Application of pepper ethanol extract in preparation of medicines and health-care products for preventing and treating alcoholic liver injury

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a pepper ethanol extract in preparation of medicines and health-care products for preventing and treating alcoholic liver injury. The method has the beneficial effects that the liver index, the liver injury score, the liver tissue TG content, the AST content and the ALT content of a pepper ethanol extract sample group mouse are remarkably reduced compared with those of a model group mouse, and a remarkable acute alcoholic liver injury prevention and treatment effect of the sample is shown.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of a pepper ethanol extract in the preparation of drugs and health products for the prevention and treatment of alcoholic liver damage. Background Art

[0002] Alcoholic liver damage is a serious public health problem. Long-term heavy drinking can cause alcoholic liver disease (ALD), which can progress to alcoholic hepatitis and alcoholic liver fibrosis, and eventually even develop into alcoholic cirrhosis and liver cancer. ALD has become the second leading cause of liver damage after viral hepatitis. (Biomed Pharmacother. 2018, 99: 537-542) The pathogenesis of ALD is not fully understood. It is currently believed that its pathogenesis is mainly related to oxidative stress and lipid peroxidation, cytokine and mitochondrial dysfunction, among which alcohol metabolism and the resulting oxidative stress play a key role in the occurrence and development of alcoholic liver damage. (Int J Mol Sci. 2015, 16(11): 26087-124; World J Gastroenterol. 2014, 20(47): 17756-72) The main metabolic pathway of alcohol is the oxidation of ethanol to acetaldehyde through the oxidation mediated by alcohol dehydrogenase (ADH) activity and CYP2E1. Acetaldehyde is then converted to acetate by aldehyde dehydrogenase 2 (ALDH2) in the mitochondria and cytoplasm. This reaction increases the amount of NADH and decreases NAD + / NADH ratio, leading to the accumulation of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anions. ROS produced by alcohol metabolism are continuously neutralized by glutathione (GSH) derived from the endoplasmic reticulum and mitochondria. When long-term exposure to alcohol exceeds the production of GSH, GSH will be depleted, and ROS will interact with ethanol and iron to form reactive metabolites, all of which will lead to lipid peroxidation of cell membranes. (Rev EspEnferm Dig.2020,112(11):869-878; Nutrients,2021,13(5):1612) So far, there are no clear-cut drugs and health products for the treatment of acute alcoholic liver injury on the market. Therefore, it is of great significance to study drugs and health products for the prevention and treatment of acute alcoholic liver injury. Summary of the Invention

[0003] The present invention aims to provide a method for preparing a pepper ethanol extract for the preparation of a drug and a health product for the prevention and treatment of alcoholic liver injury. Experiments of the present invention show that the pepper ethanol extract has the effect of preventing and treating acute alcoholic liver injury.

[0004] The pepper ethanol extract is obtained by extracting pepper.

[0005] The first purpose of the present invention is to use the pepper ethanol extract in preparing a drug for preventing and treating alcoholic liver damage.

[0006] The second purpose of the present invention is to use the pepper ethanol extract in preparing a drug for preventing and treating acute alcoholic liver injury.

[0007] The third purpose of the present invention is to use the pepper ethanol extract in preparing health products for alcoholic liver damage.

[0008] The fourth purpose of the present invention is to use the pepper ethanol extract in preparing health products for acute alcoholic liver injury.

[0009] The method for extracting the pepper ethanol extract specifically comprises the following steps: grinding pepper fruits into pepper powder, adding the powder into an ethanol aqueous solution, soaking the powder, refluxing the powder, filtering, and collecting the filtrate and filter residue; concentrating the filtrate, adding acid to adjust the pH, standing the powder, and centrifuging to obtain a supernatant; adjusting the pH of the supernatant with an alkaline solution, adding distilled water until no yellow precipitate is precipitated, and then fully cooling the supernatant; centrifuging the obtained mixed solution, and drying the precipitate to obtain a yellow powdery solid, namely the pepper ethanol extract.

