CN111217719B - Alkaloid, and extraction method and application from black pepper - Google Patents
Alkaloid, and extraction method and application from black pepper Download PDFInfo
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Abstract
The invention belongs to the field of chemical medicine, and particularly relates to alkaloid, an extraction method of the alkaloid from black pepper and application of the alkaloid. The invention provides an alkaloid, the structural formula of which is shown as a formula I. In addition, the invention also provides an extraction method and application of the alkaloid from black pepper. The alkaloid provided by the invention has good anti-inflammatory effect.
Description
Technical Field
The invention belongs to the field of chemical medicine, and particularly relates to alkaloid, an extraction method of the alkaloid from black pepper and application of the alkaloid.
Background
Nitric oxide is both an effector of inflammatory responses and immune regulation, and a key regulator. NO is involved in a variety of inflammatory signaling, interacting with a variety of inflammatory factors. NO is produced at each stage of the inflammatory response process, and therefore, the amount of NO in the body can be measured to evaluate the anti-inflammatory activity of an anti-inflammatory agent. In addition, excessive production of NO plays a key regulatory role in the development of inflammation, tumors, infection, cardiovascular disease, autoimmune disease, and the like. Therefore, NO has become not only a new therapeutic strategy for inflammation, autoimmune diseases, etc., but also an important target for the development of new drugs.
Black pepper is used as a traditional medicinal material in many countries, is also a food used as both medicine and food in China, has an anti-inflammatory property, and is most suitable for relieving pain caused by arthritis and dyspepsia. The research finds that the active ingredient piperine (formula X) of black pepper can inhibit cell inflammation, and further is helpful for relieving arthritis.
In order to find a drug with better anti-inflammatory activity, the invention provides a novel alkaloid and a method for extracting the novel alkaloid from black pepper.
Disclosure of Invention
The invention provides an alkaloid, which has a structural formula shown as a formula I:
wherein R is 1 Is C1-C8 alkyl or
a＝0～10，b＝0～10，c＝0～10。
Preferably, the above alkaloid, R 1 Is C1-C6 alkyl or
a＝0～6，b＝0～6，c＝0～6。
Further preferably, R 1 Is C1-C6 alkyl or
a＝0～2，b＝0～6，c＝0～2。
As a preferred technical scheme of the invention, when a is 2,c is 0,R 1 Is methyl, and has a structural formula shown as the following formula II:
wherein b =0 to 10. Preferably b =0 to 6.
The structural formula of the alkaloid is as follows:
as a preferred technical scheme of the invention, when a is 2,c is 1, the structural formula is shown as the following formula III:
wherein R is 1 Is C1-C8 alkyl; b =0 to 10. Preferably R 1 Is C1-C6 alkyl; b =0 to 6. More preferably R 1 Is a pentyl group; b =3 or 4.
The structural formula of the alkaloid is as follows:
as a preferred embodiment of the present invention, when R is 1 Is composed of
And the structural formula is shown as formula IV:
wherein, a =0 to 10, b =0 to 10, c =0 to 10. Preferably, a =0 to 6, b =0 to 6, c =0 to 4. More preferably, a =0 to 2, b =0 to 4, c =0 to 2.
The structural formula is as follows:
the invention also provides pharmaceutically acceptable salts of the alkaloids.
The invention also provides a pharmaceutically acceptable hydrate of the alkaloid.
The invention also provides a pharmaceutical composition which is prepared by adding pharmaceutically acceptable auxiliary components into the alkaloid, salt or hydrate.
The invention also provides application of the alkaloid, the salt, the hydrate or the pharmaceutical composition in preparing anti-inflammatory drugs.
The alkaloid is extracted from black pepper.
The invention also provides a method for extracting the alkaloid from black pepper, which comprises the following steps:
a. extracting black pepper by using an ethanol water solution, and concentrating an extracting solution to obtain an extract;
b. dispersing the extract with water, extracting with solvent, and concentrating to obtain extract;
c. performing column chromatography separation on the extract, collecting and combining the same fractions;
d. c, performing column chromatography separation on at least one section of the fractions obtained in the step c again, and collecting and combining the same fractions;
e. and d, dissolving at least one section of the fraction obtained in the step d by using a solvent, and purifying and separating by using a semi-preparative high performance liquid chromatography to obtain the target compound.
Preferably, in the step a of the method for extracting alkaloids from black pepper, the ratio of black pepper to ethanol aqueous solution is 1-10L of ethanol aqueous solution per 1kg of black pepper.
