Application of wild jujube leaf total flavonoids in medicine for treating / preventing hepatic fibrosis

Abstract

The invention belongs to the technical field of traditional Chinese medicines and application, and relates to application of wild jujube leaf total flavonoids in a medicine for treating / preventing hepatic fibrosis. Researches find that the wild jujube leaf total flavonoids can induce the activated HSC cells to generate ferroptosis and play a role in resisting hepatic fibrosis, so that the bioavailability is improved, and the treatment effect is improved; meanwhile, the action mechanism of resisting hepatic fibrosis is defined, and a research basis is provided for clinical application of the wild jujube leaf total flavonoids in treating and preventing hepatic fibrosis.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine and its application, and relates to the application of total flavonoids from wild jujube leaves in a medicine for treating / preventing liver fibrosis. Background Art

[0002] Liver fibrosis (LF) is a common pathological feature of the progression of various chronic liver diseases. Its core mechanism is the abnormal activation of hepatic stellate cells (HSCs) and the excessive deposition of extracellular matrix (ECM), ultimately leading to structural destruction and functional failure of liver tissue. The characteristics of fibrosis progression depend primarily on four key factors: the etiology, the area of injury, the source of the involved fibrogenic cells, and the primary fibrogenic mechanism. In the chronic progressive stage of viral hepatitis (particularly hepatitis B and C as typical pathogens), characteristic pathological changes manifest as inflammation at the portal tract interface accompanied by bridging necrosis, ultimately leading to the formation of fibrous septa that extend throughout the hepatic lobules. Under normal circumstances, the progression of fibrosis is not obvious, but once it progresses to cirrhosis, patients experience high rates of disability and mortality.

[0003] In recent years, the treatment of liver fibrosis has evolved from a single approach focused on alleviating inflammatory responses to a multimodal approach. Traditional Chinese medicine (TCM) holds great potential for the treatment of liver fibrosis due to its significant biological activity. However, TCM has low bioavailability and poor therapeutic efficacy. Furthermore, due to significant differences in the pathogenesis of liver fibrosis and related clinical efficacy, the mechanisms of action of TCM anti-fibrotic drugs remain unclear, limiting their clinical application.

[0004] Ziziphi Spinosae Folium, also known as spinose leaves, are the dried leaves of the plant Ziziphi Spinosae. In recent years, Ziziphi Spinosae, a traditional medicinal herb used for both medicinal and edible purposes, has garnered widespread research attention. Its pharmacological effects include improving sleep and providing antioxidant benefits. Its fruit, seeds, leaves, stems, branches, bark, and flowers can all be used as food, medicine, and health supplements. However, compared to the medicinal value of Ziziphi Spinosae kernels, Ziziphi Spinosae Folium is currently primarily used in beverages and tea, resulting in a serious structural imbalance in its development and utilization.

[0005] Therefore, it is particularly necessary to develop the sour jujube leaves, improve their bioavailability in liver fibrosis and clarify the therapeutic mechanism. Summary of the Invention

[0006] In view of the technical problems in the background technology that existing traditional Chinese medicines for the treatment of liver fibrosis have low bioavailability, poor therapeutic effects and unclear anti-fibrosis mechanisms, the present invention provides an application of total flavonoids from Chinese jujube leaves in drugs for the treatment / prevention of liver fibrosis.

[0007] The present invention extracts total flavonoids from the leaves of Ziziphus jujuba. Through research, it is found that the total flavonoids from the leaves of Ziziphus jujuba can induce ferroptosis in activated HSC cells, exert an anti-liver fibrosis effect, thereby improving bioavailability and enhancing therapeutic effects. At the same time, the anti-liver fibrosis mechanism of action is clarified, providing a research basis for the clinical application of total flavonoids from the leaves of Ziziphus jujuba in the treatment and prevention of liver fibrosis.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] Application of total flavonoids from jujube leaves in a medicine for treating / preventing liver fibrosis.

[0010] The invention discloses a drug for treating / preventing liver fibrosis by reducing serum ALT, AST and ALP levels and reducing serum Col-Ⅳ, HA, LN and PCⅢ contents.

[0011] A method for treating / preventing liver fibrosis by using total flavonoids from Chinese jujube leaves to significantly improve abnormal organ indexes, reduce collagen fiber deposition and liver cell structural damage, and delay the progression of fibrosis.

[0012] A total flavonoid from jujube leaves is used in a drug for treating / preventing liver fibrosis by downregulating the expression of fibrosis marker proteins α-SMA, Collagen I and Collagen IV and reducing the expression of NRF2, SLC7A11 and GPX4 proteins.

[0013] The invention discloses a method for treating / preventing liver fibrosis by using total flavonoids from Chinese jujube leaves, which can significantly increase the ROS level, increase MDA and reduce GSH content in HSC-T6 cells.

[0014] A total flavonoid from jujube leaves promotes ferroptosis of activated HSC cells by inhibiting the NRF2 / SLC7A11 / GPX4 signaling pathway and is used in drugs for treating / preventing liver fibrosis.

[0015] It is further defined that the chemical components in the total flavonoids of the jujube leaves include quercetin-3-O-acaciaside, epigallocatechin, eriocitrin, quercetin-3-(2R)-rutinoside, rutin, quercetin-3-O-rutinoside, apocynin, quercetin-3-O-β-D-glucopyranoside, kaempferol-3-rutinoside, kaempferol-3-O-rutinoside, dendroicin, quercetin-3-o-β-L-arabinosyl-(1-2)-α-L-rhamnose and quercetin-3-O-β-D-xylosyl-(1-2)-AL-rhamnose.

[0016] It is further defined that among the total flavonoids of the sour jujube leaves, the mass proportion of quercetin-3-O-sophoroside is 2.84%, the mass proportion of rutin is 2.67%, and the mass proportion of caltropin is 0.89%.

[0017] It is further specified that the total flavonoids from the wild jujube leaves are extracted from the wild jujube leaves by ultrasonic method.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention extracts total flavonoids from the leaves of Chinese jujube; and through research, it is found that the total flavonoids from the leaves of Chinese jujube can induce ferroptosis in activated HSC cells, thereby playing an anti-liver fibrosis role; the total flavonoids from the leaves of Chinese jujube, as an effective ingredient extracted from medicinal materials, can improve the bioavailability of the medicinal materials, thereby enhancing the therapeutic effect; at the same time, the anti-liver fibrosis mechanism is clarified through research, providing a research basis for the clinical application of the total flavonoids from the leaves of Chinese jujube in the treatment and prevention of liver fibrosis.

