A shrub sub-chrysanthemum extract, a preparation method and application thereof

Abstract

The present application belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a shrub sub-chrysanthemum extract, a preparation method and application thereof. The preparation method of the shrub sub-chrysanthemum extract comprises the following steps: mixing shrub sub-chrysanthemum powder and water to perform extraction, to obtain a water extract; concentrating the water extract, drying the obtained extract to obtain the shrub sub-chrysanthemum extract. The present application uses a green economic extraction process, uses water as an extraction solvent to extract the medicinal components of the shrub sub-chrysanthemum, and the prepared shrub sub-chrysanthemum water extract is effective for the treatment of obesity and non-alcoholic fatty liver disease (NAFLD) and has small toxic and side effects. The present application first uses the shrub sub-chrysanthemum extract for an in-vitro and in-vivo obesity-related NAFLD model, which significantly reduces the weight of mice, reduces epididymal fat and abdominal fat, relieves liver function and blood lipid abnormalities, and inhibits the activation of inflammatory pathways and lipid metabolism disorders; the shrub sub-chrysanthemum extract of the present application can be used for preparing a medicine for preventing or treating non-alcoholic fatty liver or obesity.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction technology, specifically relating to an extract of the shrub Chrysanthemum simonii, its preparation method, and its application. Background Technology

[0002] Shrubby Aster is a plant belonging to the genus Aster in the family Asteraceae. It is mainly distributed in deserts and desert steppes at altitudes of 550–4400m in Northwest my country and Central Asia, Russia. It is widely distributed in Xinjiang, my country, and is a folk herbal medicine in Xinjiang. The whole plant is used as medicine and has the effects of stopping coughs and bleeding, calming, expectorating and fighting roundworms. It can also be used to promote blood circulation, remove blood stasis, and treat diseases such as sore throat.

[0003] Non-alcoholic fatty liver disease (NAFLD) is a series of liver abnormalities that progress from non-alcoholic fatty liver to non-alcoholic steatohepatitis (NASH). The disease course is variable, but it is a liver disease that can lead to cirrhosis and liver cancer. Studies have demonstrated that NAFLD is reversible. Early detection and intervention to reverse NAFLD in clinical practice would be of great significance for the treatment of liver diseases.

[0004] Western medicine treatments for NAFLD primarily work by inhibiting excessive lipid synthesis, promoting lipid metabolism, and reducing inflammation. However, these drugs often target specific genes or proteins, resulting in a limited number of such medications with significant side effects, falling far short of the current treatment needs for liver diseases with high morbidity and mortality rates. Finding treatments with fewer side effects and higher efficacy is urgently needed. Traditional Chinese medicine (TCM), with its multi-stage, multi-target, structurally diverse, and low-toxicity characteristics, may offer advantages over Western medicine in treating NAFLD. Therefore, discovering more effective and economical TCM treatments for NAFLD is a current research hotspot. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a shrub daisy extract, its preparation method, and its application. The shrub daisy extract obtained by this invention is effective in treating NAFLD, and the preparation method is green and economical.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing an extract of the shrub *Chrysanthemum indicum*, comprising the following steps:

[0008] The powder of the shrub *Asteris spp.* was mixed with water and extracted to obtain an aqueous extract.

[0009] The aqueous extract was concentrated, and the resulting extract was dried to obtain a shrub daisy extract.

[0010] Preferably, the ratio of the powder of the shrub *Chrysanthemum indicum* to water is 1g:15-25mL.

[0011] Preferably, the ratio of the shrub daisy powder to water is 1g:20mL.

[0012] Preferably, the extraction temperature is room temperature and the extraction time is 60-84 hours.

[0013] Preferably, the extraction time is 72 hours.

[0014] Preferably, the concentration is vacuum concentration, and the vacuum concentration temperature is 60-70°C.

[0015] Preferably, the drying is freeze drying.

[0016] The present invention also provides a shrub extract of *Chrysanthemum indicum* obtained by the preparation method described in the above technical solution.

[0017] The present invention also provides the use of the shrub daisy extract described in the above technical solution in the preparation of medicaments for the prevention and / or treatment of metabolic diseases.

[0018] Preferably, the metabolic disease includes non-alcoholic fatty liver disease or obesity.