Preferably, in the step a of the method for extracting alkaloids from black pepper, the ethanol aqueous solution has a mass concentration of 10 to 95%.
Preferably, in step a of the method for extracting alkaloid from black pepper, the extraction is performed by cold soaking, percolation or refluxing.
Preferably, in the step a of the method for extracting alkaloid from black pepper, the extraction time is 1-10 h. The extraction times are 2-4.
Preferably, in the step a of the method for extracting alkaloid from black pepper, the concentration is performed at 30-60 ℃ under reduced pressure.
Preferably, in the step b of the method for extracting alkaloid from black pepper, the solvent is any one of petroleum ether, dichloromethane, ethyl acetate or n-butanol.
Preferably, in step b of the method for extracting alkaloids from black pepper, the volume ratio of the solution obtained by dispersing the extract with water to the solvent during extraction is 1.
Preferably, in the step b of the method for extracting alkaloid from black pepper, the extraction frequency is 2-4.
Preferably, in step c of the method for extracting alkaloids from black pepper, dichloromethane is used for dissolving the extract during column chromatography.
Preferably, in step c, the column chromatography is eluted by petroleum ether-dichloromethane or dichloromethane-methanol. More preferably, the ratio of petroleum ether-dichloromethane or dichloromethane-methanol is varied in a gradient from 50. Further, the gradient from 50. Further, three column volumes were used for elution at each ratio.
Preferably, in step c, the silica gel obtained by column chromatography is 200-300 mesh.
Preferably, in step d of the method for extracting alkaloids from black pepper, at least one of the fractions obtained in step c is at least one of the fractions in the fourth or sixth stage.
Preferably, in step d of the method for extracting alkaloids from black pepper, the column chromatography is performed by dissolving with dichloromethane.
Preferably, in step d of the method for extracting alkaloids from black pepper, the column chromatography is eluted by petroleum ether-dichloromethane or dichloromethane-methanol. More preferably, the ratio of petroleum ether-dichloromethane or dichloromethane-methanol is varied in a gradient from 50. Further, the gradient from 50. Further, three column volumes were used for elution at each ratio.
Preferably, in step d of the method for extracting alkaloid from black pepper, the silica gel for column chromatography is 200-300 mesh.
Preferably, in step e of the method for extracting alkaloid from black pepper, the solvent is methanol or acetonitrile.
Preferably, in step e of the method for extracting alkaloids from black pepper, the column packing of the semi-preparative high performance liquid chromatography is any one of octadecyl bonded silica gel, octyl bonded silica gel, phenyl bonded silica gel or hydrophilic column packing. The grain diameter is 50-200 μm.
Preferably, in step e of the method for extracting alkaloids from black pepper, the mobile phase used in the semi-preparative high performance liquid chromatography is methanol-water or acetonitrile-water.
Further, in the step e of the method for extracting alkaloid from black pepper, the mobile phase is methanol-water or acetonitrile-water isocratic elution, wherein methanol: water = 10-90:90-10, and v/v. Acetonitrile: water = 10-90:90-10, v/v.
Further, when the first fraction separated in step d from the fourth fraction of step c is subjected to step e purification, the mobile phase used is methanol-water =82-88, preferably 85, to obtain the compound
Compound(s)>
Namely compounds II-1 and II-2 respectively.
Further, when the second fraction separated in step d from the fourth fraction of step c is subjected to step e purification, the mobile phase used is methanol-water =87-93, preferably 90, to obtain the compound
Compound->
Namely compounds II-3 and II-4 respectively.
Further, when the third fraction separated in step d from the fourth fraction in step c is subjected to step e purification, the mobile phase used is methanol-water =87-93, preferably 90, to obtain the compound
Compound->
Namely III-1 and III-2 respectively. />
Further, when the first fraction isolated in step d from the sixth fraction of step c is subjected to step e purification, the mobile phase used is methanol-water =67-73, preferably 70, to give the compound
Namely a compound IV-1.
Further, when the fourth fraction isolated in step d from the fourth fraction of step c is subjected to step e purification, the mobile phase used is methanol-water =82-88, preferably 85, to give the compound
Compound->
Namely a compound IV-2 and a compound IV-3 respectively.
Further, when the fifth fraction separated in step d from the fourth fraction in step c is subjected to step e purification, the mobile phase used is methanol-water =82-88, preferably 85, to give the compound
Compound->
Namely a compound IV-4 and a compound IV-5 respectively.