[0020] 2. The present invention found that total flavonoids from Chinese jujube leaves can reduce the levels of ALT, AST and ALP in serum, and reduce the contents of serum Col-Ⅳ, HA, LN and PCⅢ; significantly improve the abnormal organ index of mice with liver fibrosis, reduce collagen fiber deposition and liver cell structure damage, and delay the process of fibrosis; downregulate the expression of fibrosis marker proteins α-SMA, Collagen I and CollagenⅣ, and reduce the expression of NRF2, SLC7A11 and GPX4 proteins; significantly increase the ROS level, increase MDA and reduce the GSH content in HSC-T6 cells; and promote the ferroptosis of activated HSC cells by inhibiting the NRF2 / SLC7A11 / GPX4 signaling pathway, thereby exerting an anti-liver fibrosis effect, providing a new approach for the treatment of liver fibrosis.

[0021] 3. Through analysis, the present invention found that the chemical components of the total flavonoids in the leaves of sour jujube are 13 flavonoid compounds, and the content of total flavonoids is 610.301 mg / g. It has many chemical components and a high content of total flavonoids, which maximizes the biological activity of the total flavonoids in the leaves of sour jujube and promotes the therapeutic effect.

[0022] 4. The present invention extracts total flavonoids from the wild jujube leaves, has good safety, and deeply develops and utilizes the wild jujube leaves, thereby expanding the medical value of the wild jujube leaves and balancing the structural uses of the wild jujube leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results are for the effect of liver index on liver fibrosis in mice;

[0024] Figure 2 Results of the effect of spleen index on liver fibrosis in mice:

[0025] Figures 3 to 5 ALT in serum of LF mice ( Figure 3 )、AST( Figure 4 ) and ALP( Figure 5 ) level of impact results;

[0026] Figures 6 to 9 PCⅢ in LF mouse serum ( Figure 6 )、Col-Ⅳ( Figure 7 )、HA( Figure 8 ) and LN( Figure 9 ) level of influence;

[0027] Figures 10 to 12 The effect of pathological changes in liver tissue of LF mice; Figure 10 HE staining image (×200) The green arrow indicates fatty degeneration of hepatocytes; Figure 11 Masson staining image (×100). Yellow arrows indicate collagen deposition. Figure 12 is the collagen deposition area;

[0028] Figure 13 and Figure 14 The effect of α-SMA in liver tissue of LF mice; Figure 13 For Western blot; Figure 14 Gray value statistics for Western blot;

[0029] Figure 15 and Figure 16 The effects of NRF2, GPX4, and SLC7A11 in liver tissues of LF mice; Figure 15 For Western blot; Figure 16 Gray value statistics for Western blot;

[0030] Figure 17 The effect of TFZSF on the viability of HSC-T6 cells;

[0031] Figure 18 The effect of TFZSF on TGF-β1-induced activation of HSC-T6 cells;

[0032] Figure 19 is the effect of TFZSF on LO-2 cells;

[0033] Figure 20 and Figure 21 The effect of TGF-β1 on α-SMA protein in HSC-T6 cells; Figure 20 for Western blotting; Figure 21 Gray value statistics of Western blotting bands;

[0034] Figure 22 MDA (A), GSH (B), and iron content (C) of HSC-T6 cells induced by TGF-β1;

[0035] Figure 23 and Figure 24 The effect of TGF-β1 on the ROS level in HSC-T6 cells; Figure 23 These are ROS immunofluorescence images; Figure 24 is the statistics of ROS immunofluorescence intensity;

[0036] Figure 25 MDA (A), GSH (B), and iron content (C) in the ferroptosis pathway of HSC-T6 cells induced by TGF-β1;

[0037] Figure 26 and Figure 27 The effect of TGF-β1 on ROS levels in the ferroptosis pathway of HSC-T6 cells; Figure 26 Immunofluorescence images of ROS; Figure 27 is the statistics of ROS immunofluorescence intensity;

[0038] Figure 28 and Figure 29 The effect of α-SMA protein on the ferroptosis pathway of HSC-T6 cells induced by TGF-β1; Figure 28 For Western blotting; Figure 29 Gray value statistics of Western blotting bands;

[0039] Figure 30 and Figure 31 The effect of TGF-β1 on the NRF2 / SLC7A11 / GPX4 pathway in HSC-T6 cells; Figure 30 For Western blotting; Figure 31 Gray value statistics of Western blotting bands;

[0040] Figures 32 to 35 Effects of NRF2 inhibitors and TFZSF on TGF-β1-induced activation and ferroptosis of HSC-T6 cells; Figure 32 for α-SMA Western blotting; Figure 33 Gray value statistics of α-SMA Western blotting bands; Figure 34 Western blotting for NRF2, SLC7A11, and GPX4; Figure 35 Grayscale value statistics of NRF2, SLC7A11 and GPX4 Western blotting bands;

[0041] Figures 36 to 39 Effects of SLC7A11 inhibitor and TFZSF on TGF-β1-induced activation and ferroptosis of HSC-T6 cells; Figure 36 for α-SMA Western blotting; Figure 37 Gray value statistics of α-SMA Western blotting bands; Figure 38 Western blotting for SLC7A11 and GPX4; Figure 39 Grayscale value statistics of SLC7A11 and GPX4 Western blotting bands;

[0042] Figures 40 to 43 Effects of GPX4 inhibitor and TFZSF on TGF-β1-induced activation and ferroptosis of HSC-T6 cells; Figure 40 for α-SMA Western blotting; Figure 41 Gray value statistics of α-SMA Western blotting bands; Figure 42 for GPX4 Western blotting; Figure 43 Grayscale value statistics of GPX4 Western blotting bands;

[0043] In the above figures: # P<0.5, ## P<0.01, ### P < 0.001, vs blank group; * P<0.5, ** P<0.01, *** P<0.001, vs model group. DETAILED DESCRIPTION

[0044] The present invention will be described in further detail below with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto. Other methods for preparing the compounds of the present invention are considered to be within the scope of the present invention by making some conventional modifications to the reaction conditions of the present invention.