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

[0020] This invention provides a method for preparing an extract of *Chrysanthemum indicum*, comprising the following steps: mixing *Chrysanthemum indicum* powder with water for extraction to obtain an aqueous extract; concentrating the aqueous extract; and drying the resulting extract to obtain the *Chrysanthemum indicum* extract. This invention utilizes a green and economical extraction process, using water as the extraction solvent to extract the active ingredients of *Chrysanthemum indicum*. The resulting *Chrysanthemum indicum* extract has good therapeutic effects on NAFLD and few toxic side effects. In vitro experimental results show that the aqueous extract of *Chrysanthemum indicum* can significantly reduce triglyceride levels and inhibit lipid accumulation; in vivo experimental results show that the aqueous extract of *Chrysanthemum indicum* can reduce the body weight and the levels of EAT, AAT, AST, ALT, liver TG, and TC in mice.

[0021] This invention is the first to propose the application of *Chrysanthemum indicum* extract in the preparation of drugs for the prevention and / or treatment of metabolic diseases. In vitro and in vivo experimental results show that, for the first time, the aqueous extract of *Chrysanthemum indicum*, used in in vitro and in vivo NAFLD models, can alleviate abnormal liver function and blood lipids, inhibit the release of inflammatory factors and lipid accumulation; it can be used to prepare drugs for the prevention or treatment of NAFLD and obesity. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The effects of different extracts from the shrub *Tetrandilla spp.* on the viability of HepG2 cells;

[0024] Figure 2 The effect of different extracts of the shrub *Tetrandex chinensis* on TG expression in a NAFLD cell model;

[0025] Figure 3 The effect of aqueous extract of the shrub Chrysanthemum on cellular lipid accumulation;

[0026] Figure 4 The effect of aqueous extract of the shrub Chrysanthemum on the expression levels of intracellular inflammation-related genes and proteins;

[0027] Figure 5 The effects of aqueous extracts of the shrub Chrysanthemum on the expression levels of intracellular lipid synthesis-related genes and proteins;

[0028] Figure 6 The effect of aqueous extract of *Chrysanthemum indicum* on subacute toxicity in mice;

[0029] Figure 7 The water extract of the shrub Chrysanthemum can alleviate HFD-induced NAFLD experimental results;

[0030] Figure 8 The results are from liver pathological staining.

[0031] Figure 9 The effect of aqueous extract of the shrub Chrysanthemum morifolium on the expression levels of inflammation-related genes and proteins in the liver;

[0032] Figure 10 The effects of aqueous extracts of the shrub Chrysanthemum on the expression levels of lipid synthesis-related genes and proteins. Detailed Implementation

[0033] This invention provides a method for preparing an extract of the shrub *Chrysanthemum indicum*, comprising the following steps:

[0034] The powder of the shrub *Asteris spp.* was mixed with water and extracted to obtain an aqueous extract.

[0035] The aqueous extract was concentrated, and the resulting extract was dried to obtain a shrub daisy extract.

[0036] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0037] This invention involves mixing powdered daisy shrubs with water for extraction to obtain an aqueous extract.

[0038] In this invention, the powder of *Chrysanthemum indicum* is preferably obtained by drying the whole plant of *Chrysanthemum indicum*, cutting it, and crushing it into coarse powder.

[0039] In this invention, the preferred ratio of the shrub aster powder to water is 1g:15-25mL, more preferably 1g:20mL. When mixing the shrub aster powder and water, it is preferable to also add ethanol, preferably 3% of the water volume. The addition of ethanol in this invention prevents mold growth during the extraction process.

[0040] In this invention, the extraction temperature is preferably room temperature, and the extraction time is preferably 60-84 hours, more preferably 72 hours; the extraction is preferably soaking extraction, and the room temperature is preferably 25°C.

[0041] In this invention, the extraction process preferably further includes: performing solid-liquid separation on the mixture obtained from the extraction, and the resulting liquid is the aqueous extract; the solid-liquid separation method is preferably filtration.

[0042] After obtaining the aqueous extract, the present invention concentrates the aqueous extract and dries the resulting extract to obtain a shrub daisy extract.

[0043] In this invention, the concentration is preferably vacuum concentration, the temperature of the vacuum concentration is preferably 60-70°C, more preferably 65°C; the pressure is preferably 0.07-1 MPa, more preferably 0.8 MPa; and the vacuum concentration is preferably stopped when liquid begins to adhere to the wall.