[0010] Furthermore, the pepper powder is 80-150 mesh.

[0011] Furthermore, the acid is hydrochloric acid, which is 4-8 mol / L hydrochloric acid, and the pH is adjusted to 3-4.

[0012] Furthermore, the concentration of the sodium hydroxide solution is 10-15%, and the pH is adjusted to 11-12.

[0013] Furthermore, the cooling is ice water cooling, and the ice water cooling time is 3-6 hours.

[0014] Furthermore, the mass fraction of the ethanol aqueous solution is 85-95%, the ratio of the ethanol aqueous solution to the pepper fruit is 6:1-10:1, and the soaking and refluxing time is 3-6 hours.

[0015] The fifth object of the present invention is to provide a pharmaceutical composition comprising a therapeutic amount of pepper ethanol extract and other pharmaceutically acceptable excipients.

[0016] The pharmaceutical composition comprises a therapeutic amount of pepper ethanol extract, a therapeutic amount of a commercially available alcoholic liver injury prevention and treatment drug, and other pharmaceutically acceptable excipients.

[0017] The dosage form of the drug includes oral preparations and injections, and can be prepared into tablets, capsules or injections by conventional methods; especially oral preparations.

[0018] The sixth object of the present invention is to provide a health care product composition, comprising the pepper ethanol extract and other auxiliary materials allowed to be added to health care products.

[0019] Combinations that can be used in health supplements:

[0020] Polyphenols: curcumin, silymarin, anthocyanidin, resveratrol, coumarin, coniferyl alcohol, sinapyl alcohol, apigenin, quercetin, proanthocyanidins, tea polyphenols, apple polyphenols, honey polyphenols, pomegranate leaf polyphenols, pine polyphenols, chlorogenic acid, baicalin, astragalin, chalcones, anthraquinones, tannins, puerarin, luteolin, baicalin, 5,7-dihydroxyflavone, naringin, catechin, epigallocatechin gallate, hesperidin, ginsenosides, and notoginseng saponins;

[0021] Alkaloids: piperine, arecoline, nicotine, theophylline, theobromine, berberine, osmanthine, and caffeine;

[0022] Saponins and terpenoids: total triterpenes from apple pomace, total triterpenes from Schisandra chinensis, including cinnamaldehyde, maslinic acid, ursolic acid, gentiopicroside, and dioscin;

[0023] Polysaccharide compounds: Lentinan, mulberry polysaccharide, wolfberry polysaccharide and Ganoderma lucidum polysaccharide;

[0024] Vitamins: Vitamin B, Vitamin E, Vitamin C, Vitamin D;

[0025] Other compounds: β-sitosterol, coumarin, lycopene, zinc, coenzyme Q10, lignans, olive oil, corn oligopeptides, cod peptide, selenium-enriched peanut protein, houttuynia cordata;

[0026] Extracts: Garlic, chocolate, red dates, honey, chicken gizzard lining, nutmeg peel, turmeric, fenugreek seeds, pepper, cloves, cumin, cinnamon, kudzu flower, cornus officinalis, hovenia dulcis, tangerine peel, poria, green tangerine peel, costus root, umbellate polyporus, stir-fried Shenqu (Chinese medicine formula), oriental rhizome, dried ginger, atractylodes macrocephala, amomum villosum, honeysuckle, chrysanthemum procumbentum stem, wolfberry, gentian, astragalus, sea buckthorn, long pipe, radix scabra, and cistanche deserticola extract;

[0027] Other additives: probiotics (lactic acid bacteria, clostridium, selenium-rich probiotics), lactobacillus acidophilus, enterococcus hyae, sweet lime, pear, onion, tomato, ginseng, bupleurum, licorice, platycodon, fenugreek seeds, emblica fruit, tangerine peel, chrysanthemum, grape seed, lemon, plantain seed, dendrobium officinale, monk fruit, vine tea, kudzu root, coffee, cardamom, hawthorn, green tea.

