Macrolide sesquiterpene alkaloid compound as well as preparation method and application thereof

Abstract

The invention discloses a macrolide sesquiterpene alkaloid compound as well as a preparation method and application thereof, and belongs to alkaloid compounds, the structural formula of the macrolide sesquiterpene alkaloid compound is # imgabs0 #, and the invention adopts the macrolide sesquiterpene alkaloid compound as well as the preparation method and application thereof. According to the present invention, the LPS-induced NO release of the LPS-induced mouse mononuclear macrophage RAW264.7 can be significantly inhibited, the LPS-induced mouse primary splenic lymphocyte proliferation can be inhibited, good anti-inflammatory and immunosuppressive activity can be represented, and the LPS-induced LPS-induced mouse mononuclear macrophage can be used for preparing the anti-inflammatory immunosuppressive drugs.

Description

Technical Field

[0001] The invention relates to the technical field of alkaloid compounds, in particular to a macrolide sesquiterpene alkaloid compound and a preparation method and application thereof. Background Art

[0002] Northeast Tripterygium wilfordii is the dried root and rhizome of the plant Tripterygium wilfordii of the Celastraceae family, mainly distributed in Northeast my country. Northeast Tripterygium wilfordii has the effects of dispelling wind and dampness, promoting diuresis and reducing swelling, killing insects and detoxifying. It is often used by the people to treat rheumatoid arthritis, swelling and edema, jaundice, accumulation of mass, traumatic injury, sores and swelling, tinea capitis, skin itching and snake bites. Studies have shown that Northeast Tripterygium wilfordii mainly contains compounds such as sesquiterpenes, diterpenes, triterpenes, alkaloids, and modern pharmacological studies have shown that Northeast Tripterygium wilfordii has anti-inflammatory, immunosuppressive, and anti-cancer pharmacological effects. Therefore, analyzing the active substances that exert anti-inflammatory effects of Northeast Tripterygium wilfordii is an effective means to discover new anti-inflammatory active molecules. Summary of the invention

[0003] The purpose of the present invention is to provide a macrolide sesquiterpene alkaloid compound and a preparation method and application thereof, which embodies good anti-inflammatory and immunosuppressive activity characteristics and can be used to prepare anti-inflammatory immunosuppressive drugs.

[0004] To achieve the above object, the present invention provides a macrolide sesquiterpene alkaloid compound, the structural formula of the macrolide sesquiterpene alkaloid compound is:

[0005]

[0006] The present invention provides a method for preparing a macrolide sesquiterpene alkaloid compound, comprising the following steps:

[0007] Step 1: slice the fresh root of the sample, extract it three times with 70% ethanol by mass concentration by heating and refluxing, combine the extracts, concentrate under reduced pressure and recover the ethanol to obtain a sample extract, disperse the sample extract with water, extract it with an equal volume of ethyl acetate, and concentrate under reduced pressure to obtain an ethyl acetate extract;

[0008] Step 2: subjecting the ethyl acetate extract to alumina column chromatography for gradient elution, collecting an elution fraction for each gradient, and recording the four elution fractions obtained in sequence as HM2-1 to HM2-4;

[0009] Step 3: Tracking and separation by chemical component detection: HM2-1 obtained in step 2 is subjected to silica gel column chromatography and gradient elution is performed. Four elution fractions are collected for each gradient, and the 20 elution fractions obtained in sequence are respectively recorded as E1 to E20;

[0010] Step 4: Tracking and separation by chemical component detection: The E15 obtained in the step 3 is separated by reverse phase ODS medium pressure column chromatography, and gradient eluted with methanol-water to obtain 4 elution fractions, and the 4 elution fractions obtained in sequence are respectively recorded as DG1 to DG4;

[0011] Step 5: Using the method of chemical component detection, the DG3 obtained in step 4 is traced and separated by reverse phase semi-preparation C 18 The target compound, i.e., macrolide sesquiterpene alkaloid compound, was obtained by HPLC separation.

