CN115960068B - Chromone compound with immunosuppressive activity and preparation method and application thereof - Google Patents
Chromone compound with immunosuppressive activity and preparation method and application thereof Download PDFInfo
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- CN115960068B CN115960068B CN202210898333.5A CN202210898333A CN115960068B CN 115960068 B CN115960068 B CN 115960068B CN 202210898333 A CN202210898333 A CN 202210898333A CN 115960068 B CN115960068 B CN 115960068B
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- phomopsinol
- immunosuppressive activity
- phomopsis
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- chromone compound
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- 150000003505 terpenes Chemical class 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention provides a chromone compound with immunosuppressive activity, a preparation method and application thereof, wherein a novel chromone compound is obtained by separating phomopsis asparagi Phomopsis asparagi DHS-48, and is named as: the invention determines the planar structure and the three-dimensional structure of the phomopinol A compound, and the experimental study on the cell immunosuppressive activity shows that the phomopinol A compound has better immunosuppressive activity and low cytotoxicity, can play the role of immunosuppression by directly reducing the calcineurin activity, can be used as an immunosuppressant for inhibiting the intracellular NFAT protein expression and inhibiting the CN/NFAT signal path, and is used for preparing immunosuppressive drugs for immune diseases.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a chromone compound with immunosuppressive activity, and a preparation method and application thereof.
Background
The chromone school name benzo-gamma-pyrone, the derivative with its mother nucleus structure is called chromone compound, is a kind of biological active matter widely existing in nature, such as having various biological activities of anti-inflammatory, antibacterial, antiviral, anticancer, etc., the biological activity has close relation with the substitution condition, and different chromone structures show different biological activities. Along with the separation and identification of more and more chromone compounds, the research of the biological activity of the chromone compounds is valued by students at home and abroad, and the chromone compounds become one of hot spots for drug development, so that the continuous discovery of new chromone structures is beneficial to promoting the wide utilization of the chromone compounds in the field of drug development.
In natural microorganism, it can produce naphthol, flavonoid, alkaloid, steroid, terpenoid and other secondary metabolites. Phomopsis is a microorganism fungus of the genus Phomopsis, conidiophore sphere, convex mirror, hemisphere or cone, often formed in insignificant spots, thick and hard, most of the innominate or char. There is no report on the isolation of chromones from Phomopsis.
Disclosure of Invention
In view of the above, the present invention provides a chromone compound with immunosuppressive activity, and a preparation method and application thereof
The technical scheme of the invention is realized as follows:
the invention provides a chromone compound with immunosuppressive activity, which is named as: the chemical structural formula of the Phomopsinol A is shown as follows:
further described, the Phomopsinol A is obtained by adding the epigenetic modifier sodium butyrate to perform epigenetic regulation and control on the phomopsis asparagi Phomopsis asparagi DHS-48, and separating; the concentration of sodium butyrate was 50. Mu.M.
A preparation method of chromone compounds with immunosuppressive activity comprises the following steps:
(1) Extracting mycelium obtained by fermenting Asparagus cochinchinensis Phomopsis asparagi with ethyl acetate for 3 times each for 72 hr, concentrating and mixing the extracts to obtain crude extract;
(2) Subjecting the crude extract to chromatography on silica gel column chromatography using CH 2 Cl 2 Gradient elution with methanol binary system, CH 2 Cl 2 The volume ratio of the methanol to the methanol is as follows: the polarity is sequentially increased from 100:0 to 0:100, and Fr.1-Fr.9 components are obtained;
(3) Placing the component Fr.6 on silica gel CC, and adopting CH with volume ratio of 2:1 2 Cl 2 Isocratic elution with ethyl acetate eluent to give fr.6.1-fr.6.6 fractions;
(4) Placing the component Fr.6.3 in Sephadex LH-20CC, and adopting methanol-CH with volume ratio of 1:1 2 Cl 2 Purifying to obtain the Phomopsinol A.
Further described is the use of Phomopsinol a in the preparation of an immunosuppressant.
Application of Phomopsinol in preparing immunosuppressant for inhibiting calcineurin activity by taking calcineurin as target.
Use of Phomopsinol a in the preparation of an immunosuppressant for inhibiting expression of NFAT protein in a cell.
Application of Phomopsinol A in preparing immunosuppressant for inhibiting CN/NFAT signal path.
Use of Phomopsinol a in the preparation of an immunosuppressant which reduces the mRNA transcription level of IL-2 in a cell.
Application of Phomopsinol A in preparing medicines for inhibiting proliferation of T lymphocytes and B lymphocytes.
Use of Phomopsinol a for the preparation of an inhibitor for inhibiting the expression of cd4+ molecules in lymphocytes.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, a novel chromone compound Phomopinol A is separated from the phomopsis asparagi Phomopsis asparagi DHS-48, the planar structure and the three-dimensional structure of the Phomopinol A are determined by analyzing NMR, MS and ECD calculation data of an electronic circular dichroism spectrum, and the experimental result of mouse spleen cell immunosuppressive activity phenotype shows that the Phomopinol A can effectively inhibit T lymphocyte and B lymphocyte proliferation, and has good immunosuppressive activity and low cytotoxicity.
The research and experiment result of the mechanism of the mouse spleen cell immunosuppression activity shows that the phomopinol A can play an immunosuppression role by directly reducing the activity of the calcineurin, inhibit the expression of CD4+ molecules in lymphocytes and reduce the mRNA transcription level of IL-2 in cells, can be used as an immunosuppressant for inhibiting the expression of NFAT protein in cells, and inhibit the transfer of NFAT1 in cytoplasm into the nucleus, can be used as an immunosuppressant for inhibiting CN/NFAT signal channels, and lays a foundation for developing an immunosuppressant which takes the calcineurin as a target point with high efficiency and low toxicity.