[0028] Furthermore, the composition includes pepper ethanol extract, thiamine (vitamin B1), niacin (vitamin B3), folic acid (vitamin B9), vitamin B6, vitamin B12, vitamin C, vitamin E, vitamin K, vitamin A, carotenoids, beta-carotene, zinc and coenzyme Q10.

[0029] The present invention has the beneficial effect of discovering that pepper ethanol extract has the effect of preventing and treating acute alcoholic liver injury. The liver index, liver injury score, and liver tissue TG, AST, and ALT levels of mice in the pepper ethanol extract-treated group were significantly reduced compared with those in the model group. Furthermore, these experimental results were more significant in the pepper ethanol extract-treated group compared with pepper methanol extract and piperine monomer, indicating that pepper ethanol extract exhibits a superior preventive and therapeutic effect against acute alcoholic liver injury. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below with reference to the examples, but is not limited thereto.

[0031] Example 1: Preparation of pepper ethanol extract

[0032] 20g of pepper fruit was ground into 80-mesh pepper powder, added to 120g of 90% ethanol solution, soaked and refluxed for 4h, filtered, and the filtrate and filter residue were collected; the filtrate was concentrated by rotary evaporation, and an appropriate amount of 6.0mol / L hydrochloric acid was added to adjust the pH to 4. The mixture was allowed to stand and then centrifuged to obtain a supernatant; the supernatant was adjusted to pH 11 with 10% sodium hydroxide solution, distilled water was added until no yellow precipitate was precipitated, and the mixture was placed in ice water and fully cooled for 4h; the resulting mixture was centrifuged for 10min, and the precipitate was dried to obtain a yellow powdery solid, i.e., 0.73g of pepper ethanol extract.

[0033] Example 2: Preparation of pepper ethanol extract

[0034] Grind 20g of pepper fruit into 140-mesh pepper powder, add it to 140g of 95% ethanol solution, soak it and reflux it for 3h, filter it and collect the filtrate and filter residue; after concentrating the filtrate by rotary evaporation, add an appropriate amount of 4.0mol / L hydrochloric acid to adjust the pH to 4, let it stand and then centrifuge to obtain a supernatant; adjust the pH of the supernatant to 12 with 10% sodium hydroxide solution, add distilled water until no yellow precipitate is precipitated, and place it in ice water to fully cool for 3h; the resulting mixed solution is centrifuged for 10min, and the precipitate is dried to obtain a yellow powdery solid, i.e., 0.86g of pepper ethanol extract.

[0035] Example 3: Preparation of pepper ethanol extract

[0036] 20g of pepper fruit was ground into 150-mesh pepper powder, added to 200g of 85% ethanol solution, soaked and refluxed for 6h, and the filtrate and residue were collected by suction filtration; after rotary evaporation and concentration, an appropriate amount of 8.0mol / L hydrochloric acid was added to the concentrate to adjust the pH to 3, and the concentrate was allowed to stand and then centrifuged to obtain a filtrate; the filtrate was adjusted to pH 11 with 15% sodium hydroxide solution, distilled water was added, and the solution was placed in ice water and fully cooled for 6h; the resulting mixture was centrifuged for 10min to precipitate a yellow powdery solid, i.e., 0.82g of pepper ethanol extract.

[0037] Example 4:

[0038] The first soaking solvent in the preparation method of Example 1 was replaced with 90% methanol solution, pure acetone and pure ether to obtain pepper methanol, acetone and ether extracts respectively.

