CN110305093B

CN110305093B - Guaiane type sesquiterpenes and preparation method and application thereof

Info

Publication number: CN110305093B
Application number: CN201910288681.9A
Authority: CN
Inventors: 宋少江; 黄肖霄; 任镜先; 任强; 程卓阳
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2019-04-11
Filing date: 2019-04-11
Publication date: 2021-03-23
Anticipated expiration: 2039-04-11
Also published as: CN110305093A

Abstract

The invention belongs to the technical field of medicines, and relates to guaiane type sesquiterpenes and preparation and application thereof. In particular to 5 guaiane sesquiterpene compounds separated from Chinese medicine lilac daphne root and application thereof in the aspect of neuroprotective bioactivity. The guaiane sesquiterpene compound and the pharmaceutically acceptable salt and isomer thereof have the following structures. The compounds of the invention are prepared by the following method: extracting radix Genkwa with ethanol, concentrating the extractive solution under reduced pressure, mixing extractive solutions, concentrating to obtain extract, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and separating the ethyl acetate extract by silica gel column chromatography, macroporous resin, ODS, HPLC, etc. to obtain 5 compounds.

Description

Guaiane type sesquiterpenes and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and relates to guaiane type sesquiterpenes and preparation and application thereof. In particular to 5 guaiane sesquiterpene compounds separated from Chinese medicine lilac daphne root and application thereof in the aspect of neuroprotective bioactivity.

Background

Radix Genkwa refers to root of Genkwa of Thymelaeaceae and Thymelaeaceae, and has bitter and pungent taste, warm property, and toxic effects, and has effects of eliminating dampness, removing toxic substance, and resolving hard mass, and can be used for treating edema, lymphoid tuberculosis, acute mastitis, hemorrhoid fistula, scabies, and rheumatalgia. At present, the research on the aspects of oxidation resistance, virus resistance, tumor resistance, immune function regulation and the like is more. The lilac daphne root is rich in chemical components, including sesquiterpenes, diterpene orthoesters, flavonoids, coumarins, lignans, chlorogenic acids and phenol glycosides, wherein diterpene compounds and flavonoid compounds are main bioactive components, and recent researches show that the sesquiterpene compounds in the lilac daphne root also have good bioactivity.

Disclosure of Invention

The technical problem solved by the invention is to provide a series of guaiane type sesquiterpenoids and application thereof in preparing neuroprotective biological medicines.

The invention relates to guaiane type sesquiterpene compounds with the following structures and pharmaceutically acceptable salts and isomers thereof:

the preparation method of the compound comprises the following steps:

extracting radix Genkwa with ethanol, concentrating the extractive solution under reduced pressure, mixing extractive solutions, concentrating to obtain extract, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and separating the ethyl acetate extract by silica gel column chromatography, macroporous resin, ODS, HPLC, etc. to obtain the above 5 compounds.

Specifically, the method comprises the following steps:

(1) reflux-extracting dried radix Genkwa with 70-80% industrial ethanol for 3 times, mixing extractive solutions, and concentrating to obtain extract. The obtained extract is extracted by petroleum ether, ethyl acetate and n-butanol successively. The ethyl acetate fractions were subjected to silica gel column chromatography with gradient elution using a dichloromethane/chloroform-methanol system to collect a total of 4 fractions (A-D).

(2) Fraction A was subjected to HP20 column chromatography eluting with a gradient of ethanol water, and a total of 2 fractions (A1-A2) were collected.

(3) Fractions A1 and A2 were subjected to ODS column chromatography, eluted with a gradient of ethanol water, and 3 fractions (A1-1, A2-1, A3-1) were collected in total.

(4) Fractions A1-1, A2-1 and A3-1 were chromatographed on silica gel column, respectively, and fractions A-1-1-3, A-2-1-1-A-2-1-16 and A-3-1-1-A-3-1-14 were obtained.

(5) Fraction A-2-1-8 was subjected to reverse phase HPLC with CH₃CN-H₂Eluting with O to obtain compound 1; fraction A-2-1-4 was subjected to reverse phase HPLC with CH₃CN-H₂O elution to give

compounds

2 and 4; fraction A-3-1-9 was subjected to reverse phase HPLC with CH₃CN-H₂Eluting with O to obtain a compound 3; fraction A-3-1-12 was subjected to reverse phase HPLC with CH₃CN-H₂Elution with O affords Compound 5.