Drawings
FIG. 1 is a diagram showing the correlation between COSY and HMBC of the Phomopsinol A compound of the present invention;
FIG. 2 is a schematic representation of NOESY correlation of the Phomopsinol A compound of the present invention;
FIG. 3 is an Electronic Circular Dichroism (ECD) spectrum of the Phomopsinol A compound of the present invention;
FIG. 4 is a graph showing the analysis of the toxic effect of Phomopsinol A of the present invention on mouse spleen cells; DD-42 is the number of the compound Phomopsin A; csA is a positive control; concentration; splenocyte viability (spleen cell viability);
FIG. 5 is a graph showing the analysis of the effect of Phomopsinol A in inhibiting Con A induced splenocyte proliferation in accordance with the present invention; cell viability;
FIG. 6 is a graph showing the analysis of the proliferation of LPS-induced B lymphocytes inhibited by Phomopsinol A according to the present invention; inhibition rate (Inhibition rate);
FIG. 7 is a graph showing the inhibition of calcineurin A by Phomopsinol A of the present invention; CN Inhibition rate (CN enzyme inhibition);
FIG. 8 is a graph showing the effect of Phomopsinol A of the present invention on apoptosis of spleen cells; apoptosis percentage (percent apoptosis);
FIG. 9 is a graph showing the analysis of the effect of Phomopsinol A of the present invention on intracellular calcium ion concentration;
FIG. 10 is a graph showing the analysis of the effect of Phomopsinol A of the invention on the proportion of CD4+ and CD8+ T cells in Con A-induced spleen lymphocytes; t cell subsets (T cell populations);
FIG. 11 is a graph showing the analysis of the inhibition of NFAT protein expression in spleen lymphocytes by Phomopsinol A of the present invention; relative expression level of protein (protein expression level);
FIG. 12 is a graph showing the analysis of the effect of Phomopsinol A of the present invention on cell supernatant cytokines IL-2, IL-4, IL-6;
FIG. 13 is a graph showing the analysis of the results of the decrease in mRNA level of IL-2 gene in cells by Phomopsinol A of the present invention;
FIG. 14 is a graph showing analysis of the results of the expression level of NFAT protein in the nucleus of a cell inhibited by Phomopsinol A of the present invention;
FIG. 15 is a hydrogen spectrum of NMR of Phomopsinol A of the present invention;
FIG. 16 is a carbon spectrum of NMR of Phomopsinol A of the invention
FIG. 17 is a DEPT spectrum of Phomopsinol A of the present invention;
FIG. 18 is a graph of the H-H COSY spectrum of Phomopsinol A of the present invention;
FIG. 19 is a HSQC spectrum of Phomopsinol A of the present invention;
FIG. 20 is a HMBC spectrum of Phomopsinol A of the present invention;
FIG. 21 is a NOESY spectrum of Phomopsinol A of the present invention;
FIG. 22 is a HR-ESI-MS spectrum of Phomopsinol A of the present invention;
FIG. 23 is a graph showing colony morphology of Phomopsis asparagopsis Phomopsis asparag cultured on different PDA plates in an embodiment of the invention; a is added with 5-Ac; b is adding sodium butyrate; c is adding sodium butyrate and a 5-Ac combined inhibitor;
FIG. 24 is a graph showing the analysis of the results of various epigenetic regulators on colony growth and mycelium biomass in the examples of the present invention; sodium butyrate; 5-AZA is 5-Ac;5-AZA and Sodium butyrate are Sodium butyrate and 5-Ac combined inhibitors;
FIG. 25 is a TLC comparison of bacterial metabolites in PDA containing different inhibitors according to the present invention; a is adding sodium butyrate; b is added with 5-Ac; c is adding sodium butyrate and a 5-Ac combined inhibitor;
FIG. 26 is an HPLC plot of cultures of different concentrations of different chemical appearance inhibitors in an example of the invention; a is adding sodium butyrate; b is added with 5-Ac; c is the addition of 5-Ac and sodium butyrate.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
The phomopsis asparagi Phomopsis asparagi is preserved in China Center for Type Culture Collection (CCTCC) No. M2021318, and the preservation date is 2021, 3 months and 31, and the fungus is disclosed in patent CN 113603629A.
Example 1
According to the invention, the sodium butyrate serving as an epigenetic modifier is added to perform epigenetic regulation and control on the phomopsis asparagi Phomopsis asparagi DHS-48, so that a chromone compound with immunosuppressive activity is separated, and the chromone compound is named as: phomopsinol A (1) has the following chemical structural formula:
the present invention determines the planar and steric structures of the new compounds by resolving their NMR, MS and Electronic Circular Dichroism (ECD) calculation data.
Phomopsinol a (1), pale yellow amorphous powder. Specific optical rotation [ alpha ]] 25 D=160 (c 0.001, meOH), UV absorbing UV (MeOH) λmax 213, 214, 215nm, cationic HR-ESI-MS gives [ M+Na ] at M/z 331.0781] + Peak, thus the compound has the formula C 28 H 35 NO 3 The unsaturation was 8. Hydrogen spectrum data, carbon spectrum data of compound 1 H- 1 The H COSY and HSQC spectral data show a series of characteristic signals [ delta ] of 1,2, 3-trisubstituted benzene rings H 6.43(d,J＝8.2Hz),δ C 109.3,d,CH-2；δ H 7.38(t,J＝8.2Hz),δ C 138.9,d,CH-3；δ H 6.52(d,J＝8.2Hz),δ C 108.7,d,CH-4]Signal [ delta ] of olefinic protons of trisubstituted double bonds H 5.61(q,J＝1.7Hz)；δ C 122.3,d,CH-7]Signal of one methyl group (delta) H 1.86,3H,s；δ C 19.2,q,CH 3 -11) a signal [ delta ] of an oxymethylene group H 4.12,(d,J＝13.2Hz),δ H 4.03(d,J＝13.2Hz)；δ C 64.1,t,CH 2 -12]And the signal of two oxidized methines (delta H 4.69,1H,s,δ C 109.3,d,CH-5；4.55,1H,d,J＝4.8Hz,δ C 67.9, d, CH-8). Comparing the compound to phaseolorin D 1 H and 13 c NMR (Table 1) found that both had the same core of chromone except that the compound had a trisubstituted double bond at C-6 (δ140.5) and C-7 (δ122.3) and that C-6 of phaseolorin D was an sp 3 Methine, C-7 is an sp 3 A methylene group. 1 H- 1 Olefin protons (delta) were observed in the H COSY spectrum H 5.61, H-7) and oxygen-containing methine (delta) H 4.69, H-8) and H was observed in HMBC spectra 3 Correlation of 11 with C-5, C-6 and C-7 (see FIG. 1). Thus, the planar structure of Phomopsinol A was determined in conjunction with HR-ESI-MS data.
The invention obtains H through analysis of NOESY spectrum 2 NOE correlation between-12/H-5, from NOESY of H-5/H-8, H-8 and H-5 and H 2 -12 having opposite spatial orientations. In view of the evidence above, it is assumed that the compound has the same stereochemical structure as phaseolorin D (see fig. 2) based on the fact that it has the same biological genetic origin as phaseolorin D in theory.
The absolute configuration of compound Phomopsinol a (1) was determined by comparing experimental and calculated ECD spectra using time-dependent density functional theory (TDDFT). Two possible configurations 5S,5aS,8S,8aR and 5R,5aR,8R,8aS (1 and ent-1) were calculated using the MeOH PCM solvent model at B3LYP/6-31+G (d, p) concentrations, respectively. The ECD spectrum calculated by the compound Phomopsinol A (1) is very consistent with the experimental spectrum (as shown in figure 3), which shows that the absolute configuration is 5S,5aS,8S and 8aR. Thus, the complete structure 1 is established.
