Medicine for dispelling effects of alcohol and / or protecting gastric mucosa as well as preparation method and application of medicine

Abstract

The invention relates to a medicine for alleviating hangover and / or protecting gastric mucosa as well as a preparation method and application thereof, and relates to the technical field of medicines. The medicine comprises active pharmaceutical ingredients and pharmaceutically acceptable auxiliary materials, wherein the active pharmaceutical ingredients comprise at least one of a fomes fomentarius ethyl acetate extract, a fomes fomentarius enzymolysis extract, a fermentation composition and a phellinus igniarius extract. The medicine not only can be used for effectively dispelling the effects of alcohol, but also can be used for protecting gastric mucosa of drinkers.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, in particular to a drug for sobering up and / or protecting gastric mucosa and a preparation method and application thereof. Background Art

[0002] The relationship between alcohol and human culture is closely connected and inextricably linked. However, we have to face the fact that long-term and excessive drinking can cause serious harm to the human body, resulting in more than 200 diseases in many organs such as the brain, heart, gastrointestinal tract and liver. After alcohol enters the digestive tract, about 10% is metabolized in the stomach, and the remaining 90% quickly enters the blood. In addition to a small amount that can be excreted from urine, sweat and exhaled breath, the remaining 90% of alcohol needs to be metabolized in the liver. The liver metabolizes alcohol in the following three steps: ① Under the action of alcohol dehydrogenase, alcohol is converted into acetaldehyde; ② Under the action of acetaldehyde dehydrogenase, acetaldehyde is converted into acetic acid; ③ Through the biochemical reaction of the tricarboxylic acid cycle, acetic acid is converted into carbon dioxide and water and excreted from the body. The content of acetaldehyde dehydrogenase in different human bodies is different.

[0003] People who are relatively deficient in acetaldehyde dehydrogenase cannot continue to completely convert acetaldehyde into acetic acid, and acetaldehyde will accumulate in the body. People who are relatively rich in acetaldehyde dehydrogenase can quickly metabolize acetaldehyde into non-toxic acetic acid, decompose it into carbon dioxide and water and excrete it from the body. Such people are really "drinkers" and are not easy to get drunk. Ethanol metabolism causes oxidative stress in the body, which can damage mitochondria. Ethanol can also directly cause endoplasmic reticulum stress, which in turn causes mitochondrial dysfunction, lipid accumulation, inflammatory response and cell apoptosis. Long-term drinking can cause damage to multiple systems in the body, especially to the gastrointestinal tract and liver. Alcohol can directly or indirectly cause chronic gastritis, peptic ulcers and alcoholic cirrhosis. In recent years, the incidence of adult alcohol poisoning has also increased year by year. Acute alcohol poisoning can cause damage to multiple organs, especially acute gastric mucosal erosion and bleeding. In recent years, due to the increasing consumption of alcoholic beverages, alcohol-induced diseases have become increasingly serious.

[0004] Most of the existing alcohol sobering drugs have the effect of sobering up, but they are limited to delaying the drunkenness time of drinkers, relieving symptoms such as headaches and nausea of ​​drunkards, and shortening the drunken sleep time of drunkards, but lack the effect of protecting the gastric mucosa while sobering up. Summary of the invention

[0005] In view of the above problems, the present invention provides a drug for sobering up and / or protecting gastric mucosa, which can not only effectively sober up and sober up, but also protect the gastric mucosa of drinkers.

[0006] In order to achieve the above-mentioned purpose, the present invention provides a medicine for sobering up and / or protecting gastric mucosa, comprising a pharmaceutically active ingredient and a pharmaceutically acceptable excipient, wherein the pharmaceutically active ingredient comprises at least one of an ethyl acetate extract of Myrica chinensis, an enzymatic hydrolysis extract of Myrica chinensis, a fermentation composition, and a Phellinus igniarius extract.

[0007] The above ingredients, when used alone or in combination as active ingredients of the medicine, can achieve good effects of sobering up and sobering up, and can protect the gastric mucosa.

[0008] In one embodiment, the excipients include at least one of a solvent, a disintegrant, a flavoring agent, a colorant, a lubricant, an antioxidant, a preservative, a binder, a filler or a thickener.

[0009] In one embodiment, the ethyl acetate extract of Ipomoea batryticatus is obtained by ultrasonic extraction of Ipomoea batryticatus with ethyl acetate;

[0010] The enzymatic hydrolysis extract of bitter white ti is obtained by reflux extraction of bitter white ti with an organic solvent after enzymatic hydrolysis;

[0011] The fermentation composition is obtained by reflux extraction after substrate fermentation, and the substrate includes Inonotus maculatus, Ganoderma lucidum, and Ganoderma lucidum;

[0012] The mulberry linterus extract is obtained from mulberry linterus by an ultrasonic-flash combined extraction method.

[0013] The above enzymatic hydrolysis can dissolve more medicinal ingredients in bitter white hoof, making it easier to obtain more triterpenoid components through subsequent reflux extraction with organic solvents.

[0014] In one embodiment, the Kubaiti extract includes Kubaiti ethyl acetate extract and Kubaiti enzymatic extract, and the mass ratio of the Kubaiti ethyl acetate extract to the Kubaiti enzymatic extract is 1:(0.5-1.5).

[0015] In one embodiment, the mass ratio of Inonotus maculata, Ganoderma lucidum, and Ganoderma lucidum is 1:(0.5-1.5):(0.5-1.5).

[0016] In one embodiment, the active ingredients of the medicine include ethyl acetate extract of Ipomoea batryticatus, enzymatic hydrolysis extract of Ipomoea batryticatus, fermentation composition and mulberry lindera extract, and the dosage ratio of the ethyl acetate extract of Ipomoea batryticatus, enzymatic hydrolysis extract of Ipomoea batryticatus, fermentation composition and mulberry lindera extract is 1:(0.8-1.5):(0.8-1.5).

