Forsythia suspensa alkaloid B and its preparation method and anti-inflammatory and antiviral applications

Abstract

The invention discloses a forsythia alkaloid B and its preparation method and anti-inflammatory and antiviral applications, belonging to the technical field of pharmaceutical chemistry. The forsythia alkaloid B is a norcisan-type diterpene alkaloid with a molecular formula of C 17 H 23 NO2, the structural formula is as follows. The invention is beneficial in that: the forsythia alkaloid B is separated from the fruit of forsythia by an innovative extraction and separation process, the forsythia alkaloid B has a novel structure, and has good anti-inflammatory and anti-H9N2, H5N1 and H1N1 influenza virus activities, providing a new material basis for the research on the anti-inflammatory and antiviral activities of forsythia. #imgabs0#Forsythia alkaloid B.

Description

Technical Field

[0001] The invention relates to alkaloids and preparation methods and applications thereof, and in particular to an alkaloid (Forsythia suspensa alkaloid B) separated from the fruit of Forsythia suspensa and a preparation method thereof as well as anti-inflammatory and antiviral applications thereof, belonging to the technical field of pharmaceutical chemistry. Background Art

[0002] Forsythia suspensa Forsythia suspensa ) is a shrub of the genus Forsythia in the family Oleaceae, and its fruit is a commonly used Chinese herbal medicine included in the Chinese Pharmacopoeia. Forsythia has good anti-inflammatory and antiviral effects, but the material basis of its efficacy is not completely clear. Regarding the research on the material basis of the anti-inflammatory and antiviral efficacy of Forsythia, domestic and foreign researchers mainly focus on compounds with high content of phenylpropanoids, flavonoids, triterpenes and phenylethanol, but there is little research on alkaloid compounds with low content but high anti-inflammatory and antiviral activity. Summary of the invention

[0003] To overcome the deficiencies of the prior art, the present invention aims to separate an alkaloid compound with high anti-inflammatory and anti-influenza virus activities from the fruit of Forsythia suspensa by adopting an innovative extraction and separation process.

[0004] In order to achieve the above object, the present invention adopts the following technical solution:

[0005] Forsythia suspensa alkaloid B, the Forsythia suspensa alkaloid B is a norfloscan-type diterpene alkaloid, the molecular formula is C 17 H 23 NO2, the structural formula is as follows:

[0006] .

[0007] The preparation method of the forsythia suspensa alkaloid B comprises the following steps:

[0008] (1) Take the fruit of Forsythia suspensa, extract it with methanol or 95% ethanol under reflux, recover the organic solvent, and obtain extract I;

[0009] (2) dispersing extract I with deionized water, then extracting with chloroform or ethyl acetate, combining the organic solvent extraction parts, recovering the organic solvent, and obtaining extract II;

[0010] (3) Dissolve extract II with n-butanol, then extract with 3-4% hydrochloric acid, and combine the acid-water extracts;

[0011] (4) adjusting the pH value of the acid water extract to 8-9 with aqueous ammonia, then extracting with chloroform or ethyl acetate, combining the organic solvent extraction parts, recovering the organic solvent, and obtaining the alkaloid fraction;

[0012] (5) placing the alkaloid fraction on a normal phase silica gel chromatography column and gradient eluting the fraction using a cyclohexane-acetone system as an eluent, detecting the eluate by TLC, intercepting the eluate containing the alkaloid and concentrating it under reduced pressure to obtain a concentrate I;

[0013] (6) The concentrate I is subjected to further chromatography separation using a preparative thin layer chromatography, with 5-8% acetic acid added as the developing solvent, the alkaloid band is intercepted, eluted with acetone and concentrated under reduced pressure to obtain a concentrate II;

[0014] (7) Separate the concentrate II by preparative HPLC, C 18 Chromatographic column, size 22mm×250mm, filler particle diameter 5μm, mobile phase V 乙腈 :V 20mmol / L乙酸铵-0.1%甲酸缓冲液 =65:35, flow rate 7.0mL / min, injection volume 2mL, detection wavelength 345nm, collect the components with retention time between 13min and 15min, concentrate under reduced pressure, crystallize, and dry to obtain Forsythia suspensa alkaloid B.