Wherein:

the gradient of dichloromethane/trichloromethane-methanol in the step (1) is as follows: 100:1-1:1, specifically 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1: 1.

The concentration of the ethanol water in the step (2) is as follows: 50 to 90 percent.

The concentration of the ethanol water in the step (3) is as follows: 30-90%, specifically 30%, 40%, 50%, 60%, 70%, 80%, 90%.

The conditions of the silica gel column chromatography in the step (4) are as follows: gradient elution with petroleum ether-ethyl acetate at ratio of 50:1-1:1, specifically 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 1: 1).

The chromatographic conditions in the step (5) are as follows: 40% -50% of CH₃CN-H₂O。

The structure of the novel guaiane sesquiterpene compounds (Genkwanonid G, Genkwanonid H, Genkwanonid I, Genkwanonid J and Genkwanonid K) is analyzed.

Compound 1: a yellow oil (dichloromethane) was obtained,

HRESIMS gave the peak of the excimer ion [ M + Na ]]⁺Peak m/z 273.1463(calcd for C)₁₅H₂₂O₃Na,273.1461), combined¹H-NMR、¹³C-NMR presumed to be of the formula C₁₅H₂₂O₃The unsaturation was calculated to be 5.¹H-NMR(600MHz,CDCl₃) Middle, delta_H 1.70(3H,s),δ_H0.81(3H, d, J ═ 7.1Hz) is the two methyl hydrogen signals, δ_H3.58(1H, dd, J ═ 12.3,3.1Hz),4.02(1H, dd, J ═ 12.3,3.5Hz),3.63(1H, o) are two groups of CH with two groups of connected oxygen magnetism unequal₂Of the geminal hydrogen signal of_H4.34(1H, o) is a tertiary carbon with oxygen and no coupled hydrogen signal.¹³C-NMR(100MHz,CDCl₃) A 15 carbon signal was shown, suggesting that compound 1 is a sesquiterpene. Delta_C210.6 isolated carbonyl carbon signals, δ_C122.3,149.3 is the double bond carbon signal, δ_C58.7,63.5,87.0 are the continuous oxygen carbon signals. The compound 1 is supposed to have no terminal double bond, one hydroxymethyl group is added, and all the direct hydrocarbon related signals are subjected to full attribution through HSQC spectrum. In HMBC spectra, H₃-15(δ_H0.81) and C-3 (. delta.))_C 31.9),C-4(δ_C 38.3),C-5(δ_C43.2) related, H₃-14(δ_H1.70) and C-1 (. delta.))_C 149.3),C-9(δ_C 87.0),C-10(δ_C122.3) and thereby the position of the methyl group is determined. H-7 (delta)_H 2.05),H-9(δ_H4.25) and C-8 (. delta.))_C210.6) there is a correlation, confirming the position of the carbonyl group. H₂-12(δ_H3.63) and C-11 (. delta.))_C 47.6),C-13(δ_C58.7) there is a correlation, confirming the position of the hydroxymethyl group. According to the related information, a planar structure of the compound 1 is established, and the compound 1 is guaiane type sesquiterpene. The relative configuration of Compound 1 was determined by NOESY spectroscopy, as shown in FIG. 3, H₃-15(δ_H0.81) and H-3 beta (. delta.)_H1.41) related, H-3 α (δ)_H1.60) and H-5 (. delta.))_H2.26) related, H-5 (. delta.))_H2.26) and H-7 (. delta.))_H2.05) related, H-7 (. delta.))_H2.05) and H-12 (. delta.))_H3.63) are correlated. The relative configuration of compound 1 was thus determined to be 4S,5S,7R,8R, 11S. The absolute configuration of the compound 1 is determined by comparing the calculated specific optical rotation with the actually measured specific optical rotation by adoptingDensity Functional Theory (DFT), the (4S,5S,7R,8R,11S) -1 configuration and its enantiomer were calculated at the level of B3LYP/6-311+ + G (2d, p), respectively. The measured optical rotation of compound 1 was +22.0, the calculated optical rotation of (4S,5S,7R,8R,11S) -1 was +24.5, and the corresponding isomer was-24.5. Thus, the absolute configuration of compound 1 is further defined as 4S,5S,7R,8R, 11S.