Further, when the sixth fraction isolated in step d from the fourth fraction of step c is subjected to step e purification, the mobile phase used is methanol-water =82-88, preferably 85, to give the compound
Namely the compound IV-6.
The invention separates a plurality of alkaloids from black pepper and measures the inhibition effect of the alkaloids on the nitric oxide production of RAW 264.7 cells, wherein the alkaloids show superior effect to piperine (IC) 50 =53.45 mu M), and the anti-inflammatory drug which has high efficiency, low toxicity and can be orally taken is expected to be developed.
Detailed Description
A method for extracting alkaloid from black pepper comprises the following steps:
1) Crushing black pepper, cold soaking, percolating or reflux extracting with 10-95% concentration water solution of alcohol for 1-10 hr for 2-4 times, and vacuum concentrating the extracted liquid at 30-60 deg.c to obtain extractum;
2) Dispersing the extract with water, adding petroleum ether, dichloromethane, ethyl acetate or n-butyl alcohol according to the volume ratio of 1.5-2 for extraction for 2-4 times, and concentrating to obtain an extract;
3) Dissolving petroleum ether, dichloromethane, ethyl acetate or n-butanol extract with dichloromethane, adding silica gel, stirring, oven drying, loading on silica gel column, eluting with petroleum ether-dichloromethane or dichloromethane-methanol at different ratios, collecting fractions, and mixing the fractions by thin layer chromatography;
4) Further subdividing at least one fraction of the fourth or sixth stage with silica gel column, eluting with petroleum ether-dichloromethane or dichloromethane-methanol at different ratio, collecting fractions, and combining the same fractions by thin layer chromatography;
5) Dissolving the fraction with methanol or acetonitrile, and purifying with semi-preparative high performance liquid chromatography (NovaSep, pompey, france) with 50-200 μm octadecyl bonded silica gel, octyl bonded silica gel, phenyl bonded silica gel or hydrophilic column filler; the mobile phase is methanol-water or acetonitrile-water, and the proportion of the mobile phase is methanol-water (10-90; separating to obtain compounds II-1-II-4, III-1-III-2 and IV-1-IV-6.
EXAMPLE 1 extraction of alkaloid II-1-II-4
1) Pulverizing fructus Piperis 5kg, reflux-extracting with 20L 95% ethanol water solution for 8 hr for 3 times, and concentrating the extractive solution at 45 deg.C under reduced pressure to obtain extract;
2) Dispersing the extract with water, extracting with 1:1 volume ratio of dichloromethane for 3 times, and concentrating to obtain extract;
3) Dissolving the dichloromethane extract with dichloromethane, adding silica gel, stirring, oven drying, loading on 200-300 mesh silica gel column, performing gradient elution with petroleum ether-dichloromethane (50;
4) The fourth fraction was further subdivided on a silica gel column, gradient elution was performed with petroleum ether-dichloromethane (50;
5) The fractions were dissolved in methanol and purified by semi-preparative high performance liquid chromatography using 50 μm octadecyl-bonded silica gel as column packing and methanol-water as mobile phase, with fraction Fr 4.1 having a mobile phase ratio methanol-water =85 to give compounds II-1 and II-2, and fraction Fr 4.2 having a mobile phase ratio methanol-water =90 to give compounds II-3 and II-4.
Alkaloid II-1:
by high-resolution mass spectrometry, 1 H NMR and 13 c NMR identified the compound and the experimental data were as follows:
1 H NMR(400MHz,CD 3 OD):δppm:7.13(1H,dd,J＝15.2Hz,J＝10.4Hz),6.21(1H,dd,J＝15.2Hz,J＝10.4Hz),6.10(1H,dt,J＝15.2Hz,J＝6.8Hz),5.95(1H,d,J＝15.2Hz),3.08(2H,d,J＝6.8Hz),2.19(2H,q,J＝7.2Hz),1.82(1H,9,J＝6.8Hz),1.46(2H,quint,J＝7.2Hz),1.35(4H,m),0.94(6H,d,J＝6.8Hz),0.93(3H,t,J＝6.8Hz). 13 C NMR(400MHz,CD 3 OD):δppm:167.78,142.59,140.75,128.42,121.67,46.64,32.51,31.15,28.36,28.31,22.14,19.13,12.97.HRESIMS(m/z):224.2013([M+H] + ).
