CN114984064B - Morchella fruiting body active site and its preparation method and application - Google Patents
Morchella fruiting body active site and its preparation method and application Download PDFInfo
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- CN114984064B CN114984064B CN202210720545.4A CN202210720545A CN114984064B CN 114984064 B CN114984064 B CN 114984064B CN 202210720545 A CN202210720545 A CN 202210720545A CN 114984064 B CN114984064 B CN 114984064B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the field of microorganisms and fungi, and particularly relates to a Morchella sporophore active site, a preparation method and application thereof. The invention aims to provide a new active part of Morchella, which has the effect of treating ulcerative colitis, and the preparation method comprises the steps of A, taking Morchella fruit bodies, crushing, decocting in water, and collecting decoction; B. the decoction obtained in the step A is concentrated to obtain water extract; C. b, adding water into the water extract obtained in the step B for dispersion to obtain a suspension, adding petroleum ether for extraction, and collecting petroleum ether extract; D. adding dichloromethane into the residual aqueous solution in the step C for extraction, collecting dichloromethane extract, concentrating to dryness to obtain dichloromethane extract; and adding ethyl acetate into the residual aqueous solution for extraction, collecting ethyl acetate methane extract, concentrating to dryness, and obtaining ethyl acetate extractum. Eluting the dichloromethane extract and the ethyl acetate extract by a silica gel column to obtain dichloromethane parts A-D and ethyl acetate parts E, G.
Description
Technical Field
The invention belongs to the field of microorganisms and fungi, and particularly relates to a Morchella sporophore active site, a preparation method and application thereof.
Background
Morchella (Morchella) belongs to the fungus kingdom, ascomycotina, pantoea (Discomycetes), pantoea (Pezizales), morchelidae (Morchella) and was described in "Bencaogang mu" written by Ming Dynasty of Litsea (published in 1590). The appearance of the bamboo hat is similar to that of Morchella, so that the bamboo hat is named as Morchella, bamboo hat, corn, sparrow, morchella, etc. All varieties of Morchella are rare edible and medicinal fungi, have delicious taste and rich nutrition, contain various active substances such as amino acids, proteins, vitamins, polysaccharides and the like, have higher medicinal value, and have long history in China. Modern researches have proved that Morchella esculenta has effects of protecting cardiovascular and cerebrovascular, reducing blood lipid, regulating immunity, resisting oxidation, resisting bacteria and tumor, and protecting liver and kidney. Because of difficulty in artificial propagation of Morchella, few researches on chemical components of Morchella are reported at present, and researches on mycelium and Morchella polysaccharide are concentrated.
The research of chemical components of Morchella esculenta in the academic paper [ Tu Xiaoman ] discloses that the antioxidant activity of petroleum ether cold-leaching extract of Morchella esculenta is examined by taking Morchella esculenta as a research object, and ethyl acetate is adopted for extraction from ethanol extract, and the obtained ethyl acetate part is separated and purified by various chromatographic techniques (positive and reverse phase silica gel column chromatography, sephadex column chromatography, preparation of thin layer chromatography) and recrystallization and other methods to obtain a plurality of monomer compounds. The same technical content is also recorded in Bolus chemical composition of Morchella (journal of fungus society) scribbled Xiaoman, xie Guangbo, tang Lan, deng Kejun, xie Liyuan published by the same authors.
The active site prepared by the invention and the report on the efficacy of the active site in treating ulcerative colitis are not seen at present.
Disclosure of Invention
The invention solves the technical problem of providing a novel active site of Morchella, which has the effect of treating ulcerative colitis.
The preparation method of the novel active site of the Morchella esculenta fruiting body comprises the following steps:
A. pulverizing Morchella fruit body, decocting in water, and collecting decoction;
B. the decoction obtained in the step A is concentrated to obtain water extract;
C. b, adding water into the water extract obtained in the step B for dispersion to obtain a suspension, adding petroleum ether for extraction for multiple times until the petroleum ether extract is clear, collecting and combining to obtain the petroleum ether extract and an aqueous solution after petroleum ether extraction;
D. adding dichloromethane into the aqueous solution subjected to petroleum ether extraction in the step C for multiple extraction until the dichloromethane extract is clear, collecting and combining the dichloromethane extracts, and concentrating to dryness to obtain dichloromethane extract; adding ethyl acetate into the rest aqueous solution for multiple extraction until ethyl acetate extract liquid is clear, collecting and combining ethyl acetate methane extract liquid, concentrating to dryness to obtain ethyl acetate extract;
E. dissolving the dichloromethane extract obtained in the step D with dichloromethane/methanol, adding silica gel, drying the solvent, and grinding uniformly to obtain sample-mixing silica gel; adding the sample-mixed silica gel into a silica gel column, eluting with a dichloromethane-petroleum ether solution with the volume ratio of 3-8:7-2 in sequence, and collecting the eluent; eluting with methanol, and collecting eluate; obtaining a dichloromethane part A, a dichloromethane part B, a dichloromethane part C and a dichloromethane part D;
F. dissolving the ethyl acetate extract obtained in the step D with ethyl acetate/methanol fully, adding silica gel, drying the solvent, and grinding uniformly to obtain sample-mixing silica gel; adding the sample-mixed silica gel into a silica gel column, eluting with ethyl acetate/petroleum ether solution with the volume ratio of 3-8:7-2 in sequence, and collecting eluent; eluting with methanol, and collecting eluate; ethyl acetate fraction E and ethyl acetate fraction G were obtained.
