CN109810087B - Compound for preventing and treating alcoholism and preparation method thereof - Google Patents
Compound for preventing and treating alcoholism and preparation method thereof Download PDFInfo
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- CN109810087B CN109810087B CN201711187012.XA CN201711187012A CN109810087B CN 109810087 B CN109810087 B CN 109810087B CN 201711187012 A CN201711187012 A CN 201711187012A CN 109810087 B CN109810087 B CN 109810087B
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 91
- 208000007848 Alcoholism Diseases 0.000 title claims abstract description 56
- 201000007930 alcohol dependence Diseases 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 76
- 238000012360 testing method Methods 0.000 claims abstract description 31
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 230000000694 effects Effects 0.000 claims abstract description 24
- 238000002386 leaching Methods 0.000 claims abstract description 20
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- KQILIWXGGKGKNX-UHFFFAOYSA-N dihydromyricetin Natural products OC1C(=C(Oc2cc(O)cc(O)c12)c3cc(O)c(O)c(O)c3)O KQILIWXGGKGKNX-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
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- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a compound for preventing and treating alcoholism and a preparation method thereof, wherein the preparation method of the compound comprises the following steps: the method comprises the steps of manually picking, cleaning and removing impurities from whole herb of medicinal materials, drying in the shade indoors to obtain a whole herb dried sample, and crushing and sieving to obtain medicinal material powder; leaching the medicinal material powder with ethanol, grinding the leaching solution by a colloid mill, homogenizing under high pressure, and centrifugally separating to obtain supernatant and filter residues; adsorbing the supernatant by macroporous resin, eluting with ethanol solution, concentrating the obtained ethanol solution into extract, extracting with petroleum ether, ethyl acetate and n-butanol, and collecting the extraction parts; fourthly, treating the extracted part by using microporous resin (MCI) and repeated silica gel column, and combining a two-dimensional reversed phase/hydrophilic chromatographic analysis preparation method to obtain a monomer compound; fifthly, screening the anti-alcoholism and alcohol dependence activities of the monomer compounds based on gammA-Aminobutyric acid protein A receptor, and screening and preparing 6 active compounds through animal behavioural experiments such as a positive-turning reflection loss test, a rotating rod test, a movement coordination test and the like. The invention has scientific and reasonable technical design, low cost, high extraction rate and preparation efficiency, stable property and high content of the obtained compound, and has stronger anti-alcoholism and alcohol dependence effects.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to a compound for preventing and treating alcoholism and a preparation method thereof.
Background
The snowy ganoderma school name Gansu flea prefix (Arenaria kansuensis maxim.) is a perennial pad-like herb of the genus flea of family phyllostachys, mainly from mountain meadows and gravel belts at the Qinghai-Tibet plateau elevations of 3700-5500 m. Herba Arenariae Kansuensis is used as a drug, has bitter and cold nature, has effects of clearing heat, benefiting lung, relieving cough, eliminating swelling, lowering blood pressure, and relieving stranguria, and can be used for treating jaundice, gonorrhea, hysteromyopathy, pneumonia and various pulmonary diseases. Aster falcatum (Asterothamnus centrali-Asteracus) is a plant of Asteraceae, mainly grown in grasslands and desert areas at altitudes of 1,300 to 3,900 meters, and is currently used mainly as forage due to its high nutritional value. Modern researches show that the chemical components of the Arenaria kansuensis and the Aster tataricus mainly comprise flavone and alkaloid, the flavone and alkaloid are known by people in the aspect of strong anti-inflammatory activity, and the research on the activity of preventing and treating alcoholism/alcohol dependence is not reported in the literature.
At present, the literature of separating and preparing 6 flavonoid compounds (shown in figure 1) from Arenaria kansuensis and Aster tatariasis has not been reported, and the research on the activity of the compounds for preventing and treating alcoholism/alcohol dependence has not been reported.
