阅读说明：本技术 地胆草中吉玛烷型倍半萜内酯类化合物及其制备方法和应用 (Gilmaxane type sesquiterpene lactone compound in elephantopus scaber and preparation method and application thereof ) 是由 宋少江 黄肖霄 白明 徐伟 于 2021-10-19 设计创作，主要内容包括：地胆草中吉玛烷型倍半萜内酯类化合物及其制备方法和应用,属于医药技术领域,涉及从菊科地胆草属植物地胆草(Elephantopus scaber L.)中提取分离的六个新吉玛烷型倍半萜内酯类化合物scaberxone A,scaberxone B,scaberxone C,scaberxone D,scaberxone E,scaberxone F。上述6种化合物均具有相同的吉玛烷型倍半萜内酯母核。本发明还提供所述6种新化合物的制备方法和在制备抗炎药物方面的应用。本发明制备方法简单易行,重现性比较好,纯度较高。(A germarine type sesquiterpene lactone compound in Elephantopus scaber, a preparation method and an application thereof belong to the technical field of medicines, and relate to six novel germarine type sesquiterpene lactone compounds, namely scaberxone A, scaberxone B, scaberxone C, scaberxone D, scaberxone E and scaberxone F, which are extracted and separated from Elephantopus scaber L (Elephantopus scaber L.) of Elephantopus in the compositae. The 6 compounds all have the same parent nucleus of the germacrane type sesquiterpene lactone. The invention also providesProvides a preparation method of the 6 new compounds and application in preparing anti-inflammatory drugs. The preparation method is simple and feasible, and has good reproducibility and high purity.)

1. A germacane-type sesquiterpene lactone compound in elephantopus scaber is characterized in that the germacane-type sesquiterpene lactone compound is any one of 6 compounds shown as follows:

2. the gigaalkane-type sesquiterpene lactone compounds in Elephantopus scaber according to claim 1, wherein the 6 compounds are isolated from Elephantopus scaber L (Elephantopus scaber L.) of Elephantopus of the family compositae.

3. The preparation method of the gemma alkane type sesquiterpene lactone compound in the elephantopus scaber as claimed in claim 1, which is characterized by comprising the following steps:

(1) extracting dry herba Ajugae Bracteosae with 70-80% ethanol, mixing extractive solutions, concentrating to obtain extract, extracting with n-butanol, subjecting the obtained components to silica gel column chromatography, performing gradient elution with dichloromethane-methanol system at 50:1-1:2 at equal degree, and collecting 5 fractions I-V;

(2) subjecting fraction I to HP20 column chromatography, and gradient eluting with ethanol-water system to obtain 3 fractions A1, A2 and A3;

(3) fraction A2 is eluted by open ODS column chromatography with ethanol-water system to obtain 4 fractions Fr.1-Fr.4;

(4) fr.2 was eluted with a dichloromethane-methanol system 50:1-0:1 by silica gel column chromatography to give 5 subfractions Fr.21-Fr.25 on the basis of TLC analysis;

(5) separating Fr.23 on preparative reverse phase high performance liquid chromatography using a methanol-water mobile phase to obtain fractions Fr.231-Fr.236;

(6) fr.233 was isolated using acetonitrile-water mobile phase on semi-preparative reverse phase high performance liquid chromatography to afford compounds 1-6.

4. The method for preparing the gimerane type sesquiterpene lactone compounds in the elephantopus scaber as claimed in claim 3, wherein in the step (1), the ethanol concentration is 70-80%, the extraction is reflux extraction, and the extraction is carried out 3-4 times for 2-3 hours each time; the herba Ajugae is herba Ajugae (Elephantopus scaber L.) of Elephantopus of Compositae.

5. The method for preparing the gimerane-type sesquiterpene lactone compounds in the elephantopus scaber according to claim 3, wherein the ethanol-water system in the step (2) is a 10-50% ethanol-water system; in the step (3), the ethanol-water system is a 20-60% ethanol-water system.

6. The method for preparing the gimerane type sesquiterpene lactones in elephantopus scaber according to claim 3, wherein in the step (5), the content of methanol in the mobile phase of methanol-water is 33%.

7. The method for preparing the gimerane type sesquiterpene lactone compounds in the elephantopus scaber as claimed in claim 3, wherein in the step (6), the acetonitrile content in the acetonitrile-water mobile phase is 15-35%.

