Application of fructus aurantii flavonoid compound

Abstract

The invention discloses application of a fructus aurantii flavonoid compound, and particularly relates to application of the fructus aurantii flavonoid compound or physiologically acceptable salt of the fructus aurantii flavonoid compound in preparation of drugs for resisting inflammation and / or treating lactic dehydrogenase release related diseases, the structural formula of the fructus aurantii flavonoid compound is shown in the formula I, and the structural formula of the fructus aurantii flavonoid compound is shown in the description. R1 is selected from methoxy, hydrogen, ethyoxyl or hydroxyl; r2 is selected from methoxy, hydrogen, ethyoxyl or hydroxyl; r3 is selected from methoxy, hydrogen, ethyoxyl or hydroxyl; r4 is selected from methoxy, hydrogen, ethyoxyl or hydroxyl; r5 is selected from methoxy, hydrogen, ethyoxyl or hydroxyl; r6 is selected from methoxy, hydrogen, ethyoxyl or hydroxyl; and the C2-C3 bond is a double bond or a saturated bond. It is clear that in the fructus aurantii-derived flavonoid compound, isoaeschynosin, apigenin, nobiletin, hesperetin and isoaurantiol can significantly inhibit LDH release in LPS-induced HL-1 cells, and can be used as a lead compound of a sepsis myocardial injury resisting drug. # imgabs0 #

Description

Technical Field

[0001] The invention relates to an application of a fructus aurantii flavonoid compound and belongs to the field of medicine. Background Art

[0002] Sepsis cardiomyopathy (SCM) is one of the common complications caused by sepsis, which leads to reversible left ventricular systolic dysfunction, resulting in decreased cardiac output, shock, left ventricular dilatation with or without right heart failure, and increases the mortality rate of sepsis patients to 70% to 90%, which is one of the main causes of death in sepsis patients. In recent years, under the guidance of traditional Chinese medicine theory and the continuous updating of modern drug research methods, traditional Chinese medicine has achieved remarkable results in the treatment of sepsis and its related complications through the mechanisms of clearing away heat and detoxification, strengthening the body and consolidating the body, and attacking the interior.

[0003] Citrus belongs to the subfamily Citrus of the Rutaceae family. Common Chinese medicinal materials in the genus, such as dried tangerine peel, immature orange, fructus aurantii, and red orange, are commonly used in clinical Chinese medicines for regulating qi. According to the theory of Chinese medicine dialectics, the dried immature fruit of the Rutaceae plant Citrus aurantium L. and its cultivated varieties, Fructus aurantium, can be used as a key drug for resolving phlegm and regulating qi to treat myocardial injury caused by sepsis. Domestic and foreign studies have shown that the main components of Fructus aurantium are chemical components such as flavonoids, alkaloids, coumarins, and volatile oils. Flavonoids are its main components. Previous studies have confirmed that flavonoids have pharmacological activities such as anti-tumor, anti-inflammatory, and antioxidant effects. However, it is still unclear which Chinese medicine monomer among the main therapeutic components of Fructus aurantium, flavonoids, has the main effect. Patients with septic cardiomyopathy often have other organ dysfunctions in addition to myocardial injury on the basis of sepsis. In order to reduce the secondary damage to target organs caused by drugs with ineffective ingredients or weak effective ingredients, the commonly used Chinese medicines in treatment were separated and screened, and the key flavonoid monomers in Citrus aurantium that have therapeutic effects on septic myocardial injury were found. This provides scientific evidence for the prevention and treatment of septic myocardial injury by Citrus aurantium and its main flavonoid components, which has great clinical and translational significance. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a use of a Citrus aurantium flavonoid compound or a physiologically acceptable salt thereof in the preparation of a drug for anti-inflammatory and / or treatment of diseases associated with lactate dehydrogenase release.

