CN111514151B - Application of three compounds in myrobalan in preparing anti-inflammatory drugs and preparation method thereof - Google Patents
Application of three compounds in myrobalan in preparing anti-inflammatory drugs and preparation method thereof Download PDFInfo
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Abstract
The invention provides application of a compound with anti-inflammatory activity in terminalia chebula miq in preparing an anti-inflammatory drug and a preparation method thereof. The invention obtains 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose from fresh fruits of Terminalia microcarpa; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; the Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether is prepared into solid, liquid or paste dosage forms by adopting a conventional anti-inflammatory activity evaluation model and adopting a thin-layer chromatography method for guidance and combining other phytochemical research means. The preparation method of the three chemical components is simple and convenient, the yield is high, the purity is excellent, and the obtained compound is a natural secondary metabolite and has obvious anti-inflammatory activity.
Description
The technical field is as follows:
the invention belongs to the field of medicines, and particularly relates to a compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; preparation of Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether, application of Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether in anti-inflammatory pharmaceutical compositions and application of Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether in medicines.
Background art:
the inflammatory reaction can occur in tissues and organs of various parts of the body, is an important immune and epidemic prevention mechanism generated after the body acts on an inflammatory factor, and is helpful for alleviating the infection of pathogenic microorganisms. However, when the inflammatory reaction is disordered, the tissue cells are damaged, so that the tissue cells are degenerated and necrotized, tissue and organ damage is caused, and the disease is promoted. Invasion of pathogenic factors such as avian influenza, SARS and 2019-novel coronavirus can induce over-excitation of inflammatory reaction of organisms, induce cytokine storm, and seriously threaten life safety of human beings. At present, hormone medicines are mostly adopted for clinically inhibiting excessive immune cell activation and cytokine generation, and long-term large-scale hormone treatment easily causes adverse reactions such as body fatness, blood sugar rise, digestive tract ulcer, electrolyte disorder and osteoporosis to further cause femoral head necrosis. The wide exploration and directed excavation of the micromolecular compound with high-efficiency anti-inflammatory activity and low toxic and side effect is a new idea for regulating inflammatory reaction.
Terminalia chebula is a tree of Terminalia of Combretaceae, the fruit of the plant is a traditional common medicine for minority nationalities such as Tibetan, Mongolia, Dai and Uygur in China, has the effects of astringing intestines to stop diarrhea, astringing lung to stop cough, reducing pathogenic fire to relieve sore throat, clearing heat and removing toxicity and the like, and is one of the essential medicines of Tibetan medicine 'three-fruit decoction'. To explore the pharmacological action mechanism of the national essential drug, at present, the related research results have reported that 110 compounds are separated from the fruits of myrobalan plants, including
1,2,3,4,6-penta-O-galloyl- β -D-glucopyranose; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; erythro-guaiac-glycerol-beta-coniferyl alcohol ether, but the application of the compound in anti-inflammatory activity and medicaments thereof is not researched and reported, and no related preparation method is reported.
The invention content is as follows:
the invention aims to provide 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; preparation of Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether, application of Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether in anti-inflammatory drug composition and application of Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether in preparation of anti-inflammatory drugs.
In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:
a compound 1,2,3,4,6-penta-O-galloyl- β -D-glucopyranose represented by the following structural formula; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; the application of Erythro-guaiacyl-glycerol-beta-coniferyl aldehyde ether in preparing anti-inflammatory drugs,
1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose
4-O-(3”,4”-di-O-galloyl-α-L-rhamnopyranosyl)ellagic acid
Erythro-guaiacyl-glycerol-β-coniferyl aldehyde ether
the compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; the Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether is derived from Terminalia and other plant fruits and other parts of plants containing the compound, and has remarkable anti-inflammatory activity.
The compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; application of Erythro-guaiacyl-glycerol-beta-coniferyl aldehyde ether in preparing medicines for preventing or treating inflammatory diseases.
The compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; application of Erythro-guaiac-glycerol-beta-coniferyl alcohol ether in preparing a medicament for preventing or treating superficial and deep inflammation.
The compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; a pharmaceutical composition consisting of Erythro-guaiacyl-glycerol-beta-coniferyl aldehyde ether and a pharmaceutically acceptable carrier.
The application of the pharmaceutical composition in preparing a medicament for treating inflammatory diseases and the application thereof in preparing a medicament for preventing or treating superficial and deep inflammations.
The 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; the Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether is prepared by separating and purifying the natural product from the Terminalia genus and other plant fruits and other parts of the plant containing the compound. The method specifically comprises the steps of crushing fresh fruits and other plant materials of any one plant containing the above three compounds, such as Terminalia chebula, Terminalia chebula and the like, cold-soaking and extracting for 3 times at room temperature by using 70% acetone aqueous solution with the volume being 3 times that of the plant, filtering for 5 days each time, concentrating the filtrate under reduced pressure at 50 ℃ to remove an organic solvent, carrying out Diaion HP-20 column chromatography on the residual aqueous solution under the detection guidance of thin layer chromatography, and carrying out gradient elution by using 35% and 60% methanol/water volume ratio solution to obtain Fr. 35 ，Fr. 60 。Fr. 35 Loading on Sephadex LH-20 chromatographic column with MeOH/H 2 Eluting with 80% and 90% of O by volume, wherein the elution part with 90% of methanol/water by volume is subjected to preparative liquid phase chromatography, eluting with a mobile phase containing 20% of acetonitrile + 0.15% of trifluoroacetic acid/water by volume to obtain a compound 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid, and detecting the content by HPLC, wherein the purity is not less than 90% and the yield is 0.03% of the dry weight; performing MCI-gel CHP20P column chromatography on an elution part with the methanol/water volume ratio of 90%, then performing liquid phase column chromatography on the elution part, eluting the elution part in acetonitrile and a mobile phase with the trifluoroacetic acid/water volume ratio of 0.15% of 22% to obtain a compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose, and detecting the content by HPLC, wherein the purity is more than or equal to 90% and the yield is 0.02% of the dry weight; fr. 60 Eluting with MCI-gel CHP20P chromatography column with 20-100% methanol/water volume ratio solution, wherein the methanol/water volume ratio is 60-70%, concentrating, subjecting to silica gel column chromatography, eluting with chloroform/methanol volume ratio of 15: eluting with the solution of 1 to obtain a compound Erythro-guaiac-glycerol-beta-coniferyl alcohol ether, and detecting the content of the compound Erythro-guaiac-glycerol-beta-coniferyl alcohol ether by HPLC to obtain the product with the purity of more than or equal to 90 percentThe rate was 0.03% of dry weight.
The compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; evaluation of anti-inflammatory Activity of Erythro-guaiacyl-glycerol-beta-coniferyl aldehyde ether A mouse monocyte macrophage strain RAW264.7 was inoculated in a 96-well plate at an inoculation density of 1.5X 10 5 Dissolving compounds 1-3 in DMSO solvent respectively, adding into cell culture medium to give final concentration of 50uM and continuous gradient concentration, treating cells with 3 times of each treatment, stimulating with botulinum toxin LPS with final concentration of 1 μ g/mL, evaluating generation of nitrogen monoxide in supernatant after 18 hr with Griess reagent, detecting absorbance at 570nm, and calculating half inhibitory concentration IC of each compound by Reed-Muench method 50 A value; at the same time, the half-inhibitory concentration IC of the nitric oxide synthase inhibitor L-NMMA is adopted 50 47.34 ± 0.66 μ M as a positive control.
The compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose of the invention; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; erythro-guaiacyl-glycerol- β -coniferyl alcohol ether or a salt thereof may be administered orally or parenterally, and the amount administered varies depending on the drug, and is preferably 1 to 20mg per day for adults.
