Rosa roxburghii extract having liver protection effect, preparation method therefor, and use thereof

Abstract

Disclosed is a Rosa roxburghii extract having a liver protection effect. By removing ascorbic acid from Rosa roxburghii, the Rosa roxburghii extract is superior to Rosa roxburghii dry powder in both the taste in direct administration and the application field as an additive, and can thus be applied to drugs, health foods or food additives for protecting the liver or treating chemical liver injury, wherein the dosage form of the drugs or health foods may be hard capsules, tablets, oral liquids, granules or soft capsules. A preparation method for the Rosa roxburghii extract having the liver protection effect is characterized by comprising the steps of: purification treatment: by using reversed phase silica gel C18 as a stationary phase and an aqueous solution of methanol as a mobile phase, performing column chromatography on Rosa roxburghii dry powder, wherein the Rosa roxburghii dry powder is rinsed with the aqueous solution of methanol to obtain a purified Rosa roxburghi eluate; and freeze drying treatment: freeze drying the purified Rosa roxburghii eluate at a temperature ranging from -50°C to -60°C and a pressure ranging from 0.1 Mbar to 0.2 Mbar for 48 h to obtain a purified Rosa roxburghi dry powder.

Description

A hepatoprotective extract, its preparation method and uses Technical Field

[0001] This invention relates to a prickly pear extract, and more particularly to a prickly pear extract with liver-protective effects, its preparation method, and its uses. Background Technology

[0002] Rosa roxburghii Tratt., commonly known as prickly pear, is a perennial deciduous shrub belonging to the genus Rosa in the Rosaceae family. It is widely cultivated in the mountainous and hilly areas of southwestern and central-southern my country, growing at altitudes of 500 to 2,500 meters. Known as the "King of Vitamin C," prickly pear contains 841–3500 mg of ascorbic acid per 100 grams of fresh fruit, ranking first among fruits and vegetables. Currently, prickly pear varieties include Guinong No. 1, Guinong No. 2, Guinong No. 5, Guinong No. 7, and K7. Fresh prickly pear fruit is not easily eaten directly; the small thorns on its surface must be removed and the seeds dug out. Prickly pear can be used both as food and medicine. According to the *Encyclopedia of Guizhou* and *Compendium of Materia Medica Supplement*, the fruit, leaves, and roots of prickly pear are widely used for digestion, spleen strengthening, relieving summer heat, and stopping diarrhea. It is currently believed that prickly pear contains phenolic compounds, polysaccharides, ascorbic acid, triterpenoids, organic acids, and superoxide dismutase (SOD), etc. These compounds have been proven to have antioxidant, anti-atherosclerotic, hypoglycemic, anti-aging and anti-tumor effects (Zhang Chunni and Zhou Yu, "New Progress in Pharmacological Research of Prickly Pear").

[0003] The liver, a vital detoxification organ, is supplied with blood by both the hepatic artery and hepatic vein, performing essential physiological functions. Hepatocytes are highly susceptible to damage due to hypoxia. Liver disease caused by alcohol is called alcoholic liver disease (ALD). The pathogenesis of ALD is complex, involving multiple factors. Possible causes include hepatocyte damage caused by ethanol and its metabolites, increased reactive oxygen species, decreased antioxidants, inflammation caused by Kupffer cells, altered intestinal permeability, and gut microbiota dysbiosis. The initial manifestation of ALD is fatty liver, which can lead to cirrhosis, hepatitis, liver fibrosis, and even liver cancer.

[0004] There is a growing interest among those skilled in the art in novel strategies and effective methods for ALD intervention. However, the pathogenesis of ALD is not yet fully understood and requires further research. Some studies have shown that the conversion of alcohol into acetaldehyde by cells leads to the formation of DNA adducts, thereby causing DNA damage. Some scholars speculate that drugs with antioxidant functions may be helpful in treating ALD.

[0005] There is a lack of drugs in the current technology for treating chemically induced liver injury. The U.S. Food and Drug Administration (FDA) only recently approved drugs and treatments for ALD patients. Therefore, natural sources with anti-ALD properties are receiving increasing attention. Existing technologies disclose some plant extracts with anti-ALD properties, such as extracts from black fungus and Ganoderma lucidum. However, the therapeutic efficacy of these extracts for chemically induced liver injury remains to be verified.

[0006] The high ascorbic acid content in prickly pear results in a poor taste and makes it difficult to use prickly pear juice or freeze-dried juice as an additive in the food industry or health care field. However, those skilled in the art generally believe that the ascorbic acid in prickly pear has antioxidant effects and is an important component of its health benefits. Therefore, those skilled in the art lack the motivation to research how to remove ascorbic acid from prickly pear extracts and what effects the extracts would have after ascorbic acid removal. Summary of the Invention

[0007] The main objective of this invention is to concentrate and purify prickly pear extract to enhance its efficacy in treating chemically induced liver injury and reduce its toxicity to humans. This also improves the taste while reducing the dosage required by the user.

[0008] The objective of this invention is achieved through the following technical solution. This invention discloses a prickly pear extract with liver-protective effects, characterized in that it is obtained by removing ascorbic acid from prickly pear, and the removal rate of ascorbic acid in the prickly pear extract is greater than 95%.

[0009] This invention discloses a method for preparing a prickly pear extract with liver-protecting effects, characterized by comprising: purification treatment, which involves using C18 reverse-phase silica gel powder as the stationary phase and methanol aqueous solution as the mobile phase to perform column chromatography on prickly pear powder (PPE), and washing the prickly pear powder with methanol aqueous solution to obtain a purified prickly pear eluent; and freeze-drying treatment, which involves freeze-drying the purified prickly pear eluent at a temperature range of -50℃ to -60℃ and a pressure range of 0.1Mbar to 0.2Mbar for 48 hours to obtain purified prickly pear powder.

[0010] Preferably, the purification process includes the following steps: After packing the column with methanol at a ratio of 100g of C18 reverse-phase silica gel powder per 5g of dried prickly pear powder, equilibrate the silica gel column with a packing system of two column volumes (methanol:water volume ratio of 4:6). Then, load the column using a methanol:water ratio of 4:6. Subsequently, perform gradient washing with methanol:water at volume ratios of 4:6, 6:4, and 8:2. Taking a 5g sample loading amount as an example, the volume of each system in the gradient is 400mL, and the column is monitored by TLC every ten minutes. Once ascorbic acid is detected, maintain the same column system, replace the sample vial, and continue until the TLC no longer shows ascorbic acid.

[0011] More preferably, the TLC uses L-ascorbic acid as a standard to determine the content of ascorbic acid in the sample by thin-layer chromatography.

[0012] Furthermore, this invention also discloses a method 2 for preparing a prickly pear extract with liver-protective effects, characterized by comprising the following steps:

[0013] Step 1: Juice the prickly pear, separate the supernatant, and dehydrate the supernatant to obtain prickly pear powder;

[0014] Step 2: Dissolve the dried prickly pear powder completely in methanol, separate the solid phase that is insoluble in methanol, and obtain G1;

[0015] Step 3: After evaporating and concentrating the liquid phase (methanol-containing solution) obtained in Step 2, add excess ethyl acetate to precipitate a solid phase; after filtration, the solid phase is dissolved in water, insoluble impurities are filtered out, and then concentrated and dried to obtain G2;

[0016] Step 4: Add water to the liquid phase (a solution containing methanol and ethyl acetate) obtained in Step 3, and evaporate the oil phase organic solvent on the upper layer of the aqueous phase. Then extract the aqueous phase with excess ethyl acetate, separate the ethyl acetate from the aqueous phase and evaporate it to dryness to obtain G3.

[0017] Preferably, the dehydration of the supernatant in step 1 is performed by freeze-drying.

[0018] Preferably, the mass of methanol added in step 2 is 10 times or more the mass of the prickly pear powder.

