阅读说明：本技术 岷江瑞香中的愈创木烷倍半萜衍生物及其应用 (Guaiane sesquiterpene derivative in regachia and application thereof ) 是由 宋少江 黄肖霄 赵鹏 李志远 于 2021-08-24 设计创作，主要内容包括：岷江瑞香中的愈创木烷倍半萜衍生物及其应用,属于医药科技领域,涉及从瑞香科瑞香属植物岷江瑞香中提取分离的三个结构新颖的愈创木烷倍半萜衍生物。本发明所述化合物是采用硅胶、ODS柱层析、HP20柱层析、HPLC等色谱方法得到的。本发明还提供所述三个化合物在制备抗神经炎症药物中的应用。本发明制备方法简单,重现性好,所得化合物纯度高,获得的化合物具有较好的对LPS诱导的BV-2细胞系抗神经炎症活性。(Guaiane sesquiterpene derivatives in regaining daphne and application thereof, belong to the field of medical science and technology, and relate to three guaiane sesquiterpene derivatives with novel structures extracted and separated from the regaining daphne of the family Thymelaeaceae. The compound is obtained by adopting chromatographic methods such as silica gel, ODS column chromatography, HP20 column chromatography, HPLC and the like. The invention also provides application of the three compounds in preparing anti-neuritis medicines. The preparation method is simple, the reproducibility is good, the purity of the obtained compound is high, and the obtained compound has good anti-neuritis activity on BV-2 cell line induced by LPS.)

1. A guaiane sesquiterpene derivative in regained jiang daphne is characterized by having the following structure:

2. the process for the preparation of guaiane sesquiterpene derivatives according to claim 1 comprising the steps of:

(1) extracting dried whole herbs of regale daphne with ethanol, combining extracting solutions and concentrating to obtain an extract; sequentially extracting the obtained extract by using ethyl acetate and n-butanol, performing gradient elution on the ethyl acetate part and the n-butanol part by silica gel column chromatography respectively and dichloromethane/methanol according to the volume ratio of 100: 1-1: 1, and collecting 4 fractions Fr.A-Fr.D;

(2) subjecting the fraction Fr.A to polyamide column chromatography, eluting with ethanol water in a gradient manner, and collecting 2 fractions Fr.A 1-Fr.A 2;

(3) the fraction Fr.A1 is subjected to HP20 column chromatographic separation and ODS column separation sequentially, and is subjected to gradient elution by ethanol water, and 5 fractions Fr.A1-I, Fr.A1-II, Fr.A1-III, Fr.A1-IV and Fr.A1-V are collected;

(4) subjecting the fraction Fr.A1-I to silica gel column chromatography, performing gradient elution by petroleum ether/ethyl acetate according to the volume ratio of 100: 1-5: 1, subjecting the 2 nd fraction Fr.A1-I-2 obtained by elution to preparative HPLC, eluting by methanol/water to obtain 8 fractions Fr.A1-I-2-1-Fr.A1-I-2-8, subjecting the Fr.A1-I-2-3 to semi-preparative HPLC chromatography, eluting by acetonitrile/water, separating and purifying to obtain a compound 1, and subjecting the Fr.A1-I-2-7-5 to semi-preparative HPLC chromatography, eluting by methanol/water, and separating and purifying to obtain a compound 2 and a compound 3.

3. The method according to claim 2, wherein the ethanol in step (1) is 70-80% industrial ethanol, and the reflux extraction is performed 2-3 times.

4. The method according to claim 2, wherein the gradient concentration of ethanol/water in the step (2) is: 20% and 70%.

5. The process according to claim 2, wherein the gradient concentrations of the ethanol water separated by the HP20 column chromatography and the ODS column chromatography in step (3) are as follows: 20%, 50%, 90% and 40%, 50%, 60%, 70%, 80%, 90%.

6. A pharmaceutical composition comprising the guaiane sesquiterpene derivatives of claim 1 in association with a pharmaceutically acceptable excipient or/and a carrier.

7. Use of the guaiane sesquiterpene derivatives according to claim 1 or the pharmaceutical composition according to claim 6 in the preparation of anti-neuritis drugs.

8. A regadenox extract comprising the guaiane sesquiterpene derivative of claim 1.

9. The application of the Thamnolia regale extract as claimed in claim 8, which is used for preparing the anti-neuritis medicine.