After the database search of the scifinider, the compound 1 is an unreported new compound and is named Genkwanoid G. It is composed of¹An H NMR spectrum of the sample was obtained,¹³the signals of the C NMR spectrum are assigned in the following table, and the related spectra are shown in the attached figures 1-2.

Of Genkwanoid G¹H NMR spectrum and¹³c NMR spectral data

Compound 2: a yellow oil (dichloromethane) was obtained,

HRESIMS gave the peak of the excimer ion [ M + Na ]]⁺Peak m/z 289.1411(calcd for C)₁₅H₂₂O₄Na,289.1410), combined¹H-NMR、¹³C-NMR presumed to be of the formula C₁₅H₂₂O₄The unsaturation was calculated to be 5.¹H-NMR(600MHz,CDCl₃) Middle, delta_H 1.70(3H,s),δ_H0.82(3H, d, J ═ 7.0Hz) is the two methyl hydrogen signals, δ_H3.48(1H, dd, J ═ 12.4,1.8Hz),3.87(1H, m),3.53(1H, o) are two groups of CH with two groups of connected oxygen and magnetic inequality₂Upper geminal hydrogen signal, delta_H4.34(1H, s) is a tertiary carbon with oxygen and no coupled hydrogen signal.¹³C-NMR(100MHz,CDCl₃) A 15 carbon signal was shown, suggesting that compound 2 is a sesquiterpene. Delta_C208.8 isolated carbonyl carbon signal, delta_C122.0,149.5 is the double bond carbon signal, δ_C61.9,63.9,78.8,86.3 are continuous oxygen carbon signals. The above hydrocarbon signals are very similar to compound 1, and presumably differ in that compound 2 increases an oxygen-linked carbon signal, decreases an aliphatic carbon signal,the molecular weight is increased by 16 mass units relative to compound 1, and the 7-position of compound 1 is presumed to be substituted by hydroxyl, so that all the direct correlation signals of the hydrocarbon are fully assigned through HSQC spectrum. In HMBC spectra, H₃-15(δ_H0.82) and C-3 (. delta.))_C 32.0),C-4(δ_C 38.3),C-5(δ_C42.7) related, H₃-14(δ_H1.70) and C-1 (. delta.))_C 149.5),C-9(δ_C 86.3),C-10(δ_C122.0) and thereby the position of the methyl group is determined. H-7 (delta)_H 2.82),H-9(δ_H4.34) and C-8 (. delta.))_C208.8) there is a correlation confirming the position of the carbonyl group. H₂-13(δ_H3.48,3.87) and C-11 (. delta.))_C 78.8),C-12(δ_C63.9) there is a correlation, confirming the position of the hydroxyl and hydroxymethyl groups. According to the related information, a planar structure of the compound 2 is established, and the compound 2 is guaiane type sesquiterpene. The relative configuration of Compound 2 was determined by NOESY spectroscopy, as shown in FIG. 6, H₃-15(δ_H0.82) and H-3 beta (. delta.)_H1.39) related, H-3 α (δ)_H1.61) and H-5 (. delta.))_H2.29) related, H-5 (. delta.))_H2.29) and H-7 (. delta.))_H2.82) correlation, H-7 (. delta.))_H2.82) and H-12 (. delta.))_H3.53) correlation. The relative configuration of compound 2 was thus determined to be 4S,5S,7R,8R, 11R. The absolute configuration of compound 2 was determined by comparing the calculated specific optical rotation with the measured specific optical rotation, the measured optical rotation of compound 2 was +25.0, the calculated optical rotation of the (4S,5S,7R,8R,11R) -2 configuration was +6.0, and the corresponding isomer was-6.0. Thus, the absolute configuration of compound 2 is further determined to be 4S,5S,7R,8R, 11R.

Through the database search of the scifinider, the compound 2 is an unreported new compound and is named Genkwanoid H. It is composed of¹An H NMR spectrum of the sample was obtained,¹³the signals of the C NMR spectrum are shown in the following table.