The new active site of Morchella esculenta fruiting body comprises at least one of the residual aqueous solution of step C, the dichloromethane extract of step D, the ethyl acetate extract of step D, the dichloromethane part A of step E, the dichloromethane part B of step E, the dichloromethane part C of step E, the dichloromethane part D of step E, the ethyl acetate part E of step F, and the ethyl acetate part G of step F or their mixture.
When in alternative use, the method comprises the following steps: the aqueous solution extracted by petroleum ether, which can be prepared by the preparation method of the invention, is the active site of Morchella sporophore;
or the dichloromethane extract prepared by the preparation method is the active part of the Morchella fruit body;
or the ethyl acetate extractum prepared by the preparation method is the active part of the Morchella sporophore;
or the methylene dichloride part A prepared by the preparation method is the active part of the Morchella sporophore;
or the methylene dichloride part B prepared by the preparation method is the active part of the Morchella sporophore;
or the methylene dichloride part C prepared by the preparation method is the active part of the Morchella sporophore;
or the methylene dichloride part D prepared by the preparation method is the active part of the Morchella sporophore;
or the ethyl acetate part E prepared by the preparation method is the active part of Morchella sporophore;
or the ethyl acetate part G prepared by the preparation method is the active part of Morchella sporophore.
The technical scheme is as follows:
and (C) drying the Morchella fruit bodies in an oven at 60 ℃ until the weight of the Morchella fruit bodies is constant.
The crushing in the step A is crushing into coarse powder.
Wherein, the coarse powder can pass through the second sieve completely, but is mixed with powder which can pass through the fourth sieve by no more than 40 percent.
The water adding and decocting condition in the step A is that 14-20 times of water is added, and the water is decocted for 2-5 times, and each time lasts for 1-2.5 hours; the preferred water-adding decoction conditions are as follows: adding 16 times of water, and decocting for 3 times each for 1.5 hr.
The concentration in the step B is reduced pressure concentration.
C, adding water to disperse, wherein the water adding amount is 6-10 times of that of the step C; preferably, the water addition amount of the water dispersion in the step C is 8 times of the water addition amount.
The adding amount of petroleum ether in the step C is 0.8-1.2 times of that of the aqueous solution in each extraction; preferably, the petroleum ether of step C is added in an amount of 1 time of the aqueous solution per extraction.
The addition amount of dichloromethane in the step D is 0.8-1.2 times of the amount of the residual aqueous solution in the step C; preferably, the addition of step D dichloromethane per extraction is 1 time the amount of aqueous solution remaining in step C.
The addition amount of ethyl acetate in each extraction is 0.8-1.2 times of the amount of the water solution remained after the extraction of dichloromethane; preferably, the ethyl acetate added per extraction in step D is 1 times the amount of aqueous solution remaining after the dichloromethane extraction.
Step E, the dichloromethane/methanol is dichloromethane/methanol solution with the volume ratio of 1-2:2-1; the method comprises the step E, wherein the dichloromethane/methanol is a dichloromethane/methanol solution with the volume ratio of any one of 1:1, 1:2 and 2:1; preferably, the dichloromethane/methanol in the step E is a dichloromethane/methanol solution with the volume ratio of 1:1.
In the step E, in the sample mixing silica gel, the weight ratio of the dichloromethane extract to the silica gel is 1-1:1-3; the method comprises the step E of mixing the sample with silica gel, wherein the weight ratio of dichloromethane extract to the silica gel is any one of 1:1, 1:2 and 1:3; preferably, in the sample-mixing silica gel in the step E, the weight ratio of the dichloromethane extract to the silica gel is 1:2.
E, the silica gel in the sample mixing silica gel is 100-400 meshes; the method comprises the step E that the silica gel in the sample mixing silica gel is any one of 100-200 meshes, 200-300 meshes and 300-400 meshes; preferably, the silica gel in the sample mixing silica gel in the step E is 200-300 meshes.
In the step E, the weight of the silica gel in the silica gel column is 5-20 times of that of the sample-mixed silica gel; preferably, the silica gel in the silica gel column in the step E is 10 times of the weight of the sample-mixed silica gel.
E, the silica gel in the silica gel column is 200-600 meshes; the method comprises the step E that silica gel in a silica gel column has any particle size of 200-300 meshes, 300-400 meshes and 400-600 meshes; preferably, the silica gel in the silica gel column in the step E is 400-600 meshes.
In the step E, the eluent of the dichloromethane part A is a 3:7 dichloromethane-petroleum ether solution, and the eluent of the dichloromethane part B is a 5:5 dichloromethane-petroleum ether solution; the eluent of the dichloromethane part C is 8:2 dichloromethane-petroleum ether solution; the eluent of the dichloromethane part D is methanol solution.
The ratio of the eluent amount and the dichloromethane extract amount of each part in the step E is as follows: the eluent adopted by each 50g of dichloromethane extract is 1-5L. Preferably, in the step E, the ratio of the eluent amount of each part to the dichloromethane extract amount is: the eluent used per 50g of dichloromethane extract was 3L.
The ethyl acetate/methanol in the step F is ethyl acetate/methanol solution with the volume ratio of 1-1:1-3; the method comprises the step F, wherein the ethyl acetate/methanol is an ethyl acetate/methanol solution with the volume ratio of any one of 1:1, 1:2 and 1:3; preferably, the ethyl acetate/methanol in the step F is ethyl acetate/methanol solution with the volume ratio of 1:1.