GammA-Aminobutyric acid protein a receptor (GABA-A receptor) is one of the members of the ligand gate ion channel superfamily, and is a pentagonal heterogeneous polypeptide oligomer composed of 5 subunits embedded in the cytoplasmic membrane of nerve cells. Since its existence was confirmed at the end of the 70 s, studies on the GABA-A receptor have attracted considerable attention. The reason for this is: (1) GABA is the major inhibitory neurotransmitter in the mammalian central nervous system, and about 50% of the central synaptic sites use GABA as a transmitter, and plays an important role in controlling neuronal excitability through the mediation of its specific receptors; (2) At least 4 mutually allosteric drug binding sites exist on the same receptor complex, including sedative benzodiazepines, inhibitors barbiturates, spasmodics stephanins, neuroactive steroids, with a rich pharmacological connotation.
The interaction of alcohol with GABA-A receptors plays an important role in alcoholism and alcohol dependence, and chinese patent CN201180048110 reports methods of treating alcoholism, alcohol use disorders, and alcohol abuse with dihydromyricetin, which mainly involves the competitive inhibition or antagonism of alcohol activity on GABA-A receptors, thereby exhibiting anti-alcoholism activity and preventing alcohol abuse. Among the drugs currently used for treating alcoholism/alcohol dependence, there are mainly 3, which are naltrexone, canpral, disulfiram, but all have serious side effects such as muscle relaxation, anxiety, depression and other unpleasant sensations, headache, nausea, flushing, heart rate acceleration, shortness of breath, confusion, and circulatory failure, so that the application of these three drugs is greatly limited. Therefore, it is necessary to find natural, highly effective and low-toxic active compounds and pharmaceutical compositions for the prevention and treatment of alcoholism and alcohol dependence.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a compound for preventing and treating alcoholism and a preparation method thereof, which take Qinghai special medicinal plants Arenaria kansui or Aster tatarian as raw materials and successfully screen out a medicinal composition for preventing and treating alcoholism and alcohol dependence by adopting a colloid mill high-pressure homogenization combined technology.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a compound for preventing and treating alcoholism and its preparation method are provided, wherein the structural formula of any one of the following six compounds is as follows:
wherein compound one: r1=och 3 ,R2＝OCH 3 ,R3＝OH,R4＝OH,R5＝H,R6＝OCH 3 ；
Compound two: r1=och 3 ,R2＝OH,R3＝H,R4＝OH,R5＝H,R6＝OCH 3 ；
And (3) a compound III: r1=och 3 ,R2＝OCH 3 ,R3＝H,R4＝H,R5＝H,R6＝H；
Compound four: r1=och 3 ,R2＝OH,R3＝H,R4＝H,R5＝H,R6＝H；
Compound five: r1=och 3 ,R2＝OH,R3＝OCH 3 ,R4＝H,R5＝H,R6＝H；
Compound six: r1=h, r2=oh, r3=h, r4=h, r5=och 3 ,R6＝OCH 3 ；
A compound for preventing and treating alcoholism and a preparation method thereof comprise the following steps:
the method comprises the steps of manually picking, cleaning and removing impurities from whole herb of Arenaria kanbana or Aster tatariasis, drying in the shade indoors to obtain a whole herb dried sample, crushing, and sieving with a 20-320 mesh sieve to obtain medicinal powder;
mixing the medicinal powder with ethanol with the volume fraction of 10-100% (g/mL) according to the ratio of 1: heating and extracting the mixture at the temperature of 20-90 ℃ for 0.5-72 h according to the volume ratio of 5-100 to obtain leaching liquor;
grinding the leaching solution for 1-20 times by a colloid mill to obtain a colloid leaching solution, homogenizing under the pressure of 1-200 Mpa, and performing centrifugal separation to obtain supernatant and filter residues;
removing filter residues, passing the obtained supernatant through macroporous resin, eluting with deionized water until no sugar exists, eluting with ethanol solution with the volume of 2-40 times of the bed layer and the volume fraction of 20-100%, concentrating the obtained ethanol solution into extract, extracting with petroleum ether, ethyl acetate and n-butanol, and collecting all the extraction parts;
fifthly, treating the extracted part by using microporous resin (MCI) and repeated silica gel column, and combining a two-dimensional reversed phase/hydrophilic chromatographic analysis preparation method to obtain a monomer compound;
the six-step method is characterized in that the monomer compounds are screened for anti-alcoholism and alcohol dependence activity based on gammA-Aminobutyric acid protein A receptor (GABA-A receptor), and 6 active compounds are screened and prepared through a positive reflection loss test (Loss of Righting Reflex, LORR), a rotating rod test (Rotarod test) and a movement coordination test (Motor coordination test).
