Application of immature bitter orange extract in preparation of medicine for preventing and treating non-alcoholic fatty liver disease

Abstract

The invention provides an application of an immature bitter orange extract in preparation of a medicine for preventing and treating a non-alcoholic fatty liver disease. An appropriate immature bitter orange extract intervention concentration is selected through a CCK-8 experiment, in the concentration range of 25-100 micrograms / mL, the immature bitter orange extract has no obvious toxic or side effect on cells and can remarkably reduce the TC and TG content in NAFLD model cells, and oil red O staining shows that the extract can reduce lipid droplet accumulation in the cells and has certain concentration dependence. According to the present invention, the activity level of intracellular antioxidant proteases (CAT, GSH and SOD) and the content of lipid peroxide (MDA) are determined, the intracellular reactive oxygen species (ROS) level is detected by using a DCFH-DA probe method, and the results show that the intracellular antioxidant protein level after treatment with the immature bitter orange extract increases, the final toxic product MDA content decreases, and the ROS fluorescence intensity decreases, it is shown that the extract can relieve cell oxidative damage, and the oxidation resistance of cells is improved. The immature bitter orange extract plays a role in improving the NAFLD by activating a Keap1 / Nrf2 signal channel.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of an extract of Citrus aurantium immaturus in preparing medicines for preventing and treating non-alcoholic fatty liver disease. Background Art

[0002] Citrus aurantium is a traditional Chinese medicine for regulating qi. It is the dried young fruit of the plant Citrus aurantium and its cultivated varieties or sweet orange of the Rutaceae family. It has the effects of breaking qi and eliminating accumulation, resolving phlegm and dispersing lumps. In the era of precision medicine and under the new guidance of "new uses for old drugs", it is very necessary to improve the extraction method of Citrus aurantium, conduct further research on the extracted active substances, and explore its new applications. Summary of the invention

[0003] The purpose of the present invention is to provide an application of an extract of Citrus aurantium in preparing a medicine for preventing and treating non-alcoholic fatty liver disease.

[0004] Specifically, the present invention adopts the following technical solutions:

[0005] The present invention provides application of a Citrus aurantium immaturus extract in preparing a medicine for preventing and treating non-alcoholic fatty liver disease.

[0006] Furthermore, the Citrus aurantium extract is used to activate the Keap1 / Nrf2 signaling pathway.

[0007] Furthermore, the Citrus aurantium extract is used to increase the expression level of antioxidant enzymes in HepG2 to maintain the redox balance in cells, thereby reducing oxidative damage to cells caused by lipid peroxidation.

[0008] Furthermore, the Citrus aurantium extract is used to promote the phosphorylation and activation function of AMPK, thereby reducing sterol synthesis and inhibiting the expression of the lipid synthesis protein SREBP1-c sterol regulatory element to inhibit fatty acid synthesis in HepG2 cells.

[0009] Furthermore, the Citrus aurantium extract is used to reduce fat accumulation caused by a high-fat diet, alleviate liver function damage induced by a high-fat diet, and regulate the liver's ability to metabolize lipids to inhibit abnormal fat accumulation in the liver.

[0010] Furthermore, the intervention concentration of the Citrus aurantium extract is in the range of 25 to 100 μg / mL.

[0011] Furthermore, the Citrus aurantium extract includes Citrus aurantium active ingredients of different polarities; the preparation method of the Citrus aurantium active ingredients of different polarities includes: grinding the Citrus aurantium into powder after drying, passing through a 50-mesh sieve to obtain a sample; weighing 5 portions of the sample, mixing them with 1 portion of the sample at a solid-liquid ratio of 1:10 using 70% ethanol, pure water, ethyl acetate, n-butanol, and n-hexane as solvents, respectively, using an ultrasonic cleaner to perform ultrasonic extraction at a frequency of 53 kHz, and filtering to obtain solvent extracts of different polarities; concentrating the solvent extracts of different polarities under reduced pressure and then drying them to constant weight to obtain extracts extracted with different solvents, namely, the Citrus aurantium active ingredients of different polarities.

[0012] Furthermore, the Citrus aurantium extract is obtained by extracting Citrus aurantium using 70% ethanol or pure water as an extraction solvent.

[0013] Furthermore, the Citrus aurantium extract includes total flavonoids from Citrus aurantium; the method for extracting total flavonoids from Citrus aurantium includes: using ultrasonic extraction, using 63.01% ethanol as the extraction solvent, a solid-liquid ratio of 1:31.91, an extraction temperature of 41.88°C, and an extraction time of 29.71 min.

[0014] Furthermore, the average extraction rate of total flavonoids in the method for extracting total flavonoids from Citrus aurantium is 2.912%.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] The present invention provides the use of Citrus aurantium extract in the preparation of drugs for preventing and treating non-alcoholic fatty liver disease. The appropriate Citrus aurantium extract intervention concentration is selected by CCK-8 experiment. Within the concentration range of 25 to 100 μg / mL, the Citrus aurantium extract has no obvious toxic side effects on cells, and can significantly reduce the TC and TG contents in NAFLD model cells. Oil red O staining shows that the extract can reduce the accumulation of lipid droplets in cells, and has a certain concentration dependence. The intracellular antioxidant protease (CAT, GSH, SOD) activity level and lipid peroxide (MDA) content are measured, and the intracellular reactive oxygen (ROS) level is detected using the DCFH-DA probe method. The results show that after treatment with Citrus aurantium extract, the level of intracellular antioxidant proteins increases, the content of the final toxic product MDA decreases, and the ROS fluorescence intensity decreases, indicating that the extract can alleviate cell oxidative damage and improve the antioxidant capacity of cells. Citrus aurantium extract exerts its improvement effect on NAFLD by activating the Keap1 / Nrf2 signaling pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of FFA on HepG2 cell viability;

[0018] Figure 2The effect of FFA on the content of TG and TC in HepG2 cells;

[0019] Figure 3 The effect of FFA on intracellular lipid accumulation;

[0020] Figure 4 Toxicity effects of different extracts on HepG2 cells;

[0021] Figure 5 The ethanol extract of Citrus aurantium reduced the activities of TC and TG;

[0022] Figure 6 The water extract of Citrus aurantium reduced the activity of TC and TG;

[0023] Figure 7 The effect of Citrus aurantium extract on intracellular lipid accumulation;

[0024] Figure 8 The effect of Citrus aurantium extract on the antioxidant capacity of HepG2 cells;

[0025] Fig. 9 Effects of Citrus aurantium extract on ROS in HepG2 cells;

[0026] Fig.10 This is the protein band of Keap1 / Nrf2 signaling pathway in HepG2 cells;

[0027] Fig.11 This is the HepG2 cell antioxidant protein band;

[0028] Fig.12 This is the protein band of AMPK pathway in HepG2 cells;

[0029] Fig.13 This is the protein band of HepG2 cell lipid metabolism;

[0030] Fig.14 The changes of body weight and organs of mice;

[0031] Fig.15 The histomorphology of epididymal fat and liver in mice;

[0032] Fig.16 The changes in kidney and spleen mass of mice;

[0033] Fig.17 Changes in liver function in mice. DETAILED DESCRIPTION

[0034] The present invention is described below in conjunction with specific embodiments.

