Malus sieversii extract with liver protection efficacy, preparation method and use thereof

Abstract

The roxburgh rose extract with liver protection effect is prepared by removing ascorbic acid in roxburgh rose, and has better taste than PPE when directly taken or is applied in more application fields as an additive. The roxburgh rose extract can be applied in medicines, health foods or food additives for protecting liver or treating chemical liver injury, wherein the medicine or health food is in the form of hard capsule, tablet, oral liquid, granule or soft capsule. The preparation method of the roxburgh rose extract with liver protection effect comprises the following steps: purification treatment: using C18 reverse silica gel powder as a stationary phase and methanol aqueous solution as a mobile phase, performing column chromatography on roxburgh rose freeze-dried powder, and washing the roxburgh rose juice freeze-dried powder with methanol aqueous solution to obtain purified roxburgh rose eluent; and freeze-drying treatment: freeze-drying the purified roxburgh rose eluent at a temperature range of-50 DEG C to-60 DEG C and a pressure range of 0.1 Mbar to 0.2 Mbar for 48 h to obtain purified roxburgh rose freeze-dried powder.

Description

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] The liver is the largest solid organ in the abdominal cavity, performing vital physiological functions. As a crucial detoxification organ, the liver is supplied with blood by both the hepatic artery and hepatic vein. However, liver cells have poor tolerance to hypoxia and are easily damaged. Chemical liver injury is liver damage caused by hepatotoxic chemicals, including alcohol, environmental toxic chemicals, and certain medications.

[0003] Alcohol-induced liver disease is called alcoholic liver disease (ALD), and the main cause is long-term excessive alcohol consumption. The prevalence of ALD is significantly higher in men than in women, at 7.80% (95% confidence interval: 5.70% - 0.19%) and 0.88% (95% confidence interval: 0.35% - 1.64%), respectively. Furthermore, fatty liver is the initial manifestation of alcoholic liver disease and can lead to cirrhosis, hepatitis, liver fibrosis, and even liver cancer. In China, alcohol consumption is the second leading cause of liver damage after hepatitis virus infection. For patients with compensated or decompensated liver disease at any stage, abstinence from alcohol is considered the best option. However, progress in other areas remains limited. For the prevention or treatment of ALD, dietary support is as important as clinical treatment and abstinence from alcohol. Therefore, there is increasing interest in new strategies and effective methods for ALD intervention.

[0004] The pathogenesis of ALD is complex and involves multiple factors, including hepatocellular damage caused by ethanol and its metabolites, increased reactive oxygen species, decreased antioxidants, inflammation caused by Kupffer cells, changes in intestinal permeability, and intestinal flora imbalance.

[0005] The pathogenesis of ALD is not yet fully understood and requires further research. Studies have shown that the conversion of alcohol into acetaldehyde by cells leads to the formation of DNA adducts, causing DNA damage. This damage activates the Fanconi anemia pathway (FA pathway), which is responsible for DNA repair. The 22 genes that make up the FA pathway form a metabolic network supporting DNA replication, repair, and other cellular functions. Their primary purpose is to eliminate ICLs—covalent linkages between two complementary DNA strands that hinder DNA strand separation during replication. Furthermore, with further research into the FA pathway, researchers have discovered its significant role in cellular antioxidant processes. Proteins in this pathway specifically bind to the promoters of major antioxidant defense genes, protecting these genes from oxidative damage and thus exerting their antioxidant function.

[0006] There are currently no drugs available 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. Some plant extracts with anti-ALD properties have been disclosed in the literature, 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.