Technical Field

The invention belongs to the technical field of medicines, relates to a method for preparing guaiane sesquiterpene derivatives from regaining senegal and application of the derivatives, and particularly relates to application of 3 guaiane sesquiterpene derivatives extracted and separated from regaining senegal in preparing anti-neuritis drugs.

Background

Neuroinflammation is a condition observed in the Central Nervous System (CNS) under the stimulation of infection, toxic metabolites, trauma or autoimmunity; the main characteristics of the disease are symmetric sensory movement and vegetative nerve dysfunction, and a plurality of common diseases are related to neuroinflammation, wherein the common diseases comprise Alzheimer Disease (AD), cerebral hemorrhage, traumatic brain injury and the like. The method for treating neuroinflammation mainly comprises supplementing vitamin B₁₂Administration of some neuroprotective drugs, etc.

During the previous research on the chemical composition of Daphne genkwa sieb.et Zucc, some guaiane sesquiterpene derivatives with better neuroprotective activity were found, and then during the research on other Daphne plants, 3 guaiane sesquiterpene derivatives with neuritis resistance were found from Minjiang Daphne, one area from Wentang to Mao county, widely distributed in Sichuan province. The sesquiterpene derivative is prepared by extracting and extracting the regadeno regale, and has good activity of resisting neuritis. The compounds and the activities thereof related to the present invention have not been reported in patents or literatures so far.

Disclosure of Invention

The primary object of the present invention is to provide 3 guaiane sesquiterpene derivatives prepared from Min jiang daphne.

The invention also provides a preparation method of the 3 guaiane sesquiterpene derivatives and application of the guaiane sesquiterpene derivatives in preparation of anti-neuritis drugs.

The 3 guaiane sesquiterpene derivatives extracted and separated from daphne plant regadenoides of daphne of Thymelaeaceae have the following structures:

the method for extracting and separating the 3 guaiane sesquiterpene derivatives from the Thamnolia regale comprises the following steps:

(1) extracting dried whole herbs of regale daphne with ethanol, combining extracting solutions and concentrating to obtain an extract; sequentially extracting the obtained extract by using ethyl acetate and n-butanol, performing gradient elution on the ethyl acetate part and the n-butanol part by silica gel column chromatography respectively and dichloromethane/methanol according to the volume ratio of 100: 1-1: 1, and collecting 4 fractions Fr.A-Fr.D;

(2) subjecting the fraction Fr.A to polyamide column chromatography, eluting with ethanol water in a gradient manner, and collecting 2 fractions Fr.A 1-Fr.A 2;

(3) the fraction Fr.A1 is subjected to HP20 column chromatographic separation and ODS column separation sequentially, and is subjected to gradient elution by ethanol water, and 5 fractions Fr.A1-I, Fr.A1-II, Fr.A1-III, Fr.A1-IV and Fr.A1-V are collected;

(4) subjecting the fraction Fr.A1-I to silica gel column chromatography, performing gradient elution by petroleum ether/ethyl acetate according to the volume ratio of 100: 1-5: 1, subjecting the 2 nd fraction Fr.A1-I-2 obtained by elution to preparative HPLC, eluting by methanol/water to obtain 8 fractions Fr.A1-I-2-1-Fr.A1-I-2-8, subjecting the Fr.A1-I-2-3 to semi-preparative HPLC chromatography, eluting by acetonitrile/water, separating and purifying to obtain a compound 1, and subjecting the Fr.A1-I-2-7-5 to semi-preparative HPLC chromatography, eluting by methanol/water, and separating and purifying to obtain a compound 2 and a compound 3.

The above extraction method, wherein:

the ethanol in the step (1) is 70-80% industrial ethanol, and reflux extraction is carried out for 2-3 times.

The gradient concentration of the ethanol/water in the step (2) is as follows: 20% and 70%.

The gradient concentrations of the ethanol water separated by the HP20 column chromatography and the ODS column chromatography in the step (3) are respectively as follows: 20%, 50%, 90% and 40%, 50%, 60%, 70%, 80%, 90%.