[0012] Preferably, the sample in step one is Tripterygium wilfordii.

[0013] Preferably, in step 2, ethyl acetate-methanol is eluted in a gradient ratio of 100:1, 40:1, 1:1 and 1:100.

[0014] Preferably, in step three, dichloromethane-methanol is eluted in a gradient ratio of 100:1, 50:1, 15:1, 10:1 and 5:1.

[0015] Preferably, the ratio of methanol to water in step 4 is 50%-100%, wherein the methanol-water contains 0.03% by volume of trifluoroacetic acid.

[0016] Preferably, the flow rate of the gradient elution in step 4 is 10-20 ml / min, the elution time is 4-6 h, and the volume of the eluted fraction obtained is 1000-1500 ml.

[0017] Preferably, in the step 5, during separation by reverse phase semi-preparative C18 HPLC column, methanol:water=3:2, wherein the mixture contains 0.03% trifluoroacetic acid by volume.

[0018] Preferably, the flow rate during separation in step 5 is 2-4 ml / min.

[0019] The present invention also provides an application of a macrolide sesquiterpene alkaloid compound in an anti-inflammatory immunosuppressive drug.

[0020] Therefore, the present invention adopts the above-mentioned macrolide sesquiterpene alkaloid compound and its preparation method and application, which can significantly inhibit the NO release of LPS-induced mouse mononuclear macrophage RAW264.7 and inhibit the LPS-induced proliferation of mouse primary spleen lymphocytes, showing good anti-inflammatory and immunosuppressive activity, and can be used to prepare anti-inflammatory immunosuppressive drugs.

[0021] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the HRESIMS spectrum of compound A of a macrolide sesquiterpene alkaloid compound and its preparation method and application example of the present invention;

[0023] Figure 2 The key to compound A of the macrolide sesquiterpene alkaloid compound and its preparation method and application example of the present invention is 1 H- 1 Schematic diagram of H COSY, HMBC and NOESY related signals;

[0024] Figure 3 It is an X-ray single crystal diffraction pattern of compound A of a macrolide sesquiterpene alkaloid compound and a preparation method and application example of the present invention;

[0025] Figure 4 It is a macrolide sesquiterpene alkaloid compound of the present invention and its preparation method and application example of compound A 1 H NMR spectrum;

[0026] Figure 5 It is a macrolide sesquiterpene alkaloid compound of the present invention and its preparation method and application example of compound A 13 C NMR spectrum;

[0027] Figure 6 It is a macrolide sesquiterpene alkaloid compound of the present invention and its preparation method and application example of compound A 1 H- 1 H COSY spectrum;

[0028] Figure 7 It is the HSQC spectrum of compound A of a macrolide sesquiterpene alkaloid compound and its preparation method and application example of the present invention;

[0029] Figure 8 This is the HMBC spectrum of compound A of a macrolide sesquiterpene alkaloid compound and a preparation method and application example of the present invention;

[0030] Fig. 9 It is a NOESY spectrum of compound A of a macrolide sesquiterpene alkaloid compound and its preparation method and application example of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0032] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0033] Example

[0034] The present invention provides a macrolide sesquiterpene alkaloid compound, and the macrolide sesquiterpene alkaloid compound has the structural formula:

[0035]

[0036] The present invention provides a method for preparing a macrolide sesquiterpene alkaloid compound, comprising the following steps:

[0037] Step 1: Slice the fresh root tuber of Tripterygium wilfordii, extract it three times with 70% ethanol by heating and refluxing, combine the extracts, concentrate under reduced pressure and recover ethanol to obtain a sample extract, disperse the sample extract with water, extract it with an equal volume of ethyl acetate, and concentrate under reduced pressure to obtain an ethyl acetate extract;

[0038] Step 2: subjecting the ethyl acetate extract to alumina column chromatography, using ethyl acetate-methanol to gradient elution at 100:1, 40:1, 1:1 and 1:100, collecting one elution fraction for each gradient, and recording the four elution fractions obtained in sequence as HM2-1 to HM2-4;