[0044] In this invention, the drying is preferably freeze-drying, the freeze-drying temperature is preferably -43.4°C, the vacuum degree is preferably 4.6 Pa, and the time is preferably 6 to 10 hours, more preferably 8 hours.

[0045] The present invention also provides a shrub daisy extract prepared by the preparation method described in the above technical solution.

[0046] In this invention, the yield of the shrub daisy extract is preferably 15-25%, more preferably 21.72%.

[0047] In this invention, the shrub daisy extract is a shrub daisy aqueous extract, which contains phenols and sesquiterpene lactones.

[0048] The present invention also provides the use of the shrub daisy extract described in the above technical solution in the preparation of medicaments for the prevention and / or treatment of metabolic diseases.

[0049] In this invention, the metabolic disease preferably includes liver disease or obesity, and the liver disease is preferably non-alcoholic fatty liver disease.

[0050] In this invention, through in vitro screening, the optimal concentration of FFA (free fatty acids) for in vitro modeling was determined to be 0.75 mM, and the modeling time was 24 h. The *Chrysanthemum indicum* extract (aqueous extract) described in this invention is the extract with the strongest in vitro anti-NAFLD effect. The *Chrysanthemum indicum* aqueous extract of this invention exerts its pharmacological effect in the in vitro NAFLD model by reducing lipid synthesis and the release of inflammatory factors. The *Chrysanthemum indicum* aqueous extract can improve abnormalities in serum liver function and lipid-related indicators in mouse NAFLD models, exerting its anti-NAFLD pharmacological effect by reducing lipid deposition and the release of inflammatory factors.

[0051] To further illustrate the present invention, the shrub chrysanthemum extract, its preparation method, and its application provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1 Water Extract

[0053] Accurately weigh 100g of chamomile powder, add 20 times its weight of water (containing 3% ethanol by volume) and soak for 72h. Filter, concentrate under reduced pressure (65℃, 0.8MPa) to make an extract, and freeze-dry (-43.4℃, vacuum 4.6Pa, drying for 8h) to make a powder, thus obtaining the chamomile water extract.

[0054] The yield of the aqueous extract of the shrub *Chrysanthemum indicum* was 21.72%.

[0055] Comparative Example 1: Alcoholic extract (30% ethanol solution by volume)

[0056] The difference from Example 1 is that water (containing 3% ethanol by volume) was replaced with a 30% ethanol solution by volume. After the extract was prepared, it was vacuum dried (45°C, drying for 4 hours) to obtain a powder, thus obtaining a 30% ethanol extract of chrysanthemum shrub.

[0057] The yield of the 30% ethanol extract of *Aster spp.* was 13.97%.

[0058] Comparative Example 2: Alcoholic extract (60% ethanol solution by volume)

[0059] The difference from Comparative Example 1 is that the 30% ethanol solution was replaced with a 60% ethanol solution to obtain a 60% ethanol extract of chrysanthemum shrub.

[0060] The yield of the 60% ethanol extract of *Aster spp.* was 18%.

[0061] Comparative Example 3: Alcoholic extract (90% ethanol solution by volume)

[0062] The difference from Comparative Example 1 is that the 30% ethanol solution was replaced with a 90% ethanol solution to obtain a 90% ethanol extract of chrysanthemum shrub.

[0063] The yield of the 90% ethanol extract of *Aster spp.* was 12.58%.

[0064] Example 2 Cell Experiment

[0065] 1. Cytotoxicity detection

[0066] HepG2 cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 pcs·mL -1 Cell suspensions were seeded into 96-well plates. When the cells reached 50% confluence, serum-free medium was added for starvation treatment. After 9 hours of culture, the medium was discarded, and extracts of *Asteris spp.* (500, 250, 125, 62.5, 31.75, 15.5, and 7.75 μg / mL) from *Asteris spp.* (Examples 1 and 3, respectively) were added. -1 The culture medium contained the drug was used, with a solvent group (cell-free) and a blank control group (drug-free), in 6 replicates. After 24 hours of culture, the culture medium was discarded, and the cells were washed twice with PBS. 100 μL of 5% CCK-8 reagent was added to each well, and the cells were incubated for 1–4 hours. The absorbance (A) of each well was measured at 450 nm using a microplate reader, and the average value was used to calculate cell viability. The experiment was repeated in triplicate. The toxicity of the drug to normal cells was then assessed.

