Preparation method of hovenia dulcis thunb active component extract with liver protection effect as well as obtained product and application of hovenia dulcis thunb active component extract

Abstract

The invention belongs to the field of biological medicine, and relates to a hovenia dulcis thunb extraction process, in particular to a preparation method of a hovenia dulcis thunb liver-protecting active component extract with a liver-protecting effect, an obtained product and application. According to the hovenia dulcis thunb extract provided by the invention, active components with specified components can be more efficiently obtained by adopting a two-dimensional high-throughput separation analysis technology; according to the method, the flavonoid components in the hovenia dulcis thunb crude extract are accurately obtained, the content of the flavonoid components is larger than 80%, the extraction time is shortened by 60%, and manpower and reagent use are saved. The hovenia dulcis thunb active component obtained by the method provided by the invention is clear in substance composition, high in extraction rate of flavonoid components and obvious in biological activity in liver protection, and in-vitro cell experiments prove that the hovenia dulcis thunb extract can obviously reduce the ROS level of cells, improve the survival rate of the cells and obviously reduce the ALT and AST levels in cell supernatant. The hovenia dulcis thunb active component extract prepared by the invention lays an experimental foundation for developing a new drug for treating alcoholic liver diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, relates to a Hovenia dulcis fruit extraction process, and specifically relates to a preparation method of a Hovenia dulcis fruit liver-protecting active component extract having a liver-protecting effect, and the obtained product and application. Background Art

[0002] Alcoholic liver disease (ALD) refers to liver lesions caused by excessive drinking. The initial symptoms are mainly fatty hepatitis, liver fibrosis and cirrhosis. In severe cases, it can cause liver cancer and even liver failure. In 2019, about 25% of deaths from cirrhosis worldwide were related to alcohol. Currently, its treatment methods are mostly strict abstinence from alcohol, reduced glutathione therapy and other drug treatments, and nutritional support therapy, which are used in the middle and late stages of the disease. Traditional Chinese medicine has made effective progress in the treatment of all stages of the disease.

[0003] Hovenia dulcis was first recorded in Tang Materia Medica. It has the effects of quenching thirst, relieving restlessness, clearing dampness and heat, and detoxifying alcohol. The Medical Compilation records that "for detoxifying alcohol, Hovenia dulcis is the best". Modern medical research has confirmed that Hovenia dulcis has the effects of detoxifying alcohol, protecting the liver, and anti-tumor. There are currently a number of alcohol detoxification and liver protection products based on Hovenia dulcis on sale. However, its liver protection components are unclear, which limits the use of products and quality upgrades. Therefore, the extraction and research of active components with liver protection effects in Hovenia dulcis can provide theoretical and technical support for the clinical application and resource development of Hovenia dulcis. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a method for preparing an active component extract of Hovenia dulcis with liver protection effect. The present invention also provides an active component extract of Hovenia dulcis prepared by the above preparation method.

[0005] Another object of the present invention is to provide the use of the active component extract of Hovenia dulcis.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: The present invention provides a method for preparing an active component extract of Hovenia dulcis with liver protection effect, comprising the following steps: S1. Grind the Hovenia dulcis into coarse powder, add ethanol solution to reflux extraction, filter, collect the filtrate, combine and recover ethanol, and concentrate to obtain a thick paste; S2. The obtained thick paste was suspended in water, extracted with an organic solvent until the organic layer became pale in color, the organic solvent was recovered under reduced pressure, and the extracts of each part were obtained by freeze-drying; S3. Weigh the dry extract of ethyl acetate, dissolve it in methanol, filter it through a membrane, add C4 powder for adsorption, stir evenly, and recover the solvent under reduced pressure; dry column loading, use an eluent to perform primary separation and secondary separation according to the elution procedure, and automatically take the fractions; S4. Recover the solvent under reduced pressure to a small volume and freeze-dry it.

[0007] Furthermore, in S1, the volume fraction of the ethanol solution is 75%; the mass volume ratio of the Hovenia dulcis to the ethanol solution is 1:10; and the reflux extraction is performed twice, each time for 1.5 hours.

[0008] Furthermore, in S2, during the extraction process, dichloromethane and ethyl acetate are used sequentially for extraction.

[0009] Furthermore, in S3, the mass volume ratio of the dry extract to methanol is 0.1-0.5:1; the mass volume ratio of the dry extract to C4 powder is 1-2:1.