8. A pharmaceutical composition comprising one or more of the gemma alkane type sesquiterpene lactones of claim 1.

9. The use of the germacrane type sesquiterpene lactone compounds of elephantopus scaber as claimed in claim 1 or 2, wherein the germacrane type sesquiterpene lactone compounds are used in the preparation of anti-inflammatory drugs.

10. Use of a pharmaceutical composition according to claim 8 for the preparation of an anti-inflammatory agent.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to novel germacrane type sesquiterpene lactone in a plant elephantopus scaber, a preparation method thereof and application of the compound in preparation of anti-inflammatory drugs.

Background

Herba elephantopi: herba Elephantopi scaberis (Elephantopus scaber L.) also called herba Elephantopi scaberis, is a plant of the genus Elephantopi in the family of Compositae, and is widely distributed in tropical regions of America, Asia, and Africa; is distributed in provinces such as Zhejiang, Jiangxi, Fujian, Guizhou and Yunnan in China. There are more than 30 kinds of elephantopus, and there are only two kinds in our country, namely elephantopus scaber and elephantopus tomentosus. Herba Ajugae Bracteosae is used as medicine mainly at root, has bitter taste and cold nature, has effects of clearing heat, cooling blood, removing toxic substance, and promoting diuresis, and can be used for treating common cold, tonsillitis, conjunctivitis, jaundice, etc. In addition, elephantopus scaber is also widely used as a soup cooking food material. In recent years, various chemical components such as sesquiterpene lactones, flavonoids, triterpenes, lignans, aromatic compounds and the like have been separated from elephantopus scaber.

Inflammation is a defense response of higher animals against damaging stimuli, however, excessive inflammatory responses can cause severe damage to various tissues and organs, and chronic diseases such as asthma, cancer, cardiovascular diseases and neurodegenerative diseases belong to the category of inflammatory diseases, which are spreading worldwide and have evolved into important public health problems seriously jeopardizing human health and sustainable socioeconomic development. Thus, natural anti-inflammatory compounds derived from plants have attracted the attention of many researchers and have become an important field of research that has attracted attention in recent years.

Disclosure of Invention

The invention provides six new germacrane type sesquiterpene lactones separated from Elephantopus scaber L (Elephantopus scaber L.) of Elephantopus of Compositae:

the method for preparing the novel germacrane type sesquiterpene lactone compounds by using the elephantopus scaber comprises the following steps:

(1) extracting dry herba Ajugae Bracteosae with 70-80% ethanol, mixing extractive solutions, concentrating to obtain extract, extracting with n-butanol, subjecting the obtained components to silica gel column chromatography, performing gradient elution with dichloromethane-methanol system at 50:1-1:2 at equal degree, and collecting 5 fractions (I-V).

(2) Subjecting fraction I to HP20 column chromatography, and gradient eluting with ethanol-water system to obtain 3 fractions A1, A2 and A3.

(3) The obtained fraction A2 was eluted through an open ODS column chromatography using an ethanol-water system to give 4 fractions Fr.1-Fr.4.

(4) Fr.2 was eluted by silica gel column chromatography with a dichloromethane-methanol system 50:1-0:1, giving 5 subfractions Fr.21-Fr.25 on the basis of TLC analysis.

(5) Separation of Fr.23 on preparative reverse phase high performance liquid chromatography using a methanol-water mobile phase gave fractions Fr.231-Fr.236.

(6) Fr.233 was isolated using acetonitrile-water mobile phase on semi-preparative reverse phase high performance liquid chromatography to afford compounds 1-6.

The method for preparing the novel germacrane type sesquiterpene lactone compounds by using the elephantopus scaber comprises the following steps:

in the step (1), the concentration of the ethanol is 70-80%, and the extraction is ethanol reflux extraction for 3-4 times, and each time lasts for 2-3 hours. The herba Ajugae is herba Ajugae (Elephantopus scaber L.) of Elephantopus of Compositae.

In the step (2), the ethanol-water system is a 10-50% ethanol-water system.

In the step (3), the ethanol-water system is a 20-60% ethanol-water system.

In the step (5), the content of methanol in the mobile phase of methanol-water is 33%.

In the step (6), the acetonitrile content in the acetonitrile-water mobile phase is 15-35%.

The results of systematic structural identification of the obtained compounds 1 to 6 are as follows:

identifying the structure of compound 1-6 by high resolution mass spectrometry, NMR, ECD calculation, and X-single crystal diffraction, and the corresponding spectrogram is shown in figure 1-30.