[0005] Technical solution: The present invention provides an application of a Citrus aurantium flavonoid compound or a physiologically acceptable salt thereof in the preparation of a drug for anti-inflammatory and / or treatment of diseases related to lactate dehydrogenase release, wherein the Citrus aurantium flavonoid compound has a structural formula as shown in Formula I:

[0006]

[0007] Among them, R1 is selected from methoxy, hydrogen, ethoxy or hydroxyl; R2 is selected from methoxy, hydrogen, ethoxy or hydroxyl; R3 is selected from methoxy, hydrogen, ethoxy or hydroxyl; R4 is selected from methoxy, hydrogen, ethoxy or hydroxyl; R5 is selected from methoxy, hydrogen, ethoxy or hydroxyl; R6 is selected from methoxy, hydrogen, ethoxy or hydroxyl; the C2-C3 bond is a double bond or a saturated bond.

[0008] Furthermore, the Citrus aurantium flavonoid compound is selected from isosarcinolide, apigenin, nobiletin, hesperidin or isoauricularia.

[0009] Furthermore, the diseases associated with lactate dehydrogenase release include sepsis and myocardial injury.

[0010] Furthermore, the medicine also includes other pharmaceutically acceptable excipients.

[0011] Furthermore, the concentration of the Fructus Aurantii Immaturus flavonoids or the physiologically acceptable salts thereof is 10-50 μM / L.

[0012] Furthermore, the concentration of the Fructus Aurantii Immaturus flavonoids or the physiologically acceptable salts thereof is 10-25 μM / L.

[0013] Furthermore, the fructus aurantii flavonoids are obtained by the following extraction method:

[0014] (1) crushing the fructus aurantii, extracting it with an organic solvent, and recovering the solvent to obtain an extract;

[0015] (2) the extract was separated by silica gel column chromatography to obtain four components, and the mobile phase used was dichloromethane-methanol 100:1-:1;

[0016] (3) Components 1 to 3 are separated and purified by silica gel column chromatography, gel column chromatography or recrystallized to obtain the Citrus aurantium flavonoids;

[0017] The mobile phase used for silica gel column chromatography separation was petroleum ether-ethyl acetate 10:1-1:5.

[0018] Furthermore, in step (3), component 1 obtained in step (2) is separated by silica gel column chromatography to obtain three subcomponents, and the second subcomponent is purified by gel column chromatography to obtain isochlorothiazolidine, and the mobile phase used for purification is dichloromethane-methanol.

[0019] Furthermore, in step (3), component 2 obtained in step (2) is separated by silica gel column chromatography to obtain 6 subcomponents, and the second subcomponent is dissolved in petroleum ether-ethyl acetate and recrystallized to obtain apigenin.

[0020] Furthermore, the fourth subcomponent was separated again by silica gel column chromatography to obtain three subcomponents, and the second subcomponent was purified by gel column chromatography to obtain nobiletin.

[0021] Furthermore, in step (3), component 3 obtained in step (2) is separated and purified by silica gel column chromatography, and the component obtained by elution with the mobile phase of petroleum ether-ethyl acetate 3:1 is analyzed to obtain tangeretin.

[0022] Furthermore, in step (3), component 3 obtained in step (2) is separated and purified by silica gel column chromatography, and the component obtained by elution with the mobile phase of petroleum ether-ethyl acetate 2:1 is analyzed to obtain isoauricularia flavonoids.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention clarifies that among the flavonoid compounds derived from Fructus Aurantii Immaturus, isosarcinolide, apigenin, nobiletin, tangeretin and isoauricularia flavonoids can significantly inhibit the release of LDH in HL-1 cells induced by LPS, indicating that the above compounds play a protective effect on septic myocardial cell damage and can be used as lead compounds for drugs against septic myocardial damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the results of different concentrations of Fructus Aurantii Immaturus flavonoids inhibiting the release of lactate dehydrogenase. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0026] Example 1 Preparation of 95% ethanol extract of Fructus Aurantii Immaturus

[0027] Take 1.5 kg of dried raw material of Citrus aurantium (purchased from the herbal medicine room of the Affiliated Hospital of Yangzhou University), cut into pieces, and crush into 5-20 mesh. Use 25L of 95% ethanol to reflux extract for 1.5 hours, repeat 3 times, combine the extracts and concentrate under reduced pressure until there is no ethanol smell, thus obtaining 167 g of 95% ethanol extract.