alkaloid II-2:
by high-resolution mass spectrometry, 1 H NMR and 13 c NMR identified the compound and the experimental data were as follows:
1 H NMR(400MHz,CDCl 3 ):δppm:7.19(1H,dd,J＝15.2Hz,J＝10.0Hz),6.13(1H,dd,J＝15.2Hz,J＝10.0Hz),6.06(1H,m),5.76(1H,d,J＝15.2Hz),5.60(1H,brs),3.16(2H,t,J＝6.8Hz),2.14(2H,q,J＝6.8Hz),1.80(1H,9,J＝6.8Hz),1.41(2H,quint,J＝6.8Hz),1.28(8H,m),0.92(6H,d,J＝6.8Hz),0.88(3H,t,J＝6.8Hz). 13 C NMR(400MHz,CDCl 3 ):δppm:166.43,143.19,141.29,128.21,121.76,46.94,32.95,31.78,29.13,29.10,28.82,28.63,22.63,20.13,14.07.HRESIMS(m/z):252.2338([M+H] + ).
alkaloid II-3:
by high-resolution mass spectrometry, 1 H NMR and 13 c NMR identified the compound, with the following experimental data:
1 H NMR(400MHz,CDCl 3 ):δppm:7.02(1H,dd,J＝15.2Hz,J＝10.4Hz),6.10(1H,dd,J＝15.2Hz,J＝10.4Hz),6.00(1H,dt,J＝15.2Hz,J＝6.8Hz),5.84(1H,d,J＝15.2Hz),2.98(2H,d,J＝7.2Hz),2.08(2H,q,J＝6.8Hz),1.71(1H,9,J＝6.8Hz),1.35(2H,m),1.20(16H,m),0.83(6H,d,J＝6.8Hz),0.81(3H,t,J＝6.8Hz). 13 C NMR(400MHz,CD 3 OD):δppm:167.79,142.58,140.76,128.44,121.67,46.64,32.55,31.28,29.37,29.32,29.16,29.09,28.95,28.85,28.62,28.36,22.24,19.13,13.05.HRESIMS(m/z):308.2595([M+H] + ).
alkaloid II-4:
by high-resolution mass spectrometry, 1 HNMR and 13 c NMR identified the compound, with the following experimental data:
1 H NMR(400MHz,CDCl 3 ):δppm:7.19(1H,dd,J＝15.2Hz,J＝10.0Hz),6.13(1H,dd,J＝15.2Hz,J＝10.0Hz),6.06(1H,dt,J＝15.2Hz,J＝6.8Hz),5.77(1H,d,J＝15.2Hz),5.63(1H,brs),3.16(2H,t,J＝6.4Hz),2.14(2H,q,J＝6.8Hz),1.80(1H,9,J＝6.8Hz),1.41(2H,quint,J＝6.8Hz),1.26(20H,m),0.92(6H,d,J＝6.8Hz),0.88(3H,t,J＝6.8Hz). 13 C NMR(400MHz,CDCl 3 ):δppm:166.44,143.18,141.27,128.21,121.77,46.94,32.96,31.92,29.68,29.67,29.65,29.58,29.45,29.36,29.19,28.83,28.63,22.69,20.13,14.11.HRESIMS(m/z):336.3264([M+H] + ) EXAMPLE 2 extraction of alkaloid III-1-III-2
1) Pulverizing 5kg of fructus Piperis, reflux extracting with 20L of 95% ethanol water solution for 8 hr, treating for 3 times, and concentrating the extractive solution at 45 deg.C under reduced pressure to obtain extract;
2) Dispersing the extract with water, extracting with 1:1 volume ratio of dichloromethane for 3 times, and concentrating to obtain extract;
3) Dissolving the dichloromethane extract with dichloromethane, adding silica gel, stirring, oven drying, loading on 200-300 mesh silica gel column, performing gradient elution with petroleum ether-dichloromethane (50;
4) The fourth fraction was further subdivided on a silica gel column, gradient elution was performed with petroleum ether-dichloromethane (50;
5) Dissolving the fraction with methanol, and purifying by semi-preparative high performance liquid chromatography, wherein the column packing material is octadecyl bonded silica gel with the size of 50 μm, the mobile phase is methanol-water, and the mobile phase ratio of the fraction Fr 4.3 is methanol-water =90, so as to obtain compounds III-1 and III-2 by separation.