[0035] 1. Study on the improvement effect of Citrus aurantium extract on NAFLD cell model

[0036] 1 Experimental Materials

[0037] HepG2 cell-specific culture medium, DMEM high-glucose culture medium, FBS fetal bovine serum, penicillin / streptomycin: Wuhan Punosai Biotechnology Co., Ltd.; Improved Oil Red O kit, BCA detection kit: Shanghai Bio-Technology Co., Ltd.; Trypsin, BSA (fatty acid-free), lysis buffer: Beijing Solebow; Oleic acid, palmitic acid: Aladdin; TG, TC, ROS, CAT, SOD, MDA, GSH, ROS detection kit: Nanjing Jiancheng.

[0038] 2 Experimental instruments

[0039] BSA124S electronic analytical balance, Beijing Sartorius Instrument System Co., Ltd.; TI-S inverted fluorescence biological microscope, Nikon Corporation; cell incubator, -80℃ refrigerator, Multiskan Go ELISA analyzer, Thermo Fisher Scientific; TGL-16G high-speed centrifuge, Shanghai Anting Scientific Instrument Co., Ltd.

[0040] 3 Experimental methods

[0041] 3.1 Establishment of NAFLD cell model

[0042] 1) FFA cytotoxicity assay

[0043] HepG2 cells in the logarithmic growth phase were seeded in a 96-well plate at 8000 cells per well. After the cells adhered to the wall and grew stably for 48 hours, 200 mL of DMEM medium containing 5% serum was replaced in the blank control group, and medium containing different concentrations of FFA was added to the FFA group to make the final concentrations of 0.25, 0.5, 1.0, and 2.0 mmol / L, respectively. The culture was continued for 24 hours, the medium in the wells was aspirated, and 100 μL of 10% CCK-8 solution diluted with 5% serum medium was added to each well under light-proof conditions, and the plates were cultured in an incubator at a constant temperature of 37°C and 5% CO2 for 1 hour.

[0044] 2) Determination of triglyceride and cholesterol content

[0045] HepG2 cells were seeded in 6-well plates at a density of 600,000 / well and treated with different concentrations of free fatty acids (FFA) to model the cells. The cells were divided into five groups: control group (blank control group) and 0.25, 0.5, 1.0, and 2.0 mmol / L FFA treatment groups. After 24 hours of treatment, the cell culture medium was removed, the cells were washed three times with PBS buffer, and the cells were lysed with RIPA cell lysis buffer. The lysed cell samples were used to directly determine the TC and TG contents according to the kit.

[0046] 3) Oil Red O staining

[0047] HepG2 cells were planted in 12-well plates at a density of 300,000 / well, and treated with different concentrations of free fatty acids (FFA) to model the cells, and divided into five groups: Control group (blank control group) and 0.25, 0.5, 1.0, 2.0 mmol / L FFA treatment groups. After 24 hours, the cell culture medium was removed, and the cells were washed three times with PBS buffer, followed by adding an appropriate amount of 4% paraformaldehyde for fixation at room temperature for 30 minutes. After washing the residual liquid with PBS, Oil Red O dye was added and allowed to stand for 20 minutes, and then rinsed with PBS three times. Finally, the accumulation of intracellular lipid droplets was observed under a microscope, and photos were recorded.

[0048] 3.2 Determination of lipid-lowering activity of Citrus aurantium extract in NAFLD cell model

[0049] 1) Cytotoxicity assay of Citrus aurantium extract

[0050] HepG2 cells in the logarithmic growth phase were inoculated into 96-well plates at 8,000 cells / well. After the cells adhered to the wall and grew stably for 24 hours, the culture medium was removed. The blank control group was replaced with 200 μL of DMEM culture medium containing 5% serum, and the extract and positive drug groups were added with DMEM culture medium containing different concentrations of Citrus aurantium extract to make the final concentrations of 25, 50, 100, 200, and 400 μg / mL.

[0051] 2) Determination of triglyceride and cholesterol content

[0052] HepG2 cells were seeded in 6-well plates at a density of 600,000 / well and divided into six groups: Control group (blank control group), model group, extract intervention group (containing extract 25, 50, 100 μg / mL), and positive drug group (Silymarin 25 μg / mL). After 24 hours of treatment, the cell culture medium was removed, the cells were washed three times with PBS buffer, and the cells were lysed with RIPA cell lysis buffer. The lysed cell samples were used to directly determine the TC and TG contents according to the kit.

[0053] 3) Oil Red O staining

[0054] HepG2 cells were planted in a 12-well plate at a density of 300,000 / well. After 24 hours of culture, they were divided into six groups: control group, model group (0.5mM / LFFA), dose group (0.5mM / LFFA+25, 50, 100μg / mL extract), and positive control group (0.5mM / LFFA+25μg / mL Silymarin). After 24 hours, the cell culture medium was removed, and the cells were washed three times with PBS buffer, followed by adding an appropriate amount of 4% paraformaldehyde for fixation at room temperature for 30 minutes. After washing the residual liquid with PBS, Oil Red O dye was added and allowed to stand for 20 minutes. Rinse with PBS three times. Finally, the accumulation of intracellular lipid droplets was observed under a microscope and photos were recorded.

[0055] 3.3 Determination of the antioxidant activity of Citrus aurantium extract on NAFLD cell model

[0056] According to the above experimental results, the ethanol extract of Citrus aurantium was selected for subsequent experiments. HepG2 cells were planted in a 6-well plate at a density of 600,000 / well and divided into six groups: Control group (blank control group), model group, Citrus aurantium extract group (containing extract 25, 50, 100 μg / mL), and positive drug group (Silymarin 25 μg / mL). After 24 hours of treatment, the cell culture medium was removed, the cells were washed three times with PBS buffer, and the cells were lysed with RIPA cell lysis buffer. The lysed cell samples were used to determine the content of CAT, GSH, MDA, and SOD according to the kit.

[0057] HepG2 cells were inoculated into 6-well culture plates. They were placed in a cell incubator for 24 hours. The cells were grouped and administered as above. After discarding the culture medium, they were washed twice with PBS. A 10 μM DCFH-DA fluorescent probe solution was added. After incubation for 1 hour, the cells were washed twice with PBS and placed in a dark room. The cell fluorescence was observed and photographed using a fluorescent inverted microscope.

[0058] 4 Experimental results

[0059] 4.1 Establishment of NAFLD cell model

[0060] 1) FFA cytotoxicity CCK-8 experiment

[0061] from Figure 1It can be seen that with the increase of FFA concentration, the activity of HepG2 cells showed a gradual downward trend. The cell survival rate of HepG2 cells treated with 0.5mM FFA was 97.48%±6.59%, which was not significantly different from the blank group (P>0.05). After being treated with 1.0mM and 2.0mM FFA, the cell survival rate was significantly reduced (P<0.001). In summary, when the FFA concentration is lower than 0.5mM, it has no obvious toxic effect on HepG2 cells.