[0015] Preferably, in step (1), the ratio of Forsythia suspensa fruit to methanol or 95% ethanol is 1 kg: 10 L to 23 L, the reflux extraction time is 3 h, and the reflux extraction times are 2 to 3 times.

[0016] Preferably, in step (2), the extract I is dispersed with an equal volume of deionized water and extracted three times with an equal volume of chloroform or ethyl acetate.

[0017] Preferably, in step (3), extract II is dissolved with 2 to 3 volumes of n-butanol and extracted 5 times with an equal volume of hydrochloric acid.

[0018] Preferably, in step (4), extraction is performed three times with equal volumes of chloroform or ethyl acetate.

[0019] Preferably, in step (5), cyclohexane and acetone are gradient eluted in a volume ratio of 100:0 to 80:20 or 95:5 to 75:25.

[0020] Preferably, in step (6), the developing solvent is a mixture of methanol, chloroform and ethyl acetate, V 甲醇 :V 氯仿 :V 乙酸乙酯 =0.6:10:4.

[0021] The application of the forsythia suspensa alkaloid B in the preparation of anti-inflammatory drugs, and the application of the forsythia suspensa alkaloid B in the preparation of anti-H9N2, H5N1 and H1N1 influenza virus drugs.

[0022] The invention is beneficial in that: an innovative extraction and separation process is adopted to separate the forsythia suspensa alkaloid B from the fruit of the forsythia suspensa, the forsythia suspensa alkaloid B has a novel structure and has good anti-inflammatory and anti-H9N2, H5N1 and H1N1 influenza virus activities, and provides a new material basis for the research on the anti-inflammatory and antiviral activities of forsythia suspensa. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the forsythia suspensa alkaloid B 1 H-NMR spectrum;

[0024] Figure 2 It is the forsythia suspensa alkaloid B 13 C-NMR spectrum;

[0025] Figure 3 This is the HMBC spectrum of Forsythia suspensa alkaloid B;

[0026] Figure 4 yes Figure 3 A partial enlarged view, where f1: 20ppm-160ppm, f2: 6.80ppm-7.70ppm;

[0027] Figure 5 yes Figure 3 A partial enlarged view, where f1: 10ppm-55ppm, f2: 0.4ppm-3.4ppm;

[0028] Figure 6 yes Figure 3 A partial enlarged view, where f1: 115ppm-185ppm, f2: 0.8ppm-4.0ppm;

[0029] Figure 7 This is the HSQC spectrum of Forsythia suspensa alkaloid B;

[0030] Figure 8 yes Figure 7 A partial enlarged view, where f1: 115ppm-140ppm, f2: 6.90ppm-7.70ppm;

[0031] Fig. 9 yes Figure 7 A partial enlarged view, where f1: 10ppm-50ppm, f2: 0.6ppm-3.2ppm;

[0032] Fig.10 This is the ROESY spectrum of Forsythia suspensa alkaloid B;

[0033] Fig.11 yes Fig.10A partial enlarged view, where f1: 1.0ppm-8.0ppm, f2: 6.70ppm-7.75ppm;

[0034] Fig.12 yes Fig.10 A partial enlarged view, where f1: 0.4ppm-3.0ppm, f2: 0.5ppm-3.0ppm;

[0035] Fig.13 yes Fig.10 A partial enlarged view, where f1: 6ppm-14ppm, f2: 0.7ppm-2.9ppm;

[0036] Fig.14 This is the HR-MS spectrum of Forsythia suspensa alkaloid B;

[0037] Fig.15 This is the UV spectrum of Forsythia suspensa alkaloid B;

[0038] Fig.16 This is the HMBC correlation diagram of Forsythia suspensa alkaloid B;

[0039] Fig.17 This is the main NOESY correlation diagram of Forsythia suspensa alkaloid B. DETAILED DESCRIPTION

[0040] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] 1. Structure of Forsythia suspensa alkaloid B

[0042] The forsythia suspensa alkaloid B provided by the present invention is a norfloscan-type diterpene alkaloid, and its molecular formula is C 17 H 23 NO2, the structural formula is as follows:

[0043] .

