A pyrrolidone compound with anti-inflammatory activity and its preparation method and application

Abstract

The present invention discloses a pyrrolidone compound with anti-inflammatory activity, a preparation method thereof, and an application thereof. The pyrrolidone compound is extracted from Indian truffles. The beneficial effects of the present invention are as follows: the pyrrolidone compound of the present invention can inhibit the mRNA expression of inflammatory mediators TNF-α, IL-1β, IL-6, and iNOS in LPS-induced RAW264.7 macrophages by reducing the production of inflammatory mediators such as NO, ROS, IL-6, and TNF-α, and can reduce the production of LPS-induced inflammatory mediators COX-2 and iNOS protein content, and can also inhibit the NLRP3-related protein pathway, that is, the pyrrolidone compound of the present invention has a good anti-inflammatory effect.

Description

Technical Field

[0001] The invention belongs to the technical field of truffle extracts, and particularly relates to a pyrrolidone compound with anti-inflammatory activity, a preparation method and an application thereof. Background Art

[0002] Indian truffles (Tuber indicum) belong to the order Tuberales, family Tuberaceae, and genus Tuber. They are found within 5 cm of the ground, beneath pines on Mount Huashan at altitudes of 1200-2800 meters. As a rare edible fungus, truffles are highly nutritious, boasting immune-boosting, antioxidant, anti-inflammatory, anti-tumor, stomach-tonifying, mentally soothing, hemostatic, and hemorrhoidal properties. However, current research on truffles is relatively limited, both in terms of their chemical composition and pharmacological activity. Therefore, we isolated Indian truffles, which are the most abundant in the Panzhihua region. Through comprehensive analysis of spectral data, we determined their relative structure; and by matching their CD spectra with calculated ECDs, we determined the absolute configuration of compound 1. Summary of the Invention

[0003] The main purpose of this application is to provide a pyrrolidone compound with anti-inflammatory activity and its preparation method and application.

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

[0005] A pyrrolidone compound with anti-inflammatory activity, wherein the structural formula of the pyrrolidone compound is:

[0006]

[0007] The above-mentioned pyrrolidone compound with anti-inflammatory activity is extracted from Indian truffle.

[0008] In a second aspect of the present application, a method for extracting pyrrolidone compounds having anti-inflammatory activity is provided, comprising the following steps:

[0009] (1) crushing fresh Indian truffles, extracting with ethanol reflux, concentrating the extract, and treating the concentrated extract into a fluid extract;

[0010] (2) The fluid extract obtained in step (1) was dispersed with distilled water, purified on D101 adsorption resin, and gradient eluted with methanol-distilled water eluent to obtain a 30% methanol elution portion, a 60% methanol elution portion, and a 90% methanol elution portion; since this application only describes the 30% methanol elution portion in detail, the 60% methanol elution portion and the 90% methanol elution portion are not described in detail in this application.

[0011] (3) The 30% methanol elution fraction was purified on HP-20 macroporous resin using a gradient elution of distilled water and methanol, and the fractions A, B, and C were obtained by combining.

[0012] (4) Component A obtained in step (3) was purified by MCI reverse phase chromatography column, using distilled water and methanol for gradient elution, and combined under the guidance of TLC and HPLC to obtain 12 fractions, namely A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, and A12. Fraction A4 was used to prepare the compound by semi-preparative liquid phase.

[0013] The above-mentioned method for extracting pyrrolidone compounds with anti-inflammatory activity is a preferred embodiment, wherein in step (1), 95% ethanol in an amount of 8-10 times the weight of Indian truffles is used for reflux extraction;

[0014] The fluid extract is processed as follows: using distilled water to disperse the concentrated liquid, and the dispersion is placed in a dialysis bag and soaked in distilled water for 12 days, with the water being changed twice a day. After the soaking is completed, the dialysate is concentrated to obtain the fluid extract.

[0015] The above-mentioned method for extracting pyrrolidone compounds with anti-inflammatory activity is a preferred embodiment, in step (2), the methanol-distilled water gradient elution method is:

[0016] The elution time was 0-40 hours, the eluent was distilled water, and the eluent flow rate was 5 ml / min;

[0017] The elution time is 40-80 hours, the eluent is methanol-distilled water with a volume ratio of 30:70, and the eluent flow rate is 5 ml / min;

[0018] The elution time is 80-120 hours, the eluent is methanol-distilled water with a volume ratio of 60:40, and the eluent flow rate is 5 ml / min;

[0019] The elution time is 120-160 hours, the eluent is methanol-distilled water with a volume ratio of 90:10, and the eluent flow rate is 5 ml / min.