Scaberoxone A (1) white noneShaping the powder;-70.0(c 0.1, MeOH); HR-ESI-MS shows that the peak of the quasi-molecular ion is [ M + H ]]⁺295.1178(cal.C₁₅H₁₇O₆295.1176) determining the molecular formula C by combining a hydrogen spectrum and a carbon spectrum₁₅H₁₈O₆The unsaturation degree is 7. Process for preparation of Compound 1¹H-NMR(600MHz in DMSO-d₆) And (3) displaying spectral data: delta_H7.48(1H, s) and 4.74(1H, dt, J ═ 10.0,1.5Hz) are characteristic olefinic proton signals in the 1,5 positions of the Gilmane type sesquiterpene lactones, in addition to the presence of a methyl signal delta_H1.56(3H, d, J ═ 1.5Hz), three connected oxygen proton signals [ (δ [, ]_H 5.42,1H,dt,J＝4.7,1.7Hz；4.27,1H,td,J＝10.0,4.8Hz；3.81,1H,m)]A pair of continuous oxygen methylene proton signals [ delta ]_H3.79(1H,dd,J＝6.4,3.0Hz),3.51(1H,ddd,J＝9.8,4.8,3.0Hz)]One methine proton signal δ_H2.39(1H, m), two pairs of methylene proton signals δ_H2.62(1H, dt, J ═ 14.5,1.7 Hz); 2.23(1H, dd, J ═ 14.5,4.7Hz) and 2.79(1H, m); 2.46(1H, t, J ═ 11.6 Hz). In addition, two hydroxyl signals δ are present in the hydrogen spectrum_H5.00(1H, d, J ═ 4.8Hz) and 4.92(1H, t, J ═ 4.8 Hz).¹³C-NMR(150MHz in DMSO-d₆) Spectra showing 15 carbon signals, including two lactone carbonyl carbon signals (delta)_C177.1,174.0), four olefin carbon signals (δ)_C150.4,130.4,130.0,129.3), four signals containing oxygen and carbon (delta)_C80.9,79.5,70.5,60.5), two methylene signals (delta)_C39.4,29.8), two methine signals (. delta.))_C48.4,48.0) and a methyl signal (. delta.))_C21.0). In HMBC spectra, Compound 1 was determined to have a Δ by correlation of H-1 with C-2, C-3, C-9, C-10, C-15, H-5 with C-3, C-7, C-14, H-8 with C-6, C-12, H-7 with C-9⁷⁽⁸⁾-the structure of a germacrane type sesquiterpene lactone; by passing¹H-¹The H COSY spectrum also confirms the framework [ H-5/H-6/H-7(H-11/H-13)/H-8/H-9]. H-13 was associated with HMBC at C-11, demonstrating attachment of the hydroxymethyl group at position 11. The relative configuration of Compound 1 was determined by NOESY Spectroscopy, H-1/H-14, H-6NOE correlation between H-8, H-6/H-11, H-8/H-14, determined that H-2, H-6, H-8, H-11 is in the beta configuration, and NOE correlation between H-5 and H-7, determined that H-7 is in the alpha configuration. The absolute configuration of 1 was determined by single crystal X-ray diffraction method by obtaining crystals of compound 1 from methanol. In addition, experimental and calculated ECD data also support the 2S,6R,7S,8S,11S configuration of 1.

In conclusion, the structure of the compound is finally determined to be scaberxone A (1).

Process for preparation of Compound 1¹H (600MHz) and¹³c (150MHz) NMR data (DMSO-d)₆)

