CN114748566B - Anti-alcohol liver-protecting composition and application thereof - Google Patents

Anti-alcohol liver-protecting composition and application thereof Download PDF

Info

Publication number
CN114748566B
CN114748566B CN202210365794.6A CN202210365794A CN114748566B CN 114748566 B CN114748566 B CN 114748566B CN 202210365794 A CN202210365794 A CN 202210365794A CN 114748566 B CN114748566 B CN 114748566B
Authority
CN
China
Prior art keywords
liver
extract
effervescent tablet
alcohol
yeast
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210365794.6A
Other languages
Chinese (zh)
Other versions
CN114748566A (en
Inventor
宋立孝
谭余庆
杨米一
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Enzhong Shandong Digital Health Development Co ltd
Original Assignee
Enzhong Shandong Digital Health Development Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Enzhong Shandong Digital Health Development Co ltd filed Critical Enzhong Shandong Digital Health Development Co ltd
Priority to CN202210365794.6A priority Critical patent/CN114748566B/en
Publication of CN114748566A publication Critical patent/CN114748566A/en
Application granted granted Critical
Publication of CN114748566B publication Critical patent/CN114748566B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/488—Pueraria (kudzu)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • A23L33/145—Extracts
    • 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
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/287—Chrysanthemum, e.g. daisy
    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0007—Effervescent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • 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
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • 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

Abstract

The invention provides an anti-alcoholic liver protection composition and application thereof, belonging to the technical field of medicines and foods, wherein the anti-alcoholic liver protection composition comprises 35-45 parts of traditional Chinese medicine extracts, 0.2-0.4 part of yeast extract I, 0.2-0.4 part of yeast extract II and 0.3-0.5 part of garlic extract, and the traditional Chinese medicine comprises radix puerariae: flower of kudzu vine: the mass ratio of the chrysanthemum is 1-3: 1 to 3:2 to 6. The composition can effectively remove oxygen free radicals, reduce the harm of the oxygen free radicals to liver cells, has antibacterial, anti-inflammatory and immunity-enhancing physiological activities, and achieves good effects of dispelling the effects of alcohol and protecting the liver.

