eluting mixture c on a small-pore resin column using a methanol aqueous solution with a volume percentage of 58-62%.

6. The method for preparing the extract of Alpinia galanga according to claim 5, characterized in that: Step S4 specifically involves: eluting mixture A onto an ODS reversed-phase column using a methanol-water solution, collecting the product obtained from elution with a methanol-water solution of 60-80% (v / v), then eluting it onto a Sephadex LH-20 gel column using a methanol-water solution of 85-95% (v / v), followed by elution onto a semi-preparative high-performance liquid chromatography (HPLC) column using a methanol-water solution of 70-80% (v / v), and finally eluting it onto a second semi-preparative HPLC column using a methanol-water solution of 85-95% (v / v) to obtain the product with the following structural formula: Alpinia galanga extract; Alternatively, step S4 is as follows: Elute mixture A on an ODS reversed-phase column using a methanol-water solution, collect the product obtained by elution with a methanol-water solution of 50-80% (v / v), and then elute with a methanol-water solution of 60-70% (v / v) on a semi-preparative high-performance liquid chromatography (HPLC) column to obtain the product with the following structural formula: Alpinia galanga extract; Alternatively, step S4 is as follows: Elute mixture B on an ODS reversed-phase column using a methanol-water solution, collecting the product obtained by elution with a methanol-water solution at a volume ratio of 50-80%. Then, elute with cyclohexane and ethyl acetate on a silica gel column, collecting the product obtained by elution with cyclohexane and ethyl acetate at a volume ratio of 3:1 to 1:

1. Finally, elute with a methanol-water solution at a volume fraction of 60-80% on a semi-preparative high-performance liquid chromatography (HPLC) column to obtain the product with the following structural formula: and Alpinia galanga extract; Alternatively, step S4 is as follows: Elute mixture C on an ODS reversed-phase column using a methanol-water solution, collect the product obtained by elution with 60-80% methanol-water solution (v / v), then elute with 60-80% methanol-water solution (v / v) on a semi-preparative high-performance liquid chromatography (HPLC) column, and then elute with 85-95% methanol-water solution (v / v) on a semi-preparative HPLC column to obtain the product with the following structural formula: Alpinia galanga extract; Alternatively, step S4 is as follows: Elute mixture D on an ODS reversed-phase column using a methanol-water solution, collect the product obtained from the methanol-water elution with a volume fraction of 60-80%, and then elute it with a methanol-water solution with a volume fraction of 70-90% on a semi-preparative high-performance liquid chromatography (HPLC) column to obtain the product with the following structural formula: Alpinia galanga extract; Alternatively, step S4 is as follows: Elute mixture C on an ODS reversed-phase column using a methanol-water solution, collect the product obtained by elution with a methanol-water solution of 60-80% (v / v), then elute with a methanol-water solution of 65-75% (v / v) on a semi-preparative high-performance liquid chromatography (HPLC) column, and then elute with a methanol-water solution of 85-95% (v / v) on a semi-preparative HPLC column to obtain the product with the following structural formula: Alpinia galanga extract; Alternatively, step S4 is as follows: Elute mixture B on an ODS reversed-phase chromatography column using a methanol-water solution, collect the product obtained by elution with a 50-70% (v / v) methanol-water solution, and then elute with a 50-70% (v / v) methanol-water solution on a semi-preparative high-performance liquid chromatography column to obtain the product with the following structural formula: Alpinia galanga extract; Alternatively, step S4 may be as follows: Elute mixture E on an ODS reversed-phase chromatography column using a methanol-water solution, collect the product obtained from elution with a methanol-water solution of 50-80% (v / v), and then elute with a methanol-water solution of 60-70% (v / v) on a semi-preparative high-performance liquid chromatography column to obtain the product with the following structural formula: Alpinia galanga extract; Alternatively, step S4 is as follows: Elute mixture B on an ODS reversed-phase chromatography column using a methanol-water solution, collecting the product obtained by elution with a methanol-water solution of 40-70% (v / v). Then elute on a silica gel column using a mixture of cyclohexane and ethyl acetate, collecting the product obtained by elution with cyclohexane and ethyl acetate at a v / v ratio of 50:1 to 5:

1. Finally, elute on a semi-preparative high-performance liquid chromatography column using a methanol-water solution of 60-70% (v / v) to obtain the product with the structural formula […]. Alpinia galanga extract; Alternatively, step S4 is as follows: The mixture F is eluted on an ODS reversed-phase column using a methanol-water solution with a volume fraction of 40-70%. The resulting product is then eluted on a semi-preparative high-performance liquid chromatography column using a methanol-water solution with a volume fraction of 60-70%, followed by elution on a semi-preparative high-performance liquid chromatography column using a methanol-water solution with a volume fraction of 50-60%, yielding a product with the following structural formula: Alpinia galanga extract.

7. A pharmaceutical composition, characterized in that: It includes the Alpinia galanga extract as described in claim 1 and pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that: The dosage form of the pharmaceutical composition is tablets, capsules, pills, granules, oral solutions, oral suspensions, syrups, injections, sterile powders for injection, aerosols, powder sprays, or sprays.

9. The use of the Alpinia galanga extract according to claim 1 in the preparation of a medicament for the prevention or treatment of inflammatory diseases.

10. The application according to claim 9, characterized in that: The inflammatory diseases mentioned include dermatitis, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, frozen shoulder, tendinitis, tenosynovitis, tendinitis, myositis, hepatitis, cystitis, nephritis, Sjögren's syndrome, multiple sclerosis, or acute and chronic inflammatory diseases.

Images