R-type fructus forsythiae alkaloid A as well as preparation method and anti-inflammatory application thereof

Abstract

The invention discloses fructus forsythiae alkaloid A as well as a preparation method and anti-inflammatory application thereof. The fructus forsythiae alkaloid A is a norlabdane type diterpenoid alkaloid compound, the molecular formula is C17H25NO, and the structural formula is shown in the specification. The method has the beneficial effects that the fructus forsythiae alkaloid A is separated from fructus forsythiae fruits by adopting an innovative extraction and separation process, the structure of the fructus forsythiae alkaloid A is novel, the fructus forsythiae alkaloid A has good anti-inflammatory activity, and a new material basis is provided for research on the anti-inflammatory activity of fructus forsythiae. # imgabs0 #

Description

Technical Field

[0001] The invention relates to alkaloids and preparation methods and applications thereof, and in particular to an alkaloid (R-type forsythia suspensa alkaloid A) separated from the fruit of forsythia suspensa and a preparation method and anti-inflammatory application thereof, belonging to the technical field of pharmaceutical chemistry. Background Art

[0002] Forsythia suspensa is a shrub of the genus Forsythia in the family Oleaceae. Its fruit has been called "the holy medicine for sores" by doctors of all dynasties. It is a commonly used bulk medicinal material in my country and an important raw material for more than 40 classic Chinese patent medicines (Lianhua Qingwen, Shuanghuanglian, Huanglian Shangqing and Vitamin C Yinqiao Tablets, etc.). Forsythia suspensa has good anti-inflammatory effects. Regarding the research on the material basis of the anti-inflammatory efficacy of Forsythia suspensa, 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 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 activity 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] R-type Forsythia suspensa alkaloid A, the R-type Forsythia suspensa alkaloid A is a norfloscan-type diterpene alkaloid, the molecular formula is C 17 H 25 NO, the structural formula is as follows:

[0006]

[0007] Among them, the absolute configuration of C4 is R type.

[0008] The preparation method of the aforementioned R-type Forsythia suspensa alkaloid A comprises the following steps:

[0009] (1) taking Forsythia suspensa fruit, extracting it with methanol or 95% ethanol under reflux, recovering the organic solvent, and obtaining extract I;

[0010] (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;

[0011] (3) dissolving extract II with n-butanol, then extracting with 3% to 4% hydrochloric acid, and combining the acid-water extracts;

[0012] (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;

[0013] (5) placing the alkaloid fraction on a normal phase silica gel chromatography column, gradient eluting with 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;

[0014] (6) Using preparative thin layer chromatography to separate the concentrate I again, adding 5% to 8% acetic acid as the developing solvent, intercepting the alkaloid band, eluting with acetone and concentrating under reduced pressure to obtain a concentrate II;

[0015] (7) Separating the concentrated solution II by preparative high performance liquid chromatography, 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 7.0 mL / min, flow rate 7.0 mL / min, detection wavelength 345 nm, collect the components with retention time between 16 min and 18 min, concentrate under reduced pressure, crystallize, and dry to obtain R-type Forsythia suspensa alkaloid A.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] Preferably, in step (4), extraction is performed three times with equal volumes of chloroform or ethyl acetate.

[0020] 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.

[0021] Preferably, 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.

[0022] The use of the aforementioned R-type Forsythia suspensa alkaloid A in the preparation of anti-inflammatory drugs.

[0023] The benefit of the present invention lies in that an innovative extraction and separation process is adopted to separate R-type Forsythia suspensa alkaloid A (the absolute configuration of C4 is R-type) from the fruit of Forsythia suspensa. It is verified that the R-type Forsythia suspensa alkaloid A has good anti-inflammatory activity, which provides a new material basis for the research on the anti-inflammatory activity of Forsythia suspensa. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the R-type of Forsythia suspensa alkaloid A 1 H-NMR spectrum;

[0025] Figure 2 It is the R-type of Forsythia suspensa alkaloid A 13 C-NMR spectrum;

[0026] Figure 3 It is the R-type of Forsythia suspensa alkaloid A 1 H- 1 H COSY spectrum;

[0027] Figure 4 This is the HMBC spectrum of R-type Forsythia suspensa alkaloid A;

[0028] Figure 5 This is the HSQC spectrum of R-type Forsythia suspensa alkaloid A;

