Application of chrysanthemum extract in prevention and treatment of hepatic fibrosis

Abstract

The invention discloses an application of a chrysanthemum extract in prevention and treatment of hepatic fibrosis, and relates to the field of biological medicines. The natural medicine for effectively improving the NAFLD is wide in source, low in price, safe and free of toxic and side effects, and has great value in prevention and treatment of the hepatic fibrosis caused by the NAFLD.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of a chrysanthemum extract in preventing and treating liver fibrosis. Background Art

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD), also known as nonalcoholic fatty liver disease (NAFLD), is an acquired metabolic stress-induced liver injury. NAFLD includes nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH), the latter of which will continue to progress to liver fibrosis and then induce more serious cirrhosis and hepatocellular carcinoma. As a liver metabolic dysfunction disease, NAFLD is usually associated with insulin resistance and liver lipogenesis. It is one of the most common chronic diseases in contemporary society, with a global incidence rate of up to 25%. Therefore, effective prevention and treatment of NAFLD to slow down or block the process of liver fibrosis is a key step in affecting its development into terminal diseases such as cirrhosis and liver cancer. Excessive fat accumulation and metabolic disorders are the prominent characteristics of NAFLD, which affect the generation of related inflammation and the development of subsequent diseases. At present, there is only one drug approved by the authorities for the treatment of NAFLD with liver fibrosis. Although the targets of most other NAFLD drugs under development that have entered clinical trials are different, they have been forced to suspend research due to the complex pathogenesis of NAFLD and the safety issues of the drugs themselves. Therefore, compared with chemical drugs with a single target, for complex diseases such as NAFLD, multi-component, multi-target, and multi-pathway natural medicines that conform to traditional medical concepts have unique advantages in the prevention and treatment of NAFLD. Therefore, the development of a natural medicine that can effectively delay the fibrosis process of NAFLD and is highly safe is of great value for the prevention and treatment of NALFD. Summary of the invention

[0003] Based on the technical problems existing in the background technology, the present invention proposes an application of chrysanthemum extract in preventing and treating liver fibrosis.

[0004] The invention provides application of chrysanthemum extract in preparing medicine for preventing and treating liver fibrosis.

[0005] Preferably, the chrysanthemum extract is a water extract of chrysanthemum.

[0006] Preferably, the preparation method of the chrysanthemum extract comprises: adding chrysanthemum slices into water and soaking them for 30 minutes, adding water equivalent to 7 to 9 times the mass of the chrysanthemum slices, boiling and decocting for 20 to 40 minutes, filtering after cooling, taking the filter residue, adding water equivalent to 5 to 7 times the mass of the chrysanthemum slices, boiling and decocting for 15 to 30 minutes, filtering after cooling, combining the obtained filtrates, concentrating under reduced pressure at 50 to 70° C. to obtain a fluid extract with a relative density of 1.2 to 1.25, and drying the obtained fluid extract.

[0007] Preferably, the components of the chrysanthemum extract include chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, luteolin and luteolin.

[0008] Preferably, the components of the chrysanthemum extract include 2.2-3.2 mg / g of chlorogenic acid, 3.5-4.5 mg / g of isochlorogenic acid A, and 2.2-3.1 mg / g of olibanum.

[0009] Preferably, the liver fibrosis is liver fibrosis caused by NALFD.

[0010] The beneficial effects of the present invention are as follows:

[0011] The present invention proposes a new use of chrysanthemum extract in preventing and treating liver fibrosis, and provides a natural medicine that is widely available, low in price, safe, and has no toxic side effects and can effectively improve NAFLD, which is of great value for preventing and treating the process of liver fibrosis caused by NAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The following are the analysis results of the chemical components of the chrysanthemum extract prepared in Example 1.

[0013] Figure 2 The protein expression of PPARα / δ / γ and the mRNA expression of PPARα / δ / γ in mice.

[0014] Figure 3 These are the expression detection results of lipid metabolism genes downstream of PPARα / γ / δ in mice.

[0015] Figure 4 This is the Oil Red staining result of mouse liver tissue.