Description

Anti-alcohol liver-protecting composition and application thereof
Technical Field
The invention belongs to the technical field of medicines and foods, and particularly relates to an anti-alcohol and liver-protecting composition and application thereof.
Background
Alcoholic Liver Disease (ALD) is a liver disease resulting from long-term, high-volume alcohol consumption, and includes 4 major classes of Alcoholic Fatty Liver (AFL), alcoholic Hepatitis (AH), alcoholic liver fibrosis (AHF), and Alcoholic Cirrhosis (AC). The number of deaths from alcoholism is 250 tens of thousands worldwide, accounting for 4% of deaths, with 40% of deaths due to cirrhosis being due to ALD. Alcohol abuse, particularly ALD caused by alcoholism in young people, has become a major public health problem throughout society.
Oxidative Stress (OS) produced during ethanol metabolism plays a critical role in the pathogenesis of alcoholic liver injury, and hepatocyte injury is closely related to Reactive Oxygen Species (ROS). Ethanol metabolism produces large amounts of Nicotinamide Adenine Dinucleotide (NADH), resulting in an increase in the ratio of ADH/NAD+ in the cytoplasm or mitochondria, thereby promoting an increase in the mitochondrial respiratory chain electron transport stream, producing large amounts of ROS in the liver. The large accumulation of ROS can lead to unbalanced oxidation-reduction reaction in liver, so that lipid peroxidation damage is caused to liver cells, and ROS can attack DNA chains to cause breakage, fragment deletion or insertion and other mutations to cause possible mutation to the liver cells. ROS react with unsaturated fatty acids in the biological membrane, so that the unsaturated fatty acids in the membrane are reduced, the permeability of the membrane is enhanced, and thus the structure and function of liver cells are damaged, and when the liver cells are damaged, AST (and in mitochondria) and ALT in the cells enter the blood through the cell membrane; lipid peroxidation products such as malondialdehyde, etc., polymerize with proteins or DNA to form adducts, inducing the expression of a large number of cellular inflammatory factors, resulting in hepatocellular injury.
The researches prove that the kudzuvine root, the kudzuvine flower and the chrysanthemum contain rich flavonoid substances, the flavonoid compounds belong to phenolic acid compounds, have the functions of capturing oxygen free radicals, removing the oxygen free radicals, reducing the harm of the oxygen free radicals to organisms, and have good physiological activities of inhibiting the free radicals, resisting DNA damage, resisting bacteria, resisting inflammation, protecting liver, enhancing immunity and the like. The bioactive components such as zinc, B vitamins, and reduced glutathione contained in yeast can be used as synthetic raw materials of organism reaction enzymes, and can effectively remove oxygen ions and free radicals in vivo to protect liver cells. Garlic is one kind of bulb formed in Allium plant of Liliaceae, and is one kind of vegetable with rich phenolic acid, flavone, flavonol, flavone and other bioactive compounds with liver protecting, immunity regulating, antioxidant, anticoagulant, cardiac muscle protecting, antiviral, hypolipemic, intestinal flora regulating, etc. and has garlic polysaccharide content over 70%, small molecular heteropolysaccharide and excellent biological function. However, up to now, it has not been reported whether the Chinese medicinal composition with the functions of dispelling the effects of alcohol and protecting liver can be prepared by mixing the extracts of kudzuvine root, kudzuvine flower and chrysanthemum, the yeast extract and the garlic extract.
Disclosure of Invention
Therefore, the invention aims to provide the anti-alcoholic liver protection composition which has remarkable anti-alcoholic liver protection effect through the synergistic effect of the compositions.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an anti-alcohol liver-protecting composition which comprises the following components in parts by mass:
35-45 parts of traditional Chinese medicine extract, 0.2-0.4 part of yeast extract I, 0.2-0.4 part of yeast extract II and 0.3-0.5 part of garlic extract;
the Chinese medicinal materials are prepared from radix Puerariae: flower of kudzu vine: the mass ratio of the chrysanthemum is 1-3: 1 to 3:2 to 6.
Preferably, the preparation method of the traditional Chinese medicine extract comprises the following steps: mixing the Chinese medicinal materials with 60-85% ethanol solution according to a feed-liquid ratio of 1:8-15, soaking for 7-12 h, heating and refluxing for 1.5-3 h to obtain a liquid medicine, concentrating and drying.
Preferably, the preparation method of the yeast extract I comprises the following steps: dry yeast and Na of 0.032-0.066 mol/L 2 HPO 4 Mixing the solutions according to a feed-liquid ratio of 1:3-5, extracting for 2.5-4 h, filtering with 200-400 meshes to obtain supernatant, and freeze-drying.
Preferably, the preparation method of the yeast extract II comprises the following steps: mixing dry yeast and water according to a feed-liquid ratio of 1:8-12, incubating in a water bath at 35-40 ℃ for 15-30 min to obtain bacterial suspension, filtering the bacterial suspension by 80-200 meshes, and taking a precipitate; suspending the precipitate in buffer solution again according to the mass-volume ratio of 1:3-5, homogenizing and crushing yeast at high speed, centrifuging to obtain supernatant, and freeze-drying the supernatant at-20 ℃ to-40 ℃ for 48-72 h.
Preferably, the crushing conditions are: stirring for 4-5 min under 6000-8000 r/min, and then continuing stirring for 6-8 min under 15000-20000 r/min.
Preferably, the preparation method of the garlic extract comprises the following steps: after the garlic is primarily ground and crushed, adding buffer solution with the volume of 4-6 times, and continuously grinding for 15-30 min; and then heating the mixed solution at 50-60 ℃ for 15-30 min, filtering with 200-400 meshes to obtain supernatant, namely the garlic extract, and freeze-drying the garlic extract at-20-40 ℃ for 48-72 h.
The invention also provides application of the composition in preparation of food, health-care product or medicine for dispelling effects of alcohol and protecting liver.
The invention also provides application of the composition in preparing a medicament for treating or preventing liver injury, wherein the liver injury is caused by alcohol.
Preferably, the composition is added with pharmaceutically acceptable auxiliary materials to prepare a medicament, wherein the medicament comprises tablets, pills and powder.
The invention also provides an anti-alcohol liver-protecting effervescent tablet which comprises the composition and pharmaceutically acceptable auxiliary materials.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the kudzu root, the kudzu flower and the chrysanthemum extract are mixed with the yeast extract and the garlic extract in proportion, so that the raw materials are synergistic, the obtained composition can promote the elimination of acetaldehyde, reduce the concentration of ethanol in blood, shorten the drunk sleeping time and achieve the effect of rapidly dispelling the effects of alcohol. Meanwhile, the composition can effectively remove oxygen free radicals, improve the activity of antioxidant enzyme, reduce lipid peroxidation products, further reduce the harm of the oxygen free radicals to organisms, play a role in protecting liver cells, and can be used for preventing or relieving liver injury caused by alcohol.
The composition has the advantages of simple and easily obtained raw materials, low cost, obvious effects of dispelling the effects of alcohol and protecting liver, few side effects and safe and stable drug effect.
The effervescent tablet is prepared by adding pharmaceutically acceptable auxiliary materials, is convenient to take and carry, and can achieve good effects of dispelling the effects of alcohol and protecting the liver.
Drawings
FIG. 1 shows the activity of glutamic-pyruvic transaminase (AST) and glutamic-oxaloacetic transaminase (ALT) in mouse serum;
FIG. 2 shows Triglyceride (TG) content in liver tissue of mice;
FIG. 3 shows reduced Glutathione (GSH) content in liver tissue of mice;
FIG. 4 shows Malondialdehyde (MDA) content in liver tissue of mice;
FIG. 5 shows changes in liver pathology in mice (HE X100); A. blank group, model group, compound liver benefiting tablet group, effervescent tablet No.1 dose group, effervescent tablet No. E dose group, effervescent tablet No. 2 dose group, effervescent tablet No. 3 dose group, effervescent tablet No. G dose group, effervescent tablet No. 4 dose group;
FIG. 6 shows changes in liver pathology in mice (HE X400); A. blank group, model group, compound liver benefiting tablet group, effervescent tablet No.1 dose group, effervescent tablet No. E dose group, effervescent tablet No. 2 dose group, effervescent tablet No. 3 dose group, effervescent tablet No. G dose group, effervescent tablet No. 4 dose group;
FIG. 7 shows changes in liver pathology in mice (oil red staining X100); A. blank group, model group, compound liver benefiting tablet group, effervescent tablet No.1 dose group, effervescent tablet No. E dose group, effervescent tablet No. 2 dose group, effervescent tablet No. 3 dose group, effervescent tablet No. G dose group, effervescent tablet No. 4 dose group;
FIG. 8 shows changes in liver pathology in mice (oil red staining X400); A. blank group, model group, compound liver benefiting tablet group, effervescent tablet No.1 dose group, effervescent tablet No. E dose group, effervescent tablet No. 2 dose group, effervescent tablet No. 3 dose group, effervescent tablet No. G dose group, effervescent tablet No. 4 dose group;