[0067] The effects of different extracts of *Chrysanthemum indicum* on the viability of HepG2 cells are as follows: Figure 1 As shown, A is the aqueous extract of *Chrysanthemum indicum*, B is the 30% ethanol extract of *Chrysanthemum indicum*, C is the 60% ethanol extract of *Chrysanthemum indicum*, and D is the 90% ethanol extract of *Chrysanthemum indicum*. The safe range of the drug was determined as follows: 0–250 μg / mL for the aqueous extract, 0–125 μg / mL for the 30% ethanol extract, 0–62.5 μg / mL for the 60% ethanol extract, and 0–125 μg / mL for the 90% ethanol extract. The dosage concentration was determined based on these values ​​later.

[0068] 2. Measurement of intracellular TG (triglyceride) levels

[0069] Cells were cultured in 6-well plates, with three groups: a control group, a model group (FFA, free fatty acids), and a treatment group (FFA + chamomile extract). After culturing at the selected modeling concentration, time (0.75 mM, 24 h), and drug concentration, the cell supernatant was discarded, and the cells were washed 2–3 times with PBS. Cells were then digested with trypsin, centrifuged, and the supernatant was discarded. 200 μL of lysis buffer was added to lyse the cells. The cell lysates were collected and incubated on ice for 15–30 min. TC (total cholesterol) and TG (triglycerides) levels were measured according to the instructions of the respective kits.

[0070] No changes in TC were detected at the cellular level, but significant changes in TG were observed. The effects of different extracts of *Chrysanthemum indicum* on TG expression in a NAFLD cell model are shown below. Figure 2 As shown, A represents the effect of the aqueous extract of *Chrysanthemum indicum* on TG content, B represents the effect of the 30% ethanol extract of *Chrysanthemum indicum* on TG, C represents the effect of the 60% ethanol extract of *Chrysanthemum indicum* on TG, and D represents the effect of the 90% ethanol extract of *Chrysanthemum indicum* on TG (Note: FFA is a modeling agent, free fatty acid, at a concentration of 0.75 mM). Four groups (A, B, C, and D) were tested on different batches, with a blank group and a model group set up for each group.

[0071] Depend on Figure 2 It can be seen that after administering different extracts of *Chrysanthemum indicum*, the aqueous extract of *Chrysanthemum indicum* significantly inhibited TG expression. The TG content in the model group reached 2.62 mmol / gprot, significantly increased compared to the blank group. However, after administration, the TG content in the administered groups decreased in a dose-dependent manner, with the high-dose group showing a level as low as 1.42 mmol / gprot, significantly lower than the model group (P < 0.01). The ethanolic extract of *Chrysanthemum indicum* had no inhibitory effect on TG expression.

[0072] 3. Effects of water extracts of the shrub Chrysanthemum on lipid accumulation in vitro

[0073] According to the grouping settings, place coverslips at the bottom of the 6-well plates. After HepG2 cells reach the logarithmic growth phase, digest them and scientifically count the single-cell suspensions. Each well contains 0.5 mL of culture medium with a cell count of 1 × 10⁶ cells. 5 Add the cells to a coverslip, wait for them to stabilize, then add 1.5 mL of culture medium and gently transfer them to an incubator. After 24 hours of stable cell culture, discard the supernatant. Add incomplete culture medium to the control wells (normal group), administer 0.75 mM FFA to the model group, and dilute the drug-treated groups with 0.75 mM FFA solution at concentrations of 250 μg / mL (high-dose group), 62.5 μg / mL (medium-dose group), and 15.5 μg / mL (low-dose group), respectively. Observe cell morphology and perform Oil Red O staining.

[0074] Effects of water extracts of chrysanthemum shrub on cellular lipid accumulation, such as Figure 3 As shown in the figure, Figure A shows the Oil Red O staining results of the normal group (CON), model group (FFA), low-dose group, medium-dose group and high-dose group, respectively, and Figure B is a statistical graph of the Oil Red O staining results. The results show that the water extract of Chrysanthemum indicum can inhibit lipid accumulation in a dose-dependent manner.

[0075] 4. Study on the in vitro anti-NAFLD effect and mechanism of aqueous extract of chrysanthemum shrub

[0076] After six-well plate formation and drug administration, cells were collected, lysis buffer was added to lyse the cells, and cellular RNA and proteins were extracted. Real-time quantitative PCR and Western blotting experiments were used to verify whether the aqueous extract of Chrysanthemum indicum exerts its efficacy by regulating lipid metabolism and inflammatory response.