[0017] In one embodiment, the fermented fermentation seed liquid includes at least one of Lactobacillus casei seed liquid and Bacillus subtilis seed liquid.

[0018] In one embodiment, the dosage form of the drug includes an oral preparation or an injectable preparation.

[0019] The present invention also provides an application of a Kubaiti extract in preparing a drug for sobering up and / or protecting gastric mucosa, wherein the Kubaiti extract comprises at least one of a Kubaiti ethyl acetate extract and a Kubaiti enzymatic hydrolysis extract.

[0020] The present invention also provides an application of a fermentation composition in the preparation of a drug for sobering up and / or protecting gastric mucosa. The fermentation composition is obtained by reflux extraction after substrate fermentation. The substrate includes Inonotus maculata, Ganoderma lucidum, and Ganoderma lucidum.

[0021] The present invention also provides an application of a mulberry linterus extract in the preparation of a drug for sobering up and / or protecting gastric mucosa. The mulberry linterus extract is obtained from mulberry linterus by an ultrasonic-flash combined extraction method.

[0022] The present invention also provides the use of at least one of the following: ethyl acetate extract of Ipomoea australis, enzymatic hydrolysis extract of Ipomoea australis, fermentation composition and mulberry linterus extract in the preparation of a drug for sobering up and / or protecting gastric mucosa.

[0023] The present invention also provides a method for preparing the ethyl acetate extract of Ipomoea batryticatus in the medicine or the ethyl acetate extract of Ipomoea batryticatus in the application, comprising the following steps: crushing Ipomoea batryticatus, adding ethyl acetate, ultrasonically extracting, taking the extract, removing ethyl acetate, and obtaining the ethyl acetate extract of Ipomoea batryticatus.

[0024] In one of the embodiments, the solid-liquid ratio of the bitter white hoof to ethyl acetate is 1:(8-12), and the ultrasonic extraction time is 3-3.5h.

[0025] In one embodiment, the bitter taro includes bitter taro fruiting bodies.

[0026] The present invention also provides a method for preparing the enzymatic hydrolysis extract of the bitter white ti in the medicine or the bitter white ti in the application, comprising the following steps: mixing the bitter white ti with an enzyme, performing enzymatic hydrolysis, filtering, taking the filter residue, adding an organic solvent for reflux extraction, extracting the filtrate, and obtaining the bitter white ti enzymatic hydrolysis extract.

[0027] In one embodiment, the ratio of bitter white hoof to enzyme is 1g:(550-650)U, the temperature of enzymolysis is 38-42°C, and the time of enzymolysis is 35-45min.

[0028] In one embodiment, the enzyme includes at least one of pectinase and hemicellulase.

[0029] In one embodiment, the enzymes include pectinase and hemicellulase, and the enzyme concentration ratio of the pectinase to the hemicellulase is 1:(0.8-1.2).

[0030] In one embodiment, the organic solvent includes ethanol, the number of reflux extraction is 2-3 times, and the time of reflux extraction is 50-70 min.

[0031] The present invention also provides a method for preparing the fermentation composition in the medicine or the fermentation composition in the application, comprising the following steps: mixing Inonotus maculata, Ganoderma lucidum and Ganoderma lucidum, crushing, adding water to form a suspension, inoculating fermentation seed liquid into the suspension, oscillating and culturing, adding a solvent for reflux extraction, taking the extract and obtaining the fermentation composition.

[0032] In one embodiment, the inonotus maculatus is a fruiting body of the inonotus maculatus, the ganoderma lucidum is a fruiting body of the ganoderma lucidum, and the ganoderma lucidum is a fruiting body of the ganoderma lucidum.

[0033] In one embodiment, the fermentation seed liquid includes at least one of Lactobacillus casei seed liquid and Bacillus subtilis seed liquid.

[0034] In one embodiment, the method for preparing the Lactobacillus casei seed liquid comprises: inoculating Lactobacillus casei into a culture medium, culturing anaerobically, picking a single colony, and culturing aerobically in the culture medium to obtain the Lactobacillus casei seed liquid.

[0035] In one embodiment, in the method for preparing the Lactobacillus casei seed liquid, the culture medium includes MRS liquid culture medium, and the temperature of the anaerobic culture is 35-40°C.

[0036] In one embodiment, the method for preparing the Bacillus subtilis seed solution comprises: inoculating Bacillus subtilis into a culture medium, anaerobic culturing, picking a single colony, and shaking culturing in the culture medium to obtain the Bacillus subtilis seed solution.

[0037] In one embodiment, in the method for preparing the Bacillus subtilis seed solution, the culture medium includes a brain heart infusion liquid culture medium, the temperature of the anaerobic culture is 35-40°C, the temperature of the shaking culture is 35-40°C, and the rotation speed of the shaking culture is 160-200r / min.

[0038] The present invention also provides a method for preparing the mulberry linterus extract in the medicine or the mulberry linterus extract in the application, comprising the following steps: crushing the mulberry linterus, adding water, performing ultrasonic extraction to obtain a feed liquid, performing flash extraction to obtain an extract, centrifuging, taking the supernatant, concentrating, ethanol precipitation, standing, taking the precipitate, centrifuging, and obtaining the mulberry linterus extract.

[0039] In one embodiment, the temperature of the ultrasonic extraction is 40-50°C, and the ultrasonic power is 450-550W; the extraction voltage of the flash extraction is 130-180V, the time is 85-95s, and the number of extractions is 2-4 times.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The drug for sobering up and / or protecting gastric mucosa of the present invention and its preparation method and application can not only effectively sober up and sober up, but also protect the gastric mucosa of drinkers. DETAILED DESCRIPTION

[0042] For ease of understanding of the present invention, the present invention will be described more fully below with reference to relevant embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0044] definition:

[0045] In the present invention, fruiting body refers to: the spore-producing structure of higher fungi, i.e., fruiting body, which is composed of organized mycelium. It is also called basidiocarp in basidiomycetes and ascocarp in ascomycetes. Whether it is sexual reproduction or asexual reproduction, whether the structure is simple or complex, its spore-producing structure is called fruiting body. The fruiting body of bitter white hoof, the fruiting body of brown inonotus, the fruiting body of purple ganoderma, the fruiting body of tree tongue ganoderma, etc. can all be purchased from conventional sales channels such as Chinese herbal medicine pharmacies.