[0016] Figure 5 HE staining results and NALFD scores of mouse liver tissues.

[0017] Figure 6 These are the results of the detection of AST and ALT levels in mouse serum.

[0018] Figure 7The expression of F4 / 80 in mice and the expression of inflammatory factors in mouse liver.

[0019] Figure 8 The results of Sirius red staining of mouse liver tissue.

[0020] Fig. 9 Shown are the immunohistochemical results of α-SMA and the expression of liver fibrosis genes in mouse liver.

[0021] Fig.10 The expression of fibrosis factor α-SMA and collagen index COL1a1 in LX-2 cells. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is described in detail below through specific embodiments.

[0023] In the following examples, chrysanthemum slices were purchased from Anhui Jingdao Pharmaceutical Co., Ltd.

[0024] The human hepatic stellate cell line LX-2 was provided by Wuhan Servicebio Company.

[0025] Example 1 Preparation of Chrysanthemum Extract

[0026] Add chrysanthemum slices into water and soak for 30 minutes, add water equivalent to 8 times the mass of chrysanthemum slices, boil and decoct for 30 minutes, filter after cooling, take the filter residue, add water equivalent to 6 times the mass of chrysanthemum slices, boil and decoct for 20 minutes, filter after cooling, combine the obtained filtrates, concentrate under reduced pressure at 60°C to obtain a fluid extract with a relative density of 1.2 to 1.25, and freeze-dry the obtained fluid extract to obtain; the freeze-drying process includes: first freezing at -20°C for 12 hours, and then drying with a freeze dryer.

[0027] The chemical components of the chrysanthemum extract prepared above were analyzed by Orbitrap LC-MS. The results are as follows Figure 1 And as shown in Table 1.

[0028] Table 1

[0029]

[0030] Figure 1 Middle: A-Total ion current diagram of chrysanthemum extract, where the left side is the total ion current diagram of chrysanthemum extract in positive ion mode, and the right side is the total ion current diagram of chrysanthemum extract in negative ion mode; B-Secondary mass spectrum of chlorogenic acid; C-Secondary mass spectrum of olibanum glucoside; D-Secondary mass spectrum of isochlorogenic acid A.

[0031] Depend on Figure 1As shown in Table 1, a total of 7 chemical components were identified in the chrysanthemum extract prepared above: chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, and flavonoid components luteolin and luteolin that have the ability to lower blood lipids.

[0032] The contents of the main chemical components in the five batches of chrysanthemum extracts prepared above were determined by HPLC, and the results are shown in Table 2:

[0033] Table 2

[0034]

[0035] Example 2 Verification of the effect of preventing and treating liver fibrosis (one)

[0037] Mice fed a methionine choline deficiency diet (MCD) can develop severe liver steatosis and induce inflammation in a short period of time, leading to liver cell necrosis and even fibrosis, so they are widely used in NASH-related research. Therefore, in this part of the experiment, the MCD diet was used to induce the NASH model in mice to evaluate the improvement effect of chrysanthemum extract on liver inflammation and early fibrosis.

[0038] 8-week-old C57 mice were selected and divided into five groups, namely, chrysanthemum prevention group (PCM) (400mg / kg / d), chrysanthemum treatment group (CM) (400mg / kg / d), blank control group (NC), model group (MCD), positive control group (BZBT) (50mg / kg / d), 10 mice in each group. Mice in the blank control group were given ordinary feed, and mice in other groups were given methionine and choline deficient feed (MCD). The chrysanthemum prevention group was gavaged with chrysanthemum extract in the first week of feeding, and the remaining groups were gavaged with blank solvent. At the 5th week, the chrysanthemum treatment group began to gavage chrysanthemum extract, and the positive control group was gavaged with bezafibrate. The modeling was terminated at the end of the 8th week and the mice were killed, and serum and liver were taken for subsequent experiments. The chrysanthemum extracts used in the above experiments were all chrysanthemum extracts of batch A in Example 1.

