A new biflavonoid compound, its extraction method and use

Abstract

This invention discloses a novel biflavonoid compound, its extraction method, and its uses. The extraction method is as follows: extraction with an aqueous ethanol solution, concentration to obtain an extract, followed by extraction with ethyl acetate to obtain an extract. The extract is then subjected to one normal-phase column chromatography separation and one reverse-phase column chromatography separation to obtain a crude product. The crude product is further purified by gel column chromatography to obtain the novel biflavonoid compound. Pharmacological studies have shown that this compound has effective anti-inflammatory properties and is suitable for preparing new drugs for the prevention and treatment of inflammation, possessing broad application prospects and significant potential value.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the extraction of ephedra sinica from the legume plant Ephedra sinica. Shuteria involucrata A method for isolating a new biflavonoid compound and its application in the preparation of anti-inflammatory drugs. Background Technology

[0002] Ephedra sinica is a species of leguminous plant belonging to the genus Ephedra (Fabaceae). Shuteria Plants of the Chinese genus *Stromberaria* S. involucrata The dried root of *Vigna angularis*, also known as wild pea, is mainly distributed in the dry and hot river valleys of central and southern Yunnan at altitudes of 500-2300 m. In Dai medicine, it is called "Tuoyenuteng." Dai medical theory holds that this herb is bitter and cool in nature, and when added to the earthenware pot, it has the effects of clearing heat and detoxifying, dispelling wind and relieving pain. Clinically, it is used to treat "Bingwahuang, Aihuhuojie" (a type of cold caused by wind-heat, cough, and sore throat). Besides its use in Dai medicine, this herb has also been used medicinally by several other ethnic minorities. For example, in Yi medicine, it is called "Yesuonu," and Yi medicine believes it can clear heat and detoxify, mainly treating colds, bronchitis, tonsillitis, and acute pharyngitis; in Wa medicine, it is called "Tongqiangen," and Wa herbal medicine uses it in prescriptions to treat bronchitis.

[0003] The main active pharmaceutical components of *Ephedra sinica* are still unclear, and there are no literature reports on its effects on inflammation; further research is needed. In our laboratory's research on the flavonoid components of *Ephedra sinica*, we isolated and identified a new biflavonoid compound. Pharmacological studies have shown that this compound has significant potential for treating inflammation. Summary of the Invention

[0004] The purpose of this invention is to provide a medicinal plant, *Stachys chinensis*. S. involucrata The roots of the plant are used as raw materials to extract and separate biflavonoid compounds that have the activity of preventing or treating inflammation.

[0005] This invention is achieved through the following method:

[0006] A biflavonoid compound with anti-inflammatory effects, characterized by the following structural formula:

[0007] .

[0008] This biflavonoid compound was isolated from Ephedra sinica, and its preparation method includes the following steps:

[0009] (1) The medicinal material of Ephedra sinica was extracted by hot reflux using an aqueous ethanol solution, and the extract was concentrated to obtain an extract.

[0010] (2) After diluting the extract obtained in step (1) with water, extract it first with petroleum ether and then with ethyl acetate. Concentrate the solution after ethyl acetate extraction to obtain the extract extract.

[0011] (3) The extractive infusion obtained in step (2) is subjected to first column chromatography separation by normal phase silica gel column, the eluent is a mixed solvent of chloroform and acetone in different proportions, the eluate rich in the compound is collected, and a mixture containing the compound is obtained after concentration;

[0012] (4) The mixture obtained in step (3) is subjected to second column chromatography separation by reverse phase column, the eluent is a mixed solvent of methanol and water in different proportions, the eluate rich in the compound is collected, and a crude product containing the compound is obtained after concentration;

[0013] (5) The crude product obtained in step (4) is subjected to purification by gel column, the eluent is pure methanol, and the novel biflavonoid compound is obtained by collecting the eluate.

[0014] In step (1), the concentration of ethanol in the aqueous ethanol solution is 70-75%, and extraction is performed 2-3 times, each time for 2-3 hours; in step (3), the volume ratio of chloroform to acetone is 10:1-1:1; in step (4), the reverse phase column used is MCI, and the volume ratio of water to methanol in the mobile phase is 100:0-0:100; in step (5), the gel column used is Sephadex LH-20.