[0045] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0046] Technologies, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such technologies, methods, and equipment should be considered part of the specification.

[0047] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, which should be covered by the scope of protection of the present invention / invention.

[0048] It should be noted that the chemicals, reagents, testing equipment and instruments used in the present invention are all conventional in the art and can be purchased from the market.

[0049] The invention extracts total flavonoids from wild jujube leaves and clarifies the mechanism of action of total flavonoids from wild jujube leaves in resisting liver fibrosis through research.

[0050] 1. Extraction and determination of total flavonoids from jujube leaves

[0051] This part first extracts the total flavonoids from the leaves of sour jujube, and then determines the content of the components in the total flavonoids in the leaves of sour jujube.

[0052] 1.1 Extraction of total flavonoids from jujube leaves

[0053] Ultrasonic extraction method was used to extract total flavonoids from the leaves of Ziziphus jujuba. The specific extraction process is as follows:

[0054] S1. Ultrasonic extraction

[0055] S1.1. Pretreatment of sour jujube leaves

[0056] The above-ground leaves and stems of the sour jujube are separated to remove impurities; the sour jujube leaves are then placed in a ventilated and cool place to dry in the shade, and then crushed and sieved to obtain sour jujube leaf powder.

[0057] S1.2 Ultrasonic extraction

[0058] Weigh 500g of jujube leaf powder, add 10 times the mass of 50% (v / v) methanol, and ultrasonically extract three times for 1 hour each time. Combine the three filtrates; then vacuum filter to obtain the extract, and reduce the pressure at 60°C to recover the alcohol-free content to obtain a crude extract.

[0059] S2. Enrichment and purification of total flavonoids

[0060] S2.1. Pretreatment of AB-8 macroporous resin

[0061] First, soak the macroporous resin in a 4wt% hydrochloric acid solution for 5 hours, then rinse repeatedly with distilled water until the rinse solution is neutral. Next, prepare a 5wt% sodium hydroxide solution and soak it in it for 5 hours, then rinse again with distilled water until it is neutral. Finally, soak it in anhydrous ethanol at room temperature for 24 hours, rinse with distilled water until the alcohol smell is gone, and set aside.

[0062] S2.2. Enrichment and purification

[0063] The crude extract obtained in step S1 was suspended in an equal volume of water to obtain a suspension solution. The suspension solution was then added to a pre-treated AB-8 macroporous resin column and adsorbed overnight (with absorbent cotton plugged at the very top to prevent the macroporous resin from being washed away during elution). Five column volumes (approximately 6 L) of pure water were used to remove impurities until the eluate was transparent. Finally, elution was performed with 10 L of 70% (v / v) methanol. The eluate was collected and combined, recovered under reduced pressure at 60° C. until the alcohol content was free, and vacuum dried to obtain a powdered total flavonoid from the leaves of Ziziphus jujuba (26.74 g in total, with an extraction yield of approximately 5.35%).

[0064] 1.2 Determination of total flavonoids in jujube leaves

[0065] 1.2.1 Content of total flavonoids in jujube leaves

[0066] The total flavonoids content of the extracted jujube leaves was determined by ultraviolet spectrophotometer, and the absorbance was measured at 510 nm with rutin as the reference substance. The content of total flavonoids (active ingredients) was calculated, as shown in Table 1.

[0067] Table 1 Determination of total flavonoids in jujube leaves

[0068]

[0069] 1.2.2 Characterization of chemical components of total flavonoids in jujube leaves

[0070] UPLC-Q-TOF / MS was used to detect the chemical components of total flavonoids in the leaves of Ziziphus jujuba. Analyst TF1.7.1 was used to collect UPLC-Q-TOF / MS data, and Peakview 1.2 was used to process the data to identify the compounds in the total flavonoids in the leaves of Ziziphus jujuba.

[0071] The analytical conditions for UPLC-Q-TOF / MS were as follows:

[0072] (1) Accurately weigh the total flavonoids from the leaves of Ziziphus jujuba and dissolve them in 50% methanol. Ultra-high performance liquid chromatography (UPLC) analysis was performed using an Agilent ZORBAX RRHDSB-Aq column (2.1 × 100 mm, 1.8 μm). The mobile phase consisted of acetonitrile (A) and 0.1% formic acid in water (B). The column temperature was 30°C, the flow rate was 0.3 mL / min, and the injection volume was 3 μL. The gradient elution program is shown in Table 2.

[0073] Table 2 Gradient elution program

[0074]

[0075] (2) Mass spectrometry conditions: ESI negative and positive ion modes were used, with a TOF mass range of 50 to 1700 m / z and a MS / MS mass range of 50 to 1250 m / z. Mass spectrometry analysis was performed using a Waters G2 QTOF mass spectrometer equipped with an electrospray ionization (ESI) source. Mass spectra were obtained in ESI negative and positive ion modes over a full scan range of 100 to 1500 Da. Other parameters were: capillary voltage -2.4 kV, cone voltage 40 V, source temperature 120°C, desolvation temperature 400°C, desolvation gas flow rate 800 L / h, and cone gas flow rate 50 L / h. The identification results are shown in Table 3.

[0076] Table 3 Identification results of chemical components of total flavonoids in Ziziphus jujuba leaves

[0077]

[0078] It can be seen from Table 3 that the chemical components of the total flavonoids in the leaves of Ziziphus jujuba include 13 flavonoids, including quercetin-3-O-acaciaside, epigallocatechin, eriocitrin, quercetin-3-(2R)-rutinoside, rutin, quercetin-3-O-rutinoside, apocynin, quercetin-3-O-β-D-glucopyranoside, kaempferol-3-rutinoside, kaempferol-3-O-rutinoside, dendroicin, quercetin-3-o-β-L-arabinosyl-(1-2)-α-L-rhamnose and quercetin-3-O-β-D-xylosyl-(1-2)-AL-rhamnose.

[0079] HPLC quantitative analysis was further used to determine the contents of quercetin-3-O-sophoroside, rutin and dendrobium in the total flavonoids of Ziziphus jujuba leaves.