[0029] Figure 6 This is the NOESY spectrum of R-type Forsythia suspensa alkaloid A;

[0030] Figure 7 This is the HR-MS spectrum of R-type Forsythia suspensa alkaloid A;

[0031] Figure 8 This is the UV spectrum of R-type Forsythia suspensa alkaloid A;

[0032] Fig. 9 It is the R-type of Forsythia suspensa alkaloid A 1 H- 1 H COSY and HMBC correlation diagrams;

[0033] Fig.10 This is the main NOESY correlation diagram of R-type Forsythia suspensa alkaloid A. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0035] 1. Structure of R-type Forsythia suspensa alkaloid A

[0036] The R-type Forsythia suspensa alkaloid A provided by the present invention is a norfloscan-type diterpene alkaloid, and its molecular formula is C 17 H 25 NO, the structural formula is as follows:

[0037]

[0038] Among them, the absolute configuration of C4 is R type.

[0039] 2. Preparation method of R-type Forsythia suspensa alkaloid A

[0040] Example 1

[0041] 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 extracts are combined. The alkaloid fraction is separated by silica gel column chromatography, preparative thin layer chromatography and preparative high performance liquid chromatography. Specifically, the alkaloid fraction is firstly placed on a normal phase silica gel column, eluted with a cyclohexane-acetone system as an eluent in a gradient elution ratio of (95:5) to (75:25) by volume, and the eluent is detected by thin layer chromatography (TLC). 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 a 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, and finally separate the components in concentrated solution II by preparative high performance liquid chromatography. 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 16min and 18min, concentrate under reduced pressure, crystallize, and dry to obtain 24.7mg colorless needle-shaped crystals.

[0042] The specific rotation of this colorless needle-shaped crystal is [α] 25 D +56.0 (c 0.43, methanol).

[0043] The colorless needle-shaped crystals reacted positively with potassium bismuth iodide reagent in methanol and were preliminarily judged to be alkaloids.

[0044] In high-resolution mass spectrometry (HR-MS) detection ( Figure 7 ), at m / z 260.2005 [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 H 25 NO.

[0045] In ultraviolet spectroscopy (UV) detection ( Figure 8 ), two maximum absorption peaks were obtained at 209nm and 273nm, indicating that the colorless needle-shaped crystals have aromatic heterocyclic structure fragments.

[0046] In the H NMR spectrum ( 1 H-NMR) detection Figure 1 , 400 MHz), 3 methyl signals, 1 oxymethylene hydrogen signal, 1 hydroxyl hydrogen signal, and 2 aromatic proton signals were found.

[0047] In the carbon NMR spectrum ( 13 C-NMR) detection Figure 2 , 100MHz), 17 carbon signals were found.

[0048] The heteronuclear multi-quantum relation (HMQC) was used to detect Figure 5 ), it was determined that these 17 carbon signals belonged to 3 methyl groups, 6 methylene groups, 3 methine groups and 5 quaternary carbon groups.

[0049] In homokaryotic correlation spectroscopy ( 1 H- 1 H-COSY) detection Figure 3 ), two spin systems were found, one of which included 1 methine and 2 methylene groups (the C5-C6-C7 structural fragment could be determined), and the other spin system included 3 methylene groups (the C1-C2-C3 structural fragment could be determined).

[0050] through 1 Heteronuclear Multicarbon Correlation Spectroscopy (HMBC) Detection of H Figure 4 ), and determined that the colorless needle-shaped crystals were norfloxacin-type diterpene alkaloids.

[0051] In two-dimensional nuclear Overhauser effect spectroscopy (NOESY) detection ( Figure 6 ), it was found that H-5 was associated with CH2-18, and CH3-19 was associated with CH3-20, which indicated that CH2-18 and H-5 were coplanar and in α configuration, while CH3-19 and CH3-20 were located on the other side of the molecular plane and in β configuration.

[0052] From the above test results, it can be determined that the molecular formula of the colorless needle-shaped crystal is C 17 H 25 NO, the structural formula is:

[0053]

[0054] Among them, the absolute configuration of C4 is R type.

[0055] Because the colorless needle-shaped crystals are separated from Forsythia suspensa and the absolute configuration of C4 in the structural formula is R-type, they are named R-type Forsythia suspensa alkaloid A.