[0007] The 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," the prickly pear contains ascorbic acid (841.58–3499.84 mg per 100 grams of fresh fruit), ranking first among fruits and vegetables. Currently, the main varieties of prickly pear 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. The prickly pear can be used as food and also as a traditional medicine. According to the *Encyclopedia of Guizhou* and *Compendium of Materia Medica Supplement*, the fruit, leaves, and roots of the prickly pear are widely used for digestion, spleen strengthening, relieving summer heat, and stopping diarrhea. Currently, it is 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"). It is generally believed that the biological efficacy of prickly pear is due to the synergistic effect of its rich ascorbic acid (vitamin C) and other components. As a food, prickly pear is more easily accepted by consumers than drugs. Wu Lifu, He Gang, and others believe that prickly pear juice contains abundant vitamin C and other beneficial substances, thus having certain antioxidant and liver-protective effects. They also pointed out that "although vitamin C can prevent the occurrence of liver tumors, long-term use as a drug is not in line with people's lifestyles. Prickly pear juice is a natural beverage, and it goes without saying that regular consumption of prickly pear juice can combine cancer prevention with enjoyment" ("Protective Effect of Prickly Pear Juice on Liver Tumors Caused by Dimethylnitrosamine Precursor", Journal of Guizhou Agricultural College, 1987, No. 2).

[0008] The high ascorbic acid content in prickly pear results in a poor taste and makes it difficult to juice or freeze-dry the juice for use as an additive in the food industry or health care field. Summary of the Invention

[0009] The main objective of this invention is to concentrate and purify prickly pear extract to enhance its efficacy in treating chemically induced liver injury. This also improves the taste while reducing the dosage for the user.

[0010] 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%.

[0011] This invention discloses a method for preparing a prickly pear extract with liver-protective effects. The method is characterized by the following steps: purification: using C18 reverse-phase silica gel powder as the stationary phase and methanol-water solution as the mobile phase, column chromatography is performed on the lyophilized prickly pear powder. The lyophilized prickly pear juice is then washed with methanol-water solution to obtain a purified prickly pear eluent; and freeze-drying: the purified prickly pear eluent is freeze-dried at a temperature range of -50℃ to -60℃ and a pressure range of 0.1 Mbar to 0.2 Mbar for 48 hours to obtain purified lyophilized prickly pear powder.

[0012] In a preferred embodiment of the present invention, after obtaining the supernatant, a freeze-drying step is further included. The freeze-drying step involves freeze-drying the supernatant for 48 hours at a temperature range of -50°C to -60°C and a pressure range of 0.1 Mbar to 0.2 Mbar, and then storing it to obtain freeze-dried prickly pear powder.

[0013] In a preferred embodiment of the present invention, the method further includes a step of detecting the ascorbic acid content in the freeze-dried powder: using L-ascorbic acid as a standard, the ascorbic acid content in the freeze-dried prickly pear powder is determined by thin-layer chromatography.

[0014] In a preferred embodiment of the present invention, the ascorbic acid content in the freeze-dried prickly pear powder is 28.25 mg / 100 mg to 32.33 mg / 100 mg, and the residual ascorbic acid content in the purified freeze-dried prickly pear powder is 1.03 mg / 100 mg to 2.12 mg / 100 mg.

[0015] In a preferred embodiment of the present invention, the volume ratio of methanol to water in the mobile phase is 4:6, 6:4, or 8:2.

[0016] This invention also discloses a method for preparing purified lyophilized prickly pear powder using prickly pear extract, characterized in that the ascorbic acid removal rate is above 95%. When the concentration of the purified lyophilized prickly pear powder is 35 μg / mL, the scavenging rate of ABST free radicals is 65.23% and the scavenging rate of DPPH free radicals is 56.69%; when the concentration of the purified lyophilized prickly pear powder is 50 μg / mL, the scavenging rate of ABST free radicals is 92.5% and the scavenging rate of DPPH free radicals is 82.58%; when the concentration of the purified lyophilized prickly pear powder is 125 μg / mL, the scavenging rate of ABST free radicals is 98.56% and the scavenging rate of DPPH free radicals is 91.63%.

[0017] The present invention also discloses the use of a prickly pear extract with hepatoprotective effects for protecting the liver or treating chemically induced liver damage, characterized in that the amount of purified prickly pear freeze-dried powder used is 1 gram / (kg body weight·day).

[0018] This invention also discloses the application of a prickly pear extract with hepatoprotective effects as a drug for protecting the liver or treating chemically induced liver damage, in health foods or as a food additive.