In the step F, the weight ratio of the ethyl acetate extract to the silica gel is 1-1:1-3; the method comprises the step F of mixing the sample with silica gel, wherein the weight ratio of the ethyl acetate extract to the silica gel is any one of 1:1, 1:2 and 1:3; preferably, in the sample-mixing silica gel in the step F, the weight ratio of the ethyl acetate extract to the silica gel is 1:2.
F, the silica gel in the sample mixing silica gel is 100-400 meshes; the method comprises the step F that the silica gel in the sample mixing silica gel is any one of 100-200 meshes, 200-300 meshes and 300-400 meshes; preferably, the silica gel in the sample mixing silica gel in the step F is 200-300 meshes.
The weight of the silica gel in the silica gel column in the step F is 5-20 times of that of the sample-mixed silica gel; preferably, the silica gel in the silica gel column in the step F is 10 times of the weight of the sample-mixed silica gel.
F, the silica gel in the silica gel column is 200-600 meshes; the method comprises the step of setting the particle size of silica gel in a silica gel column to any one of 200-300 meshes, 300-400 meshes and 400-600 meshes; preferably, the silica gel in the silica gel column in the step F is 400-600 meshes.
The eluent of the ethyl acetate part E in the step F is ethyl acetate with the ratio of 3:7: petroleum ether solution; the eluent of the ethyl acetate part F is ethyl acetate to petroleum ether solution in a ratio of 5:5; the eluent of the ethyl acetate part G is ethyl acetate-petroleum ether solution with the ratio of 8:2; the eluent of the ethyl acetate part H is methanol solution. The effective active sites obtained by the invention are an ethyl acetate site E and an ethyl acetate site G.
The ratio of the eluent amount and the ethyl acetate extract amount of each part in the step F is as follows: the eluent adopted by each 50g of ethyl acetate extract is 1-5L. Preferably, the ratio of the eluent amount to the ethyl acetate extract amount of each part in the step F is: the eluent adopted for each 50g of ethyl acetate extract is 3L.
The second technical problem to be solved by the invention is to provide a new active site of Morchella sporophore, which comprises at least one of the residual water solution in the step C, the dichloromethane extract obtained in the step D, the ethyl acetate extract obtained in the step D, the dichloromethane part A obtained in the step E, the dichloromethane part B obtained in the step E, the dichloromethane part C obtained in the step E, the dichloromethane part D obtained in the step E, the ethyl acetate part E obtained in the step F and the ethyl acetate part G obtained in the step F or the mixture thereof.
The active site of the Morchella esculenta fruiting body is an aqueous solution extracted by petroleum ether prepared by the method.
The active site of the Morchella esculenta fruiting body is dichloromethane extract prepared by the method.
The active site of the Morchella esculenta fruiting body is the ethyl acetate extract obtained by the preparation method.
The active site of the Morchella esculenta fruiting body is a methylene dichloride site A obtained by the preparation method.
The active site of the Morchella fruit body is a methylene dichloride site B obtained by adopting the preparation method.
The active site of the Morchella esculenta fruiting body is a methylene dichloride site C obtained by the preparation method.
The active site of the Morchella esculenta fruiting body is a methylene dichloride site D obtained by the preparation method.
The active site of the Morchella esculenta fruiting body is the ethyl acetate site E obtained by the preparation method.
The active site of the Morchella fruit body is an ethyl acetate site G obtained by the preparation method
The third technical problem to be solved by the invention is to provide a derivative product of an active site of Morchella fruit body, which is an oral preparation prepared by adding pharmaceutically or health-care acceptable auxiliary materials or auxiliary components into at least one of aqueous solution after petroleum ether extraction, dichloromethane extract, ethyl acetate extract, dichloromethane site A, dichloromethane site B, dichloromethane site C, dichloromethane site D, ethyl acetate site E and ethyl acetate site G or any mixture thereof as an active component.
The fourth technical problem to be solved by the invention is to provide the use of at least one of aqueous solution after petroleum ether extraction, dichloromethane extract, ethyl acetate extract, dichloromethane part A, dichloromethane part B, dichloromethane part C, dichloromethane part D, ethyl acetate part E and ethyl acetate part G or any mixture thereof in preparing medicines and health care products for treating ulcerative colitis.
The inventor of the invention has the activity of resisting ulcerative colitis in water solution, methylene dichloride extract and ethyl acetate extract which are obtained by extracting and separating water extract of Morchella fruit bodies by petroleum ether, and methylene dichloride part A, methylene dichloride part B, methylene dichloride part C, methylene dichloride part D, ethyl acetate part E and ethyl acetate part G which are further separated from the extract.
Drawings
FIG. 1 effect of different treatments on the intestinal length of mice.
Remarks: lower case letters indicate the level of significant differences between treatments (p < 0.05), the same letter differences between samples were not significant, and different letters reached significant differences.
FIG. 2 Morchella serum DAO activity of mice treated differently,
remarks: lower case letters indicate the level of significant differences between treatments (p < 0.05), the same letter differences between samples were not significant, and different letters reached significant differences.
FIG. 3 Morchella is a graph showing MPO activity in colon tissue of mice treated differently.
Remarks: lower case letters indicate the level of significant differences between treatments (p < 0.05), the same letter differences between samples were not significant, and different letters reached significant differences.
FIG. 4T-SOD activity of colon tissue of mice treated with Morchella.
Remarks: lower case letters indicate the level of significant differences between treatments (p < 0.05), the same letter differences between samples were not significant, and different letters reached significant differences.
Fig. 5 variation of MDA content in colon tissue of mice treated with morchella.