[0028] Example 2 Preparation of Fructus Aurantii Immaturus Flavonoids Monomer Compounds (1)

[0030] The 95% ethanol extract obtained in Example 1 was mixed with silica gel (100-200 mesh) (the ratio of sample to silica gel was 1:1.5) and then subjected to silica gel column chromatography; dichloromethane-methanol (100:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 0:1) was used for elution, and three retention volumes (each volume was 2 L) were used for each gradient elution. The obtained fractions were concentrated under reduced pressure and combined after TLC detection to obtain 4 components, namely Fr.1 (100:1-30:1), Fr.2 (20:1-10:1), Fr.3 (8:1-5:1), and Fr.4 (3:1-0:1). (2)

[0032] (2.1) Preparation of isochara (Compound 1)

[0033] The Fr.1 component was separated by silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase according to the gradient elution of (10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5) to obtain three subcomponents, namely Fr.1.1 (10:1-5:1), Fr.1.2 (3:1-2:1), and Fr.1.3 (1:1-1:5). Fr.1.2 was repeatedly purified by Sephadex LH-20 gel column chromatography with dichloromethane-methanol (1:1) as the mobile phase to obtain 2.3 mg of iso-sakurain (compound 1).

[0034] The structure of the compound was confirmed by NMR: 1 H-NMR (CDCl3, 400MHz): δ: 7.43 (2H, d, J = 8.7Hz, H-2′, H-6′), 6.98 (2H, d, J = 8.8Hz, H-3′, H-5′), 5.91 (1H, d, J = 2.1Hz, H-6), 5.91 (1H, d, J = 2. 1Hz,H-8),5.41(1H,dd,J=3.0,13.1Hz,H-2),3.13(1H,dd,J=13.1,17.2Hz,H-3α),2.74(1H,dd,J=3.0,17.0Hz,H-3β),3.83(3H,s,4′-OCH3); 13 C-NMR (100MHz, CDCl3) δ: 197.1 (C-4), 168.3 (C-5), 165.5 (C-9), 164.8 (C-7), 161.4 (C-4′), 132.4 (C-1′), 128. 9(C-2′,6′),115.0(C-3′,-5′),103.3(C-10),97.0(C-6),96.1(C-8),80.2(C-2),55.7(4′-OCH3),44.0(C-3).

[0035] (2.2) Preparation of apigenin (Compound 2)

[0036] The Fr.2 component obtained in (1) was separated by silica gel column chromatography using petroleum ether-ethyl acetate as the mobile phase according to the gradient elution of (10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5) to obtain 6 subcomponents, namely Fr.2.1 (10:1), Fr.2.2 (5:1), Fr.2.3 (3:1), Fr.2.4 (2:1), Fr.2.5 (1:1), and Fr.2.6 (1:3-1:5). Fr.2.2 was dissolved in petroleum ether:ethyl acetate at a ratio of 1:3, and 3.2 mg of apigenin (compound 2) was obtained by recrystallization.

[0037] The structure of the compound was confirmed by NMR: 1 H-NMR (CDCl3, 400MHz): δ: 12.96 (1H, s, 5-OH), 7.91 (2H, d, J = 8.6 Hz, H-2', 6'), 6.92 (2H, d, J =8.6Hz,H-3',5'),6.77(1H,s,H-3),6.47(1H,d,J=2.0Hz,H-8),6.19(1H,d,J=2.0Hz,H-6); 13 C-NMR (100MHz, CDCl3) δ: 181.7 (C-4), 164.1 (C-2), 163.7 (C-7), 161.4 (C-4''), 161.1 (C-9), 157.3 (C-5 ),128.4(C-2',6'),121.1(C-1'),115.9(C-3',5'),103.7(C-10),102.8(C-3),98.8(C-6),93.9(C-8).

[0038] (2.3) Preparation of Nobiletin (Compound 3)

[0039] The subcomponent Fr.2.4 obtained in (2.2) was again purified by silica gel column chromatography using petroleum ether-ethyl acetate as the mobile phase according to the gradient elution ratio of (10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5) to further obtain three subcomponents, namely Fr.2.4.1 (10:1-5:1), Fr.2.4.2 (3:1-2:1), and Fr.2.4.3 (1:1-1:5). Among them, Fr.2.4.2 was purified repeatedly by Sephadex LH-20 gel column chromatography using methanol as the mobile phase to obtain 1.9 mg of citrus nobilis (compound 3).