Of Genkwanoid H¹H NMR spectrum and¹³c NMR spectral data

Compound 3 yellow oil (dichloromethane),

HRESIMS gave the peak of the excimer ion [ M + Na ]]⁺Peak m/z 289.1413(calcd for C)₁₅H₂₂O₄Na,289.1410), combined¹H-NMR、¹³C-NMR presumed to be of the formula C₁₅H₂₂O₄The unsaturation was calculated to be 5.¹H-NMR(600MHz,CDCl₃) Middle, delta_H 1.69(3H,s),δ_H0.88(3H, d, J ═ 7.1Hz) is the two methyl hydrogen signals, δ_H3.47(1H, o),3.71(1H, o),3.49(1H, o),3.71(1H, o) are two groups of CH with oxygen and magnetism unequal₂Upper geminal hydrogen signal, delta_H4.24(1H, s) is a tertiary carbon with oxygen and no coupled hydrogen signal.¹³C-NMR(100MHz,CDCl₃) A 15 carbon signal was shown, suggesting that compound 3 is a sesquiterpene. Delta_C207.9 isolated carbonyl carbon signal, delta_C121.3,150.2 is the double bond carbon signal, δ_C60.9,66.6,75.0,85.7 are continuous oxygen carbon signals. The hydrocarbon signals are very similar to that of the compound 2, the hydrocarbon signals of the hydroxymethyl group are obvious, and the molecular weight of the hydroxymethyl group is the same as that of the compound 2. Therefore, 3 is presumed to be an epimer of compound 2. All the directly related signals of the hydrocarbon are subjected to full attribution through HSQC spectrum. In HMBC spectra, H₃-15(δ_H0.88) and C-3 (. delta.))_C 32.1),C-4(δ_C 38.5),C-5(δ_C42.3) correlation, H₃-14(δ_H1.69) and C-1 (. delta.))_C 150.2),C-9(δ_C 85.7),C-10(δ_C121.3) to determine the position of the methyl group, H-7 (. delta.),_H 2.87),H-9(δ_H4.24) and C-8 (. delta.))_C207.9) there was a correlation confirming the position of the carbonyl group. H₂-13(δ_H3.49,3.71) and C-11 (. delta.))_C 75.0),C-12(δ_C66.6) the presence of correlation confirms the position of the hydroxyl and hydroxymethyl groups. Establishing a compound 3 planar structure according to the related informationCompound 3 is a guaiane-type sesquiterpene. The relative configuration of Compound 3 was determined by NOESY spectroscopy, as shown in FIG. 9, H₃-15(δ_H0.88) and H-3 beta (. delta.))_H1.42) related, H-3 α (δ)_H1.64) and H-5 (. delta.))_H2.33) related, H-5 (. delta.))_H2.33) and H-7 (. delta.)_H2.87) related, H-7 (. delta.))_H2.87) and H-6 alpha (. delta.) (delta.)_H1.82) related, H₂-12(δ_H3.47,3.71) and H-6 beta (. delta.) (delta.)_H1.47). The relative configuration of compound 3 was thus determined to be 4S,5S,7R,8R, 11S. The absolute configuration of compound 3 was determined by comparing the calculated specific optical rotation with the measured specific optical rotation, the measured optical rotation of compound 3 was +22.0, the calculated optical rotation of the (4S,5S,7R,8R,11S) -3 configuration was +22.3, and the corresponding isomer was-22.3. Thus, the absolute configuration of compound 3 is further determined to be 4S,5S,7R,8R, 11S.

After the database search of the scifinider, the compound 3 is an unreported new compound and is named Genkwanoid I. It is composed of¹An H NMR spectrum of the sample was obtained,¹³the signals of the C NMR spectrum are shown in the following table.