[0044] 2. Preparation method of Forsythia suspensa alkaloid B Example 1

[0045] Take 10kg of Forsythia suspensa fruit, extract it twice with 100L 95% (v / v) ethanol, the first reflux extraction is 2h, the second reflux extraction is 1h, the extracts are combined, and then the organic solvent is recovered by vacuum concentration to obtain extract I; extract I is dispersed with an equal volume of deionized water, and then extracted with an equal volume of ethyl acetate for 3 times, the organic solvent extraction parts are combined, and then the organic solvent is recovered by vacuum concentration to obtain extract II; extract II is dissolved with 2 times the volume of n-butanol, and then extracted with an equal volume of 3% (v / v) hydrochloric acid for 5 times, and the acid water extracts are combined; the pH value of the acid water extract is adjusted to 9 with ammonia water, and then extracted with an equal volume of ethyl acetate for 3 times, and the organic solvent is combined. The solvent extraction part is then concentrated under reduced pressure to recover the organic solvent to obtain the alkaloid part; the alkaloid part is subjected to silica gel column chromatography, preparative thin layer chromatography and preparative high performance liquid chromatography. Specifically, the alkaloid part is firstly placed on a normal phase silica gel column, and a cyclohexane-acetone system is used as an eluent, and the gradient elution is performed according to a volume ratio of (95:5) to (75:25), and the eluent is detected by TLC, and the eluent containing the alkaloid is intercepted and concentrated under reduced pressure to obtain a concentrated solution I, and then the concentrated solution I is chromatographed again by a preparative thin layer, and the developing solvent (a mixture of methanol, chloroform and ethyl acetate, V 甲醇 :V 氯仿 :V 乙酸乙酯 =0.6:10:4) add 5% (v / v) acetic acid, intercept the alkaloid band, elute with acetone and concentrate under reduced pressure to obtain concentrated solution II. Finally, use preparative high performance liquid chromatography to separate the components in concentrated solution II. The chromatographic conditions are: C 18 Chromatographic column, size 22mm×250mm, filler particle diameter 5μm, mobile phase V 乙腈 :V 20mmol / L乙酸铵-0.1%甲酸缓冲液 =65:35, flow rate 7.0mL / min, injection volume 2mL, detection wavelength 345nm, collect components with retention time between 13min and 15min, concentrate under reduced pressure, crystallize, and dry to obtain 35.87mg colorless needle-shaped crystals.

[0046] The specific rotation of this colorless needle-shaped crystal (acetone) is [α] 25 D +32.0 (c 0.39, methanol).

[0047] The colorless needle-shaped crystals reacted positively with potassium bismuth iodide reagent in methanol and were preliminarily judged to be alkaloids.

[0048] In high-resolution mass spectrometry (HR-MS) detection ( Fig.14 ),exist m / z 274.1807[M+H] + The quasi-molecular ion peak of the compound was obtained, and the molecular formula of the colorless needle-shaped crystal was determined to be C 17 H23 NO2.

[0049] In ultraviolet spectroscopy (UV) detection ( Fig.15 ), two maximum absorption peaks were obtained at 204nm and 273nm, indicating that the structure of the colorless needle-shaped crystals contains aromatic heterocyclic fragments.

[0050] In the H NMR spectrum ( 1 H-NMR) detection ( Figure 1 , 400MHz), three methyl signals, one carboxyl hydrogen signal and two aromatic proton signals were found.

[0051] In the carbon NMR spectrum ( 13 C-NMR) detection ( Figure 2 , 100MHz), and 17 carbon signals were found.