[0015] The biflavonoid compound is used for preparing an anti-inflammatory drug.

[0016] The inflammation is chronic bronchitis, pharyngolaryngitis, tonsillitis or mastitis.

[0017] The anti-inflammatory drug is a preparation containing the novel biflavonoid compound.

[0018] The preparation containing the novel biflavonoid compound is prepared from the novel biflavonoid compound alone or from the novel biflavonoid compound and a pharmaceutically acceptable carrier.

[0019] The preparation is a tablet, a capsule, a pill, an oral solution, a granule, a suspension, an injection, an aerosol, a sustained-release preparation or a sublingual tablet, and is administered orally, by spray inhalation, rectally, nasally, parenterally such as subcutaneously, intravenously or intramuscularly.

[0020] The pharmaceutically acceptable carrier refers to a conventional drug carrier in the pharmaceutical field, and is selected from one or more of a filler, a binder, a disintegrant, a solubilizer, a suspending agent, a wetting agent, a pigment, an essence, a solvent, a surfactant or a flavoring agent.

[0021] The clinical dosage of the biflavonoid compound is 100-800 mg / d.

[0022] The present application has the following advantages:

[0023] (1) The new use of the compound provided by the application, that is, for the prevention and treatment of inflammation, brings new drug options for patients with inflammation;

[0024] (2) The pharmacodynamic experiment results show that the compound can obviously improve the inflammation model caused by lipopolysaccharide (LPS) induction, and has good drug efficacy, indicating that the compound of the application can be used for preparing a therapeutic drug for preventing or treating diseases caused by inflammasome.

[0025] (3) The compound in the application is obtained by chiral resolution into two enantiomers, which have the same pharmacological effect as before resolution, and both have certain anti-inflammatory effect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a chemical structural formula of the compound a of the application;

[0027] Figure 2 is a preparation process of the compound a of the application;

[0028] Figure 3 is a high-resolution mass spectrum of the compound a of the application;

[0029] Figure 4 is an ultraviolet spectrum of the compound a of the application;

[0030] Figure 5 is an infrared spectrum of the compound a of the application;

[0031] Figure 6 is an ECD spectrum of the enantiomer of the compound a of the application after resolution; 1 H-NMR spectrum;

[0032] Figure 7 is a C-NMR spectrum of the compound a of the application; 13

[0033] Figure 8 is a H- 1 H COSY spectrum of the compound a of the application; 1

[0034] Figure 9 is a HSQC spectrum of the compound a of the application;

[0035] Figure 10 is a HMBC spectrum of the compound a of the application;

[0036] Figure 11 is a resolution ratio of the compound a of the application on chiral HPLC;

[0037] Figure 12 is an ECD spectrum of the enantiomer of the compound a of the application after resolution;​​

[0038] Figure 13 the major conformation of the Boltzmann weighted TDDFT ECD spectrum of compound a of the present invention;

[0039] Figure 14 the absolute configuration structure of compound a of the present invention;

[0040] Figure 15 the effect of compound a and its enantiomers on IL-6 level in LPS-induced macrophage RAW264.7-based inflammation model;

[0041] Figure 16 the effect of compound a and its enantiomers on TNF-α level in LPS-induced macrophage RAW264.7-based inflammation model. α DETAILED DESCRIPTION

[0042] The method of the present invention is further illustrated in detail by the following examples, but the scope of protection of the present invention is not limited to the content described, and the reagents used in the examples are conventional commercially available reagents, or reagents prepared according to conventional methods, unless otherwise specified.

[0043] Example 1: Preparation of compound a

[0044] The preparation flow chart of compound a is shown in Figure 2 .

[0045] Dry copper money ephedra root (5 kg) was crushed and extracted with 10 L of 70% ethanol by heating and refluxing for 3 times, 2 h each time. The extract was concentrated under reduced pressure until no alcohol smell was left, and ethanol extract (1.8 kg) was obtained. The extract was dispersed with 1 L of water, defatted with 1 L of petroleum ether for 1 time, and then extracted with ethyl acetate for 3 times, 2 L each time. The ethyl acetate extract was combined and recovered under reduced pressure to obtain ethyl acetate extract (0.9 kg).