[0019] Preferably, the evaporation and concentration system in step 3 adopts rotary evaporation, with a rotary evaporation temperature of 40-50℃ and a rotary evaporation pressure of atmospheric pressure.

[0020] Preferably, the mass of ethyl acetate added in step 3 is 9 times or more the mass of the dried prickly pear powder used in step 2.

[0021] Preferably, the method for drying the aqueous solution containing G2 in step 3 is freeze drying, which is carried out for 48 hours under the conditions of a temperature range of -20℃ to -40℃ and a pressure range of 0.1Mbar to 0.2Mbar.

[0022] Preferably, in step 4, the aqueous phase is extracted with excess ethyl acetate in three separate extractions. The mass of ethyl acetate used in each extraction is 4.5 times or more the mass of the dried prickly pear powder used in step 2. The ethyl acetate obtained from the three extractions is then combined.

[0023] Preferably, the ascorbic acid removal rate of the three prickly pear extracts G1, G2, and G3 obtained through the above steps is greater than 95%.

[0024] Another objective of this invention is to provide a prickly pear extract with hepatoprotective effects. This hepatoprotective prickly pear extract is obtained by dehydrating prickly pear juice to obtain prickly pear powder (PPE), then sequentially extracting the PPE with methanol, ethyl acetate, and water to remove ascorbic acid and obtain prickly pear extract G1 and prickly pear extract G2; or by using C18 reversed silica gel powder as the stationary phase and methanol-water solution as the mobile phase, performing column chromatography on the prickly pear powder (PPE) with methanol-water solution to obtain a purified prickly pear eluent, which is then freeze-dried to obtain purified prickly pear powder.

[0025] Preferably, the prickly pear variety is any one or a mixture of several of the following: Guinong No. 1, Guinong No. 5, or Guinong No. 7.

[0026] Preferably, the prickly pear extract G1 contains components of prickly pear powder that are insoluble in methanol, and the ascorbic acid content is 5% or less of the ascorbic acid content in the prickly pear powder, and its toxicity at the same dosage is lower than that of the prickly pear powder.

[0027] Preferably, the prickly pear extract G2 contains components of prickly pear powder that are soluble in methanol but insoluble in ethyl acetate, and the ascorbic acid content is 5% or less of the ascorbic acid content in the prickly pear powder, and its toxicity at the same dosage is lower than that of the prickly pear powder.

[0028] Another objective of this invention is to disclose the use of a prickly pear extract with hepatoprotective effects for protecting the liver or treating chemically induced liver injury, which has a protective effect against alcoholic liver injury. The prickly pear extract comprises components of prickly pear powder that are insoluble in methanol, or components of prickly pear powder that are soluble in methanol and insoluble or slightly soluble in ethyl acetate, or components of prickly pear powder that are insoluble in methanol and components of prickly pear powder that are soluble in methanol and insoluble or slightly soluble in ethyl acetate.

[0029] Furthermore, the present invention provides the use of the prickly pear extract in the preparation of medicaments for protecting the liver or treating chemically induced liver injury.

[0030] Preferably, the drug comprises an effective amount of the prickly pear extract and pharmaceutical excipients.

[0031] Preferably, the maximum single oral dose of the prickly pear extract is 20-25 g / kg body weight.

[0032] Preferably, the maximum single intraperitoneal injection dose of the prickly pear extract is 0.5 to 1.0 g / kg body weight.

[0033] Preferably, the amount of the prickly pear extract used is no more than 1 gram per kilogram of body weight per day.

[0034] Preferably, the dosage form of the drug is selected from hard capsules, tablets, oral liquids, granules, or soft capsules. Attached Figure Description

[0035] Figure 1 is a flowchart illustrating the steps of a method for preparing prickly pear extract according to the technology disclosed in this invention.

[0036] Figure 2 is a flowchart illustrating the steps of another method for preparing prickly pear extract according to the technology disclosed in this invention.

[0037] Figure 3 is a schematic diagram illustrating how each component mitigates ethanol-induced cytotoxicity according to the technology disclosed in this invention. A: Cell viability assay. B: Representative light microscopic images of cells from each treatment group.

[0038] Figure 4 is a schematic diagram illustrating the effects of each component on cellular antioxidant indices according to the technology disclosed in this invention. A: MDA content in each treatment group. B: GSH content in each treatment group. C: ALT activity in each treatment group. D: AST activity in each treatment group.

[0039] Figure 5 is a schematic diagram illustrating the mitigation of ethanol-induced liver injury in an animal model of ALD according to the technology disclosed in this invention. AD: Detection of MDA (A), GSH (B), ALT (C), and AST (D) in the liver of each treatment group.

[0040] Figure 6 is a schematic diagram illustrating how each component alleviates ethanol-induced liver pathological damage according to the technology disclosed in this invention.

[0041] Figure 7 is a schematic diagram illustrating the function of screening and predicting 12 PPE metabolites that directly enter the liver according to the technology disclosed in this invention.

[0042] Figure 8 illustrates a protein-protein interaction (PPI) network of potential ALD targets according to the technology disclosed in this invention. Detailed Implementation

[0043] To enable those skilled in the art to better understand the objectives, technical features, and advantages of this invention and to implement it, the technical features and embodiments of this invention are specifically illustrated in conjunction with the accompanying drawings, and preferred embodiments are further described. The drawings used in the following text are illustrative of the features of this invention and are not, and need not be, drawn in complete accordance with actual circumstances. Furthermore, technical content well-known to those skilled in the art is not described in the description of the embodiments of this invention.

[0044] Fresh prickly pear fruits were harvested from Liupanshui City, Guizhou Province, China. HepG2 cells (XY9106H) were provided by Shanghai Xinyu Biotechnology Co., Ltd. DMEM medium, fetal bovine serum (FBS), streptomycin, and penicillin were purchased from Gibco Life Sciences. DPPH and ABTS free radical scavenging activity assay kits and Cell Count Kit-8 (CCK-8) were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd. All antioxidant assay kits, including those for alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), and glutathione (GSH) activities, were provided by Solarbio Science & Technology Co., Ltd. RIPA lysis buffer (strong) and protease inhibitor mixture (100×) were purchased from Jiangsu Keying Biotechnology Co., Ltd. C18 reverse silica gel powder and thin-layer chromatography silica gel plates were purchased from Shanghai Haohong Pharmaceutical Technology Co., Ltd.

[0045] Example 1.

[0046] Step S10: Raw material pretreatment. In this step, the skin of the Guinong No. 5 prickly pear from Liupanshui City, Guizhou Province, is washed and crushed. The juice is then extracted using a juicer to obtain a solid-liquid mixture containing prickly pear pulp. Alternatively, a 10% (w / w) sodium bicarbonate solution can be used to wash the prickly pear skin, but this has no effect on the experimental results. Next, the solid-liquid mixture containing prickly pear pulp is centrifuged for 10-15 minutes at a temperature below 4℃ and a rotation speed between 5,000 r / min and 6,000 r / min. After centrifugation and allowing it to stand for a period of time, the supernatant of the prickly pear juice is collected.

[0047] Step S11: Freeze-drying. In this step, the supernatant of the prickly pear juice obtained in step S10 is freeze-dried for 48 hours at a temperature range of -20℃ to -40℃ and a pressure range of 0.1Mbar to 0.2Mbar to obtain prickly pear powder (PPE).

[0048] Purification can be performed directly after step S10, but purification using the prickly pear powder (PPE) obtained in step S11 is more efficient.