Of Genkwanoid I¹H NMR spectrum and¹³c NMR spectral data

Compound 4: a yellow oil (dichloromethane) was obtained,

HRESIMS gave the peak of the excimer ion [ M + Na ]]⁺Peak m/z 273.1462(calcd for C)₁₅H₂₂O₃Na,273.1461), combined¹H-NMR、¹³C-NMR presumed to be of the formula C₁₅H₂₂O₃The unsaturation was calculated to be 5.¹H-NMR(600MHz,CDCl₃) Middle, delta_H 1.06(3H,d,J＝6.8Hz)，δ_H 1.59(3H,s),δ_H1.87(3H, s) is the three methyl hydrogen signals, δ_H3.65(1H, d, J ═ 11.1Hz),3.90(1H, d, J ═ 11.1Hz), 4.57(1H, d, J ═ 16.8Hz), 4.63(1H, d, J ═ 16.8Hz) are two groups of vicinal hydroperoxidesSignal at delta_HA series of lipid hydrogen signals are shown in the range of 1.5-2.9.¹³C-NMR(100MHz,CDCl₃) A 15 carbon signal was shown, suggesting that compound 4 is a sesquiterpene. Delta_C125.9,158.4,175.6 is the carbon signal, delta, of an alpha, beta-unsaturated ketone_C126.5,142.4 is the double bond carbon signal, δ_CAnd the oxygen-linked carbon signals are 64.8 and 72.9. All the directly related signals of the hydrocarbon are subjected to full attribution through HSQC spectrum. While in HMBC spectra, H₃-14(δ_H1.59) and C-1 (. delta.))_C 142.4),C-9(δ_C 64.8),C-10(δ_C126.5) correlation, H₃-12(δ_H1.87) and C-7 (. delta.))_C 125.9),C-11(δ_C 158.4),C-13(δ_C72.9) correlation, H₃-15(δ_H1.59) and C-3 (. delta.))_C 30.1),C-4(δ_C 38.6),C-5(δ_C43.8), H-6(1.92,2.36) and C-5 (. delta.))_C43.8),C-7(δ_C 125.9),C-8(δ_C175.6), which establishes the planar structure of compound 4, compound 4 is guaianolide. The relative configurations of C-4, C-5 and C-1, C-10 of Compound 4 were determined by NOESY spectroscopy. H₃-15(δ_H1.06) and H-6 alpha (. delta.) (delta.)_H2.36) there is a correlation, H₃-15(δ_H1.06) and H-5 (. delta.))_H2.86) No correlation was observed, indicating that H-5 and H ₃15 in the opposite direction. Thus, the relative configuration of C-4, C-5 is designated 4S, 5S. H₃-14(δ_H1.59) and H-2 (. delta.))_H2.26.2.33) determines Δ^1,10Is in the Z configuration. The absolute configuration of compound 4 was determined by comparing the calculated specific optical rotation with the measured specific optical rotation, and the measured optical rotation of compound 4 was +9.0, and the calculated optical rotation of the (4S,5S) -4 configuration was +80.9, and the enantiomer thereof was-80.9. Thus, the absolute configuration of compound 4 was further determined to be 4S, 5S.

Compound 4 was an unreported new compound, named Genkwanoid J, by scifinider database search. It is composed of¹An H NMR spectrum of the sample was obtained,¹³the signals of the C NMR spectrum are shown in the following table.

Of Genkwanoid J¹H NMR spectrum and¹³c NMR spectral data

Compound 5: a yellow oil (dichloromethane) was obtained,

HRESIMS gave the peak of the excimer ion [ M + Na ]]⁺Peak m/z 289.1404(calcd for C)₁₅H₂₂O₄Na,289.1410), combined¹H-NMR、¹³C-NMR presumed to be of the formula C₁₅H₂₂O₄The unsaturation was calculated to be 4.¹H-NMR(600MHz,CDCl₃) Middle, delta_H 0.95(3H,s),δ_H1.06(3H,d,J＝6.1Hz),δ_H1.47(3H, s) is the three methyl hydrogen signals, δ_H3.61(1H, dd, J ═ 11.5,7.0Hz),3.67(1H, d, J ═ 11.5Hz) are a set of connected oxygen-hydrogen signals, δ_H2.15(1H, d, J ═ 19.3Hz),2.51(1H, d, J ═ 19.3Hz) show a set of geminal CH₂A hydrogen signal.¹³C-NMR(100MHz,CDCl₃) Shows a 15 carbon signal, suggesting that compound 5 is a sesquiterpene. Delta_C217.2,218.6 for the two carbonyl carbon signals, delta_C61.4,83.5 are the continuous oxygen carbon signals. While in the HMBC spectrum, H-1 (. delta.)_H 2.02),H-3(δ_H2.27,2.37) and C-2 (. delta.))_C217.2) confirmed double bond at position 2, H₃-14(δ_H1.59) and C-1 (. delta.))_C 142.4),C-9(δ_C 64.8),C-10(δ_C126.5) correlation, H₃-15(δ_H1.59) and C-3 (. delta.))_C 30.1),C-4(δ_C 38.6),C-5(δ_C43.8) correlation, confirming the position of the two methyl groups, H-6 (. delta.))_H2.43,1.47) and H-5 (. delta.))_H 2.27),H-7(δ_H1.91) correlation with confirmation of 7-position as vicinal oxygen carbon, H₂-13(δ_H3.61,3.67) and C-10 (. delta.))_C43.3),C-11(δ_C49.5) correlation confirms the position of the hydroxymethyl group, the above HMBC correlation establishes the plane of compound 5. Relative configuration of Compound 5, the relative configuration of Compound 5, H, was determined from the NOESY spectra₃-15(δ_H1.06) and H-6 beta (. delta.)_H1.91) related, H-9 beta (. delta.))_H2.51) and H-13 alpha (. delta.) (delta.)_H3.67) related, H-1 (. delta.))_H2.02) and H-5 (. delta.))_H2.43) and H-9 alpha (. delta.) (delta.)_H2.15) related, H-5 (. delta.))_H2.43) and H-9 alpha (. delta.))_H2.15) correlation, no H-5 (. delta.) was observed_H2.43) with H₃-15(δ_H1.06). Thus, the relative configuration of compound 5 was designated 1S,4S,5S,7S,10S, 11R. The absolute configuration of compound 5 was determined by comparing the calculated specific optical rotation with the measured specific optical rotation, and the calculated optical rotation of (1S,4S,5S,7S,10S,11R) -5 was +113.2 and the measured optical rotation was + 68.0. Thus, the absolute configuration of compound 5 is designated 1S,4S,5S,7S,10S,11R, which is designated genkwanoid K.

