Coreopsis tinctoria extract for liver protection and preparation method and application thereof

Abstract

The invention relates to a coreopsis tinctoria extract for liver protection as well as a preparation method and application thereof, and relates to the technical field of medicines. The senecio cineraria extract is obtained by alcohol extraction of senecio cineraria and solvent extraction. The coreopsis tinctoria extract acts on a PI3K / AKT pathway, regulates MMP9 active protein at the downstream of the pathway and delays liver fibrosis, so that the liver protection effect is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a snow chrysanthemum extract for liver protection and a preparation method and application thereof. Background Art

[0002] Snow chrysanthemum is an annual herb native to North America and is currently cultivated in various parts of China. Snow chrysanthemum has a long flowering period, with a variety of bright colors, and is quite ornamental. It also has medicinal value and has been used as a tea for hundreds of years. The active compounds such as flavonoids, polysaccharides, and polyphenols in snow chrysanthemum have multiple pharmacological activities such as lipid-lowering, blood pressure-lowering, anti-inflammatory, and antioxidant, and are often used for the prevention and health care of cardiovascular diseases.

[0003] The liver has many important physiological functions such as metabolism, detoxification, transformation, synthesis, and immune defense. Pathogenic factors such as chemical drugs, alcoholism, and bad living habits can cause liver damage. Many natural medicines have a certain protective effect on chemical liver damage, but the mechanism of action remains to be clarified. How to use drugs to protect the liver has always been a focus of attention in the medical field. At the same time, as a complex mixed system containing multiple ingredients, the efficacy of natural medicines is often the result of the synergistic effect of multiple active ingredients. In order to exert a certain therapeutic effect, there are often problems such as large dosage and slow effectiveness, which is not conducive to the development and application of modern products; therefore, it is necessary to further extract, separate and screen the activity of natural medicines to obtain one or more groups of active components with fewer ingredients and higher activity for subsequent development and utilization. Summary of the invention

[0004] In view of the above problems, the present invention provides an application of a snow chrysanthemum extract for liver protection in the preparation of a liver protection drug. The snow chrysanthemum extract acts on the PI3K / AKT pathway, regulates the MMP9 active protein downstream of the pathway, and delays liver fibrosis, thereby achieving a liver protection effect.

[0005] In order to achieve the above object, the present invention provides an application of a snow chrysanthemum extract for liver protection in the preparation of a liver protection drug. The snow chrysanthemum extract is obtained by extracting snow chrysanthemum with alcohol and then extracting with a solvent.

[0006] The inventors found during the research process that although most of the active substances in the natural medicine snow chrysanthemum can be extracted by ethanol extraction and separation, the ethanol-extracted species contain various compounds, and the composition is still complex, and the respective effects are unclear. Therefore, on the basis of ethanol extraction, the inventors used solvents for extraction, and screened and obtained active components with liver protective effects using an in vitro hepatocyte injury model, and verified through a carbon tetrachloride-induced animal model that the snow chrysanthemum extract acts on the PI3K / AKT pathway, regulates the downstream MMP9 active protein of the pathway, delays liver fibrosis, and has a good protective effect on hepatocyte damage, and can be used to prepare liver protection drugs.

[0007] In one embodiment, the liver protection drug further includes a pharmaceutically acceptable excipient, and the solvent includes ethyl acetate.

[0008] The inventors used a variety of solvents for extraction during the research process, such as petroleum ether, ethyl acetate, and n-butanol, to obtain extracts of solvents with different polarities. Through in vitro liver cell damage model experiments, it was found that the ethyl acetate extract had better activity and had a good protective effect on liver cell damage.

[0009] In one embodiment, the liver-protecting drug protects the liver by down-regulating the expression of proteins related to the PI3K / AKT pathway and down-regulating the expression of MMP9 active protein downstream of the PI3K / AKT pathway.

[0010] In one embodiment, the dosage of the snow chrysanthemum extract in the liver protection drug is ≥ 200 mg·kg -1 ·d -1 .

[0011] The present invention also provides a method for preparing the snow chrysanthemum extract in the application, comprising the following steps: extracting the snow chrysanthemum with alcohol and then extracting the extract with a solvent.