[0046] The ethyl acetate extract was subjected to silica gel column chromatography (chloroform-acetone system) gradient elution (40:1-2:1), and each fraction was identified by thin layer chromatography. The fractions containing compound a (21 g) were combined and subjected to MCI column chromatography, gradient elution with methanol-water (30:70-1000), and each fraction was identified by thin layer chromatography. The fractions containing only compound a were combined to obtain crude compound a (1.1 g), which was purified by Sephadex LH-20 (methanol) to obtain pure compound a (981 mg).

[0047] Structure identification: the structure was identified by spectroscopy, including mass spectrometry, ultraviolet, infrared, nuclear magnetic resonance spectroscopy (H-NMR, C-NMR, 2D-NMR). 1 H-NMR, 13 C-NMR, 2D-NMR).​

[0048] Compound a is a white amorphous powder, as determined by high-resolution mass spectrometry (see [link to mass spectrometry]). Figure 3 The molecular weight is 677.2417.

[0049] Ultraviolet spectrum (see) Figure 4 The absorption peaks at 207, 276, 311 and 375 nm in the sample indicate that the compound is a flavonoid.

[0050] Infrared spectrum (see) Figure 5 The carbonyl absorption peak is shown at 1601 cm⁻¹. -1 ), aromatic ring resonance peaks (1166, 1252, 1461 cm⁻¹) -1 ), hydroxyl absorption peak (3390 cm⁻¹) -1 ).

[0051] 1 H-NMR spectrum ( Figure 6 ) showed delta H 7.68 (1H,d, J =8.8 Hz, H-5), 6.48 (1H,dd, J =8.7, 2.3 Hz, H-6), and 6.22 (1H, d, J =2.3 Hz, H-8') ABX spin system and delta H 6.62 (1H,d, J = 8.3 Hz, H-5') and 6.26 (1H, d, J AB spin system (=8.4 Hz). 1 H-NMR shows delta H 4.98 (1H,s, H-8') is attributed to the olefin proton, along with 1.61 (3H,s, H-10'), 1.44 (3H,s, H-11'), and 3.37 (2H,m, H-7') to the isoprene proton signal.

[0052] 13 C-NMR spectrum (see) Figure 7 The spectrum showed 20 carbon signals, including 2 methyl groups, 1 methylene group, 8 methyl groups, and 9 quaternary carbons. Preliminary inference suggests that the compound structure is a symmetrical biflavonoid with C-3' / C-3'' linkages. Detailed analysis of the key two-dimensional NMR spectrum of compound a further confirmed the planar structure of 1. 1 H- 1 H COSY spectrum (see) Figure 8 ) can be derived delta H7.68 (H-5 / 5") / 6.48 (H-6 / 6"), 6.62 (H-5' / 5"') / 6.26 (H-6' / 6"') in HMBC (see Fig. 2) and HMQC (see Fig. 3) spectra. Figure 10 Figure 9 delta C 143.9 (C-3' / C-3"), 146.4 (C-4' / C-4"), 165.2 (C-7 / C-7") in HMBC (see Fig. 2) and HMQC (see Fig. 3) spectra. delta C 81.4 (C-2 / C-2") and 146.4 (C-4' / C-4") in HMBC (see Fig. 2) and HMQC (see Fig. 3) spectra. C 194.6 (C-4 / C-4") and 165.2 (C-7 / C-7") in HMBC (see Fig. 2) and HMQC (see Fig. 3) spectra. delta C 143.9 (C-3' / C-3"), 146.4 (C-4' / C-4"), and 165.2 (C-7 / C-7") in HMBC (see Fig. 2) and HMQC (see Fig. 3) spectra. 1 H‒ 1 HCOSY (see Fig. 1) also showed the correlation of delta H 6.19 (H-2 / 2") / 2.84 (H-3 / 3") and 3.35 (H-7' / 7") / 4.98 (H-8' / 8") in HMBC (see Fig. 2) and HMQC (see Fig. 3) spectra. delta H 1.61 (H-10' / 10") and 1.44 (H-11' / 11") with delta C 124.5 (C-8' / 8") in HMBC (see Fig. 2) and HMQC (see Fig. 3) spectra. delta H 6.19 (H-2 / 2") and 3.35 (H-7' / 7") in HMBC (see Fig. 2) and HMQC (see Fig. 3) spectra. delta C 128.3 (C-2' / 2") in HMBC (see Fig. 2) and HMQC (see Fig. 3) spectra. delta C 51.9) in HMBC (see Fig. 2) and HMQC (see Fig. 3) spectra. The trans configuration of H-2 / H-3 was further confirmed by comparing the coupling constants of H-2 / H-3 (H-2" / H-3") and the data of Involucrasin B as a symmetric unit in the previous literature. ​​