For oral administration, the compound is first mixed with conventional pharmaceutical adjuvants such as excipient, disintegrant, binder, lubricant, antioxidant, coating agent, colorant, aromatic agent, surfactant, etc., and made into granule, capsule, tablet, etc.; the non-oral administration can be in the form of injection, infusion solution, suppository or liniment. In preparing the above formulation, conventional formulation techniques may be used.
The specific implementation mode is as follows:
the invention will be further illustrated by the following examples which are intended to be purely exemplary of the preferred embodiments of the invention and are not intended to limit the scope of protection of the invention in any way.
Example 1:
1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose, its anti-inflammatory medicinal composition and its application in medicine.
1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose
The method comprises the following steps: preparation of 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose:
50kg of fresh fruits of Terminalia chebula Retz, which is produced by Yongde, is crushed, is extracted by cold soaking for 3 times at room temperature by using acetone aqueous solution with the volume being 3 times of that of the fresh fruits of Terminalia chebula Retz for 5 days each time, is filtered, the filtrate is decompressed and concentrated at 50 ℃ to remove the organic solvent, the residual aqueous solution is subjected to Diaion HP-20 column chromatography, and is subjected to gradient elution by using solution with the volume ratio of methanol to water being 35 percent and 60 percent to obtain Fr. 35 ，Fr. 60 。Fr. 35 Loading on Sephadex LH-20 chromatographic column with MeOH/H 2 Eluting with 80% and 90% of O by volume, performing MCI-gel CHP20P column chromatography on the 90% methanol/water by volume elution part, performing preparative liquid phase column chromatography, eluting with acetonitrile + 0.15% trifluoroacetic acid/water by volume of 22% mobile phase to obtain 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose, detecting by HPLC content, the purity is not less than 90%, and the yield is 0.02% of dry weight
Step two: evaluation of anti-inflammatory Activity of 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose
Inoculating mouse mononuclear macrophage strain RAW264.7 into 96-well plate at the inoculation density of 1.5 × 10 5 Dissolving compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose in DMSO solvent, adding into cell culture medium to make compound final concentration 50uM and continuous gradient concentration, performing cell treatment, each treatment is 3 times repeated, stimulating with botulinum toxin LPS with final concentration of 1 μ g/mL, evaluating generation of nitrogen monoxide in supernatant with Griess reagent after 18 hours, detecting absorption value at 570nm, calculating half inhibitory concentration IC of each compound by Reed-Muench method 50 36.43 ± 0.21 μ M; simultaneously, using oxygenHalf inhibitory concentration IC of nitric oxide synthase inhibitor L-NMMA 50 47.34 ± 0.66 μ M as a positive control. The nitric oxide generation inhibition activity of the compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose is obviously better than that of L-NMMA.
Example 2
4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid, its anti-inflammatory pharmaceutical composition, and its application in medicine are provided.
4-O-(3”,4”-di-O-galloyl-α-L-rhamnopyranosyl)ellagic acid
The method comprises the following steps: preparation of 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid
50kg of fresh fruits of Terminalia chebula Retz, which is produced by Yongde, is crushed, is extracted by cold soaking for 3 times at room temperature by using acetone aqueous solution with the volume being 3 times of that of the fresh fruits of Terminalia chebula Retz for 5 days each time, is filtered, the filtrate is decompressed and concentrated at 50 ℃ to remove the organic solvent, the residual aqueous solution is subjected to Diaion HP-20 column chromatography, and is subjected to gradient elution by using solution with the volume ratio of methanol to water being 35 percent and 60 percent to obtain Fr. 35 ，Fr. 60 。Fr. 35 Loading on Sephadex LH-20 chromatographic column with MeOH/H 2 And (3) eluting with 80% and 90% of O by volume, wherein the elution part with 90% of methanol/water by volume is subjected to preparative liquid phase chromatography, is eluted by a mobile phase with 20% of acetonitrile + 0.15% of trifluoroacetic acid/water by volume, and the eluent is concentrated and dried to obtain the compound 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid, wherein the purity is more than or equal to 90% by HPLC content detection, and the yield is 0.03% of the dry weight.