Alkaloid III-1:
by high resolution mass spectrometry, 1 H NMR and 13 c NMR identified the compound, with the following experimental data:
1 H NMR(400MHz,CD 3 OD):δppm:7.02(1H,dd,J＝14.8Hz,J＝10.4Hz),6.10(1H,dd,J＝14.8Hz,J＝10.4Hz),6.00(1H,dt,J＝14.8Hz,J＝6.8Hz),5.84(1H,d,J＝14.8Hz),5.25(2H,m),2.97(2H,d,J＝6.8Hz),2.08(2H,q,J＝6.8Hz),1.94(4H,m),1.71(1H,9,J＝6.8Hz),1.35(2H,quint,J＝6.8Hz),1.24(12H,m),0.83(6H,d,J＝6.4Hz),0.81(3H,t,J＝6.8Hz). 13 C NMR(400MHz,CD 3 OD):δppm:167.78,142.54,140.74,129.44,129.40,128.45,121.69,46.64,32.55,31.75,29.42,29.02,28.88,28.85,28.62,28.36,26.71,26.50,21.97,19.14,12.97.HRESIMS(m/z):334.3108([M+H] + ).
alkaloid III-2:
by high-resolution mass spectrometry, 1 HNMR and 13 c NMR identified the compound, with the following experimental data:
1 H NMR(400MHz,CDCl 3 ):δppm:7.19(1H,dd,J＝15.2Hz,J＝10.0Hz),6.13(1H,dd,J＝15.2Hz,J＝10.0Hz),6.06(1H,dt,J＝15.2Hz,J＝6.8Hz),5.77(1H,d,J＝15.2Hz),5.65(1H,brs),5.35(2H,m),3.16(2H,t,J＝6.4Hz),2.14(2H,q,J＝6.8Hz),2.01(4H,m),1.80(1H,9,J＝6.8Hz),1.41(2H,quint,J＝6.8Hz),1.28(16H,m),0.92(6H,d,J＝6.4Hz),0.88(3H,t,J＝6.8Hz). 13 C NMR(400MHz,CDCl 3 ):δppm:166.47,143.12,141.26,129.86,129.79,128.23,121.81,46.94,32.95,31.78,29.73,29.51,29.33,29.28,29.19,28.98,28.81,28.63,27.22,27.17,22.65,20.12,14.10.HRESIMS(m/z):362.3424([M+H] + ).
EXAMPLE 3 extraction of alkaloids IV-1-IV-6
1) Pulverizing fructus Piperis 5kg, reflux-extracting with 20L 95% ethanol water solution for 8 hr for 3 times, and concentrating the extractive solution at 45 deg.C under reduced pressure to obtain extract;
2) Dispersing the extract with water, extracting with 1:1 volume ratio of dichloromethane for 3 times, and concentrating to obtain extract;
3) Dissolving the dichloromethane extract with dichloromethane, adding silica gel, stirring, oven drying, loading on 200-300 mesh silica gel column, performing gradient elution with petroleum ether-dichloromethane (50;
4) The fourth fraction was further subdivided on a silica gel column, gradient elution was performed with petroleum ether-dichloromethane (50; the sixth fraction was further subdivided on a silica gel column, gradient elution was performed with petroleum ether-dichloromethane (50;
5) Dissolving the fraction with methanol, and purifying by semi-preparative high performance liquid chromatography, wherein the chromatographic column is packed by 50 μm octadecyl bonded silica gel, the mobile phase is methanol-water, the mobile phase ratio of the fraction Fr 6.1 is methanol-water =70, and the compound IV-1 is obtained by separation, the mobile phase ratio of the fraction Fr 4.4 is methanol-water =85, the compound IV-2 and IV-3 are obtained by separation, the mobile phase ratio of the fraction Fr 4.5 is methanol-water =85, the compound IV-4 and IV-5 are obtained by separation, and the mobile phase ratio of the fraction Fr 4.6 is methanol-water = 85.
Alkaloid IV-1:
by high resolution mass spectrometry, 1 H NMR and 13 c NMR identified the compound and the experimental data were as follows:
1 H NMR(400MHz,CDCl 3 ):δppm:7.36(1H,dd,J＝14.8Hz,J＝10.8Hz),6.96(1H,s),6.87(1H,d,J＝7.6Hz),6.76(2H,m),6.67(1H,dd,J＝14.8Hz,J＝10.8Hz),5.97(2H,s),5.95(1H,d,J＝14.8Hz),5.77(1H,brs),3.19(2H,t,J＝6.0Hz),1.82(1H,9,J＝6.4Hz),0.94(6H,d,J＝6.4Hz). 13 C NMR(400MHz,CDCl 3 ):δppm:166.24,148.20,140.94,138.77,130.90,124.70,123.33,122.57,108.48,105.72,101.30,47.03,28.65,20.17.HRESIMS(m/z):274.1441([M+H] + ).