The rotating speed of the rotor of the colloid mill in the step III is 2000 r/min-20000 r/min; the high-pressure homogenizer is a continuous correlation type (shown in figure 2) which is obtained by modifying the impact valve body type (shown in figure 3); the centrifugal speed is 2000-20000 r/min, and the centrifugal time is 10-60 min.
The macroporous adsorbent resins used in the steps are HPD300, D101, AB-8, NKA-9 and HP-20.
The microporous resin (MCI) particle size involved in step II is 4-300 μm; the chromatographic columns include Unitary C18, megress C18, hedera-ODS-C18, dubhe C18, RP-C18HCE, XAMide, xaqua C3.
In step ii, alcohol exhibits alcoholism symptoms due to interaction between ethanol and the GABA-A receptor, and the screened compound can competitively inhibit or antagonize the activity of ethanol on the GABA-A receptor, thereby exhibiting anti-alcoholism and alcohol-dependent activity.
Compared with the prior art, the invention has the following advantages:
1. according to the invention, a colloid mill high-pressure homogenization combined technology is combined with an extraction preparation method, at present, a high-pressure homogenizer used by a production enterprise is mainly of an impact valve type (figure 2), and because materials in solution are impacted with the impact valve severely, a plurality of metal particles can be formed, so that the produced product is very unfavorable for human health; the high-pressure homogenizer used in the invention is a continuous correlation type (figure 1), is obtained by modification on the basis of the impact valve body type, mainly utilizes the correlation flow principle and a unique lampshade-shaped structure to ensure that high-speed moving material particles in high-pressure solution collide with each other, and simultaneously, the homogenization efficiency is obviously improved through continuous twice homogenization. Therefore, the continuous correlation high-pressure homogenizer not only solves the problem of metal particle residue, prolongs the service life of the cavity, but also obviously increases the homogenization performance, ensures more stable active ingredients and greatly improves the working efficiency. In addition, the two-dimensional reversed phase/hydrophilic chromatographic system established based on different chromatographic column selections and reversed/hydrophilic mixing modes can play a great role in purifying and separating complex samples, particularly comprising a large number of difficult-to-separate compounds, and meanwhile, the limitations of the conventional separation and preparation method in terms of sample preparation, selectivity and separation efficiency are overcome.
2. The invention combines the colloid mill running-in technology and the high-pressure homogenizing technology, thus not only effectively shortening the extraction time and reducing the consumption of extraction solvent and energy consumption in the process, but also ensuring stable property, high content and high biological activity of the obtained flavone.
3. The invention adopts the snowmelt ganoderma lucidum and the aster tataricus with abundant resources as raw materials, thereby effectively reducing the production cost.
4. The established two-dimensional reversed phase/hydrophilic chromatographic system can purify and separate complex samples, particularly contains a large number of difficult-to-separate compound samples, and the purity of the obtained compound is high (more than 98 percent), and meanwhile, the limitations of the conventional separation preparation method in terms of sample preparation, selectivity and separation efficiency are overcome.
5. The 6 flavone compounds screened can be easily separated and prepared by utilizing a two-dimensional reversed phase/hydrophilic chromatographic system, have high activity of resisting alcohol toxicity/alcohol dependence, and do not produce side effects such as muscle relaxation, anxiety, depression and the like.
6. The instrument, equipment and consumable related by the invention are simple and convenient to operate, the technical parameters are easy to control, the application range is wide, and the instrument, equipment and consumable related by the invention can be used for industrialized mass production to form a biological medicine product industrial chain.
7. The invention comprehensively utilizes the combined technology of colloid mill and high-pressure homogenization to be applied to the extraction of the flavone, thereby not only greatly improving the working efficiency, but also ensuring stable property and high content of the obtained flavone; through system comparison research, the modified continuous correlation high-pressure homogenizer is obviously improved in the aspects of homogeneity, working efficiency, production, product safety and the like as compared with the impact valve body homogenizer, and is suitable for large-scale production.