[0062] 2) Determination of triglyceride and cholesterol content

[0063] from Figure 2 It can be seen that with the increase of FFA concentration, the content of TG and TC in HepG2 cells gradually increased. When the FFA concentration was 0.5mM, the content of TG and TC in HepG2 cells showed a very significant difference compared with the blank group (P<0.0001). Based on the above cytotoxicity data, 0.5mM FFA was selected as the modeling concentration.

[0064] 3) Cell Oil Red O staining

[0065] Oil Red O staining is a method to evaluate the degree of intracellular lipid denaturation. Figure 3 It can be seen that no obvious red lipid droplets were observed in the HepG2 cells in the blank control group. When different concentrations of FFA were added, the number of red lipid droplets in the cells increased, and there was a significant difference compared with the blank group. With the increase of FFA concentration, the number of red lipid droplets increased significantly, indicating that the number of red lipid droplets in the cells was concentration-dependent with FFA.

[0066] 4.2 Lipid-lowering activity of Citrus aurantium extract on NAFLD cells

[0067] 1) Cytotoxicity of Citrus aurantium extract

[0068] from Figure 4 It can be seen that after the extract intervened in the cells for 24 hours, the CCK-8 method found that the extract at a concentration of 25-400 μg / mL had no effect on the cell survival rate, indicating that the extract had no obvious toxic side effects on the cells. Therefore, 25-100 μg / mL was selected for the subsequent experimental concentration.

[0069] 2) Determination of triglyceride and cholesterol content

[0070] from Figure 5 and Figure 6It can be seen that after adding FFA, the total cholesterol and total triglyceride contents in the cells increased significantly compared with the control group. After intervention with Citrus aurantium extract, the total cholesterol and total triglyceride showed a significant downward trend. The ethanol extract of Citrus aurantium was able to exert lipid-lowering activity at 50 μg / mL, which was better than the water extract.

[0071] 3) Oil Red O staining

[0072] Oil red O staining was used to evaluate the improvement effect of Citrus aurantium extract on intracellular lipid droplet accumulation. Figure 7 It can be seen that the number of red lipid droplets in cells treated with Citrus aurantium extract was significantly reduced compared with the model group. The average optical density value was analyzed by Image J software and it was found that the number of lipid droplets was concentration-dependent. As the treatment concentration increased, the number of lipid droplets gradually decreased. This shows that Citrus aurantium extract can reduce the accumulation of lipid droplets in NAFLD cell models.

[0073] 4.3 Determination of the antioxidant activity of Citrus aurantium extract in NAFLD cell model

[0074] from Figure 8 It can be seen that the catalase activity of cells was measured by CAT kit, and it was found that the CAT activity in the cells of the Citrus aurantium extract group was significantly higher than that in the model group, indicating that the Citrus aurantium extract can reduce the hydrogen peroxide content in cells and increase CAT activity.

[0075] The results of the SOD experiment showed that in HepG2 cells treated with FFA, the SOD level decreased significantly compared with the blank group, while the SOD level in the extract-treated group increased significantly compared with the model group. This indicates that the Fructus Aurantii Immaturus extract can effectively increase the level of SOD and remove excess free radicals in the body, thereby resisting the oxidative stress caused by FFA-induced fatty degeneration of HepG2 cells.

[0076] GSH is an important reducing agent that participates in a variety of redox reactions in vivo. The results of the GSH experiment showed that in HepG2 cells treated with FFA, the GSH level was significantly decreased compared with the blank group. However, the GSH level in the Citrus aurantium group was significantly increased compared with the model group. This means that Citrus aurantium extract has the effect of reducing cell oxidative stress damage.

[0077] By measuring MDA, it was found that the extract of Citrus aurantium could significantly reduce the content of malondialdehyde in cells, reduce the toxic effect of the final product MDA on cells, and protect cells. In summary, the extract of Citrus aurantium can effectively enhance the antioxidant capacity of cells and alleviate cell oxidative damage caused by peroxidation.

[0078] By observation Fig. 9It can be found that compared with the Control group, the cells in the FFA group showed high-intensity green fluorescence, indicating that the ROS content in this model group was high and the cells were in a state of severe oxidative damage. On the contrary, the cells in the three concentration groups of Citrus aurantium extract showed lower green fluorescence intensity, indicating that Citrus aurantium extract can significantly reduce the ROS content, thereby reducing the degree of oxidative damage.

[0079] 2. Study on the protective mechanism of Citrus aurantium extract on NAFLD cells

[0080] 1 Experimental Materials

[0081] Broad-spectrum Maker (11-180Kda), Tris, PMSF, ammonium persulfate, TEMED, SDS-PAGE gel preparation solution, skim milk powder, 30% acrylamide, Solebo; p-AMPK, SREBP-1c antibodies, Affinity; HO-1, Nrf2, NQO1, Keap1, rabbit anti-, mouse anti-, GAPDH, Wuhan Mitsubishi; ECL chemiluminescent developer, BSA, Biolight.

[0082] 2 Experimental instruments

[0083] SK-D3309-Pro decolorization shaker, Thermo Fisher Technology Co., Ltd.; XH-D vortex mixer, Jiangsu Tianling Instrument Co., Ltd.; S1010E handheld centrifuge, Cellocec Company; Bio-rad transfer electrophoresis system, Beijing Berland Company; FC3 gel imaging system, ProteinSimple Company.

[0084] 3 Experimental methods

[0085] Cell lysis: discard the culture medium and wash each well with PBS three times. Place the cells on ice at 4°C and lyse them with RIPA, vortex every five minutes, and repeat three times;

[0086] Protein content determination: Use the BCA method to establish a protein standard for selection and determine the intracellular protein content in each well;

[0087] SDS-PAGE gel electrophoresis: Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis was used to separate the proteins in Hepg2 cells. A 10% SDS-PAGE gel was selected. First, a separation gel was prepared and poured into the gel tank. Next, 1 mL of ethanol was added to remove bubbles. After standing for 30 minutes to allow the separation gel to solidify, a 5% concentration gel was prepared and poured into the gel tank. Next, a comb was inserted and allowed to stand at room temperature for 30 minutes. Subsequently, the prepared gel plate was placed in the electrophoresis tank and a sufficient amount of electrophoresis solution was poured in. Then, the sample was loaded. After the power was turned on, electrophoresis was performed at a constant voltage of 80 V for 30 minutes, and then the voltage was adjusted to 120 V and maintained at a constant voltage for about 1 hour.

[0088] Transfer: Cut the corresponding PVDF membrane according to the size of the gel and activate it with methanol. Prepare the transfer solution according to the instructions and store at 4°C. Take out the gel after electrophoresis, gently pry open the glass plate, cut off the excess gel, and soak the gel in the transfer solution. Soak the transfer plate in the transfer solution, select one side of the white transfer plate, mark the PVDF membrane to distinguish the front and back, stack them in the order of PVDF membrane and gel, remove the bubbles between the layers, and gently stack the filter paper to ensure that the membrane and gel will not move. Clip the transfer clip, pour the electrophoresis buffer solution to cover the gel, 210mA, 60-80min. Place ice bags in the electrophoresis tank, cover the outside of the tank with ice cubes, and transfer at low temperature.