[0046] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0047] Example 1

[0048] Ethyl acetate extract of Psoralea corylifolia.

[0049] Ultrasonic extraction with ethyl acetate was performed as follows:

[0050] Air-dry the fruiting bodies of the bitter white hoof, crush them, and pass them through a 60-mesh sieve. Weigh 100 g of the powder, add ethyl acetate at a solid-liquid ratio of 1:10, and ultrasonically extract twice, each time for 3 hours. Combine the extracts, remove the solvent by rotary evaporation under reduced pressure, and naturally dry to constant weight to obtain the bitter white hoof ethyl acetate extract.

[0051] Example 2

[0052] Bitter white tilapia enzymatic extract

[0053] 1. The enzymatic hydrolysis extracts of bitter white hoof were prepared by the following 4 methods, as follows:

[0054] 1. Enzymatic hydrolysis with pectinase.

[0055] Weigh 5.0g of bitter white hoof, put it in a 150ml ground conical flask, add 15 times the amount of pure water, adjust the pH to 4.0, add pectinase, add 3000U of enzyme, fix the enzymatic hydrolysis temperature to 40°C, and the enzymatic hydrolysis time to 40min. After the enzymatic hydrolysis treatment, filter, take the filter residue and add 12 times the amount of 70% ethanol solution, reflux extract twice, each time for 60min, combine the filtrate, and determine the total volume. Determine the total terpene content in the enzymatic hydrolysis extract 1 of bitter white hoof.

[0056] 2. Enzymatic hydrolysis with hemicellulase.

[0057] Weigh 5.0g of bitter white hoof, put it in a 150ml ground conical flask, add 15 times the amount of pure water, adjust the pH to 4.0, add hemicellulase, add 3000U of enzyme, fix the enzymatic hydrolysis temperature to 40°C, and the enzymatic hydrolysis time to 40min. After the enzymatic hydrolysis treatment, filter, take the filter residue and add 12 times the amount of 70% ethanol solution, reflux extract 2 times, each time for 60min, combine the filtrate, and determine the total volume. Determine the total terpene content in the enzymatic hydrolysis extract 2 of bitter white hoof.

[0058] 3. Enzymatic hydrolysis with pectinase and hemicellulase.

[0059] Weigh 5.0g of bitter white hoof, put it in a 150ml ground conical flask, add 15 times the amount of pure water, adjust the pH to 4.0, add pectinase: hemicellulase (1:1), add 3000U of enzyme, fix the enzymatic hydrolysis temperature to 40℃, and the enzymatic hydrolysis time to 40min. After the enzymatic hydrolysis treatment, filter, take the filter residue and add 12 times the amount of 70% ethanol solution, reflux extract 2 times, each time for 60min, combine the filtrate, and determine the total volume. Determine the total terpene content in the enzymatic hydrolysis extract 3 of bitter white hoof.

[0060] 4. Weigh 5.0 g of Kubaiti, add 15 times the amount of 70% ethanol solution, reflux extract twice, each time for 60 minutes, combine the filtrates, and determine the total volume. Determine the total terpene content in Kubaiti enzymatic extract 4.

[0061] 2. Determine the total terpene content in the enzymatic hydrolyzed extracts 1, 2, 3, and 4 of the bitter white ti.

[0062] Accurately weigh an appropriate amount of Sebiferenic acid reference substance, place it in a volumetric flask, add pure methanol to the mark, and fully dissolve to obtain a 0.224mg / mL Sebiferenic acid reference substance solution. Accurately measure 0.1, 0.2, 0.3, 0.4, and 0.5mL of the reference substance solution and place them in 10mL stoppered test tubes, evaporate them in a water bath, add 0.2mL of freshly prepared 5% vanillin-glacial acetic acid solution and 0.8mL of perchloric acid, heat them in a 60℃ water bath for 15min, take them out, cool them in an ice bath for 4min, and then pipette 5.0mL of glacial acetic acid into the stoppered test tube and mix them. Use the corresponding reagent as a blank, measure the absorbance at the maximum absorption wavelength of 544nm, repeat the experiment 3 times, and take the average value. A standard curve was drawn with absorbance A as the ordinate and Sebiferenic acid mass concentration C (μg / mL) as the abscissa, and the linear regression equation was A=0.0761C+0.0214 (r=0.9992).

[0063] Table 1 Total terpenoid content in enzymatic hydrolyzed extracts 1, 2, 3 and 4 of bitter white tilapia

[0064] Total triterpenes Enzymatic Extract of Kubaiti 1 2.07％ Bitter white tilapia enzymatic extract 2 2.17％ Bitter white tilapia enzymatic extract 3 3.04％ Enzymatic Extract of Kubaiti 1.84％

[0065] From the data in the above table, it can be seen that the triterpene extraction amount in the extract obtained by enzyme treatment was higher than that in the non-enzyme control group, among which the triterpene content in the extract of hemicellulase: pectinase group was the highest, higher than that of the traditional ethanol extraction group.

[0066] Example 3

[0067] Fermented compositions.

[0068] 1. This embodiment prepares the fermentation composition by bidirectional fermentation. Specifically, the following four methods are used:

[0069] 1. Use Lactobacillus casei fermentation liquid and Bacillus subtilis fermentation liquid as fermentation seed liquid.