[0012] In one embodiment, the reaction conditions of the alcohol extraction include: reflux extraction with ethanol having a volume concentration of 50-70% as the extractant, the temperature of the reflux extraction is 65-75° C., and the solid-liquid ratio of the reflux extraction is 1:(25-35).

[0013] In one embodiment, the reflux extraction time is 1.5-2.5h.

[0014] In one embodiment, the solvent extraction step includes: mixing the ethanol extract of snow chrysanthemum with water, dissolving, and extracting with a solvent to obtain the snow chrysanthemum extract.

[0015] In one embodiment, the ratio of the ethanol extract of snow chrysanthemum to the solvent is 1 g: (700-800) mL.

[0016] The invention also provides the snow chrysanthemum extract for liver protection obtained by the preparation method.

[0017] The present invention also provides a medicine for liver protection, comprising a medicine active ingredient and a pharmaceutically acceptable auxiliary material, wherein the medicine active ingredient is the snow chrysanthemum extract in the application.

[0018] In one embodiment, the drug is in the form of oral solution, pills, granules, capsules, tablets, pellets, injections or powders.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention discloses a snow chrysanthemum extract for liver protection, a preparation method thereof and an application thereof. The snow chrysanthemum extract acts on the PI3K / AKT pathway, regulates the MMP9 active protein downstream of the pathway, delays liver fibrosis, and thus achieves liver protection effect. The snow chrysanthemum extract can be used to prepare liver protection drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the results of the in vitro liver protection activity screening of the snow chrysanthemum extract in the examples;

[0022] Figure 2 This is the UPLC-MS positive ion chromatogram of the ethyl acetate fraction (EACT) component analysis in the example;

[0023] Figure 3 UPLC-MS negative ion chromatogram of the ethyl acetate fraction (EACT) component analysis in the example;

[0024] Figure 4 The figure is the staining result of pathological sections of animal experiments in the embodiment;

[0025] Figure 5 This is a diagram of the AST measurement results of the animal experiment in the embodiment;

[0026] Figure 6 ALT measurement results of animal experiments in the examples;

[0027] Figure 7 This is a diagram showing the IL-1β determination results of the animal experiment in the embodiment;

[0028] Figure 8 The TNF-α determination result diagram of the animal experiment in the embodiment;

[0029] Fig. 9 This is a diagram showing the IL-6 assay results of the animal experiment in the example;

[0030] Fig.10The results of SOD activity determination in animal experiments in the examples are shown in FIG.

[0031] Fig.11 This is a diagram showing the MDA measurement results of the animal experiment in the embodiment;

[0032] Fig.12 The figure is the measurement result of protein expression in the example. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] source:

[0036] Snow chrysanthemum (Heilongjiang Province, China); anhydrous ethanol (Jilin Xinli Chemical Reagent Co., Ltd.); ALT, AST, MDA, SOD, TNF-α, IL-6 and IL-1β assay kits (Nanjing Jiancheng Bioengineering Institute). AL204 analytical balance (Shanghai Mettler-Toledo Instrument Co., Ltd.); rotary evaporator (Shanghai Quanjie Instrument Co., Ltd.); low-temperature high-speed centrifuge (Eppendorf, USA); Infinite M200 microplate reader (TECAN, Switzerland); desktop centrifuge (Eppendorf, USA); U3000 ultra-high performance liquid chromatograph (ThermoScientific, USA); TripleTOF5600; mass spectrometer (ABSCIEX, USA).

[0037] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0038] Example

[0039] 1. Preparation of snow chrysanthemum extract.

[0040] Take an appropriate amount of snow chrysanthemum, use 60% ethanol by volume as solvent, solid-liquid ratio 1:30, heat and reflux at 70°C for 2 times, 1 hour each time, combine the filtrate to obtain a crude extract; extract the crude extract in batches, about 20g each time, add 500mL of water, dissolve it by ultrasonic for 20min, and then extract it with petroleum ether, ethyl acetate, and n-butanol in order from low to high polarity. Extract with each solvent 3 times, 500mL each time, combine the concentrated solvents, evaporate and weigh, and obtain the petroleum ether part (PCT), ethyl acetate part (EACT) and n-butanol part (NBCT) of snow chrysanthemum respectively.