[0039] Example 5: Evaluation of the preventive and therapeutic effects of acute alcoholic liver injury in mice

[0040] The mice in the sample group were given 40 mg / kg of pepper ethanol extract by gavage for 30 days, while the blank control group and the model control group were given distilled water. After that, the mouse liver injury model was established by gavage with 12 mL / kg of 50% alcohol (equivalent to an alcohol dose of 6000 mg / kg) once. The animals were fasted for 16 hours and then killed. The prevention and treatment effects of acute alcoholic liver injury were evaluated by detecting the mouse liver index and liver tissue section injury score, as well as the levels of triglycerides (TG), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the liver tissue. The results showed that the mice in the sample group given 40 mg / kg of pepper ethanol extract had no obvious adverse reactions after 30 days, showing that the sample had good safety.

[0041] The test results are shown in the table below. The livers of mice in the model group were swollen. The liver index, calculated as the ratio of liver weight to body weight, increased in the model group compared to the control group, while administration of pepper ethanol extract significantly reduced the liver index. Pathological tissue sections examined under a light microscope revealed numerous lipid droplets of varying sizes in the cytoplasm of the model group, with clear boundaries. Hepatocytes were enlarged and swollen, indicating fatty degeneration of the liver following modeling. Inflammation was also observed in some cells in the model group. A liver injury score was calculated based on the distribution, extent, and area of ​​lipid droplets within the liver. A higher score indicates more severe damage. The score increased in the model group compared to the control group, while administration of pepper ethanol extract significantly reduced the score. Triglycerides (TG), a marker of lipid metabolism disorders, reflect the early biochemical and pathological changes of alcoholic liver injury. TG levels were significantly elevated in the model group compared to the control group, while administration of pepper ethanol extract significantly decreased TG levels. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are commonly used indicators of liver function in clinical practice. Compared with the control group, the levels of AST and ALT in the model group were significantly increased, while the levels of AST and ALT in the pepper ethanol extract were significantly decreased.

[0042] The above experimental results demonstrate the significant preventive and therapeutic efficacy of pepper ethanol extract in acute alcoholic liver injury. Parallel experiments conducted at equivalent doses of pepper methanol, acetone, and ether extracts, as well as piperine monomer, demonstrated that the pepper ethanol extract significantly outperformed the other samples in the preventive and therapeutic efficacy. In a mouse model of acute alcoholic liver injury, liver index, liver injury score, and liver tissue TG, AST, and ALT levels were measured following administration of 40 mg / kg of pepper methanol extract and piperine monomer, as shown in the table below.

[0043]

[0044] Compared with the blank control, ## p<0.01, # p<0.05;

[0045] Compared with the model group, **p<0.01, *p<0.05

[0046] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Application of pepper ethanol extract in the preparation of drugs for the prevention and treatment of alcoholic liver damage.

2. Application of pepper ethanol extract in the preparation of drugs for the prevention and treatment of acute alcoholic liver injury.

3. Application of pepper ethanol extract in the preparation of health products for alcoholic liver damage.

4. Application of pepper ethanol extract in the preparation of health products for acute alcoholic liver injury.

5. The method for extracting the pepper ethanol extract according to any one of claims 1 to 4, wherein The method specifically comprises the following steps: grinding pepper fruit into pepper powder, adding the powder to an ethanol aqueous solution, soaking the mixture, refluxing the mixture, and collecting a filtrate and a filter residue by suction filtration; rotary evaporating the filtrate, concentrating the filtrate, adding hydrochloric acid to the concentrate, adjusting the pH, allowing the concentrate to stand, and then centrifuging to obtain a filtrate; The pH of the filtrate was adjusted with sodium hydroxide solution, distilled water was added, and the solution was then placed in ice water to be fully cooled; the obtained mixed solution was centrifuged, and the precipitation was dried to obtain a yellow powdery solid, i.e., the pepper ethanol extract.

6. The method according to claim 5, characterized in that The pepper powder has a mesh size of 80-150.

7. The method according to claim 5, characterized in that The hydrochloric acid is 4-8 mol / L hydrochloric acid, and the pH is adjusted to 3-4.

8. The method according to claim 5, characterized in that The concentration of the sodium hydroxide solution is 10-15%, and the pH is adjusted to 11-12.