[0053] The optical rotation of compound a is [α]20 D = 0 ( c 1.0, CH3OH) indicates that compound a may be an enantiomer. Furthermore, based on the weak cotton-like effect and weak optical activity exhibited by compound a in CD spectroscopy, its racemic nature was confirmed by chiral HPLC, and (+)-a and (−)-a were separated in a 3:2 ratio. Figure 11 By calculating the circular dichroism spectrum ( Figure 12 Their absolute configurations were further determined by methods comparing with literature. For example... Figure 13 As shown, the Boltzmann-weighted TDDFT ECD spectrum reveals seven dominant conformations (over 1%). (+)-a, i.e., (2R, 3S, 2"R, 3"S), exhibits a positive cotton-cotton effect at 218 nm and a negative cotton-cotton effect at 318 nm, while (-)-a shows the opposite. This result reasonably indicates that the absolute configuration of (+)-a is 2R, 3S, 2"R, 3"S, while the absolute configuration of (-)-a is 2S, 3R, 2"S, 3"R. Therefore, the absolute configuration of compound a is determined, as shown below. Figure 14 As shown.

[0054] The spectral data of compound a obtained in the above embodiments are as follows:

[0055] The molecular formula is C 40 H 38 O 10 Its molecular weight is 678.2465. 1 H NMR (400 MHz, CD3OD) delta H 7.68 (1H,d, J =8.8 Hz, C-5), 6.62 (1H,d, J =8.3 Hz, C-5'), 6.48 (1H, dd, J =8.7, 2.3 Hz, C-6), 6.26 (1H, d, J =8.4Hz,C-6'),6.22(1H,d, J =2.3Hz,C-6),6.19(1H,m,C-2),4.98(1H,s,C-8'),3.35(2H,m,C-7'), 2.84(1H,d, J =12.3 Hz,C-3), 1.61(3H,s,C-11'), 1.44 (3H,s,C-10'); 13 C NMR (100 MHz, CD3OD) delta C194.6 (C-8), 166.4 (C-9), 165.2 (C-7), 146.4 (C-4'), 143.9 (C-3'), 132.1 (C-9'), 130.9 (C-1'), 130.1 (C-5), 128.3 (C-2'),124.5 (C-8'), 120.5(C-6'), 115.5 (C-10), 114.1 (C-5'), 111.5 (C-6), 103.6 (C-8), 81.4 (C-2), 51.6 (C-3), 25.8 (C-10'), 25.4 (C-7'), 18.1 (C-11').

[0056] Example 2: Anti-inflammatory effect of compound a on LPS-induced RAW264.7 cells

[0057] 1. Reagents: Lipopolysaccharide (LPS) (0111:B4) and dexamethasone (batch number: BCBV3214) were purchased from Sigma-Aldrich; TNF-α α The detection kit (batch number: A221215-102a) and the IL-6 ELISA detection kit (batch number: M230404-005a) were purchased from Xinbosheng Biotechnology Co., Ltd.; DMEM medium; 10% fetal bovine serum (Spectramaxplus384); the complete medium ratio was 10% fetal bovine serum: 90% DMEM medium.

[0058] 2. Instruments: Multifunctional microplate reader (Spectra max plus 384, Beijie Technology); CO2 incubator (Panasonic, MCO-18AC); inverted microscope (Leica, LEICADMi1); centrifuge (Sichuan Shuke Instrument Co., Ltd., 20072804); -80℃ ultra-low temperature freezer (8920, Thermo Fisher Scientific); electronic analytical balance (AR224CN, Ohaus Instrument Co., Ltd.).