Phomopsinol A(1), 1 H NMR(400MHz,CD 3 OD)δ H 7.38(1H,t,J＝8.2Hz,H-3),6.52(1H,d,J＝8.2Hz,H-4),6.43(1H,d,J＝8.2Hz,H-2),5.61(q,J＝1.7Hz,H-7),4.69(1H,s,H-5),4.55(1H,d,J＝4.8Hz,H-8),4.12(d,J＝13.2Hz,Ha-12),4.03(d,J＝13.2Hz,Hb-12),1.86(3H,s)； 13 C NMR(100MHz,CD 3 OD)δ C 197.2(C-9),163.2(C-1),160.8(C-4a),140.5(C-6),138.9(C-3),122.3(C-7),109.3(C-2),108.7(C-4),108.4(C-9a),86.5(C-8a),74.5(C-10a),74.3(C-5),67.9(C-8),64.1(C-12),19.2(C-11)。
Example 2 epigenetic Regulation experiments
The invention regulates and controls the endophytic fungi Phomopsis asparagi by a chemical epigenetic modification method, and observes and compares the bacterial growth condition before and after adding small molecular substances and the change of metabolites.
1.1 preparation of epigenetic modifier and Small Scale fermentation
(1) Purchase of the small molecule inhibitors 5-azacytidine (5-AC) (DNA methyltransferase inhibitor), sodium butyrate (histone deacetylase inhibitor) from saengorgement;
(2) The epigenetic reagent is dissolved by dimethyl sulfoxide to prepare mother solution with certain concentration, agar, glucose and potato broth are mixed according to fixed proportion to prepare PDA solid culture medium, 12 groups of culture dishes are prepared, and each group is 5 dishes. Adding small molecule inhibitor into culture medium after high temperature sterilization, adding epigenetic reagent mother liquor with different volumes according to calculation, and making 5-azacytidine (5-AC) final concentration (μM) in culture medium (1-4 groups) be 0 (control), 10,50,100; sodium butyrate (5-7 groups) final concentration (μm) was 0,10,50,100; final concentration (μm), 0,10,50,100 of 5-azacytidine and sodium butyrate combination (groups 8-10); in addition, a blank control is added, and when the culture medium is solidified, the target strain is inoculated into the culture medium, and the culture is performed in an inverted manner after sealing.
(3) Simultaneously preparing 1000mL of LPDB liquid culture medium, dividing into 10 bottles, sterilizing at high temperature for 100mL of each bottle, and repeating the above operation, wherein each group of prepared inhibitor is respectively added into PDB culture medium to make the final concentration (mu M) of 5-azacytidine (5-AC) in the culture medium (1-4 bottles) be 0 (control), 10,50,100; sodium butyrate (5, 8 bottles) final concentration (μm) was 0,10,50,100; 5-azacytidine and sodium butyrate in combination, 9-12 bottles) at a final concentration (μΜ) of 0,10,50,100;
(4) Inoculating the bacteria into the culture medium, and culturing for 10 days at the constant temperature of 28 ℃;
(5) The above experiment was performed in triplicate.
1.2 determination of colony growth and mycelium dry weight for solid and liquid cultures
Phomopsis asparagi DHS-48 were grown in PDA for 5 days to allow colonies to grow into stable colonies. Thereafter, small pieces of agar were picked in PDA and inoculated into 500 mLErlenmer flasks of 100mL potato dextrose broth (24 g/L), continuously shaken at 150rpm for 10 days, PDA (15 mL agar medium) was incubated upside down at 28℃for 5 days, and each PDB flask and PDA contained 5-azacytidine (5-ac) at concentrations (0, 10,50, 100. Mu.M), sodium butyrate, and a combination of both inhibitors. Each PDB flask and PDA was extracted three times with 250mL and 50mL EtOAc. After filtering the liquid medium, the mycelium pellet was washed three times with distilled water and dried to constant weight at 45 ℃. Since fungi are grown on PDA, mycelium is not easily separated from the solid culture medium and nucleic acid can be used to indirectly determine total mycelium dry weight. Pure mycelia with weights of 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3g were added with 25 ml of 5% trichloroacetic acid solution in a water bath at 80℃and stirred for 25 minutes, cooled in an ice bath at 8000r/min, centrifuged at 4℃for 15 minutes, diluted 5 times, and OD values were measured at 260nm using 1% trichloroacetic acid as a blank. Calibration equations and regression coefficients between nucleic acid content and mycelium weight were obtained. For each condition, three PDA plates were ground and dried, nucleic acids were extracted and measured in the same manner as described above, with unfermented solid matrix as a blank. OD values of PDA extracts were measured at 260nm and converted to the number of mycelia by calibration equation. The experiment is carried out in three times of parallel experiments, the average value of the biological metabolism quantity called by the three times of parallel experiments is taken, and the standard deviation is calculated for analysis.
Analysis of results:
(1) Colony morphology: the target strain was inoculated with 5-azacytidine (5-Ac) at a final concentration (. Mu.M) of 0 (control), 10,50,100; the final concentration of sodium butyrate (μM) was 0,5,10,30,50,100; the final nicotinamide concentration (. Mu.M) was 0,5,10,30,50,100 in the center of the PDA plate, which was incubated upside down in an incubator at 28℃for 5 days, and the colony color, size and hypha growth were observed. The results are shown in FIG. 23: under the same culture environment, when the 5-Ac inhibitor, the 5-Ac and the sodium butyrate combined inhibitor are added into the culture medium, the colony diameter is obviously smaller than that of a control group, and the hypha grows more slowly with the increase of the concentration; when sodium butyrate was added to the medium at a concentration of less than 50. Mu.M, the colony diameter increased with increasing concentration, and when sodium butyrate was at a concentration of 100. Mu.M, the colony diameter was significantly smaller than that of the control group.
(2) Analysis of the effect of different epigenetic modulators on colony growth and mycelium biomass: chemical epigenetic manipulations of Phomopsis asparagi DHS-48 were performed using different concentrations (0,10,50,100. Mu.M) of histone deacetylase inhibitor, 5-azacytidine (5-ac), DNA methyltransferase inhibitor, sodium butyrate, and combinations of these inhibitors in PDB and PDA. We assessed mycelium dry weight and colony growth in PDB and PDA (liquid and solid fermentations) with cultures without epigenetic modifications as blank controls. As a result, as shown in FIG. 24, colony growth and mycelium dry weight were both dependent on the concentration of epigenetic agent. Colony size and mycelium dry weight decreased with increasing concentrations of 5-AC inhibitor and sodium butyrate in combination with 5-AC inhibitor. Surprisingly, sodium butyrate proved to increase colony size and mycelium dry weight at an optimal concentration of 50 μm. The advantage of 50. Mu.M sodium butyrate is more pronounced in PDA. Thus, it was concluded that solid fermentation is preferable to liquid fermentation.