Remarks: lower case letters indicate the level of significant differences between treatments (p < 0.05), the same letter differences between samples were not significant, and different letters reached significant differences.
Detailed Description
The following description of the embodiments of the invention illustrates, but does not limit, the invention.
The preparation method of the novel active site of the Morchella esculenta fruiting body comprises the following steps:
A. pulverizing Morchella fruit body, decocting in water, and collecting decoction;
B. the decoction obtained in the step A is concentrated to obtain water extract;
C. b, adding water into the water extract obtained in the step B for dispersion to obtain a suspension, adding petroleum ether for extraction for multiple times until the petroleum ether extract is clear, collecting and combining to obtain the petroleum ether extract and an aqueous solution after petroleum ether extraction;
D. adding dichloromethane into the aqueous solution subjected to petroleum ether extraction in the step C for multiple extraction until the dichloromethane extract is clear, collecting and combining the dichloromethane extracts, and concentrating to dryness to obtain dichloromethane extract; adding ethyl acetate into the rest aqueous solution for multiple extraction until ethyl acetate extract liquid is clear, collecting and combining ethyl acetate methane extract liquid, concentrating to dryness to obtain ethyl acetate extract;
E. dissolving the dichloromethane extract obtained in the step D with dichloromethane/methanol, adding silica gel, drying the solvent, and grinding uniformly to obtain sample-mixing silica gel; adding the sample-mixed silica gel into a silica gel column, eluting with a dichloromethane-petroleum ether solution with the volume ratio of 3-8:7-2 in sequence, and collecting the eluent; eluting with methanol, and collecting eluate; obtaining a dichloromethane part A, a dichloromethane part B, a dichloromethane part C and a dichloromethane part D;
F. dissolving the ethyl acetate extract obtained in the step D with ethyl acetate/methanol fully, adding silica gel, drying the solvent, and grinding uniformly to obtain sample-mixing silica gel; adding the sample-mixed silica gel into a silica gel column, eluting with ethyl acetate/petroleum ether solution with the volume ratio of 3-8:7-2 in sequence, and collecting eluent; eluting with methanol, and collecting eluate; ethyl acetate fraction E and ethyl acetate fraction G were obtained.
The preparation method comprises the following steps:
and (C) drying the Morchella fruit bodies in an oven at 60 ℃ until the weight of the Morchella fruit bodies is constant.
The crushing in the step A is crushing into coarse powder.
Wherein, the coarse powder can pass through the second sieve completely, but is mixed with powder which can pass through the fourth sieve by no more than 40 percent.
The water adding and decocting condition in the step A is that 14-20 times of water is added, and the water is decocted for 2-5 times, and each time lasts for 1-2.5 hours; the preferred water-adding decoction conditions are as follows: adding 16 times of water, and decocting for 3 times each for 1.5 hr.
The concentration in the step B is reduced pressure concentration.
C, adding water to disperse, wherein the water adding amount is 6-10 times of that of the step C; preferably, the water addition amount of the water dispersion in the step C is 8 times of the water addition amount.
The adding amount of petroleum ether in the step C is 0.8-1.2 times of that of the aqueous solution in each extraction; preferably, the petroleum ether of step C is added in an amount of 1 time of the aqueous solution per extraction.
The addition amount of dichloromethane in the step D is 0.8-1.2 times of the amount of the residual aqueous solution in the step C; preferably, the addition of step D dichloromethane per extraction is 1 time the amount of aqueous solution remaining in step C.
The addition amount of ethyl acetate in each extraction is 0.8-1.2 times of the amount of the water solution remained after the extraction of dichloromethane; preferably, the ethyl acetate added per extraction in step D is 1 times the amount of aqueous solution remaining after the dichloromethane extraction.
Step E, the dichloromethane/methanol is dichloromethane/methanol solution with the volume ratio of 1-2:2-1; the method comprises the step E, wherein the dichloromethane/methanol is a dichloromethane/methanol solution with the volume ratio of any one of 1:1, 1:2 and 2:1; preferably, the dichloromethane/methanol in the step E is a dichloromethane/methanol solution with the volume ratio of 1:1.
In the step E, in the sample mixing silica gel, the weight ratio of the dichloromethane extract to the silica gel is 1-1:1-3; the method comprises the step E of mixing the sample with silica gel, wherein the weight ratio of dichloromethane extract to the silica gel is any one of 1:1, 1:2 and 1:3; preferably, in the sample-mixing silica gel in the step E, the weight ratio of the dichloromethane extract to the silica gel is 1:2.
E, the silica gel in the sample mixing silica gel is 100-400 meshes; the method comprises the step E that the silica gel in the sample mixing silica gel is any one of 100-200 meshes, 200-300 meshes and 300-400 meshes; preferably, the silica gel in the sample mixing silica gel in the step E is 200-300 meshes.
In the step E, the weight of the silica gel in the silica gel column is 5-20 times of that of the sample-mixed silica gel; preferably, the silica gel in the silica gel column in the step E is 10 times of the weight of the sample-mixed silica gel.
E, the silica gel in the silica gel column is 200-600 meshes; the method comprises the step E that silica gel in a silica gel column has any particle size of 200-300 meshes, 300-400 meshes and 400-600 meshes; preferably, the silica gel in the silica gel column in the step E is 400-600 meshes.
In the step E, the eluent of the dichloromethane part A is a 3:7 dichloromethane-petroleum ether solution, and the eluent of the dichloromethane part B is a 5:5 dichloromethane-petroleum ether solution; the eluent of the dichloromethane part C is 8:2 dichloromethane-petroleum ether solution; the eluent of the dichloromethane part D is methanol solution.