[0020] The above-mentioned method for extracting pyrrolidone compounds with anti-inflammatory activity is a preferred embodiment. In step (3), the gradient elution method of distilled water and methanol is as follows:

[0021] The elution time was 0-6 hours, the eluent was distilled water, and the eluent flow rate was 5 ml / min;

[0022] The elution time is 6-12 hours, the eluent is methanol-distilled water with a volume ratio of 5:95, and the eluent flow rate is 5 ml / min;

[0023] The elution time is 12-18 hours, the eluent is methanol-distilled water with a volume ratio of 10:90, and the eluent flow rate is 5 ml / min;

[0024] The elution time is 18-24 hours, the eluent is methanol-distilled water with a volume ratio of 15:85, and the eluent flow rate is 5 ml / min;

[0025] The elution time is 24-30 hours, the eluent is methanol-distilled water with a volume ratio of 20:80, and the eluent flow rate is 5 ml / min;

[0026] The elution time is 30-36 hours, the eluent is methanol-distilled water with a volume ratio of 25:75, and the eluent flow rate is 5 ml / min;

[0027] The elution time is 36-42 hours, the eluent is methanol-distilled water with a volume ratio of 30:70, and the eluent flow rate is 5 ml / min;

[0028] The elution time is 42-48 hours, the eluent is methanol-distilled water with a volume ratio of 35:65, and the eluent flow rate is 5 ml / min;

[0029] The elution time is 48-54 hours, the eluent is methanol-distilled water with a volume ratio of 45:55, and the eluent flow rate is 5 ml / min;

[0030] The elution time was 54-60 hours, the eluent was methanol-distilled water with a volume ratio of 55:45, and the eluent flow rate was 5 ml / min;

[0031] The elution time was 60-66 hours, the eluent was methanol-distilled water with a volume ratio of 100:0, and the eluent flow rate was 5 ml / min;

[0032] In step (3), the component A is a mixture of products obtained by eluting with distilled water as the eluent and methanol-distilled water in volume ratios of 5:95, 10:90 and 15:85.

[0033] Component B is a mixture of products obtained by elution with methanol-distilled water at volume ratios of 20:80, 25:75, and 30:70;

[0034] Component C is a mixture of products obtained by eluting with methanol and distilled water in volume ratios of 35:65, 45:55, 55:45, and 100:0.

[0035] The above-mentioned method for extracting pyrrolidone compounds with anti-inflammatory activity is a preferred embodiment. In step (4), the elution method of gradient elution with distilled water and methanol is:

[0036] The elution time was 0-1.5 hours, the eluent was distilled water, and the eluent flow rate was 2 ml / min;

[0037] The elution time is 1.5-3 hours, the eluent is methanol-distilled water with a volume ratio of 5:95, and the eluent flow rate is 2 ml / min;

[0038] The elution time is 3-4.5 hours, the eluent is methanol-distilled water with a volume ratio of 10:90, and the eluent flow rate is 2 ml / min;

[0039] The elution time is 4.5-6 hours, the eluent is methanol-distilled water with a volume ratio of 15:85, and the eluent flow rate is 2 ml / min;

[0040] The elution time was 6-7.5 hours, the eluent was methanol-distilled water with a volume ratio of 20:80, and the eluent flow rate was 2 ml / min;

[0041] The elution time was 7.5-9 hours, the eluent was methanol-distilled water with a volume ratio of 25:75, and the eluent flow rate was 2 ml / min;

[0042] The elution time was 9-10.5 hours, the eluent was methanol-distilled water with a volume ratio of 30:70, and the eluent flow rate was 2 ml / min;

[0043] The elution time was 10.5-12 hours, the eluent was methanol-distilled water with a volume ratio of 35:65, and the eluent flow rate was 2 ml / min;

[0044] The elution time was 12-13.5 hours, the eluent was methanol-distilled water with a volume ratio of 45:55, and the eluent flow rate was 2 ml / min;

[0045] The elution time was 13.5-15 hours, the eluent was methanol-distilled water with a volume ratio of 55:45, and the eluent flow rate was 2 ml / min;

[0046] The elution time was 15-16.5 hours, the eluent was methanol-distilled water with a volume ratio of 100:0, and the eluent flow rate was 2 ml / min;

[0047] The A4 portion is a product obtained by eluting with methanol-distilled water in a volume ratio of 5:95.

[0048] Portions A1-A3 are products obtained by elution with distilled water; portion A5 is a product obtained by elution with methanol-distilled water in a volume ratio of 10:90; portion A6 is a mixture of products obtained by elution with methanol-distilled water in a volume ratio of 10:90 and 15:85; portion A7 is a product obtained by elution with methanol-distilled water in a volume ratio of 15:85; portion A8 is a mixture of products obtained by elution with methanol-distilled water in a volume ratio of 15:85 and 20:80; portion A9 is a product obtained by elution with methanol-distilled water in a volume ratio of 20:80; portion A10 is a mixture of products obtained by elution with methanol-distilled water in a volume ratio of 25:75 and 30:70; portion A11 is a product obtained by elution with methanol-distilled water in a volume ratio of 35:65, and portion A12 is a mixture of products obtained by elution with methanol-distilled water in a volume ratio of 45:55, 55:45, and 100:0.

[0049] During the elution process, TLC is used to separate components by taking advantage of the different adsorption capacities of the components on the same adsorbent and the different solubility of the developing agent for different substances. The same components are combined by observing the spot plate results (based on UV, iodine development, and color development).