[0070] Weigh 20.0g of Ganoderma lucidum fruiting body, 20.0g of Ganoderma lucidum fruiting body, and 20.0g of Inonotus maculatus fruiting body. Place in an oven and dry at 50°C, crush with a multifunctional Chinese medicine grinder, pass through an 80-mesh sieve, and place in a cool dryer; prepare the crushed mixed powder with water in a ratio of 3:7 to form a suspension, sterilize at 121°C for 15 minutes, cool to room temperature, and sterilize with ultraviolet light in a biological safety cabinet for 30 minutes, inoculate the two kinds of bacteria composite fermentation liquid in the suspension at an inoculum amount of 5%, and shake and culture in a constant temperature shaker for 5 days; reflux extract twice with water in a ratio of 1g:10L for 2 hours each time, combine the extracts, and finally freeze-dry the dialyzate to obtain a fermentation composition 1. The two kinds of bacteria composite fermentation liquid are composed of Lactobacillus casei fermentation liquid and Bacillus subtilis fermentation liquid in a mass ratio of 1:1.

[0071] In the present embodiment, the preparation method of Lactobacillus casei fermentation broth:

[0072] An ampoule bottle containing Lactobacillus casei strain CICC 20990 (strain CICC 20990, Lactobacillus casei Lactobacillus) was placed in a biosafety cabinet and disinfected with an alcohol cotton ball. 1 ml of MRS liquid culture medium was placed in the ampoule bottle and mixed by oscillation. The bacterial suspension was inoculated into the MRS liquid culture medium at an inoculum size of 5%, and cultured anaerobically at 37° C. for 18 h. Three-zone lines of the bacterial suspension were inoculated into MRS solid culture medium to purify the strain, and cultured anaerobically at 37° C. for 18 h. Single colonies were picked and cultured in the MRS liquid culture medium for 18 h to obtain Lactobacillus casei fermented liquid.

[0073] In this embodiment, the preparation method of Bacillus subtilis fermentation broth is:

[0074] The ampoule containing Bacillus subtilis CICC10446 (Bacillus subtilis CICC10446) was placed in a biological safety cabinet and disinfected with an alcohol cotton ball. 1 ml of brain heart infusion liquid culture medium was placed in the ampoule, and the mixture was shaken and mixed. The bacterial suspension was inoculated into the brain heart infusion liquid culture medium at an inoculum of 5%, and after anaerobic culture at 37° C. for 18 h, the bacterial suspension was inoculated into the brain heart infusion liquid culture medium by three-zone streaking to purify the strain, and the culture was anaerobically cultured at 37° C. for 18 h. A single colony was picked and cultured in a constant temperature shaker at 180 r / min at 37° C. for 24 h in the brain heart infusion liquid culture medium to obtain Bacillus subtilis fermentation liquid.

[0075] 2. Use Lactobacillus casei fermentation liquid as the fermentation seed liquid.

[0076] Weigh 20.0g of Ganoderma lucidum fruiting body, 20.0g of Ganoderma lucidum fruiting body, and 20.0g of Inonotus maculatus fruiting body. Dry in an oven at 50°C, crush with a multifunctional Chinese medicine grinder, pass through an 80-mesh sieve, and place in a cool dryer; prepare the crushed mixed powder with water in a ratio of 3:7 to form a suspension, sterilize at 121°C for 15min, cool to room temperature, sterilize with ultraviolet light in a biosafety cabinet for 30min, inoculate Lactobacillus casei fermentation liquid at an inoculation rate of 5%, and culture in a constant temperature shaker for 5d; extract with water at a ratio of 1g:10L to the solid-liquid ratio twice, each time for 2h, combine the extracts, and finally freeze-dry to obtain a fermentation composition 2.

[0077] 3. Use Bacillus subtilis fermentation liquid as the fermentation seed liquid.

[0078] Weigh 20.0g of Ganoderma lucidum fruiting body, 20.0g of Ganoderma lucidum fruiting body, and 20.0g of Inonotus maculatus fruiting body. Dry in an oven at 50°C, crush with a multifunctional Chinese medicine grinder, pass through an 80-mesh sieve, and place in a cool dryer; prepare the crushed mixed powder with water in a ratio of 3:7 to form a suspension, sterilize at 121°C for 15min, cool to room temperature, sterilize with ultraviolet light in a biological safety cabinet for 30min, inoculate Bacillus subtilis fermentation liquid at an inoculation rate of 5%, and culture in a constant temperature shaker for 5d; extract twice with water in a ratio of 1g:10L to solid-liquid ratio, each time for 2h, combine the extracts, and finally freeze-dry to obtain a fermentation composition 3.

[0079] 4. Add 20.0g of Ganoderma lucidum fruiting body, 20.0g of Ganoderma lucidum fruiting body, and 20.0g of Inonotus maculatus fruiting body to 100mL of deionized water, reflux extract at 100℃ for 1h, extract twice, combine the two extracts, centrifuge at 10000r / min for 10min, take the supernatant, and obtain Ganoderma lucidum + Ganoderma lucidum + Inonotus maculatus water extract, freeze-dry after concentration, and set aside. After freeze-drying, Ganoderma lucidum + Ganoderma lucidum + Inonotus maculatus fermentation freeze-dried powder was obtained.

[0080] 2. Determine the total polysaccharides and total triterpenes in the water extracts of the above fermentation compositions 1, 2, 3, Ganoderma lucidum + Ganoderma lucidum + Inonotus macrocarpon.

[0081] 1. Using glucose as the standard, the total sugar content was determined by sulfuric acid-anthrone colorimetry. The absorbance was measured at a wavelength of 625 nm, and the absorbance was linearly regressed against the mass, and the regression equation was y = 0.0276x + 0.0105 (R 2 =0.9993), the curve fitting is good. The total sugar content is calculated according to the glucose standard solution curve.