The obtained compound is identified by a system structure, and the result is as follows, and the corresponding map is shown in figures 2-26:

daphenonid a (1): a colorless crystal (methanol),HRESIMS gave the excimer peak [ M + H ]]⁺Peak m/z 291.1220(calcd for C)₁₆H₁₉O₅291.1227), in combination¹H-NMR、¹³C-NMR presumed to be of the formula C₁₆H₁₈O₅The unsaturation was calculated to be 8. An absorption peak at a wavelength of 238nm, UV (MeOH). lambda._max(logε):238nm(0.81)。

¹H-NMR(600MHz,CDCl₃) Middle, delta_H6.16(1H, d, J ═ 1.5Hz) is an olefinic hydrogen proton signal, δ_H3.93(1H, s) is a hydroxyl signal, δ_H2.11(1H, brd, J. 5.3Hz) is a methine proton signal, δ_H3.63(3H, s) is the proton signal on the methoxy radical, δ_H2.02(3H,d,J＝1.4Hz)，δ_H1.23(3H, s) is the hydrogen signal on two methyl carbons. Combined with HSQC spectrum, pair¹³C-NMR(150MHz,CDCl₃) Analysis of the spectra showed 16 carbon signals, of which there were four sp³Hybrid quaternary carbon signal (delta)_C84.7,68.2,60.5, 59.0), four sp)²Hybrid quaternary carbon signal (delta)_C212.6,198.0,171.0, 158.9), a double bond carbon signal (. delta.) with attached protons_C125.4), one methine carbon signal (δ)_C53.5), three methylene carbon signals (. delta.))_C38.6, 33.0, 32.2) and three methyl carbon signals (δ)_C52.5, 22.7, 11.9, wherein_C52.5 carbon-to-oxygen). Consider that there is one pairBond, three carbonyl groups and eight unsaturations, it is concluded from the above information that compound 1 may be a sesquiterpene possessing a tetracyclic system.

By analysis of the HMBC spectra, H-5 (. delta.)_H2.11) and C-3 (. delta.)_C212.6) correlation, H-2 (. delta.))_H2.98,δ_H2.57) and C-5 (. delta.)_C53.5) correlation and H₃-15(δ_H1.23) and C-3 (. delta.)_C212.6)，C-4(δ_C59.0) and C-5 (. delta.))_C53.5) confirms the presence of the five-membered carbocyclic ring A consisting of C-1, C-2, C-3, C-4, C-5 and its presence of the ketone at C-3 and of the methyl group at C-4. H₃-16(δ_H3.63) and C-13 (. delta.))_C171.0) confirmed the presence of methoxyacyl groups. To further determine the planar structure of compound 1, we obtained a series of possible planar structures that fit the 2D nuclear magnetic data of compound 1 using a computer-aided structure analysis system (CASE), and then selected the most likely structure based on empirical NMR chemical shift predictions. "CASE results and key NOESY related and crystal structure of compound 1" as shown below, 4 structures were generated within 1.0s and based on¹³The average deviations of C were ranked. Compound 1 was determined to match the best structure according to the lowest standard deviation value. In the MCD diagram, methylene hydrogen (. delta.) at the C-12 position_H2.31, 1.82) associated with the presence of five quaternary carbons (C-1, C-3, C-4, C-7, C-11) and one tertiary carbon (C-5), determined the bridging of C-1 and C-7 together by C-11 and the linkage of methoxyacyl groups at the C-11 position, and the presence of a five-membered carbocyclic ring B consisting of C-1, C-4, C-5, C-12 and C-11. OH in position 7 (. delta.)_H3.93) and C-6 (. delta.))_C32.3)，C-8(δ_C198.0)，C-11(δ_C68.2) there is a correlation, indicating that the 7-OH is attached to C-7 and that a ketocarbonyl group is present at the C-8 position. Based on the above information, in combination¹H-¹The spin-coupled system shown in the H COSY spectra, illustrates the presence of a cage-like structure. Finally, it was concluded that the planar structure of Compound 1 is a tetracyclic [5.3.2.0 ]^1,6.0^4,11]Novel caged sesquiterpenes with dodecane skeleton.

The relative configuration of Compound 1, H-5 (. delta.) is next determined by NOESY spectroscopy_H2.11) and H-15 (. delta.))_H1.23) NOE, indicating that H-5 is on the same side as H-15, ascribed to the α orientation; the above inference is further confirmed by the correlation of H-2 β/H-12 β and H-6 β/H-12 α. The relative configuration was assigned as 1R,4R,5R,7S,11S due to the steric hindrance of the 5/5/5/6 caged four-ring system and the rigid connection of the five-membered carbocyclic ring C and ring D. Then, the planar structure and relative configuration of compound 1 were further determined using NMR chemical shift calculations. To determine its absolute configuration, compound 1 was subjected to computational ECD at B3LYP/6-311+ + G (2d, p) levels, where the calculated ECD spectrum of (1R,4R,5R,7S,11S) -1 matched the experimental ECD spectrum of compound 1. After many attempts, single crystals of compound 1 were obtained in a mixed solvent of dichloromethane/methanol (1:1), and X-ray diffraction results confirmed assignment of absolute configuration and presumption of steric structure (fig. 10, CCDC 1876453, flip parameter ═ 0.04 (3)). After the database search of the scifinider, the compound 1 is an unreported new compound and is named as Daphnenoid A.