[0089] Blocking: After the transfer, wash the PVDF membrane with 1×TBST three times, 5 min each time. After washing, add the milk shaker with the protein facing up to block for 1 h. After blocking, recover the skim milk (stored at 4°C) and wash briefly with 1×TBST.

[0090] Primary antibody incubation: prepare the antibody (dilution ratio according to the requirements of the antibody manual, dilute with 1×TBST, with the band protein facing up, and shake at 4°C overnight.

[0091] Secondary antibody incubation: prepare antibody (dilution ratio according to the requirements of antibody manual, dilute with 1×TBST, about 20 mL), recover primary antibody (store at -20℃), wash the membrane 3 times with 1×TBST buffer, 5 min each time, add secondary antibody with protein facing up, shake at room temperature for 1 h, recover secondary antibody after incubation (store at -20℃), wash the membrane 3 times with 1×TBST buffer, 5 min each time.

[0092] Exposure: Prepare the reagent kit A solution + B solution (A:B=1:1) for immediate use, about 4mL. Turn on the instrument. Evenly drop the prepared developer onto the developer plate, clamp the marker position of the membrane, and evenly contact the protein front with the developer to ensure that the developer has soaked all parts of the membrane, then turn the front side up. Turn on the software and perform exposure and development.

[0093] 4 Experimental results

[0094] 4.1 Regulatory effect of Citrus aurantium extract on Keap1 / Nrf2 signaling pathway

[0095] from Fig.10 and Fig.11 It can be seen that the Kelch-like epichlorohydrin-related protein 1 / nuclear factor E2-related factor 2 (Keap1 / Nrf2) signaling pathway is an important endogenous antioxidant signaling pathway in cells and is involved in the regulation of antioxidant responses in cells. The activation of Nrf2 can regulate downstream antioxidant proteins such as superoxide dismutase (SOD), heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 [NAD(P)H:quinone oxidoreductase 1, NQO1], etc., thereby regulating the level of oxidative stress to alleviate the damage of oxidative damage to cells. Western blot experiments showed that compared with the control group, the expression of Keap1 protein in the FFA group increased significantly, while the expression of Keap1 protein was significantly reduced after treatment with the extract of Citrus aurantium. In addition, compared with the control group, the expression level of Nrf2 in the FFA group was significantly reduced, and the expression of Nrf2 protein was significantly increased after intervention with the extract, indicating that the Nrf2 protein was activated in large quantities. By detecting downstream antioxidant proteins, it was found that compared with the FFA group, the HO-1 and NQO1 proteins in the Citrus aurantium extract group were significantly increased, which can alleviate cellular oxidative damage and improve the antioxidant capacity of cells.

[0096] The results showed that the extract of Citrus aurantium could significantly increase the expression level of antioxidant enzymes in HepG2, which helps to maintain the intracellular redox balance and reduce the oxidative damage of cells caused by lipid peroxidation.

[0097] 4.2 Effects of Citrus aurantium extract on AMPK pathway and its related protein expression

[0098] Depend on Fig.12It was found that the p-AMPK / AMPK ratio increased after intervention with Citrus aurantium extract. In addition, compared with the con group, the expression of SREBP1c protein in HepG2 cells in the FFA group was significantly increased (P<0.05), and the relative expression of SREBP-1c protein was significantly reduced after intervention, indicating that Citrus aurantium extract can inhibit the expression of SREBP-1c protein, thereby reducing lipid accumulation in HepG2 cells. Citrus aurantium extract can promote the phosphorylation of AMPK and activate its function, thereby reducing sterol synthesis and inhibiting the expression of the lipid synthesis protein SREBP1-c sterol regulatory element, inhibiting fatty acid synthesis in HepG2 cells.

[0099] Depend on Fig.13 The results showed that the expression of CPT1a and PPAR-α proteins in the FFA group was significantly decreased compared with the con group, and returned to normal levels after treatment with the extract of Citrus aurantium. The increased expression levels of CPT1a and PPAR-α proteins can promote the β-oxidation of fatty acids in the mitochondria of HepG2 cells.

[0100] 3. Study on the protective effect and mechanism of Citrus aurantium extract on NAFLD animal model

[0101] 1 Experimental animals

[0102] Male C57BL / 6J mice (6 weeks old, body weight 20 ± 2 g) were purchased from Hangzhou Muhao Biotechnology Co., Ltd.

[0103] 2 Experimental Materials

[0104] 60% high-fat experimental mouse diet (D12492) was purchased from Suzhou Synergy Pharmaceutical Bioengineering Co., Ltd.; Yishanfu (PPc) was purchased from Sanofi (Beijing) Pharmaceutical Co., Ltd.; triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) kits were purchased from Nanjing Jiancheng Bioengineering Institute; hematoxylin stain was purchased from Servicebio; other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0105] 3 Experimental instruments

[0106] HH-W420 constant temperature water tank, Jintan Baita Xinbao Instrument Factory; BSA124S electronic analytical balance, Beijing Sartorius Instrument System Co., Ltd.; TGL-16G high-speed centrifuge, Shanghai Anting Scientific Instrument Co., Ltd.; CX31 optical microscope, Olympus (China) Co., Ltd.; -80℃ refrigerator, Multiskan Go enzyme label analyzer, Thermo Fisher Scientific.

[0107] 4 Experimental methods

[0108] 4.1 Adaptive feeding of mice

[0109] In the experiment, 6-8 week old male C57BL / 6 mice weighing about 20-24g were selected. The breeding conditions were that the indoor temperature was controlled at 22±2℃, the relative humidity was controlled between 40% and 60%, the light was alternating between day and night for 12 hours, and sterile drinking water and food were provided to ensure that the animals had adequate nutrition. One week before the experiment, all mice were adapted to the experimental environment to reduce the impact of the experiment on them.

[0110] 4.2 Establishment of non-alcoholic fatty liver disease mouse model

[0111] 48 SPF healthy male C57BL / 6 mice were randomly divided into a normal group (n=8) and a high-fat diet group (n=40) after one week of adaptive feeding. The normal group was fed with ordinary feed, and the high-fat diet group was fed with high-fat feed for a total of 5 weeks to establish the NAFLD model.

[0112] 4.3 Grouping and Dosing

[0113] After successful modeling, the high-fat diet group was randomly divided into the model group, the low-dose Zhishi group (L-ZS), the medium-dose Zhishi group (M-ZS), the high-dose Zhishi group (H-ZS) and the positive control atorvastatin group (Ator), with 8 mice in each group. Except for the normal group, the other groups continued to be fed with high-fat feed. After grouping, the corresponding drugs were given by gavage for 4 weeks, while the normal group and the model group were given the corresponding volume of normal saline, as shown in Table 1.