[0080] HPLC quantitative analysis conditions were as follows: an Agilent-1260 high-performance liquid chromatograph was used, with an Agilent C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of 0.05% aqueous phosphoric acid (A) and acetonitrile (B), using a gradient elution program. The detection wavelength was 254 nm, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the injection volume was 10 μL. Elution conditions are shown in Table 4.

[0081] Table 4 Gradient elution program

[0082] Time / min 0.05% phosphoric acid water / % Acetonitrile / % 0 90 10 10 85 15 25 82 18 35 75 25 40 60 40 45 90 10

[0083] The test results showed that among the total flavonoids in the leaves of sour jujube, quercetin-3-O-sophoroside accounted for 2.84%, rutin accounted for 2.67%, and dendrobium accounted for 0.89%.

[0084] 2. Study on the effect of total flavonoids from jujube leaves on CCl4-induced liver fibrosis in mice

[0085] This study systematically evaluated the effects of total flavonoids from jujube leaves (TFZSF) on organ indices, serum liver function markers (ALT, AST, and ALP), and liver fibrosis in a CCl4-induced mouse liver fibrosis model. Hepatic histopathology (HE and Masson staining) and Western blot were used to examine the expression levels of fibrosis markers (α-SMA, Collagen I and Collagen IV) and ferroptosis-related proteins (GPX4, SLC7A11, and NRF2). This study revealed the molecular mechanism by which total flavonoids from jujube leaves improve liver fibrosis, providing a theoretical basis for the in-depth development of jujube leaves and multi-target therapeutic strategies for liver fibrosis.

[0086] 2.1 Animals and husbandry

[0087] SPF-grade male C57BL / 6 mice, 6–8 weeks old, weighing 20 ± 2 g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (SCXK (Sichuan) 2020-030) and housed in the SPF-grade animal laboratory of the Shaanxi Provincial Collaborative Innovation Center for the Industrialization of Traditional Chinese Medicine Resources (SYXK (Shaanxi) 2022-008). All experiments were conducted in accordance with the Guiding Principles for Animal Experimentation of Shaanxi University of Chinese Medicine and the University Ethics Committee (SUCMDL20240604001).

[0088] 2.2 Methods

[0089] 2.2.1 Animal grouping and modeling

[0090] After one week of adaptive feeding, the mice were randomly divided into a blank group (Control), a model group (Model), a silymarin group (Silymarin), a low-dose TFZSF-L group (TFZSF-L), and a high-dose TFZSF-H group (TFZSF-H), with 10 mice in each group. Except for the blank group, all other groups received intraperitoneal injections of 10% CCl₄ (prepared in olive oil, injection volume: 10 mL / kg, 0.1 mL / 10 g) twice weekly for 8 consecutive weeks. During this period, the silymarin group and the TFZSF-H group received oral gavage of silymarin (100 mg / kg) and TFZSF-H (10 mg / kg, 20 mg / kg). The blank and model groups received oral gavage of the same amount of olive oil solution once daily for 6 consecutive weeks. 16 hours after the last administration, blood was collected from the fundus venous plexus of the mice, and the mice were killed by cervical dislocation. The serum was collected and allowed to stand for 2 hours. The serum was then centrifuged at 4°C and 12,000 rpm for 10 minutes, separated, aliquoted, and frozen at -80°C.

[0091] 2.2.2 Determination of animal organ index

[0092] After the experiment, the liver and spleen of each group of mice were taken out, washed with physiological saline, dried with filter paper, and the wet weight of the liver and spleen was measured, and the organ index of each group was calculated.

[0093] 2.2.3. Determination of liver function indicators

[0094] The serum AST, ALT and ALP levels of mice in each group were detected by automatic biochemical analyzer.

[0095] 2.2.4 Determination of Col-Ⅳ, HA, LN, and PCⅢ in mouse serum

[0096] According to the instructions of Nanjing Jiancheng Elisa kit, the levels of Col-Ⅳ, HA, LN and PCⅢ in mouse serum were detected.

[0097] 2.2.5. Liver tissue pathological observation

[0098] Liver tissue samples were fixed with 4% paraformaldehyde for 48 hours, then dehydrated and embedded in paraffin, followed by preparation of 5μm serial sections. Following standard dewaxing procedures, hematoxylin and eosin (HE) and Masson's staining were performed, and the histopathological features of the liver tissue were systematically observed under a light microscope. Quantitative analysis of Masson's staining was performed using ImageJ software. Blue indicates positive staining areas.

[0099] 2.2.6. Western blot detection of key proteins

[0100] 2.2.6.1. Sample protein extraction process

[0101] 50 mg of liver tissue from each group was taken and added with 1400 μL RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors (1:50). After grinding, the tissue was fully lysed on ice for 60 min and centrifuged at 12000 r / min at 4°C for 15 min. The supernatant was aspirated and the protein concentration was determined using a BCA protein quantification kit.

[0102] 2.2.6.2 Immunoblotting Procedure

[0103] Prepare SDS-PAGE gels of corresponding proportions (as shown in Table 5).

[0104] 1) Prepare SDS-PAGE gel of corresponding proportion;

[0105] 2) Loading the sample;

[0106] 3) The protein was electrophoresed in the stacking gel and the separating gel at 60V and 90V respectively;

[0107] 4) Transfer the membrane at 204 mA, and determine the transfer time based on the protein molecular weight;

[0108] 5) Prepare 5% skim milk in 1×TBST and block the PVDF membrane at room temperature for 1 h;

[0109] 6) Dilute the primary antibody with 5% BSA solution prepared in 1×TBST and incubate overnight at 4°C. See Table 6 for the primary antibody dilution ratio.

[0110] 7) Wash the membrane three times with 1× TBST, each time for 15 min.

[0111] 8) Incubate with secondary antibody at room temperature for 1 hour;

[0112] 9) Wash the membrane three times with 1× TBST, 15 min each time;

[0113] 10) After ECL development, the PVDF membrane was exposed using a gel imaging system;

[0114] 11) Using GAPDH as an internal reference, the grayscale values of the protein bands after imaging were quantitatively analyzed using ImageJ software (the above experiment was repeated 3 times).