[0082] Table 2 Total polysaccharides in the aqueous extracts of fermented compositions 1, 2, 3, Ganoderma lucidum + Ganoderma lucidum + Inonotus maculatus

[0083]

[0084]

[0085] The data in the above table show that under the same crude drug dosage conditions of Ganoderma lucidum + Ganoderma lucidum + Inonotus maculatus, the total polysaccharide content in the fermentation composition was significantly increased compared with the water extract of Ganoderma lucidum + Ganoderma lucidum + Inonotus maculatus (P<0.01).

[0086] 2. Drawing of standard curve: Using oleanolic acid as standard, the total triterpene content was determined by vanillin-acetic acid-perchloric acid spectrophotometry. The absorbance was measured at a wavelength of 550 nm. The standard curve was drawn with the mass x of oleanolic acid as the horizontal coordinate and the absorbance y as the vertical coordinate. The regression equation was: Y = 6.3045x + 0.1672, r 2 =0.9998. w = af / m×100%

[0087] Wherein w is the total triterpenoid extraction rate, %;

[0088] a——the mass of triterpenoid in the sample to be tested, mg;

[0089] f——dilution multiple;

[0090] m——sample mass, mg.

[0091] Table 3 Total triterpenes in the aqueous extracts of fermentation compositions 1, 2, 3, Ganoderma lucidum + Ganoderma lucidum + Inonotus maculatus

[0092] Total triterpenoid yield Fermentation composition 1 12.38％ Fermentation composition 2 9.21％ Fermentation composition 3 8.49％ Ganoderma lucidum + Ganoderma lucidum + Inonotus maculatus water extract 3.24％

[0093] The data in the above table show that under the same crude drug amount of Ganoderma lucidum + Ganoderma lucidum + Inonotus punctatus fruiting body, the total triterpenoid extraction rate in the fermentation composition was significantly increased compared with the water extract of Ganoderma lucidum + Ganoderma lucidum + Inonotus punctatus (P<0.01). The reason may be that the mixed strains promote the decomposition of the cell walls of Ganoderma lucidum and Ganoderma lucidum + Inonotus punctatus during the fermentation process, and promote the dissolution of polysaccharides and total triterpenoid substances.

[0094] Example 4

[0095] Phellinus linteus extract.

[0096] In this embodiment, an ultrasonic-flash combined extraction method is used to obtain a mulberry linterus extract from mulberry linterus. The ultrasonic-flash combined extraction method has the advantages of high efficiency, short time, simple operation, and performance saving. Ultrasonic waves use its cavitation, shearing and mechanical effects to break the cell wall and dissolve its effective components. The flash extraction method uses stirring, shearing and osmotic effects to quickly crush the raw materials into fine particles at room temperature and under the action of solvents, and dissolve the active ingredients through local negative pressure osmosis to achieve efficient extraction.

[0097] 1. The Phellinus linteus extract was prepared by the following 4 methods, as follows:

[0098] 1. Dry the Phellinus igniarius fruiting bodies to constant weight, crush them, and pass them through a 60-mesh sieve. Obtain Phellinus igniarius fruiting body powder for later use. Mix a certain mass of Phellinus igniarius powder with purified water at a solid-liquid ratio of 1:20 (g / mL), and then place it in an ultrasonic cell crusher for ultrasonic extraction. (The ultrasonic temperature is 40-50 degrees Celsius, and the ultrasonic power is 500W). Place the ultrasonically extracted liquid in a multifunctional flash extractor for extraction. (Fixed extraction voltage 150V, flash extraction time 90s, extraction times 2 times.

[0099] The extract after flash extraction was placed in a centrifuge for centrifugation at a speed of 5000 r / min for 10 min. The supernatant after centrifugation was placed in a rotary evaporator for reduced pressure concentration, and the concentrated material was added with 8 times the volume of 95% ethanol for alcohol precipitation, and then placed in a refrigerator at 4°C for 12 h.

[0100] The supernatant of the alcohol-precipitated feed solution was removed, and the precipitate was placed in a centrifuge and centrifuged at 7000 r / min for 20 min. Vacuum drying: The precipitate after centrifugation was placed in a vacuum drying oven for drying to obtain the Phellinus igniarius extract 1.

[0101] 2. Dry the Phellinus igniarius fruiting bodies to constant weight, crush them, and pass them through a 60-mesh sieve. Obtain Phellinus igniarius fruiting body powder for later use. Mix a certain mass of Phellinus igniarius powder with purified water at a solid-liquid ratio of 1:20 (g / mL), and then place it in an ultrasonic cell crusher for ultrasonic extraction. (The ultrasonic temperature is 40-50 degrees Celsius, and the ultrasonic power is 500W). Place the ultrasonic extract in a centrifuge for centrifugation at a speed of 5000r / min for 10min. Place the supernatant after centrifugation in a rotary evaporator for reduced pressure concentration, add 8 times the volume of 95% ethanol to the concentrated material for alcohol precipitation, and let it stand in a 4°C refrigerator for 12h.

[0102] The supernatant of the alcohol-precipitated feed solution was removed, and the precipitate was placed in a centrifuge and centrifuged at 7000 r / min for 20 min. Vacuum drying: The precipitate after centrifugation was placed in a vacuum drying oven for drying to obtain the Phellinus igniarius extract 2.

[0103] 3. Dry the Phellinus linteus fruiting bodies to constant weight, crush them, and pass them through a 60-mesh sieve. Obtain Phellinus linteus fruiting body powder for later use. Mix a certain amount of Phellinus linteus powder with purified water at a solid-liquid ratio of 1:20 (g / mL), and place it in a multifunctional flash extractor for extraction. (Fixed extraction voltage 150V, flash extraction time 90s, extraction times 2 times)

[0104] The extract after flash extraction was placed in a centrifuge for centrifugation at a speed of 5000 r / min for 10 min. The supernatant after centrifugation was placed in a rotary evaporator for reduced pressure concentration, and the concentrated material was added with 8 times the volume of 95% ethanol for alcohol precipitation, and then placed in a refrigerator at 4°C for 12 h.