[0039] Step 3: Tracking and separation by chemical component detection: HM2-1 obtained in step 2 was subjected to silica gel column chromatography, and was eluted with dichloromethane-methanol at a gradient ratio of 100:1, 50:1, 15:1, 10:1 and 5:1, and four elution fractions were collected for each gradient. The 20 elution fractions obtained in sequence were respectively recorded as E1 to E20;

[0040] Step 4: Tracking and separation by chemical component detection. The E15 obtained in step 3 was separated by reverse phase ODS medium pressure column chromatography, and gradient elution was performed with methanol-water (50%-100%, 0.03% trifluoroacetic acid v / v) to obtain 4 elution fractions. The 4 elution fractions obtained in sequence were respectively recorded as DG1 to DG4, wherein the flow rate of the gradient elution was 10-20 ml / min, the elution time was 4-6 h, and the volume of the elution fraction obtained was 1000-1500 ml.

[0041] Step 5: Using the method of chemical composition detection, the DG3 obtained in step 4 is traced and separated by reverse phase semi-preparative C 18 The target compound, i.e., macrolide sesquiterpene alkaloid compound, was obtained by HPLC separation with methanol-water gradient elution (50%-100%, 0.03% trifluoroacetic acid v / v) at a flow rate of 2-4 ml / min.

[0042] The present invention also provides an application of a macrolide sesquiterpene alkaloid compound in an anti-inflammatory immunosuppressive drug.

[0043] Example 1

[0044] The present invention provides a macrolide sesquiterpene alkaloid compound, and a preparation method thereof comprises the following steps:

[0045] Step 1, slice the fresh root tuber of Northeast Tripterygium wilfordii, extract it three times with ethanol at a mass concentration of 70%, combine the extracts, concentrate under reduced pressure and recover the ethanol to obtain Northeast Tripterygium wilfordii extract; disperse the Northeast Tripterygium wilfordii extract with water, extract it with petroleum ether, ethyl acetate and n-butanol in equal volumes for multiple times, and concentrate under reduced pressure to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively;

[0046] Step 2: subjecting the ethyl acetate extract to alumina column chromatography, using ethyl acetate-methanol as gradient elution at 100:1, 40:1, 1:1, and 1:100, collecting one elution fraction for each gradient, and recording the four elution fractions obtained in sequence as HM2-1 to HM2-4;

[0047] Step 3, the eluted fraction HM2-1 was subjected to silica gel column chromatography, and eluted with dichloromethane-methanol at a gradient ratio of 100:1, 50:1, 15:1, 10:1, and 5:1, and 4 eluted fractions were collected for each gradient. The 20 eluted fractions obtained in sequence were respectively recorded as E1 to E20;

[0048] Step 4: Separate the obtained elution fraction (E15) by reverse phase ODS medium pressure column chromatography, elute with methanol-water gradient (50%-100%, 0.03% trifluoroacetic acid v / v), flow rate 15 ml / min, elution for 5 hours, and obtain 4 elution fractions, each elution fraction is 1125 ml, and the 4 elution fractions obtained in sequence are respectively recorded as DG1 to DG4;

[0049] Step 5: The eluted fraction (DG3) was subjected to reverse phase semi-preparative C 18 The product was separated by HPLC with methanol:water=3:2 (0.03% trifluoroacetic acid v / v) and a flow rate of 3.0 ml / min to obtain compound A.