The ratio of the eluent amount and the dichloromethane extract amount of each part in the step E is as follows: the eluent adopted by each 50g of dichloromethane extract is 1-5L. Preferably, in the step E, the ratio of the eluent amount of each part to the dichloromethane extract amount is: the eluent used per 50g of dichloromethane extract was 3L.
The ethyl acetate/methanol in the step F is ethyl acetate/methanol solution with the volume ratio of 1-1:1-3; the method comprises the step F, wherein the ethyl acetate/methanol is an ethyl acetate/methanol solution with the volume ratio of any one of 1:1, 1:2 and 1:3; preferably, the ethyl acetate/methanol in the step F is ethyl acetate/methanol solution with the volume ratio of 1:1.
In the step F, the weight ratio of the ethyl acetate extract to the silica gel is 1-1:1-3; the method comprises the step F of mixing the sample with silica gel, wherein the weight ratio of the ethyl acetate extract to the silica gel is any one of 1:1, 1:2 and 1:3; preferably, in the sample-mixing silica gel in the step F, the weight ratio of the ethyl acetate extract to the silica gel is 1:2.
F, the silica gel in the sample mixing silica gel is 100-400 meshes; the method comprises the step F that the silica gel in the sample mixing silica gel is any one of 100-200 meshes, 200-300 meshes and 300-400 meshes; preferably, the silica gel in the sample mixing silica gel in the step F is 200-300 meshes.
The weight of the silica gel in the silica gel column in the step F is 5-20 times of that of the sample-mixed silica gel; preferably, the silica gel in the silica gel column in the step F is 10 times of the weight of the sample-mixed silica gel.
F, the silica gel in the silica gel column is 200-600 meshes; the method comprises the step of setting the particle size of silica gel in a silica gel column to any one of 200-300 meshes, 300-400 meshes and 400-600 meshes; preferably, the silica gel in the silica gel column in the step F is 400-600 meshes.
The eluent of the ethyl acetate part E in the step F is ethyl acetate with the ratio of 3:7: petroleum ether solution; the eluent of the ethyl acetate part F is ethyl acetate to petroleum ether solution in a ratio of 5:5; the eluent of the ethyl acetate part G is ethyl acetate-petroleum ether solution with the ratio of 8:2; the eluent of the ethyl acetate part H is methanol solution. The effective active sites obtained by the invention are an ethyl acetate site E and an ethyl acetate site G.
The ratio of the eluent amount and the ethyl acetate extract amount of each part in the step F is as follows: the eluent adopted by each 50g of ethyl acetate extract is 1-5L. Preferably, the ratio of the eluent amount to the ethyl acetate extract amount of each part in the step F is: the eluent adopted for each 50g of ethyl acetate extract is 3L.
The new active site of Morchella esculenta fruiting body comprises at least one of the residual aqueous solution of step C, the dichloromethane extract of step D, the ethyl acetate extract of step D, the dichloromethane part A of step E, the dichloromethane part B of step E, the dichloromethane part C of step E, the dichloromethane part D of step E, the ethyl acetate part E of step F, and the ethyl acetate part G of step F or their mixture.
The following experiments were performed using the most preferred steps and parameters of the preparation method of the present invention to prepare the methylene chloride sites a, B, C, D, E, G:
taking 4.0kg of dried Morchella fruit bodies, pulverizing, adding 16 times of water, decocting for three times each for 1.5 hours, mixing decoctions, and concentrating under reduced pressure to obtain Morchella water extract.
The Morchella extract is fully dissolved and dispersed by 5L of water, then is extracted by petroleum ether for multiple times, and is extracted by petroleum ether for 5L each time until the petroleum ether extract is clear, the petroleum ether extracts are combined and concentrated to obtain 107g of oily component. The aqueous solution was extracted with 5L of dichloromethane several times until the dichloromethane extract was clear, the dichloromethane extracts were combined and concentrated to dryness to give 65g of black extract. The remaining aqueous solution was extracted with ethyl acetate 5L for a number of times until the ethyl acetate extract was clear, and the ethyl acetate extracts were combined and concentrated to dryness to give 80g of a blackish brown extract.
50g of dichloromethane extract is weighed and fully dissolved by dichloromethane/methanol, 100g of 200-300 mesh silica gel is weighed and evenly stirred, and the solvent is dried and evenly ground. 1kg of 400-600 mesh silica gel is taken and evenly filled into a silica gel column, and then the mixed sample silica gel is evenly put into the silica gel column for compaction. Elution was then performed with A3:7, B5:5, C8:2 (dichloromethane: petroleum ether), respectively, 3L for each gradient. Finally, the remaining material was eluted with a 3L methanol column. Four components, A12 g, B7 g, C9 g, D10 g, were obtained.
Weighing 50g of ethyl acetate extract, fully dissolving the ethyl acetate extract with ethyl acetate/methanol, weighing 100g of 200-300 mesh silica gel, uniformly stirring, drying the solvent, and uniformly grinding. 1kg of 400-600 mesh silica gel is taken and evenly filled into a silica gel column, and then the mixed sample silica gel is evenly put into the silica gel column for compaction. Elution was then performed with E3:7, F5:5, G8:2 (ethyl acetate: petroleum ether), respectively, 3L for each gradient. Finally, the remaining material was eluted with a 3L methanol column. Four components, E15G, F2G, G12G, H4G, were obtained.