[0010] Further, in S3, the elution procedure used in the main separation is: In the main separation stage, the elution portion from 33 to 36 minutes was collected, and then secondary separation was performed.

[0011] Further, in S3, the elution procedure used in the secondary separation is: In the secondary separation stage, the elution portion from 26.7 to 30.1 min was collected to obtain fraction trap-48.

[0012] Furthermore, in S4, the freeze-drying operation is as follows: pre-freeze in a -40 to -50°C cold trap for 2 to 4 hours, and then freeze-dry at -45 to -55°C for 8 to 24 hours.

[0013] The present invention also provides an active component extract of Hovenia dulcis prepared by the above preparation method, wherein the content of flavonoids in the active component extract of Hovenia dulcis is greater than 80%.

[0014] The present invention further provides the use of the active component extract of Hovenia dulcis in preparing products for treating and / or preventing alcoholic liver damage.

[0015] The active component extract of Hovenia dulcis prepared by the present invention can be used to prepare any form of products such as medicines, health foods and foods for auxiliary liver protection. The active component extract of Hovenia dulcis prepared by the present invention can preventively protect alcoholic liver damage, reduce oxidative stress response, and reduce ALT and AST levels.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The Hovenia dulcis extract provided by the present invention can more efficiently obtain active components of a specified composition by adopting a two-dimensional high-throughput separation and analysis technology; accurately obtain the flavonoid components in the crude extract of Hovenia dulcis, wherein the content of flavonoid components is greater than 80%, shorten the extraction time by 60%, and save manpower and reagents.

[0017] (2) The active components of Hovenia dulcis obtained by the method provided by the present invention have a clear material composition, a high extraction rate of flavonoid components, and obvious biological activity in protecting the liver. In vitro cell experiments have shown that the Hovenia dulcis extract can significantly reduce the level of cellular ROS, improve cell survival rate, and significantly reduce the levels of ALT and AST in the cell supernatant.

[0018] (3) The active component extract of Hovenia dulcis prepared by the present invention lays an experimental foundation for developing a new drug for treating alcoholic liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of the effects of different extracts on cell proliferation rate; Figure 2 Effects of the active component extract of Hovenia dulcis (trap-48) prepared in Example 1 on ROS; wherein a is a normal group, b is a positive probe group, c is a model group, and d is a drug-administered group; Figure 3 Effects of the active component extract of Hovenia dulcis (trap-48) prepared in Example 1 on ALT and AST. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0021] Example 1 This example prepares an extract of liver-protective active components from Hovenia dulcis, and the specific process is as follows: 1. Extraction and separation of active parts Grind 500g of Hovenia dulcis into coarse powder, add 10 times the amount of 75% ethanol and reflux extract twice, each time for 1.5h, filter, collect the filtrate, combine and recover ethanol, and concentrate to obtain a thick paste. The obtained thick paste is suspended in water, extracted with dichloromethane and ethyl acetate in sequence until the color of the organic layer is light, and the ethyl acetate part is recovered under reduced pressure, and freeze-dried to obtain a dry extract of the ethyl acetate part. Weigh, add appropriate amount of methanol to dissolve, pass through a 0.45µm filter membrane, add appropriate amount of C4 powder for adsorption, stir evenly, and recover the solvent under reduced pressure. Dry column loading. A two-dimensional high-throughput separation and analysis system (Sepbox 2D-2000) was used, and the eluent was used according to the elution program for primary separation (filler C4 powder) and secondary separation (filler C18). The fractions were automatically collected (TrapColunm 11-Second Step9-26.7~30.1min), and the solvent was recovered to a small volume under reduced pressure and freeze-dried to obtain 11.54 mg of extract (trap-48). The samples were stored at -20°C.

[0022] The specific process is as follows: the main separation is performed using the eluent according to the elution procedure of the main separation (Table 1), and the capture column Trap Column 11 (elution part of 33 to 36 min) is used for secondary separation using the secondary separation column and the secondary separation procedure (Table 2). The elution procedures are shown in Tables 1 and 2.

[0023] Table 1 Main separation and elution schedule Note: Wavelength:252nm; appendix No.8 Table 2 Second Stap9 conditions Note: Wavelength:220nm; appendix No.17 2. Mass spectrometry analysis of active site components Fraction trap-48 was subjected to UPLC-MS / MS analysis according to the following method.