[0040] The structure of the compound was confirmed by NMR: 1H-NMR (CDCl3, 400MHz): δ: 7.68 (1H, dd, J = 2.0, 8.5Hz, H-6′), 7.56 (1H, d, J = 2.0Hz, H-2′), 7.15 (1H, d, J = 8.9Hz, H-5′), 6.72 (1H, s, H-3 ),4.12(3H,s,5-OCH3),4.09(3H,s,6-OCH3),4.05(3H,s,7-OCH3),3.95(3H,s,8-OCH3),3.91(3H,s,3′-OCH3),3.90(3H,s,4′-OCH3); 13 C-NMR (100MHz, CDCl3) δ: 163.6 (C-2), 107.4 (C-3), 179.6 (C-4), 144.6 (C-5), 132 .1(C-6),151.8(C-7),145.5(C-8),145.5(C-9),112.8(C-10),124.8(C-1′),109 .6(C-2′),150.9(C-3′),153.9(C-4′),110.3(C-5′),121.2(C-6′),56.0(3′-OCH 3), 56.0(4′-OCH3), 62.5(5-OCH3), 62.0(6-OCH3), 61.9(7-OCH3), 61.7(8-OCH3).

[0041] (2.4) Preparation of tangeretin (Compound 4)

[0042] The Fr.3 component obtained in (1) was eluted by silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase at a gradient ratio of (10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5), and the component eluted with the mobile phase 3:1 analyzed the yellow powder compound, which was 1.3 mg of tangeretin (compound 4).

[0043] The structure of the compound was confirmed by NMR: 1 H-NMR (CDCl3, 400MHz): δ: 7.99-7.91 (2H, d, J = 8.4Hz, H-2′ / H-6′), 7.12-7.06 (2H, m, J = 8.4Hz, H-3′ / H-5′), 6.6 9(1H,s,H-3),4.12(3H,s,5-OCH3),4.04(3H,s,7-OCH3),3.94(3H,s,6-OCH3),3.91(6H,s,4′-OCH3 / 3′-OCH3); 13C-NMR(100MHz, CDCl3)δ:178.2(C-4),163.8(C-2),162.4(C-4′),151.9( C-9),147.9(C-5),147.8(C-7),145.6(C-6),138.3(C-8),129.1(C-2′ / C- 6′),124.5(C-1′),115.7(C-3′ / C-5′),114.0(C-10),106.7(C-3),62.6( 5-OCH3), 62.6(7-OCH3), 62.1(6-OCH3), 62.1(4′-OCH3), 56.0(3′-OCH3).

[0044] (2.5) Preparation of isoauricularia flavonoid (Compound 5)

[0045] The Fr.3 component obtained in (1) was eluted by silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase at a gradient ratio of (10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5), and the component eluted with the mobile phase 2:1 was analyzed to obtain a powdery compound, namely 1.5 mg of isocinerin (compound 5).

[0046] The structure of the compound was confirmed by NMR: 1 H-NMR (CDCl3, 400MHz): δ: 7.67 (1H, dd, J = 8.6, 2.1Hz, H-6′), 7.56 (1H, d, J = 2.1Hz, H-2′), 7.14 (1H, d, J = 8.6Hz, H-5′), 6.70 (1H, s, H -3),6.67(1H,s,H-6),4.05(3H,s,7-OCH3),3.96(3H,s,4′-OCH3),3.96(3H,s,3′-OCH3),3.96(3H,s,8-OCH3),3.96(3H,s,5-OCH3); 13 C-NMR(100MHz, CDCl3)δ:160.2(C-2),105.6(C-3),175.6(C-4),151.7(C-5) ,97.2(C-6),157.3(C-7),140.4(C-8),153.8(C-9),111.6(C-10),123.0(C-1 ′),109.1(C-2′),148.9(C-3′),151.5(C-4′),111.8(C-5′),119.6(C-6′),55 .6(5-OCH3), 61.9(7-OCH3), 55.8(8-OCH3), 60.1(3′-OCH3), 56.5(4′-OCH3).