[0019] In a preferred embodiment of the present invention, the dosage form of the drug or health food is a hard capsule, tablet, oral liquid, granule, or soft capsule. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the steps of a method for preparing prickly pear extract according to the technology disclosed in this invention. Figure 2 This is a flowchart illustrating the steps of another method for preparing prickly pear extract according to the technology disclosed in this invention.

[0021] Figure 3 This is a schematic diagram illustrating how the components mitigate 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.

[0022] Figure 4 This 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.

[0023] Figure 5 This diagram illustrates the mitigation of ethanol-induced liver injury in an animal model of ALD, based on the technology disclosed in this invention. AD: Detection of MDA (A), GSH (B), ALT (C), and AST (D) levels in the liver of each treatment group. Figure 6 This is a schematic diagram illustrating how each component alleviates ethanol-induced liver pathological damage, based on the technology disclosed in this invention.

[0024] Figure 7 This is a schematic diagram illustrating the function of screening and predicting 12 PPE metabolites that directly enter the liver, based on the technology disclosed in this invention.

[0025] Figure 8 This is a protein-protein interaction (PPI) network representing potential ALD targets, based on the technology disclosed in this invention. Detailed Implementation

[0026] 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.

[0027] 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) activity, 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.

[0028] Example 1.

[0029] Please refer to the following first. Figure 1 . Figure 1 This is a flowchart illustrating the steps of a method for preparing prickly pear extract according to the technology disclosed in this invention. Figure 1 As shown, step S10: Raw material pretreatment. In this step, the outer skin of the Guinong No. 5 prickly pear from Liupanshui City, Guizhou Province, is washed and crushed, then juiced 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 ranging from 5,000 g / min to 6,000 g / min. After centrifugation and standing for a period of time, the supernatant of the prickly pear juice is collected.

[0030] 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 freeze-dried powder (PPE).

[0031] Purification can be performed directly after step S10, but purification is more efficient using the lyophilized prickly pear powder (PPE) obtained in step S11.

[0032] 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 for column chromatography of the prickly pear lyophilized powder obtained in step S11. The mobile phase (methanol:water) is used in gradient washing at a volume ratio 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 lyophilized powder, the column is packed with methanol and then equilibrated with a column volume of 2 column volumes (methanol:water volume ratio of 4:6). Then, a methanol:water ratio of 4:6 is used as the loading system for column loading. Subsequently, gradient washing is performed 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. The column is monitored by TLC every ten minutes. After ascorbic acid is detected, the column loading system is kept constant, and the sample vial is replaced until ascorbic acid is no longer detected by TLC. After purification, the silica gel column was washed with three column volumes of methanol solution, and the components other than ascorbic acid were collected. Then, the methanol in the sample vial was removed by rotary evaporation, and the remaining solution was cold-dried at -60°C and 0.1 Mbar for 48 hours to obtain ascorbic acid-free lyophilized prickly pear powder.

[0033] Step S14: Freeze-drying. In this step, the purified prickly pear eluent obtained in step S12 is freeze-dried at a temperature range of -40℃ to -60℃ and a pressure range of 0.1Mbar to 0.2Mbar for 48 hours to obtain purified prickly pear freeze-dried powder (nVc-PPE).

[0034] For subsequent testing, the purified freeze-dried prickly pear powder can be mixed with deionized water to obtain a purified prickly pear aqueous solution.

[0035] Step S15, Ascorbic Acid Content Detection. Using L-ascorbic acid (vitamin C) as a standard, the ascorbic acid content in the lyophilized prickly pear powder (PPE) was determined by thin-layer chromatography (TLC). The lyophilized prickly pear powder obtained in step S11 and the purified lyophilized prickly pear powder obtained in step S12 were mixed thoroughly with deionized water. The detection was performed according to GB5009.86-2016 Method I (High Performance Liquid Chromatography). The results showed that the ascorbic acid content in the lyophilized prickly pear powder (PPE) was 28.25 mg / 100 mg, and the ascorbic acid content in the purified lyophilized prickly pear powder was 1.03 mg / 100 mg. This confirms that most of the ascorbic acid in the purified lyophilized 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.