(3) TLC comparison: after the strain grows for 14d in the culture medium, soaking and extracting metabolites of the strain with ethyl acetate, placing each group of extracts into a sample collecting small bottle, using a silica gel plate for display, observing dark spots on the plate under ultraviolet, using different color developing agents for color development reaction, and coating bismuth potassium iodide and FeCl in a TLC plate 3 When the color developing agent with the same characteristics is used, no obvious color developing characteristic exists, and the chemical composition displayed by the strain can be seen through a baking mode (figure 25) under the action of the concentrated sulfuric acid color developing agent. 1. In the TLC plates of groups 2,3 and 4, the color of the concentrated sulfuric acid baking plate is more vivid and various, and compared with the control group, the polarity of the chemical substances is different, and the color of the concentrated sulfuric acid baking plate of groups 5-9 is single and dark compared with the control group, so that the addition of sodium butyrate in the culture medium is more favorable for activating the silent genes of the strain, and the strain metabolizes more novel monomer compounds.
(4) HPLC comparative analysis: according to the invention, HPLC spectrograms of metabolites of bacteria under different culture conditions are respectively detected by adopting a gradient elution mode, and the results are shown as 26: when the sodium butyrate inhibitor was added at a concentration of less than 50 μm, the metabolites showed more diversity in the HPLC profile, and new peaks appeared at retention times of 20min and 30min, and at retention times of 38min, the height of the HPLC peaks was higher than that of the control group, and when the additive was added at a concentration of 50 μm, the heights and abundance of the peaks were more than 10 μm and 100 μm, whereas when the other two inhibitors were added, the metabolite peaked significantly less than that of the control group, and with increasing inhibitor concentration, the peak height also decreased, thus it was seen that the liquid profile peaks were more complex than that of the control group, indicating a more abundant metabolite content when sodium butyrate was added to the medium.
In combination with the above experiment, under the same conditions, the difference in morphology of the bacteria was observed, and when sodium butyrate was added to the medium, the hypha diameter was larger than that of the control group, the growth rate of the bacteria was increased, and at a concentration of 50. Mu.M, the colony diameter was larger than that of the medium at concentrations of 10. Mu.M and 100. Mu.M. From this, it was initially deduced that the growth of this strain was more favored in a 50. Mu.M sodium butyrate-conditioned PDA medium. The metabolites of the bacteria regulated by sodium butyrate are compared and analyzed, and when the plates are baked by concentrated sulfuric acid, chemical substances with polarity different from that of a control group are observed; and the HPLC spectrum shows that the new peaks appear at 20min and 30min when the metabolites regulated and controlled by sodium butyrate are added, so that the new metabolites can be judged to be more beneficial to the bacteria under the culture condition, and the spectrum peaks appearing in the regulation group are higher than those of the control group when the culture condition is about 38min, and the sodium butyrate can be inferred to stimulate the bacteria to cause the increase of the content of the metabolites, so that the method has important significance for finding new stimulated metabolites. In connection with the above analysis, sodium butyrate medium added at a concentration of 50. Mu.M was selected as an optimal medium, and large-scale fermentation was performed with the medium.
Example 3
A preparation method of chromone compounds with immunosuppressive activity comprises the following steps:
(1) Extracting mycelium obtained by fermenting Phomopsis asparagi Phomopsis asparagi with ethyl acetate for 3 times each for 72 hours, concentrating and mixing the extracts, and weighing to obtain 20g of crude extract;
(2) The crude extract (65 g) was chromatographed on a silica gel column using CH 2 Cl 2 Gradient elution is carried out on a methanol binary system (100:0-0:100, v/v), and the polarity is sequentially increased to obtain Fr.1-Fr.9 components;
(3) Placing Fr.6 component on silica gel CC with CH 2 Cl 2 Isocratic elution with ethyl acetate (2:1, v/v) eluent to give the fr.6.1-fr.6.6 fraction;
(4) Placing the component Fr.6.3 on Sephadex LH-20CC with methanol-CH 2 Cl 2 (1:1, v/v) to give Phomopsinol A (4.6 mg).
EXAMPLE 4 phenotypic Studies of mouse spleen cell immunosuppressive Activity by Phomopsinol A
According to the invention, the isolated compound phomopinol A is evaluated for cytotoxicity, con A induced splenocyte proliferation and LPS induced splenocyte proliferation immunosuppressive activity, and experimental results show that the compound phomopinol A has immunosuppressive activity on mouse splenocytes and has lower cytotoxicity on the mouse splenocytes.
Animal material: female BLAB/c mice raised under SPF conditions for 6-8 weeks.
Experimental reagent: RPMI1640 medium (Boster), fetal bovine serum (Sichuan green), CCK-8 reagent (Biosharp), LPS (Sigma), calcineurin (CaN) test kit (Nanjing built), annexin V-FITC/PI apoptosis kit (Vazyme Cat. A211-01), cell-specific DMSO (Boster), (Con A) Canavalin A (Sigma), erythrocyte lysate (Biosharp), phomopsinol A compounds and the like.
1.1, preparation of mouse spleen cell suspension:
1) A dig-off box, a 5mL injection needle, etc. are prepared in the ultra-near workbench. The mice were sacrificed by cervical vertebra removal, soaked in alcohol, sprayed with alcohol, placed on a bench, and then a small opening was cut from the middle of the left abdominal side of the mice, the abdominal wall was exposed, the peritoneum was lifted, the spleen was exposed, the spleen was removed with forceps, and placed in a 15mL centrifuge tube containing 5mL of culture medium.
2) Spleen was cut into sections on a 200 mesh stainless steel mesh, the spleen was gently ground with a syringe needle and the mesh was gently rinsed with culture medium to obtain spleen cell suspension.
3) Centrifuging at 1000rpm for 5min, removing supernatant, lysing erythrocytes with erythrocyte lysate for 5min, centrifuging for 5min, and adding 4ml RPM 1640 complete medium to obtain cell suspension. Then counting to obtain a density of 1 x 10 7 Cell suspensions per ml.
1.2 toxicity experiments
1) Taking 96-well cell culture plate, wherein the inoculation concentration of each well is 1-10 7 mu.L of lymphocyte suspension per mL, 37℃and 5% CO 2 The cells were cultured overnight in an incubator to stabilize the cells.
2) Phomopsinol A or positive control (CsA) diluted to different concentrations with complete medium was added per well to give final concentrations of 1, 5,10, 15, 20, 30 and 40. Mu.M, respectively. The blank was added to complete medium containing 0.2% dmso, 3 duplicate wells per concentration. After 72h incubation in the incubator, 20. Mu.L of CCK-8 reagent was added to each well. Incubation is carried out for 4h at 37 ℃, and OD is read by an enzyme label instrument 450 Cell viability was calculated. The calculation formula is as follows:
spleen cell viability = OD Experimental group /OD Blank control group ×100％。
Analysis of experimental results: as shown in FIG. 4, the compound Phomopsinol A has spleen lymphocyte cytotoxicity IC on normal mice 50 124.87 + -8.74 μm, while CsA has strong cytotoxicity, IC 50 =10.89±0.78 μm, indicating that Phomopsinol a has low toxicity to spleen cells over a range of concentrations.