Scaberxone B (2) white amorphous powder;13.0(c 0.12, MeOH); HR-ESI-MS shows that the peak of the quasi-molecular ion is 311.1123[ M + H ]]⁺(cal.C₁₅H₁₉O₇311.1125) determining the molecular formula C by combining a hydrogen spectrum and a carbon spectrum₁₅H₁₈O₇The unsaturation degree was 7. The nuclear magnetic data of the compound 2 is very similar to that of the compound 1, two hydroxyl signals exist in a hydrogen spectrum, and the carbon spectrum is carefully analyzed to find that one quaternary carbon connected with oxygen exists in the compound 2 and combines 11-OH with C-7, C-11, C-12 and C-13; the association of 13-OH with C-11, C-13 HMBC confirms the attachment of a hydroxyl group at C-11, C-13, respectively, in Compound 2. Thus, the main difference between compound 2 and 1 is that 2 has one more hydroxyl group attached at C-11. The relative configuration of Compound 2 was determined by NOESY spectroscopy, NOESY correlation between H-1/H-14, H-6/H-8, H-6/H-14, H-6/H-13, determining that H-2, H-6, H-8, H-13 is in the beta configuration and NOE correlation between H-5 and H-7, determining that H-7 is in the alpha configuration. The planar structure and the structure of 2 were further verified by single crystal X-ray diffraction method by obtaining crystals of Compound 2 from methanolRelative configuration, due to poor crystal quality, the absolute configuration of compound 2 was finally determined by calculating ECD, and experimental and calculated ECD data support the absolute configuration of 2 as 2S,6R,7S,8S, 11S.

In conclusion, the structure of the compound is finally determined to be scaberxone B (2).

Process for preparation of Compound 2¹H (600MHz) and¹³c (150MHz) NMR data (DMSO-d)₆)

Scaberoxone C (3) white amorphous powder;28.6(c 0.12, MeOH); HR-ESI-MS shows that the peak of the quasi-molecular ion is 279.1229[ M + H ]]⁺(cal.C₁₅H₁₉O₅279.1227) determining the molecular formula C by combining a hydrogen spectrum and a carbon spectrum₁₅H₁₉O₅The unsaturation degree was 7. By comparing mass spectra with nuclear magnetic data, it was found that compound 3 was highly similar in structure to 1, with methyl rather than hydroxymethyl being observed attached at C-11 in 3, by delta_H1.49(H-13) and δ_C38.5(C-11) HMBC correlation was confirmed. The relative configuration was determined by NOESY spectroscopy, with key correlation peaks for H-1/H-5, H-5/H-7 and H-6/H-13 indicating that H-5, H-7 and H-11 are coplanar, tentatively alpha-oriented; the NOESY correlation between H-6/H-8, H-6/H-14 confirms that H-2, H-6 and H-8 are in the beta-orientation. Through comparison of ECD spectra obtained through experiments and calculation, the absolute configuration of 3 is determined to be 2S,6R,7S,8S and 11S.

In conclusion, the structure of the compound is finally determined to be scaberxone C (3).

Process for preparation of Compound 3¹H (600MHz) and¹³c (150MHz) NMR data (CDCl)₃)

Scaberxone D (4) white amorphous powder;28.8(c 0.12, MeOH); HR-ESI-MS shows that its excimer peak separation is 279.1228[ M + H ]]⁺(cal.C₁₅H₁₉O₅279.1227), analysis of the combined HSQC and HMBC data by comparison of the 1D NMR spectral data, showed that 4 and 3 were nearly identical except for the slight difference in NMR data around C-1. Thus, the C-2 epimer with 4 being 3 was identified. Further confirmation was made by NOESY experiments, where the correlation of H-1 and H-5 indicates that H-2 is alpha oriented. Comparative analysis of the experimental and calculated ECD curves showed that the absolute configuration of 4 was 2R,6R,7S,8S, 11S.

In conclusion, the structure of the compound is finally determined to be scaberxoneD (4).

Process for preparation of Compound 4¹H (600MHz) and¹³c (150MHz) NMR data (DMSO-d)₆)

Scaberxone E (5) white amorphous powder;22.6(c 0.12, MeOH); HR-ESI-MS shows that the peak of the quasi-molecular ion is 463.1572[ M + Na ]]⁺(cal.C₂₁H₂₈O₁₀Na, 463.1575). 5 and 4 nuclear magnetic resonanceComparison of the data shows that the main differences between these two compounds are: in compound 5 is Δ⁶⁽⁷⁾Structure of-germacrane type sesquiterpene lactones instead of Δ⁷⁽⁸⁾The germacrane type sesquiterpene lactones and one additional glucose present in 5. With the aid of HSQC experiments, a partial glucose signal [ delta ] was observed_H 4.44(1H,d,J＝7.9Hz),3.68(1H,ddd,J＝11.9,6.0,2.2Hz),3.46(1H,dd,J＝11.9,4.2Hz),3.17(1H,m),3.15(1H,d,J＝8.8Hz),3.05(1H,dd,J＝8.8,3.0Hz),3.02(1H,dt,J＝7.9,4.2Hz)]And (delta)_C103.9,77.1,76.9,73.7,70.1,61.1). The correlation of HMBC at H-1'/C-8 and C-1'/H-8 indicates that the sugar moiety is attached at the C-8 position. NOESY correlation indicates that the relative configurations of C-2, C-6, C-8 and C-11 are identical to 1, while the beta-configuration of the sugar moiety is due to the coupling constant delta of the terminal proton signal_H4.44(1H, d, J ═ 7.9Hz, H-1'). After acid hydrolysis, HPLC analysis showed D-glucose. ECD calculations were performed on 5 to determine the absolute configuration. The results show that the calculation results of (2S,6R,7S,8S,11S) -5 are consistent with the experiments. Finally, the complete structure and absolute configuration of 5 are further verified through an X-ray diffraction experiment.