8. The two-dimensional reversed phase/hydrophilic chromatographic system established based on different chromatographic column selections and reversed/hydrophilic mixing modes not only enables complex samples (containing a large amount of difficult-to-separate compounds) to be separated and purified, but also has high purity (more than 98 percent), and simultaneously overcomes the limitations of the conventional separation preparation method in terms of sample preparation, selectivity and separation efficiency;
9. the natural active compounds for preventing and treating alcoholism/alcohol dependence are screened from Qinghai special medicinal plants based on the GABA-A receptor, are easy to separate and prepare, have high activity and do not produce side effects such as muscle relaxation, anxiety, depression and the like.
Drawings
FIG. 1 is a schematic view of a high pressure homogenizer in continuous correlation
FIG. 2 is a schematic view of a high pressure homogenizer striking a valve body
FIG. 3 is a schematic representation of the DMB inhibition of [3H ] flunitrazepam binding by the active compound
FIG. 4 is a schematic diagram of a loss of specular reflection experiment (LORR)
FIG. 5 is a schematic diagram of a spin lever experiment 1 (rotating test 1)
FIG. 6 is a schematic diagram of a horizontal line grabbing experiment (Horizontal wire test)
FIG. 7 is a schematic diagram of spin lever experiment 2 (rotating test 2)
FIG. 8 is a schematic diagram of an activity test (Locomotor activity test)
Detailed Description
Example 1
A compound for preventing and treating alcoholism and a preparation method thereof are characterized in that any one of the following six compounds has the following structural formula:
wherein compound one: r1=och3, r2=och3, r3=oh, r4=oh, r5=h, r6=och3;
compound two: r1=och3, r2=oh, r3=h, r4=oh, r5=h, r6=och3;
and (3) a compound III: r1=och3, r2=och3, r3=h, r4=h, r5=h, r6=h;
compound four: r1=och3, r2=oh, r3=h, r4=h, r5=h, r6=h;
compound five: r1=och3, r2=oh, r3=och3, r4=h, r5=h, r6=h;
compound six: r1=h, r2=oh, r3=h, r4=h, r5=och3, r6=och3.
A compound for preventing and treating alcoholism and a preparation method thereof comprise the following steps:
the method comprises the steps of manually picking, cleaning and removing impurities from whole herb, drying in the shade indoors to obtain a whole herb dried sample, crushing, and sieving with an 80-mesh sieve to obtain medicinal powder;
secondly, mixing the medicinal powder with 80% ethanol according to the volume fraction of 1:20 Heating and extracting the feed liquid volume ratio of (g/mL) at 70 ℃ for 4 hours to obtain leaching liquor;
grinding the leaching solution for 3 times by a colloid mill to obtain a colloid leaching solution, homogenizing under 100Mpa pressure, and centrifuging to obtain supernatant and filter residues;
removing filter residues, passing the supernatant through AB-8 macroporous resin, eluting with deionized water until no sugar exists, eluting with ethanol solution with the volume of 6 times of the bed layer and the volume fraction of 80%, concentrating the obtained ethanol solution into extract, extracting with petroleum ether, ethyl acetate and n-butanol, and collecting all the extraction parts;
dissolving ethyl acetate part with 5 times of 75% ethanol by volume fraction, filtering, eluting filtrate with 4 times of 85% ethanol solution by volume of microporous resin (MCI) with particle size of 100 μm, concentrating the eluate under reduced pressure, subjecting to silica gel column treatment (eluting with 3 times of ethyl acetate-n-butanol mixed solvent by volume of column), and combining two-dimensional reversed phase/hydrophilic chromatographic analysis preparation method established by reversed phase C18 and hydrophilic XAMide chromatographic column to obtain monomer compound;
the above monomer compounds were screened for anti-alcoholism/alcohol-dependent activity based on gammA-Aminobutyric acid protein a receptor (GABA-A receptor), and 6 active compounds, which were compounds 1, 2, 3, 4, 5, and 6, were selected and prepared through animal behavioral experiments such as a loss of anti-reflection test (Loss of Righting Reflex, lor), a rotating rod test (Rotarod test), and a movement coordination test (Motor coordination test).