1.3 Effect of Phomopsinol A on the immunosuppressive Activity of Con A-induced mouse spleen cells
1) Spleen cell suspension was prepared in the same manner as 1.1 by taking 96-well cell culture plates at an inoculation concentration of 5×10 6 Spleen cell suspension per ml, 100. Mu.l per well, 37 ℃,5% CO 2 Culturing in an incubator.
2) Grouping:
blank control group: 200 μl of RPMI-1640 complete medium; negative control group: 100. Mu.l of cell suspension and 100. Mu.l of complete RPMI-1640 medium; con A positive control group: 100. Mu.l of cell suspension and 100. Mu.l of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL); csA positive control group: 100. Mu.l of cell suspension, 50. Mu.l of complete RPMI-1640 medium containing Con A (final concentration 5. Mu.g/mL), 50. Mu.l of complete CsA (CsA concentration 5. Mu.M); drug control group: 100. Mu.L of complete RPMI-1640 medium and 50. Mu.L of Con A (final concentration 5. Mu.g/mL) and 50. Mu.L of Phomopsinol A at different concentrations;
drug group: 100. Mu.l of cell suspension, 50. Mu.l of Con A-containing (final concentration 5. Mu.g/mL), 50. Mu.l of complete RPMI-1640 medium containing Phomopsinol A (final concentration 20. Mu.M, 35. Mu.M, 50. Mu.M, 70. Mu.M, 100. Mu.M), three duplicate wells were placed per well, 37℃and 5% CO 2 After culturing in an incubator for 48 hours, taking out the 96-well plate, adding 20ul of CCK-8 reagent into each well, and placing the mixture in the incubator to keep out of the sun for further incubation for 4 hours. Absorbance was measured at 450nm using a microplate reader.
Analysis of experimental results: as shown in FIG. 5, in the experiment of proliferation of Con A-induced spleen cells, the control group, namely the negative control group, was set to have a cell survival rate of 100%, and the other groups were all induced to proliferate with Con A having a final concentration of 5. Mu.g/ml, and as shown in FIG. 5, phomopinol A had a certain inhibitory effect on Con A-induced spleen lymphocytes, and the higher the concentration of Phomopinol A, the lower the cell survival rate, and the concentration dependence was exhibited. The concentration of the Phomopsinol A for half-inhibiting the spleen lymphocytes induced by Con A is IC 50 ＝42.35±2.49μM。
1.4.Phomopsinol A determination of immunosuppressive Activity of LPS-induced mouse spleen cells
1) The spleen cell suspension of mice was prepared as in 1.1. Taking 96-well cell culture plate, inoculating 100 μl of spleen cell suspension per well at a concentration of 5×10 6 Individual/ml, 5% CO at 37℃ 2 Culturing in an incubator.
2) Grouping: blank control group: 200 μl of RPMI-1640 complete medium; negative controlGroup: 100. Mu.l of cell suspension and 100. Mu.l of complete RPMI-1640 medium; LPS positive control group: 100. Mu.l of cell suspension and 100. Mu.l of RPMI-1640 complete medium containing LPS (final concentration 10. Mu.g/mL); drug control group: 100. Mu.L of complete RPMI-1640 medium and 50. Mu.L of LPS (final concentration 10. Mu.g/mL) and 50. Mu.L of Phomopsinol A at different concentrations; drug group: 100. Mu.l of cell suspension, 50. Mu.l of LPS-containing (final concentration 10. Mu.g/mL), 50. Mu.l of complete RPMI-1640 medium containing Phomopsinol A (final concentration 20. Mu.M, 35. Mu.M, 50. Mu.M, 70. Mu.M, 100. Mu.M), three duplicate wells were placed per well, 37℃and 5% CO 2 After culturing in an incubator for 48 hours, taking out the 96-well plate, adding 20ul of CCK-8 reagent into each well, and placing the mixture in the incubator to keep out of the sun for further incubation for 4 hours. Absorbance was measured at 450nm using a microplate reader.
Analysis of experimental results:
as shown in fig. 6, LPS can induce proliferation of B lymphocytes in spleen cells, and as shown in fig. 6, phomopinol a has a certain inhibitory effect on LPS-induced spleen lymphocytes, and the higher the concentration of phomopinol a, the better the inhibitory effect, and the concentration dependence is exhibited. The half-inhibitory concentration of Phomopsinol A on LPS-induced spleen lymphocytes is IC 50 ＝88.19±2.59μM。
Furthermore, as compared with fig. 5 and 6 above, phomopsinol a has a relatively better effect on inhibiting T cell proliferation than B cells.
1.5.Phomopsinol A Effect on the inhibitory Activity of CN enzymes in vitro
1) The operation steps are as follows: the sample to be tested is Phomopsinol A, and the final concentration is as follows: 1. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M, 50. Mu.M, 70. Mu.M, 90. Mu.M, 120. Mu.M.
2) The calculation formula is as follows: relative inhibition (%) = [1- (OD drug + enzyme-OD drug control)/OD enzyme ] ×100%
Analysis of experimental results: as shown in FIG. 7, in vitro enzyme activity experiments were performed using pNPP as a substrate, and the relative inhibition ratio of the monomeric compound, phomopsinol A, to the CN target enzyme was measured, and as can be seen from FIG. 7, the higher the concentration of Phomopsinol A, the higher the inhibition ratio to CN, the higher the concentration dependence, and the IC 50 48.48.+ -. 0.23. Mu.M.
1.6.Phomopsinol A effects on apoptosis of spleen cells
1) Spleen cell suspension was prepared in the same manner as 1.1, with final cell concentration of 1×10 7 Cell suspension per ml. Added to 24-well plates at 37℃in 500. Mu.l each well with 5% CO 2 Culturing in an incubator overnight.
2) Other reagent groups were as follows: negative control group: 1mL of RPMI-1640 complete medium was added. Con A positive control group: 1mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL). CsA positive control group: 100. Mu.l of cell suspension, 50. Mu.l of complete RPMI-1640 medium containing Con A (final concentration 5. Mu.g/mL), 50. Mu.l of complete CsA (CsA concentration 5. Mu.M); drug group: 1mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL) and Phomopsinol A (final concentrations 30. Mu.M, 45. Mu.M, 60. Mu.M, respectively). 37 ℃,5% CO 2 After 48h of culture in an incubator, the cells were collected, stained and examined by flow cytometry.
Analysis of experimental results: as shown in fig. 8, after 48 hours of Phomopsinol a treatment, apoptosis was detected by flow cytometry, and as shown in fig. 8, spleen cells had almost no apoptosis with increasing concentration, i.e., no toxic effect on cells within the selected concentration.
EXAMPLE 5 mechanism of Phomopsinol A on mouse spleen cell immunosuppressive Activity
Animal material: female BLAB/c mice raised under SPF conditions for 6-8 weeks.