[0105] The supernatant of the alcohol-precipitated liquid was removed, and the precipitate was placed in a centrifuge and centrifuged at 7000 r / min for 20 min. Vacuum drying: The precipitate after centrifugation was placed in a vacuum drying oven for drying to obtain Phellinus igniarius extract 3.

[0106] 4. Dry the Phellinus igniarius fruiting bodies to constant weight, crush them, and pass through a 60-mesh sieve. Obtain Phellinus igniarius fruiting body powder for later use. Mix a certain mass of Phellinus igniarius powder with purified water at a solid-liquid ratio of 1:20 (g / mL), and reflux extract twice, each time for 2 hours. Put the extract after reflux extraction into a centrifuge for centrifugation at a speed of 5000r / min for 10 minutes. Put the supernatant after centrifugation into a rotary evaporator for reduced pressure concentration, add 8 times the volume of 95% ethanol to the concentrated material for alcohol precipitation, and let it stand in a refrigerator at 4°C for 12 hours.

[0107] The supernatant of the alcohol-precipitated feed solution was removed, and the precipitate was placed in a centrifuge and centrifuged at 7000 r / min for 20 min. Vacuum drying: The precipitate after centrifugation was placed in a vacuum drying oven for drying to obtain Phellinus igniarius extract 4.

[0108] 2. Determine the total polysaccharides in the above-mentioned Phellinus igniarius extracts 1, 2, 3, and 4.

[0109] Preparation of standard curve: Take 2mL glucose solution of different concentrations as standard, add 50μL 80% phenol and 3mL concentrated sulfuric acid respectively, mix well, cool to room temperature, and measure the absorbance at 490nm. Sample determination: weigh 10mg sample, add 1mL 72% H 2 SO 4 , stir at room temperature for 30 minutes to completely dissolve the sample, dilute to 0.2 mg / mL, take 2 mL of diluent, add 50 μL 80% phenol solution, 3 mL concentrated sulfuric acid, mix well, cool to room temperature and measure the absorbance, calculate the total sugar purity in the sample according to the following formula:

[0110] Total sugar purity (%) = C × V / m × 100

[0111] Where: C is the total sugar concentration (mg / mL) obtained from the standard curve; V is the volume of the sample during measurement (mL); m is the mass of the sample (mg).

[0112] The calculation formula of polysaccharide yield is as follows: Polysaccharide yield (%) = m 1 ×C 1 / m×100.

[0113] Table 4 Total polysaccharides in Phellinus linteus extracts 1, 2, 3, and 4

[0114]

[0115]

[0116] Experimental example

[0117] 1. In vitro experiments.

[0118] 1. Cell culture.

[0119] HepG2 cells were cultured in DMEM complete medium at 37°C and 5% CO 2 In a constant temperature incubator. Change the culture medium every 2 days. When the cells grow to 80% to 90% of the culture medium, discard the culture medium, add sterile PBS to gently rinse twice, discard, add trypsin to digest the cells, discard the digestion solution and add DMEM complete culture medium to terminate the digestion, centrifuge and discard the supernatant, add DMEM complete culture medium, gently blow to form a single cell suspension, subculture in a new culture bottle, and take the logarithmic growth phase HepG2 cells for the experiment.

[0120] 2. Establish a HepG2 cell acute alcohol injury model.

[0121] 2.1 HepG2 cells were cultured at 2×10 5 The cells were inoculated at a concentration of 100 / mL in a 12-well plate and placed in a 37°C, 5% CO 2 Culture in an incubator, and when the cells grow to 70% to 90% of the culture medium, aspirate the culture medium;

[0122] 2.2 The normal control group was not treated with any treatment, the model control group was treated with 1% ethanol for 6 hours, the ethyl acetate extract group of bitter white ti, the enzymatic extract group of bitter white ti, the combined group of bitter white ti (the mass ratio of ethyl acetate extract of bitter white ti and enzymatic extract of bitter white ti was 1:1), the fermentation composition group, the mulberry lindera extract group, the total extract group (the mass ratio of ethyl acetate extract of bitter white ti, enzymatic extract of bitter white ti, fermentation composition, and mulberry lindera extract was 1:1:1:1), and the decoction group were pre-cultured in a medium with a concentration of 100 μg / mL, 100 μg / mL, 200 μg / mL, 100 μg / mL, 100 μg / mL, 300 μg / mL, and 300 μg / mL for 24 hours and then treated with 1% ethanol for 6 hours. In this experimental example, the above-mentioned ethyl acetate extract of bitter white ti, enzymatic extract of bitter white ti, fermentation composition, and mulberry lindera extract were all prepared by the optimal method in the above-mentioned embodiments.

[0123] Preparation method of the above decoction group: weigh appropriate amounts of bitter white hoof fruiting bodies, purple ganoderma fruiting bodies, tree tongue ganoderma fruiting bodies, spot brown inonotus fruiting bodies and phellinus linteus fruiting bodies in a mass ratio of 1:1:1:1:1. Cut the decoction pieces according to the pharmacopoeia standard, and then decoct them in water at a material-liquid ratio of 1:20 for 2 hours. After filtering, concentrate to a relative density of 1.0g / ml. After centrifugation, take the supernatant.

[0124] 2.3 After 6 hours, dilute the cell suspension with PBS (PH7.2-7.4) to a cell concentration of about 1 million / mL. Repeatedly freeze and thaw to destroy the cells and release the intracellular components. Centrifuge at 2000-3000 rpm for about 20 minutes. Collect the supernatant carefully.

[0125] 3. Determination of alcohol dehydrogenase (ADH) content in cells.

[0126] 3.1 Take the ADH kit out of the refrigerated environment and equilibrate it at room temperature for 15-30 minutes. Take out the cell supernatant sample to be tested and equilibrate it at room temperature for 60 minutes.