alkaloid IV-2:
by high resolution mass spectrometry, 1 H NMR and 13 c NMR identified the compound, with the following experimental data:
1 H NMR(400MHz,CD 3 OD):δppm:6.91(1H,d,J＝1.6Hz),6.78(1H,dt,J＝15.2Hz,J＝7.2Hz),6.77(1H,dd,J＝8.0Hz,J＝1.2Hz),6.72(1H,d,J＝8.0Hz),6.30(1H,d,J＝15.6Hz),6.08(1H,dt,J＝15.6Hz,J＝7.2Hz),5.95(1H,d,J＝15.2Hz),5.91(2H,s),3.07(2H,d,J＝6.8Hz),2.20(4H,m),1.81(1H,9,J＝6.8Hz),1.48(4H,m),1.39(4H,m),0.93(6H,d,J＝6.8Hz). 13 C NMR(400MHz,CD 3 OD):δppm:167.45,148.00,146.64,144.18,132.47,129.43,128.53,123.35,119.92,107.64,104.85,100.82,46.54,32.54,31.60,29.13,28.72,28.69,28.32,28.05,19.13.HRESIMS(m/z):358.2377([M+H] + ).
alkaloid IV-3:
by high-resolution mass spectrometry, 1 H NMR and 13 c NMR identified the compound and the experimental data were as follows:
1 H NMR(400MHz,CD 3 OD):δppm:7.14(1H,dd,J＝15.2Hz,J＝10.4Hz),6.91(1H,d,J＝1.2Hz),6.78(1H,dd,J＝8.0Hz,J＝1.2Hz),6.73(1H,d,J＝8.0Hz),6.34(1H,d,J＝15.6Hz),6.26(1H,dd,J＝15.2Hz,J＝10.4Hz),6.14(1H,dt,J＝16.0Hz,J＝6.4Hz),6.08(1H,dt,J＝16.0Hz,J＝6.4Hz),5.96(1H,d,J＝14.8Hz),5.92(2H,s),3.08(2H,d,J＝6.8Hz),2.33(4H,m),1.81(1H,9,J＝6.8Hz),0.93(6H,d,J＝6.8Hz). 13 C NMR(400MHz,CD 3 OD):δppm:167.75,148.02,146.79,141.64,140.62,132.22,130.12,128.86,127.26,121.98,120.09,107.66,104.90,100.86,46.65,32.53,31.94,28.35,19.13.HRESIMS(m/z):328.1913([M+H] + ).
alkaloid IV-4:
by high resolution mass spectrometry, 1 H NMR and 13 c NMR identified the compound and the experimental data were as follows:
1 H NMR(400MHz,CDCl 3 ):δppm:7.18(1H,dd,J＝15.2Hz,J＝10.4Hz),6.88(1H,d,J＝0.4Hz),6.75(1H,dd,J＝8.0Hz,J＝1.2Hz),6.72(1H,d,J＝8.0Hz),6.28(1H,d,J＝15.6Hz),6.13(1H,dd,J＝15.2Hz,J＝10.4Hz),6.06(1H,dt,J＝15.6Hz,J＝6.4Hz),6.02(1H,dt,J＝16.0Hz,J＝6.8Hz),5.92(2H,s),5.77(1H,d,J＝15.2Hz),5.64(1H,brs),3.16(2H,m),2.17(4H,m),1.80(1H,9,J＝6.8Hz),1.46(4H,m),0.92(6H,d,J＝6.8Hz). 13 C NMR(400MHz,CDCl 3 ):δppm:166.40,147.94,146.60,142.70,141.14,132.36,129.57,128.95,128.44,121.97,120.24,108.22,105.40,100.92,46.95,32.78,32.68,28.95,28.63,28.32,20.14.HRESIMS(m/z):356.2229([M+H] + ).