Experimental reagent: RPMI1640 medium (Boster), fetal bovine serum (Siraitia), cell-specific DMSO (Boster), (Con A) Canavalia (Sigma), erythrocyte lysate (Biosharp), fluo-4 AM calcium ionFluorescent probes (Biyunshan S1060), tris-Glycine-SDS electrophoresis buffer (Boster), tris-Glycine-SDS transfer buffer (Boster), SDS-PAGE gel preparation kit (Boster), super-sensitive ECL chemiluminescent ready-to-use substrate (Boster), 10 XTBST buffer (Solaro), 45 μm PVDF membrane (Biosharp), NFAT1 (D43B 1) XP (R) Rabbit mAb (CST), 4% paraformaldehyde (Biosharp), enhanced RIPA lysate (Boster), perCP/cyanine5.5 Anti-Mouse CD3 (Elabscience), FITC Conjugate-Anti-Mouse CD4 (Elabscience), PE Conjugate-Anti-Mouse CD8 (Elabscience), ELISA kit Mouse interleukin 4 (IL-4), ELISA kit Mouse interleukin 6 (IL-6), ELISA kit Mouse interferon-gamma (IFN-gamma), ELISA kit Mouse interleukin 2 (IL-2) (Shanghai pacifying), Q711 ChamQ Universal SYBR qPCR Master Mix, RC101 FastPure Cell/Tissue Total RNA Isolation Kit, R323-01HiScriptAll in one RT SuperMix pefect for qPCR (Vazyme), 4% paraformaldehyde (Biosharp), triton X-100 (Biyun sky), cy 3-conjugated IgG (Boster), phomopsinol A compounds, and the like.
2.1.Phomopsinol A effect on intracellular calcium ion concentration
1) Spleen cell suspension was prepared in the same manner as 1.1, with final cell concentration of 1×10 7 Cell suspension per ml. Into 24-well plates, 500. Mu.l each at 37℃and 5% CO 2 Culturing in an incubator overnight.
2) Other reagent groups were as follows: negative control group: 1mL of RPMI-1640 complete medium was added. Con A positive control group: 1mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL). Drug group: 1mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL) and Phomopsinol A (final concentrations 30. Mu.M, 45. Mu.M, 60. Mu.M, respectively). 37 ℃,5% CO 2 After culturing for 48h in an incubator, the cells are collected, washed three times with PBS, and then washed 3 times with PBS after being stained for 30min in the dark at 37 ℃ by Fluo-4 AM, and incubated for 15min in the dark, and the intracellular calcium ion condition is detected by a flow cytometer.
Analysis of experimental results:
as shown in fig. 9, the target cell population is subjected to the process. Calculating the average fluorescence intensity which can be detected by the FITC channel, wherein the average fluorescence intensity of only cells, FITC-A, is 121344; after Con A stimulation, the average fluorescence intensity FITC-A is 157602, and only cells have se:Sub>A significant difference (P < 0.05) from the average fluorescence intensity of Con A group, namely calcium ions are in inflow; and after being treated by the phomopinol A, the average fluorescence intensity has no statistical significance (P is more than 0.05) compared with that of Con A group, so that the phomopinol A does not influence the calcium ion inflow, and the phomopinol A plays an immunosuppressive role by directly reducing the calcineurin activity, which is obtained by combining with FIG. 7.
2.2.Phomopsinol A effect on the proportion of CD4+ and CD8+ T cells in Con A-induced spleen lymphocytes
1) Spleen cell suspension was prepared in the same manner as 1.1, with final cell concentration of 1×10 7 Cell suspension per ml. Into 24-well plates, 500. Mu.l each at 37℃and 5% CO 2 Culturing in an incubator overnight.
2) Other reagent groups were as follows: negative control group: 1mL of RPMI-1640 complete medium was added. Con A positive control group: 1mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL). CsA positive control group: 100. Mu.l of cell suspension, 50. Mu.l of complete RPMI-1640 medium containing Con A (final concentration 5. Mu.g/mL), 50. Mu.l of complete CsA (CsA concentration 5. Mu.M); drug group: 1mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL) and Phomopsinol A (final concentrations 30. Mu.M, 45. Mu.M, 60. Mu.M, respectively). 37 ℃,5% CO 2 The incubator was cultured for 48 hours.
3) Dyeing: the three blank groups were respectively labeled with the fluorescent-labeled antibodies CD3/Percp-Cy5.5, CD4/FITC, CD8/PE; taking one antibody without adding any antibody; the other samples need to be added with the three antibodies simultaneously. After dyeing at 4℃for 30min in the absence of light, the sample was taken out and washed three times with PBS. Finally, 500. Mu.l of PBS was added and the mixture was examined by flow cytometry. Dyeing ratio: 100 μl PBS+5 μl antibody.
Analysis of experimental results:
as shown in fig. 10, the expression level of cd4+ molecules in Con a group was significantly increased compared to the blank group, while Phomopsinol a significantly inhibited the expression of cd4+ molecules induced by Con a, but no change was apparent to the cd8+ T cell subset, resulting in a significantly reduced cd4+/cd8+ T cell ratio.
Western blot detection of NFAT protein expression
1) Spleen cell suspension was prepared in the same manner as 1.1, with final cell concentration of 1×10 7 Cell suspension per ml. 2mL of each well was added to a 6-well plate at 37℃with 5% CO 2 Culturing in an incubator overnight.
2) Other reagent groups were as follows: negative control group: adding 2mL of RPMI-1640 complete culture solution; con A positive control group: 2mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL); csA group: 2mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL) and CsA (final concentration 5. Mu.M); drug group: 2mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL) and Phomopsinol A (final concentrations 30. Mu.M, 45. Mu.M, 60. Mu.M, respectively). 37 ℃,5% CO 2 The incubator was cultured for 48 hours.
3) Cells were collected and the supernatant was removed by centrifugation. The pre-chilled PBS was washed twice and transferred to the EP tube.
4) Protein extraction and concentration determination: extracting protein with strong lysis solution of Boshide, carefully blowing off cells per tube, reacting on ice for 13min, centrifuging at 14000g for 5min, and collecting supernatant. Protein concentration was determined using BCA protein quantification kit, and then adjusted with 5 x SDS-PAGE Loading Buffer and pre-chilled PBS. Boiling at 100deg.C for 8min, cooling, and storing in a refrigerator at-20deg.C. The protein loading was 30. Mu.g and the loading volume was 15. Mu.l.
5) Electrophoresis and transfer: the gel was prepared according to SDS-PAGE gel preparation kit, a comb was inserted, the gel was allowed to solidify, and the comb was carefully pulled out. Connecting an electrophoresis device, adding electrophoresis liquid, and carefully adding the protein sample into the slot. The power supply was connected (parameters: u=80v; i=120ma; p=50w; t=15min) and the electrophoresis conditions were changed when the proteins were electrophoresed to the edge of the separation gel (parameters: u=120v; i=150ma; p=50w; t=50min). And after the isoprotein sample is electrophoresed to the bottom of the gel plate, closing electrophoresis, taking out the gel, cutting the PVDF film according to the size of the gel, and soaking the PVDF film in methanol for 1min. The materials were placed in this order from negative to positive (sponge-filter paper-gel-PVDF membrane-filter paper-sponge). Transfer membrane was performed in transfer membrane solution (parameters: u=220v; i=250ma; p=50w; t=90 min).