Step two: evaluation of anti-inflammatory Activity of 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid
Inoculating mouse mononuclear macrophage strain RAW264.7 into 96-well plate at the inoculation density of 1.5 × 10 5 The compound 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid is dissolved in DMSO solvent and added to the cell culture medium to give a final concentration of 50uM and its continuous ladderConcentration, cell treatment was then carried out, 3 replicates for each treatment, while stimulating with botulinum toxin LPS at a final concentration of 1. mu.g/mL, 18 hours later the nitric oxide production in the supernatant was evaluated using Griess reagent, absorbance was measured at 570nm, and the half inhibitory concentration IC of each compound was calculated using Reed-Muench method 50 42.28 ± 0.09 μ M; at the same time, the half-inhibitory concentration IC of the nitric oxide synthase inhibitor L-NMMA is adopted 50 47.34 ± 0.66 μ M as a positive control. The nitric oxide generation inhibiting activity of the compound 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyrano-syl) ellagic acid is superior to that of L-NMMA.
Example 3
Preparation of Erythro-guaiac-glycerol-beta-coniferyl alcohol ether, anti-inflammatory pharmaceutical composition thereof, and application thereof in medicine.
Erythro-guaiacyl-glycerol-β-coniferyl aldehyde ether
The method comprises the following steps: preparation of Erythro-guaiac-glycerol-beta-coniferyl aldehyde ether
50kg of fresh fruits of Terminalia chebula Retz, which is produced by Yongde, is crushed, is extracted by cold soaking for 3 times at room temperature by using acetone aqueous solution with the volume of 3 times and 70 percent, each time is 5 days, the filtration is carried out, the filtrate is decompressed and concentrated at 50 ℃ to remove the organic solvent, the residual aqueous solution is processed by Diaion HP-20 column chromatography under the detection guidance of thin layer chromatography, and gradient elution is carried out by using solution with the volume ratio of methanol to water of 35 percent and 60 percent to obtain Fr. 35 ，Fr. 60 。Fr. 60 Eluting with MCI-gel CHP20P chromatography column with 20-100% methanol/water volume ratio solution, wherein the methanol/water volume ratio is 60-70%, concentrating, subjecting to silica gel column chromatography, eluting with chloroform/methanol volume ratio of 15: the compound Erythro-guaiac-glycerol-beta-coniferyl alcohol ether is obtained by eluting the solution of 1, and the purity is more than or equal to 90 percent and the yield is 0.03 percent of the dry weight by HPLC content detection.
Step two: evaluation of anti-inflammatory Activity of Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether
Inoculating mouse mononuclear macrophage strain RAW264.7 into 96-well plate at the inoculation density of 1.5 × 10 5 Dissolving Erythro-guaiac-glycerol-beta-coniferyl alcohol ether in DMSO solvent, adding into cell culture medium to give compound final concentration of 50uM and continuous gradient concentration, treating cells with 3 times of each treatment, stimulating with botulinum toxin LPS (1 μ g/mL) for 18 hr, evaluating the generation of nitrogen monoxide in the supernatant with Griess reagent, detecting absorbance at 570nm, and calculating half inhibitory concentration IC of each compound by Reed-Muench method 50 18.17 ± 0.57 μ M; at the same time, the half-inhibitory concentration IC of the nitric oxide synthase inhibitor L-NMMA is adopted 50 47.34 ± 0.66 μ M as a positive control. The inhibiting activity of the compound Erythro-guaiacyl-glycerol-beta-coniferyl aldehyde ether on the generation of nitric oxide is remarkably superior to that of L-NMMA.
Formulation example 1:
the compound 1,2,3,4, 6-penta-O-galloyl-. beta. -D-glucopyranose was prepared according to the procedure of examples 1-3; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid; erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether. Adding water for injection, fine filtering, bottling, and sterilizing to obtain injection.
Formulation example 2:
the compound 1,2,3,4,6-penta-O-galloyl- β -D-glucopyranose was prepared according to the procedure of examples 1 to 3; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid;
erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether is dissolved in sterile water for injection, stirred to be fully dissolved, filtered by a sterile suction filter funnel, then sterile and fine filtered, subpackaged in 2 ampoules, frozen and dried at low temperature, and then sterile melt-sealed to obtain the powder injection.