[0049] Step S12: Purification. In this step, C18 reverse-phase silica gel powder is used as the stationary phase, and methanol-water solution is used as the mobile phase. The prickly pear powder obtained in step S11 is subjected to column chromatography, with the mobile phase (methanol:water) used in gradient washes at ratios of 4:6, 6:4, and 8:2. The specific operation is as follows: Using 100g of C18 reverse-phase silica gel powder per 5g of prickly pear powder, the column is packed with methanol and then equilibrated with a column volume of two column volumes (methanol:water volume ratio of 4:6). Then, a methanol:water ratio of 4:6 is used as the loading system. Subsequently, gradient washes are performed with methanol:water at volume ratios of 4:6, 6:4, and 8:2. For a 5g sample loading amount, the volume of each system in the gradient is 400mL. L-ascorbic acid (vitamin C) is used as a standard, and the ascorbic acid content in the liquid phase is determined by thin-layer chromatography (TLC). The column was monitored by TLC every ten minutes. Once ascorbic acid was detected, the column chromatography system was kept constant, and the sample vial was replaced until ascorbic acid was no longer detected by TLC. After purification, the silica gel column was washed with three column volumes of methanol solution, and all components except ascorbic acid were collected. The purified prickly pear eluent was obtained.

[0050] Step S14: Freeze-drying. In this step, the purified prickly pear eluent obtained in step S12 is freeze-dried for 48 hours at a temperature range of -40℃ to -60℃ and a pressure range of 0.1 Mbar to 0.2 Mbar to obtain purified prickly pear powder (nVc-PPE).

[0051] In another embodiment of this application, the purified prickly pear eluent obtained in step S12 is subjected to a rotary evaporator to remove methanol, and the remaining solution is cold-dried for 48 hours at a temperature of -60°C and a pressure of 0.1 Mbar to obtain purified prickly pear freeze-dried powder.

[0052] For subsequent testing, the purified prickly pear powder can be mixed with deionized water to obtain a purified prickly pear aqueous solution.

[0053] Step S15: Ascorbic acid content detection. The prickly pear powder obtained in step S11 and the purified prickly pear powder obtained in step S14 were mixed evenly with deionized water. Using GB5009.86-2016 Method I (High Performance Liquid Chromatography) as the detection standard, the ascorbic acid content in the prickly pear powder (PPE) was found to be 28.25 mg / 100 mg, and the ascorbic acid content in the purified prickly pear powder was 1.03 mg / 100 mg. This confirms that most of the ascorbic acid in the purified prickly pear powder was removed through the above extraction steps, with a removal rate of 97.36%. The calculation formula is as follows: Formula I.

[0054] Removal rate = 1 - a / (b * (1 - a) / (1 - b)) Equation I

[0055] Where a = ascorbic acid content (w / w) in purified prickly pear powder nVc-PPE.

[0056] b = Ascorbic acid content (w / w) in prickly pear powder (PPE)

[0057] Example 2.

[0058] Figure 2 is a flowchart illustrating another method for preparing prickly pear extract according to the technology disclosed in this invention. Step S20: Raw material pretreatment. In this step, the outer skin of Guinong No. 1 prickly pear from Liupanshui City, Guizhou Province is washed and crushed, and then juiced using a juicer to obtain a solid-liquid mixture containing prickly pear pulp. Alternatively, a 5% (w / w) acetic acid solution can be used to wash the prickly pear skin, but this has no effect on the experimental results. Next, the solid-liquid mixture containing prickly pear pulp is centrifuged for 10-15 minutes at a temperature below 4°C and a rotation speed ranging from 5,000 r / min to 6,000 r / min. After centrifugation and standing for a period of time, the supernatant of the prickly pear juice is collected.

[0059] Step S21: Freeze-drying. In this step, the supernatant of the prickly pear juice obtained in step S20 is freeze-dried for 48 hours at a temperature range of -30℃ to -60℃ and a pressure range of 0.1Mbar to 0.2Mbar to obtain prickly pear powder (PPE).

[0060] Step S22: Purification. Mix 30 g of the dried prickly pear powder obtained in step S21 with 90 mL of a methanol-water solution (methanol:water ratio 4:6). Perform column chromatography using C18 reverse-phase silica gel as the stationary phase and the methanol-water solution as the mobile phase. Wash the dried prickly pear powder with the methanol-water solution to obtain a purified prickly pear eluent. The purification process in step S22 is the same as in step S12 described above, and will not be elaborated further here.

[0061] Step S23: Freeze-drying. In this step, the purified prickly pear eluent obtained in step S22 is freeze-dried for 48 hours at a temperature range of -40℃ to -60℃ and a pressure range of 0.1Mbar to 0.2Mbar to obtain purified prickly pear powder.

[0062] For subsequent testing, the purified prickly pear powder can be mixed with deionized water to obtain a purified prickly pear aqueous solution.

[0063] Step S24, Ascorbic acid content detection. The prickly pear powder (PPE) obtained in step S21 and the purified prickly pear powder obtained in step S23 were respectively mixed evenly with deionized water. Using GB5009.86-2016 Method I (High Performance Liquid Chromatography) as the detection standard, the ascorbic acid content in the prickly pear powder in this embodiment was 32.33 mg / 100 mg, and the ascorbic acid content in the purified prickly pear powder was 2.12 mg / 100 mg. This confirms that through the above extraction steps, most of the ascorbic acid in the purified prickly pear powder has been removed, with a removal rate of 95.47%.

[0064] Example 3.

[0065] The steps are the same as in Example 1, except that acetonitrile is used instead of methanol aqueous solution as the mobile phase.

[0066] After testing for ascorbic acid content, it was found that the ascorbic acid content in the dried prickly pear powder was 28.25 mg / 100 mg, and the ascorbic acid content in the purified dried prickly pear powder was 1.57 mg / 100 mg. This confirms that most of the ascorbic acid in the purified dried prickly pear powder has been removed through the above extraction steps, with a removal rate of 95.95%.

[0067] Example 4

[0068] In this embodiment, methanol, ethyl acetate and water are used as solvents to remove ascorbic acid from PPE.

[0069] Please refer to Figure 1. Step S10: Raw material pretreatment. The outer skin of the Guinong No. 5 prickly pear from Liupanshui City, Guizhou Province, was washed. In different embodiments of this application, distilled water and a 10% (w / w) sodium bicarbonate solution were used to wash the prickly pear skin, but this did not affect the experimental results. After washing the prickly pear skin, it was crushed and juiced using a juicer to obtain a solid-liquid mixture containing prickly pear pulp. The solid-liquid mixture containing prickly pear pulp was then centrifuged for 10-15 minutes at a temperature below 4°C and a rotation speed of 5,000 r / min to 6,000 r / min. After centrifugation and standing for a period of time, the supernatant of the prickly pear juice was taken out.

[0070] Step S11: Freeze-drying treatment. The supernatant of the prickly pear juice obtained in step S10 is freeze-dried for 48 hours at a temperature range of -20℃ to -40℃ and a pressure range of 0.1Mbar to 0.2Mbar to obtain dehydrated prickly pear powder (PPE).

[0071] Step S401: Accurately weigh 100.0g of PPE dry powder and place it in a container. Add 1000mL of anhydrous methanol in batches (the amount should be sufficient to ensure that all methanol-soluble substances in the PPE are completely dissolved). Stir thoroughly at room temperature until fully dissolved, then let stand for 10 minutes and filter. The resulting solid filter residue, G1, is the methanol-insoluble component of the PPE. Wash with anhydrous methanol, filter, dry, and weigh. The mass of G1 is 7.9g. The liquid filtrate (methanol) is then processed in the next step.

[0072] Step S402: The filtrate obtained in step S401 was rotary evaporated at 40-50℃ and normal pressure until solid precipitated, then the rotary evaporation was stopped. Subsequently, 1000 mL of ethyl acetate was slowly added while stirring. As the ethyl acetate was added and mixed, the temperature gradually decreased and solid began to slowly precipitate. After all the ethyl acetate was added, the mixture was allowed to stand for 10 minutes, followed by filtration. The resulting solid residue was G2, a highly polar component of PPE, soluble in methanol or water but with low solubility in ethyl acetate. The obtained G2 solid residue was fully dissolved in distilled water, filtered again, and freeze-dried for 48 hours at a temperature range of -20℃ to -40℃ and a pressure range of 0.1 Mbar to 0.2 Mbar. The mass of G2 was 31.4 g. The liquid filtrate (methanol + ethyl acetate) was then processed in the next step.