[0115] 2.2.6.3. SDS-PAGE gel configuration

[0116] Table 5 Reference table for separation gel and stacking gel formula

[0117] Reagents Separation gel 12% Concentrated gel 5% <![CDATA[ddH2O]]> 8mL 5.5mL 30% Acr / Bis (29:1) 6.6mL 1.3mL 1M Tris-HCl (pH 6.8) 0 1.0 1.5M Tris-HCl (pH 8.8) 5mL 0 10% SDS 200 μL 80 μL 10% PAGE gel coagulant 200 μL 120 μL PAGE gel coagulant 20 μL 12 μL Optimal separation range 20~80kDa

[0118] Table 6 Western blot detection indicators

[0119]

[0120]

[0121] 2.2.7 Statistical methods

[0122] The results were analyzed and statistically analyzed using IBM SPSS Statistics 20 and GraphPad Prism 8. Values are expressed as mean ± standard deviation (mean ± SD). One-way analysis of variance was used for comparisons between multiple groups, and Student's ...

[0123] 2.3 Results

[0124] 2.3.1 Effects on the liver and spleen index of mice

[0125] like Figure 1 、 Figure 2 As shown in Table 7, the liver and spleen index of the model group was significantly increased compared with the blank group (P<0.05). Compared with the model group, the liver and spleen index of the silybin group and the total flavonoids of jujube leaves were significantly decreased (P<0.05).

[0126] Table 7 Effect of liver and spleen index on mice (x±SD, n=10)

[0127] Group Liver index (%) Spleen index (%) Control 4.4±0.39 0.3±0.08 Model 4.5±0.18# 0.4±0.21# Silymarin 4.3±0.18** 0.4±0.08* TFZSF-L 4.4±0.32* 0.4±0.05* TFZSF-H 4.4±0.34* 0.3±0.1*

[0128] Note: # P<0.5, ## P<0.01, ### P<0.001, vs. blank group; *P<0.5, **P<0.01, ***P<0.001, vs. model group.

[0129] 2.3.2 Effects on Serum Biochemical Parameters in Mice

[0130] like Figure 3 、 Figure 4 、 Figure 5 As shown in Table 8, compared with the blank group, the ALT, AST, and ALP levels of the model group mice were increased (P < 0.0001). Compared with the model group, the ALT, AST, and ALP levels of the silybin group and the high and low doses of total flavonoids from jujube leaves were significantly reduced (P < 0.05).

[0131] Table 8 Effects of serum biochemical parameters in mice (x±SD, n=10)

[0132] Group ALT(U / L) AST(U / L) ALP(U / L) Control 74.3±8.8 212.4±42.3 86.2±27.7 Model <![CDATA[8264.6±1675 #### ]]> <![CDATA[6006.1±1206.9 #### ]]> <![CDATA[143.9±27.6 #### ]]> Silymarin <![CDATA[4420.1±981.8 **** ]]> <![CDATA[2544.1±361.6 **** ]]> <![CDATA[103.3±18.8 *** ]]> TFZSF-L 6978±1198.2* 5106.1±1052.1* 102.5±15.1** TFZSF-H 4725.4±1635**** 3549.2±1319.3*** 96.9±15.5**

[0133] Note: # P<0.5, ## P<0.01, ###P<0.001, vs. blank group; *P<0.5, **P<0.01, ***P<0.001, vs. model group.

[0134] 2.3.3 Effects on Col-Ⅳ, HA, LN, and PCⅢ in mouse serum

[0135] like Figures 6 to 9 As shown in the results, compared with the blank group, the serum levels of Col-Ⅳ, HA, LN, and PCⅢ in the model group mice were significantly increased (P<0.05). Compared with the model group, oral administration of TFZSF and silybin significantly reduced the serum levels of Col-Ⅳ, HA, LN, and PCⅢ in the model group mice (P<0.05). This suggests that total flavonoids from Ziziphus jujuba leaves have a protective effect on liver function in mice with hepatic fibrosis.

[0136] 2.3.4 Effects on Pathological Changes in Mice

[0137] See also Figure 10 HE staining showed that compared with normal mice, the model group mice had obvious hepatocyte fatty degeneration and inflammatory cell infiltration. Treatment with total flavonoids from jujube leaves and silybin significantly reduced hepatocyte damage and inflammatory infiltration.

[0138] See also Figure 11 Masson staining results showed that liver tissue sections from mice in the blank group had normal cellular structure and no obvious collagen fiber production. However, abundant proliferating collagen fibers were observed in liver tissue sections from mice in the model group. A small amount of collagen fibers was still present in liver tissue sections from mice in the TFZSF group, but the staining was lighter than in liver tissue sections from mice in the model group.

[0139] See also Figure 12 After quantifying the collagen fiber-positive area in the liver of mice in each group, it was found that compared with the model group, the collagen fiber-positive area in the liver of mice in the TFZSF group was significantly reduced (P<0.05).

[0140] 2.3.5 Effects on the Expression of α-SMA, Collagen I, and Collagen IV Proteins in Mice

[0141] like Figure 13 and Figure 14 As shown in the results, compared with the blank group, the protein levels of α-SMA, Collagen I, and Collagen IV in the model group were significantly increased (P<0.05); compared with the model group, the protein levels of α-SMA, Collagen I, and Collagen IV in the TFZSF group were significantly decreased (P<0.05), indicating that TFZSF can reduce the activation of HSCs in liver tissue.

[0142] 2.3.6 Effects on NRF2, GPX4, and SLC7A11 Protein Expression in Mice

[0143] like Figure 15 and Figure 16 As shown in the results, compared with the blank group, the NRF2, GPX4, and SLC7A11 protein levels in the model group were significantly increased (P<0.05); compared with the model group, the NRF2, GPX4, and SLC7A11 protein levels in the TFZSF group were significantly decreased (P<0.05). This suggests that total flavonoids from jujube leaves may exert their anti-hepatic fibrosis effects by inhibiting the NRF2 / SLC7A11 / GPX4 pathway.