The effects of dichloromethane sites a, B, C, D, E, G on ulcerative colitis were examined in the following experiments, collectively referred to as A, B, C, D, E, G. The preparation method comprises the following steps: accurately weighing crude extract A, B, C, D of dichloromethane extraction part, preparing 2.5% and 5% concentration solutions of ethyl acetate extraction part crude extract E, G with redistilled water respectively, respectively referred to as low concentration A, B, C, D, E, G and high concentration A, B, C, D, E, G, and refrigerating for use.
1. Establishment of ulcerative colitis model in mice
Ulcerative colitis is clinically manifested by reduced body mass, fecal bloody stool, and shortened colon. Thus, the changes of the weight and the intestinal length of the mice and the fecal condition can intuitively reflect the severity of the inflammation of the mice.
112 male C57BL/6 mice of 6-8 weeks old were selected, adaptively fed for 1 week, and randomly divided into a blank group (Control), dextran sodium sulfate (DSS, dextran sulphate sodium, colitis modeling) group, A, B, C, D, E, G low concentration group, A, B, C, D, E, G high concentration group, 8 mice each, and weights of the mice of each group were recorded. All groups of mice in the first week were freely drunk deionized water, and the blank group and the DSS group were filled with 2mL of deionized water on time every day, and the administration groups were respectively filled with 2mL of 2.5% and A, B, C, D, E, G sample aqueous solutions with 5% concentration. Seven days later, the mice body weight was weighed and recorded. The second week was followed by daily on-time dosing of 2mL deionized water in the stomach blank and DSS groups, 2mL of 2.5% and 5% strength in A, B, C, D, E, G sample water solution respectively, and the remaining time, free drinking deionized water in the blank mice, free drinking 3% DSS water solution in DSS groups and dosing group mice, regular daily water intake recordings, and daily observations recorded mouse fecal and hematochezia status. After 24h of the last gastric lavage, the mice were sacrificed by weighing, blood was taken, the whole colon was taken and intestinal length was measured as well as fresh feces. The temperature in the room is kept at 25 ℃ and the relative humidity is 50-70%, the illumination is controlled, the day and night is carried out for 12 hours, and the padding is replaced every three days.
After daily observation of the fecal condition of the mice, except for the normal group, the mice started to have fecal unshaped in the third day and hematochezia in the fifth day after freely drinking the DSS aqueous solution. As DSS caused colitis in mice, mice in the model had an average decrease in weight of 27.88%, mortality of 40%, control had an increase in weight of 2.84% and mortality of 0. After administration, the weight loss rate and mortality rate of the administered group were lower than those of the model building group, indicating that the samples can alleviate colitis to some extent. The study also found that the high concentration group had a lower rate of change in body weight and lower mortality than the low concentration group, indicating that the sample concentration was proportional to the effect of colitis alleviation (Table 1).
TABLE 1 mouse ulcerative colitis disease index table
As can be seen from fig. 1, the colon length of the control group is about 2cm longer than the average length of the modeling group, and the colon of the modeling group is reddish due to the bleeding of the colonitis of the mice caused by DSS, and the control group is white, thus indicating that the modeling of the colonitis model of the mice is successful. As can be seen from fig. 2, the length of the intestines of the high-concentration and low-concentration samples is significantly longer than that of the model group after administration, and the length of the intestines of the high-concentration sample is higher than that of the low-concentration sample after administration, which again proves that the high-concentration and low-concentration samples can effectively relieve colonitis. Sample G showed the best relief at high concentrations among 6 samples, and bleeding was also more moderate.
2. Determination of DAO Activity in mouse serum
DAO activity accurately reflects the integrity, lesion and proliferation of intestinal mucosa.
Treatment of serum samples: the heart of the mice was sampled and placed in an EP tube, centrifuged at 8000rpm for 15min in a high-speed centrifuge pre-chilled at 4℃and the supernatant carefully removed and frozen at-20 ℃. The measuring method comprises the following steps: reference is made to DAO assay kit instructions.
As can be seen from fig. 2, the DAO activity of the high-low concentration sample is significantly higher than that of the modeling module, the DAO activity of the high-concentration sample is superior to that of the low concentration sample, and the DAO activity of the high-concentration sample G in all the high-low samples is significantly superior to that of the rest samples, which indicates that the high-low concentration sample has significant effect of alleviating and protecting acute colitis in mice, and the effect of the sample G is most obvious.
3. Determination of Myeloperoxidase (MPO) content in colon tissue of mice
MPO can reflect the infiltration number and activity of neutrophils and is an important index for measuring the degree of inflammation.
Precisely weighing colon tissue, adding corresponding volume of homogenizing medium according to the weight-volume ratio, homogenizing by an automatic homogenizer (4 ℃ C., 5s of homogenizing stop 2s, 60 cycles total), centrifuging at 4 ℃ C., 12000r/min for 10min, collecting supernatant, and measuring the content according to MPO kit instruction.
As can be seen from FIG. 3, the MPO value of the model is significantly higher than that of the control group due to the inflammatory reaction of the colon of the mice caused by DSS. The MPO activity of the high-low concentration group samples after the gastric lavage is obviously lower than that of the molding group, and the high concentration value is obviously lower than that of the low concentration, which indicates that the high-low concentration has a certain relieving effect on acute colitis, and the high concentration G effect is most obvious.
4. Determination of T-SOD Activity and MDA content in colon tissue of mice
MDA content and T-SOD activity react with lipid peroxidation, and can remove excessive oxygen free radicals, reduce tissue damage and ulcer formation.
Precisely weighing colon tissue, adding corresponding volume of homogenizing medium according to the weight-volume ratio, homogenizing by an automatic homogenizer (4 ℃ C., 5s of homogenizing stop 2s and 60 cycles), centrifuging at 4 ℃ C., 12000r/min for 10min, collecting supernatant, and measuring the content according to the specification of the T-SOD and MDA kit.
As can be seen from fig. 4, the T-SOD value of the control group is significantly higher than that of the modeling group, the T-SOD activity of the low concentration sample C, D, G is significantly higher than that of the modeling group, and the T-SOD activity of the low concentration sample A, B, E is not significantly different from that of the modeling group. The high concentration samples were all significantly higher than the modeling modules, indicating that the high concentration samples had higher radical scavenging than the low concentrations. It was also found that the T-SOD values of the high concentration samples C and G were significantly higher than the remaining samples, with the most significant radical scavenging effect.
As can be seen from fig. 5, the activity of the model group MDA is significantly higher than that of the control group, and the activity of the high-low concentration sample MDA is significantly lower than that of the model group, which indicates that the high-low concentration sample has a certain effect of alleviating the damage to acute colitis, and the high-concentration sample G has a more obvious effect of alleviating.
Thus, the following extraction sites of Morchella fruit body extract were found: the method comprises the steps of residual aqueous solution in the step C, dichloromethane extract obtained in the step D, ethyl acetate extract obtained in the step D, dichloromethane part A obtained in the step E, dichloromethane part B obtained in the step E, dichloromethane part C obtained in the step E, dichloromethane part D obtained in the step E, ethyl acetate part E obtained in the step F, at least one or a mixture of ethyl acetate part G obtained in the step F has obvious effect of relieving colonitis of mice, and the ethyl acetate part G obtained in the step F of a high-concentration sample has the most obvious effect of relieving colonitis of mice.
Claims (3)
1. The preparation method of the active site of the morchella sporophore for treating ulcerative colitis is characterized by comprising the following steps: the method comprises the following steps:
A. drying Morchella fruit body in oven at 60deg.C until weight is constant, pulverizing into coarse powder, decocting with 14-20 times of water for 2-5 times each for 1-2.5 hr, and collecting decoction;
B. the decoction obtained in the step A is decompressed and concentrated to obtain water extract;
C. b, adding water into the water extract obtained in the step B for dispersion to obtain a suspension, adding water with the water addition amount of 6-10 times of that of the water extract, adding petroleum ether for multiple extraction, wherein the addition amount of petroleum ether for each extraction is 0.8-1.2 times of that of the water solution until the petroleum ether extract is clear, and collecting and combining to obtain the petroleum ether extract and the water solution after petroleum ether extraction;
D. adding dichloromethane into the aqueous solution subjected to petroleum ether extraction in the step C for multiple extraction until the dichloromethane extract is clear, collecting and combining the dichloromethane extracts, and concentrating to dryness to obtain dichloromethane extract; adding ethyl acetate into the rest aqueous solution for multiple extraction until ethyl acetate extract liquid is clear, collecting and combining ethyl acetate methane extract liquid, concentrating to dryness to obtain ethyl acetate extract;
E. dissolving the dichloromethane extract obtained in the step D with dichloromethane/methanol, adding silica gel, drying the solvent, and grinding uniformly to obtain sample-mixing silica gel; adding the sample-mixed silica gel into a silica gel column, eluting with a dichloromethane-petroleum ether solution with the volume ratio of 3-8:7-2 in sequence, and collecting the eluent; eluting with methanol, and collecting eluate; obtaining a dichloromethane part A, a dichloromethane part B, a dichloromethane part C and a dichloromethane part D;
F. dissolving the ethyl acetate extract obtained in the step D with ethyl acetate/methanol fully, adding silica gel, drying the solvent, and grinding uniformly to obtain sample-mixing silica gel; adding the sample-mixed silica gel into a silica gel column, eluting with ethyl acetate/petroleum ether solution with the volume ratio of 3-8:7-2 in sequence, and collecting eluent; eluting with methanol, and collecting eluate; obtaining an ethyl acetate part E and an ethyl acetate part G;
wherein, the coarse powder in the step A is powder which can pass through a second sieve completely but is mixed with not more than 40% of powder which can pass through a fourth sieve;
the addition amount of dichloromethane in the step D is 0.8-1.2 times of the amount of the residual aqueous solution in the step C;
the addition amount of ethyl acetate in each extraction is 0.8-1.2 times of the amount of the water solution remained after the extraction of dichloromethane;
step E, the dichloromethane/methanol is dichloromethane/methanol solution with the volume ratio of 1-2:2-1;
in the step E, the weight ratio of the dichloromethane extract to the silica gel is 1-1:1-3;
in the step E, the silica gel in the sample mixing silica gel is 100-400 meshes;
in the step E, the weight of the silica gel in the silica gel column is 5-20 times of that of the sample-mixed silica gel;
e, the silica gel in the silica gel column is 200-600 meshes;
in the step E, the eluent of the dichloromethane part A is a 3:7 dichloromethane-petroleum ether solution, and the eluent of the dichloromethane part B is a 5:5 dichloromethane-petroleum ether solution; the eluent of the dichloromethane part C is 8:2 dichloromethane-petroleum ether solution; the eluent of the dichloromethane part D is methanol solution;
the ratio of the eluent amount and the dichloromethane extract amount of each part in the step E is as follows: the eluent adopted by each 50g of dichloromethane extract is 1-5L;
the ethyl acetate/methanol in the step F is ethyl acetate/methanol solution with the volume ratio of 1-1:1-3;
in the step F, the weight ratio of the ethyl acetate extract to the silica gel is 1-1:1-3;
in the step F, the silica gel in the sample mixing silica gel is 100-400 meshes;
in the step F, the weight of the silica gel in the silica gel column is 5-20 times that of the sample-mixed silica gel;
f, the silica gel in the silica gel column is 200-600 meshes;
the eluent of the ethyl acetate part E in the step F is ethyl acetate with the ratio of 3:7: petroleum ether solution;
the eluent of the ethyl acetate part F is ethyl acetate to petroleum ether solution in a ratio of 5:5;
the eluent of the ethyl acetate part G is ethyl acetate-petroleum ether solution with the ratio of 8:2;
the eluent of the ethyl acetate part H is methanol solution;
the ratio of the eluent amount and the ethyl acetate extract amount of each part in the step F is as follows: the eluent adopted by each 50g of ethyl acetate extract is 1-5L.