[0073] Step S403: The liquid filtrate obtained from step S402 is rotary evaporated at 40-50℃ and normal pressure to a volume of approximately 500 mL. Rotary evaporation is stopped, and 500 mL of distilled water is added. Rotary evaporation is then continued at 40-50℃ and normal pressure until all the organic solvent in the upper layer of the liquid phase has evaporated and a solid precipitate has formed. Rotary evaporation is then stopped. 500 mL of ethyl acetate is added, stirred thoroughly, and extracted. This process is repeated three times to ensure complete disappearance of the solid precipitate at the bottom of the liquid phase. The organic phases (ethyl acetate) from the three extractions are combined and concentrated. The precipitate is G3, a less polar fraction of PPE, which is sparingly soluble in water but soluble in ethyl acetate. After rinsing with distilled water, filtering, drying, and weighing, the mass of G3 is 6.3 g. The aqueous filtrate is then processed in the next step.

[0074] Step S404: The aqueous filtrate obtained in step S403 is concentrated and dried to obtain a viscous solid substance, G4, which is a component of PPE that exhibits certain solubility in both water and organic reagents, including most of the vitamin C. After lyophilization, the mass of G4 is weighed to obtain 49.6 grams.

[0075] Step S45: Mix the dried prickly pear powder obtained in step S11 with deionized water until homogeneous; mix all G1 obtained in step S401, all G2 obtained in step S402, and all G3 obtained in step S403 (total weight of G1+G2+G3: 45.6g) to obtain vitamin C-removed dried prickly pear powder, and mix it with deionized water until homogeneous. The detection method is based on GB 5009.86-2016 "National Food Safety Standard - Determination of Ascorbic Acid in Food" Method I (High Performance Liquid Chromatography). The results show that 100g of dried prickly pear powder (PPE) contains 27.36g of ascorbic acid, and the vitamin C-removed dried prickly pear powder contains 0.32g of ascorbic acid, with a removal rate of 98.83%. The calculation formula is shown in Formula II below. This confirms that most of the ascorbic acid in the vitamin C-removed dried prickly pear powder has been removed through the above extraction steps.

[0076] Removal rate = 1 - b / a (Equation II)

[0077] Where a = ascorbic acid content (g) in 100g of prickly pear powder (PPE),

[0078] b = Ascorbic acid content (g) in 100g of dried prickly pear powder (without vitamin C).

[0079] Example 5

[0080] In this embodiment, methanol, ethyl acetate and water are used as solvents to remove ascorbic acid from PPE.

[0081] Step S10: Raw material pretreatment. The outer skin of the Guinong No. 7 prickly pear from Liupanshui City, Guizhou Province, was washed. In different embodiments of this application, distilled water and a 5% (w / w) acetic acid solution were used to wash the prickly pear skin, but this had no effect on the experimental results. After washing, the prickly pears were crushed and juiced using a juicer to obtain a solid-liquid mixture containing prickly pear pulp. The solid-liquid mixture containing prickly pear pulp was then centrifuged for 10-15 minutes at a temperature below 4°C and a rotation speed between 5,000 and 6,000 r / min. After centrifugation and standing for a period of time, the supernatant of the prickly pear juice was collected.

[0082] Step S11: Freeze-drying treatment. The supernatant of the prickly pear juice obtained in step S10 is freeze-dried for 48 hours at a temperature range of -20℃ to -40℃ and a pressure range of 0.1Mbar to 0.2Mbar to obtain dehydrated prickly pear powder (PPE).

[0083] Step S501: Accurately weigh 100.0g of PPE dry powder into a container, add 1000mL of anhydrous methanol in batches (the amount should be sufficient to ensure that all methanol-soluble substances in the PPE are dissolved in methanol), stir thoroughly at 40℃, and after complete dissolution, let stand for 10 minutes, then filter to obtain solid filter residue G1, which is the methanol-insoluble component of PPE. After drying, weigh G1 to obtain a mass of 7.2g. The liquid filtrate (methanol) is then processed in the next step.

[0084] Step S502: The filtrate obtained in step S501 is rotary evaporated at 40-50℃ and normal pressure until solids precipitate, then the rotary evaporation is stopped. Then, 1000 mL of ethyl acetate is slowly added while stirring. As the ethyl acetate is added and mixed, the temperature gradually decreases and solids begin to precipitate slowly. After all the ethyl acetate has been added, the mixture is allowed to stand for 10 minutes, followed by filtration. The solid residue obtained after filtration is G2, a highly polar component of PPE, soluble in methanol or water but with low solubility in ethyl acetate. After drying, the mass of G2 is 28.1 g. The liquid filtrate (methanol + ethyl acetate) is then processed in the next step.

[0085] Step S503: The liquid filtrate obtained from step S502 is rotary evaporated at 40-50℃ and normal pressure to a volume of approximately 500 mL. Rotary evaporation is stopped, and 500 mL of distilled water is added. Rotary evaporation is then continued at 40-50℃ and normal pressure until all the organic solvent in the upper layer of the liquid phase evaporates and a solid precipitate forms. Rotary evaporation is then stopped. 500 mL of ethyl acetate is added, stirred thoroughly, and extracted. This process is repeated three times to ensure complete disappearance of the solid precipitate at the bottom of the liquid phase. The organic phases (ethyl acetate) from the three extractions are combined and concentrated. The precipitate is G3, a less polar fraction of PPE, which is sparingly soluble in water but soluble in ethyl acetate. After drying, the mass of G3 is 6.8 g. The aqueous filtrate is then processed further.

[0086] Step S504: The aqueous filtrate obtained in step S503 is concentrated and dried to obtain a viscous solid substance, G4, which is a component of PPE that exhibits certain solubility in both water and organic reagents, including most of the vitamin C. After lyophilization, G4 is weighed to obtain a mass of 54.2 grams.

[0087] Step S55: Ascorbic acid content detection. The prickly pear powder obtained in step S21 is mixed evenly with deionized water; G1 obtained in S501, G2 obtained in S502, and G3 obtained in S503 are mixed to obtain vitamin C-removed prickly pear powder (total weight of G1+G2+G3 42.1g), which is then mixed evenly with deionized water. The detection is based on Method I (High Performance Liquid Chromatography) of GB 5009.86-2016 "National Food Safety Standard - Determination of Ascorbic Acid in Food". The results show that the ascorbic acid content in 100g of prickly pear powder (PPE) is 29.53g, and the ascorbic acid content in the vitamin C-removed prickly pear powder is 0.56g, with a removal rate of 98.10%. The calculation formula is shown in Formula II below. This confirms that most of the ascorbic acid in the vitamin C-removed prickly pear powder has been removed through the above extraction steps.

[0088] Removal rate = 1 - b / a (Equation II)

[0089] Where a = ascorbic acid content (g) in 100g of prickly pear powder (PPE),

[0090] b = Ascorbic acid content (g) in 100g of dried prickly pear powder (without vitamin C).

[0091] Experimental Example 1

[0092] The following experiments were conducted using the PPE and nVc-PPE obtained in Example 1 as experimental samples.

[0093] Six-week-old male C57BL / 6J mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. and housed in a sterile environment with a 12-hour light-dark cycle, humidity between 40% and 60%, and temperature of 22℃±2℃. After one week of acclimatization, the mice were randomly divided into the following four groups, as shown in Table 1.

[0094] Table 1: Treatment methods for different groups of animals

[0095] The PPE and nVc-PPE were obtained according to the method described in Example 1. After gavage, the mice were allowed free movement and free feeding.