9. The method according to claim 5, characterized in that The ice water cooling time is 3-6 hours.

10. The method according to claim 5, characterized in that The mass fraction of the ethanol aqueous solution is 85-95%, the ratio of the ethanol aqueous solution to the pepper fruit is 6:1-10:1, and the soaking and refluxing time is 3-6 hours.

11. A pharmaceutical composition, characterized in that The invention comprises a therapeutic amount of pepper ethanol extract and other pharmaceutically acceptable excipients.

12. The pharmaceutical composition according to claim 11, characterized in that The pharmaceutical composition comprises a therapeutic amount of pepper ethanol extract, a therapeutic amount of a commercially available alcoholic liver injury prevention and treatment drug, and other pharmaceutically acceptable excipients.

13. A health care product composition, characterized in that Including pepper ethanol extract and other auxiliary materials.

14. The health care product composition according to claim 13, characterized in that The health care product composition also includes one or more of polyphenol compounds, alkaloids, saponins and terpenoid compounds, polysaccharide compounds, vitamins, other compounds, extracts, and other additives; specifically, The polyphenolic compounds are selected from the following ingredients: curcumin, silymarin, anthocyanidin, resveratrol, coumarin, coniferyl alcohol, sinapyl alcohol, apigenin, quercetin, proanthocyanidins, tea polyphenols, apple polyphenols, honey polyphenols, pomegranate leaf polyphenols, pine polyphenols, chlorogenic acid, baicalin, astragalin, chalcone, anthraquinone, tannin, puerarin, luteolin, baicalin, 5,7-dihydroxyflavone, naringin, catechin, epigallocatechin gallate, hesperidin, ginsenosides and notoginseng saponins; The alkaloids are selected from the group consisting of piperine, arecoline, nicotine, theophylline, theobromine, berberine, osmanthine, and caffeine; The saponins and terpenoids are selected from the following ingredients: total triterpenes from apple pomace, total triterpenes from Schisandra chinensis var. antrodiaceticum, hawthorn acid, ursolic acid, gentiopicroside and dioscin; The polysaccharide compound is selected from the following ingredients: lentinan, mulberry polysaccharide, wolfberry polysaccharide and ganoderma lucidum polysaccharide; Vitamins are selected from the following ingredients: vitamin B, vitamin E, vitamin C, vitamin D; The other compounds are selected from the following ingredients: β-sitosterol, coumarin, lycopene, zinc, coenzyme Q10, lignans, olive oil, corn oligopeptides, cod polypeptide, selenium-enriched peanut protein, and houttuynia cordata; The extract is selected from the following ingredients: garlic, chocolate, red dates, honey, chicken gizzard lining, nutmeg peel, turmeric, fenugreek seeds, pepper, cloves, cumin, cinnamon, kudzu flower, cornus officinalis, hovenia dulcis, tangerine peel, poria, green peel, costus root, umbellate, stir-fried Shenqu, oriental rhizome, dried ginger, atractylodes macrocephala, amomum villosum, honeysuckle, chrysanthemum procumbentum stem, wolfberry, gentian, astragalus, sea buckthorn, long pipe, radix scabra and cistanche deserticola extract; Other additives are selected from the following ingredients: lactic acid bacteria, Clostridium, Lactobacillus, Enterococcus hyardii, sweet lime, pear, onion, tomato, ginseng, bupleurum, licorice, platycodon, fenugreek seeds, emblica fruit, tangerine peel, chrysanthemum, grape seed, lemon, plantain seed, Dendrobium officinale, monk fruit, vine tea, kudzu root, coffee, cardamom, hawthorn, green tea.

15. The health care product composition according to claim 13, characterized in that The health product composition comprises pepper ethanol extract, thiamine, niacin, folic acid, vitamin B6, vitamin B12, vitamin C, vitamin E, vitamin K, vitamin A, carotenoids, beta-carotene, zinc and coenzyme Q10.

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