alkaloid IV-5:
by high resolution mass spectrometry, 1 H NMR and 13 c NMR identified the compound and the experimental data were as follows:
1 H NMR(400MHz,CDCl 3 ):δppm:7.19(1H,dd,J＝14.8Hz,J＝10.0Hz),6.89(1H,d,J＝0.8Hz),6.75(1H,dd,J＝8.0Hz,J＝1.2Hz),6.73(1H,d,J＝8.0Hz),6.28(1H,d,J＝16.0Hz),6.13(1H,dd,J＝15.2Hz,J＝10.0Hz),6.07(1H,dt,J＝15.2Hz,J＝6.8Hz),6.02(1H,dt,J＝15.6Hz,J＝6.8Hz),5.93(2H,s),5.75(1H,d,J＝14.8Hz),5.56(1H,brs),3.16(2H,m),2.15(4H,m),1.80(1H,9,J＝6.8Hz),1.43(4H,m),1.33(4H,m),0.92(6H,d,J＝6.8Hz). 13 C NMR(400MHz,CDCl 3 ):δppm:166.40,147.92,146.54,143.04,141.25,132.48 129.35,129.34,128.29,121.81,120.20,108.22,105.40,100.90,46.94,32.90,32.85,29.34,29.00,28.95,28.73,28.64,20.13.HRESIMS(m/z):384.2543([M+H] + ).
alkaloid IV-6:
by high-resolution mass spectrometry, 1 H NMR and 13 c NMR identified the compound and the experimental data were as follows:
1 H NMR(400MHz,CDCl 3 ):δppm:7.19(1H,dd,J＝14.8Hz,J＝10.0Hz),6.89(1H,d,J＝1.2Hz),6.75(1H,dd,J＝8.0Hz,J＝1.6Hz),6.73(1H,d,J＝8.0Hz),6.28(1H,d,J＝16.0Hz),6.12(1H,dd,J＝15.2Hz,J＝10.0Hz),6.08(1H,m),6.04(1H,dt,J＝16.0Hz,J＝6.8Hz),5.92(2H,s),5.75(1H,d,J＝14.8Hz),5.56(1H,brs),3.16(2H,t,J＝6.4Hz),2.15(4H,m),1.80(1H,9,J＝6.8Hz),1.42(4H,m),1.29(8H,m),0.92(6H,d,J＝6.8Hz). 13 C NMR(400MHz,CDCl 3 ):δppm:166.51,147.92,146.52,143.24,141.39,132.52,129.49,129.25,128.22,121.69,120.17,108.21,105.3,100.89,46.96,32.94,32.90,29.43,29.41,29.37,29.35,29.16,28.79,28.62,20.12.HRESIMS(m/z):412.2845([M+H] + ).
example 4 detection of nitric oxide production by Griess reaction
The nitrate concentration in the medium was measured by Griess reaction to investigate nitric oxide production. Macrophage RAW 264.7 (purchased from American Type Culture Collection, usa) (8000/well) was transferred to a 96-well plate and cultured with RPMI 1640 medium. The compound solutions of each concentration gradient were added to give final concentrations of 40. Mu.M, 20. Mu.M, 10. Mu.M, 5. Mu.M, 2.5. Mu.M, and 1.25. Mu.M, respectively, and the mixture was pretreated for 2 hours. Nitric oxide production in cells was induced by addition of LPS at a final concentration of 1 μ g/mL to 96-well plates for 22 hours. Subsequently, the supernatant was aspirated and an equal volume of Griess reagent was added for a 10 minute co-incubation. The absorbance of the mixture was measured at 540nm using SpectraMax M5. Comparing the measured value with the measured value of physiological saline to calculate the inhibition rate of each compound on the generation of nitric oxide under different concentrations, and further calculating the IC 50 The value is obtained.
TABLE 1 inhibition of nitric oxide production by alkaloids
As can be seen from Table 1, a plurality of alkaloids of the invention have certain anti-inflammatory activity, so that the derivatives have wide application prospect in medicaments for treating inflammation.