[0096] In this invention, the dosage of Erguotou liquor was determined according to the "Evaluation and Testing Methods for Functional Functions of Health Foods (2023 Edition)". The research of this invention shows that edible alcohol is better able to simulate real-life ALD in humans than industrial alcohol. This invention established an alcohol-induced liver injury model using Erguotou (53% alcohol by volume, 12 mL / kg). All animals received their respective treatments for 8 days, with mice euthanized on the last day. Liver tissue and blood were collected and stored at -80°C.

[0097] Next, in order to demonstrate that nVc-PPE has significant liver-protective and antioxidant effects, the present invention further conducted the following analyses.

[0098] Biochemical analysis:

[0099] To separate serum, whole blood samples from the four groups of mice were collected in blood collection tubes without anticoagulants. The four samples were allowed to stand at room temperature for 30-60 minutes without shaking or disturbance, allowing the blood to clot naturally. Next, each of the four samples was centrifuged at 4°C and 4,000 rpm for 10 minutes. After standing, the supernatant was collected; this supernatant was the serum. 1.0 g of liver tissue from each of the four groups of mice was homogenized in 900 μL of protein extraction buffer (as per the corresponding kit) to prepare a liver homogenate. Then, each of the four homogenates was centrifuged at 4°C and 3,000 rpm for 5 minutes, followed by standing to extract the supernatant. Subsequently, using this supernatant, various antioxidant markers in the liver and serum were detected using a test kit, including: malondialdehyde (MDA) (BC0025, Solarbio), superoxide dismutase (SOD) (BC5165, Solarbio), glutathione (GSH) (BC1175, Solarbio), aspartate aminotransferase (AST) (BC1565, Solarbio), alanine aminotransferase (ALT) (BC1555, Solarbio), alcohol dehydrogenase (ADH) (BC1085, Solarbio), and acetaldehyde dehydrogenase (ALDH) (BC0755, Solarbio).

[0100] Histological analysis:

[0101] Immediately after euthanasia, the left lobe of the liver was removed and fixed in 4% paraformaldehyde at 4°C for 24 hours. The liver tissue was then cut into 5μm thick sections and embedded in paraffin. Finally, the degree of liver damage was assessed by H&E staining.

[0102] Cell culture and processing:

[0103] HepG2 cells were cultured in a 37°C, 5% CO2 incubator, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in DMEM medium. Cells were passaged at 70%-80% confluence. To determine the PPE concentration without negative impact on HepG2 cells, the protective effect of PPE was investigated in concentrations ranging from 0 μg / mL to 300 μg / mL (in increments of 50 μg / mL). Results showed that PPE concentrations between 0 μg / mL and 250 μg / mL had a positive effect on HepG2 cells. Simultaneously, the half-maximal inhibitory concentration (IC50) of ethanol on HepG2 cells was evaluated, measured at concentrations from 0% to 10% (in increments of 1%) to determine the optimal concentration for establishing a liver injury model. Then, cells were treated with 250 μg / ml PPE and nVc-PPE containing IC50 concentration ethanol (3.7% ethanol) for 24 hours to evaluate the protective effect of each component against ethanol-induced HepG2 cell damage, as shown in Figure 3C.

[0104] Assess free radical scavenging ability:

[0105] ABTS and DPPH were used to determine the free radical scavenging capacity or hydrogen donor capacity of different components.

[0106] Cell viability assessment:

[0107] Cell viability was assessed using a CCK-8 assay kit. 8 x 10⁸ cells were seeded in 96-well plates. 4 HepG2 cells / well. After 24 hours of cell growth, cells were co-treated with PPEE and nVc-PPE containing 3.7% ethanol for another 24 hours. Cell viability was then assessed using a cell counting kit (CCK-8).

[0108] Evaluation of antioxidant system biomarkers:

[0109] HepG2 cells were seeded into 6-well plates (8 x 10 cells per well). 5 Cells were grown for 24 hours and then treated with PPEE and nVc-PPE (50 μg / mL and 250 μg / mL) containing 3.7% ethanol for 24 hours to assess ALT and AST activities, as well as GSH and MDA levels. Subsequently, the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione (GSH), and malondialdehyde (MDA) were measured according to the kit instructions.

[0110] Determination of ADH and ALDH activity:

[0111] To determine the enzyme activities of ADH and ALDH and their changes over time, HepG2 cells were seeded in 6-well plates (8 x 10 cells per well). 5Cells were cultured at 37°C for 24 hours. Afterwards, the cells were treated with PPEE and nVc-PPE (50 μg / mL and 250 μg / mL) containing 3.7% ethanol for 0.25, 0.5, 1, 3, 7, or 24 hours.

[0112] Based on the above experiments and analyses, the results are as follows. It should be noted that all experimental results were presented using GraphPad Prism 9.4.1 software; SPSS software was used for all statistical analyses; and R language was used for bioinformatics analysis. Furthermore, p < 0.05 is defined as statistically significant in this invention.

[0113] The antioxidant capacity of PPE, Vc, and nVc-PPE was preliminarily studied using DPPH and ABTS free radical scavenging experiments. The results showed that PPE has a strong antioxidant capacity, as listed in Table 2 for comparison of free radical scavenging effects. The concentration of PPE showed a significant dose-dependent relationship in the range of 0-125 μg / mL, and nVc-PPE had a stronger free radical scavenging ability.

[0114] Table 2

[0115] Please refer to Figure 3. Figure 3 is a schematic diagram illustrating how the components of PPE alleviate ethanol-induced cytotoxicity according to the technology disclosed in this invention. To investigate the ability of PPE and nVc-PPE to counteract ethanol-induced cytotoxicity in vitro, HepG2 cells were co-treated with 250 μg / mL of PPE and nVc-PPE and 3.7% ethanol for 24 hours. The reduction of ethanol-induced cytotoxicity by each component is listed in Table 3. Statistical analyses in Table 3 were performed using one-way ANOVA. Compared with the ETOH group, *p<0.05, **p<0.01.

[0116] Table 3

[0117] As shown in Figure 3A, both PPE and nVc-PPE treatments significantly restored cell viability. Furthermore, under a 4X objective, ethanol-treated HepG2 cells exhibited dispersion and suspension, with fewer cells and lower confluence compared to other groups. Under a 20X objective, ethanol-treated HepG2 cells were largely suspended, with rounded cell morphology, unable to adhere properly, and exhibiting poor cell aggregation (as indicated by circles). In contrast, after treatment with each component of PPE, cell aggregation was enhanced, boundaries were clear, and both morphology and number were close to normal cells (Figure 3B). Overall, the purified nVc-PPE did not diminish its effectiveness in protecting HepG2 cells from ethanol-induced cytotoxicity.

[0118] Figure 4 is a schematic diagram illustrating the effects of each component on cellular antioxidant indices according to the technology disclosed in this invention. Table 4 shows the results of cellular antioxidant indicators such as MDA (mean), MDA (standard deviation), GSH (mean), GSH (standard deviation), ALT (mean), ALT (standard deviation), AST (mean), and AST (standard deviation) for each group. All data in Table 4 were analyzed using one-way ANOVA. Compared with the ETOH group, *p<0.05, **p<0.01, ***p<0.001.

[0119] Table 4

[0120] As shown in Figure 4, the levels of relevant antioxidant system indicators were measured in an ethanol-induced cell damage model to investigate the effects of various interventions on ethanol-induced oxidative stress in HepG2 cells. Figures A and B in Figure 4 show that, compared to the control group, ethanol treatment increased MDA content while decreasing GSH content. PPE and nVc-PPE treatments both decreased MDA content and increased GSH content. Figures C and D show that acute ethanol exposure significantly increased the enzyme activities of AST and ALT, while PPE and nVc-PPE inhibited the ethanol-induced increase in AST and ALT levels. Overall, both PPE and nVc-PPE significantly improved the antioxidant capacity of cells and alleviated ethanol-induced oxidative damage.