[0144] The experiments described above in this study demonstrated that total flavonoids from jujube leaves significantly improved abnormal organ indices in mice with liver fibrosis and reduced serum levels of ALT, AST, and ALP. Four serological tests for liver fibrosis revealed that total flavonoids from jujube leaves reduced serum levels of Col-IV, HA, LN, and PC III. Liver histopathological analysis (HE and Masson staining) further confirmed that total flavonoids from jujube leaves significantly reduced collagen deposition and hepatocyte structural damage, thereby delaying the progression of fibrosis. Western blot analysis revealed that total flavonoids from jujube leaves inhibited HSC activation by downregulating the expression of fibrosis markers α-SMA, Collagen I, and Collagen IV, suggesting that total flavonoids from jujube leaves may reduce HSC activation in liver tissue. Western blot analysis also revealed that total flavonoids from jujube leaves significantly reduced the expression of GPX4, SLC7A11, and NRF2 proteins, suggesting that total flavonoids from jujube leaves may exert anti-hepatic fibrosis effects by inhibiting the NRF2 / SLC7A11 / GPX4 pathway. This study improved liver fibrosis through the antioxidant-antifibrotic synergistic effect of total flavonoids from Chinese jujube leaves and the molecular mechanism of regulating the ferroptosis pathway, providing a theoretical basis for the in-depth development of Chinese jujube leaf resources and multi-target treatment strategies for liver fibrosis.

[0145] 3. Effects of total flavonoids from jujube leaves on TGF-β1-induced HSC cells

[0146] TGF-β1 is a key driving factor for HSC activation. Therefore, this study takes the TGF-β1-induced HSC-T6 cell fibrosis model as the core, combined with ferroptosis inhibitors (Ferrostatin-1, Fer-1) and NRF2, SLC7A11, and GPX4 specific inhibitors, to systematically explore the regulatory effects of total flavonoids from Chinese jujube leaves (denoted as TFZSF) on HSC activation and ferroptosis pathways. The purpose is to reveal the multi-target molecular mechanism of total flavonoids from Chinese jujube leaves in anti-liver fibrosis, provide new evidence for the regulation of ferroptosis pathways by total flavonoids from Chinese jujube leaves, and lay a theoretical foundation for the development of precise intervention strategies for liver fibrosis.

[0147] The cell line used in this study was rat hepatic stellate cell HSC-T6, which was provided by the Beina Biotechnology Cell Bank.

[0148] 3.1 Methods

[0149] 3.1.1 Preparation of total flavonoids working solution from jujube leaves

[0150] Weigh 10 mg of total flavonoids powder from the leaves of Solanaceae jujuba into a 2 mL centrifuge tube, add 1 mL of DMEM high-glucose medium to dissolve it, and prepare a working stock solution of total flavonoids from the leaves of Solanaceae jujuba with a concentration of 10 mg / mL.

[0151] 3.1.2. Culture of HSC-T6 cells

[0152] HSC-T6 cells were cultured in DMEM high glucose complete medium supplemented with 10% fetal bovine serum and 1% blue chain double antibody, 5% CO2 and 37℃ saturated humidity.

[0153] 3.1.3 Effect of TFZSF on HSC-T6 Cell Proliferation

[0154] A blank group and groups with different concentrations of TFZSF working solution were set up; HSC-T6 cells in the logarithmic growth phase were taken and 5×10 4 A cell suspension of 100 μg / mL was seeded into a 96-well plate and cultured for 24 h to allow attachment. Different concentrations of TFZSF working solution (0.1, 0.2, 0.5, 1, 5, 10, 20, 40, and 80 μg / mL) were added and cultured for 24 h. After that, 10 μL of CCK-8 reagent was added. Following the kit instructions, absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0155] 3.1.4 Effect of TFZSF on TGF-β1-induced HSC-T6 cell viability

[0156] A blank group, a TGF-β1 group, and groups with different concentrations of TFZSF working solution were set up. HSC-T6 cells in the logarithmic growth phase were taken, and 5×10 4 Cells were seeded in 96-well plates with 100 μg / mL of TGF-β1 and cultured for 24 h. Then, TFZSF working solution with concentrations of 0.1, 0.2, 0.5, 1, 2, 5, and 10 μg / mL was added and cultured for 24 h. Then, 10 μL of CCK-8 reagent was added to each well. The absorbance at 450 nm was measured using a microplate reader according to the instructions of the kit to calculate the cell viability.

[0157] 3.1.5. Determination of MDA, GSH, and Iron Contents in HSC-T6 Cells Induced by TGF-β1 by TFZSF

[0158] Set up blank group, TGF-β1 group and TFZSF working solution group; HSC-T6 cells in logarithmic growth phase were taken, 1×10 3 Cells were seeded in 6-well plates with 10 μg / mL of TGF-β1 and incubated for 24 hours. TGF-β1 was then added at a concentration of 10 ng / mL for another 24 hours. TFZSF working solution was then added at concentrations of 0.2 and 0.5 μg / mL. Activated HSC-T6 cells were harvested 48 hours after treatment, and absorbance was measured according to the manufacturer's instructions to calculate MDA, GSH, and iron levels.

[0159] 3.1.6 ROS detection

[0160] Set up blank group, TGF-β1 group and TFZSF working solution group; HSC-T6 cells in logarithmic growth phase were taken, 1×10 3 Cells were seeded in 6-well plates at a concentration of 100 μg / mL. After incubation for 24 hours, 10 ng / mL TGF-β1 was added for another 24 hours. TFZSF working solution was then added at concentrations of 0.2 and 0.5 μg / mL. The reactive oxygen species probe (DCFH-DA) was diluted 1:1000 in PBS to a final concentration of 10 μmol / L. After removing the culture medium from HSC cells, 300 μL of the diluted DCFH-DA was added and incubated in an incubator for 30 minutes. The cells were washed three times with PBS to remove any remaining DCFH-DA and then imaged under an inverted fluorescence microscope.

[0161] 3.1.7 Western Blot Analysis of α-SMA, NRF2, SLC7A11, and GPX4 Protein Expression in TGF-β1-Induced HSC-T6 Cells

[0162] Set up blank group, TGF-β1 group and TFZSF working solution group; HSC-T6 cells in logarithmic growth phase were taken, 1×10 3 Cells were inoculated with 10 μg / mL of TGF-β1 in 6-well plates and incubated for 24 h. TGF-β1 at a concentration of 10 ng / mL was added and incubated for 24 h. TFZSF working solution at concentrations of 0.2 and 0.5 μg / mL was then added. After culturing for 24 h, the supernatant from the 6-well plates was aspirated. Cell protein extraction, protein quantification, and detection were performed using the same methods as for Western blot detection of key proteins in 2.2.6. The antibody dilution ratios are shown in Table 6.