[0052] HSQC (Heteronuclear Single Quantum Correlation) Figure 7 , Figure 8 , Fig. 9 ), it was determined that these 17 carbon signals belonged to 3 methyl groups, 5 methylene groups, 3 methine groups and 6 quaternary carbon groups.

[0053] through 1 Heteronuclear multicarbon correlation spectroscopy (HMBC) detection of H Figure 3 , Figure 4 , Figure 5 , Figure 6 ), the colorless needle-shaped crystals were determined to be norcanane-type diterpene alkaloids.

[0054] In the rotating frame Overhauser effect spectroscopy (ROESY) detection ( Fig.10 , Fig.11 , Fig.12 , Fig.13 ), it was found that H-5 was associated with CH3-19, and C18-OH was associated with CH3-20, which indicated that CH3-19 and H-5 were in the same plane and in α configuration, while C18-OH and CH3-20 were located on the other side of the molecular plane and in β configuration.

[0055] From the above test results, it can be determined that the molecular formula of the colorless needle-shaped crystal is C 17 H 23 NO2, the structural formula is:

[0056] .

[0057] Because the colorless needle-shaped crystals were separated from Forsythia suspensa, they were named Forsythia suspensa alkaloid B.

[0058] Forsythia suspensa alkaloids B 1 H-NMR and 13C-NMR data are shown in Table 1 and related spectra are shown in Figures 1 to 9 .

[0059] Table 1 NMR data of Forsythia suspensa alkaloid B

[0060]

[0061]

[0062] Example 2

[0063] Take 10 kg of Forsythia suspensa fruit, extract it with 100 L, 80 L and 50 L of methanol for 3 times, each time for 1 hour, combine the extracts, and then concentrate under reduced pressure to recover the organic solvent to obtain extract I; disperse extract I with an equal volume of deionized water, and then extract it with an equal volume of chloroform for 3 times, combine the organic solvent extraction parts, and then concentrate under reduced pressure to recover the organic solvent to obtain extract II; dissolve extract II with 3 times the volume of n-butanol, and then extract it with an equal volume of 4% (v / v) hydrochloric acid for 5 times, and combine the acid water extracts; adjust the pH value of the acid water extract to 8 with ammonia water, and then extract it with an equal volume of chloroform for 3 times, and combine the The organic solvent extraction part is then concentrated under reduced pressure to recover the organic solvent to obtain the alkaloid part; the alkaloid part is subjected to silica gel chromatography column chromatography, preparative thin layer chromatography and preparative high performance liquid chromatography separation. Specifically, the alkaloid part is firstly placed on a normal phase silica gel chromatography column, and a cyclohexane-acetone system is used as an eluent, and the gradient elution is performed according to a volume ratio of (100:0) to (80:20), and the eluent is detected by TLC, and the eluent containing the alkaloid is intercepted and concentrated under reduced pressure to obtain a concentrated solution I, and then the concentrated solution I is chromatographed again by a preparative thin layer, and the developing solvent (a mixture of methanol, chloroform and ethyl acetate, V 甲醇 :V 氯仿 :V 乙酸乙酯 =0.6:10:4) add 8% (v / v) acetic acid, use bismuth potassium iodide reagent to develop color, intercept the alkaloid band, elute with acetone and concentrate under reduced pressure to obtain concentrated solution II, and finally use preparative high performance liquid chromatography to separate the components in concentrated solution II. The chromatographic conditions are: C 18 Chromatographic column, size 22mm×250mm, filler particle diameter 5μm, mobile phase V 乙腈 :V 20mmol / L乙酸铵-0.1%甲酸缓冲液 =65:35, flow rate 7.0mL / min, injection volume 2mL, detection wavelength 345nm, collect components with retention time between 13min and 15min, concentrate under reduced pressure, crystallize, and dry to obtain 38.46mg colorless needle-shaped crystals.

[0064] After testing, the colorless needle-shaped crystals were found to be Forsythia suspensa alkaloid B.