[0050] HPLC uses the method of combining components dissolved in the mobile phase and the stationary phase. Due to the different sizes and strengths of the interactions with the stationary phase, the components stay in the stationary phase for different periods of time, and thus flow out of the stationary phase in sequence. The same components can be combined by observing the peak patterns.

[0051] The above-mentioned method for extracting pyrrolidone compounds with anti-inflammatory activity is a preferred embodiment. In step (4), the semi-preparative liquid phase adopts a YMC-Pack ODS-A semi-preparative column, the eluent is 13% methanol, the flow rate is 3 ml / min, the detection wavelength is 254 nm, and the column temperature is 30°C.

[0052] The third aspect of the present application provides a composition with anti-inflammatory effect, comprising the pyrrolidone compound.

[0053] The beneficial effects of the present application are as follows: the present invention provides a method for isolating a new pyrrolidone compound from Indian truffles, and characterizes the structure of the new compound by using nuclear magnetic resonance, electrospray ionization mass spectrometry, nuclear magnetic resonance, etc.

[0054] The present invention verifies that the pyrrolidone compound can reduce the production of inflammatory mediators such as NO, ROS, IL-6 and TNF-α, can inhibit the mRNA expression of inflammatory mediators TNF-α, IL-1β, IL-6, and iNOS in LPS-induced RAW264.7 macrophages, can reduce the production of LPS-induced inflammatory mediators COX-2 and iNOS protein content, and can also inhibit the NLRP3-related protein pathway, showing anti-inflammatory properties in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The pyrrolidone compound with anti-inflammatory activity described in Example 2 1 H- 1 H COSY, HMBC, NOESY correlation;

[0056] Figure 2 This is the HRESIMS spectrum of the pyrrolidone compound with anti-inflammatory activity described in Example 2;

[0057] Figure 3 This is the UV spectrum of the pyrrolidone compound with anti-inflammatory activity described in Example 2;

[0058] Figure 4 This is the infrared spectrum of the pyrrolidone compound with anti-inflammatory activity described in Example 2;

[0059] Figure 5 The pyrrolidone compound with anti-inflammatory activity described in Example 2 in D2O 1 HNMR spectrum;

[0060] Figure 6 The pyrrolidone compound with anti-inflammatory activity described in Example 2 in D2O 13 CNMR images;

[0061] Figure 7 This is the HSQC spectrum of the pyrrolidone compound with anti-inflammatory activity in D2O described in Example 2;

[0062] Figure 8 The pyrrolidone compound with anti-inflammatory activity described in Example 2 in D2O 1 H- 1 HCOSY spectrum;

[0063] Figure 9 This is the HMBC spectrum of the pyrrolidone compound with anti-inflammatory activity in D2O described in Example 2;

[0064] Figure 10 This is the NOESY spectrum of the pyrrolidone compound with anti-inflammatory activity in D2O described in Example 2;

[0065] Figure 11 The most stable conformation of the pyrrolidone compound with anti-inflammatory activity described in Example 2 calculated using TD-SCF at the CAM-B3LYP / DGDZVP level;

[0066] Figure 12 This is the experimental ECD spectrum of the pyrrolidone compound with anti-inflammatory activity in MeOH as described in Example 2;

[0067] Figure 13 The effect of different concentrations of pyrrolidone compounds on the viability of RAW264.7 macrophages in the logarithmic growth phase;

[0068] Figure 14 The effect of different concentrations of pyrrolidone compounds on NO production in macrophages stimulated by LPS;

[0069] Figure 15 The effect of different concentrations of pyrrolidone compounds on the ROS content produced by RAW264.7 cells after LPS stimulation;

[0070] Figure 16 The effects of different concentrations of pyrrolidone compounds on the expression of inflammatory mediator iNOS, inflammatory factors TNF-α, IL-1β and IL-6 mRNA in LPS-induced RAW264.7 macrophages;

[0071] Figure 17 Effects of different concentrations of pyrrolidone compounds on the protein expression levels of inflammatory mediators iNOS, COX-2 and inflammation-related pathways NLRP3, caspase1, ASC / TMS1 in LPS-induced RAW264.7 macrophages;

[0072] Figure 18 The effect of different concentrations of pyrrolidone compounds on the expression levels of inflammatory factors TNF-α and IL-6 in the supernatant of RAW264.7 macrophages induced by LPS. DETAILED DESCRIPTION

[0073] In order to help those skilled in the art better understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to examples. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0074] The experimental instruments used in the examples of this application are shown in Table 1:

[0075] Table 1

[0076]

[0077]

[0078]

[0079] The materials and reagents used in the examples of this application are shown in Table 2:

[0080] Table 2

[0081]

[0082]

[0083] Example 1

[0084] A pyrrolidone compound with anti-inflammatory activity, wherein the structural formula of the pyrrolidone compound is:

[0085]

[0086] The pyrrolidone compound is extracted from Indian truffles collected from the pines of Mount Hua in Panzhihua City.