[0050] Determination of Compound A:

[0051] Compound A was measured. Figure 1-Figure 9 They are the HRESIMS spectra of compound A, 1 H- 1 Schematic diagram of H COSY, HMBC and NOESY related signals, X-ray single crystal diffraction diagram, 1 H NMR spectrum, 13 C NMR spectrum, 1 H- 1 The H COSY spectrum, HSQC spectrum, HMBC spectrum and NOESY spectrum, the nuclear magnetic resonance hydrogen spectrum and carbon spectrum data of compound A are shown in Table 1:

[0052] Table 1 H NMR and C NMR data of compound A

[0053]

[0054] Table 1 H NMR and C NMR data of compound A

[0055]

[0056] Note: 1 H-NMR (600 MHz, CD3OD), 13 C-NMR (150 MHz, CD3OD)

[0057] According to (+)-HR-ESIMS m / z 840.2645[M+H] + (calcd for C 41 H 46 NO 18 + , 840.2709) combined with NMR data and 20 degrees of unsaturation to determine its molecular formula is C 41 H 45 NO 18 ( Figure 1 ). Compound A 1 H NMR spectrum showed the presence of four acetyl units [δ H 2.17 (3H, s, OAc-2), 1.94 (3H, s, OAc-9), 1.88 (3H, s, OAc-15), 1.67 (3H, s, OAc-1)], three methyl hydrogen signals [δ H 1.86 (3H, s, H3-14), 1.80 (3H, s, H3-10'), 1.20 (3H, s, H3-12)], two aliphatic methylene hydrogen signals [δ H3.72 (1H, m, H-7'a) and 2.94 (1H, m, H-7'b); 2.79 (1H, m, H-8'a) and 2.32 (1H, m, H-8'b)], two oxymethylene hydrogen signals [δ H 5.85 (1H, d, J = 12.6 Hz, H-13a) and 3.89 (1H, d, J = 12.6 Hz, H-13b); 4.89 (1H, d, J = 13.2 Hz, H-15a) and 4.39 (1H, d, J = 13.2 Hz, H-15b)], an aliphatic methine hydrogen signal [δ H 3.12 (1H, br.s, H-7)], five oxygen-linked methylene hydrogen signals [δ H 5.64 (1H, d, J = 3.0 Hz, H-1), 5.28 (1H, br.s, H-6), 5.40 (1H, s, H-9), 5.10 (1H, t, J = 3.0 Hz, H-2), 4.96 (1H, d, J = 3.0 Hz, H-3)], a 2,3-disubstituted pyridine unit [δ H 8.73 (1H, dd, J = 4.8, 1.8 Hz, H-6'), 8.18 (1H, dd, J = 7.8, 1.8 Hz, H-4'), 7.48 (1H, dd, J = 7.8, 4.8 Hz, H-5')], one benzoyl unit [δ H 7.75(2H,d,J=7.8Hz,H-2″,6″), 7.54(1H,t,J=7.8Hz,H-4″), 7.35(2H,t,J=7.8Hz,H-3″,5″)] (Table 1).

[0058] 13 C NMR spectrum showed 41 resonant carbon signals, including one keto carbonyl carbon signal [δ C 198.8 (C-8)], seven ester carbonyl carbon signals [δ C 167.4 (C-7″), 169.2 (C-12′), 169.7 (OAc-1), 170.1 (OAc-2), 170.5 (OAc-15), 171.1 (OAc-15), 172.6 (C-7″)], an aliphatic quaternary carbon signal [δ C 53.7(C-10)], four oxygen-linked quaternary carbon signals [δ C 95.4 (C-5), 87.1 (C-11), 82.9 (C-9'), 72.1 (C-4)], a monosubstituted benzene ring carbon signal [δ C131.2 (C-1″), 131.2 (C-2″, 6″), 129.4 (C-3″, 5″), 134.4 (C-4″)] and a 2,3-disubstituted pyridine ring carbon signal [δ C 162.1 (C-2'), 128.2 (C-3'), 139.9 (C-4'), 123.1 (C-5'), 152.8 (C-6')] (Table 1).

[0059] The above spectral data combined with HSQC, 1 H- 1 The structure of the compound was determined by H COSY, HMBC, NOESY and X-ray single crystal diffraction (CuKα radiation) experiments as shown below:

[0060] It was named (1R,2R,3S,4S,5S,6R,7R,9S,10S,11R,9′R)-1,2,9,15-tetracetoxy-4,6-dihydroxy-8-oxo-3,13-[2′-(3-carboxy-3-benzoyloxybutyl)]nicotinic acid-dicarbolactone-dihydro-β-agarofuran.