[0114] Table 1 Mouse grouping and drug administration

[0115]

[0116]

[0117] 4.4 Collection of materials

[0118] At the end of the 10-week experiment, the eyeballs were removed to collect blood, the mice were killed by spinal dislocation, and fresh feces from the liver, fat, and cecum of each group of mice were collected. The collected blood was centrifuged at 4000r / min at 4°C for 15min, and the supernatant was taken, aliquoted, and stored in a -80°C refrigerator until use. The separated liver was weighed, and the left lobe was fixed in 4% paraformaldehyde for histopathological analysis. The feces and the rest of the liver were aliquoted and quickly frozen in liquid nitrogen and stored at -80°C for subsequent analysis.

[0119] 4.5 Experimental index determination

[0120] Observation of daily status of mice: During the experiment, the coat color, food intake, activity, mental state, etc. of each group of mice were observed daily, and the weight of mice was monitored weekly during the drug administration experiment.

[0121] 4.6 Serum biochemical index determination

[0122] Kits were used to measure serum lipid indexes including triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c) and liver function indexes including alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor (TNF-α) and interleukin-6 (IL-6).

[0123] 4.7 Antioxidant indexes of liver tissue

[0124] The enzyme activities of MDA, CAT, SOD and GSH-Px were determined according to the kit.

[0125] 4.8 Observation of mouse liver pathology

[0126] (1) Remove the liver tissue from the fixative, trim it flat with a scalpel in a fume hood, and place it in a dehydration box.

[0127] (2) Dehydration was performed by sequentially placing in gradient ethanol, i.e., 70% ethanol (10 h) - 80% ethanol (12 h) - 90% ethanol (2 h) - 95% ethanol (1 h) - 100% ethanol I (25 min) - 100% ethanol II (25 min).

[0128] (3) Then immerse in xylene to make it transparent, i.e. xylene I (15 min) - xylene II (30 min).

[0129] (4) Wax dipping and subsequent embedding.

[0130] (5) The trimmed wax block was sliced ​​into 4-mm thick sections using a paraffin slicer.

[0131] (6) Place the slices in 40°C warm water, place the flattened tissue on a glass slide, and bake in a 60°C constant temperature oven for 4 h.

[0132] (7) After taking out, store at room temperature for later use.

[0133] H&E staining of liver tissue:

[0134] (1) Dewaxing and dehydration: Immerse the sections in xylene I and II for 30 minutes each; immerse the slides in 100%, 95%, 75% and 70% gradient ethanol in sequence to ensure complete penetration.

[0135] (2) Staining: Place the slides in a hematoxylin solution for 5 minutes; in a 1% hydrochloric acid ethanol solution for 2 seconds, then rinse with running water until the sections turn blue, and finally stain with eosin for 10 seconds.

[0136] (3) Dehydration, transparency, and sealing: Dehydration is performed by using ethanol from low concentration to high concentration to finally achieve transparency and sealing. Xylene I and II are immersed in their respective containers, and then neutral gum is added and fixed with a coverslip. The pathological characteristics of the liver tissue are observed under a microscope at 200 times magnification.

[0137] Oil Red O staining of liver:

[0138] (1) Fixation: Soak the paraffin sections in PBS and rinse three times, 5 minutes each time.

[0139] (2) Staining: Soak the sections in Oil Red O working solution, rinse three times with PBS, and then stain with Oil Red O staining solution for 1 to 2 hours.

[0140] (3) Differentiation: Differentiate with 75% alcohol for 2 seconds and wash with water for 1 minute.

[0141] (4) Re-staining of cell nuclei: stain with hematoxylin, wash with tap water, then differentiate with 1% hydrochloric acid alcohol, wash with 0.6% ammonia water to turn blue, and finally rinse with running water.

[0142] (5) Sealing: Use neutral resin as sealing agent for sealing.

[0143] (6) Microscopic examination: Place the slices under an optical microscope, collect images and analyze the results.

[0144] 4.9Westernbolt (Keap1, Nrf2, ARE, HO-1, NQO1; p-AMPK, AMPK, SREBP-1c, CPT-1, PPAR-α)

[0145] (1) Liver tissue protein extraction: About 60 mg of liver tissue samples were taken from each group, lysed with RIPA lysis buffer (containing 1% PMSF) and centrifuged at 12,000 rpm for 30 minutes. The supernatant was taken to measure the protein concentration, diluted with buffer and then centrifuged at 100°C.

[0146] Heat in a water bath for 10 minutes and set aside.

[0147] (2) Gel preparation: According to the molecular weight of the target protein, select different concentrations of concentrated gel and separation gel to obtain the best separation effect. Pour the separation gel evenly into the glass plate, compact it with double distilled water, dry it with filter paper after gelation, insert a comb into it, and then add concentrated gel to fill it up to prevent bubble formation.

[0148] (3) Sample loading: Pour the electrophoresis working solution into the electrophoresis tank, place a protein marker on one side of the sample, adjust the voltage of the electrophoresis instrument to 90 V, and continue for 30 minutes. Then adjust the voltage to 120 V and maintain a constant voltage for one hour until the protein is electrophoresed to the bottom of the entire glass plate.

[0149] (4) Transfer: After electrophoresis is completed, extract the gel from the target protein, then place the filter paper, gel and membrane in order, then install the transfer clamp in the electrotransfer tank, pour in the electrotransfer working solution, set the output power of the electrophoresis instrument to 350mA, and transfer the membrane at a constant current of 4°C for 90 minutes.

[0150] (5) Blocking: After transfer, label the membrane and wash it three times with 1× TBST buffer, each time for 10 minutes. After washing, aspirate the liquid, pour in the blocking solution, and shake slowly on a shaker at room temperature for 2 hours.

[0151] (6) Incubation with antibodies: Aspirate the blocking liquid, then add the corresponding primary antibody, and then incubate overnight at 4°C. After incubation, wash the membrane three times with 1×TBST buffer, each time for 10 minutes. After washing, aspirate the liquid, pour the corresponding secondary antibody, and shake slowly on a shaker at room temperature for 1 hour. Finally, wash the membrane three times with 1×TBST buffer, each time for 10 minutes.

[0152] (7) Exposure: Add Bio ECL ultrasensitive luminescent reagent (A and B solutions mixed in a ratio of 1:1) onto the membrane and expose it to obtain the desired bands.

[0153] 4.10 Immunofluorescence detection of Nrf2 protein expression in liver tissue

[0154] (1) Frozen sections of liver tissue were prepared. After washing with PBS, an appropriate amount of 4% paraformaldehyde solution was added and fixed at room temperature for 30 minutes.

[0155] (2) Treat with 0.5% TritonX-100 / PBS for 15 min, then rinse with PBS for 5 min × 3 times.

[0156] (3) Block with 10% Normal Donkey Serum / PBS at room temperature for 30 min.

[0157] (4) Dilute the fluorescently labeled primary antibody in 1% Normal Donkey Serum / PBS, incubate at 4°C overnight, and then rinse with PBS for 5 min × 3 times.