Daphnenoid B and Daphnenoid C (2-3): a colorless oil, compound 2 having an optical rotation value of The optical rotation value of the compound 3 isHRESIMS gave the excimer peak M/z273.1464[ M + Na ] of Compound 2]⁺(calcd for C₁₅H₂₂O₃Na,273.1461), excimer peak M/z273.1462[ M + Na ] of Compound 3]⁺(calcd for C₁₅H₂₂O₃Na, 273.1461). Bonding of¹H-NMR、¹³C-NMR presumed that both of the compounds 2 and 3 have the formula C₁₅H₂₂O₃The unsaturation was calculated to be 5. For compounds 2 and 3¹³C-NMR(150MHz,CDCl₃) And analysis of the HSQC spectra showed 15 carbon signals, including one sp³Hybridized quaternary carbon signal, three sp²A hybridized quaternary carbon signal, one protonated olefinic hydrogen carbon signal, three methine carbon signals, five methylene carbon signals, and two methyl carbon signals. In all the observed signals, one double bond and two carbonyl groups occupy three unsaturations, the remaining two unsaturations possibly constituting two carbocyclic skeletons, indicating, according to the above information, that compounds 2 and 3 are a pair of sesquiterpenes having the same bicyclic skeleton.

For HMBC spectral analysis, H₂Correlation of-2 with C-1, C-4 and C-5 and H₂The correlation of-3 with C-1 and C-5 indicates the presence of a five-membered carbocyclic ring A, and the presence of a ketocarbonyl group in the C-1 position and a methyl group in the C-4 position. H₂The correlation of-13 with HMBC at C-7, C-11 and C-12 also indicates the presence of a terminal double bond. HMBC's with H-4 and C-6 and C-12, H-12 and C-1 and C-6 and H-6 and C-1 and C-12 illustrate bicyclic ring systems linked together by a spiro carbon atom, C-5. Assignment of Ring B is made difficult by the critical HMBC-related deletion of H-7 and the overlap of chemical shift values of H-8 and H-3, although H-6 α is associated with C-11, there is not enough evidence to justify the presence of the five-membered carbocyclic ring B. Furthermore, the peculiar 5/5 spiro sesquiterpene in nature has not been discovered, and even the 5/6 spiro system sesquiterpenes are rare in plants and marine microorganisms. To further confirm the structure of Ring B accurately, a computer-aided Structure analysis System (CASE) was used to generate all possible structures, with structures having 5/5 fused spiro ring systems having the lowest possible structure compared to other candidate structures¹³C mean deviation, is considered the most reasonable structure. This was confirmed by NMR chemical shift calculations. Finally, the plane structures of Daphnenoid B and Daphnenoid C (2-3) were determined and their spiro [4.4 ]]The nonane skeleton is also determined.