[0024] 2.1 Establishment of mass spectrometry database By consulting the literature, the chemical composition information of Hovenia dulcis was collected and a database containing Compound, Formula, and t R , Calculated mass (m / z), MS / MS and other information.

[0025] 2.2 Sample preparation Take the main fraction trap-48, recover the solvent under reduced pressure, weigh the residue, add an appropriate amount of methanol, dissolve and shake well, transfer to a 1mL volumetric flask, make up to volume, shake well, and filter through a 0.22µm microporous membrane to obtain the fraction with a concentration of 0.44mg / mL.

[0026] 2.3 Preparation of reference solution Weigh appropriate amount of myricetin, dihydromyricetin, gallic acid, and protocatechuic acid reference substances, dissolve in methanol, dilute to volume in a 5 ml volumetric flask, shake well, and filter through a 0.22 µm microporous membrane. The concentrations of each reference substance are 0.09 mg / mL, 0.12 mg / mL, 0.095 mg / mL, and 0.23 mg / mL, respectively.

[0027] 2.4 LC-MS test conditions Column: Thermo Acclaim TM RSLC 120 C18 column (100 mm×2.1 mm, 2.2 μm); mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution (0 min, 90% B; 2 min, 90% B; 5 min, 80% B; 8 min, 80% B; 12 min, 70% B; 14 min, 65% B; 19 min, 10% B; 23 min, 10% B; 27 min, 90% B; 30 min, 90% B); flow rate: 0.30 mL min -1 ; Injection volume: 3 µl; Column temperature: 35 ℃.

[0028] The mass spectrometry conditions used for sample detection are shown in Table 3.

[0029] Table 3 Mass spectrometry conditions 2.5 Sample determination and result analysis The determined analytical conditions were used for the determination, and the result data were analyzed using Xcalibur 4.3 software to infer the composition of the components. The results are shown in Table 4.

[0030] Application Example 1 The Hovenia dulcis extract trap-48 of Example 1 of the present invention was compared with other Hovenia dulcis extracts trap-29 (Trap Column 6- Second Step 5-3.5-4.4 min) and trap-45 (Trap Column 11-Second Step 9-3.2-5 min) to conduct a pharmacodynamic experiment on the proliferation rate of HepG2 cells.

[0031] Separation conditions of Trap29: Take the 25-27min section (TrapColumn6) of the main separation for secondary separation. The specific separation conditions are shown in Table 5. Take the 3.5-4.4min section fraction and reduce the pressure to recover the solvent to dryness, which is Trap29.

[0032] Table 5 Secondary separation chromatographic conditions (step 5) Note: Wavelength: 220nm; appendix No. 13 Separation conditions of Trap45: Take the 33-36 min section of the main separation (TrapColumn 11) for secondary separation. The specific separation conditions are shown in Table 2. Take the 3.3-5.0 min section fraction and reduce the pressure to recover the solvent to dryness, which is Trap45.

[0033] 1. Research Methods HepG2 cells were inoculated in 96-well plates and cultured in a CO2 incubator at 37°C for 24 hours. The culture medium was replaced in the control group and the model group. Each drug group (trap-29, trap-45, and trap-48 groups) was pretreated with drugs at a concentration of 30 µg / mL for 24 hours, and 6 replicates were set up in each group. The culture medium was replaced in the control group, and the model group and the drug group were cultured with a culture medium containing an alcohol concentration of 0.4 mol / L for another 24 hours. The CCK-8 method was used to detect and calculate the cell survival rate.

[0034] 2. Research Results Depend on Figure 1 As shown, there was a significant difference between the model group and the control group (p < 0.01); compared with the model group, the cell survival rate of the drug trap-48 pretreatment group was significantly increased, and the survival rate increased to about 80% at 30μg / mL, with a significant difference (p < 0.01), indicating that it has a protective effect on alcoholic damage to HepG2 cells; while the cell survival rates of the trap-29 and trap-45 groups were significantly lower than those of the trap-48 group, and there was no significant change compared with the model group, indicating that the protective effect of trap-48 extract on alcoholic damage to HepG2 cells is significantly better than that of trap-29 and trap-45 extracts.