In conclusion, the structure of the compound is finally determined to be scaberxone E (5).

Process for preparation of Compound 5¹H (600MHz) and¹³c (150MHz) NMR data (DMSO-d)₆)

ScaberxoneF (6) white amorphous powder;69.4(c 0.12 MeOH); HR-ESI-MS shows that the peak of the quasi-molecular ion is 441.1761[ M + H ]]⁺(cal.C₂₁H₂₉O₁₀,441.1755). Process for preparation of Compound 6¹H and¹³C-NMR SpectroscopySignals are shown for a typical germacrane-type sesquiterpene lactone, along with a glucosyl moiety structurally identical to compound 5. The only difference is the relative configuration of the sesquiterpene aglycone, whose structure is described below, the beta-orientation of H-2 was deduced from the NOESY cross-peaks of H-1/H-5 and H-1/H-7, thus determining the C-2 epimer of structure 5 of Compound 6. The absolute configuration of 6 was determined by calculation of ECD to be 2R,6R,7S,8S, 11S.

In conclusion, the structure of the compound is finally determined to be scaberxoneF (6).

Process for preparation of Compound 6¹H (600MHz) and¹³c (150MHz) NMR data (DMSO-d)₆)

The six compounds in the invention are examined for anti-inflammatory activity, and all six compounds show certain anti-inflammatory activity. Among them, compound 6 was most effective. Therefore, the germacrane type sesquiterpene lactone compound has the prospect of further developing anti-inflammatory drugs.

The invention has the advantages that the compounds are all novel compounds, have novel structures, are all optically pure compounds with determined stereo configuration, have anti-inflammatory activity, enrich structural diversity of the germacrane type sesquiterpene lactone, provide clues for developing new drugs and have further development value.

Drawings

Process for preparation of compound 1 of FIG. 1¹H-NMR spectra (600MHz, DMSO-d)₆)；