FIG. 9 is a morphology of mouse liver cells; A. blank group, model group, compound liver benefiting tablet group, effervescent tablet No.1 dose group, effervescent tablet No. E dose group, effervescent tablet No. 2 dose group, effervescent tablet No. 3 dose group, effervescent tablet No. G dose group, effervescent tablet No. 4 dose group;
FIG. 10 is a morphology of mouse liver subcellular; A. blank group, model group, compound liver benefiting tablet group, effervescent tablet No.1 dose group, effervescent tablet No. E dose group, effervescent tablet No. 2 dose group, effervescent tablet No. 3 dose group, effervescent tablet No. G dose group, effervescent tablet No. 4 dose group;
FIG. 11 is a mitochondrial morphology of mouse liver; A. blank group, model group, compound liver benefiting tablet group, effervescent tablet No.1 dose group, effervescent tablet No. E dose group, effervescent tablet No. 2 dose group, effervescent tablet No. 3 dose group, effervescent tablet No. G dose group, effervescent tablet No. 4 dose group.
Detailed Description
The invention provides an anti-alcohol liver-protecting composition, which comprises the following components: 35-45 parts of traditional Chinese medicine extract, 0.2-0.4 part of yeast extract I, 0.2-0.4 part of yeast extract II and 0.3-0.5 part of garlic extract; the Chinese medicinal materials are prepared by mixing radix Puerariae, flos Puerariae Lobatae and flos Chrysanthemi.
The traditional Chinese medicine raw materials and the pharmacological activity of the traditional Chinese medicine raw materials used in the invention are as follows:
radix Puerariae is dry root of Pueraria lobata Ohwi of Pueraria of Leguminosae. Mainly contains flavonoid substances such as daidzin, daidzein, puerarin, etc., and also contains daidzein-4, 7-diglucoside, puerarin-7-xyloside, puerarin, isoflavone glycoside and starch. Has effects of relieving fever, promoting eruption, promoting salivation, quenching thirst, and invigorating yang. Is mainly used for treating exterior syndrome fever, strong pain of neck and back, measles, thirst due to heat disease, diabetes due to yin deficiency, heat-clearing dysentery, spleen deficiency diarrhea.
Flos Puerariae Lobatae is flower of Pueraria lobata Ohwi of Leguminosae. Mainly contains irisin, genistein, tricin, kudzuvine flower glycoside, triterpene saponin and volatile oil. Has effects of relieving alcoholic intoxication, activating spleen, stopping bleeding, and can be used for treating alcoholic intoxication, dysphoria, thirst, headache, dizziness, abdominal distention, vomiting, anorexia, hematemesis, and intestinal wind.
Flos Chrysanthemi is dry head flower of Chrysanthemum morifolium Ramat of Chrysanthemum of Compositae. The essential oil contains borneol, camphor, chrysanthenone, etc., and also contains chrysantheside, adenine, choline, flavone, stachydrine, vitamin A, vitamin B1, vitamin E, amino acid, locust element, etc. Has effects of dispelling pathogenic wind and heat, suppressing liver yang, removing liver fire for improving eyesight, and clearing heat and toxic materials.
The invention uses kudzuvine root: flower of kudzu vine: the mass ratio of the chrysanthemum is 1-3: 1 to 3: 2-6, and extracting active ingredients from the mixture, wherein a certain synergistic effect exists between active substances in the extract obtained by the mixture ratio, so that the scavenging capability of oxygen free radicals can be obviously improved, and a better alcohol dispelling and liver protecting effect is achieved. Radix Puerariae: flower of kudzu vine: the mass ratio of the chrysanthemum is further preferably 1:1:2.
the invention does not limit the specific sources of the kudzuvine root, the kudzuvine flower and the chrysanthemum.
In the invention, the preparation method of the traditional Chinese medicine extract comprises the following steps: mixing the Chinese medicinal materials with 60-85% ethanol solution according to a feed-liquid ratio of 1:8-15, soaking for 7-12 h, and heating and refluxing for 1.5-3 h to obtain the medicinal liquid. Wherein the concentration of ethanol is preferably 80%, the feed-liquid ratio is preferably 1:10-12 g/mL, the soaking time is preferably 8-10 h, and the heating reflux time is preferably 2-2.5 h. The heating reflux operation of the invention adopts a conventional heating reflux device, and the heating temperature is 40-60 ℃, and more preferably 50-55 ℃. By the treatment, the flavonoid compounds contained in the traditional Chinese medicine mixture can be effectively extracted, the extraction rate and the flavonoid content are high, and the impurity content in the extract can be reduced, so that the extract is easy to concentrate and dry.
The invention filters the Chinese medicine extract to remove residue, as an optional implementation mode, the invention directly passes the liquid medicine obtained by reflux through a 200-300 mesh sieve, and filters the residue; as another alternative, the invention directly adopts vacuum filtration to remove the residue. The invention is not limited to a specific filtering and deslagging mode.
The invention carries out concentration and drying on the obtained traditional Chinese medicine extract. As an alternative implementation mode, the invention carries out alcohol recovery treatment on the obtained liquid medicine, further concentrates residual liquid, prepares dry paste by vacuum drying concentrated liquid, and grinds the dry paste and screens the dry paste with 40-60 meshes for standby. The specific concentrating and drying mode of the Chinese medicine extract is not limited.
In the present invention, the preparation method of the yeast extract I comprises: dry yeast and Na of 0.032-0.066 mol/L 2 HPO 4 Mixing the solutions according to a feed-liquid ratio of 1:3-5, extracting for 2.5-4 h, and filtering to obtain supernatant. Wherein Na is 2 HPO 4 The concentration of the solution is preferably 0.04-0.06 mol/L, the feed-liquid ratio is preferably 1:4g/mL, and the extraction time is preferably 3h.
The extraction method comprises the following steps: the feed liquid mixture is put in a water bath with the temperature of 36-37 ℃ for heat preservation for 2-4 hours, and is continuously stirred in the heat preservation process, and the operation is favorable for the dispersion of yeast in the solution and the transfer of active substances.
The invention screens the mixed extract by a 200-400 mesh sieve, keeps the supernatant at 50-55 ℃ for 15-20 min, continuously stirs, immediately cools the mixed extract to room temperature in cold water at 0 ℃ after the heat preservation is finished, and filters the mixed extract again at 200-400 mesh (4 ℃) and collects filtrate. Preferably, the invention screens the mixed extracts through 300 meshes respectively to obtain the supernatant. The invention can separate the extracting solution by using a filter screen or a plate filter and other instruments, does not require the specific used instruments, and can purify the active ingredients in the extracting solution by the method so as to improve the pharmacological action of the yeast extract I.
The supernatant obtained by filtration is freeze-dried for 48-72 hours at the temperature of minus 20 ℃ to minus 40 ℃, and the dried paste is ground into powder and sieved by a sieve with 40-60 meshes for standby. The temperature is more preferably-30 ℃ to-35 ℃, the time is more preferably 55 to 60 hours, and the particle size is more preferably 45 to 55 meshes.
The preparation method can obtain the yeast extract with higher activity, can effectively promote the decomposition of ethanol in the organism, reduce the concentration of ethanol in blood, and can realize good anti-alcohol effect after being matched with other components.
In the present invention, the preparation method of the yeast extract II comprises: mixing dry yeast and water according to a feed-liquid ratio of 1:8-12, incubating in a water bath at 35-40 ℃ for 15-30 min to obtain bacterial suspension, centrifuging the bacterial suspension, and taking a precipitate; suspending the precipitate in buffer solution again according to the mass-volume ratio of 1:3-5, crushing yeast suspension in ice-water bath by using a high-speed homogenizer, and filtering to obtain supernatant. Wherein the feed liquid ratio is preferably 1:9-10 g/mL, the water temperature is preferably 37-38 ℃, the incubation time is preferably 20-25 min, and the mass-volume ratio is preferably 1:4g/mL.
The invention carries out centrifugal treatment on bacterial suspension, and abandons supernatant and leaves sediment. The filtering conditions of the invention are as follows: 80 to 200 mesh, preferably 100 to 150 mesh. As an alternative implementation mode, the sediment obtained by centrifugation can be washed for 1-3 times by distilled water, filtered again under the condition of 80-200 meshes after washing, and the sediment is reserved and stored in a refrigerator at 4 ℃ for standby. The invention removes the upper layer liquid through the filtering treatment of the step to obtain a large number of complete yeast cells.
The invention resuspends the yeast cell pellet in buffer. As an alternative embodiment, the buffer of the present invention is Na 2 HPO 4 -KH 2 PO 4 ，pH 8.0。
The invention utilizes a high-speed homogenizer to crush cell sap, wherein the crushing conditions are as follows: crushing for 4-5 min at the temperature of 4 ℃ and the speed of 6000-8000 r/min, and then continuously crushing for 6-8 min at the speed of 15000-20000 r/min. Further preferably, the crushing is carried out for 4.5min under 6500-7000 r/min, and then the crushing is continued for 7min under 17000-18000 r/min. The invention breaks the yeast cells through high-speed homogenization treatment, and the yeast cytoplasmic matrix components flow out so as to facilitate the subsequent separation and extraction.