2. The method for preparing the active site of the fruiting body of Morchella for treating ulcerative colitis according to claim 1, wherein: at least any one of the following is satisfied:
the water adding and decocting conditions in the step A are as follows: adding 16 times of water, and decocting for 3 times each for 1.5 hours;
c, adding water to disperse, wherein the water adding amount is 8 times of that of the step C;
step C, petroleum ether is added in an amount which is 1 time of the amount of the aqueous solution in each extraction;
the addition amount of dichloromethane in the step D is 1 time of the amount of the residual aqueous solution in the step C;
step D, the addition amount of ethyl acetate in each extraction is 1 time of the amount of the aqueous solution remained after the extraction of dichloromethane;
the methylene dichloride/methanol in the step E is a methylene dichloride/methanol solution with the volume ratio of any one of 1:1, 1:2 and 2:1;
in the step E, the weight ratio of the dichloromethane extract to the silica gel is any one of 1:1, 1:2 and 1:3;
the silica gel in the sample mixing silica gel in the step E has any particle size of 100-200 meshes, 200-300 meshes and 300-400 meshes;
the weight of the silica gel in the silica gel column in the step E is 10 times that of the sample-mixed silica gel;
the silica gel in the silica gel column in the step E has any particle size of 200-300 meshes, 300-400 meshes and 400-600 meshes;
the ratio of the eluent amount and the dichloromethane extract amount of each part in the step E is as follows: the eluent adopted by each 50g of dichloromethane extract is 3L;
the ethyl acetate/methanol in the step F is an ethyl acetate/methanol solution with the volume ratio of any one of 1:1, 1:2 and 1:3;
in the sample mixing silica gel in the step F, the weight ratio of the ethyl acetate extract to the silica gel is any one of 1:1, 1:2 and 1:3;
the silica gel in the sample mixing silica gel in the step F has any particle size of 100-200 meshes, 200-300 meshes and 300-400 meshes;
the weight of the silica gel in the silica gel column in the step F is 10 times of that of the sample-mixed silica gel;
the silica gel in the silica gel column in the step F has any particle size of 200-300 meshes, 300-400 meshes and 400-600 meshes;
the ratio of the eluent amount and the ethyl acetate extract amount of each part in the step F is as follows: the eluent adopted for each 50g of ethyl acetate extract is 3L.
3. The method for preparing the active site of the fruiting body of Morchella for treating ulcerative colitis according to claim 2, wherein: at least any one of the following is satisfied:
step E, dichloromethane/methanol is dichloromethane/methanol solution with volume ratio of 1:1;
step E, 200-300 meshes of silica gel in the sample mixing silica gel;
in the step E, the weight ratio of the dichloromethane extract to the silica gel is 1:2;
e, silica gel in the silica gel column is 400-600 meshes;
f, the ethyl acetate/methanol is ethyl acetate/methanol solution with volume ratio of 1:1;
in the step F, the weight ratio of the ethyl acetate extract to the silica gel is 1:2;
step F, 200-300 meshes of silica gel in the sample mixing silica gel;
and F, the silica gel in the silica gel column is 400-600 meshes.
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| US5411935A (en) * | 1993-03-17 | 1995-05-02 | Sumitomo Chemical Co., Ltd. | Dihydrobenzofuran derivatives, their production and use |
| WO2005026269A1 (en) * | 2003-09-04 | 2005-03-24 | Mcdaniel C Steven | Microorganism coating components, coatings, and coated surfaces |
| CN113150181A (en) * | 2021-05-06 | 2021-07-23 | 四川省农业科学院土壤肥料研究所 | Morchella extract and preparation method and application thereof |
| CN113332317A (en) * | 2021-07-07 | 2021-09-03 | 西安文理学院 | Extraction method and application of morchella esculenta extract |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5411935A (en) * | 1993-03-17 | 1995-05-02 | Sumitomo Chemical Co., Ltd. | Dihydrobenzofuran derivatives, their production and use |
| WO2005026269A1 (en) * | 2003-09-04 | 2005-03-24 | Mcdaniel C Steven | Microorganism coating components, coatings, and coated surfaces |
| CN113150181A (en) * | 2021-05-06 | 2021-07-23 | 四川省农业科学院土壤肥料研究所 | Morchella extract and preparation method and application thereof |
| CN113332317A (en) * | 2021-07-07 | 2021-09-03 | 西安文理学院 | Extraction method and application of morchella esculenta extract |
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