[0039] Graphpad Prism 8.0 software was used for statistical analysis, and all experimental data were expressed as mean ± standard deviation. The data were expressed in the form of . One-way ANOVA was used for the analysis among multiple groups. Tukey test was used for further pairwise comparison. Dunnett-t test was used for comparison between multiple experimental groups and the same control group. p<0.05 indicated significant difference.

[0040] (1) Effect of chrysanthemum extract on liver lipid metabolism in MCD mice

[0041] In order to explore the mechanism of chrysanthemum extract in preventing and treating NASH, the protein expression of PPARα / δ / γ was detected by Western Blotting, and the mRNA expression of PPARα / δ / γ was further detected by RT-PCR. Figure 2 The protein expression of PPARα / δ / γ and the mRNA expression of PPARα / δ / γ in mice, among which, Figure 2 -A. Figure 2 -B shows the protein expression of PPARα / δ / γ in mice, Figure 2 -C shows the expression of PPARα / δ / γ mRNA in mice. Figure 2 -A and Figure 2 -B, compared with the blank control group mice, the expression of PPARα / β / γ protein in the MCD group was significantly decreased, and after preventive administration of chrysanthemum, the expression of PPARα / δ / γ protein was significantly increased (P<0.05). Figure 2 -C results showed that compared with the blank control group mice, the expression of PPARα / δ / γ in the liver of the MCD group mice was reduced, and after preventive administration of chrysanthemum, PPARα / γ was significantly upregulated compared with the MCD group (P<0.05).

[0042] To evaluate the effect of chrysanthemum extract on lipid metabolism, the expression of lipid metabolism genes downstream of PPARα / γ / δ was detected by RT-PCR. Figure 3 As shown. SREBP-1C, FASN and ACC genes are important genes for the synthesis of fatty acids, and CPT1a, CD36 and FATP1 are proteins related to the oxidation and transport of fatty acids. Figure 3 It can be seen that compared with the blank control group mice, the mRNA levels of SREBP-1C, FASN, CD36, and ACC in the MCD group mice were significantly increased, and the levels of CPT1a and FATP1 were significantly downregulated. The chrysanthemum extract can significantly reduce the expression of lipid synthesis genes and significantly increase the expression of CD36 and FATP1 (P<0.01). Among them, the chrysanthemum prevention group had the best therapeutic effect, which indicates that chrysanthemum extract can improve lipid metabolism.

[0043] (2) Effect of chrysanthemum extract on lipid accumulation in the liver of MCD mice

[0044] Figure 4 This is the oil red staining result of mouse liver tissue. Figure 4It can be seen that the MCD-induced mice had severe fatty degeneration in the liver, with a large number of lipid droplets deposited in the hepatocytes and spread around the liver sinusoids. Chrysanthemum extract also had a good preventive and therapeutic effect on MCD-induced fatty degeneration in the liver. Compared with the MCD group mice, the chrysanthemum prevention group had fewer lipid droplets in the liver and significantly decreased lipid deposition. The chrysanthemum prevention group and the chrysanthemum treatment group had better improvement effects on lipid accumulation than the lipid-lowering drug bezafibrate.

[0045] Figure 5 HE staining results and NALFD scores of mouse liver tissues. Figure 5 -A is the HE staining result, Figure 5 -B is the NALFD score. Figure 5 -A and Figure 5 -The results in B showed that the liver tissue structure of the blank control group was intact, with clear edges, regular arrangement of hepatocytes, and no vacuoles. The liver of MCD mice showed obvious lesions, fatty degeneration, a large number of fat vacuoles, severe ballooning degeneration, and the NALFD score was higher than that of the blank control group. Chrysanthemum extract can significantly reduce fat vacuolar lesions and ballooning degeneration, with intact morphology and neat arrangement, and reduced NAFLD scores, which further indicates that chrysanthemum extract can improve HFD-induced liver fatty degeneration.

[0046] AST represents whether mitochondrial function is intact, and ALT represents whether liver function is normal. In order to evaluate the protective effect of chrysanthemum extract on the liver of MCD mice, the levels of AST and ALT in mouse serum were tested. The test results are as follows: Figure 6 shown. Figure 6 The results showed that compared with the blank control group mice, the ALT and AST levels in the MCD group mice were significantly increased, and the AST and ALT levels were significantly decreased after the administration of chrysanthemum extract (P<0.05), indicating that chrysanthemum extract can improve liver damage.