[0061] The Griess reduction method was used to evaluate the effect of compound A on LPS-induced NO production in mouse mononuclear macrophage RAW264.7 cells.

[0062] The specific method is as follows:

[0063] Cell culture:

[0064] Mouse mononuclear macrophage RAW264.7 cells were placed in a constant temperature incubator at 37°C and 5% CO2.

[0065] Reagent configuration:

[0066] Indomethacin mixed with DMSO (10 mM), compound A mixed with DMSO (10 mM, stored at -20°C), lipopolysaccharide LPS (final concentration of 1 μg / mL), cell culture medium, PBS, and Griess reaction reagent.

[0067] Experimental methods:

[0068] The experiment was divided into a control group (only cell culture medium was added), an LPS group, a drug group (LPS + drug with a final concentration of 100 μM), and an indomethacin group (LPS + indomethacin with a final concentration of 20 μM). Each experiment had three replicate wells. RAW264.7 mononuclear macrophages were taken, counted, and plated. The blank control group only added culture medium, and the drug group added LPS and the drug to be tested, and cultured at 37°C and 5% CO2 for 24 hours. The supernatant was added with Griess reagent, and the OD value was measured with an enzyme reader at 550nm. According to NO2 – The NO content was calculated based on the standard curve.

[0069] Standard curve drawing:

[0070] The graph is drawn based on the absorbance values ​​obtained after color development with Griess reagent using different concentrations of NaNO2.

[0071]

[0072] The inhibition rate of compound A on LPS-induced NO production in mouse macrophage RAW264.7 cells was calculated.

[0073] The compound A prepared in Example 1 and indomethacin were used as inhibitors to detect their inhibition rates on NO production in mouse mononuclear macrophage RAW264.7 cells induced by LPS. The results are shown in Table 2.

[0074] Table 2 Compound A inhibits NO release from macrophages

[0075] Compound NO inhibition rate (%) A 62.4％ Indomethacin 28.9％

[0076] Note: Compound A (100 μM), indomethacin (20 μM)

[0077] As can be seen from Table 2, compound A has a significant inhibitory effect on the NO production induced by LPS in mouse mononuclear macrophage RAW264.7 cells.

[0078] The immunosuppressive activity of compound A was evaluated by:

[0079] After anesthetization with chloral hydrate, C57BL / 6 mice were killed by dislocating the neck, soaked in 75% alcohol for 1 minute, and the spleen was removed by opening the abdomen in the clean bench and placed in a 1.5mL sterile centrifuge tube. Wet the 200-mesh sieve with HanK's solution, place the spleen on it, cut it into pieces, and grind it with the piston of the syringe until the spleen tissue is completely ground. Mix the separation liquid and the grinding liquid in a volume ratio of 1:1, place it in a centrifuge, and centrifuge it at 216g for 20min. After the centrifugation, aspirate the upper liquid and carefully aspirate the white film layer, which is the lymphocyte layer. Resuspend the separated lymphocytes with complete culture medium and inoculate them in a 96-well plate, and add compound A (final concentration 25μM) and LPS (final concentration 1μg / ml) at the same time. The well plate was placed in an incubator at 5% CO2 and 37°C for 48 h. 10 μL of CCK-8 solution was added to each well under light-proof conditions. After 2 h of incubation, the absorbance at 450 nm was measured by an enzyme-labeled instrument to calculate the cell survival rate.

[0080] Table 3 Compound A inhibits the proliferation of spleen lymphocytes

[0081]

[0082] As shown in Table 3, the proliferation of primary splenic lymphocytes of mice in the LPS-induced group was more significant, showing immune activation; after the addition of compound A at a certain concentration (25 μM), it can effectively inhibit the proliferation of primary splenic lymphocytes, showing a therapeutic effect, which reflects that compound A has a significant effect on inhibiting lymphocyte proliferation and an immunosuppressive effect.