[0035] Application Example 2 The Hovenia dulcis extract trap-48 of Example 1 of the present invention was subjected to an in vitro pharmacodynamics experiment to detect its physiological and biochemical indicators. 1. Research Methods HepG2 cells were inoculated in 96-well plates and cultured in a CO2 incubator at 37°C for 24 hours. The culture medium was replaced in the control group (blank group) and the model group, and the trap-48 administration group was administered at a concentration of 30µg / mL to pretreat the cells for 24 hours. Six replicates were set up in each group. The culture medium was replaced in the control group, and the model group and the drug group were cultured in a culture medium containing an alcohol concentration of 0.4mol / L for another 24 hours.

[0036] 2. Determination of test indicators Reactive oxygen species (ROS), AST, ALT and other indicators were measured for each group, and the in vitro efficacy of the fractions was tested according to the instructions of the kit.

[0037] 3. Research Results 3.1 Results of reactive oxygen species detection ROS detection can analyze the true level of reactive oxygen species in cells, showing that alcohol metabolism causes an increase in the content of reactive oxygen species in cells, such as Figure 2 In the model group treated with 0.4 mol / L alcohol for 24 h, the amount of ROS generated in HepG2 cells was significantly higher than that in the control group. After drug pretreatment, the amount of ROS generated was significantly lower than that in the model group.

[0038] 3.2 AST and ALT test results Elevated AST and ALT levels are conventional indicators for evaluating liver cell damage and are also important markers of drug-induced liver cell necrosis. The content of AST and ALT in the cell supernatant after alcohol modeling and drug treatment was affected as follows: Figure 3 The results showed that AST and ALT in the model group increased significantly, while those in the drug-treated group decreased significantly compared with the model group. This indicates that trap-48 can significantly reverse the increase in ALT and AST levels in HepG2 cells caused by alcohol.

Claims

1. A method for preparing an active component extract of Hovenia dulcis with liver protection effect, characterized in that: The following steps are involved: S1. Grind the Hovenia dulcis into coarse powder, add ethanol solution to reflux extraction, filter, collect the filtrate, combine and recover ethanol, and concentrate to obtain a thick paste; S2. The obtained thick paste was suspended in water, extracted with an organic solvent until the organic layer became pale in color, the organic solvent was recovered under reduced pressure, and the extracts of each part were obtained by freeze-drying; S3. Weigh the dry extract of ethyl acetate, dissolve it in methanol, filter it through a membrane, add C4 powder for adsorption, stir evenly, and recover the solvent under reduced pressure; dry column loading, use an eluent to perform primary separation and secondary separation according to the elution procedure, and automatically take the fractions; S4. Recover the solvent under reduced pressure to a small volume and freeze-dry it.

2. The preparation method according to claim 1, characterized in that: In S1, the volume fraction of the ethanol solution is 75%; the mass volume ratio of the Hovenia dulcis to the ethanol solution is 1:10; and the reflux extraction is performed twice, each time for 1.5 hours.

3. The preparation method according to claim 1 or 2, characterized in that: In S2, during the extraction process, dichloromethane and ethyl acetate are used in sequence for extraction.

4. The preparation method according to claim 1, characterized in that: In S3, the mass volume ratio of the dry extract to methanol is 0.1-0.5:1; the mass volume ratio of the dry extract to C4 powder is 1-2:

1.

5. The preparation method according to claim 1 or 4, characterized in that: In S3, the elution procedure used in the main separation is: In the main separation stage, the elution portion from 33 to 36 minutes was collected, and then secondary separation was performed.

6. The preparation method according to claim 1 or 5, characterized in that: In S3, the elution procedure used in the secondary separation is: In the secondary separation stage, the elution portion from 26.7 to 30.1 min was collected to obtain fraction trap-48.

7. The preparation method according to any one of claims 1, 4-6, characterized in that: In S4, the freeze-drying operation is as follows: after pre-freezing in a -40 to -50°C cold trap for 2 to 4 hours, freeze-drying at -45 to -55°C for 8 to 24 hours.

8. An active component extract of Hovenia dulcis L. prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The content of flavonoids in the active component extract of Hovenia dulcis is greater than 80%.

9. Use of the active component extract of Hovenia dulcis L. according to claim 8 in the preparation of a product for treating and / or preventing alcoholic liver damage.

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