Claims (6)
1. The method for extracting alkaloid from black pepper is characterized by comprising the following steps: the method comprises the following steps:
a. extracting black pepper by using an ethanol water solution, and concentrating an extracting solution to obtain an extract;
b. dispersing the extract with water, extracting with solvent, and concentrating to obtain extract;
c. performing column chromatography separation on the extract, collecting and combining the same fractions;
d. c, performing column chromatography separation on at least one section of the fractions obtained in the step c, and collecting and combining the same fractions;
e. d, dissolving at least one section of the fraction obtained in the step d by using a solvent, and purifying and separating by adopting a semi-preparative high performance liquid chromatography to obtain a target substance;
in the step a, the ratio of the black pepper to the ethanol aqueous solution is that 1-10L of ethanol aqueous solution is added into every 1kg of black pepper;
in the step a, the mass concentration of the ethanol water solution is 10-95%;
in the step b, the solvent is dichloromethane;
in the step b, the volume ratio of the solution obtained by dispersing the extract with water to the solvent during extraction is 1;
in the step c or d, the column chromatography is eluted by petroleum ether-dichloromethane or dichloromethane-methanol; the ratio of petroleum ether-dichloromethane or dichloromethane-methanol is varied in a gradient from 50;
in step d, the fraction is at least one of the fourth stage or the sixth stage;
when the first fraction separated in step d from the fourth fraction of step c is subjected to step e purification, the mobile phase used is methanol-water =82-88, and compound is obtained
Compound (I)
When the second fraction separated in step d from the fourth fraction in step c is subjected to step e purification, the mobile phase used is methanol-water =87-93, yielding the compound
Compound (I)
When for the fourth stage of step cAnd e, when the fraction is purified in the step e by the third fraction separated in the step d, the mobile phase adopted is methanol-water =87-93, and the compound is obtained
Compound (I)
When the first fraction isolated in step d from the sixth fraction of step c is subjected to step e purification, the mobile phase used is methanol-water =67-73, yielding compounds
When the fourth fraction isolated in step d from step c is subjected to step e purification, the mobile phase used is methanol-water =82-88, yielding the compound
Compound(s)>
When the fifth fraction separated in step d from the fourth fraction in step c is subjected to step e purification, the mobile phase used is methanol-water =82-88, yielding the compound
Compound(s)>
When the sixth fraction isolated in step d from the fourth fraction of step c is subjected to step e purification, the mobile phase used is methanol-water =82-88, yielding the compound
2. The method for extracting alkaloid from black pepper according to claim 1, wherein: in the step a, at least any one of the following items is satisfied:
the extraction adopts a cold soaking, percolating or refluxing mode;
the extraction times are 2-4 times;
the extraction time is 1-10 h for each extraction;
the concentration is carried out at 30-60 ℃ under reduced pressure.
3. The method for extracting alkaloid from black pepper according to claim 1, wherein: in the step b, the extraction times are 2-4 times.
4. The method for extracting alkaloid from black pepper according to claim 1, wherein: in step c or d, the gradient change from 50 gradient 1 to 1:5 is in the order of 50.
5. The method for extracting alkaloid from black pepper according to claim 1, wherein: in the step e, at least any one of the following conditions is satisfied:
the solvent is methanol;
the chromatographic column filler of the semi-preparative high performance liquid chromatography is any one of octadecyl bonded silica gel, octyl bonded silica gel, phenyl bonded silica gel or hydrophilic chromatographic column filler;
the particle size of the chromatographic column filler of the semi-preparative high performance liquid chromatography is 50-200 mu m.
6. The method for extracting alkaloid from black pepper according to any one of claims 1-5, wherein:
when the first stage fraction obtained by separating the fourth stage fraction in the step c through the step d is subjected to step e purification separation, the mobile phase adopted is methanol-water = 85;
when the second-stage fraction obtained by separating the fourth-stage fraction in the step c in the step d is subjected to step e purification separation, the mobile phase adopted is methanol-water = 90;
when the third fraction obtained by separating the fourth fraction in the step c through the step d is subjected to step e purification separation, the mobile phase adopted is methanol-water = 90;
when the first stage fraction obtained by separating the sixth stage fraction in the step c in the step d is subjected to step e purification separation, the mobile phase adopted is methanol-water = 70;
when the fourth fraction obtained by separating the fourth fraction in the step c in the step d is subjected to step e purification separation, the mobile phase adopted is methanol-water = 85;
when the fifth fraction obtained by separating the fourth fraction in the step c in the step d is subjected to step e purification separation, the mobile phase adopted is methanol-water = 85;
when the sixth fraction separated in step d from the fourth fraction in step c is subjected to step e purification separation, the mobile phase used is methanol-water = 85.
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Non-Patent Citations (3)
| Title |
|---|
| CYP3A4 Inhibitory Activity of New Bisalkaloids, Dipiperamides D and E, and Cognates from White Pepper;Sachiko Tsukamoto;《Bioorganic & Medicinal Chemistry》;20021231;第2981-2985页 * |
| Hepatoprotective amide constituents from the fruit of Piper chaba: Structural requirements, mode of action, and new amides;Hisashi Matsuda et al.;《Bioorganic & Medicinal Chemistry》;20090829;第17卷(第20期);第7313-7323页 * |
| STN Columbus;Registry;《STN Columbus》;20150314 * |
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