[0056] R-type Forsythia suspensa alkaloid A 1 H-NMR and 13 C-NMR data are shown in Table 1 and related spectra are shown in Figures 1 to 10 .

[0057] Table 1 NMR data of R-type Forsythia suspensa alkaloid A

[0058]

[0059]

[0060] Example 2

[0061] Take 10 kg of Forsythia suspensa fruit, extract it with 100 L, 80 L and 50 L 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, intercept the alkaloid band, elute with acetone and concentrate under reduced pressure to obtain concentrated solution II, and finally separate the concentrated solution II by preparative high performance liquid chromatography. 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 the components with retention time between 16min and 18min, concentrate under reduced pressure, crystallize, and dry to obtain 22.4mg colorless needle-shaped crystals.

[0062] After testing, the colorless needle-shaped crystals were found to be R-type Forsythia suspensa alkaloid A.

[0063] 3. Anti-inflammatory activity of R-type Forsythia suspensa alkaloid A

[0064] 1. Inhibitory effect of R-type Forsythia suspensa alkaloid A on the activity of β-glucosidase released by neutrophils induced by platelet-activating factor

[0065] Rat neutrophils were taken and suspended in Dulbecco's phosphate buffered saline (DPBS) to a cell concentration of 2.5 × 10 6 / mL to obtain a cell suspension.

[0066] Take a 10mL test tube, add 5μL of different concentrations (0.1μmol / L, 1μmol / L, 10μmol / L, 100μmol / L) of R-type Forsythia suspensa alkaloid A and 245μL of cell suspension, and set up 4 replicates for each concentration. Place all test tubes in a 37℃ water bath, shake and incubate for 20 minutes, then add 2.5μL of 1mmol·L -1 Cytochalasin B was added for 5 min, and then 2.5 μL of 20 μmol·L -1 Platelet activating factor (PAF) was added and the incubation was continued for 10 min. Then the test tube was moved into an ice bath to terminate the reaction. The tube was then centrifuged for 5 min and the supernatant was used as the enzyme release solution.

[0067] Take 1 mL of cell suspension, add 10 μL of 10% Triton-x100 and centrifuge for 2 min, and take the supernatant as the total enzyme solution.

[0068] 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.

[0069] A 96-well ELISA plate was used, and each well was added with 25 μL of released enzyme solution, total enzyme solution or inactivated enzyme solution, 25 μL of phenolphthalein glucuronic acid with a concentration of 2.5 mmol / L, and 100 μL of 0.1 mol / L acetate buffer (pH = 4.6), and incubated in a 37°C incubator for 18 hours, and then 150 μL of 0.3 mol / L NaOH was added to each well to terminate the reaction and develop color, and finally the absorbance value of each ELISA well was measured at 550 nm using an ELISA reader. The data obtained in the experiment were statistically processed and expressed as mean ± SD.

[0070] The inhibition rate of different concentrations of R-type Forsythia suspensa alkaloid A on β-glucuronidase activity was calculated according to the following formula:

[0071] Inhibition rate = [(OD 刺激组 -OD 给药组 ) / (OD 刺激组 -OD 溶剂对照组 )]×100%

[0072] 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.

[0073] After calculation, the inhibition rates of different concentrations of R-type Forsythia suspensa alkaloid A on β-glucuronidase activity are as follows:

[0074] Table 2 Inhibition rate of β-glucuronidase activity by different concentrations of R-type Forsythia suspensa alkaloid A

[0075] Concentration of R-type Forsythia suspensa alkaloid A (μmol / L) n Inhibition rate (%) 0.1 4 22.3±1.68 1 4 38.4±2.16 10 4 58.7±2.42 100 4 61.5±1.95

[0076] As shown in Table 2, R-type Forsythia suspensa alkaloid A has a good inhibitory effect on the activity of β-glucuronidase released by neutrophils induced by platelet activating factor.

[0077] 2. Inhibitory effect of R-type Forsythia suspensa alkaloid A on the release of ROS, TNF-α, IL-1β, and IL-6 inflammatory cytokines from RAW264.7 macrophages induced by lipopolysaccharide

[0078] Mouse macrophage RAW264.7 was prepared by conventional methods, and then the cells were washed twice with RPMI-1640 culture medium, counted, and the cell concentration was adjusted to 2×10 6 / mL to obtain a cell suspension.