The invention carries out centrifugal treatment on the yeast crushed liquid and collects supernatant. The filtering condition of the invention is 200-400 mesh filtering to obtain supernatant, and more preferably 300-350 mesh filtering. The purpose of the filtration herein of the invention is to discard the organelle and nuclear components of the precipitated yeast cells and collect the cytoplasmic matrix components of the yeast cells.
The invention freeze-dries the supernatant fluid obtained by separation for 48-72 hours at the temperature of minus 20 ℃ to minus 40 ℃, and grinds the dry paste and screens the dry paste with a sieve of 40-60 meshes for standby. The temperature is more preferably-30 ℃ to-35 ℃, the time is more preferably 55 to 60 hours, and the particle size is more preferably 45 to 55 meshes.
The yeast extract II can be obtained by adopting the preparation method. The yeast extract II contains a large amount of active ingredients such as intracellular enzymes, has the effect of promoting the decomposition of acetaldehyde in the organism, can reduce the damage of acetaldehyde to liver cells, and can realize good effects of dispelling the effects of alcohol and protecting liver after being matched with other components.
In the present invention, the preparation method of the garlic extract comprises: grinding and crushing garlic, and then adding buffer solution with the volume of 4-6 times to continuously grind for 15-30 min; then heating the mixed solution at 50-60 ℃ for 15-30 min, and filtering with 200-400 meshes to obtain supernatant. Wherein, the buffer solution is added in an amount of preferably 5 times, the grinding time is preferably 20-25 min, the heating temperature is preferably 55 ℃, the treatment time is preferably 20-25 min, and the filtration pore diameter is preferably 300 meshes.
The invention adds buffer to continue grinding garlic, as an alternative embodiment, the buffer is phosphate buffer (pH 7.8). The invention adds buffer solution for grinding, which can dissolve active substances (especially superoxide dismutase) in garlic into buffer solution, thus facilitating the subsequent extraction of pharmacological active substances.
The invention carries out centrifugation on the ground garlic-buffer mixture, abandons the sediment, and leaves the supernatant containing the active ingredients. The filtering conditions are as follows: the supernatant is collected by filtration through 200 to 400 mesh, more preferably 300 to 350 mesh.
The garlic extract obtained by the invention has rich active enzymes, micromolecular heteropolysaccharide, flavone, phenolic acid and other substances with pharmacological activity, so as to improve the antioxidant, immunity enhancing and blood fat reducing capabilities of the composition.
The invention carries out freeze drying on the supernatant obtained by separation at the temperature of minus 20 ℃ to minus 40 ℃ for 48 to 72 hours, and the dried paste is ground into powder and passes through a 40 to 60-mesh sieve for standby. The temperature is more preferably-35 ℃ to-38 ℃, the time is more preferably 50 to 65 hours, and the particle size is more preferably 45 to 55 meshes.
The preparation method of the anti-alcohol liver-protecting composition comprises the following steps: mixing the above Chinese medicinal materials extract, yeast extract I, yeast extract II, and Bulbus Allii extract uniformly according to mass parts. Preferably, the composition comprises the following components in percentage by weight: 38-42 parts of traditional Chinese medicine extract, 0.25-0.35 part of yeast extract I, 0.25-0.35 part of yeast extract II and 0.35-0.45 part of garlic extract; more preferably, the Chinese medicinal material extract is 40 parts, the yeast extract I is 0.3 part, the yeast extract II is 0.3 part, and the garlic extract is 0.4 part.
The anti-alcoholic liver protection composition can be directly used as a main component in food, health products or medicines for anti-alcoholic liver protection, is used for rapidly dispelling the effects of alcohol after drunk, and is used for preventing, relieving or treating liver injury caused by alcohol.
The anti-alcohol liver-protecting composition is taken as a main medicinal component, and pharmaceutically acceptable auxiliary materials are added to prepare medicaments, wherein the medicaments comprise, but are not limited to, tablets, pills and powder. As an alternative implementation mode, the effervescent tablet can be prepared, is convenient to carry and take, and has good effects of dispelling effects of alcohol and protecting liver.
The invention provides an anti-alcohol liver-protecting effervescent tablet, which comprises the following components in percentage by mass: 35 to 45 percent of traditional Chinese medicine extract, 0.2 to 0.4 percent of yeast extract I, 0.2 to 0.4 percent of yeast extract II, 0.3 to 0.5 percent of garlic extract and the balance of common auxiliary materials of effervescent tablets. The invention does not limit the selection of specific auxiliary materials.
The invention utilizes the anti-alcohol liver-protecting composition to prepare the anti-alcohol liver-protecting effervescent tablet. As an alternative implementation mode, the invention weighs Chinese medicinal material extract, yeast extract I, yeast extract II, garlic extract and auxiliary materials (acid source, alkali source, lactose and sweetener) are evenly mixed, 5-10% absolute ethyl alcohol PVP solution is used for preparing soft materials, 10-14 mesh sieve granulation is carried out, 50-70 min baking is carried out in a baking oven at 45-50 ℃, the soft materials are taken out, and the soft materials are granulated again through 10-14 mesh sieve and then tabletting is carried out. The invention does not limit the specific preparation process of the effervescent tablet.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the specific embodiment of the invention, animals are adopted as follows: ICR male mice 70, SPF grade, 14-16g, purchased from Peking Vitre Liwa laboratory animal technologies Co., ltd., animal quality eligibility number: no.1100112111113775142.
Triglyceride kit (TG, single reagent GPO-PAP method): south Beijing institute of biological engineering, lot number: 20210911; glutamic-oxaloacetic transaminase (AST, trace enzyme labeling method): south Beijing institute of biological engineering, lot number: 20211117; glutamic pyruvic transaminase (ALT, trace enzyme labeling method): south Beijing institute of biological engineering, lot number: 20210728; malondialdehyde test box (MDA): south Beijing institute of biological engineering, lot number: 20210728; reduced glutathione (GSH, microplate method): south Beijing institute of biological engineering, lot number: 20210827; electron microscope fixing liquid, hundred kilowatt biology, lot number: b0012;4% paraformaldehyde fixing solution, beijing Soy Bao technology Co., ltd. Compound Yiganling tablet with the specification of 0.32g multiplied by 100 tablet is prepared from Jiangsu Zhongxing pharmaceutical industry Co., ltd., national medicine standard character: z32020664.
In the invention, SPSS 20.0 statistical software is adopted for data processing, metering data is represented by mean ± standard deviation, a plurality of groups of samples meet the normal test and the variance alignment, variance analysis is adopted, pairwise comparison between groups adopts paired t test, and P <0.05 is used as difference, thus having statistical significance.
In the present invention, P <0.05 compared to the blank group, P <0.01 compared to the blank group, P <0.001 compared to the blank group; # indicates P <0.05 compared to the model group, # indicates P <0.01 compared to the model group, and # indicates P <0.001 compared to the model group.
Example 1
In this embodiment, the method for extracting the extract of the Chinese medicinal material is designed in an orthogonal manner.
Mixing radix Puerariae, flos Puerariae Lobatae and flos Chrysanthemi powder at a mass ratio of 1:1:2 with ethanol solution, soaking for 10 hr, and heating and refluxing to obtain medicinal liquid. Wherein the extraction process parameters are subjected to orthogonal experiments according to table 1, and the optimal extraction method is subsequently determined by evaluating the extraction rate of the active ingredients.
TABLE 1 extraction method orthogonal design table
The content of the above effective substances in the extract was measured by high performance liquid chromatography using puerarin, tectoridin, and rutin as representative components, and the results are shown in Table 2.
Table 2 active ingredient content (%, w/w) in the extract
As can be seen from Table 2, the content of puerarin, tectoridin and rutin in the traditional Chinese medicine extract obtained by the method in experiment No. 9 is significantly higher than that of other experiment groups, which shows that the traditional Chinese medicine extract with higher flavonoid content can be obtained by extracting for 1 time only by adopting 80% ethanol solution with the feed liquid ratio of 1:15 for 3 hours, so the method in experiment No. 9 is the optimal extraction process.
Example 2
The anti-hangover and liver-protecting composition is prepared in the embodiment.
(1) Preparation of traditional Chinese medicine extract:
radix Puerariae: flower of kudzu vine: the chrysanthemum is prepared from the following components in percentage by mass: 1:2, adding 80% ethanol solution according to the feed-liquid ratio of 1:15g/mL, soaking for 10h, and heating (55 ℃) and refluxing for 3h. Directly sieving the obtained medicinal liquid with 200 mesh sieve, filtering to remove residue, and concentrating and drying the obtained Chinese medicinal extractive solution. Grinding the dry paste, and sieving with a 40-mesh sieve for standby.
(2) Preparation of Yeast extract I:
adding 0.05mol/L Na into the dry yeast according to the feed liquid ratio of 1:4g/mL 2 HPO 4 The solution is kept in a water bath at 37 ℃ for 3 hours, stirring is continued during the period, and the solution is extracted for 3 hours at normal temperature after the heat preservation is finished. The combined extracts were filtered using a 200 mesh screen. The obtained solution is kept at 55 ℃ for 15min, and is continuously stirred, and is immediately placed in cold water at 0 ℃ to be cooled to room temperature after the heat preservation is finished, and is filtered again under 400 meshes, the obtained solution is frozen and dried for 48h at-40 ℃, and the obtained dry paste is ground into powder and is sieved by a 40-mesh sieve for standby.
(3) Preparation of Yeast extract II:
mixing dry yeast and water according to a feed liquid ratio of 1:10g/mL, incubating in a water bath at 37 ℃ for 20min to obtain a bacterial suspension, filtering the bacterial suspension by 100 meshes, and discarding the supernatant; the precipitate was washed 1 time with distilled water, and after washing, filtered again (100 mesh) to leave the precipitate.
The pellet was resuspended in 0.05mol/L PBS (Na) at a mass-to-volume ratio of 1:4g/mL 2 HPO 4 -KH 2 PO 4 pH 8.0) in a buffer solution, crushing the obtained yeast suspension by a high-speed refiner in an ice-water bath (crushing is divided into two steps, stirring is carried out for 4min under 8000r/min, then stirring is continued for 8min under 18000 r/min), filtering by a 400-mesh screen after crushing, and collecting supernatant. Freeze drying supernatant at-30deg.C for 72 hr, grinding into powder, and sieving with 40 mesh sieve.
(4) Preparation of garlic extract:
grinding peeled garlic cloves, crushing, adding 0.05mol/L phosphate buffer (pH 7.8) with 5 times of volume, and continuously grinding for 20min; and then heating the mixed solution at 55 ℃ for 20min, sieving with a 400-mesh sieve to obtain supernatant, freeze-drying at-30 ℃ for 72h, and grinding the obtained dry paste and sieving with a 40-mesh sieve for later use.
(5) Taking 40 parts of the traditional Chinese medicine extract obtained in the step (1), 0.3 part of the yeast extract I obtained in the step (2), 0.3 part of the yeast extract II obtained in the step (3) and 0.4 part of the garlic extract obtained in the step (4) according to parts by weight; mixing well.
Example 3
In the embodiment, the adding proportion of each raw material of the effervescent tablet is optimally designed. Weighing Chinese medicinal materials extract, yeast extract I, yeast extract II, mixing Bulbus Allii extract with adjuvants (acid source, alkali source, lactose, erythritol), making into soft mass with anhydrous ethanol PVP solution, granulating with 14 mesh sieve, oven drying at 48deg.C for 60min, taking out, sieving with 14 mesh sieve again, and tabletting.
In this example, the Chinese medicinal material extract, yeast extract I, yeast extract II, and garlic extract were obtained by extraction in example 2, respectively. In this example, yeast extract I: yeast extract ii: garlic extract = 3:3:4 to give a mixture. The dispersing agent comprises an acid source and a carbon source in proportion, wherein the acid source is citric acid, and the carbon source is sodium bicarbonate.
In this example, the experimental design was optimized according to table 3 for each raw material addition ratio.
Table 3 effervescent tablet raw material formulation (%, w/w)
The physical properties of the effervescent tablets obtained in each experimental group were determined according to the following criteria,
(1) And (3) pH value measurement: taking 1 effervescent tablet, putting into a 50mL beaker, adding 45 ℃ 20mL of water, standing until the effervescent tablet is completely disintegrated, and measuring the pH value of the solution.
(2) Determination of disintegration time: 1 tablet of effervescent tablet was put into 200mL of hot water at 45℃and the time from when the release of air bubbles began to time when the complete dissolution of the tablet dispersed and the escape of gas from the water ceased.
(3) And (3) gas production amount measurement: the mass of a 100mL beaker (50 mL of water at 20.+ -. 5 ℃ C.) was precisely determined by mass loss. Precisely weighing 1 piece of wine-transported effervescent tablet, placing in the beaker until gas around the tablet stops escaping, completely dissolving or dispersing the tablet in water, and weighing the total mass of the beaker and the solution.
The pH, disintegration time and gas yield of the effervescent tablets obtained for each group were measured 3 times in parallel and the average value was calculated, and the results are shown in table 4.
Table 4 physicochemical Properties of the effervescent tablets of each group
As can be seen from Table 4, the effervescent tablet obtained in experiment group 7 has an average disintegration time of 76s, a gas yield of 0.051g and a pH value of 5.45, and the overall situation is better than that of other experiment groups. Therefore, the relevant properties of the anti-alcoholic liver-protecting effervescent tablet prepared according to the proportion of 41 percent of anti-alcoholic liver-protecting composition (Chinese medicinal material extract: mixture=40:1), 45 percent of dispersing agent (citric acid: sodium bicarbonate 1:1.5), 5 percent of PVP, 2 percent of erythritol and 7 percent of lactose are the optimal combination.
Example 4
The traditional Chinese medicine composition obtained in the example 2 is adopted in the example to prepare the anti-alcohol liver-protecting effervescent tablet.
The effervescent tablet comprises the following raw materials in percentage by weight: 41% of an anti-alcohol liver-protecting composition, 45% of a dispersing agent, 5% of PVP, 2% of erythritol and 7% of lactose. The dispersing agent is formed by mixing citric acid and sodium bicarbonate according to the mass ratio of 1:1.5.
Respectively weighing the anti-hangover and liver-protecting composition, the dispersing agent, the erythritol and the lactose according to a certain proportion, uniformly mixing, preparing a soft material by using an absolute ethyl alcohol PVP solution, and granulating by a 14-mesh sieve. Drying soft material granule in oven at 50deg.C for 60min, sieving with 14 mesh sieve, and tabletting directly in tablet press.
Example 5
The experimental animals were randomly divided into 7 groups, which were blank control group, model group, compound YIGANLING tablet group, effervescent tablet No.1 dose group, effervescent tablet No. 2 dose group, effervescent tablet No. 3 dose group, and effervescent tablet No. 4 dose group, respectively.
Each group of mice is filled with gastric drug at 8 am every experimental day, wherein the experimental animals of the blank control group and the model group are filled with gastric purified water according to the administration volume of 15 ml/kg; the positive medicine group is subjected to stomach infusion according to the administration volume of 15ml/kg, and the administration dosage of the corresponding compound Yiganling tablet is 0.52g/kg (body weight); the effervescent tablet No.1 dose group, no. 2 dose group, no. 3 dose group and No. 4 dose group are respectively irrigated with stomach according to the administration volume of 15ml/kg, and the corresponding administration doses are 1.0g/kg (body weight), 2.0g/kg (body weight), 4.0g/kg (body weight) and 8.0g/kg (body weight). Each group was continuously intragastrically for 28 days. The method comprises the steps of pouring 53-degree white spirit into the stomach according to the proportion of 20mL/kg at 14 points, pouring 53-degree white spirit according to the proportion of 15mL/kg at 20 points, weighing the mice at the 30 th day of the experiment after 12 hours, taking out eyeballs, collecting blood, collecting the blood in a 2mL polyethylene centrifuge tube, taking liver tissues, and weighing.
1. Measurement of organ index of mice
All mice were fasted 8 pm before the day of sampling, fasted for 12h, and weighed the next day. The whole liver was taken out after the blood was taken out of the eyeball, and after washing with physiological saline, the fresh weight of the liver was weighed after the filter paper was dried, and the liver index was calculated, and the results are shown in Table 5.
Liver index = fresh liver weight (g)/body weight (g) ×100%
TABLE 5 liver index of mice
From table 5, it can be seen that modeling of alcohol significantly increases liver index of experimental animals, which is consistent with the side effects of alcohol in practice.
2. Determination of serum glutamic pyruvic transaminase (AST) and serum glutamic oxaloacetic transaminase (ALT) Activity in mice
Fresh blood from the mice collected was left at an incubator at 37℃for 2 hours and centrifuged (3000 rpm, i.e., 0 min), serum was separated, and absorbance (OD value) of each well was measured at wavelengths of 510nm and 505nm, respectively, according to the procedure of the corresponding kit, to thereby obtain corresponding AST and ALT activity data.
Mice AST and ALT activity assays are shown in FIG. 1. As can be seen from fig. 1, the activity of AST and ALT in serum of the model group was significantly increased compared to that of the blank group, and it was found that the model formation method using the alcohol lavage did cause clear liver damage to the experimental animals. When the compound YIGANLING and the effervescent tablet are used for treatment with different dosages, the activity of both transaminases is inhibited to a certain extent, and the results prove that the effervescent tablet has a certain protection effect on liver injury caused by alcohol.
3. Determination of protein content in mouse liver tissue
Accurately weighing liver tissue of a certain mass, adding physiological saline of which the volume is (mL) =1:9 according to the weight (g), mechanically homogenizing under the ice water condition to prepare 10% tissue homogenate, 2500 rpm, centrifuging for 10min, taking supernatant, diluting 30 times, mixing the diluted tissue homogenate with working solution according to the instruction in the kit, adding an orifice plate, setting a blank hole and a standard hole, carrying out color comparison by an enzyme-labeling instrument at 562nm wavelength, and reading absorbance. The total protein concentration of the sample was calculated according to the following formula:
protein concentration (μg/mL) = (measured OD value-blank OD value)/(standard OD value-blank OD value) ×standard concentration×dilution of sample before test
Standard samples were measured and a standard curve was constructed according to the instructions, the functional relationship between uv absorbance (Y) and protein concentration (C) y=0.7509c+0.0928 (R 2 ＝0.9954)。
4. Determination of triglyceride content in liver tissue of mice (TG)
Liver tissue homogenates were taken and OD values of each well were measured at 510nm wavelength using an enzyme-labeled instrument according to the kit instructions, and the results are shown in fig. 2.
Triglyceride content (mmol/L) = (measured OD value-blank OD value)/(standard OD value-blank OD value) ×standard substance concentration/protein concentration of sample to be measured
As shown in FIG. 2, the triglyceride content of the animals in the model and each treatment group was significantly increased as compared with that in the blank group, and it was confirmed that the study obtained an experimental animal model of alcoholic liver injury. After the compound Yiganling tablet and the effervescent tablet are used for preventive treatment for 28 days at different dosages, the TG content of each group is reduced compared with that of untreated model groups, and the effervescent tablet has a certain protection effect on alcoholic liver injury.
5. Determination of reduced glutathione content (GSH) in liver tissue of mice
Liver tissue homogenates were taken and OD values of each well were measured at a wavelength of 405nm using a microplate reader, according to the kit instructions, and the results are shown in fig. 3.
GSH content (nmol/gprot) = (measured OD value-blank OD value)/(standard OD value-blank OD value) ×standard substance concentration×test sample protein concentration in tissue
As shown in fig. 3, GSH content was reduced in each experimental group compared to the blank group, which is due to liver damage caused by alcohol modeling. After 30 days of preventive treatment, modeling, it was seen that the GSH content of each treatment group was increased relative to the model group, and likewise with increasing dose of effervescent tablets.
6. Measurement of malondialdehyde content in liver tissue of Mice (MDA)
Liver tissue homogenates were taken and OD values of each well were measured at 532nm wavelength with an enzyme-labeled instrument, according to the kit instructions, and the results are shown in fig. 4.
MDA content (nmol/gprot) in tissue= (measured OD value-blank OD value)/(standard OD value-blank OD value) ×standard substance concentration/protein concentration of sample to be measured
As shown in FIG. 4, after the alcohol gastric lavage, MDA content of each group was increased. After the compound YIGANLING tablet and the effervescent tablet with different dosages are used for treatment, the MDA content of the compound YIGANLING tablet is reduced relative to a model group, and the prevention and treatment method is proved to be adopted to relieve liver injury caused by alcohol, and the effervescent tablet No. 2 dose group has the most obvious effect of reducing the MDA content, and the data has obvious difference compared with the model group.
7. Histopathological examination
Taking a part of tissue of left liver leaves respectively, preparing slices according to different requirements, performing HE staining, and observing by using an optical microscope, wherein the HE staining results are shown in fig. 5 and 6; the oil red staining results are shown in fig. 7 and 8.
(1) HE staining: a part of left hepatic leaf tissue was cut into 1cm x 1cm pieces and placed in 4% paraformaldehyde for fixation. Three days later, liver tissue was removed for sampling and dehydration: washing with running water, alcohol overnight, alcohol placing dehydrated tissue into xylene, and observing transparency of tissue in xylene; placing the transparent tissue into melted paraffin, and then placing into paraffin; pouring the dissolved paraffin into an embedding frame, sequentially placing the tissue blocks into the embedding frame by using heated forceps, and marking; after cooling, the wax block was trimmed. Slicing in a slicing machine, spreading with warm water, taking clean glass slide, and collecting. The slice with the adhered tissue was placed in an oven for baking for about 4 hours.
1) Paraffin sections dewaxed to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethanol I10 min-absolute ethanol II 10min-95% alcohol 5min-90% alcohol 5min-80% alcohol 5min-70% alcohol 5 min-distilled water for washing.
2) Hematoxylin-stained nuclei: the slices are stained with Harris hematoxylin for 3-8min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for several seconds, washed with tap water, and returned to blue with 0.6% ammonia water, and washed with running water.
3) Eosin-stained cytoplasm: the slices are dyed in eosin dye solution for 1-3min.
4) And (3) removing the water sealing piece: sequentially placing the slices into 95% alcohol I5 min-95% alcohol II 5 min-absolute alcohol I5 min-absolute alcohol II 5 min-xylene I5 min-xylene II 5min for dehydration and transparency, taking out the slices from the xylene, slightly airing, and sealing the slices with neutral resin.
As can be seen from the HE staining of the liver tissue shown in FIG. 5 and FIG. 6, the blank liver cells are complete in morphology and the hepatic cables are orderly arranged. The model group can be used for obviously forming liver cell vacuoles, liver cable disorder, diffuse water sample denaturation, cell nucleus blurring and irregular eosinophilic block-shaped substance deposition (Mallly-Denk corpuscles) in cells. The swelling of the cells in each group given the drug treatment was reduced, the denaturation of the water sample was reduced and the cell arrangement was relatively ordered. The compound liver benefiting tablet and the effervescent tablet are obviously lower than the model group and the effervescent tablet low-dose group in all dosage groups and pathological conditions.
(2) Oil red dyeing
1) Freezing slices and airing at normal temperature;
2) Saturated oil red O dye liquor A: pure water = 3:2 preparing oil red working solution, and filtering for later use.
3) Slicing, and dyeing the oil red O working solution prepared in the step 2) for 7-10min (in dark place), and preparing the oil red O working solution for use.
4) Slicing into differentiation solution B, and differentiating for 5-10s.
5) Soaking and washing with distilled water for 2 times.
6) The sections are stained with hematoxylin staining solution C for 2-4min, soaked in distilled water for 2 times, differentiated with hematoxylin differentiation solution D for 2-3S, soaked in distilled water, and soaked in tap water for 2-3S, and microscopic examination.
7) And (5) sealing with glycerol gelatin.
From fig. 7 and 8, it can be seen that the model group has obvious steatosis, dense round lipid droplets are visible under the lens, lipid droplets exist in liver tissues of other groups of mice, but the lipid droplets in the liver tissues are reduced compared with the model group after 28 days of dry prognosis of the compound Yiganling tablet group and the effervescent tablet group with different doses, particularly, the dosage group lipid droplets in the effervescent tablet are obviously reduced, and the low dosage group lipid droplets of the effervescent tablet are not obviously changed.
8. Morphology of liver tissue
Taking partial tissue of left liver leaves of each group of mice, about grain size, and fixing the partial tissue in glutaraldehyde and 0.1% mol/L phosphate buffer solution precooled at 4 ℃.2 days later, the block is trimmed, about 1mm multiplied by 1mm, and the block is put into 0.01M PBS for 3 times, 15min-1% osmium acid is fixed for 2h-0.01M PBS for cleaning times, 15 min-ethanol gradient dehydration-100% acetone for 15min is carried out for 2 times, and acetone is carried out for 2 times: resin V/V1:1, 1 h-acetone: the V/V ratio of the resin is 1:2, and the resin is 2h embedded in the pure resin. Polishing the embedded resin blocks, slicing, staining with uranyl acetate and lead citrate, and microscopic examination.
The intervention effect of the effervescent tablet on acute alcoholic liver injury is observed from two angles of nucleus and mitochondrial micro morphology. As can be seen from fig. 9 and 10, the outline of the cells in the blank group is smooth, the nuclei are round and regular, and the nucleoli are clear; the cell outline of the model group is irregular, the cell nucleus is shrunken, nucleolus is fuzzy, and a large number of vacuoles are arranged in the plasma; after the drug treatment is given, the cell morphology of the compound Yiganling tablet group and the effervescent tablet No. 2-4 dose group is improved and changed, the vacuoles in the plasma are reduced, the nucleus is rounded, and the nucleolus is relatively clear. As can be seen from FIG. 11, the mitochondria of the blank group have smooth outline and clear kurtosis structure; the mitochondrial cristae of the model group is swollen, the outline disappears, and floccules appear in cytoplasm; however, this phenomenon was alleviated after treatment, and especially the improvement of effervescent tablet No. 3 and No. 4 dose groups was relatively remarkable, although the mitochondria were slightly swollen, the outline was smooth and the cristae structure was clear.
The results show that the effervescent tablet has a certain protection effect on liver tissue lesions, fat accumulation and other phenomena caused by alcoholic liver injury, and can effectively relieve liver tissue cavitation and reduce fat deposition in liver cells.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (3)