6) Closing: PVDF membranes were placed in an incubator with 5% nonfat milk powder in TBST, and incubated overnight at 4℃on a shaker.
7) Antibody adding and incubation: the PVDF membrane was removed the next day and washed three times for ten minutes each. The primary antibody and the secondary antibody are diluted according to the requirements of the specification, after the primary antibody is incubated for 2 hours at room temperature, the PVDF membrane is washed three times for ten minutes each time, then the secondary antibody is incubated for 1 hour at room temperature, and the PVDF membrane is washed three times.
8) Developing: and (3) 3mL each of ECL luminous solution A and ECL luminous solution B are taken and mixed in equal volume, the front surface of the PVDF film is downward and protected from light for 1min, and then the PVDF film is placed into a cassette to be exposed for 10-15s.
9) Data were processed and Image J was subjected to gray scale analysis.
Analysis of experimental results:
as shown in FIG. 11, the expression level of NFAT was significantly increased after spleen cells were induced by Con A, as can be seen from the bar graph. After addition of various concentrations of Phomopsinol a and positive control (CsA) treatments, NFAT protein expression levels were reduced and exhibited concentration dependence. From this, it is clear that the process of NFAT expression can be inhibited by Phomopsinol A. Thus, phomopsinol a is a potential immunosuppressant capable of inhibiting intracellular NFAT protein expression.
ELISA method for detecting cell supernatant cytokines
1) Spleen cell suspension was prepared in the same manner as 1.1, with final cell concentration of 1×10 7 Cell suspension per ml. Added to a 24-well plate, 500. Mu.L of each well was incubated at 37℃with 5% CO 2 Culturing in an incubator overnight.
2) The group addition reagents were as follows: negative control group: adding 1mL of RPMI-1640 complete culture solution; con A positive control group: 1mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL); csA group: 1mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL) and CsA (final concentration 5. Mu.M); drug group: 1mL contains Con A (final concentration 5. Mu.g/mL) and Phomopsinol A (final concentration)30. Mu.M, 45. Mu.M, 60. Mu.M) of RPMI-1640 complete medium. 37 ℃,5% CO 2 The incubator was cultured for 48 hours.
3) Sterile EP tubes were collected, 3000 rpm/heart was separated for 20min, the collected supernatants were aspirated, and each cytokine standard was diluted as required in the specification.
4) Sample adding: blank holes are respectively arranged: no sample or enzyme-labeled reagent is added, 50 mu L of sample diluent is added, and the rest steps are unchanged; standard well: 50 μl of diluted standard; sample well to be measured: 40. Mu.L of sample dilution+10. Mu.L of sample. After incubation for 30min at 37 ℃ in dark, the liquid is discarded, and after spin-drying, the liquid is washed five times with diluted washing liquid. And removing blank holes, adding 50 mu L of enzyme-labeled reagent into the rest holes, incubating for 30min at 37 ℃ in a dark place, throwing away liquid, washing for five times, then adding 50 mu L of color reagent A, 50 mu L of color reagent B and 10min at 37 ℃ in a dark place into each hole, stopping the reaction by using 50 mu L of stop solution, and detecting OD (optical density) values at 450nm after the blank Kong Diaoling.
Analysis of experimental results:
as shown in FIG. 12, after spleen cells are induced by Con A, the cells are activated rapidly, the content of IL-2, IL-4 and IL-6 is obviously increased, and cells are treated by adding the phomopinol A with different concentrations and positive control (CsA), so that the result shows that the phomopinol A can obviously reduce the content of IL-2, and the greater the concentration, the greater the inhibition effect and the concentration dependence. Phomopsinol A can inhibit the production of IL-2, IL-4 and IL-6 in spleen cells, thereby having better immunosuppressive activity.
2.5. Real-time fluorescent quantitative PCR detection
1) Spleen cell suspension was prepared in the same manner as 1.1, with final cell concentration of 1×10 7 Cell suspension per ml. 2mL of each well was added to a 6-well plate at 37℃with 5% CO 2 Culturing in an incubator overnight.
2) Other reagent groups were as follows: negative control group: adding 2mL of RPMI-1640 complete culture solution; con A positive control group: 2mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL); control group: 2mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL) and CsA (final concentration 5. Mu.M); drug group: 2mL contains Con A (final concentration 5. Mu.g/mL) and Phomopsinol A (final concentrationRPMI-1640 complete medium at 30. Mu.M, 45. Mu.M, 60. Mu.M, respectively). 37 ℃,5% CO 2 The incubator was cultured for 48 hours.
3) Cells were collected, total RNA was extracted using the FastPure Cell/Tissue Total RNA Isolation Kit kit, and then concentration was measured to adjust the total RNA concentration to 1. Mu.g/. Mu.L. Then use HiScriptAll in one RT SuperMix pefect for qPCR reverse transcription was performed. The following mixtures were placed in RNase-free centrifuge tubes: 14. Mu.L RNase-free ddH 2 O, 4. Mu.L of 5 Xall-in-one Supermix, 1. Mu.L of Enzyme Mix, 1. Mu.L of total RNA. The reverse transcription procedure was: 42 ℃,60min,70 ℃ and 5min.
4) Real-time fluorescent quantitative PCR
The IL-2 target gene primer is as follows: an upstream primer 5'-CCTGAGCAGGATGGAGAATTACA-3', a downstream primer 5'-CCGCAGAGGTCCAAGTTCA-3'; internal reference beta-actin primer: upstream primer 5'-CCTCTGACGTCCATCATCTA-3', downstream primer: 5'-ATCTTCTGCTGCCGTCGCTT-3'.
The sterile and enzyme-free qPCR tube is configured as follows:
2X ChamQ Universal SYBR qPCR Master Mix plus 2. Mu.L; 2.5. Mu.M Gene product plus 1.5. Mu.L; adding 2 mu L of reverse transcription product; ddH 2 O was added 4. Mu.L. The reaction procedure: pre-denaturation at 95℃for 10min; circulating for 40 times at 95 ℃ for 15 s-60 ℃ for 1min; the dissolution profile was 60℃to 95℃and the temperature was raised by 0.3℃every 15s. And finally, carrying out data analysis according to the Ct value of the result.
Analysis of experimental results:
as shown in FIG. 13, the effect of Phomopsinol A on the mRNA level of the cytokine IL-2 downstream of NFAT in cells was examined by real-time quantitative PCR. The results in FIG. 13 show that Phomopsinol A can effectively reduce the mRNA transcription level of IL-2 in cells, and finally reduce the expression of IL-2, thereby achieving the immunosuppressive effect.
2.6. Immunofluorescence detection
1) Spleen cell suspension was prepared in the same manner as 1.1, with final cell concentration of 1×10 7 Cell suspension per ml. AddingInto 6-well plates, 2mL of each well at 37℃and 5% CO 2 Culturing in an incubator overnight.