[0041] 2. In vitro hepatocyte viability experiment.

[0042] 1. Carbon tetrachloride-induced HepG2 cell injury model.

[0043] The model was established according to the method in the literature (Gao Jian, Dong Lingyue, An Wei. GFER inhibits the damage of carbon tetrachloride to HepG2 cells [J]. Chinese Journal of Histochemistry and Cytochemistry, 2015, 24(05): 447-451.).

[0044] 2. Determine the effects of solvent extraction parts with different polarities on the viability of hepatocytes in vitro.

[0045] Methods: According to the MTT method in the literature (Yan Yuhui, Li Shaoheng, Kong Liang, et al. Study on the neuroprotective effect of Angelica dahurica coumarin on Aβ-induced neuronal damage [J]. Liaoning Journal of Traditional Chinese Medicine, 2016, 43(08): 1714-1717.), the effects of petroleum ether fraction (PCT), ethyl acetate fraction (EACT) and n-butanol fraction (NBCT) on the viability of hepatocytes in vitro were determined, and the active fraction of snow chrysanthemum with hepatocyte protective effect was screened.

[0046] 3. UPLC-MS analysis.

[0047] Methods: Take 0.1 g of the active extract of snow chrysanthemum, add 1 mL of 50% methanol aqueous solution, and ultrasonicate for 30 min; centrifuge at 1200 r / min for 10 min at 4 ° C, take 10 μL of the supernatant for injection, and analyze the active sites with the help of UPLC-MS. Through the search and comparison of the mass spectrum library, according to the matching degree and other information, after comprehensive analysis, the component composition information was obtained.

[0048] 3. Animal experiments

[0049] 1. Experimental animals.

[0050] 60 SPF-grade healthy ICR male mice (Changchun Yisi Experimental Animal Technology Co., Ltd., animal certificate number; SCXK (Ji) -2020-0002), 6-8 weeks old, body weight (20±2) g, were fed in separate cages at a temperature of 20-25°C and a humidity of 50%-60%, with a 12-hour day and night alternation. The experiment began after 3 days of adaptive feeding. The experiment complies with relevant regulations on animal ethics.

[0051] 2. Animal grouping and drug administration method.

[0052] Sixty ICR mice (half male and half female) were randomly divided into a blank group (distilled water), a model group (distilled water), a positive drug group (bifendate dripping pill solution 200 mg kg -1 ·d -1 ), EACT administration group (200 mg·kg -1 ·d -1 ) 4 groups were given drugs by gavage for 7 days. One hour after the last drug administration, mice in other groups were injected with 0.2% CCl4 olive oil solution intraperitoneally to establish the model except the blank group, which was injected with the same amount of olive oil solution. The mice in each group were fasted but not watered, and eyeball blood and liver tissue were collected 24 hours after model establishment.

[0053] Kits were used to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, superoxide dismutase (SOD), malondialdehyde (MDA), and three inflammatory factors (TNF-α, IL-6, and IL-1β) in liver tissue, and the liver tissue was stained with HE sections for observation.

[0054] 3. Western Blot experiment.

[0055] Western Blot was used to analyze the expression of AKT, PI3K and MMP9 proteins in mouse liver tissue.

[0056] 4. Statistical analysis.

[0057] SPSS 22.0 was used to process and analyze the data in this example. The measurement data were expressed as mean ± standard deviation, and the sample means among multiple groups were compared using one-way analysis of variance, and the pairwise comparisons between groups were compared using t-test. P < 0.05 was considered statistically significant.

[0058] 4. Experimental results.

[0059] 1. Results of in vitro hepatocyte viability experiments.

[0060] The viability of HepG2 cells in the EACT group was significantly higher than that in the model group (P < 0.01); while the cell viability in the PECT and NBCT groups was not significantly different from that in the model group (P > 0.05). It can be seen that EACT has a certain protective effect on hepatocytes in vitro and is the active site of snow chrysanthemum in protecting the liver. After UPLC-MS determination results were retrieved by mass spectrometry database and comprehensively analyzed and screened, it was shown that EACT contained 20 main active ingredients.