1. An effervescent tablet for treating or preventing liver injury is characterized by being prepared from 41% of an anti-alcohol liver-protecting composition, 45% of a dispersing agent, 5% of PVP, 2% of erythritol and 7% of lactose;
the dispersing agent is prepared from citric acid: sodium bicarbonate is obtained according to the proportion of 1:1.5;
the anti-alcohol liver-protecting composition is prepared from traditional Chinese medicine extracts: mixture=40:1 ratio;
the mixture is composed of yeast extract I: yeast extract ii: garlic extract = 3:3:4;
the Chinese medicinal materials are prepared from radix Puerariae: flower of kudzu vine: the mass ratio of the chrysanthemum is 1-3: 1 to 3:2 to 6, mixing to obtain;
the preparation method of the yeast extract I comprises the following steps: dry yeast and Na of 0.032-0.066 mol/L 2 HPO 4 Mixing the solutions according to a feed-liquid ratio of 1:3-5, extracting for 2.5-4 h, filtering with 200-400 meshes to obtain a supernatant, and freeze-drying;
the preparation method of the yeast extract II comprises the following steps: mixing dry yeast and water according to a feed-liquid ratio of 1:8-12, incubating in a water bath at 35-40 ℃ for 15-30 min to obtain bacterial suspension, filtering the bacterial suspension by 80-200 meshes, and taking a precipitate; suspending the precipitate in buffer solution again according to the mass volume ratio of 1:3-5, homogenizing and crushing yeast at high speed, centrifuging to obtain supernatant, and freeze-drying the supernatant at-20 ℃ to-40 ℃ for 48-72 h;
the preparation method of the garlic extract comprises the following steps: after the garlic is primarily ground and crushed, adding buffer solution with the volume of 4-6 times, and continuously grinding for 15-30 min; then heating the mixed solution at 50-60 ℃ for 15-30 min, filtering with 200-400 meshes to obtain supernatant, namely garlic extract, and freeze-drying the garlic extract at-20-40 ℃ for 48-72 h;
the liver injury is liver injury caused by alcohol.
2. The effervescent tablet of claim 1, wherein the method for preparing the extract of the chinese medicinal material comprises: mixing the Chinese medicinal materials with 60-85% ethanol solution according to a feed-liquid ratio of 1:8-15, soaking for 7-12 h, heating and refluxing for 1.5-3 h to obtain a liquid medicine, concentrating and drying.
3. The effervescent tablet of claim 1, wherein the crushing conditions are: stirring for 4-5 min under 6000-8000 r/min, and then continuing stirring for 6-8 min under 15000-20000 r/min.
CN202210365794.6A 2022-04-08 2022-04-08 Anti-alcohol liver-protecting composition and application thereof Active CN114748566B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210365794.6A CN114748566B (en) 2022-04-08 2022-04-08 Anti-alcohol liver-protecting composition and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210365794.6A CN114748566B (en) 2022-04-08 2022-04-08 Anti-alcohol liver-protecting composition and application thereof