[0083] Therefore, the present invention adopts the above-mentioned macrolide sesquiterpene alkaloid compound and its preparation method and application, which can significantly inhibit the NO release of LPS-induced mouse mononuclear macrophage RAW264.7 and inhibit the LPS-induced proliferation of mouse primary spleen lymphocytes, showing good anti-inflammatory and immunosuppressive activity, and can be used to prepare anti-inflammatory immunosuppressive drugs.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A macrolide sesquiterpene alkaloid compound, characterized in that: The structural formula of the macrolide sesquiterpene alkaloid compound is:

2. A method for preparing the macrolide sesquiterpene alkaloid compound as claimed in claim 1, characterized in that: The following steps are involved: Step 1: slice the fresh root of the sample, extract it three times with 70% ethanol by mass concentration by heating and refluxing, combine the extracts, concentrate under reduced pressure and recover the ethanol to obtain a sample extract, disperse the sample extract with water, extract it with an equal volume of ethyl acetate, and concentrate under reduced pressure to obtain an ethyl acetate extract; Step 2: subjecting the ethyl acetate extract to alumina column chromatography for gradient elution, collecting an elution fraction for each gradient, and recording the four elution fractions obtained in sequence as HM2-1 to HM2-4; Step 3: Tracking and separation by chemical component detection: HM2-1 obtained in step 2 is subjected to silica gel column chromatography and gradient elution is performed. Four elution fractions are collected for each gradient, and the 20 elution fractions obtained in sequence are respectively recorded as E1 to E20; Step 4: Tracking and separation by chemical component detection: The E15 obtained in the step 3 is separated by reverse phase ODS medium pressure column chromatography, and gradient eluted with methanol-water to obtain 4 elution fractions, and the 4 elution fractions obtained in sequence are respectively recorded as DG1 to DG4; Step 5: Using the method of chemical component detection, the DG3 obtained in step 4 is traced and separated by reverse phase semi-preparation C 18 The target compound, i.e., macrolide sesquiterpene alkaloid compound, was obtained by HPLC separation.

3. The method for preparing a macrolide sesquiterpene alkaloid compound according to claim 2, characterized in that: The sample in the step 1 is Tripterygium wilfordii.

4. The method for preparing a macrolide sesquiterpene alkaloid compound according to claim 2, characterized in that: In the step 2, ethyl acetate-methanol is eluted according to a gradient ratio of 100:1, 40:1, 1:1 and 1:

100.

5. The method for preparing a macrolide sesquiterpene alkaloid compound according to claim 2, characterized in that: In the step 3, dichloromethane-methanol is eluted in a gradient ratio of 100:1, 50:1, 15:1, 10:1 and 5:

1.

6. The method for preparing a macrolide sesquiterpene alkaloid compound according to claim 2, characterized in that: The ratio of methanol to water in step 4 is 50%-100%, wherein the methanol-water contains 0.03% trifluoroacetic acid by volume.

7. The method for preparing a macrolide sesquiterpene alkaloid compound according to claim 2, characterized in that: The flow rate of the gradient elution in step 4 is 10-20 ml / min, the elution time is 4-6 h, and the volume of the eluted fraction obtained is 1000-1500 ml.

8. The method for preparing a macrolide sesquiterpene alkaloid compound according to claim 2, characterized in that: In the step 5, when the separation is performed by reverse phase semi-preparative C18 HPLC column, the ratio of methanol to water is 3:2, wherein the ratio contains 0.03% trifluoroacetic acid by volume.

9. The method for preparing a macrolide sesquiterpene alkaloid compound according to claim 2, characterized in that: The flow rate during separation in step 5 is 2-4 ml / min.

10. Use of the macrolide sesquiterpene alkaloid compound as claimed in claim 1 in anti-inflammatory immunosuppressive drugs.

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