[0158] (5) Dilute the fluorescently labeled secondary antibody in 1% Normal Donkey Serum / PBS, incubate at room temperature in the dark for 1 h, and then rinse with PBS for 5 min × 3 times.

[0159] (6) Incubate with DAPI working solution at room temperature for 2 min, then rinse with PBS for 5 min × 2 times.

[0160] (7) Antifade sealing agent was added to seal the slices, and then the slices were photographed using a laser scanning confocal microscope (×630).

[0161] 5 Experimental results

[0162] 5.1 Effect of Citrus aurantium extract on body weight of mice

[0163] from Fig.14It can be seen that as the experiment progressed, the body weight of the high fat diet (HFD) group was significantly higher than that of the CON group, proving that the NAFLD model was successfully established. After the intervention of the Citrus aurantium extract in the 5th week, the weight gain rate of the mice in the drug-treated group slowed down, and the high-dose drug-treated group also had a certain weight loss effect. These results show that Citrus aurantium extract can effectively reduce the rate of weight gain in mice, slowing down the rate of increase, has a significant preventive effect, and has potential application value. Compared with mice receiving a normal diet, the liver weight of mice in the HFD group was significantly higher than that of the CON group. However, after treatment with Citrus aurantium extract, the liver weight of mice was significantly reduced. And the epididymal fat weight of mice was significantly reduced. This finding suggests that Citrus aurantium extract helps to reduce fat accumulation caused by a high-fat diet.

[0164] 5.2 Effects of Citrus aurantium extract on the appearance and weight of mouse organs

[0165] from Fig.15 It can be seen that by observing the epididymal fat and liver of mice with the naked eye, it was found that in terms of epididymal fat volume, the epididymal fat volume of the HFD group increased, and the volume of the CON group was smaller, while the epididymal fat volume was significantly reduced after the intervention of the Citrus aurantium extract. The liver of the mice in the CON group was maroon, shiny, and soft in texture. After being fed with a high-fat diet, the liver of the mice in the HFD group was beige, with obvious white fat deposits, increased volume, tight skin membranes, rounded edges, and greasy cross-sections. After gavage with Citrus aurantium extract, the liver volume of the mice in the HFD group was smaller, and the color was maroon and shiny. This shows that Citrus aurantium extract can improve the tissue morphological changes in mice caused by a high-fat diet to a certain extent.

[0166] from Fig.16 It can be seen that compared with the CON group, the kidney and spleen weights of the mice in the HFD group increased significantly. However, after treatment with the Citrus aurantium extract, the kidney and spleen weights of the mice decreased significantly.

[0167] 5.2 Effects of Citrus aurantium extract on liver function in mice

[0168] from Fig.17 It can be seen that compared with the CON group, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice in the HFD group were significantly increased, indicating that the intake of a high-fat diet affects the liver function of mice. After treatment with the extract of Citrus aurantium, the ALT activity and AST activity of mice decreased significantly. And it showed a certain dose dependence. Therefore, the extract of Citrus aurantium may alleviate the liver damage induced by a high-fat diet.

[0169] 5.3 Biochemical analysis of mouse serum by extract of Citrus aurantium

[0170] Table 2 Blood lipid levels of mice in each group (mmol / L)

[0171]

[0172] After testing the blood lipid levels in the mouse serum, it was found that the serum TG content of the HFD group mice was significantly increased compared with the CON group. After treatment with the Citrus aurantium extract, the TC and TG content in the serum decreased significantly. In addition, the LDL-C level of the HFD group mice was significantly higher than that of the CON group, but the drug treatment could significantly reduce the LDL-C level in the mouse serum and increase the HDL-C level, thereby increasing the production of endogenous lipoproteins. The results show that Citrus aurantium extract can inhibit abnormal fat accumulation in the liver by regulating the liver's ability to metabolize lipids.

[0173] It should be noted that the preparation of active ingredients with different polarity of Citrus aurantium and the screening of their in vitro antioxidant activity specifically include:

[0174] 1 Experimental Materials

[0175] Citrus aurantium powder, Dazhou, Sichuan; anhydrous ethanol, ethyl acetate, n-butanol, n-hexane, sodium nitrite, aluminum nitrate, sodium hydroxide, Shanghai Sinopharm Chemical Reagent Co., Ltd.; 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), reduced coenzyme I (NADH), nitro blue tetrazolium (NBT), phenazine methyl sulfate (PMS), rutin standard, ascorbic acid, Shanghai MacLean Biochemical Technology Co., Ltd.

[0176] 2 Experimental instruments

[0177] HH-W420 constant temperature water tank, Jintan Baita Xinbao Instrument Factory; BSA124S electronic analytical balance, Beijing Sartorius Instrument System Co., Ltd.; ultra-micro UV spectrophotometer, Thermo Fisher Scientific; electric constant temperature blast drying oven, Shanghai Senxin Experimental Instrument Co., Ltd.; ultrasonic cleaner, Shanghai Kedao Ultrasonic Instrument Co., Ltd.

[0178] 3 Experimental methods

[0179] After drying, the immature bitter orange was ground into powder and passed through a 50-mesh sieve. Five samples were accurately weighed and mixed with 70% ethanol, pure water, ethyl acetate, n-butanol, and n-hexane as solvents in a solid-liquid ratio of 1:10. Ultrasonic extraction was performed using an ultrasonic cleaner at a frequency of 53 kHz. Extracts with different polar solvents were filtered out, concentrated under reduced pressure, and dried in an oven to constant weight to obtain extracts extracted with different solvents. The extracts were dissolved in 60% ethanol as the sample solution for subsequent antioxidant experiments and stored at 4°C.

[0180] 4 Experimental results

[0181] The weight of the extracts from different polar solvents was the highest in 70% ethanol, which was 7.61 g, followed by pure water extract, which was 6.32 g, and the weight of n-hexane was the lowest, which was 0.37 g. This shows that highly polar compounds are the main components of Citrus aurantium, and the content of non-polar compounds is relatively small.

[0182] The process of total flavonoids extraction from Citrus aurantium is optimized, including:

[0183] 1 Experimental Materials

[0184] Citrus aurantium powder, Dazhou, Sichuan; anhydrous ethanol, sodium nitrite, aluminum nitrate, sodium hydroxide, Shanghai Sinopharm Chemical Reagent Co., Ltd.

[0185] 2 Experimental instruments

[0186] HH-W420 constant temperature water tank, Jintan Baita Xinbao Instrument Factory; BSA124S electronic analytical balance, Beijing Sartorius Instrument System Co., Ltd.; ultra-micro UV spectrophotometer, Thermo Fisher Scientific; ultrasonic cleaner, Shanghai Kedao Ultrasonic Instrument Co., Ltd.

[0187] 3 Experimental methods

[0188] 3.1 Single Factor Experiment

[0189] The solid-liquid ratio was fixed at 1:10 g / mL, the extraction time was 30 min, and the extraction temperature was room temperature. The effects of different ethanol concentrations (50%, 60%, 70%, 80%, 90%) on the total flavonoids content were investigated.