[0077] Figure 4 The results show the effects of aqueous extracts of the shrub *Chrysanthemum indicum* on the expression levels of inflammatory genes and proteins in cells. A represents IL-1β mRNA expression; B represents MCP-1 mRNA expression. After FFA modeling, the mRNA expression levels of the inflammatory factors IL-1β and MCP-1 in HepG2 cells were significantly upregulated (p<0.05 or p<0.001). After drug intervention, the expression levels of both inflammatory factors were significantly downregulated, with MCP-1 showing a more significant downregulation trend (p<0.001). C represents the expression levels of P-NF-κB and NF-κB proteins; D represents the expression levels of P-IKKβ and IKKβ proteins; E represents the expression level of P-NF-κB / NF-κB proteins; and F represents the expression level of P-IKKβ / IKKβ proteins. As can be seen from the figure, inflammatory proteins were significantly upregulated in the FFA group, while different concentrations of WEAF significantly reduced the abnormal expression of these two inflammatory proteins (p<0.05). Mean±SEM, n=5, ***p<0.001, **p<0.01, *p<0.05VS FFA group. ###p<0.001, ##p<0.01, #p<0.05VS WEAF group.

[0078] Figure 5The results show the effects of aqueous extracts of the shrub *Chrysanthemum indicum* on the expression levels of lipid synthesis genes and proteins in cells. A represents SREBP1 mRNA expression; B represents PPARγ mRNA expression; and C represents ACCs mRNA expression. As shown in the figure, compared to the control group, after FFA modeling, the expression levels of lipid synthesis-related genes were significantly upregulated (p<0.05, p<0.001). After drug intervention, the expression levels of all three lipid synthesis genes were significantly downregulated, with SREBP1 and PPARγ (p<0.05, p<0.01) showing a dose-dependent effect. D represents SREBP1 protein expression; E represents PPARγ protein expression; F represents SREBP1 protein expression level; and G represents PPARγ protein expression level. In the FFA group, the expression levels of both SREBP1 and PPARγ proteins were upregulated. However, after drug administration, the expression level of SREBP1 protein in the FFA group was significantly downregulated (p<0.01), while PPARγ showed a relatively obvious downregulation trend, but the effect was not statistically significant (p>0.05). Mean±SEM, n=5, ***p<0.001, *p<0.05VS FFA group. ###p<0.001, ##p<0.01, #p<0.05VS WEAF group.

[0079] After administration of the aqueous extract of Chrysanthemum indicum, compared with the model group, the expression of intracellular lipids and inflammation-related genes and proteins was significantly inhibited, suggesting that the aqueous extract of Chrysanthemum indicum may exert its pharmacological effects by inhibiting lipid synthesis and the release of inflammatory factors.

[0080] 5. In vivo safety evaluation

[0081] Following in vitro pharmacodynamic studies, in vivo pharmacodynamic studies will be conducted. However, there are no literature or experimental reports regarding the drug safety of WEAF, making further in vivo drug safety evaluation of WEAF particularly important. Therefore, a 14-day subacute toxicity test was performed on mice to observe the drug's toxic effects on mice after administration. Figure 6Tables 1-3 show the effects of WEAF on mice in the subacute toxicity test, where A represents the change in body weight in male mice after administration; B represents the change in body weight in female mice after administration; C represents HE staining images of the heart, liver, spleen, and kidney of male mice; and D represents HE staining images of the heart, liver, spleen, and kidney of female mice. Table 1 shows the effects of WEAF on the organ indices of the heart, liver, spleen, lung, and kidney in mice in the subacute toxicity test (n=10); Table 2 shows the effects of WEAF on the complete blood count in mice in the subacute toxicity test; and Table 3 shows the effects of WEAF on the blood biochemical indicators in mice in the subacute toxicity test. The tests on various indicators showed no significant changes in mouse body weight or organ indices compared to the control group. Therefore, further studies on complete blood count, blood biochemistry, and pathological staining were conducted. Results showed no significant differences in white blood cells, red blood cells, platelets, hemoglobin, and neutrophils between male and female mice and the control group. Blood biochemistry indicators (ALT, AST, BUN, CREA, TP, ALB, GLO) showed no significant changes, and pathological staining revealed no organ lesions. No significant toxicity of WEAF was observed after administration, indicating good tolerability and high safety. This material can be used as a non-toxic medicinal resource for further research and has good development value. It can provide experimental evidence for further in vivo pharmacodynamic studies.