[0059] 3. Experimental methods:

[0060] [1] Cell resuscitation and culture: The cryovial containing 1.5 ml of RAW264.7 cell suspension was removed from liquid nitrogen and rapidly thawed in a 37°C water bath. After complete thawing, it was transferred to the operating table. The pre-prepared cell culture medium was added to the centrifuge tube, and the cell suspension in the cryovial was quickly transferred to the centrifuge tube using a pipette. The centrifuge tube was equilibrated, centrifuged at 1000 r / min for 3 min, the supernatant was discarded, and the cells were resuspended using complete culture medium. The cell suspension in the centrifuge tube was transferred to the culture flask using a pipette, and the centrifuge tube was rinsed 1-2 times with culture medium. The cells were then transferred to the culture flask and mixed by pipetting. The culture flask was placed horizontally and shaken from side to side to ensure that the concentration of the culture medium was uniform. The cell morphology was examined under an inverted microscope, and then the flask was placed in a 37°C, 5% CO2 incubator. On the second day, the culture medium was replaced and the cell morphology was observed to be good. RAW264.7 cells were cultured in complete medium at 37°C and grown in a 5% CO2 atmosphere at 37°C. When the cell density reached 90%, the cells were passaged. The cells were pipetted off the medium and uniformly suspended into a single suspension. 3 mL of the suspension was then transferred to a pre-sterilized culture medium.

[0061] [2] CCK-8 cytotoxicity assay: Log-growing cells were taken and subjected to a reaction at a rate of 2 × 10⁻⁶. 5 Cells / mL were added to each well of a 96-well plate, with a total of 100 μL of cell suspension added. The plates were incubated at 37°C and 5% CO2 for 24 hours. The following groups were established: a blank control group (with an equal volume of culture medium), a normal control group, a dimethyl sulfoxide (DMSO) group (specifically set according to pharmacopoeia requirements), and an SBD-16 administration group. Except for the blank control group, which had 5 replicates, all other groups had 6 replicates. After 24 hours of cell adhesion, the supernatant was discarded. 100 μL of fresh culture medium was added to the blank control and normal control groups to dissolve the compound in DMSO, and then diluted to different concentrations with culture medium. Compound a with final concentrations of 200, 100, 50, 25, 12.5, and 6.25 μmol / L was added to each well of the drug group and cultured for 24 h. After discarding the supernatant, 100 μL of CCK-8 reagent was added to each well, and the cells were incubated in a cell culture incubator for another 1.5 h. The absorbance (OD) was measured at 450 nm using a microplate reader. Cell viability was calculated, and high, medium, and low drug concentrations that had no significant effect on cell activity were selected. These concentrations were used in subsequent experiments.

[0062] [3] Establishing an inflammation model: Log-growing cells were taken and processed at a ratio of 2 × 10⁻⁶ cells. 5μL, 100 μL per well was inoculated in 96-well plates, and normal group (NC), model group (Model) and positive drug group (DEX) were set up. Compound a, (+) -a and (-) -a were set up as drug groups according to the high, medium and low doses obtained in [2] under "3. Experimental method". Each group had 3 replicate wells. After the cells were attached and grew for 24 h, the supernatant was discarded. 50 μL of culture medium was added to the normal group and the model group, and an equal amount of 4 μg / mL dexamethasone solution was added to the positive drug group. 50 μL of culture medium containing the corresponding drug concentration was added to the drug groups. After 2 h of culture, 50 μL of culture medium was added to the normal group, and an equal amount of 2 μg / mL LPS reagent was added to the other groups. After 12 h of continuous culture, the morphological structure changes of the cells in each group were observed, and the supernatant was collected for testing of inflammatory factors.

[0063] [4] Detection of inflammatory factors IL-6, TNF- α in cell supernatant: prepare all reagents and standards, prepare standards according to the method shown in the ELISA kit, and set up blank wells, standard wells and sample wells. 100 μl of standard diluent was added to the blank wells, 2 times diluted standard solution was added to the standard wells, and 100 μl of diluted sample was added to the sample wells. Incubate at 37°C for 1.5 h; wash and tap the plate 5 times, add 100 μl of biotinylated antibody diluent to each well, avoid light, and incubate at 37°C for 1 h; wash and tap the plate 5 times, add 100 μl of enzyme-conjugated diluent to each well, avoid light, and incubate at 37°C for 30 min; wash and tap the plate 5 times, add 100 μl of color developing solution to each well, add reaction termination solution after 15 min, and detect OD value at 450 nm wavelength within 3 min. Draw the standard curve with OD value as the abscissa and standard concentration as the ordinate. Determine the content of tumor necrosis factor- α (tumornecrosis factor-α, TNF- α ) and interleukin-6 (IL-6) in the culture medium according to the OD value of the sample.