[0047] The general substitution of the flavonoid compounds prepared in this example is shown in Table 1.

[0048] Table 1 Substitution of the general formula of flavonoid compounds

[0049]

[0050] The specific structural formula is as follows:

[0051]

[0052] Example 3 Study on the Inhibitory Effect of Fructus Aurantii Immaturus Flavonoids on LPS-Induced LDH Release in HL-1 Cells The information of drugs and reagents used in this example is shown in Table 2, and the information of experimental instruments is shown in Table 3.

[0053] Table 2 Drugs and reagents

[0054]

[0055] Table 3 Experimental instruments

[0056]

[0057] Experimental methods:

[0058] 1. Cultivation of HL-1 Cell Line

[0059] (1) Cell recovery

[0060] HL-1 cells were taken out of liquid nitrogen, placed in a 37°C constant temperature water bath, and bathed until completely dissolved. The dissolved cells were transferred to DMEM high-glucose medium and centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was removed, and the cells were resuspended in complete medium (DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% antibiotics), and the cell suspension was inoculated into a culture bottle, mixed and placed in an incubator at 37°C and 5% CO2 saturated humidity.

[0061] (2) Cell passaging and culture

[0062] Observe the cells. When the cell density is below 50%, replace with complete medium, discard the medium, wash with PBS, and replace with new complete medium. When the cell density is above 80%, perform cell subculture. Discard the medium, wash with PBS. Add trypsin to digest the cells for 3-4 minutes. Observe the detachment of adherent cells and add an equal ratio of complete medium to terminate digestion. Transfer to a centrifuge tube and centrifuge at 1000 rpm and 4°C for 5 minutes. Remove the supernatant and add complete medium to make a cell suspension. Subculture at a ratio of 1:2. Mix well and culture in an incubator at 37°C and 5% CO2 saturated humidity.

[0063] 2. Cell Grouping and Processing

[0064] (1) Preparation of Fructus Aurantii Immaturus Flavonoids Monomer Solution

[0065] 5 mg of traditional Chinese medicine monomers (the five kinds of Fructus Aurantii Immaturus flavonoid monomers prepared in Example 2) were dissolved in DMSO solution to a final concentration of 200 mM, and stored at -20°C in the dark after subpackaging.

[0066] (2) Grouping of different doses of Citrus aurantium flavonoid monomer

[0067] NC group: HL-1 cells + 200 μl DMEM;

[0068] LPS group: HL-1 cells + 200 μl DMEM + dimethyl sulfoxide (DMSO) were incubated for 1 h and then LPS (20 μg / ml) was added;

[0069] Fructus Aurantii Immaturus flavonoids monomer group: HL-1 cells + 200 μl DMEM + Fructus Aurantii Immaturus flavonoids monomers (concentrations were 10 μM / L, 25 μM / L and 50 μM / L, respectively), incubated for 1 h, and then LPS (20 μg / ml) was added.

[0070] 3.LDH detection

[0071] The LDH cytotoxicity assay kit was used to detect the cell activity of each group. Specifically, after the grouping treatment according to the above scheme, the treated HL-1 cells were cultured for 24 hours. LDH release reagent was added to one well of the NC group 1 hour in advance (23 hours). After 1 hour, 120 μl of the supernatant from each well was aspirated to a transparent 96-well plate, and the LDH reaction solution was prepared using the LDH cytotoxicity assay kit with 1x enzyme solution: 1xint reagent: 1x lactic acid = 1:1:1. 60 μl of LDH reaction solution was added to each well of the transparent 96-well plate, and then placed in a 37°C cell culture incubator for incubation for 15 minutes. Finally, the absorbance value was measured at a wavelength of 490 nm using an enzyme reader. The average absorbance value of the 6 wells in the NC group was taken, and the cell activity was calculated according to the following formula. Cell activity % = (absorbance value of each well - average absorbance value of the NC group) / (absorbance value of the release well - average absorbance value of the NC group) × 100%. Each experiment was repeated 3 times.

[0072] 4. Statistical analysis

[0073] Graphpad prism 8.0 software was used to analyze the statistical data, and the data were expressed as mean ± SD. One-way analysis of variance was used for comparison between groups, and repeated measurement analysis of variance was used for comparison within groups. P < 0.05 was considered statistically significant.