[0087] Example 2

[0088] Example 2 provides a method for extracting pyrrolidone compounds with anti-inflammatory activity, comprising the following steps: (1) crushing fresh Indian truffles, performing reflux extraction with 95% ethanol in an amount 8 times the weight of the Indian truffles, concentrating the extract, and treating the concentrated extract to form a fluid extract; the fluid extract treatment specifically comprises: dispersing the concentrate with distilled water, placing the dispersed solution in a dialysis bag, and soaking the dispersed solution in distilled water for 12 days, changing the water twice a day, and concentrating the dialyzate after soaking to obtain a fluid extract;

[0089] (2) The fluid extract obtained in step (1) was dispersed with distilled water, purified by D101 adsorption resin, and gradient eluted with methanol-distilled water eluent to obtain a 30% methanol elution fraction, a 60% methanol elution fraction, and a 90% methanol elution fraction;

[0090] The methanol-distilled water gradient elution method is:

[0091] The elution time was 0-40 hours, the eluent was distilled water, and the eluent flow rate was 5 ml / min;

[0092] The elution time is 40-80 hours, the eluent is methanol-distilled water with a volume ratio of 30:70, and the eluent flow rate is 5 ml / min;

[0093] The elution time is 80-120 hours, the eluent is methanol-distilled water with a volume ratio of 60:40, and the eluent flow rate is 5 ml / min;

[0094] The elution time is 120-160 hours, the eluent is methanol-distilled water with a volume ratio of 90:10, and the eluent flow rate is 5 ml / min;

[0095] (3) The 30% methanol elution fraction was purified on HP-20 macroporous resin using a gradient elution of distilled water and methanol, and the fractions were combined under the guidance of TLC and HPLC to obtain fractions A, B, and C;

[0096] The gradient elution method of distilled water and methanol is as follows:

[0097] The elution time was 0-6 hours, the eluent was distilled water, and the eluent flow rate was 5 ml / min;

[0098] The elution time is 6-12 hours, the eluent is methanol-distilled water with a volume ratio of 5:95, and the eluent flow rate is 5 ml / min;

[0099] The elution time is 12-18 hours, the eluent is methanol-distilled water with a volume ratio of 10:90, and the eluent flow rate is 5 ml / min;

[0100] The elution time is 18-24 hours, the eluent is methanol-distilled water with a volume ratio of 15:85, and the eluent flow rate is 5 ml / min;

[0101] The elution time is 24-30 hours, the eluent is methanol-distilled water with a volume ratio of 20:80, and the eluent flow rate is 5 ml / min;

[0102] The elution time is 30-36 hours, the eluent is methanol-distilled water with a volume ratio of 25:75, and the eluent flow rate is 5 ml / min;

[0103] The elution time is 36-42 hours, the eluent is methanol-distilled water with a volume ratio of 30:70, and the eluent flow rate is 5 ml / min;

[0104] The elution time is 42-48 hours, the eluent is methanol-distilled water with a volume ratio of 35:65, and the eluent flow rate is 5 ml / min;

[0105] The elution time is 48-54 hours, the eluent is methanol-distilled water with a volume ratio of 45:55, and the eluent flow rate is 5 ml / min;

[0106] The elution time was 54-60 hours, the eluent was methanol-distilled water with a volume ratio of 55:45, and the eluent flow rate was 5 ml / min;

[0107] The elution time was 60-66 hours, the eluent was methanol-distilled water with a volume ratio of 100:0, and the eluent flow rate was 5 ml / min;

[0108] The component A is a mixture of products obtained by eluting with distilled water as the eluent and methanol-distilled water in volume ratios of 5:95, 10:90, and 15:85;

[0109] (4) Component A obtained in step (3) was purified by MCI reverse phase chromatography column, and gradient elution was performed using distilled water and methanol in sequence. The fractions A1-A12 were combined under the guidance of TLC and HPLC to obtain 12 fractions. Fraction A4 was prepared by semi-preparative liquid phase; the semi-preparative liquid phase was performed using a YMC-Pack ODS-A semi-preparative column, the eluent was 13% methanol, the flow rate was 3 ml / min, the detection wavelength was 254 nm, and the column temperature was 30°C.

[0110] The elution mode for gradient elution with distilled water and methanol is as follows:

[0111] The elution time was 0-1.5 hours, the eluent was distilled water, and the eluent flow rate was 2 ml / min;

[0112] The elution time is 1.5-3 hours, the eluent is methanol-distilled water with a volume ratio of 5:95, and the eluent flow rate is 2 ml / min;

[0113] The elution time is 3-4.5 hours, the eluent is methanol-distilled water with a volume ratio of 10:90, and the eluent flow rate is 2 ml / min;

[0114] The elution time is 4.5-6 hours, the eluent is methanol-distilled water with a volume ratio of 15:85, and the eluent flow rate is 2 ml / min;

[0115] The elution time was 6-7.5 hours, the eluent was methanol-distilled water with a volume ratio of 20:80, and the eluent flow rate was 2 ml / min;

[0116] The elution time was 7.5-9 hours, the eluent was methanol-distilled water with a volume ratio of 25:75, and the eluent flow rate was 2 ml / min;

[0117] The elution time was 9-10.5 hours, the eluent was methanol-distilled water with a volume ratio of 30:70, and the eluent flow rate was 2 ml / min;

[0118] The elution time was 10.5-12 hours, the eluent was methanol-distilled water with a volume ratio of 35:65, and the eluent flow rate was 2 ml / min;

[0119] The elution time was 12-13.5 hours, the eluent was methanol-distilled water with a volume ratio of 45:55, and the eluent flow rate was 2 ml / min;

[0120] The elution time was 13.5-15 hours, the eluent was methanol-distilled water with a volume ratio of 55:45, and the eluent flow rate was 2 ml / min;

[0121] The elution time was 15-16.5 hours, the eluent was methanol-distilled water with a volume ratio of 100:0, and the eluent flow rate was 2 ml / min.