[0127] 3.2 Set up standard wells, add 50μL of standard of different concentrations to each. Set up blank wells and test sample wells respectively. First add 40μL of sample diluent to the test sample well on the ELISA plate, and then add 10μL of the test sample. Add the sample to the bottom of the ELISA plate well, try not to touch the well wall, and gently shake to mix.

[0128] 3.3 Add 100 μL of enzyme-labeled reagent to each well except the blank well. Seal the plate with a sealing film and incubate at 37°C for 60 minutes.

[0129] 3.4 Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry. First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color at 37℃ in the dark for 15 minutes.

[0130] 3.5 Add 50 μL of stop solution to each well to stop the reaction. Use the blank well as zero and measure the absorbance of each well at 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0131] Table 5 ADH content in each group

[0132] Group ADH content (U / mg prot) n=3, x±s Normal control group 44.87 Model control group 17.56 Ethyl acetate extract of Psoralea corylifolia <![CDATA[29.88 ※ ]]> Enzymatic hydrolysis extract of bitter white ti <![CDATA[31.44 ※※ ]]> Kubaiti Merged Group <![CDATA[31.92 ※※ ]]> Fermentation composition group <![CDATA[36.21 ※※※ ]]> Phellinus linteus extract group <![CDATA[30.77 ※※ ]]> Total extract group <![CDATA[46.58 ※※※ ]]> Decoction group <![CDATA[22.04 ※ ]]>

[0133] Compared with the model control group, ※※※ P < 0.001 ※※ Indicates P < 0.01 ※ Indicates P < 0.05

[0134] 2. Alcohol detoxification experiment in mice model of chronic alcoholism.

[0135] 1. Establishment and grouping of the chronic alcoholism model in mice.

[0136] 1.1 Fifty C57BL / 6 mice were adaptively fed for 1 week and randomly divided into 11 groups, namely: blank control group (normal saline), alcohol model group, Kubaiti ethyl acetate extract group, Kubaiti enzymatic extract group, Kubaiti combined group, fermentation composition group, Phellinus lintei extract group, total extract high-dose group, total extract medium-dose group, total extract low-dose group, decoction group, 10 mice in each group. The blank control group and the alcohol model group were intragastrically administered with 10 mL / kg (based on body weight) of normal saline, the Kubaiti ethyl acetate extract group, Kubaiti enzymatic extract group, Kubaiti combined group, fermentation composition group, Phellinus lintei extract group, and decoction group were all administered with 50 mg / kg, and the total extract low-dose group, total extract medium-dose group, and total extract high-dose group were administered with 25 mg / kg, 50 mg / kg, and 100 mg / kg, respectively, by intragastrically administering.

[0137] 1.2 30 minutes after each gavage, the alcohol model group, low-dose group, medium-dose group and high-dose group were gavaged with 10 mL / kg (based on body weight) of 52° liquor, and the blank control group was gavaged with an equal dose of normal saline to establish a chronic alcohol poisoning model in mice, and the gavage was continued for 42 days.

[0138] 1.3 Collection and processing of animal samples.

[0139] At the end of the sixth week of the experiment, the mice were fasted but not watered for 12 h after the last oral gavage. After weighing, blood was collected from the eyeballs into EP tubes and centrifuged at 4°C, 3000 r / min for 15 min. After separation, serum was collected and placed in 1.5 mL centrifuge tubes, which were aliquoted and stored at -80°C.

[0140] 1.4 Determination of alcohol dehydrogenase (ALDH) content in mouse serum.

[0141] 1.4.1 Take out the ALDH kit from the refrigerated environment and equilibrate it at room temperature for 15-30 minutes. Take out the serum supernatant sample to be tested and equilibrate it at room temperature for 60 minutes.

[0142] 1.4.2 Set up standard wells, add 50μL of standard of different concentrations to each. Set up blank wells and test sample wells respectively. First add 40μL of sample diluent to the test sample well on the ELISA plate, and then add 10μL of the test sample. Add the sample to the bottom of the ELISA plate well, try not to touch the well wall, and gently shake to mix.

[0143] 1.4.3 Add 100 μL of enzyme-labeled reagent to each well except the blank well. Seal the plate with a sealing film and incubate at 37°C for 60 minutes.

[0144] 1.4.4 Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry. First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color at 37℃ in the dark for 15 minutes.

[0145] 1.4.5 Add 50 μL of stop solution to each well to stop the reaction. Use the blank well as zero and measure the absorbance of each well at 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0146] Table 6 ALDH content in each group

[0147] Group ALDH content (U / mg prot) n=5 Blank control group 27.96 Alcohol model group 10.62 Ethyl acetate extract of Psoralea corylifolia <![CDATA[12.12 ※ ]]> Enzymatic extract of bitter white tilapia <![CDATA[12.97 ※ ]]> Kubaiti Merged Group <![CDATA[13.43 ※ ]]> Fermentation composition group <![CDATA[15.86 ※ ]]> Phellinus linteus extract group <![CDATA[13.03 ※ ]]> Total extract high dose group <![CDATA[24.53 ※※※ ]]> Total extract medium dose group <![CDATA[20.57 ※※ ]]> Total extract low dose group <![CDATA[15.66 ※ ]]> Decoction group <![CDATA[11.21 ※ ]]>

[0148] Compared with the normal control group, the ALDH activity of mice in the model control group was decreased, which was statistically significant (P < 0.001); compared with the model control group, the ALDH activity of the total extract high, medium and low groups and the decoction group was increased, which was statistically significant (P < 0.001, P < 0.01, P < 0.05).

[0149] 2. Take the stomach of mice from the blank control group (normal saline), alcohol model group, Ipomoea batryticatus ethyl acetate extract group, Ipomoea batryticatus enzymatic extract group, Ipomoea batryticatus combined group, fermentation composition group, Phellinus igniarius extract group, total extract high-dose group, total extract medium-dose group, total extract low-dose group, and decoction group, accurately weigh the mouse stomach tissue and add 9 times the volume of homogenate medium (0.9% normal saline) at a ratio of weight (mg): volume (μL) = 1:9, grind at high speed in an ice water bath at 2500-3000r / min, centrifuge for 10min, and take the supernatant.