[0163] 3.1.8 Statistical methods

[0164] The results were analyzed and statistically analyzed using IBM SPSS Statistics 20 and GraphPad Prism 8. Values are expressed as mean ± standard deviation (mean ± SD). One-way analysis of variance was used for comparisons between multiple groups, and Student's ...

[0165] 3.2 Results Analysis

[0166] 3.2.1 Effect of TFZSF on TGF-β1-induced HSC-T6 cell viability

[0167] Figure 17 The CCK-8 results showed that compared with the blank group, TFZSF concentrations in the range of 0.1-20 μg / mL inhibited the viability of HSC-T6 cells. HSC-T6 cells were cultured with 10 ng / mL TGF-β1 and different concentrations of TFZSF. Figure 18 The results showed that compared with the blank group, the cell viability of the TGF-β1 group increased significantly (P<0.05), while TFZSF (0.1-5μg / mL) could inhibit the viability of HSC-T6 cells treated with TGF-β1 (P<0.05). The results showed that total flavonoids from the leaves of Ziziphus jujuba could reduce the proliferation of HSC-T6 cells induced by TGF-β1 and inhibit the viability of HSC-T6 cells. In normal hepatocytes LO-2, LO-2 was cultured with different concentrations of TFZSF, see Figure 19 The CCK-8 results showed that compared with the blank group, the total flavonoids of Ziziphus jujuba leaves had no toxicity to LO-2 cells in the range of 0.1-0.5 μg / mL. Therefore, 0.2 and 0.5 μg / mL were selected as the concentrations for subsequent studies.

[0168] 3.2.2 TFZSF inhibits TGF-β1-induced HSC-T6 cell activation

[0169] α-SMA is an effective indicator of HSC cell activation. When HSC is activated, the expression of α-SMA increases and ECM is synthesized. Figure 8 Western Blot results showed that compared with the blank group, the protein level of α-SMA in HSC-T6 cells treated with TGF-β1 was significantly increased (P<0.05); compared with the model group, total flavonoids from jujube leaves significantly reduced the protein level of α-SMA in HSC-T6 cells treated with TGF-β1 (P<0.05) ( Figure 20 and Figure 21 ).

[0170] 3.2.3 TFZSF promotes TGF-β1-induced ferroptosis in HSC-T6 cells

[0171] The levels of MDA, GSH and iron in cells were detected. Figure 22 The results showed that compared with the blank group, under the stimulation of TGF-β1, MDA and iron levels were significantly increased (P<0.05), and GSH content was significantly decreased (P<0.05); compared with the model group, TFZSF incubation significantly increased MDA ( Figure 22 A) and iron ( Figure 22B) level (P<0.05), significantly reduced GSH ( Figure 22 C) content (P<0.05).

[0172] The ROS in cells were detected. Figure 23 and Figure 24 As shown in the results, compared with the blank group, the ROS level was significantly increased under the stimulation of TGF-β1 (P<0.05); compared with the model group, TFZSF incubation could significantly increase the ROS level in HSC-T6 cells (P<0.05).

[0173] 3.2.4. Verification of the Effect of TFZSF on Ferropoptosis in Activated HSC-T6 Cells Using Fer-1

[0174] Figure 25 The results showed that compared with the blank group, under the stimulation of TGF-β1, MDA and iron levels were significantly increased (P<0.05), and GSH content was significantly decreased (P<0.05); compared with the model group, TFZSF incubation significantly increased MDA ( Figure 25 A) and iron ( Figure 25 B) level (P<0.05), significantly reduced GSH ( Figure 25 C) levels (P<0.05), while Fer-1 administration reversed the changes in the above indicators.

[0175] The ROS in cells were detected. Figure 26 and Figure 27 As shown in the results, compared with the blank group, the ROS level was significantly increased under the stimulation of TGF-β1 (P<0.05); compared with the model group, TFZSF incubation could significantly increase the ROS level in HSC-T6 cells (P<0.05), while the ROS level was significantly decreased after Fer-1 administration (P<0.05), further verifying that TFZSF can induce iron overload and oxidative stress in activated HSCs, thereby promoting ferroptosis and improving liver fibrosis.

[0176] 3.2.5 Effect of TFZSF on α-SMA Protein in the Ferroptosis Pathway of HSC-T6 Cells Induced by TGF-β1

[0177] Figure 28 and Figure 29 Western blot results showed that compared with the blank group, the protein level of α-SMA in HSC-T6 cells treated with TGF-β1 was significantly increased (P<0.05); compared with the model group, TFZSF could significantly reduce the protein level of α-SMA in HSC-T6 cells treated with TGF-β1 (P<0.05), while the protein level of α-SMA was significantly increased after Fer-1 administration (P<0.05).

[0178] 3.2.6 Effect of TFZSF on the NRF2 / SLC7A11 / GPX4 pathway in HSC-T6 cells induced by TGF-β1

[0179] The NRF2 / SLC7A11 / GPX4 pathway is an antioxidant system that targets lipid peroxide accumulation. Inhibition of the NRF2 / SLC7A11 / GPX4 pathway can lead to cell membrane peroxidation damage and ferroptosis. Western blot results showed that compared with the blank group, the protein levels of NRF2, SLC7A11, and GPX4 in TGF-β1-treated HSC-T6 cells were significantly increased (P<0.05); compared with the model group, TFZSF significantly reduced the protein levels of NRF2, SLC7A11, and GPX4 in TGF-β1-treated HSC-T6 cells (P<0.05) ( Figure 30 and Figure 31 These results suggest that TFZSF may inhibit the NRF2 / SLC7A11 / GPX4 pathway, promote HSC ferroptosis, and exert its anti-LF effect.

[0180] 3.2.7 Effects of NRF2 inhibitors and TFZSF on TGF-β1-induced activation and ferroptosis of HSC-T6 cells

[0181] Figures 32 and 33 Western blot results showed that compared with the blank group, the protein level of α-SMA in HSC-T6 cells treated with TGF-β1 was significantly increased (P<0.05); compared with the model group, incubation with ML385 and TFZSF significantly reduced the protein level of α-SMA in HSC-T6 cells treated with TGF-β1 (P<0.05).