[0079] The cell suspension was inoculated in a 24-well plate, 1 mL per well, and placed in a 37°C, 5% CO2 incubator for 2 hours, then washed with phosphate buffered saline (PBS) to remove non-adherent cells, and then 1 mL of RPMI-1640 culture medium containing different concentrations (0.1 μmol / L, 1 μmol / L, 10 μmol / L, 100 μmol / L) of R-type Forsythia alkaloid A was added to each well, and the wells without R-type Forsythia alkaloid A were used as solvent control groups, placed in a 37°C, 5% CO2 incubator for 0.5 hours, and then 1 μL of lipopolysaccharide (LPS) with a concentration of 1 mg / mL was added, and placed in a 37°C, 5% CO2 incubator for 6 hours. After the culture was completed, the supernatant was collected and the corresponding ELISA kits were used to determine the contents of TNF-α, IL-1β, IL-6 and ROS. The experimental data were statistically processed and expressed as mean ± SD.

[0080] The inhibition rate of different concentrations of R-type Forsythia suspensa alkaloid A on the release of ROS, TNF-α, IL-1β, and IL-6 by mouse macrophages RAW264.7 was calculated according to the following formula:

[0081] Inhibition rate = [(C 刺激组 -C 给药组 ) / (C 刺激组 -C 溶剂对照组 )]×100%

[0082] Among them, C 刺激组 is the amount of TNF-α, IL-1β, IL-6 or ROS contained in the supernatant collected from the wells added with LPS, C 给药组 is the amount of TNF-α, IL-1β, IL-6 or ROS contained in the supernatant collected from the wells added with R-type Forsythia suspensa alkaloid A and lipopolysaccharide, C 溶剂对照组 It is the amount of TNF-α, IL-1β, IL-6 or ROS contained in the supernatant collected from well A without adding R-type Forsythia suspensa alkaloids.

[0083] After calculation, the inhibition rates of different concentrations of R-type Forsythia suspensa alkaloid A on the release of ROS, TNF-α, IL-1β, and IL-6 by mouse macrophages RAW264.7 were as follows:

[0084] Table 3 Inhibition rate of different concentrations of R-type Forsythia suspensa alkaloid A on the release of ROS, TNF-α, IL-1β, and IL-6 in mouse macrophages RAW264.7

[0085]

[0086] As shown in Table 3, R-type Forsythia suspensa alkaloid A has a good inhibitory effect on the release of ROS, TNF-α, IL-1β, and IL-6 from mouse macrophages RAW264.7 induced by lipopolysaccharide.

[0087] The above results show that R-type Forsythia suspensa alkaloid A has good anti-inflammatory activity. As a natural product, it can be developed into a new anti-inflammatory Chinese medicine and has certain application prospects.

[0088] 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. R-type Forsythia suspensa alkaloid A, characterized in that: The R-type Forsythia suspensa alkaloid A is a norfloscan-type diterpene alkaloid with a molecular formula of C 17 H 25 NO, the structural formula is as follows: Among them, the absolute configuration of C4 is R type.

2. The method for preparing the R-type Forsythia suspensa alkaloid A according to claim 1, characterized in that: The following steps are involved: (1) taking Forsythia suspensa fruit, extracting it with methanol or 95% ethanol under reflux, recovering the organic solvent, and obtaining 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) dissolving extract II with n-butanol, then extracting with 3% to 4% hydrochloric acid, and combining 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, gradient eluting with 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% to 8% acetic acid as the developing solvent, intercepting the alkaloid band, eluting with acetone and concentrating under reduced pressure to obtain a concentrate II; (7) Separating the concentrated solution II by preparative high performance liquid chromatography, 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 7.0 mL / min, injection volume 2 mL, detection wavelength 345 nm, collect the components with retention time between 16 min and 18 min, concentrate under reduced pressure, crystallize, and dry to obtain R-type Forsythia suspensa alkaloid A.

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

4. The method for preparing R-type Forsythia suspensa alkaloid A 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 R-type Forsythia suspensa alkaloid A 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 R-type Forsythia suspensa alkaloid A according to claim 2, characterized in that: In step (4), extraction is performed three times with an equal volume of chloroform or ethyl acetate.

7. The method for preparing R-type Forsythia suspensa alkaloid A 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 R-type Forsythia suspensa alkaloid A 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 R-type Forsythia suspensa alkaloid A according to claim 1 in the preparation of anti-inflammatory drugs.

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