Formulation example 3:
the compound 1,2,3,4, 6-penta-O-galloyl-beta-D-glucopyranose was prepared according to the method of examples 1-3; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid;
adding excipient into Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether and the excipient in the weight ratio of 9:1, and preparing into powder.
Formulation example 4:
the compound 1,2,3,4,6-penta-O-galloyl- β -D-glucopyranose was prepared according to the procedure of examples 1 to 3; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid;
adding excipient into Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether according to the weight ratio of the Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether to the excipient of 1:5-1:10, granulating and tabletting.
Formulation example 5:
the compound 1,2,3,4,6-penta-O-galloyl- β -D-glucopyranose was prepared according to the procedure of examples 1 to 3; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid;
erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether, or making into oral liquid by conventional oral liquid preparation method.
Formulation example 6:
the compound 1,2,3,4,6-penta-O-galloyl- β -D-glucopyranose was prepared according to the procedure of examples 1 to 3; 4-O- (3 ', 4' -di-O-galloyl-alpha-L-rhamnopyranosyl) ellagic acid;
adding excipient, smearing agent or cleaning agent into Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether according to the weight ratio of the Erythro-guaiacyl-glycerol-beta-coniferyl alcohol ether to the excipient of 5: 1.
Formulation example 7:
the procedure of examples 1 to 3 was followed to prepare 1,2,3,4, 6-penta-O-galloyl-D-glucopyranose compound; 4-O- (3 ', 4' -di-O-galloyl-L-rhodopyranyl) ellagic acid; adding excipient into Erythro-guaiacyl-glycerol-coniferyl alcohol ether according to the weight ratio of the Erythro-guaiacyl-glycerol-coniferyl alcohol ether to the excipient of 3:1, and preparing into smearing preparation or cleaning agent.
Claims (6)
1. The application of the compound 2 or the compound 3 shown in the following structural formula in preparing anti-inflammatory drugs,
2. use according to claim 1, wherein the compound is derived from a secondary metabolite of a fresh plant fruit of the plant Terminalia microcarpa.
3. The use of claim 1, wherein the inflammation is superficial and deep inflammation.
4. A process for preparing compound 2 or compound 3 represented by the following structural formula, comprising the steps of: crushing fresh fruits of Terminalia microcarpa, extracting at room temperature for 5 days by cold soaking with 3 times volume of 70% acetone aqueous solution for 3 times, filtering, concentrating the filtrate at 50 deg.C under reduced pressure to remove organic solvent, subjecting the residual aqueous solution to Diaion HP-20 column chromatography under the detection guidance of thin layer chromatography, and performing gradient elution with 35% methanol/water and 60% solution to obtain Fr. 35 ，Fr. 60 ；Fr. 35 Loading on Sephadex LH-20 chromatographic column with MeOH/H 2 Eluting with 80% and 90% of O by volume, wherein the 90% of methanol/water by volume is subjected to preparative liquid column chromatography, eluting with mobile phase containing acetonitrile and 0.15% trifluoroacetic acid/water by volume of 20% to obtain compound 2, and detecting by HPLC content to obtain compound 2 with purity of more than or equal to 90%; fr. 60 Eluting with MCI-gel CHP20P chromatography column with 20-100% methanol/water volume ratio solution, wherein the methanol/water volume ratio is 60-70%, concentrating, subjecting to silica gel column chromatography, eluting with chloroform/methanol volume ratio of 15: eluting with the solution of 1 to obtain a compound 3, and detecting the content of the compound 3 by HPLC, wherein the purity of the compound is more than or equal to 90%;
5. the application of a pharmaceutical composition consisting of a compound 2 or a compound 3 shown in the following structural formula and a medicinal carrier in preparing anti-inflammatory drugs,
6. the use of claim 5, wherein the inflammation is superficial and deep inflammation.
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