[0065] 3. Anti-inflammatory activity of Forsythia suspensa alkaloid B

[0066] Polymorphonuclear leukocytes of rats were obtained and suspended in Dulbecco's phosphate buffered saline (DPBS) to a cell concentration of 2.5 × 10 6 / mL to obtain a cell suspension.

[0067] Take a 10mL test tube, add 5mL of different concentrations (0.1μmol / L, 1μmol / L, 10μmol / L, 100μmol / L) of Forsythia suspensa alkaloid B and 245mL of cell suspension, and set up 4 replicates for each concentration. Place all test tubes in a 37℃ water bath, shake and incubate for 20min, then add 2.5mL of 1mmol / L cytochalasin B, incubate for 5min, then add 2.5mL of 20μmol / L platelet activating factor (PAF), continue incubation for 10min, then move the test tube into an ice bath to terminate the reaction, then centrifuge for 5min, and take the supernatant as the enzyme release solution.

[0068] Take 1 mL of cell suspension, add 10 mL of 10% Triton-x100, centrifuge for 2 min, and take the supernatant as the total enzyme solution.

[0069] Take 1 mL of the total enzyme solution and inactivate it in a water bath at 80°C for 10 min to use as the inactivated enzyme solution.

[0070] A 96-well ELISA plate was used. 25 mL of released enzyme solution, total enzyme solution or inactivated enzyme solution, 25 mL of 2.5 mmol / L phenolphthalein glucuronic acid and 100 mL of 0.1 mol / L acetate buffer (pH 4.6) were added to each well. The plate was incubated at 37°C for 18 h. Then 150 mL of 0.3 mol / L -1 The reaction was terminated by adding NaOH and the color was developed. Finally, the absorbance of each well was measured at 550 nm using an enzyme-labeled instrument. The data obtained from the experiment were statistically processed and expressed as mean ± SD.

[0071] The effects of different concentrations of Forsythia suspensa alkaloid B on β -Inhibition rate of glucuronidase activity:

[0072] Inhibition rate = [(OD 刺激组 -OD 给药组 ) / (OD 刺激组 -OD 溶剂对照组 )]×100%

[0073] Among them, OD 刺激组 OD is the absorbance value measured at 550nm after the color development of the ELISA wells with the total enzyme solution added. 给药组 OD is the absorbance value measured at 550nm after the enzyme-labeled wells are colored after adding the released enzyme solution.溶剂对照组 It is the absorbance value measured at 550nm after the enzyme-labeled wells are colored after adding the inactivated enzyme solution.

[0074] The results show that different concentrations of Forsythia suspensa alkaloid B have an effect on β -The inhibition rates of glucuronidase activity are as follows:

[0075] Table 2 Effects of different concentrations of Forsythia suspensa alkaloid B on β -Inhibition rate of glucuronidase activity

[0076]

[0077] As shown in Table 2, Forsythia suspensa alkaloid B has a good inhibitory effect on the activity of platelet activating factor-induced polymorphonuclear leukocytes releasing β-glucuronidase. This shows that Forsythia suspensa alkaloid B has good anti-inflammatory activity, and as a natural product, it can be developed into a new anti-inflammatory Chinese medicine, which has certain application prospects.

[0078] 4. Antiviral activity of Forsythia suspensa alkaloid B

[0079] 1. Cell lines and virus strains

[0080] MDCK cells (dog kidney culture cells) were infected with H9N2, H5N1, and H1N1 influenza viruses respectively.

[0081] 2. Cytotoxicity test

[0082] MDCK cells (not infected with influenza virus) were densely seeded in 96-well cell culture plates at a cell density of 1.0 × 10 per well. 4 After culturing for 24 h, MTT solution (5 mg / mL) was added to dilute the Forsythia suspensa alkaloid B or ribavirin (control). The concentrations of Forsythia suspensa alkaloid B and ribavirin were 1×10 -4 mmol / L、1×10 -3 mmol / L, 0.01mmol / L, 0.1mmol / L, 1mmol / L, 10mmol / L, 50μL per well, and a blank control group (only MTT solution was added), 4 replicates per group, cultured at 37℃, 5% CO2 for 48h, the absorbance of each well (A) was measured, and the Reed-Muench method was used to calculate the median toxic concentration (CC) of Forsythia suspensa alkaloid B and ribavirin 50 ).