Example 2
A compound for preventing and treating alcoholism and a preparation method thereof are characterized in that any one of the following six compounds has the following structural formula:
wherein compound one: r1=och 3 ,R2＝OCH 3 ,R3＝OH,R4＝OH,R5＝H,R6＝OCH 3 ；
Compound two: r1=och 3 ,R2＝OH,R3＝H,R4＝OH,R5＝H,R6＝OCH 3 ；
And (3) a compound III: r1=och 3 ,R2＝OCH 3 ,R3＝H,R4＝H,R5＝H,R6＝H；
Compound four: r1=och 3 ,R2＝OH,R3＝H,R4＝H,R5＝H,R6＝H；
Compound five: r1=och 3 ,R2＝OH,R3＝OCH 3 ,R4＝H,R5＝H,R6＝H；
Compound six: r1=h, r2=oh, r3=h, r4=h, r5=och 3 ,R6＝OCH 3 。
A compound for preventing and treating alcoholism and a preparation method thereof comprise the following steps:
the method comprises the steps of manually picking, cleaning and removing impurities from whole herb, drying in the shade indoors to obtain a whole herb dried sample, crushing, and sieving with a 20-mesh sieve to obtain medicinal powder;
secondly, mixing the medicinal powder with ethanol with the volume fraction of 100% according to the ratio of 1:100 Heating and extracting the feed liquid volume ratio of (g/mL) at 20 ℃ for 72 hours to obtain leaching liquor;
grinding the leaching solution for 20 times by a colloid mill to obtain a colloid leaching solution, homogenizing under the pressure of 1Mpa, and centrifuging to obtain supernatant and filter residues;
removing filter residues, passing the supernatant through HP-20 macroporous resin, eluting with deionized water until no sugar exists, eluting with ethanol solution with the volume of 2 times of the bed layer and the volume fraction of 20%, concentrating the obtained ethanol solution into extract, extracting with petroleum ether, ethyl acetate and n-butanol, and collecting all the extraction parts;
dissolving n-butanol part with 60% ethanol with 3 times volume fraction, filtering, eluting filtrate with 70% ethanol solution with 3 times column volume after microporous resin (MCI) with 50 μm particle size, concentrating eluate under reduced pressure, subjecting to silica gel column treatment (eluting with 4 times column volume of ethyl acetate-n-butanol mixed solvent), and combining two-dimensional reversed phase/hydrophilic chromatography established by RP-C18HCE, xaqua C3 and hydrophilic Xamide chromatographic column to obtain monomer compound;
the above monomer compounds were screened for anti-alcoholism/alcohol-dependent activity based on gammA-Aminobutyric acid protein a receptor (GABA-A receptor), and 5 active compounds were screened and prepared through animal behavioral experiments such as a loss of anti-alcoholism/alcohol-dependent activity test (Loss of Righting Reflex, lor), a rotating rod test (Rotarod test), a movement coordination test (Motor coordination test) and the like.