[0036] Removal rate = 1 - a / (b * (1 - a) / (1 - b)) (Equation I)

[0037] Where a = ascorbic acid content (w / w) in purified prickly pear freeze-dried powder nVc-PPE.

[0038] b = Ascorbic acid content (w / w) in PPE of freeze-dried prickly pear powder

[0039] Example 2.

[0040] Figure 2 This 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 in the range of 5,000 g / min to 6,000 g / min. After centrifugation and standing for a period of time, the supernatant of the prickly pear juice is collected.

[0041] 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, and then stored to obtain freeze-dried prickly pear powder (PPE).

[0042] Step S22: Purification. 30 g of the lyophilized prickly pear powder obtained in step S21 was mixed with 90 mL of a methanol-water solution (methanol:water ratio 4:6). Column chromatography was performed using C18 reverse-phase silica gel as the stationary phase and the methanol-water solution as the mobile phase. The lyophilized prickly pear powder was washed 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.

[0043] 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 freeze-dried powder.

[0044] For subsequent testing, the purified freeze-dried prickly pear powder can be mixed with deionized water to obtain a purified prickly pear aqueous solution.

[0045] Step S24, Ascorbic Acid Content Detection. Using L-ascorbic acid (vitamin C) as a standard, the ascorbic acid content in the lyophilized prickly pear powder was determined by thin-layer chromatography (TLC). The lyophilized prickly pear powder (PPE) obtained in step S21 and the purified lyophilized prickly pear powder obtained in step S23 were mixed thoroughly with deionized water. The detection was performed according to GB5009.86-2016 Method I (High Performance Liquid Chromatography). The results showed that the ascorbic acid content in the lyophilized prickly pear powder in this embodiment was 32.33 mg / 100 mg, and the ascorbic acid content in the purified lyophilized prickly pear powder was 2.12 mg / 100 mg. This confirms that most of the ascorbic acid in the purified lyophilized prickly pear powder was removed through the above extraction steps, with a removal rate of 95.47%.

[0046] Example 3.

[0047] The steps are the same as in Example 1, except that acetonitrile is used instead of methanol aqueous solution as the mobile phase.

[0048] After testing for ascorbic acid content, it was found that the ascorbic acid content in the freeze-dried prickly pear powder was 28.25 mg / 100 mg, and the ascorbic acid content in the purified freeze-dried prickly pear powder was 1.57 mg / 100 mg. This confirms that most of the ascorbic acid in the purified freeze-dried prickly pear powder has been removed through the above extraction steps, with a removal rate of 95.95%.

[0049] Experimental example.

[0050] 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.

[0051] Table 1: Treatment methods for different groups of animals

[0052]

[0053] 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.

[0054] 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.

[0055] Next, in order to demonstrate that nVc-PPE has significant liver-protective and antioxidant effects, the present invention further conducted the following analyses.

[0056] Biochemical analysis:

[0057] To separate serum, whole blood from the four groups of rats was 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 g / min 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 rats 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 g / min 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).

[0058] Histological analysis:

[0059] Immediately after euthanasia of rats, 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.

[0060] Cell culture and processing:

[0061] HepG2 cells were cultured in a 37°C, 5% CO2 incubator, with DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were passaged at 70%-80% confluence. To determine the PPE concentration without negative impact on HepG2 cells, the protective effect of PPE in the range of 0 μg / mL to 300 μg / mL (in increments of 50 μg / mL) was investigated. 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 assessed, detecting concentrations from 0% to 10% (in increments of 1%) to determine the optimal concentration for establishing a liver injury model. Cells were then treated for 24 hours with 250 μg / mL PPE and nVc-PPE containing the IC50 concentration of ethanol (3.7% ethanol) to evaluate the protective effect of each component against ethanol-induced HepG2 cell injury. Figure 3 As shown in Figure C.

[0062] Assess free radical scavenging ability:

[0063] ABTS and DPPH were used to determine the free radical scavenging capacity or hydrogen donor capacity of different components.

[0064] Cell viability assessment:

[0065] 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).