[0047] (3) Effect of chrysanthemum extract on inflammation in MCD mice

[0048] To verify the effect of chrysanthemum extract on liver inflammation in MCD mice, the expression of F4 / 80 in MCD mice was detected by immunohistochemistry, and the expression of inflammatory factors TNF-α, IL-1β, IL-6, INOS, and CCL2 in the liver was detected by RT-PCR. Figure 7 The expression of F4 / 80 in mice and the expression of inflammatory factors in mouse liver. Figure 7 -A is the expression of F4 / 80 in mice, Figure 7 -B is the expression of inflammatory factors in mouse liver. Figure 7-A results showed that compared with the blank control group mice, the expression of F4 / 80 in the MCD group mice increased, the activation level of macrophages increased, the expression of F4 / 80 in the chrysanthemum prevention group and the chrysanthemum treatment group decreased, and the infiltration of macrophages decreased compared with the MCD group. Figure 7 -B results showed that compared with the blank control group mice, the mRNA levels of TNFα, IL-1β, IL-6, INOS, and CCL2 in the liver of the MCD group mice were significantly increased (P<0.01). The preventive administration of chrysanthemum could inhibit the expression of inflammatory genes, while the effect of the positive drug bezafibrate was weaker than that of the chrysanthemum prevention or treatment group, indicating that chrysanthemum extract can effectively inhibit the inflammatory response.

[0049] (4) Effect of chrysanthemum extract on liver fibrosis in MCD mice

[0050] MCD diet can induce liver fibrosis in a short period of time. In order to verify the effect of chrysanthemum extract on liver fibrosis in the process of NAFLD, Sirius red staining was performed on the liver tissue of mice. The results are as follows Figure 8 shown. Figure 8 The results of Sirius red staining of mouse liver tissue. Figure 8 The results showed that compared with the blank control group mice, the livers of the MCD group mice had obvious collagen deposition. Both the chrysanthemum prevention group and the treatment group significantly reduced the collagen area, improved liver fibrosis compared with the MCD group, and the effects were better than bezafibrate.

[0051] α-SMA is a marker of hepatic stellate cell activation. The expression of α-SMA in the liver was detected by immunohistochemistry, and the expression of liver fibrosis genes TGF-β, α-SMA, TIMP1, COL1A1, and COL1A3 was further detected by RT-PCR. Fig. 9 shown. Fig. 9 The immunohistochemical results of α-SMA and the expression of liver fibrosis genes in mouse liver, among which, Fig. 9 -A is the immunohistochemical result of α-SMA in mouse liver, Fig. 9 -B is the expression of liver fibrosis gene of α-SMA in mouse liver. Fig. 9 -A results showed that compared with the MCD group, the chrysanthemum prevention and treatment groups significantly inhibited the activation of α-SMA. Fig. 9 -B results showed that compared with the blank control group, the expression of fibrosis genes in the liver of mice in the MCD group increased significantly (P<0.01), and the preventive administration of chrysanthemum could significantly inhibit the expression of fibrosis genes (P<0.05), and the inhibitory effect of the chrysanthemum treatment group was weaker than that of the chrysanthemum prevention group. The above results show that chrysanthemum extract can inhibit the progression of liver fibrosis, and its effect is better than the marketed lipid-lowering drug bezafibrate.

[0052] In summary, the above results indicate that chrysanthemum extract may alleviate the progression of liver fibrosis in NALFD by activating the expression of PPARα / γ / δ, improving lipid deposition and fibrosis, and inhibiting inflammatory response. (two)