[0182] Figure 34 and Figure 35 Western blot results showed that compared with the blank group, the protein levels of NRF2, SLC7A11, and GPX4 in TGF-β1-treated HSC-T6 cells were significantly increased (P<0.05). Compared with the model group, incubation with ML385 and TFZSF significantly reduced the protein levels of NRF2, SLC7A11, and GPX4 in TGF-β1-treated HSC-T6 cells (P<0.05). This indicates that TFZSF can inhibit the expression of NRF2 protein and the expression of NRF2 downstream target proteins SLC7A11 and GPX4, promote HSC ferroptosis, and exert an anti-LF effect.

[0183] 3.2.8 Effects of SLC7A11 Inhibitor and TFZSF on TGF-β1-induced Activation and Ferroptosis of HSC-T6 Cells

[0184] Figure 36 and Figure 37 Western blot results showed that compared with the blank group, the protein level of α-SMA in HSC-T6 cells treated with TGF-β1 was significantly increased (P<0.05); compared with the model group, incubation with Erastin and TFZSF significantly reduced the protein level of α-SMA in HSC-T6 cells treated with TGF-β1 (P<0.05).

[0185] Figure 38 and Figure 39 Western blot results showed that compared with the blank group, the protein levels of SLC7A11 and GPX4 in TGF-β1-treated HSC-T6 cells were significantly increased (P<0.05). Compared with the model group, incubation with Erastin and TFZSF significantly reduced the protein levels of SLC7A11 and GPX4 in TGF-β1-treated HSC-T6 cells (P<0.05). This suggests that TFZSF can inhibit the expression of SLC7A11 protein and the expression of SLC7A11 downstream target protein GPX4, promote HSC ferroptosis, and exert an anti-LF effect.

[0186] 3.2.9 Effects of GPX4 inhibitors and TFZSF on TGF-β1-induced activation and ferroptosis of HSC-T6 cells

[0187] Figure 40 and Figure 41 Western blot results showed that compared with the blank group, the protein level of α-SMA in HSC-T6 cells treated with TGF-β1 was significantly increased (P<0.05); compared with the model group, incubation with RSL3 and TFZSF significantly reduced the protein level of α-SMA in HSC-T6 cells treated with TGF-β1 (P<0.05).

[0188] Figure 42 and Figure 43 Western blot results showed that compared with the blank group, the protein level of GPX4 in HSC-T6 cells treated with TGF-β1 was significantly increased (P<0.05). Compared with the model group, incubation with RSL3 and TFZSF significantly reduced the protein level of GPX4 in HSC-T6 cells treated with TGF-β1 (P<0.05). This suggests that TFZSF and RSL3 have the same effect, inhibiting GPX4 expression, promoting HSC ferroptosis, and exerting an anti-LF effect.

[0189] The results of this study indicate that treatment with total flavonoids from jujube leaves significantly reduced α-SMA protein expression in HSC cells, significantly increased ROS levels in HSC-T6 cells, significantly increased MDA and iron levels, and significantly reduced GSH content. This suggests that TFZSF can inhibit the proliferation of activated HSCs, induce iron overload and oxidative stress in activated HSCs, and thereby promote ferroptosis and improve liver fibrosis. Total flavonoids from jujube leaves significantly reduced the expression of NRF2, SLC7A11, and GPX4 proteins, promoting ferroptosis through the NRF2 / SLC7A11 / GPX4 pathway and exerting an anti-hepatic fibrosis effect. Incubation with ML385 and total flavonoids from jujube leaves can significantly reduce the protein levels of NRF2, SLC7A11 and GPX4 in HSC-T6 cells treated with TGF-β1, and incubation with erastin and total flavonoids from jujube leaves can significantly reduce the protein levels of SLC7A11 and GPX4 in HSC-T6 cells treated with TGF-β1; incubation with RSL3 and total flavonoids from jujube leaves can significantly reduce the protein level of GPX4 in HSC-T6 cells treated with TGF-β1; it can be seen that total flavonoids from jujube leaves can inhibit the expression of NRF2, SLC7A11 and GPX4 proteins, respectively, inhibit the NRF2 / SLC7A11 / GPX4 signaling pathway to promote ferroptosis of activated HSC cells, and exert anti-liver fibrosis effects, providing a new approach for the treatment of liver fibrosis.

[0190] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of total flavonoids from wild jujube leaves in a drug for treating / preventing liver fibrosis.

2. A method for treating / preventing liver fibrosis by reducing serum ALT, AST and ALP levels and reducing serum Col-Ⅳ, HA, LN and PCⅢ contents.

3. A method for treating / preventing liver fibrosis by using total flavonoids from Chinese jujube leaves to significantly improve abnormal organ indexes, reduce collagen fiber deposition and liver cell structure damage, and delay the progression of fibrosis.

4. A method for treating / preventing liver fibrosis by downregulating the expression of fibrosis marker proteins α-SMA, Collagen I and Collagen IV and reducing the expression of NRF2, SLC7A11 and GPX4 proteins.

5. A method for treating / preventing liver fibrosis by significantly increasing ROS levels, increasing MDA and decreasing GSH content in HSC-T6 cells.

6. Application of total flavonoids from Chinese jujube leaves in the treatment / prevention of liver fibrosis by promoting ferroptosis of activated HSC cells by inhibiting the NRF2 / SLC7A11 / GPX4 signaling pathway.

7. The use according to any one of claims 1 to 6, characterized in that The chemical components in the total flavonoids of the jujube leaves include quercetin-3-O-acaciaside, epigallocatechin, eriocitrin, quercetin-3-(2R)-rutinoside, rutin, quercetin-3-O-rutinoside, apocynin, quercetin-3-O-β-D-glucopyranoside, kaempferol-3-rutinoside, kaempferol-3-O-rutinoside, dendroicin, quercetin-3-o-β-L-arabinosyl-(1-2)-α-L-rhamnose and quercetin-3-O-β-D-xylosyl-(1-2)-AL-rhamnose.

8. The use according to claim 7, characterized in that Among the total flavonoids of the sour jujube leaves, the mass proportion of quercetin-3-O-sophoroside is 2.84%, the mass proportion of rutin is 2.67%, and the mass proportion of calcitrinin is 0.89%.

9. The use according to any one of claims 1 to 6, characterized in that: The total flavonoids from the wild jujube leaves are extracted from the wild jujube leaves by adopting an ultrasonic method.

Images