[0082] Table 1. Effects of WEAF on organ indices of heart, liver, spleen, lung, and kidney in mice during subacute toxicity testing.

[0083]

[0084] Table 2. Effects of WEAF on blood routine tests in mice undergoing subacute toxicity testing.

[0085]

[0086] Table 3. Effects of WEAF on blood biochemical parameters in mice undergoing subacute toxicity testing.

[0087]

[0088] 6. In vivo model of NAFLD induced by high-fat diet (HFD) in mice

[0089] Six- to eight-week-old C57 mice were acclimatized for one week and then randomly divided into two groups: a control group and a model group. The control group was fed a normal diet, while the model group was fed a high-fat diet containing 60% fat for 12 weeks. At week 11, two mice were randomly selected for dissection to test various indicators and determine the model establishment. After successful model establishment, the mice were randomly divided according to body weight into a control group (normal diet), a model group (high-fat diet containing 60% fat), a low-dose group (water extract of chrysanthemum shrub, 0.5 g / kg, LD), a medium-dose group (water extract of chrysanthemum shrub, 1 g / kg, MD), a high-dose group (water extract of chrysanthemum shrub, 2 g / kg, HD), and a positive control group (ATO atorvastatin, 10 mg / kg). The control and model groups were given sodium carboxymethyl cellulose aqueous solution (CMC-Na), while the other groups were administered the drug according to the dosage determined in the subacute toxicity test.

[0090] Sample collection: After the last administration, blood was collected from the eyeballs of mice, and the mice were euthanized by cervical dislocation. The heart, liver, spleen, kidney, epididymis, and abdominal fat were harvested and weighed, and organ indices were calculated. The liver and fat were rinsed with physiological saline and stored at -80℃ for later use. The levels of four related indicators of serum liver function and blood lipids in mice were detected using a kit.

[0091] Figure 7 The results of experiments demonstrating that the aqueous extract of the shrub *Chrysanthemum indicum* can alleviate HFD-induced NAFLD are shown in Figure 1. A represents the change in mouse body weight after administration; B represents the cumulative food intake of mice after administration; C represents the change in mouse liver weight; D represents the epididymal fat index of mice; E represents the abdominal fat index of mice; F represents the change in serum ALT levels; G represents the change in serum AST levels; and HI represents the changes in liver TG and TC levels, respectively. After administration, mouse body weight decreased significantly, and not only were serum ALT and AST levels improved, but liver TC and TG levels were also significantly improved.

[0092] The livers and epididymal fat of three mice in each group were immersed in paraformaldehyde. The livers were stained with Oil Red O, HE, and Masson staining, and the epididymal fat was stained with HE staining. Figure 8 The results are as follows: A represents the statistical results of Oil Red O staining; B represents the statistical results of HE staining; C represents the statistical results of Masson staining; and D represents the HE staining results of epididymal fat. Compared with the model group (HFD), lipid accumulation was significantly reduced after drug administration, vacuolation and inflammatory cell infiltration were improved, collagen deposition was significantly reduced, and the area of ​​adipocytes was also significantly reduced.

[0093] 7. Study on the effect and mechanism of water extract of chrysanthemum shrub in anti-NAFLD in vivo

[0094] Liver tissue was weighed and homogenized to extract proteins and RNA. The expression of relevant proteins and genes after drug administration was detected by Western blotting and real-time quantitative PCR.

[0095] Figure 9 The results show the effects of aqueous extracts of the shrub *Chrysanthemum indicum* on the expression levels of inflammation-related genes and proteins in the liver. A represents IL-6 mRNA expression; B represents IL-1β mRNA expression; and C represents TNF-α mRNA expression. As can be seen from the figure, in the HFD model, the expression levels of inflammatory factors were significantly upregulated (p<0.05 or p<0.01). After intervention with different concentrations of WEAF, the expression levels of all three inflammatory factors were significantly downregulated (p<0.05 or p<0.01). D represents P-NF-κB and NF-κB protein expression; E represents P-IKBα and IKBα protein expression; F represents MYD88 protein expression; G represents TLR2 protein expression; H represents IL-1β protein expression; I represents the P-NF-κB / NF-κB protein expression level; J represents the P-IKBα / IKBα protein expression level; K represents MYD88 protein expression level; L represents TLR2 protein expression level; and M represents IL-1β protein expression level. The results showed that the liver tissue of mice in the HFD group significantly upregulated P-NF-κB, P-IKBα, MYD88, TLR2, and IL-1β. Intervention with different concentrations of WEAF significantly reduced the expression of these inflammatory proteins (p<0.05, p<0.01, or p<0.001), indicating that WEAF exerts its anti-NAFLD effect through the TLR2 / NF-κB signaling pathway. Mean±SEM, n=5, ***p<0.001, **p<0.01, *p<0.05 vs HFD group. ###p<0.001, ##p<0.01, #p<0.05 vs WEAF group.