[0054] TGF-β-induced LX2 cells (hepatic stellate cell line) are a classic in vitro model of liver fibrosis. TGF-β can activate hepatic stellate cells to increase the expression of α-SMA (fibroblast activation marker). After activation, hepatic stellate cells will overexpress extracellular matrix genes such as collagen genes to drive the process of fibrosis. The present invention uses the main components of chlorogenic acid 3mg / g, luteolin 3mg / g and isochlorogenic acid A 4mg / g in the content determination of chrysanthemum extract as the basis for dosage setting. Subsequently, 10μg / mL was administered according to the concentration of chrysanthemum extract, and the concentrations of chlorogenic acid, luteolin and isochlorogenic acid A were increased tenfold to intervene in human hepatic stellate cells LX-2 respectively. The LX-2 cells after passage were cultured at 2×10 5 The cells were plated on a 6-well plate at a density of 10 cells / well. When the cells grew to about 80% of the plate area, they were randomly divided into 7 groups, namely, blank group, model group, bezafibrate group (BZBT: 3.62μg / mL), chrysanthemum extract group (CM: 10μg / mL), chlorogenic acid group (CA: 0.3μg / mL), luteolin (LUT: 0.3μg / mL) and isochlorogenic acid A group (ICQA: 0.4μg / mL). Among them, the blank group was not treated with TGF-β1, and the other groups were treated with 10ng / mL TGF-β1 for 24h; after TGF-β1 treatment, the chrysanthemum extract group, bezafibrate group, chlorogenic acid group, luteolin group and isochlorogenic acid A group were treated with corresponding concentrations of chrysanthemum extract, chlorogenic acid, luteolin and isochlorogenic acid A for 24h, respectively, and the model group was not treated; finally, the cells were collected and treated for Western Blot and RT-PCR experimental analysis.

[0055] The expression of fibrosis factor α-SMA and collagen index COL1a1 in LX-2 cells was measured by RT-PCR experiment, and Western blot experiment was further used to verify the inhibitory effect of chrysanthemum extract on hepatic stellate cell activation. The results are as follows Fig.10 shown. Fig.10 The expression of fibrosis factor α-SMA and collagen index COL1a1 in LX-2 cells. Fig.10 -A is the mRNA expression of α-SMA and collagen index COL1a1 in LX-2 cells, such as Fig.10-A, compared with the blank group, the mRNA expression of α-SMA and COL1a1 was significantly upregulated after TGF-β1 stimulation (P<0.05), and the overexpression of α-SMA and COL1a1 mRNA was significantly inhibited after intervention with chrysanthemum extract (P<0.05). Fig.10 -B is the expression of α-SMA protein in LX-2 cells. Fig.10 -B shows that compared with the blank group, the expression of α-SMA protein in LX-2 cells under the stimulation of TGF-β1 was significantly increased (P<0.05). Compared with the model group, the expression of α-SMA protein after intervention with chrysanthemum extract, chlorogenic acid, luteolin and isochlorogenic acid A decreased to varying degrees (P<0.05), and was significantly lower than that in the bezafibrate positive control group. It is worth noting that chrysanthemum extract showed better effects than single compounds in inhibiting LX-2 cell activation and overexpression of extracellular matrix, and the difference was statistically significant, which may be related to the multi-component and multi-target mechanism of action of chrysanthemum extract. This also suggests that the effect of chrysanthemum extract in inhibiting liver fibrosis does not come from a single component, but is the result of the synergistic effect of multiple components.

[0056] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Use of a chrysanthemum extract in the preparation of a drug for preventing and treating liver fibrosis.

2. The use according to claim 1, characterized in that: The chrysanthemum extract is a water extract of chrysanthemum.

3. The use according to claim 1, characterized in that: The preparation method of the chrysanthemum extract comprises: adding chrysanthemum slices into water and soaking them for 30 minutes, adding water equivalent to 7 to 9 times the mass of the chrysanthemum slices, boiling and decocting for 20 to 40 minutes, filtering after cooling, taking the filter residue, adding water equivalent to 5 to 7 times the mass of the chrysanthemum slices, boiling and decocting for 15 to 30 minutes, filtering after cooling, combining the obtained filtrates, concentrating under reduced pressure at 50 to 70° C. to obtain a fluid extract with a relative density of 1.2 to 1.25, and drying the obtained fluid extract.

4. The use according to claim 1, characterized in that: The liver fibrosis is liver fibrosis caused by NALFD.

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