By analysis of the NOESY spectra of Compounds 2 and 3, H-6. beta. was compared with H-9 and H₃Correlation of-15 demonstrates CH₃-15 and H-7 are both in the alpha-orientation; the correlation of H-7 with H-12 was found in compound 2, indicating that H-7 is in the same side as H-12 in the alpha-orientation, whereas no correlation of H-7 with H-12 was found in compound 3, but a correlation of H-4 with H-12 was found, indicating that H-12 is in the beta-orientation in compound 3, indicating that compounds 2 and 123 is a diastereomer. In addition, to further confirm the diastereomeric differences of compounds 2 and 3, NMR chemical shifts were then calculated for the two candidate structures (A and B) in chloroform in a PCM solvent model at mPW1PW91/6-311+ G (d, p)// ω B97XD/6-31G (d) levels, first using R obtained based on quantum mechanical chemical shift analysis²Value to distinguish Compounds 2 and 3, but R²The difference in values was small and could not be used to confirm the relative configuration of H-12 for compounds 2 and 3; thereafter, MAE was used_ΔΔδFrom the configuration of H-12, lowest MAE_ΔΔδThe values indicate that compound 2 best matches the combination of structure a and compound 3 best matches structure B; finally, using another highly reliable parameter for diastereomer structural assignment CP3, CP3 calculation showed that the 4R,5R,7S, 12R configuration (i.e. structure B) belongs to compound 3, and based on experiments and calculations¹H and¹³the probability was determined to be 100% as a result of the chemical shift comparison. Accordingly, NOESY analysis, R²Value, MAE_ΔΔδAnd CP3 results were consistent, so the relative configurations of compounds 2 and 3 were 4R,5R,7S, 12S and 4R,5R,7S, 12R, respectively. But due to the flexibility of the 2-butanone moiety, no cultivation success was achieved for single crystals of compounds 2 and 3; and finally, comparing the experiment and the calculated ECD spectrogram to determine the absolute configurations of the compounds 2 and 3, wherein the absolute configurations of the compounds 2 and 3 are respectively determined as 4S,5S,7R,12R and 4S,5S,7R and 12S.

Upon database search by scifininder, compounds 2 and 3 were unreported new compounds, named daphenoid B (2) and daphenoid C (3).

Of compounds 1 to 3¹H NMR data and¹³c NMR data

HMBC and of compounds 1-2¹H-¹H COSY correlation

CASE results for Compound 1 correlate with key NOESY and crystal structure

Key NOESY correlation of Compounds 2-3

The invention also provides a pharmaceutical composition, which comprises the guaiane sesquiterpene derivative, a pharmaceutically acceptable salt of the compound, a pharmaceutically acceptable excipient or/and a carrier.

The invention also provides application of the Thamnolia regale extract in a medicament for treating the neuritis.

The invention also provides application of the 3 guaiane sesquiterpene derivatives in medicines for treating neuritis.

The 3 guaiane sesquiterpene derivatives related to the invention were evaluated for their anti-neuritic activity. Results compounds 1-3 showed comparable LPS-induced anti-neuritic activity against BV-2 mouse microglia as the positive drug Dex. Therefore, the guaiane sesquiterpene derivative has the potential of clinically treating the anti-neuritis drugs.

The guaiane sesquiterpene derivatives have the advantages that the guaiane sesquiterpene derivatives are all novel compounds, are novel in structure, are all optically pure compounds with determined stereo configuration, have good anti-neuritis activity and have further development value.

Drawings

The structures of compounds 1-3 of FIG. 1;

figure 2 mass spectrum of compound 1;

FIG. 3 preparation of Compound 1¹H-NMR Spectroscopy (600MHz, CDCl)₃)；

FIG. 4 preparation of Compound 1¹³C-NMR Spectroscopy (150MHz, CDCl)₃)；

FIG. 5 HSQC spectra (600MHz, CDCl) of Compound 1₃)；

FIG. 6 HMBC spectra (600MHz, CDCl) of Compound 1₃)；

FIG. 7 preparation of Compound 1¹H-¹H COSY spectrum;

FIG. 8 NOESY spectrum (600MHz, CDCl) of Compound 1₃)；

FIG. 9 generation of a possible structure and MCD map of Compound 1 from the CASE system;

FIG. 10 comparison of Compound 1 calculation with Experimental ECD and single crystal plot;

figure 11 mass spectrum of compound 2;

FIG. 12 preparation of Compound 2¹H-NMR Spectroscopy (600MHz, CDCl)₃)；

FIG. 13 preparation of Compound 2¹³C-NMR Spectroscopy (150MHz, CDCl)₃)；

FIG. 14 HSQC spectra (600MHz, CDCl) of Compound 2₃)；

FIG. 15 HMBC spectra (600MHz, CDCl) of Compound 2₃)；

FIG. 16 preparation of Compound 2¹H-¹H COSY spectrum;

FIG. 17 NOESY spectrum (600MHz, CDCl) of Compound 2₃)；

Figure 18 mass spectrum of compound 3;

FIG. 19 preparation of Compound 3¹H-NMR Spectroscopy (600MHz, CDCl)₃)；

FIG. 20 preparation of Compound 3¹³C-NMR Spectroscopy (150MHz, CDCl)₃)；

FIG. 21 HSQC spectra (600MHz, CDCl) of Compound 3₃)；

FIG. 22 HMBC spectra (600MHz, CDCl) of Compound 3₃)；

FIG. 23 preparation of Compound 3¹H-¹H COSY spectrum;

FIG. 24 NOESY spectrum (600MHz, CDCl) of Compound 3₃)；

FIG. 25 experiments and calculations of Compounds 2 and 3¹³Chemical potential of C NMRShifted CP3 computation and MAE_ΔΔδA parameter;

figure 26 experimental and calculated ECD spectra for compounds 2 and 3;

FIG. 27 protective activity of compounds 1-3 against LPS-induced neuroinflammatory injury of BV-2 mouse microglia; all data are expressed as means ± SD (three independent experiments); dex is a positive drug.