[0096] Figure 10 The results show the effects of aqueous extracts of the shrub *Chrysanthemum indicum* on the expression levels of lipid synthesis-related genes and proteins in the liver, where A represents PPARγ mRNA expression. Figure 10 As shown in Figure A, compared with the CON group, the expression level of PPARγ in the HFD model group was significantly upregulated (p<0.001). After drug intervention, the expression level of PPARγ gene was significantly downregulated (p<0.05, p<0.01), and this was dose-dependent. Figure B shows the SREBP1 protein expression; Figure C shows the PPARγ protein expression; Figure D shows the ACCs protein expression; Figure E shows the SOCS3 protein expression; Figure F shows the SREBP1 protein expression level; Figure G shows the PPARγ protein expression level; Figure H shows the ACCs protein expression level; Figure I shows the SOCS3 protein expression level. The results are as follows... Figure 10As shown in the B1 diagram. Lipid synthesis-related proteins SREBP1, PPARγ, and ACCs were all highly expressed in the HFD model (P<0.05, P<0.01), and their expression levels were significantly downregulated after drug administration (p<0.05, p<0.01). SOCS3, a key protein linking inflammation and lipid metabolism, was also highly expressed in the HFD group (p<0.01), and its expression was also significantly downregulated after drug administration (p<0.05), indicating that WEAF exerts its anti-NAFLD effect through the SREBP1 / PPARγ signaling pathway. Mean ± SEM, n = 5, ***p<0.001, *p<0.05 vs. FFA group. ###p<0.001, ##p<0.01, #p<0.05 vs. WEAF group.

[0097] WEAF can alleviate NAFLD in two ways: (1) by inhibiting lipid synthesis through the PPAR pathway; and (2) by inhibiting inflammatory activation and reducing liver inflammation.

[0098] This invention utilizes a green and economical extraction process to extract the medicinal components of the shrub *Chrysanthemum indicum*, conducts in vitro and in vivo NAFLD activity studies, elucidates the mechanism of action of *Chrysanthemum indicum* extract on NAFLD, provides a scientific basis for the rational utilization of *Chrysanthemum indicum* resources, and lays the foundation for the research and development of innovative drugs using *Chrysanthemum indicum* as a liver protectant and anti-NAFLD agent.

[0099] This invention investigates the role of *Asteris spp.*, a shrub of the genus *Asteris*, in the treatment of NAFLD, and holds promise as an effective treatment strategy that can overcome the shortcomings of current liver disease medications.

[0100] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a shrub daisy extract in the preparation of a medicament for the prevention and / or treatment of metabolic diseases, characterized in that, The metabolic disease is non-alcoholic fatty liver disease or obesity; the preparation method of the shrub daisy extract includes the following steps: The powder of the shrub *Asteris spp.* was mixed with water and extracted to obtain an aqueous extract. The aqueous extract was concentrated, and the resulting extract was dried to obtain a shrub daisy extract.

2. The application according to claim 1, characterized in that, The ratio of the powder of the shrub *Chrysanthemum indicum* to water is 1g:15~25mL.

3. The application according to claim 2, characterized in that, The ratio of the powder of the shrub *Asteris spp.* to water is 1 g: 20 mL.

4. The application according to claim 1, characterized in that, The extraction temperature is room temperature, and the time is 60-84 hours.

5. The application according to claim 4, characterized in that, The extraction time is 72 hours.

6. The application according to claim 1, characterized in that, The concentration is a vacuum concentration, and the vacuum concentration temperature is 60~70℃.

7. The application according to claim 1, characterized in that, The drying process is freeze-drying.

Images