[0064] [5] Statistical analysis: data were processed by Graphpad Prism 8.0.2 statistical software, and represented as mean ± standard deviation (x ± s). T test was used for comparison between groups (t test was used when variance was equal, and t' test was used when variance was not equal). The test result was considered statistically significant when P < 0.05.

[0065] 4. Experimental results

[0066] Nature Control is the normal control group, Model is the model group, Dex is the dexamethasone group, and compound a and its enantiomers enantiomers (+)-1 and (-)-1 are each set to high, medium and low dose groups (high dose: 100 mM; medium dose: 50 mM; low dose: 25 mM). Compared with the normal group, the use of LPS alone significantly increased the production of IL-6 and TNF- α . The isolated compound a and its enantiomers (+)-a and (-)-a had obvious inhibitory effect on IL-6 and TNF- α , and showed a certain linear relationship with the dose. Compared with DEX, compound a or its enantiomers had good inhibitory effect on IL-6 and TNF- α . Notably, (-)-a at 100 mM had higher inhibitory rate on IL-6 and TNF- α than DXM (p<0.0001), indicating that compound a and its enantiomers are effective for treating inflammation.

[0067] Therefore, compound a has significant anti-inflammatory activity and can be used for preparing drugs for preventing and treating inflammation.

[0068] In the cell inflammation model experiment, the low dose of compound a is 25 mM, the medium dose of compound a is 50 mM, and the high dose is 100 mM.

[0069] The embodiments involved in the present application are only used to illustrate the technical solutions of the present application and are not limited, although the above embodiments

[0070] have been described in detail, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. A biflavonoid compound having an anti-inflammatory effect, characterized in that, A structure is: 。 2. A method of preparing a biflavone according to claim 1, wherein, is isolated from the copper money Ephedra, its preparation method comprises the following steps: ​ (1) using ethanol aqueous solution on the copper money Ephedra medicinal materials are hot reflux extraction, the extract is concentrated to obtain extract; (2) the extract obtained in step (1) is diluted with water, first extracted with petroleum ether, then extracted with ethyl acetate, and the solution after concentrating the ethyl acetate extraction is obtained to extract the extract; (3) the extract extract obtained in step (2) is separated by first column chromatography with normal phase silica gel column, the eluent is a mixture of different proportions of chloroform and acetone, the eluent rich in the compound is collected, and the mixture containing the compound is obtained after concentration; (4) the mixture obtained in step (3) is separated by second chromatography with reverse phase column, the eluent is a mixture of different proportions of methanol and water, the eluent rich in the compound is collected, and the crude product containing the compound is obtained after concentration; (5) the crude product obtained in step (4) is purified by gel column, the eluent is pure methanol, and the biflavonoid compound is obtained by collecting the eluent.

3. The preparation method according to claim 2, characterized in that, The ethanol concentration of the ethanol aqueous solution used in step (1) is 70-75%, and the extraction is carried out 2-3 times, 2-3 hours each time; the volume ratio of chloroform and acetone used in step (3) is 10:1-1:1; the reverse phase column used in step (4) is MCI, and the volume ratio of water and methanol in the mobile phase is 100:0-0:100; the gel column used in step (5) is Sephadex LH-20.

4. Use of the biflavonoid compound according to claim 1 for the preparation of an anti-inflammatory medicament, characterized in that, The inflammation is the inflammation induced by lipopolysaccharide LPS.

5. Use according to claim 4, characterized in that, The drug is a preparation containing the biflavonoid compound.

6. Use according to claim 5, characterized in that, The preparation is made of the biflavonoid compound alone or the biflavonoid compound and a pharmaceutically acceptable carrier.

7. Use according to claim 5, characterized in that, The preparation is tablet, capsule, pill, oral liquid, granule, suspension, injection or aerosol.

8. Use according to claim 6, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of the following: filler, binder, disintegrant, solubilizer, suspending agent, wetting agent, pigment, essence, solvent or flavoring agent.

9. Use according to any one of claims 4 to 8, characterized in that, The clinical dosage of the biflavonoid compound is 100-800 mg / d.

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