[0122] The A4 portion is a product obtained by eluting with methanol-distilled water in a volume ratio of 5:95.

[0123] The obtained compound was a yellow solid; ESI-MS m / z: 204 [M+Na] + The molecular weight of the compound is estimated to be 181, and the molecular formula is C9H 11 NO3, unsaturation 5, UV spectrum see Figure 3 .

[0124] According to the FT-IR spectrum ( Figure 4 ) inferred that the compound contains hydroxyl groups (3436cm -1 ), methylene (2927cm -1 ), amide (1664cm -1 ).

[0125] pass 1 H-NMR ( Figure 5 ), 13 C-NMR ( Figure 6 )、HSQC( Figure 7 ) spectrum inferred that the compound contained a carbonyl group [δC:181.9]; two methylene groups [δH:2.37–2.27 (2H m), 2.21–2.17 (1H, m), 1.90 (1H, d, 4.4); δC:29.6, 22.2]; an oxidized methine group [δH:4.68 (1H, d, 6.4, Hz); δC:69.6]; two sets of conjugated double bonds [δH:7.54–7.53 (1H, m), 6.43 (1H, d, 3.2, Hz), 6.47 (1H, dd, 3.1, 1.9, Hz); δC:143.1, 107.9, 110.4, 152.7]. 1 H- 1 HCOSY spectrum ( Figure 1 、 Figure 8 ) found that H-7 / H-8, H-8 / H-9 were related, combined 1 H-NMR, 13 C-NMR and HSQC spectra inferred that the compound contained a furan ring fragment.

[0126] Through the correlation of H-2 / H-3, H-3 / H-4, H-4 / H-5, it is inferred that C-2 is connected to C-3, C-3 is connected to C-4, and C-4 is connected to C5. The HMBC spectrum ( Figure 1 、 Figure 9) found that H-9 was related to C-6 / C-7 / C-8, H-8 was related to C-6 / C-7 / C-9, and H-7 was related to C-6 / C-8 / C-9, confirming the furan ring fragment speculation;

[0127] H-5 is related to C-3 / C-4 / C-6 / C-7, H-4 is related to C-1 / C-2 / C-3 / C-5 / C-6, H-3 is related to C-1 / C-2 / C-4 / C-5, and H-2 is related to C-1 / C-3 / C-4. It is inferred that the 5-hydroxymethyl-2-pyrrolidone fragment is connected to the furan ring at the C-6 position through C-5 ( Figure 9 ). According to the above speculation process, combined with mass spectrometry data ( Figure 2 ) confirmed the planar structure of compound 1, and the relevant signals in the NOESY spectrum were shown in Figure 10 Then the experimental CD and calculated ECD of the compound were calculated ( Figure 12 ) (see Table 4, Table 5, for supplementary materials related to computational chemistry) Figure 11 ), the structure of the compound was confirmed, and the absolute configuration of the compound was determined to be 4S-(1'-hydroxy-2'S-furyl)pyrrolidone.

[0128] The pyrrolidone compound 1 HNMR (600 MHz) and 13 CNMR (150MHz) data are shown in Table 3:

[0129] Table 3

[0130]

[0131] The results of z-matrix optimization of simplified pyrrolidone compounds in WB97XD / DGDZVP level D2O are shown in Table 4:

[0132] Table 4

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] The energy analysis results of the pyrrolidone compounds are shown in Table 5:

[0142] Table 5

[0143] Spatial energy (kJ / mol) Relative energy (kJ / mol) distributed(%) 1a -629.7966934 -0.0001768 9.99 1b -629.7960739 0.0004427 5.18 1c -629.7966827 -0.0001661 9.88 1d -629.7963865 0.0001301 7.22 1e -629.7965166 0.0000000 8.28 1f -629.7952931 0.0012235 2.26 1g -629.7965673 -0.0000507 8.74 1h -629.7947851 0.0017315 1.32 1i -629.7955293 0.0009873 2.91 1j -629.7951125 0.0014041 1.87 1k -629.7951547 0.0013619 1.96 1l -629.7967784 -0.0002618 10.93 1m -629.7966828 -0.0001662 9.88 1n -629.7969553 -0.0004387 13.19 1o -629.7962723 0.0002443 6.39

[0144] Example 3

[0145] The use of the pyrrolidone compounds with anti-inflammatory activity described in Example 2.