[0150] 2.1 TNF-α was determined by enzyme-linked immunosorbent assay (ELISA).

[0151] TNF-α is one of the indicators for measuring gastritis and gastric ulcer. Increased inflammation will lead to increased TNF-α levels. If the content of the pro-inflammatory factor TNF-α decreases after administration, it proves that inflammation has decreased.

[0152] 2.1.1 Dilute the supernatant with specimen diluent at a ratio of 1:1 to prepare the sample to be tested.

[0153] 2.1.2 Add 50uL of the diluted standard to the reaction well and 50μL of the sample to be tested to the reaction well. Immediately add 50μL of the biotin-labeled antibody, cover the membrane plate, gently shake to mix, and incubate at 37℃ for 1 hour.

[0154] 2.1.3 Shake off the liquid in the wells, fill each well with washing solution, shake for 30 seconds, shake off the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, increase the number of washes by one.

[0155] 2.1.4 Add 50 μL of streptavidin-HRP to each well, gently shake to mix, and incubate at 37°C for 30 minutes.

[0156] 2.1.5 Shake off the liquid in the wells, fill each well with washing solution, shake for 30 seconds, shake off the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times. If washing with a plate washer, increase the number of washes by one.

[0157] 2.1.6 Add 50 μL of substrate A and B to each well, shake gently to mix, and incubate at 37°C for 10 minutes. Avoid exposure to light.

[0158] 2.1.7 Take out the ELISA plate and quickly add 50 μL of stop solution. The results should be measured immediately after adding the stop solution.

[0159] 2.1.8 Measure the OD value of each well at a wavelength of 450 nm.

[0160] 2.2 TNF-α determination results of each group: blank control group 55.87±14.27pg / mL; alcohol model group 98.66±18.27pg / mL, total extract low-dose group 72.56±13.45pg / mL, total extract medium-dose group 68.32±14.44pg / mL; total extract high-dose group 54.69±11.90pg / mL.

[0161] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0162] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A drug for sobering up and / or protecting gastric mucosa, characterized in that: The invention comprises active pharmaceutical ingredients and pharmaceutically acceptable auxiliary materials, wherein the active pharmaceutical ingredients comprise at least one of ethyl acetate extract of Psoralea corylifolia, enzymatic hydrolysis extract of Psoralea corylifolia, fermentation composition and mulberry linterus extract.

2. The drug according to claim 1, characterized in that The ethyl acetate extract of Ipomoea batryticatus is obtained by ultrasonic extraction of Ipomoea batryticatus with ethyl acetate; The enzymatic hydrolysis extract of bitter white ti is obtained by reflux extraction of bitter white ti with an organic solvent after enzymatic hydrolysis; The fermentation composition is obtained by reflux extraction after substrate fermentation, and the substrate includes Inonotus maculatus, Ganoderma lucidum, and Ganoderma lucidum; The mulberry linterus extract is obtained from mulberry linterus by an ultrasonic-flash combined extraction method.

3. The drug according to claim 1, characterized in that The dosage form of the drug includes oral preparations or injection preparations.

4. Use of a Kubaiti extract in the preparation of a drug for sobering up and / or protecting gastric mucosa, wherein the Kubaiti extract comprises at least one of a Kubaiti ethyl acetate extract and a Kubaiti enzymatic hydrolysis extract.

5. Use of a fermentation composition in the preparation of a drug for sobering up and / or protecting gastric mucosa, wherein the fermentation composition is obtained by reflux extraction after substrate fermentation, and the substrate comprises Inonotus maculatus, Ganoderma lucidum, and Ganoderma lucidum.

6. Use of a mulberry linterus extract in the preparation of a drug for sobering up and / or protecting gastric mucosa, wherein the mulberry linterus extract is obtained from mulberry linterus by an ultrasonic-flash combined extraction method.

7. A method for preparing the ethyl acetate extract of Ipomoea batryticatus in the medicine according to any one of claims 1 to 3 or the ethyl acetate extract of Ipomoea batryticatus in the application according to claim 4, characterized in that: The method comprises the following steps: crushing the bitter white tibia, adding ethyl acetate, performing ultrasonic extraction, taking extracting liquid, removing ethyl acetate, and obtaining the bitter white tibia ethyl acetate extract.

8. A method for preparing the enzymatic hydrolyzed extract of the herba kubaiti in the medicine according to any one of claims 1 to 3 or the enzymatic hydrolyzed extract of the herba kubaiti in the application according to claim 4, characterized in that: The method comprises the following steps: mixing bitter white hoof with enzyme, performing enzymolysis, filtering, taking filter residue, adding organic solvent for reflux extraction, extracting filtrate, and obtaining bitter white hoof enzymolysis extract.

9. A method for preparing the fermentation composition in the medicine according to any one of claims 1 to 3 or the fermentation composition in the application according to claim 5, characterized in that: The following steps are involved: Inonotus maculata, Ganoderma lucidum and Ganoderma lucidum are mixed, crushed, and water is added to form a suspension. Fermented seed liquid is inoculated into the suspension, shaken and cultured, a solvent is added for reflux extraction, and an extract is taken to obtain a fermentation composition.

10. A method for preparing the Phellinus linteus extract in the medicine according to any one of claims 1 to 3 or the Phellinus linteus extract in the application according to claim 6, characterized in that: The method comprises the following steps: crushing Phellinus igniarius, adding water, performing ultrasonic extraction to obtain a feed liquid, performing flash extraction to obtain an extract, centrifuging, taking a supernatant, concentrating, precipitating with alcohol, standing, taking a precipitate, centrifuging, and obtaining a Phellinus igniarius extract.

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