Through the database search of the sciflinder, the compound 5 is an unreported new compound and is named Genkwanoid K. It is composed of¹An H NMR spectrum of the sample was obtained,¹³the signals of the C NMR spectrum are shown in the following table.

Of Genkwanoid K¹H NMR spectrum and¹³c NMR spectral data

The 5 guaiane-type sesquiterpenes referred to in the present invention were evaluated for neuroprotective activity. The method comprises the following steps:

the cells were left to stand in DMEM complete medium for 4H and SH-SY5Y neural cells were pretreated with varying concentrations of compounds 1-5(25,50, 100. mu.M) for 1H, followed by addition of H to a final concentration of 200. mu.M₂O₂Culturing for 36 h. Subsequently, 20. mu.L/well of 0.5mg/mL MTT in phosphate buffer was added and left at 37 ℃ for 4 h. Remove supernatant and add DMSO (150 mL/well), shake on a constant temperature shaker for 10min with H₂O₂(200. mu.M) cells treated alone were used as a control group and trolox was used as a positive drug. Detection of different concentrations of treated cells was detected at 490nm using a UV spectrophotometer (Thermo Scientific Multiskan MK3, Shanghai, China). The degree of cell survival is expressed as percent survival. The results showed that compounds 1-5 showed a p-H equivalent to Trolox, a positive drug₂O₂Induced protection against SH-SY5Y neuronal damageAnd (4) activity.

The neuroprotective effect data are given in the following table:

drawings

FIG. 1 is the HSQC spectra of Genkwanoid G

FIG. 2 is an HMBC spectrum of Genkwanoid G

FIG. 3 is NOESY spectrum of Genkwanoid G

FIG. 4 is HSQC spectrum of Genkwanoid H

FIG. 5 is an HMBC spectrum of Genkwanoid H

FIG. 6 is NOESY spectrum of Genkwanoid H

FIG. 7 is HSQC spectrum of Genkwanoid I

FIG. 8 is an HMBC spectrum of Genkwanoid I

FIG. 9 is NOESY spectrum of Genkwanoid I

FIG. 10 is the HSQC spectra of Genkwanoid J

FIG. 11 is an HMBC spectrum of Genkwanoid J

FIG. 12 is NOESY spectrum of Genkwanoid J

FIG. 13 is HSQC spectrum of Genkwanoid K

FIG. 14 is an HMBC spectrum of Genkwanoid K

FIG. 15 is NOESY spectrum of Genkwanoid K

FIG. 16 shows the in vitro pairing of compounds with H₂Neuroprotective effects of O2-induced neuroblastoma SH-SY5Y cell damage

FIG. 17 is compound pair H₂O₂Effect of induced apoptosis of human neuroblastoma SH-SY5Y cells.