[0121] Effects of each component on ethanol-induced liver injury in mice. This invention constructed an ethanol-induced liver injury model in mice, detected various oxidative damage indicators in the liver and serum, and further explored the protective effects of PPE and nVc-PPE on mice with alcoholic liver injury. Table 5 shows the data of MDA (mean), MDA (standard deviation), GSH (mean), GSH (standard deviation), ALT (mean), ALT (standard deviation), AST (mean), and AST (standard deviation) in the liver of each group. Statistical analysis in Table 5 used one-way ANOVA. Compared with the ETOH group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0122] Table 5

[0123] Ethanol-induced liver MDA levels significantly increased in mice, while PPE and nVc-PPE interventions significantly inhibited the ethanol-induced increase in liver MDA (Figure 5, A). GSH levels in the livers of the intervened mice were significantly higher than in the ETOH group, while ALT and AST levels were significantly lower.

[0124] Effects of each component on ethanol-induced liver pathological damage

[0125] Next, this study further investigated the protective effect on a mouse model of alcoholic liver injury. After 8 days of ethanol treatment, the histological changes in the liver of mice were observed by H&E staining, as shown in Figure 6. In the normal group, hepatocytes were neatly arranged with clear cell boundaries and no pathological abnormalities were observed. This indicates that under normal circumstances, the liver tissue structure is good and the cells are healthy. However, in the ETOH group, obvious hepatocyte lesions were observed. Specifically, there was an increase in vacuoles in the tissue, which may be due to intracellular fat accumulation or cell damage; the hepatocytes were irregularly arranged, and the originally tightly ordered cell arrangement became disordered; the cell boundaries were blurred, and the boundaries between cells were no longer clear, which is a manifestation of cell swelling or damage.

[0126] After intervention with PPE and nVc-PPE, these pathological changes in the liver were improved. In the PPE+ETOH group, the hepatocyte boundaries became clearer, the number of vacuoles in the tissue decreased, and the hepatocytes were more tightly packed. This indicates that PPE has a certain protective effect against alcohol-induced liver damage, possibly by alleviating cell damage and maintaining the normal structure of the liver through antioxidant and anti-inflammatory mechanisms. The nVc-PPE+ETOH group also showed a similar trend of improvement.

[0127] Screening of potential active ingredients in PPE

[0128] To investigate whether certain components of PPE can directly enter the liver via gavage, this invention further evaluated the levels of 126 differentially expressed metabolites (DEMs) upregulated in the high PPE group in other treatment groups. Figure 7 illustrates the function of 12 PPE metabolites that directly enter the liver, as disclosed in this invention. The results showed that these metabolites were highly expressed in the PPE treatment groups (low / high PPE groups), primarily composed of lipids and organic acids, as shown in Figure 7A. To confirm whether the upregulated metabolites originated from PPE, these metabolites were further screened. The three groups that did not receive PPE gavage (normal, negative, and positive groups) were grouped together as the nPPE group, while the two groups that received PPE gavage (low PPE and high PPE groups) were grouped together as the PPE group. Subsequently, the FC, p.adjust, and FDR of these two groups were calculated, and the results were screened according to the criteria of FC > 1.5 and p.adjust < 0.05. This process identified a total of 29 metabolites. Further screening of these metabolites based on their overall PPE content revealed 12 DEMs that were present in high amounts in PPE and at significantly higher levels in the PPE group than in the nPPE group, as shown in Figure 7B and listed in Table 6. It is speculated that these 12 components can directly enter the liver from PPE, thereby mitigating ethanol-induced oxidative damage.

[0129] Table 6

[0130] To further understand the functions of these 12 identified metabolites, their target genes were predicted using network pharmacology, and a metabolite-gene interaction network was constructed, as shown in Figure 7, C. Next, KEGG enrichment analysis was performed on the target genes of these 12 identified metabolites. Figure 7, D, shows the 15 pathways with the highest enrichment, primarily in fatty acid degradation, glycolysis / gluconeogenesis, drug metabolism, and alcoholic liver disease pathways. Disruptions in these pathways are commonly associated with the development of ALD. Furthermore, please refer to Figure 8. Figure 8 is a schematic diagram representing the protein-protein interaction (PPI) network of potential ALD targets according to the technology disclosed in this invention. In Figure 8, the protein-protein interaction (PPI) network of target genes was created using the STRING database (minimum necessary interaction value = 0.9). The results in Table 7 show that CYP1A1 and MAOB have more interactions with other potential targets. Therefore, these 12 PPE-enriched metabolites may directly enter the liver and regulate the normal function of these pathways, thereby playing a role in the prevention and treatment of ALD.

[0131] Table 7

[0132] The present invention further verifies the protective effects of PPE, Vc and nVc-PPE against ethanol-induced liver injury.

[0133] Experimental animals: Male C57BL / 6 mice (6 weeks old, SPF grade, housed separately).

[0134] Reagents:

[0135] PPE, Vc and nVc-PPE, Red Star Erguotou (53% alcohol by volume), TNF-α / IL-6 / IL-1β ELISA kit, Oil Red O staining reagent, Masson staining reagent; triglyceride (TG), total cholesterol (TC), and total bilirubin (TBIL) detection kits.

[0136] Experimental Groups:

[0137] A liver injury model was induced using Red Star Erguotou (53% alcohol by volume), with an oral gavage volume of 12 mL / kg body weight (BW). The concentrations of PPE, vitamin C, and nVc-PPE were 1 g / kg BW·day. -1 Immediately after administering the drug (diluted with distilled water) via gavage, administer ethanol via gavage.

[0138] The administration period for the test samples was 30 days. The grouping and treatment of the experimental animals are listed in Table 8.

[0139] Table 8

[0140] Animal culling and harvesting:

[0141] After successful model establishment, mice in each group were fasted overnight for 12 hours, weighed, and then euthanized by cervical dislocation. The thoracic cavity was opened, and blood was drawn from the heart using a 1mL syringe. The blood was injected into a labeled anticoagulant tube and centrifuged at 12,000 rpm for 10 minutes at 4°C to separate the plasma, which was then stored at -80°C for later analysis. The abdomens of the experimental animals in each group were quickly dissected, and the livers were completely removed.

[0142] Experiment 1: Assessment of Liver Fibrosis and Collagen Deposition

[0143] Objective: To evaluate the inhibitory effects of PPE, Vc, and nVc-PPE on ethanol-induced liver fibrosis.

[0144] Masson staining:

[0145] The same part of the left lobe of the liver was cut from each mouse and fixed in 4% paraformaldehyde solution for more than 24 hours. Then, a third-party company was commissioned to perform Masson staining.

[0146] Experiment 2: Detection of Inflammatory Response

[0147] Objective: To verify the inhibitory effects of PPE, Vc, and nVc-PPE on ethanol-induced inflammation.

[0148] Experiment 2 included the detection of serum inflammatory factors and liver inflammatory factors.

[0149] Serum inflammatory factor detection:

[0150] Collected plasma samples were used to detect the levels of TNF-α, IL-6, and IL-1β according to the ELISA kit protocol.

[0151] Liver inflammatory factor detection:

[0152] Collected liver samples were mixed with the corresponding volume of PBS (0.1g corresponds to 900uL PBS) (1% 100× protease inhibitor was added to the PBS), and thoroughly ground on ice. Then, the samples were centrifuged at 5000r / min for 10min at 4℃. After standing, the liver supernatant was collected, and the levels of TNF-α, IL-6, and IL-1β were detected according to the ELISA kit protocol.

[0153] Experiment 3: Lipid Metabolism and Liver Function Assessment

[0154] Objective: To evaluate the effects of PPE, Vc, and nVc-PPE on lipid accumulation and liver function.

[0155] Liver lipid content determination:

[0156] Liver supernatant was prepared according to the above method, and then the TG and TC content in the liver was examined using TG and TC kits.