[0066] Evaluation of antioxidant system biomarkers:

[0067] 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.

[0068] Determination of ADH and ALDH activity:

[0069] 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).5 Cells 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.

[0070] 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.

[0071] 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.

[0072] Table 2

[0073]

[0074]

[0075] Please refer to the following: Figure 3 . Figure 3 This is a schematic diagram illustrating how the components of PPE mitigate 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 PPE and nVc-PPE with 3.7% ethanol for 24 hours. The mitigation of ethanol-induced cytotoxicity by each component is shown 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.

[0076] Table 3

[0077]

[0078] For example Figure 3Figure A shows that 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 found to be 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 morphology and number were close to normal cells. Figure 3 Figure B shows that, overall, the purified nVc-PPE did not diminish its effectiveness in protecting HepG2 cells from ethanol-induced cytotoxicity.

[0079] Figure 4 This 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 activity for each group, including MDA (mean), MDA (standard deviation), GSH (mean), GSH (standard deviation), ALT (mean), ALT (standard deviation), AST (mean), and AST (standard deviation). 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.

[0080] Table 4

[0081]

[0082] like Figure 4 As shown, in an ethanol-induced cell damage model, the levels of relevant antioxidant system indicators were measured to investigate the effects of component interventions on ethanol-induced oxidative stress in HepG2 cells. Figure 4 Figures A and B 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 AST and ALT enzyme activities, 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.

[0083] 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.

[0084] Table 5

[0085]

[0086] Ethanol-induced liver MDA levels in mice were significantly elevated, while interventions with PPE and nVc-PPE significantly inhibited the ethanol-induced increase in liver MDA. Figure 5 Figure A shows that the level of GSH in the liver of the intervened mice was significantly higher than that in the ETOH group, while the levels of ALT and AST were significantly lower.

[0087] Effects of each component on ethanol-induced liver pathological damage

[0088] 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 were observed using H&E staining, such as… Figure 6 As shown in the figure, 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, this manifested as an increase in vacuoles in the tissue, which may be due to intracellular fat accumulation or cell damage; irregular arrangement of hepatocytes, with the originally tightly ordered cell arrangement becoming disordered; and blurred cell boundaries, with the boundaries between cells no longer clear, indicating cell swelling or damage.

[0089] 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.

[0090] Screening of potential active ingredients in PPE

[0091] To investigate whether certain components of PPE could be directly introduced into 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 According to the technology disclosed in this invention, a schematic diagram illustrates the screening and prediction of the function of 12 PPE metabolites that directly enter the liver. The results show that these metabolites are highly expressed in the PPE-treated groups (low / high PPE groups), and are mainly composed of lipids and organic acids, such as... Figure 7 As shown in Figure A. To confirm whether the upregulated metabolites originated from PPE, these metabolites were further screened. The three groups that did not receive PPE gavage (normal group, negative group, and positive group) were grouped together as the nPPE group, while the two groups that received PPE gavage (low PPE group and high PPE group) 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 screened out a total of 29 metabolites. Further screening of these metabolites based on the total content of PPE metabolites revealed 12 DEMs that were themselves highly present in PPE and whose levels in the PPE group were significantly higher than those in the nPPE group, such as... Figure 7 As shown in Figure B and listed in Table 6, it is speculated that these 12 components can directly enter the liver from PPE, thereby reducing ethanol-induced oxidative damage.

[0092] Table 6

[0093]

[0094]

[0095] 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, such as... Figure 7 As shown in Figure C. Next, KEGG enrichment analysis was performed on the target genes of these 12 identified metabolites. Figure 7 The D-plot shows the 15 pathways with the highest enrichment, primarily in fatty acid degradation, glycolysis / gluconeogenesis, drug metabolism, and alcoholic liver disease. Disorders of these pathways are commonly associated with the development of ALD. Additionally, please refer to [link to relevant documentation]. Figure 8 . Figure 8 This is a schematic diagram representing the protein-protein interaction (PPI) network of potential ALD targets according to the technology disclosed in this invention. Figure 8In this study, a protein-protein interaction (PPI) network of target genes was constructed using the STRING database (minimum necessary interaction value = 0.9). The results in Table 7 revealed that CYP1A1 and MAOB had 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.