[0190] The ethanol concentration was fixed at 60%, the extraction time was 30 min, and the extraction temperature was room temperature. The effects of different solid-liquid ratios (1:10, 1:20, 1:30, 1:40, 1:50 g / mL) on the total flavonoids content were investigated.

[0191] The solid-liquid ratio was fixed at 1:10 g / mL, the ethanol concentration was 60%, and the extraction time was 30 min. The effects of different temperatures (20, 30, 40, 50, 60°C) on the total flavonoids content were investigated.

[0192] The solid-liquid ratio was fixed at 1:10 g / mL, the ethanol concentration was 60%, and the extraction temperature was room temperature. The effects of different extraction times (10, 20, 30, 40, 50 min) on the total flavonoids content were investigated.

[0193] 3.2 Response surface experimental design

[0194] On the basis of the single factor experiment, the main factors affecting the total flavonoids content of Citrus aurantium were selected as independent variables, the total flavonoids content of Citrus aurantium was selected as the response value, and the BoxBehnken test principle in the software Design-Expert.V8.0.6.1 was used to design a four-factor three-level optimization experiment. Table 3 shows the optimization experiment with the solid-liquid ratio, extraction temperature, extraction time, and ethanol concentration as the response value of the independent variable.

[0195] Table 3 Optimization experiment with solid-liquid ratio, extraction temperature, extraction time, and ethanol concentration as the response value of independent variables

[0196]

[0197] 4 Experimental results

[0198] The theoretical optimal process parameters given by the model are ethanol concentration of 64%, solid-liquid ratio of 1:33, extraction temperature of 41°C, extraction time of 30 min, and theoretical total flavonoids content of 238.75 mg / g. Considering the feasibility of practical operation, the theoretical parameters were adjusted to ethanol concentration of 65%, solid-liquid ratio of 1:30, temperature of 40°C, and extraction time of 30 min. The experiment was repeated three times, and the average extraction rate of total flavonoids was determined to be 238.93 mg / g, which is close to the theoretical predicted value, indicating that the equation is consistent with the actual situation.

[0199] In summary, the polarity of different solvents has a great influence on the content of extracts and the content of chemical components. The content of flavonoids in Citrus aurantium is relatively high, accounting for about 22.45% to 33.76%, mainly dihydroflavonoids and flavonoids, most of which are polymethoxyflavones. Flavonoids have been reported to have good antioxidant and anti-inflammatory activities. The present invention uses five different polar solvents, including n-butanol, ethyl acetate, deionized water, 70% ethanol and n-hexane, to extract Citrus aurantium, and uses three in vitro antioxidant indicators, namely, the ability to scavenge DPPH free radicals, the ability to scavenge ABTS free radicals, and the ability to scavenge superoxide anion free radicals, to evaluate each extract, and to determine its total flavonoid content. The results show that when 70% ethanol and pure water are used as solvents, the extraction rate is relatively high, indicating that the active ingredients in Citrus aurantium may mostly belong to high polarity components. The level of total flavonoid content may be the main reason affecting the strength of antioxidant activity. Some studies have reported the content of total flavonoids and antioxidant activity in different solvent extracts of blueberry leaves. The results show that the antioxidant activity of extracts obtained from different solvents is quite different, and total flavonoids are correlated with antioxidant activity. Some scholars have found that 50% ethanol extract and pure water extract of raspberry have good in vitro antioxidant activity. The five different polar solvent extracts in the present invention all show different degrees of antioxidant activity in different antioxidant detection systems, and there is a certain dose-effect relationship. The order of antioxidant activity is 70% ethanol> ethyl acetate> water> n-butanol> n-hexane, which is basically consistent with the total flavonoid content.

[0200] Lipopolysaccharide (LPS) is a prominent component of the cell wall of Gram-negative bacteria. It simulates the early stage of inflammatory response, and the body will secrete a variety of inflammatory factors TNF-α, IL-1β, and IL-6. When the body produces an inflammatory response, it will also promote the occurrence of oxidative stress. At this time, the oxidation and antioxidant effects lose balance, and the reactive oxygen in the body will be produced and accumulated in large quantities in the cells, and then oxidative stress will occur. Studies have shown that the total flavonoid extract in dandelion can significantly reduce the production of cellular NO and the expression of iNOS mRNA after LPS stimulation, and has potential anti-inflammatory activity. In the present invention, LPS is used to establish an inflammatory model to evaluate the antioxidant and anti-inflammatory effects of the extract. The results show that 70% ethanol extract and pure water extract can significantly reduce the NO, TNF-α, IL-1β, and IL-6 content released by LPS-induced macrophages, and have a certain dose-effect relationship. The cell ROS fluorescence intensity after the intervention of the extract is significantly reduced, indicating that the two types of extracts can alleviate the inflammatory response, and the anti-inflammatory effect of the 70% ethanol extract is better than that of the pure water extract.

[0201] Ethanol solution was selected as the solvent to optimize the extraction process of total flavonoids from Citrus aurantium. Ultrasonic extraction method, which is widely used for flavonoid compounds, was adopted. Through single factor and Box-Behnken response surface methodology, the optimal extraction process obtained by combining actual conditions was: ethanol concentration of 63.01%, solid-liquid ratio of 1:31.91, extraction temperature of 41.88℃, extraction time of 29.71min, theoretical total flavonoid content of 2.88%, and actual average extraction rate of total flavonoids of 2.912% (RSD=0.169%), which was close to the theoretical predicted value, indicating that the equation was consistent with the actual situation.

[0202] HepG2 cells have the advantages of continuous growth, easy culture and stable phenotype, and are often used to establish in vitro cell models to study NAFLD. The fatty acids with the highest content in NAFLD patients are palmitic acid and oleic acid, and FFA modeling solution is prepared based on them, and low concentrations have little effect on cell activity. The optimal modeling concentration was determined by CCK-8 experiment. At this concentration, FFA had no obvious effect on cell activity, and the intracellular TC and TG contents increased significantly. Oil red O staining showed that a large amount of intracellular lipid droplets accumulated after FFA treatment. The appropriate intervention concentration of Citrus aurantium extract was selected by CCK-8 experiment. In the concentration range of 25-100 μg / mL, Citrus aurantium extract had no obvious toxic side effects on cells, and could significantly reduce the TC and TG contents in NAFLD model cells. Oil red O staining showed that the extract could reduce the accumulation of intracellular lipid droplets and had a certain concentration dependence.