Example 3
A compound for preventing and treating alcoholism and a preparation method thereof are characterized in that any one of the following six compounds has the following structural formula:
wherein compound one: r1=och 3 ,R2＝OCH 3 ,R3＝OH,R4＝OH,R5＝H,R6＝OCH 3 ；
Compound two: r1=och 3 ,R2＝OH,R3＝H,R4＝OH,R5＝H,R6＝OCH 3 ；
And (3) a compound III: r1=och 3 ,R2＝OCH 3 ,R3＝H,R4＝H,R5＝H,R6＝H；
Compound four: r1=och 3 ,R2＝OH,R3＝H,R4＝H,R5＝H,R6＝H；
Compound five: r1=och 3 ,R2＝OH,R3＝OCH 3 ,R4＝H,R5＝H,R6＝H；
Compound six: r1=h, r2=oh, r3=h, r4=h, r5=och 3 ,R6＝OCH 3 。
A compound for preventing and treating alcoholism and a preparation method thereof comprise the following steps:
the method comprises the steps of manually picking, cleaning and removing impurities from whole herb, drying in the shade indoors to obtain a whole herb dried sample, crushing, and sieving with a 320-mesh sieve to obtain medicinal powder;
secondly, mixing the medicinal powder with 10% ethanol according to the volume fraction of 1: heating and extracting the feed liquid with the volume ratio of 5 (g/mL) at the temperature of 90 ℃ for 0.5h to obtain leaching liquor;
grinding the leaching solution for 1 time by a colloid mill to obtain a colloid leaching solution, homogenizing under the pressure of 200Mpa, and centrifuging to obtain supernatant and filter residues;
removing filter residues, passing the supernatant through D101 macroporous resin, eluting with deionized water until no sugar exists, eluting with 40 times of ethanol solution with 100% of bed volume and volume fraction, concentrating the obtained ethanol solution into extract, extracting with petroleum ether, ethyl acetate and n-butanol, and collecting the extraction parts;
dissolving ethyl acetate part with 5 times of 50% ethanol by volume fraction, filtering, eluting filtrate with 4 times of 75% ethanol solution by volume of microporous resin (MCI) with particle size of 200 μm, concentrating the eluate under reduced pressure, subjecting to silica gel column treatment (eluting with 2 times of ethyl acetate-n-butanol mixed solvent by volume of column), and combining with two-dimensional reversed phase/hydrophilic chromatography established by using Uniry C18 and hydrophilic XAmide chromatographic column to obtain monomer compound;
the above monomer compounds were screened for anti-alcoholism/alcohol-dependent activity based on gammA-Aminobutyric acid protein a receptor (GABA-A receptor), and 3 active compounds, which were compounds 3, 4, 5, were selected and prepared through animal behavioral experiments such as a loss of anti-reflection test (Loss of Righting Reflex, lor), a rotating rod test (Rotarod test), a movement coordination test (Motor coordination test), and the like.
Example 4
A compound for preventing and treating alcoholism and a preparation method thereof are characterized in that any one of the following six compounds has the following structural formula:
wherein compound one: r1=och 3 ,R2＝OCH 3 ,R3＝OH,R4＝OH,R5＝H,R6＝OCH 3 ；
Compound two: r1=och 3 ,R2＝OH,R3＝H,R4＝OH,R5＝H,R6＝OCH 3 ；
And (3) a compound III: r1=och 3 ,R2＝OCH 3 ,R3＝H,R4＝H,R5＝H,R6＝H；
Compound four: r1=och 3 ,R2＝OH,R3＝H,R4＝H,R5＝H,R6＝H；
Compound five: r1=och 3 ,R2＝OH,R3＝OCH 3 ,R4＝H,R5＝H,R6＝H；
Compound six: r1=h, r2=oh, r3=h, r4=h, r5=och 3 ,R6＝OCH 3 。
A compound for preventing and treating alcoholism and a preparation method thereof comprise the following steps:
the method comprises the steps of manually picking, cleaning and removing impurities from whole herb, drying in the shade indoors to obtain a whole herb dried sample, crushing, and sieving with a 70-mesh sieve to obtain medicinal powder;
the preparation method comprises the following steps of (1) mixing the medicinal powder with 95% ethanol: 30 Heating and extracting the feed liquid volume ratio of (g/mL) at 70 ℃ for 3 hours to obtain leaching liquor;
grinding the leaching solution for 5 times by a colloid mill to obtain a colloid leaching solution, homogenizing under 150Mpa pressure, and centrifuging to obtain supernatant and filter residues;
removing filter residues, passing the supernatant through AB-8 macroporous resin, eluting with deionized water until no sugar exists, eluting with ethanol solution with the volume of 3 times of the bed layer and the volume fraction of 60%, concentrating the obtained ethanol solution into extract, extracting with petroleum ether, ethyl acetate and n-butanol, and collecting the extraction parts;
dissolving ethyl acetate part with 3 times of 60% ethanol by volume fraction, filtering, eluting filtrate with 70% ethanol solution with 5 times of column volume after microporous resin (MCI) with particle size of 150 μm, concentrating eluate under reduced pressure, subjecting to silica gel column treatment (eluting with 4 times of column volume of ethyl acetate-n-butanol mixed solvent), and combining two-dimensional reversed phase/hydrophilic chromatography established by RP-C18HCE and hydrophilic XAmide chromatographic column to obtain monomer compound;
the above monomer compounds were screened for anti-alcoholism/alcohol-dependent activity based on gammA-Aminobutyric acid protein a receptor (GABA-A receptor), and 6 active compounds, which were compounds 1, 2, 3, 4, 5, and 6, were selected and prepared through animal behavioral experiments such as a loss of anti-reflection test (Loss of Righting Reflex, lor), a rotating rod test (Rotarod test), and a movement coordination test (Motor coordination test).