[0146] 3.1. CCK-8 assay for cell viability

[0147] RAW264.7 macrophages in the logarithmic growth phase were collected and 1×10 4 The cells were inoculated at a density of 100 μL / well in a 96-well cell culture plate and cultured in a cell culture incubator for 24 hours. The pyrrolidone compound described in Example 2 at a concentration of 60 mM was diluted to the corresponding concentration (0, 3.75, 7.5, 15, 30, 60, 120, 240 μM) with DMEM complete medium, and DMSO was used as a blank control. The original culture medium was replaced with 100 μL / well and cultured in a cell culture incubator for 24 hours. Under light-proof conditions, 100 μL of DMEM basal medium containing 10 μL CCK-8 was added to each well of cells and placed in a cell culture incubator for incubation in the dark for 1 hour. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader. Analysis of the results: Cell viability % = (OD value of the drug group - OD value of the blank well) / (OD value of the blank control group - OD value of the blank well) × 100%.

[0148] Test results such as Figure 13 As shown. Figure 13 It can be seen that the pyrrolidone compounds described in Example 2 have no toxic effect on macrophages at different concentrations (0, 3.75, 7.5, 15, 30, 60, 120, 240 μM).

[0149] 3.2 Determination of NO generation

[0150] RAW264.7 macrophages in the logarithmic growth phase were collected and divided into 2 × 10 5The cells were inoculated at a density of 100 μg / mL in a 6-well cell culture plate and cultured in a cell culture incubator for 24 hours. A certain amount of the pyrrolidone compound described in Example 2 was added to the cell culture medium to a final concentration of 15, 30, and 60 μM (including a single-dose group of 60 μM), and the solvent volume was adjusted with DMSO. After pre-treating the cells for 2 hours, LPS (1 μg / mL) was added. After culturing for 24 hours after administration, the cell culture plate was removed for processing. 50 μL of the cell supernatant from each group was taken out of a 96-well plate, and three replicates were made for each group. 50 μL each of Griess Reagent I and Griess Reagent II was added to each well; the absorbance was detected at 540 nm using a microplate reader.

[0151] Test results such as Figure 14 Shown: From Figure 14 As can be seen from the results, the pyrrolidone compounds described in Example 2 can inhibit the production of NO in LPS-induced RAW264.7 macrophages in a concentration-dependent manner (****P<0.0001; ***P<0.001; **P<0.01; *P<0.05 compared with the LPS group).

[0152] 3.3. Intracellular Reactive Oxygen Species (ROS) Detection

[0153] Place a 2 cm diameter glass slide in each well of a 12-well plate and add 500 μL of DMEM complete medium so that the glass slide is close to the bottom of the 12-well plate. Take RAW264.7 macrophages in the logarithmic growth phase and dilute the cell culture medium to 4 × 10 4 Cells were cultured in a complete DMEM medium (500 μL / well) containing 10 cells / mL. 500 μL of this complete medium was then added to each well and cultured for 24 hours. A certain amount of the pyrrolidone compound described in Example 2 was added to the cell culture medium to achieve final concentrations of 15, 30, and 60 μM. A positive control group was set up in this experiment. After pre-treating the cells for 2 hours with 60 μM dexamethasone (DXM), LPS (1 μg / mL) was added. 24 hours after administration, the cells were treated, the original culture medium was discarded, and the cells were washed twice with PBS. 600 μL of reaction culture medium (DCFH-DA probe: basic culture medium = 1:1000) was added to each well and incubated for 30 minutes; washed twice with PBS, 600 μL of paraformaldehyde was added and incubated for 15 minutes; the paraformaldehyde was discarded, the cells were washed twice with PBS, and 600 μL of cell nucleus staining solution (DAPI) was added and incubated for 8 minutes; the DAPI was discarded, and the cells were washed twice with PBS on a shaker. 20 μL of anti-fluorescence quenching sealing solution was dropped on the slide, the slide at the bottom of the 12-well plate was removed, and the cover was turned over and covered with the anti-fluorescence quenching sealing solution. Finally, the fluorescence intensity was detected by confocal microscopy.

[0154] Test results such as Figure 15 As shown, from Figure 15 It can be seen that the pyrrolidone compounds described in Example 2 can reduce the content of ROS produced by RAW264.7 cells after LPS stimulation.

[0155] 3.4 Reverse transcription-polymerase chain reaction (RT-PCR) analysis

[0156] RAW264.7 macrophages in the logarithmic growth phase were collected and divided into 2 × 10 5 The cells were inoculated at a density of 100 μg / mL in a 6-well cell culture plate and cultured in a cell culture incubator for 24 hours. A certain amount of the pyrrolidone compound described in Example 2 was added to the cell culture medium to a final concentration of 15, 30, and 60 μM (including a single-dose group of 60 μM), and the solvent volume was balanced with DMSO. After pre-treating the cells for 2 hours, LPS (1 μg / mL) was added. After culturing for 24 hours after administration, the cell culture plate was removed for processing. 1 mL of Trizol reagent was added to each well to extract RNA, and the RNA purity and concentration were detected using an ultramicro UV spectrophotometer. The RT Easy TM II (With gDNase) kit was used to reverse transcribe RNA into cDNA, and then Real Time PCR Easy TM cDNA was amplified using the SYBR Green I kit. The gene levels of iNOS, TNF-α, IL-6, and IL-1β in the cells were determined. The gene sequences are shown in Table 6.