[0074] 5. Results

[0075] The LDH release rate of HL-1 cells induced by LPS was measured before and after intervention with different doses of isocherrin, tangeretin, apigenin, nobiletin, and isoauricularia in the extract of Citrus aurantium. It was found that these five flavonoids could significantly inhibit the LDH release rate, P < 0.05 (the results are shown in Figure 1 As shown in the figure, the effects of apigenin and isocerasin at different doses were not much different. The effect of tangeretin at a concentration of 10 μM / L was significantly better than that at a concentration of 50 μM / L. The best effects of nobiletin and isocerasin were at concentrations of 10 μM / L and 25 μM / L.

[0076] The flavonoids isolated from the Citrus aurantium extract have a significant inhibitory effect on LPS-induced LDH release in HL-1 cells, which may be the main material basis for the anti-inflammatory effect of Citrus aurantium and is expected to become a more targeted and highly effective lead compound for anti-septic myocardial injury drugs.

Claims

1. A use of a Citrus aurantium flavonoid compound or a physiologically acceptable salt thereof in the preparation of a drug for anti-inflammatory and / or treatment of diseases related to lactate dehydrogenase release, characterized in that: The structural formula of the Citrus aurantium flavonoid compound is shown in Formula I: Among them, R1 is selected from methoxy, hydrogen, ethoxy or hydroxyl; R2 is selected from methoxy, hydrogen, ethoxy or hydroxyl; R3 is selected from methoxy, hydrogen, ethoxy or hydroxyl; R4 is selected from methoxy, hydrogen, ethoxy or hydroxyl; R5 is selected from methoxy, hydrogen, ethoxy or hydroxyl; R6 is selected from methoxy, hydrogen, ethoxy or hydroxyl; the C2-C3 bond is a double bond or a saturated bond.

2. The use according to claim 1, characterized in that: The fructus aurantii flavonoids are selected from isosarcinolide, apigenin, nobiletin, hesperidin or isoauricularia flavonoids.

3. The use according to claim 1, characterized in that: The diseases associated with lactate dehydrogenase release include sepsis and myocardial injury.

4. The use according to claim 1, characterized in that: The medicine also includes other pharmaceutically acceptable excipients.

5. The use according to claim 1, characterized in that: The fructus aurantii flavonoids are obtained by the following extraction method: (1) crushing the fructus aurantii, extracting it with an organic solvent, and recovering the solvent to obtain an extract; (2) the extract was separated by silica gel column chromatography to obtain four components, and the mobile phase used was dichloromethane-methanol 100:1-:1; (3) Components 1 to 3 are separated and purified by silica gel column chromatography, gel column chromatography or recrystallized to obtain the Citrus aurantium flavonoids; The mobile phase used for silica gel column chromatography separation was petroleum ether-ethyl acetate 10:1-1:

5.

6. The use according to claim 5, characterized in that: In step (3), component 1 obtained in step (2) is separated by silica gel column chromatography to obtain three subcomponents, and the second subcomponent is purified by gel column chromatography to obtain iso-sakurabin, and the mobile phase used for purification is dichloromethane-methanol.

7. The use according to claim 5, characterized in that: In step (3), component 2 obtained in step (2) is separated by silica gel column chromatography to obtain 6 subcomponents, and the second subcomponent is dissolved in petroleum ether-ethyl acetate and recrystallized to obtain apigenin.

8. The use according to claim 7, characterized in that: The fourth subcomponent is separated again by silica gel column chromatography to further obtain three subcomponents, and the second subcomponent is purified by gel column chromatography to obtain nobiletin.

9. The use according to claim 5, characterized in that: In step (3), component 3 obtained in step (2) is separated and purified by silica gel column chromatography, and the component obtained by elution with the mobile phase of petroleum ether-ethyl acetate 3:1 is analyzed to obtain tangeretin.

10. The use according to claim 5, characterized in that: In step (3), component 3 obtained in step (2) is separated and purified by silica gel column chromatography, and the component obtained by elution with the mobile phase of petroleum ether-ethyl acetate 2:1 is analyzed to obtain isoauricularia flavonoids.

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