Detailed Description

Example 1 preparation of New sesquiterpene Compounds 1-5 from Genkwa

Roots of lilac daphne flower bud (50kg) were ground into pieces and extracted under reflux with heating three times under 70% ethanol-water (1: 8, v/v,400L × 2 h). The crude extract was filtered and concentrated under reduced pressure. Then suspending the extract in water, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol. The ethyl acetate layer (1000g) was subjected toSilica gel column chromatography (200-300 mesh) eluting with methylene chloride/methanol afforded fractions A-D. Fraction A was separated on HP20 macroporous resin with ethanol-water (50%, 90%) to give fractions A-1 and A-2. Fractions A-1 and A-2 were further purified by ODS elution with increasing ethanol/water gradient to give fractions A-1-1, A-2-1, A-3-1. A-1-1, A-2-1 and A-3-1 are further subjected to silica gel column chromatography to obtain fractions A-1-1 to A-1-1-3, A-2-1-1 to A-2-1-16 and A-3-1-1 to A-3-1-14. Wherein the compound Genkwanoid H (24mg, t) was isolated from fraction A-2-1-4 by semi-preparative HPLC eluting with acetonitrile-water (48:52)_R 35.5min)、Genkwanoid J(13mg,t_R25.0 min); compound Genkwanoid G (55mg, t) was isolated from fraction A-2-1-8 eluting with acetonitrile-water (40:60)_R35.9 min). The compound Genkwanoid I (24mg, t) was purified from fraction A-3-1-9 using acetonitrile-water (43:57) as the mobile phase_R24.5 min); purifying fraction A-3-1-12 with acetonitrile-water (46:54) as mobile phase to obtain compound Genkwanonid K (30mg, t)_R 41.2min)。

Experimental example 2 Compounds of the invention are directed to the treatment of H in vitro₂O2-induced neuroprotective Effect assay on SH-SY5Y cell injury in neuroblastoma

(1) And (5) culturing the cells.

Neuroblastoma SH-SY5Y cell line (purchased from American model culture Collection ATCC, Manassas, USA) was cultured in DMEM medium containing 10% FBS (purchased from Rockwell Hakkrong Hyclone, Logan, USA) at 37 deg.C and 5% CO₂Culturing in the incubator, adhering the cells after 24h, and discarding the old culture solution.

(2) Grouping of cells

Blank group: without any drug, only DMEM complete medium was used.

Model group: after the cells were cultured in DMEM complete medium for 4 hours, 200. mu. M H was added₂O₂The culture was continued for 36 hours.

A positive drug group: after culturing the cells in DMEM complete medium for 4 hours, 50 mu M Trolox was added and cultured for 1 hour, and then 200 mu M H was added₂O₂The culture was continued for 36 hours.

Compound group 1: the cells were cultured in DMEM complete medium for 4hAdding compound 1 at different concentrations (12.5. mu.M, 25. mu.M, 50. mu.M), culturing for 1 hr, and adding 200. mu. M H₂O₂The culture was continued for 36 hours.

Compound group 2: after culturing the cells in DMEM complete medium for 4 hours, compound 2 was added at various concentrations (12.5. mu.M, 25. mu.M, 50. mu.M) and cultured for 1 hour, followed by addition of 200. mu. M H₂O₂The culture was continued for 36 hours.

Compound group 3: after culturing the cells in DMEM complete medium for 4 hours, compounds 3 were added at different concentrations (12.5. mu.M, 25. mu.M, 50. mu.M) and cultured for 1 hour, and 200. mu. M H was added₂O₂The culture was continued for 36 hours.

Compound 4 group: after culturing the cells in DMEM complete medium for 4 hours, compounds 4 were added at different concentrations (12.5. mu.M, 25. mu.M, 50. mu.M) and cultured for 1 hour, and 200. mu. M H was added₂O₂The culture was continued for 36 hours.

Compound 5 group: after culturing the cells in DMEM complete medium for 4 hours, compounds 5 with different concentrations (12.5. mu.M, 25. mu.M, 50. mu.M) were added and cultured for 1 hour, and 200. mu. M H was added₂O₂The culture was continued for 36 hours.

MTT assay

20 μ L wells of MTT solution (0.5mg/mL) were added and incubation continued at 37 ℃ for 4 h. The waste was then discarded, DMSO was added at 150. mu.L/well, shaken on a constant temperature shaker for 10min, and the absorbance of each well was measured at 490nm using a microplate reader (Thermo Scientific Multiskan MK3, Shanghai, China).