[0083] The median toxic concentration (CC) of forsythia suspensa alkaloid B and ribavirin 50 ) are shown in Table 3.

[0084] 3. Antiviral activity test

[0085] The antiviral activity of Forsythia suspensa alkaloid B was determined by MTT method.

[0086] MDCK cells (not infected with influenza virus) were densely seeded in 96-well cell culture plates at a cell density of 1.0 × 10 per well. 4 and cultured for 24 h.

[0087] Take appropriate amount of Forsythia suspensa alkaloid B and ribavirin (control) and dilute them into different concentrations (1×10 -4 mmol / L、1×10 -3 mmol / L, 0.01mmol / L, 0.1mmol / L, 1mmol / L, 10mmol / L) of drug-containing solutions (solution containing Forsythia suspensa alkaloid B, solution containing ribavirin).

[0088] Blank group: only containing culture medium, no cells or drug treatment, cultured at 37℃, 5% CO2 for 48h;

[0089] Normal group: cells were cultured only without virus or drug treatment at 37°C and 5% CO2 for 48 h;

[0090] Virus group: only virus solution was added for adsorption, no drug treatment was added, and cultured at 37°C and 5% CO2 for 48h;

[0091] Drug administration group: 100 TCID 50 The virus (H9N2, H5N1 or H1N1) solution was mixed with 50 μL of the solution containing Forsythia suspensa alkaloid B, cultured at 37°C and 5% CO2 for 30 min, then added to the cells in a 96-well cell culture plate and cultured at 37°C and 5% CO2 for 48 h.

[0092] Positive control group: 100 TCID 50 The virus (H9N2, H5N1 or H1N1) solution was mixed with 50 μL of ribavirin solution and cultured at 37°C and 5% CO2 for 30 min. Then, it was added to the cells in a 96-well cell culture plate and cultured at 37°C and 5% CO2 for 48 h.

[0093] After the culture, the supernatant was removed, the cells were washed twice with PBS buffer, and then 50 μL of lysate was added to each well, gently shaken, and allowed to stand for 40 min. Then 50 μL of lysate was aspirated and placed on a white plate, and an appropriate amount of luciferase colorimetric substrate was added. The absorbance (A) was measured at a wavelength of 490 nm, and the Reed-Muench method was used to calculate the half effective concentration (EC) of Forsythia suspensa alkaloid B and ribavirin against H9N2, H5N1 and H1N1 influenza viruses. 50).

[0094] The median effective concentration (EC50) of Forsythia suspensa alkaloid B and ribavirin against H9N2, H5N1 and H1N1 influenza viruses 50 ) are shown in Table 3.

[0095] According to the median toxic concentration (CC 50 ) and the median effective concentration (EC 50 ) Calculate the selectivity index (SI) of Forsythia suspensa alkaloid B and ribavirin against H9N2, H5N1 and H1N1 influenza viruses. The calculation formula of the selectivity index (SI) is as follows:

[0096] SI=CC 50 / EC 50

[0097] The calculation results of the selectivity index (SI) of Forsythia suspensa alkaloid B and ribavirin against H9N2, H5N1 and H1N1 influenza viruses are shown in Table 3.

[0098] Table 3 Antiviral activity of Forsythia suspensa alkaloid B (mean ± SD, n = 4)

[0099]

[0100] As shown in Table 3, compared with ribavirin, Forsythia suspensa alkaloid B has a significant protective effect on cells infected with three different subtypes of influenza virus, H9N2, H5N1 and H1N1. This shows that Forsythia suspensa alkaloid B has good anti-H9N2, H5N1 and H1N1 influenza virus activity, and as a natural product, it can be developed into a new Chinese medicine for anti-H9N2, H5N1 and H1N1 influenza virus, which has certain application prospects.