[0096] Table 7

[0097]

[0098]

[0099] The present invention further verifies the protective effects of PPE, Vc and nVc-PPE against ethanol-induced liver injury.

[0100] Experimental animals: Male C57BL / 6 mice (6 weeks old, SPF grade, housed separately).

[0101] Reagents:

[0102] 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.

[0103] Experimental Groups:

[0104] 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.

[0105] The administration period for the test samples was 30 days. The grouping and treatment of the experimental animals are listed in Table 8.

[0106] Table 8

[0107]

[0108]

[0109] Animal culling and harvesting:

[0110] 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.

[0111] Experiment 1: Assessment of Liver Fibrosis and Collagen Deposition

[0112] Objective: To evaluate the inhibitory effects of PPE, Vc, and nVc-PPE on ethanol-induced liver fibrosis.

[0113] Masson staining:

[0114] 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.

[0115] Experiment 2: Detection of Inflammatory Response

[0116] Objective: To verify the inhibitory effects of PPE, Vc, and nVc-PPE on ethanol-induced inflammation.

[0117] Experiment 2 included the detection of serum inflammatory factors and liver inflammatory factors.

[0118] Serum inflammatory factor detection:

[0119] Collected plasma samples were used to detect the levels of TNF-α, IL-6, and IL-1β according to the ELISA kit protocol.

[0120] Liver inflammatory factor detection:

[0121] 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 5000g / 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.

[0122] Experiment 3: Lipid Metabolism and Liver Function Assessment

[0123] Objective: To evaluate the effects of PPE, Vc, and nVc-PPE on lipid accumulation and liver function.

[0124] Liver lipid content determination:

[0125] 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.

[0126] Expanded range of serum liver function indicators:

[0127] Take the prepared plasma and detect the total bilirubin content in the plasma using the TBIL detection kit.

[0128] Experiment 4: Non-target metabolomics analysis of nVc-PPE

[0129] Objective: To compare the content of 12 potential active substances in PPE and nVc-PPE.

[0130] 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.

[0131] 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 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.

[0132] 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 freeze-dried prickly pear powder, and the removal rate of ascorbic acid from the prickly pear extract is greater than 95%.

2. The prickly pear extract as described in claim 1, characterized in that, The ascorbic acid content in the prickly pear extract is 1.03–2.12 mg / 100 mg.

3. 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: The lyophilized prickly pear powder was subjected to column chromatography using C18 reversed silica gel powder as the stationary phase and methanol aqueous solution as the mobile phase. The lyophilized prickly pear juice 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 freeze-dried prickly pear powder.

4. The method for preparing the prickly pear extract as described in claim 3, 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 g / min to 6,000 g / 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 freeze-dried prickly pear powder.

5. The method for preparing the prickly pear extract as described in claim 3, characterized in that: The process further includes an ascorbic acid content detection step following the purification and freeze-drying steps: using L-ascorbic acid as a standard, the ascorbic acid content in the freeze-dried prickly pear powder is determined by thin-layer chromatography.

6. The method for preparing the prickly pear extract as described in claim 5, characterized in that: The residual ascorbic acid content in the prickly pear freeze-dried powder after the purification process is 1.03 mg / 100 mg.

7. The method for preparing the prickly pear extract as described in claim 1, characterized in that: The volume ratio of methanol to water in the mobile phase is 4:6, 6:4, or 8:

2.

8. The use of prickly pear extract for protecting the liver or treating chemically induced liver injury, characterized in that, The dosage of the prickly pear extract is 1 gram per kilogram of body weight per day.

9. The use of the prickly pear extract with hepatoprotective effects as described in claim 1 as a drug, health food, or food additive for protecting the liver or treating chemically induced liver damage.

10. The application of the prickly pear extract as described in claim 9, characterized in that, The dosage form of the drug or the health food is a hard capsule, tablet, oral liquid, granule, or soft capsule.

Images