[0157] Expanded range of serum liver function indicators:

[0158] Take the prepared plasma and detect the total bilirubin content in the plasma using the TBIL detection kit.

[0159] Experiment 4: Non-target metabolomics analysis of nVc-PPE

[0160] Objective: To compare the content of 12 potential active substances in PPE and nVc-PPE.

[0161] 0.1g of nVc-PPE lyophilized powder was taken and subjected to non-target metabolomics sequencing. After obtaining the data, the content of 12 potential active substances was compared.

[0162] In summary, both PPE and nVc-PPE have a certain protective effect against alcoholic liver injury and can improve the pathological state of the liver. nVc-PPE, having removed ascorbic acid, is superior to PPE in both taste when taken directly and in its application as an additive. This invention further applies prickly pear extract to drugs, health foods, or food additives for protecting the liver or treating chemically induced liver injury, wherein the drug or health food dosage form is a hard capsule, tablet, oral liquid, granule, or soft capsule.

[0163] Experiment Example 2

[0164] The following experiments were conducted using the PPE and G1, G2, G3 and G4 obtained in Example 4 as experimental samples.

[0165] The experimental data in this example were analyzed using GraphPad Prism 9.4.1 software; SPSS software was used for statistical analysis; and R language was used for bioinformatics analysis. Furthermore, p < 0.05 was defined as statistically significant in this invention.

[0166] Cell culture and treatment: HepG2 cells were cultured in an incubator at 37°C and 5% CO2, supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in DMEM medium. Cells were passaged when the confluence reached 70%-80%.

[0167] Sample preparation (ethanol-free): Accurately weigh 5g each of G1, G2, G3, and G4 and dissolve them in 5mL of PBS to prepare a stock solution of 1g / mL. Filter the solution through a 0.22μm membrane for later use. Subsequently, serially dilute to prepare samples of different concentrations: 200μg / mL, 100μg / mL, 50μg / mL, 25μg / mL, and 5μg / mL (a total of 20 samples).

[0168] Cell viability assessment: Cells were seeded at a density of 8 x 10⁸ cells per well in a 96-well plate. 5 HepG2 cells were added at a density of 100 cells / mL, with 0.1 mL per well. After 24 hours of cell growth, the culture medium was discarded, and different concentrations of samples were added to each well. PBS was added to the blank control group. 100 μL of sample was added to each well, with three wells containing each sample. After another 24 hours of culture, cell viability was assessed using a cell counting kit (CCK-8). The results are shown in Table 9.

[0169] Table 9. Results of the effects of different samples on cell proliferation.

[0170] The experimental results showed that G1, G2, and G4 all exhibited significant positive effects on HepG2 cells within the concentration gradient, with G1 and G2 significantly promoting cell proliferation within specific concentration ranges. In contrast, G3 showed an inhibitory effect on HepG2 cells at high concentrations (200 μg / mL).

[0171] Sample (ethanol-containing) preparation: Accurately weigh 5g each of G1, G2, G3, and G4 and dissolve them in 5mL of PBS to prepare a 1g / mL stock solution. Filter the stock solution through a 0.22μm membrane for later use. Subsequently, serially dilute to different concentrations of 200μg / ml, 100μg / ml, 50μg / ml, 25μg / ml, and 5μg / ml (a total of 20 samples). Anhydrous ethanol was added during sample preparation to achieve an ethanol concentration of 3.7% (v / v). In addition, a 3.7% (v / v) ethanol solution was prepared using PBS as an ethanol sample for use in the ethanol model group.

[0172] Evaluation of inhibition of ethanol-induced cytotoxicity: 8 x 10⁸ cells were seeded in 96-well plates. 5 HepG2 cells were added at a density of 100 cells / mL, with 0.1 mL per well. After 24 hours of cell growth, the culture medium was discarded, and different concentrations of samples (containing ethanol) were added to each well. The ethanol model group was treated with PBS containing ethanol. 100 μL of sample was added to each well, with three wells containing each sample. After another 24 hours of culture, cell viability was assessed using a cell counting kit (CCK-8). The results are shown in Table 10.

[0173] Table 10 Results of experiments on the inhibition of ethanol-induced cytotoxicity by different samples

[0174] The data from the ethanol model group in Table 10 clearly demonstrate the inhibitory effect of ethanol toxicity on cells.

[0175] Overall, G1, G2, G3, and G4 all exhibited inhibitory effects on ethanol-induced cytotoxicity at the cellular level. Among them, G1, G2, and G3 showed significant inhibitory effects at a dose of 5 μg / mL. The inhibitory effect of G1 showed a clear dose-dependent positive correlation, while G2, G3, and G4 resulted in varying degrees of decreased cell viability at higher concentrations.

[0176] Sample (ethanol-containing) preparation: Accurately weigh 5g each of G1, G2, G3, and G4 and dissolve them in 5mL of PBS to prepare a 1g / mL stock solution. Filter the stock solution through a 0.22μm membrane for later use. Then, use DMEM medium to serially dilute to different concentrations of 200μg / ml, 100μg / ml, and 10μg / ml (a total of 12 samples). Anhydrous ethanol was added during sample preparation to achieve an ethanol concentration of 3.7% (v / v). In addition, an ethanol sample containing 3.7% (v / v) ethanol was prepared using PBS for use in the ethanol model group.

[0177] Assessment of cellular antioxidant and ethanol metabolism capacity: Cells with a density of 8×10⁶ cells were used. 5 HepG2 cells were seeded at 1 mL / well in 12-well plates. After 24 h of culture, the culture medium was discarded, and different concentrations of samples were added to each well. PBS was added to the blank control group, and ethanol was added to the ethanol model group. 1 mL of sample (containing ethanol) was added to each well, and each sample was added to 3 wells. After another 24 hours of culture, relevant indicators were measured according to the corresponding kit instructions. The measured indicators included cell viability, aspartate aminotransferase (AST) activity, and alanine aminotransferase (ALT) activity.

[0178] The activity test data of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in different samples are shown in Table 11.

[0179] The data from the ethanol model group in Table 11 clearly show that ethanol toxicity significantly increases the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

[0180] Overall, treatments with different concentrations of G1, G2, G3, and G4 all inhibited the ethanol-induced increase in AST and ALT levels, indicating that G1, G2, G3, and G4 could all alleviate ethanol-induced oxidative damage. Among them, G1 showed the most significant protective effect.

[0181] Table 11 Activity test data of AST and ALT in different samples

[0182] The procedures for animal toxicity testing were formulated in accordance with the "Technical Guidelines for Single-Dose Toxicity Studies of Drugs" (formerly State Food and Drug Administration, Announcement No. 4 of 2014). Single-dose toxicity tests were conducted on the four samples (PPE, G1, G2, and G3) obtained in Example 4. For each sample, two routes of administration (gavage and intraperitoneal injection) were required for the single-dose toxicity test, and a solvent (distilled water) control group was included. Ten animals (half male and half female) were used for each route of administration and different dosages.

[0183] ICR mice were used as the animal strain, with males weighing 32.7±2.7 grams and females weighing 26.8±3.1 grams. Dosage was calculated based on the weight of each animal. After a single administration of different doses of the sample or solvent, animals were observed for 14 consecutive days for mortality, appearance, behavior, and weight changes. Gross necropsy was performed on dead animals and at the end of the observation period, and histopathological examination was conducted if necessary.

[0184] Table 12: Single-dose toxicity studies of drugs

[0185] If at least one animal in a given dose group dies within 14 days, the dose is gradually reduced until all animals survive, and this dose is recorded as the maximum tolerated dose. After the 14-day observation period, the surviving animals in the maximum tolerated dose group are dissected to observe for organ abnormalities, and the number of animals with organ abnormalities is recorded.