[0061] The results of in vitro activity screening are shown in Figure 1 UPLC-MS chromatogram of component analysis is shown in Figure 2-Figure 3 .

[0062] 2. Animal experiment results.

[0063] Compared with the blank group, the liver tissue structure of mice in the model group showed obvious pathological changes, and the liver tissue structure of mice in the coreopsis bicolor administration group was improved compared with the model group. Compared with the blank group, the levels of ALT, AST, MDA, TNF-α, IL-6 and IL-1β in the model group were significantly increased (P<0.01), and the level of SOD was significantly decreased (P<0.01). Compared with the model group, the levels of ALT, AST, TNF-α, IL-6 and IL-1β in the EACT administration group were significantly decreased (P<0.01 or P<0.05), the content of MDA was significantly decreased (P<0.01 or P<0.05), and the activity of SOD was significantly increased (P<0.01 or P<0.05).

[0064] The results of pathological section staining are shown in Figure 4 The results of biochemical indexes are shown in Figure 5-Figure 11 .

[0065] 3. Western Blot experimental results.

[0066] Compared with the blank group, the expression levels of p-AKT, p-PI3K, and MMP9 in the liver tissue of mice in the model group were significantly increased (P < 0.05). Compared with the model group, the expression levels of p-AKT, p-PI3K, and MMP9 in the liver tissue of mice in the high-dose EACT group were significantly decreased (P < 0.05).

[0067] Western Blot results are shown in Fig.12 .

[0068] 4. Summary.

[0069] (1) The ethyl acetate fraction is the active fraction of snow chrysanthemum that has a liver-protective effect and has a protective effect against carbon tetrachloride-induced chemical liver damage in mice.

[0070] (2) The liver-protective effect of the active fraction of snow chrysanthemum is mainly through acting on the PI3K / AKT pathway, regulating the active protein of MMP9 downstream of the pathway, and delaying liver fibrosis; it also has a significant regulatory effect on oxidative stress factors and inflammatory factors.

[0071] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0072] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An application of a snow chrysanthemum extract for liver protection in the preparation of a liver protection drug, wherein the snow chrysanthemum extract is obtained by extracting snow chrysanthemum with alcohol and then extracting with a solvent.

2. The use according to claim 1, characterized in that: The liver protection drug also includes pharmaceutically acceptable excipients, and the solvent includes ethyl acetate.

3. The use according to claim 1, characterized in that: The liver-protecting drug achieves the effect of protecting the liver by down-regulating the expression of proteins related to the PI3K / AKT pathway and down-regulating the expression of MMP9 active protein downstream of the PI3K / AKT pathway.

4. The use according to claim 1, characterized in that: The dosage of the snow chrysanthemum extract in the liver protection drug is ≥200 mg·kg -1 ·d -1 .

5. The method for preparing the snow chrysanthemum extract for use in any one of claims 1 to 4, characterized in that: The following steps are involved: After the snow chrysanthemum is extracted with alcohol, it is obtained by solvent extraction.

6. The preparation method according to claim 5, characterized in that: The reaction conditions of the alcohol extraction include: using ethanol with a volume concentration of 50-70% as an extractant for reflux extraction, the temperature of the reflux extraction is 65-75° C., and the solid-liquid ratio of the reflux extraction is 1:(25-35).

7. The preparation method according to claim 5, characterized in that: The solvent extraction step comprises: mixing the snow chrysanthemum alcohol extract with water, dissolving the mixture, and extracting the mixture with a solvent to obtain the snow chrysanthemum extract.

8. The snow chrysanthemum extract for liver protection obtained by the preparation method according to any one of claims 5 to 7.

9. A drug for liver protection, characterized in that: The invention comprises a pharmaceutically active ingredient and a pharmaceutically acceptable excipient, wherein the pharmaceutically active ingredient is the snow chrysanthemum extract as claimed in claim 8.

10. The drug according to claim 9, characterized in that The dosage form of the medicine is oral liquid, pill, granule, capsule, tablet, dripping pill, injection or powder.

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