[0101] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Forsythia suspensa alkaloid B, characterized in that The forsythia suspensa alkaloid B is a norfloscan-type diterpene alkaloid with a molecular formula of C 17 H 23 NO2, the structural formula is as follows: 。 2. The method for preparing the Forsythia suspensa alkaloid B according to claim 1, characterized in that: The following steps are involved: (1) Take the fruit of Forsythia suspensa, extract it with methanol or 95% ethanol under reflux, recover the organic solvent, and obtain extract I; (2) dispersing extract I with deionized water, then extracting with chloroform or ethyl acetate, combining the organic solvent extraction parts, recovering the organic solvent, and obtaining extract II; (3) Dissolve extract II with n-butanol, then extract with 3-4% hydrochloric acid, and combine the acid-water extracts; (4) adjusting the pH value of the acid water extract to 8-9 with aqueous ammonia, then extracting with chloroform or ethyl acetate, combining the organic solvent extraction parts, recovering the organic solvent, and obtaining the alkaloid fraction; (5) placing the alkaloid fraction on a normal phase silica gel chromatography column and gradient eluting the fraction using a cyclohexane-acetone system as an eluent, detecting the eluate by TLC, intercepting the eluate containing the alkaloid and concentrating it under reduced pressure to obtain a concentrate I; (6) Using preparative thin layer chromatography to separate the concentrate I again, adding 5-8% acetic acid as the developing solvent, intercepting the alkaloid band, eluting with acetone and concentrating under reduced pressure to obtain concentrate II; (7) Separate the concentrate II by preparative HPLC, C 18 Chromatographic column, size 22mm×250mm, filler particle diameter 5μm, mobile phase V 乙腈 :V 20mmol / L乙酸铵-0.1%甲酸缓冲液 =65:35, flow rate 7.0mL / min, injection volume 2mL, detection wavelength 345nm, collect the components with retention time between 13min and 15min, concentrate under reduced pressure, crystallize, and dry to obtain Forsythia suspensa alkaloid B.

3. The method for preparing Forsythia suspensa alkaloid B according to claim 2, characterized in that: In step (1), the ratio of Forsythia suspensa fruit to methanol or 95% ethanol is 1 kg: 10 L to 23 L, the reflux extraction time is 3 h, and the reflux extraction times are 2 to 3 times.

4. The method for preparing Forsythia suspensa alkaloid B according to claim 2, characterized in that: In step (2), extract I is dispersed with an equal volume of deionized water and extracted three times with an equal volume of chloroform or ethyl acetate.

5. The method for preparing Forsythia suspensa alkaloid B according to claim 2, characterized in that: In step (3), extract II is dissolved with 2 to 3 volumes of n-butanol and extracted 5 times with an equal volume of hydrochloric acid.

6. The method for preparing Forsythia suspensa alkaloid B according to claim 2, characterized in that: In step (4), extract three times with equal volumes of chloroform or ethyl acetate.

7. The method for preparing Forsythia suspensa alkaloid B according to claim 2, characterized in that: In step (5), cyclohexane and acetone are gradient eluted in a volume ratio of 100:0 to 80:20 or 95:5 to 75:

25.

8. The method for preparing Forsythia suspensa alkaloid B according to claim 2, characterized in that: In step (6), the developing solvent is a mixture of methanol, chloroform and ethyl acetate. 甲醇 :V 氯仿 :V 乙酸乙酯 =0.6:10:

4.

9. Use of the Forsythia suspensa alkaloid B according to claim 1 in the preparation of anti-inflammatory drugs.

10. Use of the Forsythia suspensa alkaloid B according to claim 1 in the preparation of drugs against H9N2, H5N1 and H1N1 influenza viruses.

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