[0186] Gradually increase the dose from the maximum tolerated dose until all animals in the group die within 14 days, and record this dose as the minimum lethal dose. Record the number of days since death for each animal: if the animal dies on the day of administration, record the number of days since death as 0; if it dies the next day, record it as 1, and so on. Then calculate the average number of days since death for all animals that died in the minimum lethal dose group.

[0187] The experimental results are recorded in Table 12. Overall, G1 and G2 showed low toxicity, while G3 exhibited the strongest toxicity. Animals receiving intraperitoneal injection of 0.25 mg / g (dose / animal body weight) of G3 died within 14 days, suggesting that the maximum tolerated dose of G3 via intraperitoneal injection is >0.25 mg / g. Furthermore, autopsies of animals in the maximum tolerated dose group revealed no significant abnormalities in most organs; the main signs of organ abnormalities included changes in liver color or intestinal bloating.

[0188] Six-week-old male C57BL / 6J mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. and housed in a sterile environment with a 12-hour light-dark cycle, humidity between 40% and 60%, and temperature of 22℃±2℃. After one week of acclimatization, the mice were randomly divided into the following groups, with five mice in each group, as shown in Table 13.

[0189] Table 13: Treatment methods for different groups of animals

[0190] G1, G2, G3, G4, and PPE were obtained according to the method described in Example 1. Mice in each group were administered the dosages shown in Table n via gavage, after which they were allowed free movement and feeding. All animals underwent their respective treatments for 8 days. Mice were euthanized on the last day, and liver tissue and blood were collected and stored at -80°C.

[0191] This experimental example established an alcohol-induced liver injury model using Erguotou (53% alcohol by volume, 12 mL / kg). The dosage of Erguotou was determined according to the "Evaluation and Testing Methods for Functional Health Foods (2023 Edition)". The research in this application demonstrates that edible alcohol is more effective than industrial alcohol in simulating real-life ALD in humans.

[0192] Biochemical analysis: To separate serum, whole blood samples from mice in each group were collected in blood collection tubes without anticoagulants. Each sample was allowed to stand at room temperature for 30-60 minutes without shaking or disturbance, allowing the blood to clot naturally. Then, each sample was centrifuged at 4°C at 4,000 rpm for 10 minutes. After standing, the supernatant was collected; this supernatant was the serum.

[0193] Liver tissue was collected from each group of mice, 1.0 g from each mouse. The liver tissue from each mouse was homogenized in 900 μL of protein extraction buffer (as per the corresponding kit) to prepare a liver homogenate. Then, each homogenate was centrifuged at 3,000 rpm for 5 minutes at 4°C, and the supernatant was extracted. The supernatant was then used to detect various antioxidant indicators in the liver and serum using a detection kit, and the results are recorded in Table 14.

[0194] Table 14

[0195] Ethanol-induced liver MDA levels significantly increased in mice, while interventions with G1, G2, and PPE significantly inhibited the ethanol-induced increase in liver MDA. GSH levels in the livers of mice in the G1, G2, and PPE groups were significantly higher than those in the alcohol group, while ALT and AST levels were relatively lower. At the same dosage, G1, G2, and PPE showed comparable efficacy in repairing alcoholic liver damage.

[0196] In summary, G1, G2, and PPE all have protective effects against alcoholic liver injury and can improve the pathological state of the liver. G1 and G2 have removed ascorbic acid from PPE, and G1 and G2 have lower toxicity and higher tolerable doses than PPE. They are also superior to PPE in terms of both taste when taken directly and their application as additives.

[0197] The present invention further applies prickly pear extract G1 and / or G2 to health foods, drugs or food additives for protecting the liver or treating chemical liver damage, wherein the health food or drug dosage form is oral, including but not limited to hard capsules, tablets, oral liquids, granules or soft capsules.

[0198] With the same amount of PPE, Guinong 5 produces more G1 and G2 particles than Guinong 7, while the procurement cost of Guinong 5 is lower than that of Guinong 7. Therefore, in terms of economics for the aforementioned purposes, Guinong 5 is more cost-effective than Guinong 7. Furthermore, for the purposes of this application, other prickly pear varieties with low ascorbic acid content can also be used as raw materials.

[0199] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Furthermore, the above description should be clear and implementable to those skilled in the art. Therefore, any equivalent changes or modifications made without departing from the spirit disclosed in the present invention should be included within the scope of the patent application.

Claims

1. A prickly pear extract with liver-protective effects, characterized in that, The extract is obtained by removing ascorbic acid from dried prickly pear powder. The removal rate of ascorbic acid from the prickly pear extract is greater than 95%, and the prickly pear extract has the effect of repairing alcoholic liver damage.

2. The prickly pear extract as described in claim 1, characterized in that, The prickly pear extract is composed of components in prickly pear powder that are insoluble in methanol.

3. The prickly pear extract as described in claim 1, characterized in that, The prickly pear extract consists of components in prickly pear powder that are soluble in methanol and insoluble or slightly soluble in ethyl acetate.

4. [Amended according to Rule 26, 27.04.2026] The method for preparing the prickly pear extract according to any one of claims 1 to 3, characterized in that, Includes the following steps: Step 1: Juice the prickly pear, separate the supernatant, and dehydrate the supernatant to obtain prickly pear powder; Step 2: Dissolve the dried prickly pear powder completely in methanol, separate the solid phase that is insoluble in methanol, and obtain G1; Step 3: After evaporating and concentrating the liquid phase obtained in Step 2, add excess ethyl acetate to precipitate a solid phase; after filtration, the solid phase is dissolved in water, insoluble impurities are filtered out, and then concentrated and dried to obtain G2; Step 4: Add water to the liquid phase obtained in Step 3 and evaporate the oil phase organic solvent on the upper layer of the aqueous phase. Then extract the aqueous phase with excess ethyl acetate, separate the ethyl acetate from the aqueous phase and evaporate it to obtain G3.

5. The method for preparing a prickly pear extract with liver-protecting effects as described in claim 1, characterized in that, Includes the following steps: Purification process: Using C18 reversed silica gel powder as the stationary phase and methanol aqueous solution as the mobile phase, the dried prickly pear powder was subjected to column chromatography. The dried prickly pear powder was washed with the methanol aqueous solution to obtain a purified prickly pear eluent. as well as Freeze-drying: The purified prickly pear eluent was freeze-dried at a temperature range of -50℃ to -60℃ and a pressure range of 0.1Mbar to 0.2Mbar for 48 hours to obtain purified prickly pear powder.

6. The method for preparing the prickly pear extract as described in claim 5, characterized in that: It also includes raw material pretreatment: the prickly pear is washed and crushed, and then juiced using a juicer to obtain a solid-liquid mixture containing prickly pear pulp; the solid-liquid mixture containing prickly pear pulp is centrifuged at less than 4°C and at a speed range of 5,000 r / min to 6,000 r / min for 10-15 minutes, and the supernatant is taken out after standing. After obtaining the supernatant, a freeze-drying process is performed, in which the supernatant is freeze-dried for 48 hours at a temperature range of -50℃ to -60℃ and a pressure range of 0.1Mbar to 0.2Mbar, and then stored to obtain the prickly pear powder.

7. The use of the prickly pear extract according to any one of claims 1 to 3 or the prickly pear extract prepared by the method according to any one of claims 4 to 6 in the preparation of drugs and health products for protecting the liver or treating chemically induced liver injury.

8. The use according to claim 7, characterized in that, The medicine and health product include an effective amount of the prickly pear extract and excipients.

9. The use according to claim 8, characterized in that, The maximum single oral dose of the prickly pear extract is 20-25 g / kg body weight.

10. The use according to claim 8, characterized in that, The amount of the prickly pear extract used shall not exceed 1 gram per kilogram of body weight per day.

11. The use according to any one of claims 7 to 10, characterized in that, The dosage form of the drug is selected from hard capsules, tablets, oral liquids, granules, or soft capsules.

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