Claims (4)
1. An application of a compound in preparing a medicament for preventing and treating alcoholism, which is characterized in that: the preparation method of the compound comprises the following steps:
(1) Manually picking, cleaning and removing impurities from the whole herb of the Arenaria plant or the Aster tatariasis, drying in the shade indoors to obtain a whole herb dried sample, crushing, and sieving with a 20-320 mesh sieve to obtain medicinal powder;
(2) Heating and extracting the medicinal powder and ethanol with the volume fraction of 10-100% g/mL at the temperature of 20-90 ℃ according to the volume ratio of 1:5-100, and obtaining leaching liquor after 0.5-72 h;
(3) Grinding the leaching solution for 1-20 times by a colloid mill to obtain a colloid leaching solution, homogenizing under the pressure of 1-200 Mpa, and performing centrifugal separation to obtain supernatant and filter residues;
(4) Removing filter residues, passing the obtained supernatant through macroporous resin, eluting with deionized water until no sugar exists, eluting with ethanol solution with the volume of 2-40 times of the bed layer volume and the volume fraction of 20-100%, concentrating the obtained ethanol solution into extract, extracting with petroleum ether, ethyl acetate and n-butanol, and collecting all extraction parts;
(5) Treating the extracted part by microporous resin MCI and repeated silica gel column, and combining two-dimensional reversed phase/hydrophilic chromatographic analysis preparation method to obtain monomer compound;
(6) Screening the monomer compounds for anti-alcoholism and alcohol dependence activity based on gammA-Aminobutyric acid protein A receptor, and screening and preparing 6 active compounds through an anti-alcoholism loss test, a rotating rod test and a movement coordination test; the structural formula is as follows:
wherein compound one: r1=och3, r2=och3, r3=oh, r4=oh, r5=h, r6=och3;
compound two: r1=och3, r2=oh, r3=h, r4=oh, r5=h, r6=och3;
and (3) a compound III: r1=och3, r2=och3, r3=h, r4=h, r5=h, r6=h;
compound four: r1=och3, r2=oh, r3=h, r4=h, r5=h, r6=h;
compound five: r1=och3, r2=oh, r3=och3, r4=h, r5=h, r6=h;
compound six: r1=h, r2=oh, r3=h, r4=h, r5=och3, r6=och3;
the macroporous adsorption resins used in the step (4) are HPD300, D101, AB-8, NKA-9 and HP-20.
2. The use of a compound according to claim 1 for the manufacture of a medicament for the prevention and treatment of alcoholism, characterized in that: the rotating speed of the rotor of the colloid mill in the step (3) is 2000 r/min-20000 r/min; the high-pressure homogenizer is a continuous correlation type, and is obtained by modification on the basis of the impact valve body; the centrifugal speed is 2000-20000 r/min, and the centrifugal time is 10-60 min.
3. The use of a compound according to claim 1 for the manufacture of a medicament for the prevention and treatment of alcoholism, characterized in that: the microporous resin particles involved in said step (5) have a size of 4 to 300 μm; the chromatographic columns include UnitaryC18, megressC18, hedera-ODS-C18, dubheC18 and RP-C18HCE, XAmide, XAquaC.
4. The use of a compound according to claim 1 for the manufacture of a medicament for the prevention and treatment of alcoholism, characterized in that: the alcohol-mediated alcohol screening step (6) is characterized by alcohol interaction with the GABA-A receptor, and the screened compounds competitively inhibit or antagonize the activity of alcohol on the GABA-A receptor, thereby exhibiting anti-alcoholism and alcohol-dependent activity.
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