[0157] Table 6

[0158]

[0159]

[0160] The test results are as follows Figure 16 As shown, from Figure 16 It can be seen that the pyrrolidone compound described in Example 2 significantly inhibited the expression of inflammatory mediator iNOS and inflammatory factors TNF-α, IL-1β and IL-6 mRNA in LPS-induced RAW264.7 macrophages (****P<0.0001; ***P<0.001; **P<0.01; *P<0.05 compared with the LPS group).

[0161] 3.5. Western blotting to detect the expression of target protein

[0162] RAW264.7 macrophages in the logarithmic growth phase were collected and divided into 2 × 10 5Cells were seeded at a density of 100 μM / mL in a 6-well cell culture plate and cultured in a cell culture incubator for 24 hours. A certain amount of the pyrrolidone compound described in Example 2 was added to the cell culture medium to a final concentration of 15, 30, and 60 μM (including a single-dose group at 60 μM). The solvent volume was adjusted with DMSO. After pre-treating the cells for 2 hours, LPS (1 μg / mL) was added. After 24 hours of culture after administration, the cell culture plate was removed for processing. The cells were lysed with 1× SDS lysis buffer to extract total protein, heated in a 100°C constant temperature metal bath for 30 minutes, and then centrifuged at 4°C at 12,000 g / min for 15 minutes, and the supernatant was collected. The protein concentration was determined using a BCA protein assay kit, and then the concentration was adjusted with SDS lysis buffer. 1 / 4 volume of protein loading buffer (5×) was added and heated in a 95°C constant temperature metal bath for 10 minutes to prevent denaturation. Proteins were separated by 10% SDS-PAGE electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes, and incubated with primary antibodies (iNOS, COX-2, NLRP3, caspase-1, ASC / TMS-1) overnight at 4°C. The PVDF membranes were then incubated with secondary antibodies for 2 h at room temperature, and bands were detected using a high-sensitivity ECL chemiluminescence kit and analyzed using ImageJ software.

[0163] The test results are as follows Figure 17 As shown, from Figure 17 It can be seen that the pyrrolidone compounds described in Example 2 significantly inhibited the protein expression levels of inflammatory mediators iNOS, COX-2 and inflammation-related pathways NLRP3, caspase1, ASC / TMS1 in LPS-induced RAW264.7 macrophages.

[0164] 3.6 ELISA assay for TNF-α and IL-6 release

[0165] RAW264.7 macrophages in the logarithmic growth phase were collected and divided into 2 × 10 5 The cells were inoculated at a density of 100 μg / mL in 6-well cell culture plates and cultured in a cell culture incubator for 24 h. A certain amount of the pyrrolidone compound described in Example 2 was added to the cell culture medium to a final concentration of 15, 30, and 60 μM. A positive control group was set up in the experiment, with 60 μM dexamethasone and DMSO used to balance the solvent volume. After pre-treating the cells for 2 h, LPS (1 μg / mL) was added. After removing the 6-well plate, the cell supernatant was taken and the experimental operation was performed according to the ELISA kit instructions. The OD value of each group was measured at 450 nm, and the TNF-α and IL-6 cytokine release levels were calculated based on the OD value. Each value represents the mean (SEM) ± standard error (****P<0.0001; ***P<0.001; **P<0.01; *P<0.05 compared with the LPS group).

[0166] The test results are as follows Figure 18 As shown, from Figure 18 It can be seen that the pyrrolidone compound described in Example 2 significantly inhibited the expression levels of inflammatory factors TNF-α and IL-6 in the supernatant of RAW264.7 macrophages induced by LPS (****P<0.0001; ***P<0.001; **P<0.01; *P<0.05 compared with the LPS group).

[0167] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A pyrrolidone compound having anti-inflammatory activity, characterized in that: The structural formula of the pyrrolidone compound is:

2. The method for extracting pyrrolidone compounds with anti-inflammatory activity according to claim 1, characterized in that: The following steps are involved: (1) crushing fresh Indian truffles, extracting with ethanol reflux, concentrating the extract, and treating the concentrated extract into a fluid extract; (2) The fluid extract obtained in step (1) was dispersed with distilled water, purified by D101 adsorption resin, and gradient eluted with methanol-distilled water eluent to obtain a 30% methanol elution fraction, a 60% methanol elution fraction, and a 90% methanol elution fraction; (3) The 30% methanol elution fraction was purified on HP-20 macroporous resin using a gradient elution of distilled water and methanol, and the fractions A, B, and C were obtained by combining. Among them, the gradient elution method of distilled water and methanol is: The elution time was 0-6 hours, the eluent was distilled water, and the eluent flow rate was 5 ml / min; The elution time is 6-12 hours, the eluent is methanol-distilled water with a volume ratio of 5:95, and the eluent flow rate is 5 ml / min; The elution time is 12-18 hours, the eluent is methanol-distilled water with a volume ratio of 10:90, and the eluent flow rate is 5 ml / min; The elution time is 18-24 hours, the eluent is methanol-distilled water with a volume ratio of 15:85, and the eluent flow rate is 5 ml / min; The elution time is 24-30 hours, the eluent is methanol-distilled water with a volume ratio of 20:80, and the eluent flow rate is 5 ml / min; The elution time is 30-36 hours, the eluent is methanol-distilled water with a volume ratio of 25:75, and the eluent flow rate is 5 ml / min; The elution time is 36-42 hours, the eluent is methanol-distilled water with a volume ratio of 30:70, and the eluent flow rate is 5 ml / min; The elution time is 42-48 hours, the eluent is methanol-distilled water with a volume ratio of 35:65, and the eluent flow rate is 5 ml / min; The elution time is 48-54 hours, the eluent is methanol-distilled water with a volume ratio of 45:55, and the eluent flow rate is 5 ml / min; The elution time was 54-60 hours, the eluent was methanol-distilled water with a volume ratio of 55:45, and the eluent flow rate was 5 ml / min; The elution time was 60-66 hours, the eluent was methanol-distilled water with a volume ratio of 100:0, and the eluent flow rate was 5 ml / min; The component A is a mixture of products obtained by eluting with distilled water as the eluent and methanol-distilled water in volume ratios of 5:95, 10:90, and 15:85; (4) Component A obtained in step (3) was purified by MCI reverse phase chromatography using a gradient elution with distilled water and methanol to obtain 12 fractions, namely A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, and A12. Fraction A4 was used to prepare the compound by semi-preparative liquid chromatography; Among them, the gradient elution method of distilled water and methanol is: The elution time was 0-1.5 hours, the eluent was distilled water, and the eluent flow rate was 2 ml / min; The elution time is 1.5-3 hours, the eluent is methanol-distilled water with a volume ratio of 5:95, and the eluent flow rate is 2 ml / min; The elution time is 3-4.5 hours, the eluent is methanol-distilled water with a volume ratio of 10:90, and the eluent flow rate is 2 ml / min; The elution time is 4.5-6 hours, the eluent is methanol-distilled water with a volume ratio of 15:85, and the eluent flow rate is 2 ml / min; The elution time was 6-7.5 hours, the eluent was methanol-distilled water with a volume ratio of 20:80, and the eluent flow rate was 2 ml / min; The elution time was 7.5-9 hours, the eluent was methanol-distilled water with a volume ratio of 25:75, and the eluent flow rate was 2 ml / min; The elution time was 9-10.5 hours, the eluent was methanol-distilled water with a volume ratio of 30:70, and the eluent flow rate was 2 ml / min; The elution time was 10.5-12 hours, the eluent was methanol-distilled water with a volume ratio of 35:65, and the eluent flow rate was 2 ml / min; The elution time was 12-13.5 hours, the eluent was methanol-distilled water with a volume ratio of 45:55, and the eluent flow rate was 2 ml / min; The elution time was 13.5-15 hours, the eluent was methanol-distilled water with a volume ratio of 55:45, and the eluent flow rate was 2 ml / min; The elution time was 15-16.5 hours, the eluent was methanol-distilled water with a volume ratio of 100:0, and the eluent flow rate was 2 ml / min; The A4 portion is a product obtained by eluting with methanol-distilled water in a volume ratio of 5:

95.

3. The method for extracting pyrrolidone compounds with anti-inflammatory activity according to claim 2, characterized in that: In step (1), 95% ethanol in an amount of 8-10 times the weight of the Indian truffle is used for reflux extraction; The fluid extract is processed as follows: using distilled water to disperse the concentrated liquid, and the dispersion is placed in a dialysis bag and soaked in distilled water for 12 days, with the water being changed twice a day. After the soaking is completed, the dialysate is concentrated to obtain the fluid extract.

4. The method for extracting pyrrolidone compounds with anti-inflammatory activity according to claim 2, characterized in that: In step (2), the methanol-distilled water gradient elution method is: The elution time was 0-40 hours, the eluent was distilled water, and the eluent flow rate was 5 ml / min; The elution time is 40-80 hours, the eluent is methanol-distilled water with a volume ratio of 30:70, and the eluent flow rate is 5 ml / min; The elution time is 80-120 hours, the eluent is methanol-distilled water with a volume ratio of 60:40, and the eluent flow rate is 5 ml / min; The elution time is 120-160 hours, the eluent is methanol-distilled water with a volume ratio of 90:10, and the eluent flow rate is 5 ml / min.

5. The method for extracting pyrrolidone compounds with anti-inflammatory activity according to claim 2, characterized in that: In step (4), the semi-preparative liquid phase is carried out using a YMC-Pack ODS-A semi-preparative column, the eluent is 13% methanol, the flow rate is 3 ml / min, the detection wavelength is 254 nm, and the column temperature is 30°C.

6. A composition having an anti-inflammatory effect, characterized in that: The invention comprises the pyrrolidone compound according to any one of claims 1 to 5.

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