Cell viability/% (experiment a/blank a) x 100%

(3) Results of the experiment

The results of the experiment are shown in FIG. 16, where H is compared with the blank₂O₂The survival rate of the treated cells decreased. 50 μ M Compound 3 and Compound 4 vs H compared to the Positive drug control group₂O₂The induced SH-SY5Y cell damage protection effect is stronger. Experimental example 3Annexin V-FITC/PI double staining method for detecting H pairs of compound 1, compound 2, compound 3, compound 4 and compound 5₂O₂Effect of induced apoptosis of human neuroblastoma SH-SY5Y cells.

(4) Cell culture

The procedure was as in Experimental example 2

(5) Grouping of cells

The specific procedure was as in example 2, wherein the concentrations of compound 1, compound 2, compound 3, compound 4 and compound 5 were all 12.5. mu.M.

The apoptosis rate of the cells is detected by using Annexin V-FITC and PI apoptosis detection kit. The staining was first with Annexin V-FITC and then PI for 15 min at room temperature. Apoptosis rate quantification was performed using a flow cytometer (Becton Dickinson, Franklin Lakes, USA), and stained cells were immediately analyzed by FACScan flow cytometry (BD Biosciences, NJ, USA).

(6) Results of the experiment

As shown in FIG. 17, the results of the experiment were 200. mu. M H used alone, as compared with the control group₂O₂When treated, the apoptosis rate is obviously increased. And after pretreatment with 12.5. mu.M of Compound 3 and Compound 4, H₂O₂The induced apoptosis rate of SH-SY5Y cells is reduced. The above results show that Compound 3 and Compound 4 are p-H₂O₂The induced SH-SY5Y apoptosis has an inhibiting effect.

Claims

1. Guaiane-type sesquiterpene compounds of the structure:

。

2. the process for the preparation of guaiane-type sesquiterpenes compounds according to claim 1 comprising the steps of:

(1) reflux-extracting dried radix Genkwa with 70-80% ethanol for 3 times, mixing extractive solutions, concentrating to obtain extract, sequentially extracting the extract with petroleum ether, ethyl acetate and n-butanol, subjecting the ethyl acetate part to silica gel column chromatography, gradient eluting with dichloromethane/chloroform-methanol system, and collecting 4 fractions A-D;

(2) subjecting fraction A to HP20 column chromatography, eluting with ethanol water gradient, and collecting 2 fractions A1-A2;

(3) performing ODS column chromatography on fractions A1 and A2, eluting with ethanol water gradient, and collecting 3 fractions A1-1, A2-1 and A3-1;

(4) subjecting fractions A1-1, A2-1 and A3-1 to silica gel column chromatography, respectively, and collecting fractions to obtain fractions A-1-1-1 to A-1-1-3, A-2-1-1 to A-2-1-16 and A-3-1-1 to A-3-1-14;

(5) fractions A-2-1-4, A-2-1-8, A-3-1-9, A-3-1-12 were subjected to reverse phase HPLC with CH, respectively₃CN-H₂And eluting with O to obtain compounds 1 and 3-5.

3. The method according to claim 2, wherein the concentration of ethanol water in the step (2) is: 50 to 90 percent.

4. The method according to claim 2, wherein the concentration of ethanol water in the step (3) is: 30 to 90 percent.

5. The preparation method according to claim 2, wherein the conditions of the silica gel column chromatography in the step (4) are: petroleum ether-ethyl acetate 50:1-1:1 gradient elution.

6. The process according to claim 2, wherein in step (5), fractions A-2-1-4, A-2-1-8, A-3-1-9 and A-3-1-12 are subjected to reverse phase HPLC, and the mobile phase is: 40% -50% of CH₃CN -H₂O。

7. A pharmaceutical composition comprising the guaiane-type sesquiterpene compounds of claim 1 and pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier or excipient.

8. Use of the guaiane-type sesquialter compounds of claim 1 and pharmaceutically acceptable salts thereof in the manufacture of neuroprotective medicaments.

9. The use according to claim 8, characterized in that the guaiane-type sesquiterpenes and their pharmaceutically acceptable salts protect SH-SY5Y cells by reversing apoptosis.