Detailed Description

The examples set out below are intended to assist the person skilled in the art in a better understanding of the invention, but do not limit it in any way.

Example 1: the preparation method of guaiane sesquiterpene derivatives 1-3 from regained daphne comprises the following steps:

(1) taking dry whole regadenoson plants (48kg) and carrying out reflux extraction for three times by 75% industrial ethanol, combining extracting solutions and concentrating to obtain an extract. The obtained extract is extracted by ethyl acetate and n-butanol successively. The ethyl acetate layer extract (600g) and the n-butanol layer extract (1.2kg) were subjected to silica gel column chromatography under reduced pressure, and gradient elution was performed with a methylene chloride/methanol system at a volume ratio of 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1:1, and 4 fractions Fr A to Fr D were collected in total.

(2) The fraction fr.a (200g) was subjected to polyamide column chromatography and gradient elution (20%, 70%) with an ethanol water system, and 2 fractions fr.a1 to fr.a2 were collected in total.

(3) The fraction Fr.A1(90g) was first subjected to HP20 column chromatography eluting with an aqueous ethanol gradient (20%, 50%, 90%), and then subjected to ODS column chromatography eluting with an aqueous ethanol gradient (40%, 50%, 60%, 70%, 80%, 90%), and the fractions were collected to obtain 5 fractions Fr.A1-I, Fr.A1-II, Fr.A1-III, Fr.A1-IV, Fr.A1-V.

(4) Fraction fr. a1-I (12g) was subjected to silica gel column chromatography eluting with a petroleum ether/ethyl acetate gradient (volume ratio 100:1, 50:1, 30:1, 20:1, 10:1, 5: 1). The obtained 2 nd fraction Fr.A1-I-2 was subjected to preparative HPLC and eluted with methanol/water (volume ratio 40:60) to obtain 8 fractions (Fr.A1-I-2-1 to Fr.A1-I-2-8), Fr.A1-I-2-3 was subjected to semi-preparative HPLC chromatography and eluted with acetonitrile/water (volume ratio 20:80) to isolate and purify to obtain compound 1(12mg), and Fr.A1-I-2-7-5 was subjected to semi-preparative HPLC chromatography and eluted with methanol/water (volume ratio 25:75) to isolate and purify to obtain compound 2(1.2mg) and compound 3(2.2 mg). The structures of the obtained compounds 1 to 3 are shown in FIG. 1.

Example 2: and detecting the anti-neuritis activity of the obtained guaiane sesquiterpene derivatives 1-3.

BV2 mouse microglia were placed in 10% FBS-containing DMEM medium and incubated at 37 ℃ with 5% CO₂Culturing in a saturated humidity incubator. Passage was performed when the cell confluence reached 80%.

The anti-neuritis activity of the guaiane sesquiterpene derivative 1-3 obtained from regained daphne is examined through NO content detection. Seeding BV2 microglia in 96-well plate with plate density of 6 × 10⁴Each well is 100 μ L, 3 multiple wells are arranged, and the mixture is placed at 37 ℃ and 5% CO₂Culturing in a saturated humidity incubator. After 12h, the drug (10. mu.M) was dosed. After 1h, BV2 microglia cells were stimulated by the addition of 10. mu.g/mL LPS. After 24 hours, the culture supernatant was collected, and the nitric oxide content in the supernatant was measured using an NO assay kit.

First, the Griess Reagent I and Reagent II are restored to room temperature, the culture supernatant is added into a 96-well plate according to 50 mu L of each well, then the Griess Reagent I and Reagent II are sequentially added, and the absorbance is measured at 540nm by adopting an enzyme-labeling instrument. All experiments were performed in parallel and repeated three times, blanks and positive controls were set up and analyzed using GraphPad Prism 6 software, with the results shown in fig. 27.