2) Other reagent groups were as follows: negative control group: adding 2mL of RPMI-1640 complete culture solution; con A positive control group: 2mL of RPMI-1640 complete medium containing Con A (final concentration 5. Mu.g/mL); drug group: 2mL of RPMI-1640 complete medium containing Con A (final concentration of 5. Mu.g/mL) and Phomopsinol A (final concentration of 60. Mu.M). 37 ℃,5% CO 2 The incubator was cultured for 48 hours.
3) The treated cells were collected by an EP tube, centrifuged, and 1mL of 4% paraformaldehyde was added to fix the cells for 15min after discarding the supernatant. Centrifuge at 400 Xg for 5min. The PBS was washed three times for 10min each. A further 1mL of 0.1% Triton X-100 was added at room temperature for 15min to permeabilize the cell membrane. Centrifuge at 400 Xg for 5min. The PBS was washed three times for 10min each.
4) Blocking with 5% BSA for 1h reduced non-specific binding signals.
5) Centrifuge at 400 Xg for 5min. After discarding the supernatant, 500. Mu.L of diluted NFAT1 primary antibody (diluted with 1:100 PBS) was added and stained at room temperature in the absence of light for 2 hours. Centrifuge at 400 Xg for 5min. The PBS was washed three times for 10min each. mu.L of Cy 3-conjugated IgG was added as a staining secondary antibody (1:500) and incubated for 1h at room temperature in the dark. Centrifuge at 400 Xg for 5min. The PBS was washed three times for 10min each. Add 500. Mu.L DAPI nuclear stain and dye for 5min at room temperature in the dark. The PBS was washed three times, the last time left about 100. Mu.L, the cells were added dropwise to the slide glass, and the slide glass was covered with a cover glass and photographed by confocal microscopy.
Analysis of experimental results: to better investigate the expression level of NFAT in spleen cells, the effect of Phomopsinol a on NFAT expression in the nucleus was examined using immunofluorescence. The results are shown in fig. 14, with small amounts of NFAT1 protein present in the cytoplasm in the control splenocytes; after Con A stimulation, the cells proliferate rapidly and a large amount of NFAT1 protein is expressed in the nucleus. However, after acting on spleen cells with 60. Mu.M of Phomopsinol A, the NFAT1 protein was significantly reduced, indicating that Phomopsinol A was able to inhibit the transfer of intracytoplasmic NFAT1 into the nucleus. Thus, phomopsinol a is a potential immunosuppressant by inhibiting the CN/NFAT signaling pathway.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A chromone compound having immunosuppressive activity, characterized by: the chromone compounds are named: the chemical structural formula of the Phomopsinol A is shown as follows:
。
2. a chromone compound having immunosuppressive activity as recited in claim 1, wherein: the Phomopsinol A is prepared by adding 50 mu M biological epigenetic modifier sodium butyrate to phomopsisPhomopsis asparagi And performing epigenetic regulation and control on DHS-48, and separating to obtain the DHS-48.
3. The method for preparing a chromone compound having immunosuppressive activity as recited in claim 1, wherein: the method comprises the following steps:
(1) Mold of Phomopsis asparagiPhomopsis asparagiExtracting mycelium obtained by fermentation with ethyl acetate for 3 times for 72 hr each time, concentrating and mixing the extracts to obtain crude extract; said PhomopsisPhomopsis asparagiPhomopsis for epigenetic regulation of sodium butyrate as biological epigenetic modifier at 50. Mu.M concentrationPhomopsis asparagi；
(2) Subjecting the crude extract to chromatography on silica gel column chromatography using CH 2 Cl 2 Gradient elution with methanol binary system, CH 2 Cl 2 The volume ratio of the methanol to the methanol is as follows: 100:0-0:100, and sequentially increasing the polarity to obtain Fr.1-Fr. components;
(3) Placing component Fr.6 in silica gel CC, and adopting CH with volume ratio of 2:1 2 Cl 2 Acetic acid ethyl esterIsocratic elution of the ester eluent to obtain Fr. 6.1.1-Fr. 6.6.6.6 components;
(4) Placing component Fr. 6.3.3 into Sephadex LH-20CC, and adopting methanol-CH with volume ratio of 1:1 2 Cl 2 Purifying to obtain the Phomopsinol A.
4. Use of a chromone compound having immunosuppressive activity as claimed in claim 1, wherein: application of Phomopsinol A in preparation of immunosuppressant is provided.
5. The use of a chromone compound having immunosuppressive activity as recited in claim 4, wherein: application of Phomopsinol A in preparing immunosuppressant for inhibiting calcineurin activity by taking calcineurin as target.
6. Use of a chromone compound having immunosuppressive activity as recited in claim 5, wherein: use of Phomopsinol a in the preparation of an immunosuppressant for inhibiting expression of NFAT protein in a cell.
7. Use of a chromone compound having immunosuppressive activity as recited in claim 6, wherein: application of Phomopsinol A in preparing immunosuppressant for inhibiting CN/NFAT signal path.
8. The use of a chromone compound having immunosuppressive activity as recited in claim 4, wherein: use of Phomopsinol a in the preparation of an immunosuppressant which reduces the mRNA transcription level of IL-2 in a cell.
9. Use of a chromone compound having immunosuppressive activity as claimed in claim 1, wherein: application of Phomopsinol A in preparing medicines for inhibiting proliferation of T lymphocytes and B lymphocytes.
10. Use of a chromone compound having immunosuppressive activity as claimed in claim 9, wherein: use of Phomopsinol a for the preparation of an inhibitor for inhibiting the expression of cd4+ molecules in lymphocytes.
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| US3988335A (en) * | 1974-05-08 | 1976-10-26 | American Home Products Corporation | 1-Substituted-6-(2-substituted aminoethoxy)xanthen-9-ones |
| CN113603629A (en) * | 2021-06-30 | 2021-11-05 | 海南大学 | Cytochalasin compound with immunosuppressive activity and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3988335A (en) * | 1974-05-08 | 1976-10-26 | American Home Products Corporation | 1-Substituted-6-(2-substituted aminoethoxy)xanthen-9-ones |
| CN113603629A (en) * | 2021-06-30 | 2021-11-05 | 海南大学 | Cytochalasin compound with immunosuppressive activity and preparation method and application thereof |
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| A fungal metabolite which inhibits the interaction of CD4 with major histocompatibility complex-encoded class II molecules;Guy Gammon et al;Eur.J.Immunol.;第24卷;991-998 * |
| Polyketides with Immunosuppressive Activities from Mangrove Endophytic Fungus Penicillium sp. ZJ-SY2;Hongju Liu et al;Marine Drugs;第14卷;1-7 * |
| Prenylated indole alkal oids and chromone derivatives from the fungus Penicillium sp. SCSIO041218;Bin Yang et al;Tetrahedron;第74卷;77-82 * |
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