[0203] A high-fat diet can cause oxidative stress, which is one of the main mechanisms causing non-alcoholic fatty liver disease (NAFLD). When there is too much lipid accumulation in the body, cellular peroxidation will occur, and the activity of antioxidants such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) will decrease, resulting in a break in the balance between oxidation and antioxidant activity, which will lead to an increase in the content of oxidative end products such as malondialdehyde (MDA), and ultimately cell damage. Reactive oxygen species (ROS) are metabolic products of the body, including superoxide free radicals, hydrogen peroxide, and its downstream product peroxide. When FFA in liver cells increases significantly, the ROS produced during lipid metabolism will destroy the antioxidant balance in the cells, causing liver cells to enter a state of oxidative damage. The activity levels of intracellular antioxidant proteases (CAT, GSH, SOD) and the content of lipid peroxides (MDA) were measured, and the level of intracellular reactive oxygen species (ROS) was detected using the DCFH-DA probe method. The results showed that after treatment with the Citrus aurantium extract, the level of intracellular antioxidant proteins increased, the content of the final toxic product MDA decreased, and the ROS fluorescence intensity decreased, indicating that the extract can alleviate cellular oxidative damage and improve the antioxidant capacity of cells.

[0204] The Nrf2 signaling pathway is a key antioxidant pathway in cells, which maintains the redox balance in cells. Nrf2 usually combines with Keap1 in the cytoplasm to form a dimer, but under the condition of oxidative stress in the body, the dimer dissociates and Nrf2 is released, and the downstream antioxidant proteins HO-1 and NQO1 are activated, thus exerting an antioxidant effect. HO-1 and NQO1 are important antioxidant enzymes that can regulate the body's oxidative balance. Western blot experimental results showed that under the action of the extract, the expression level of Keap1 in NAFLD model cells decreased, the expression of Nrf2 and its downstream factors NQO1 and HO-1 was increased, and the antioxidant capacity of HepG2 cells was enhanced, thereby reducing the oxidative damage of cells. Therefore, the Citrus aurantium extract exerts its improvement effect on NAFLD by activating the Keap1 / Nrf2 signaling pathway.

[0205] The presence of a large amount of free fatty acids can lead to hepatocyte lipid metabolism disorders by inhibiting AMPK activation, and AMPK signal inhibition is crucial for high-fat-induced NAFLD. AMPK is an important sensor of cellular energy metabolism, and it plays a core role in maintaining the dynamic balance of cellular energy metabolism. SREBP-1 is one of the most important transcriptional regulators of fatty acid de novo synthesis downstream of AMPK signaling. Its activation can transcriptionally regulate the expression of FASN, a key enzyme in fatty acid synthesis, thereby promoting excessive new fat production in hepatocytes. Regulating the AMPK / SREBP-1 signaling pathway to inhibit hepatocyte fatty acid de novo synthesis can improve hepatocyte lipid metabolism disorders. The liver fatty acid consumption pathway is mainly mitochondrial β-oxidation. The carnitine palmitoyltransferase (CPT) system mediates the metabolism of free fatty acids, which is very critical in mitochondrial β-oxidation. In this system, CPT1 is the rate-limiting enzyme of mitochondrial β-oxidation and is responsible for the transport of free fatty acids. It has three isoforms, A, B, and C, and CPT1A is specifically expressed in the liver. Studies have shown that upregulating the expression of CPT1A can improve NAFLD. Activation of AMPK can phosphorylate downstream acetyl-CoA carboxylase (ACC), leading to ACC inactivation, and activate PPAR-α, thereby facilitating CPT1A-mediated mitochondrial β-oxidation of fatty acids. Therefore, AMPK regulation of de novo fatty acid synthesis can serve as a key molecular target for the prevention and treatment of NAFLD.

[0206] The production of lipotoxic substances after lipid peroxidation that induce liver damage is one of the causes of NAFLD. The increase in free fatty acids and cholesterol can affect the behavior and function of normal liver cells. HDL-C, LDL-C, TC and TG in serum are used as biomarkers of lipotoxicity. In related studies, the basis for measuring the changes in lipotoxicity in the in vitro model of NAFLD is measured by examining the levels of HDL-C and LDL-C. In addition, when liver cells are damaged, transaminases (such as ALT and AST) will be released from the liver into the blood, so abnormal increases in ALT and AST in serum are signs of liver damage. The present invention further studies the therapeutic effect of AFIF on NAFLD by establishing a HFD-induced NAFLD mouse model and intervening with high, medium and low doses of Aurantii Fructus Immaturus total flavonoids (AFIF). The inventors found that compared with the normal group, indicators such as ALT, AST, HDL-C, LDL-C, TC and TG were abnormally expressed in the serum of NAFLD model mice, and intervention with different concentrations of AFIF could improve the levels of the above indicators to varying degrees in a dose-dependent manner, indicating that AFIF has the effect of protecting the liver and alleviating liver lipid lesions.

[0207] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. Application of Citrus aurantium extract in the preparation of drugs for the prevention and treatment of non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: The Citrus aurantium extract is used to activate the Keap1 / Nrf2 signaling pathway.

3. The use according to claim 2, characterized in that: The Citrus aurantium extract is used to increase the antioxidant enzyme expression level of HepG2 to maintain the redox balance in cells, thereby reducing the oxidative damage of cells caused by lipid peroxidation.

4. The use according to claim 2, characterized in that: The Citrus aurantium extract is used to promote the phosphorylation and activation function of AMPK, thereby reducing sterol synthesis and inhibiting the expression of the lipid synthesis protein SREBP1-c sterol regulatory element, so as to inhibit the fatty acid synthesis of HepG2 cells.

5. The use according to claim 1, characterized in that: The Citrus aurantium extract is used to reduce fat accumulation caused by a high-fat diet, alleviate liver function damage induced by a high-fat diet, and regulate the liver's ability to metabolize lipids to inhibit abnormal fat accumulation in the liver.

6. The use according to claim 1, characterized in that: The intervention concentration of the Citrus aurantium extract is in the range of 25 to 100 μg / mL.

7. The use according to claim 1, characterized in that: The Citrus aurantium extract includes Citrus aurantium active ingredients of different polarities; the preparation method of the Citrus aurantium active ingredients of different polarities includes: grinding and crushing the Citrus aurantium into powder after drying, and passing it through a 50-mesh sieve to obtain a sample; weighing 5 portions of the sample, mixing them with 1 portion of the sample at a solid-liquid ratio of 1:10 using 70% ethanol, pure water, ethyl acetate, n-butanol, and n-hexane as solvents, respectively, using an ultrasonic cleaner to perform ultrasonic extraction at a frequency of 53 kHz, and filtering to obtain solvent extracts of different polarities; concentrating the solvent extracts of different polarities under reduced pressure and then drying them to constant weight to obtain extracts extracted with different solvents, namely the Citrus aurantium active ingredients of different polarities.

8. The use according to claim 1, characterized in that: The Citrus aurantium extract is obtained by extracting Citrus aurantium using 70% ethanol or pure water as an extraction solvent.

9. The use according to claim 1, characterized in that: The Citrus aurantium extract includes Citrus aurantium total flavonoids; the extraction method of the Citrus aurantium total flavonoids includes: using ultrasonic extraction method, using 63.01% ethanol as extraction solvent, the solid-liquid ratio is 1:31.91, the extraction temperature is 41.88°C, and the extraction time is 29.71min.

10. The use according to claim 9, characterized in that: The average extraction rate of total flavonoids from the method for extracting total flavonoids from Citrus aurantium is 2.912%.

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