FIG. 2 preparation of Compound 1¹³C-NMR spectra (150MHz, DMSO-d)₆)；

FIG. 3 HSQC spectra (600MHz, DMSO-d) of Compound 1₆)；

FIG. 4 HMBC spectra (600MHz, DMSO-d) of Compound 1₆)；

FIG. 5 NOESY spectrum of Compound 1 (600MHz, DMSO-d)₆)；

FIG. 6 preparation of Compound 2¹H-NMR spectra (600MHz, DMSO-d)₆)；

FIG. 7 preparation of Compound 2¹³C-NMR spectra (150MHz, DMSO-d)₆)；

FIG. 8 HSQC spectra (600MHz, DMSO-d) of Compound 2₆)；

FIG. 9 HMBC spectra (600MHz, DMSO-d) of Compound 2₆)；

FIG. 10 NOESY spectrum of Compound 2 (600MHz, DMSO-d)₆)；

FIG. 11 preparation of Compound 3¹H-NMR Spectroscopy (600MHz, CDCl)₃)；

FIG. 12 preparation of Compound 3¹³C-NMR Spectroscopy (150MHz, CDCl)₃)；

FIG. 13 HSQC spectra (600MHz, CDCl) of Compound 3₃)；

FIG. 14 HMBC spectra (600MHz, CDCl) of Compound 3₃)；

FIG. 15 NOESY spectrum (600MHz, CDCl) of Compound 3₃)；

FIG. 16 preparation of Compound 4¹H-NMR spectra (600MHz, DMSO-d)₆)；

FIG. 17 preparation of Compound 4¹³C-NMR spectra (150MHz, DMSO-d)₆)；

FIG. 18 HSQC spectra (600MHz, DMSO-d) of Compound 4₆)；

FIG. 19 HMBC spectra (600MHz, DMSO-d) of Compound 4₆)；

FIG. 20 NOESY spectrum (600MHz, DMSO-d) of Compound 4₆)；

FIG. 21 preparation of Compound 5¹H-NMR spectra (600MHz, DMSO-d)₆)；

FIG. 22 preparation of Compound 5¹³C-NMR spectra (150MHz, DMSO-d)₆)；

FIG. 23 HSQC spectra (600MHz, DMSO-d) of Compound 5₆)；

FIG. 24 HMBC spectra (600MHz, DMSO-d) of Compound 5₆)；

FIG. 25 NOESY spectrum (600MHz, DMSO-d) of Compound 5₆)；

FIG. 26 shows a schematic diagram ofProcess for preparation of compound 6¹H-NMR spectra (600MHz, DMSO-d)₆)；

FIG. 27 preparation of Compound 6¹³C-NMR spectra (150MHz, DMSO-d)₆)；

FIG. 28 HSQC spectra (600MHz, DMSO-d) of Compound 6₆)；

FIG. 29 HMBC spectra (600MHz, DMSO-d) of Compound 6₆)；

FIG. 30 NOESY spectrum (600MHz, DMSO-d) of Compound 6₆)；

FIG. 31 Compounds 1-6 inhibit the ability of LPS to induce NO production by BV-2 microglia.

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 the 1-6 gimerane type sesquiterpene lactone compounds in the elephantopus scaber concretely comprises the following steps:

(1) reflux-extracting whole plant of herba Ajugae Bracteosae of Elephantopus of Compositae with 70-80% industrial ethanol for 3-4 times, each for 2-3 hr. Mixing extractive solutions, concentrating to obtain extract, extracting with n-butanol, subjecting the obtained components to silica gel column chromatography, performing gradient elution with dichloromethane-methanol system at 50:0-1:2(50:1,30:1,20:1,10:1,5:1,3:1,1:1,1:2, V/V) at equal degree, and collecting 5 fractions (I-V).

(2) Subjecting fraction I to HP20 column chromatography, and gradient eluting with 10%, 30%, and 50% ethanol (ethanol-water system) to obtain 3 fractions A1, A2, and A3.

(3) The fraction A2 was eluted sequentially through an open ODS column chromatography with 20%, 40%, 50%, 60% ethanol (ethanol-water system) to give 4 fractions Fr.1-Fr.4.

(4) Fr.2 was eluted by silica gel column chromatography with a dichloromethane-methanol system 50:1-0:1(50:1,30:1,20:1,10:1,5:1,3:1,1:1,0:1, v/v) to give 5 subfractions Fr.21-Fr.25 on the basis of TLC analysis.

(5) Fr.23 was isolated on preparative reverse phase high performance liquid chromatography using a mobile phase of methanol-water (methanol 33%, v/v) to give Fr.231-Fr.236.

(6) Fr.233 was isolated on semi-preparative reverse phase high performance liquid chromatography using a mobile phase of acetonitrile-water (acetonitrile 15-35%, v/v) to give compounds 1-6.

Example 2:

and (3) examining the anti-inflammatory activity of the germacane type sesquiterpene lactone compounds 1-6 in the elephantopus scaber.

Cytotoxicity assay/cell viability assay:

BV-2 cells were plated in 96-well plates and incubated with compounds 1-6 for 24 hours. After complete removal of the medium, tetrazolium blue (MTT) was added to each well, followed by incubation in an incubator at 37 ℃ for 2-4 hours. The MTT solution was carefully removed and DMSO was added to dissolve the crystals formed well with appropriate shaking at room temperature. Finally, the absorbance was read at 490nm using a microplate reader.

Bioassay for inhibition of NO production:

BV-2 cells were seeded into 96-well plates and treated with LPS for 24 hours in the presence and absence of samples, respectively. The NO production in the supernatant was quantified by Griess reaction. The absorbance was read at 490nm with a microplate reader. The ability of compounds 1-6 to inhibit LPS-induced NO production in BV-2 microglia was assessed using Graphpad prism 7 software for analysis. Dexamethasone was used as a positive control. The specific results are shown in FIG. 31. The 6 compounds all have the capacity of inhibiting LPS (LPS) from inducing BV-2 microglia to generate NO, wherein the compound 6 has the most prominent effect.