Publications (2)

Publication Number Publication Date
CN114748566A CN114748566A (en) 2022-07-15
CN114748566B true CN114748566B (en) 2023-12-29

Family

ID=82328580

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210365794.6A Active CN114748566B (en) 2022-04-08 2022-04-08 Anti-alcohol liver-protecting composition and application thereof

Country Status (1)

Country Link
CN (1) CN114748566B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115364128A (en) * 2022-10-21 2022-11-22 北京本草源生物科技有限公司 Zinc-containing composition and liver-protecting wine and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101380433A (en) * 2007-10-22 2009-03-11 四川省宜宾五粮液集团有限公司 Combination with antialcoholism action and preparation method thereof
CN102533684A (en) * 2012-01-10 2012-07-04 华南理工大学 Method for separating acetaldehyde dehydrogenase from Xuefeng dried yeasts
CN102850245A (en) * 2012-09-04 2013-01-02 江苏大学 Method for ultrasonic assisted extraction of alliin in garlic
CN103494193A (en) * 2013-09-12 2014-01-08 北京中科邦尼国际科技有限责任公司 Plant extract for dispelling effects of alcohol and protecting liver as well as application thereof in food and healthcare food
CN105902922A (en) * 2016-04-18 2016-08-31 合肥九研医药科技开发有限公司 A composition having functions of dispelling effects of alcohol and protecting the liver and a preparing method thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101380433A (en) * 2007-10-22 2009-03-11 四川省宜宾五粮液集团有限公司 Combination with antialcoholism action and preparation method thereof
CN102533684A (en) * 2012-01-10 2012-07-04 华南理工大学 Method for separating acetaldehyde dehydrogenase from Xuefeng dried yeasts
CN102850245A (en) * 2012-09-04 2013-01-02 江苏大学 Method for ultrasonic assisted extraction of alliin in garlic
CN103494193A (en) * 2013-09-12 2014-01-08 北京中科邦尼国际科技有限责任公司 Plant extract for dispelling effects of alcohol and protecting liver as well as application thereof in food and healthcare food
CN105902922A (en) * 2016-04-18 2016-08-31 合肥九研医药科技开发有限公司 A composition having functions of dispelling effects of alcohol and protecting the liver and a preparing method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Mr.L 恩众山东数字健康发展有限公司."秋季养肝小妙招(2)".《微信公众号》.2021, *

Also Published As

Publication number Publication date
CN114748566A (en) 2022-07-15

Similar Documents

Publication Publication Date Title
CN102552549B (en) Composition for improving visual fatigue and preparation method and application thereof
CN107997048A (en) A kind of composition for having effects that to improve eyesight
CN114748566B (en) Anti-alcohol liver-protecting composition and application thereof
CN106474145B (en) Application of the Polysaccharides from Leaves of Moringa oleifera in preparation prevention and treatment alcoholic liver injury drug and food
CN106389879B (en) Application of dendrobium nobile lindl total alkaloids
CN113841894B (en) Preparation method of lycium ruthenicum anthocyanin extract and freeze-dried powder and application of lycium ruthenicum anthocyanin extract and freeze-dried powder in antioxidant and anti-aging products
CN108014150A (en) Application of the natural drug composition in anti anoxia and radiation-resistant medicine or food is prepared
CN105724642A (en) Anti-oxidation anti-aging dark tea composition and preparation method
CN106421208B (en) Pharmaceutical composition with chemical liver injury resistance function and preparation method thereof
CN110876768A (en) Traditional Chinese medicine formula, preparation method and application for losing weight, reducing fat and reducing blood fat
CN111450136A (en) Oyster and kudzu root composite particles and preparation method thereof
KR20150051597A (en) Food Composition for improving liver function containing extract of Arctiumlappa L.
CN103285123A (en) Application of Chinese Wampee Fruit extract in drug-induced hepatitis control medicines
CN109876088B (en) Natural hypoglycemic preparation capable of lowering blood sugar by regulating intestinal flora balance
CN111632082B (en) American ginseng and hovenia dulcis thunb composition and application thereof in preparing medicine for preventing and treating alcoholic liver injury
Ginting et al. In Vivo study of Antidiabetic Activity from Ethanol Extract of Clitoria ternatea L. Flower
Yadav et al. Antidiabetic activity of Plumeria rubra L. in normal and alloxan induced diabetic mice
CN111887367A (en) Loquat flower beverage and preparation method thereof
CN105963389B (en) The separation method of anti-high altitude anoxia anti-fatigue activity ingredient and its application in cape jasmine
CN105497007A (en) Medicine composition for treating gastrointestinal cancer
CN105646164A (en) Neolignan compound for the treatment of gastric cancer
CN109481502A (en) A kind of preparation method of Chinese toon extract
CN113509412B (en) Preparation method and application of sausage fruit extract
CN113967233B (en) Pharmaceutical composition for resisting pulmonary fibrosis and preparation method thereof
CN106751926A (en) The preparation method of the collagen oxidation-resistant film containing green-tea extract

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant