CN110305093A - Guainane type sequiterpene and its preparation method and application - Google Patents
Guainane type sequiterpene and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to pharmaceutical technology fields, are related to guainane type sequiterpene and its preparation and application.More particularly to the 5 guainane type sesquiterpene compounds separated from Chinese medicine root of lilac daphne and its purposes in terms of neuroprotection bioactivity.The guainane type sesquiterpenoid and its pharmaceutically acceptable salt, isomer structure are as follows: the compound of the present invention is prepared via a method which: root of lilac daphne is extracted with ethyl alcohol, extracting solution is concentrated under reduced pressure, combined extract is concentrated to give medicinal extract, medicinal extract successively uses petroleum ether, ethyl acetate, extracting n-butyl alcohol, and by acetic acid ethyl ester extract, then the means such as silica gel column chromatography, macroreticular resin, ODS, HPLC can be with isolated 5 compounds.
Description
Technical field
The invention belongs to pharmaceutical technology fields, are related to guainane type sequiterpene and its preparation and application.More particularly to from
The 5 guainane type sesquiterpene compounds separated in Chinese Drug, yuan Hua root and its purposes in terms of neuroprotection bioactivity.
Background technique
Root of lilac daphne is the root of Thymelaeceae daphne plant lilac daphne, bitter, pungent, warm-natured, toxic, has and relieves oedema or abdominal distension through diuresis or purgation, and is detoxified, and is dissipated
The effect of knot, is used for oedema, scrofula, acute mastitis, hemorrhoid complicated by anal fistula, scabies, rheumatic arthralgia.Anti-oxidant to its at present, antiviral and antitumor,
Immunity regulation etc. research is more.Chemical component is abundant in root of lilac daphne, including sesquiterpenoids, diterpene ortho-ester, Huang
Ketone, Coumarins, lignanoids, chlorogenic acid and phenolic glycoside class, wherein Diterpenes, flavone compound are that main biology is living
Property ingredient, and recent studies have shown that the sesquiterpenoids in root of lilac daphne equally has preferable bioactivity.
Summary of the invention
Technical problem solved by the invention is to provide a series of guaiaci lignum alkane type sesquiterpene compounds, and provides it
Preparing the application in neuroprotection bio-pharmaceutical.
The present invention relates to guainane type sesquiterpenoid and its pharmaceutically acceptable salt, the isomeries of such as flowering structure
Body:
The compound of the present invention preparation method passes through following steps:
Root of lilac daphne is extracted with ethyl alcohol, and extracting solution is concentrated under reduced pressure, and combined extract is concentrated to give medicinal extract, and medicinal extract successively uses petroleum
Ether, ethyl acetate, extracting n-butyl alcohol, by acetic acid ethyl ester extract then means such as silica gel column chromatography, macroreticular resin, ODS, HPLC
It can be with isolated above 5 compounds.
Specifically, the present invention includes the following steps:
(1) take dry root of lilac daphne with 70%-80% industrial alcohol refluxing extraction 3 times, combined extract is concentrated to get leaching
Cream.Gained medicinal extract successively uses petroleum ether, ethyl acetate, extracting n-butyl alcohol.Ethyl acetate portion is through silica gel column chromatography, with dichloro
Methane/chloroform-methanol system carries out gradient elution, is collected into 4 fractions (A-D) altogether.
(2) fraction A is collected into 2 fractions (A1-A2) through HP20 column chromatography with the elution of ethanol water gradient elution altogether.
(3) fraction A 1, A2 is through ODS column chromatography, with ethanol water gradient elution, be collected into altogether 3 fractions (A1-1, A2-1,
A3-1)。
(4) fraction A 1-1, A2-1 and A3-1 through silica gel column chromatography, collect fraction and obtain A-1-1-1-A-1-1-3 respectively,
A-2-1-1-A-2-1-16 and A-3-1-1-A-3-1-14.
(5) the inverted HPLC of fraction A -2-1-8, with CH3CN-H2O elution, obtains compound 1；Fraction A -2-1-4 is inverted
HPLC, with CH3CN-H2O elution, obtains compound 2 and 4；The inverted HPLC of fraction A -3-1-9, with CH3CN-H2O elution, obtains
Compound 3；The inverted HPLC of fraction A -3-1-12, with CH3CN-H2O elution, obtains compound 5.
Wherein:
Methylene chloride/chloroform-methanol gradient in step (1) are as follows: 100:1-1:1, specially 100:1,50:1,
30:1,20:1,10:1,5:1,3:1,1:1.
The concentration of ethanol water in step (2) are as follows: 50%-90%.
The concentration of ethanol water in step (3) are as follows: 30-90%, specially 30%, 40%, 50%, 60%, 70%, 80%,
90%.
The condition of silica gel column chromatography in step (4) are as follows: petroleum ether-ethyl acetate gradient elution 50:1-1:1, specially 50:
1,30:1,20:1,10:1,5:1,3:1,1:1).
Chromatographic condition in step (5) are as follows: the CH of 40%-50%3CN-H2O。
New guainane type sesquiterpenoid of the present invention (Genkwanoid G, Genkwanoid H,
Genkwanoid I, Genkwanoid J, Genkwanoid K) structure elucidation.
Compound 1: yellow oil (methylene chloride),HRESIMS provides quasi-molecule
Quasi-molecular ions [M+Na]+(the calcd for C of peak m/z 273.146315H22O3Na, 273.1461), in conjunction with1H-NMR、13C-NMR is pushed away
Surveying its molecular formula is C15H22O3, calculating degree of unsaturation is 5.1H-NMR(600MHz,CDCl3) in, δH 1.70(3H,s),δH
0.81 (3H, d, J=7.1Hz) is two methyl hydrogen signals, δH3.58 (1H, dd, J=12.3,3.1Hz), 4.02 (1H, dd, J
=12.3,3.5Hz), 3.63 (1H, o) 3.63 (1H, o) connect the incoordinate CH of oxygen magnetic for two groups2On together with even hydrogen signal, δH
4.34 (1H, o) are to connect oxygen tertiary carbon and the hydrogen signal without coupling.13C-NMR(100MHz,CDCl3) show 15 carbon signals, it mentions
Show that compound 1 is sequiterpene.δC210.6 be isolated carbonyl carbon signals, δC122.3,149.3 be double bond carbon signal, δC
58.7,63.5,87.0 for even oxygen carbon signal.Speculate that terminal double bond is not present in compound 1, increases a methylol, pass through
All hydrocarbon directly related signals have been carried out full ownership by hsqc spectrum.In HMBC spectrum, H3-15(δH0.81) with C-3 (δC
31.9),C-4(δC 38.3),C-5(δC43.2) related, H3-14(δH1.70) with C-1 (δC 149.3),C-9(δC 87.0),
C-10(δC122.3) related, so that it is determined that the position of methyl.H-7(δH 2.05),H-9(δH4.25) with C-8 (δC
210.6) there are correlations, it was confirmed that the position of carbonyl.H2-12(δH3.63) with C-11 (δC 47.6),C-13(δC58.7) deposit
In correlation, it was confirmed that the position of methylol.1 planar structure of compound is established according to the above relevant information, compound 1 is more to create
The wooden alkane type sequiterpene.The relative configuration of compound 1 is determined by NOESY spectrum, as shown in figure 5, H3-15(δH0.81) with H-3 β
(δH1.41) related, H-3 α (δH1.60) with H-5 (δH2.26) related, H-5 (δH2.26) with H-7 (δH2.05) related, H-
7(δH2.05) with H-12 (δH3.63) related.It is thus determined that the relative configuration of compound 1 is 4S*, 5S*, 7R*, 8R*, 11S*.
The absolute configuration of compound 1 be by calculate specific rotatory power with survey specific rotatory power be compared it is determining, using Density functional
Theoretical (DFT), it is different to (4S, 5S, 7R, 8R, 11S) -1 configuration and its mapping respectively in the case where B3LYP/6-311++G (2d, p) is horizontal
Structure body is calculated.The actual measurement optically-active of compound 1 is+22.0, and the calculating optical value of (4S, 5S, 7R, 8R, 11S) -1 is+24.5,
It is -24.5 that it, which corresponds to isomers,.Therefore, the absolute configuration of compound 1 is further determined as 4S, 5S, 7R, 8R, 11S.
Through scifinder database retrieval, compound 1 is the noval chemical compound that do not report, is named as Genkwanoid G.
Its1H H NMR spectroscopy,13C H NMR spectroscopy signals assignment such as following table, related map are shown in attached drawing 1- Fig. 5.
Genkwanoid G's1H H NMR spectroscopy and13C H NMR spectroscopy data
Compound 2: yellow oil (methylene chloride),HRESIMS provides quasi-molecule
Quasi-molecular ions [M+Na]+(the calcd for C of peak m/z 289.141115H22O4Na, 289.1410), in conjunction with1H-NMR、13C-NMR is pushed away
Surveying its molecular formula is C15H22O4, calculating degree of unsaturation is 5.1H-NMR(600MHz,CDCl3) in, δH 1.70(3H,s),δH
0.82 (3H, d, J=7.0Hz) is two methyl hydrogen signals, δH3.48 (1H, dd, J=12.4,1.8Hz), 3.87 (1H, m),
3.53 (1H, o) 3.53 (1H, o) connect the incoordinate CH of oxygen magnetic for two groups2On together with even hydrogen signal, δH4.34 (1H, s) are to connect oxygen uncle
Carbon and hydrogen signal without coupling.13C-NMR(100MHz,CDCl3) show that 15 carbon signals, prompt compound 2 are sequiterpene.
δC208.8 be isolated carbonyl carbon signals, δC122.0,149.5 be double bond carbon signal, δC61.9,63.9,78.8,86.3 it is
Even oxygen carbon signal.The above hydrocarbon signal and compound 1 are extremely close, thus it is speculated that the two difference is that compound 2 increases a company
Oxygen carbon signal, reduces a fatty carbon signal, and molecular weight increases 16 mass units relative to compound 1, thus it is speculated that chemical combination
7 of object 1 are optionally substituted by a hydroxyl group, and all hydrocarbon directly related signals have been carried out full ownership by hsqc spectrum.In HMBC spectrum,
H3-15(δH0.82) with C-3 (δC 32.0),C-4(δC 38.3),C-5(δC42.7) related, H3-14(δHAnd C-1 1.70)
(δC 149.5),C-9(δC 86.3),C-10(δC122.0) related, so that it is determined that the position of methyl.H-7(δH 2.82),
H-9(δH4.34) with C-8 (δC208.8) there are correlations, it was confirmed that the position of carbonyl.H2-13(δH) and C-11 3.48,3.87
(δC 78.8),C-12(δC63.9) there are correlations, it was confirmed that the position of hydroxyl and methylol.It is established according to the above relevant information
2 planar structure of compound, compound 2 are guainane type sequiterpene.The opposite structure of compound 2 has been determined by NOESY spectrum
Type, as shown in Figure 10, H3-15(δH0.82) with H-3 β (δH1.39) related, H-3 α (δH1.61) with H-5 (δH2.29) phase
It closes, H-5 (δH2.29) and H-7 (δH2.82) related, H-7 (δH2.82) with H-12 (δH3.53) related.It is thus determined that chemical combination
The relative configuration of object 2 is 4S*, 5S*, 7R*, 8R*, 11R*.The absolute configuration of compound 2 is by calculating specific rotatory power and actual measurement
Specific rotatory power is compared determining, and the actual measurement optically-active of compound 2 is+25.0, the calculating of (4S, 5S, 7R, 8R, 11R) -2 configuration
Optical value is+6.0, and corresponding to isomers is -6.0.Therefore, the absolute configuration of compound 2 is further determined as 4S, 5S, 7R,
8R,11R。
Through scifinder database retrieval, compound 2 is the noval chemical compound that do not report, is named as Genkwanoid H.
Its1H H NMR spectroscopy,13C H NMR spectroscopy signals assignment such as following table.
Genkwanoid H's1H H NMR spectroscopy and13C H NMR spectroscopy data
Compound 3: yellow oil (methylene chloride),HRESIMS provides quasi-molecule
Quasi-molecular ions [M+Na]+(the calcd for C of peak m/z 289.141315H22O4Na, 289.1410), in conjunction with1H-NMR、13C-NMR is pushed away
Surveying its molecular formula is C15H22O4, calculating degree of unsaturation is 5.1H-NMR(600MHz,CDCl3) in, δH 1.69(3H,s),δH
0.88 (3H, d, J=7.1Hz) is two methyl hydrogen signals, δH 3.47(1H,o),3.71(1H,o),3.49(1H,o),3.71
(1H, o) is two groups and connects the incoordinate CH of oxygen magnetic2On together with even hydrogen signal, δH4.24 (1H, s) are to connect oxygen tertiary carbon and the hydrogen without coupling
Signal.13C-NMR(100MHz,CDCl3) show that 15 carbon signals, prompt compound 3 are sequiterpene.δC207.9 be isolated
Carbonyl carbon signals, δC121.3,150.2 be double bond carbon signal, δC60.9,66.6,75.0,85.7 for even oxygen carbon signal.With
Upper hydrocarbon signal and compound 2 are extremely close, and the hydrocarbon signal of methylol is separate more apparent, and molecular weight is identical as compound 2.Cause
This, thus it is speculated that 3 be the epimer of compound 2.All hydrocarbon directly related signals full ownership has been subjected to by hsqc spectrum.?
In HMBC spectrum, H3-15(δH0.88) with C-3 (δC 32.1),C-4(δC 38.5),C-5(δC42.3) related, H3-14(δH
1.69) with C-1 (δC 150.2),C-9(δC 85.7),C-10(δC121.3) related, so that it is determined that the position of methyl, H-7
(δH 2.87),H-9(δH4.24) with C-8 (δC207.9) there are correlations, it was confirmed that the position of carbonyl.H2-13(δH 3.49,
3.71) with C-11 (δC 75.0),C-12(δC66.6) there is correlation and confirm the position of hydroxyl and methylol.According to the above phase
It closes information and establishes 3 planar structure of compound, compound 3 is guainane type sequiterpene.Compound has been determined by NOESY spectrum
3 relative configuration, as shown in figure 15, H3-15(δH0.88) with H-3 β (δH1.42) related, H-3 α (δH1.64) with H-5 (δH
2.33) related, H-5 (δH2.33) with H-7 (δH2.87) related, H-7 (δH2.87) with H-6 α (δH1.82) related, H2-12
(δH3.47,3.71) and H-6 β (δH1.47) related.It is thus determined that the relative configuration of compound 3 be 4S*, 5S*, 7R*, 8R*,
11S*.The absolute configuration of compound 3 is that determining, compound 3 is compared with actual measurement specific rotatory power by calculating specific rotatory power
Actual measurement optically-active be+22.0, the calculating optical value of (4S, 5S, 7R, 8R, 11S) -3 configuration is+22.3, correspond to isomers be -
22.3.Therefore, the absolute configuration of compound 3 is further determined as 4S, 5S, 7R, 8R, 11S.
Through scifinder database retrieval, compound 3 is the noval chemical compound that do not report, is named as Genkwanoid I.
Its1H H NMR spectroscopy,13C H NMR spectroscopy signals assignment such as following table.
Genkwanoid I's1H H NMR spectroscopy and13C H NMR spectroscopy data
Compound 4: yellow oil (methylene chloride),HRESIMS provide quasi-molecule from
Sub- peak [M+Na]+(the calcd for C of peak m/z 273.146215H22O3Na, 273.1461), in conjunction with1H-NMR、13C-NMR speculates
Its molecular formula is C15H22O3, calculating degree of unsaturation is 5.1H-NMR(600MHz,CDCl3) in, δH1.06 (3H, d, J=
6.8Hz), δH 1.59(3H,s),δH1.87 (3H, s) are three methyl hydrogen signals, δH3.65 (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 and connect oxygen hydrogen letter
Number, in δHA series of rouge hydrogen signals are shown within the scope of 1.5-2.9.13C-NMR(100MHz,CDCl3) show 15 carbon letters
Number, prompt compound 4 is sequiterpene.δC125.9,158.4,175.6 be α, the carbon signal of alpha, beta-unsaturated ketone, δC126.5,
142.4 be double bond carbon signal, δC64.8,72.9 for even oxygen carbon signal.By hsqc spectrum will all hydrocarbon directly related signals into
Full ownership is gone.Simultaneously in HMBC spectrum, H3-14(δH1.59) with C-1 (δC 142.4),C-9(δC 64.8),C-10(δC
126.5) related, H3-12(δH1.87) with C-7 (δC 125.9),C-11(δC 158.4),C-13(δC72.9) related, H3-15
(δH1.59) with C-3 (δC 30.1),C-4(δC 38.6),C-5(δC43.8) related, H-6 (1.92,2.36) and C-5 (δC
43.8),C-7(δC 125.9),C-8(δC175.6) related, the above correlation establishes the planar structure of compound 4, and compound 4 is
Guaianolide.C-4, C-5 and the C-1 of compound 4, the relative configuration of C-10 has been determined by NOESY spectrum.H3-15(δH
1.06) with H-6 α (δH2.36) there are correlation, H3-15(δH1.06) with H-5 (δH2.86) correlation is not observed, shows H-5
And H3- 15 are in opposite direction.Therefore, the relative configuration of C-4, C-5 are set to 4S*, 5S*.H3-14(δH1.59) with H-2 (δH
2.26.2.33 correlation) has determined Δ1,10It is Z configuration.The absolute configuration of compound 4 is by calculating specific rotatory power and actual measurement
Specific rotatory power is compared determining, and the actual measurement optically-active of compound 4 is+9.0, and the calculating optical value of (4S, 5S) -4 configuration is+
80.9, enantiomter is -80.9.Therefore, the absolute configuration of compound 4 is further determined as 4S, 5S.
Through scifinder database retrieval, compound 4 is the noval chemical compound that do not report, is named as Genkwanoid J.
Its1H H NMR spectroscopy,13C H NMR spectroscopy signals assignment such as following table.
Genkwanoid J's1H H NMR spectroscopy and13C H NMR spectroscopy data
Compound 5: yellow oil (methylene chloride),HRESIMS provides quasi-molecule
Quasi-molecular ions [M+Na]+(the calcd for C of peak m/z 289.140415H22O4Na, 289.1410), in conjunction with1H-NMR、13C-NMR is pushed away
Surveying its molecular formula is C15H22O4, calculating degree of unsaturation is 4.1H-NMR(600MHz,CDCl3) in, δH 0.95(3H,s),δH
1.06 (3H, d, J=6.1Hz), δH1.47 (3H, s) are three methyl hydrogen signals, δH3.61 (1H, dd, J=11.5,
7.0Hz), 3.67 (1H, d, J=11.5Hz) connect oxygen hydrogen signal, δ for one groupH2.15 (1H, d, J=19.3Hz), 2.51 (1H,
D, J=19.3Hz) show one group together with even CH2Hydrogen signal.13C-NMR(100MHz,CDCl3) show 15 carbon signals, it prompts
Compound 5 is sequiterpene.δC217.2,218.6 be two carbonyl carbon signals, δC61.4,83.5 for even oxygen carbon signal.Exist simultaneously
In HMBC spectrum, H-1 (δH 2.02),H-3(δH2.27,2.37) with C-2 (δC217.2) related, it was confirmed that 2 are double bond, H3-
14(δH1.59) with C-1 (δC 142.4),C-9(δC 64.8),C-10(δC126.5) related, H3-15(δHAnd C-3 1.59)
(δC 30.1),C-4(δC 38.6),C-5(δC43.8) related, it was demonstrated that the position of two methyl, H-6 (δH2.43,1.47) with
H-5(δH 2.27),H-7(δH1.91) related to confirm 7 to connect oxygen carbon, H2-13(δH3.61,3.67) with C-10 (δC
43.3),C-11(δC49.5) related to confirm the position of methylol, the above HMBC correlation establishes the plane of compound 5.Change
The relative configuration for closing object 5 has determined the relative configuration of compound 5, H by NOESY spectrum3-15(δH1.06) with H-6 β (δH 1.91)
Correlation, H-9 β (δH2.51) with H-13 α (δH3.67) related, H-1 (δH2.02) with H-5 (δHAnd H-9 α (δ 2.43)H
2.15) related, H-5 (δH2.43) with H-9 α (δH2.15) related, do not observe H-5 (δHAnd H 2.43)3-15(δH
1.06) correlation.Therefore, the relative configuration of compound 5 is set to 1S*, 4S*, 5S*, 7S*, 10S*, 11R*.Compound 5 it is absolute
Configuration be by calculate specific rotatory power with survey specific rotatory power be compared it is determining, (1S, 4S, 5S, 7S, 10S, 11R's) -5
Calculating optical value is+113.2, and actual measurement optical value is+68.0.Therefore, the absolute configuration of compound 5 is set to 1S, 4S, 5S, 7S,
10S, 11R are named as genkwanoid K.
Through scifinder database retrieval, compound 5 is the noval chemical compound that do not report, is named as Genkwanoid K.
Its1H H NMR spectroscopy,13C H NMR spectroscopy signals assignment such as following table.
Genkwanoid K's1H H NMR spectroscopy and13C H NMR spectroscopy data
Neuroprotective activity evaluation is carried out to 5 guainanes type sequiterpene of the present invention.Its step are as follows:
Cell is stood into 4h with DMEM complete culture solution, and pre- using the compound 1-5 (25,50,100 μM) of various concentration
SH-SY5Y nerve cell 1h is handled, final concentration of 200 μM of H is added afterwards2O2Cultivate 36h.Then it is added 0.5mg/mL MTT's
20 hole μ L/ of phosphate buffer solution, and 4h is placed at 37 DEG C.It removes supernatant and is added in DMSO (hole 150mL/), constant temperature vibration
It swings and vibrates 10min on device, with H2O2The cell that (200 μM) are individually handled is control group, using trolox as positive drug.Detection is different
The cell of concentration processing is detected (Thermo Scientific Multiskan using ultraviolet specrophotometer in 490nm
MK3, Shanghai, China).The survival degree of cell is indicated with percentage survival.As a result indicate that compound 1-5 is shown and positive drug
Trolox is comparable to H2O2The SH-SY5Y neural cell injury protective effect activity of induction.
The data of its neuroprotection are as follows:
Detailed description of the invention
Fig. 1 is the hsqc spectrum of Genkwanoid G
The HMBC that Fig. 2 is Genkwanoid G is composed
The NOESY that Fig. 3 is Genkwanoid G is composed
Fig. 4 is the hsqc spectrum of Genkwanoid H
The HMBC that Fig. 5 is Genkwanoid H is composed
The NOESY that Fig. 6 is Genkwanoid H is composed
Fig. 7 is the hsqc spectrum of Genkwanoid I
The HMBC that Fig. 8 is Genkwanoid I is composed
The NOESY that Fig. 9 is Genkwanoid I is composed
Figure 10 is the hsqc spectrum of Genkwanoid J
The HMBC that Figure 11 is Genkwanoid J is composed
The NOESY that Figure 12 is Genkwanoid J is composed
Figure 13 is the hsqc spectrum of Genkwanoid K
The HMBC that Figure 14 is Genkwanoid K is composed
The NOESY that Figure 15 is Genkwanoid K is composed
Figure 16 is compound in vitro to by H2The neuroprotection of the neuroblastoma SH-SY5Y cellular damage of O2 induction
Effect
Figure 17 is compound to H2O2The influence of the human neuroblastoma SH-SY5Y Apoptosis of induction.
Specific embodiment
The preparation of the new sesquiterpenoid 1-5 of 1 root of lilac daphne of embodiment
The root grinding of lilac daphne (50kg) is fragmentated, and under the conditions of 70% alcohol-water (1:8, v/v, 400L × 2h)
It is being heated to reflux extraction three times.Filtering crude extract is simultaneously concentrated under reduced pressure.Then extract is suspended in water, successively uses petroleum ether,
Ethyl acetate, extracting n-butyl alcohol.Ethyl acetate layer (1000g) is subjected to silica gel column chromatography separation (200-300 mesh), uses dichloromethane
Alkane/methanol elution, obtains flow point A-D.Flow point A is separated on HP20 macroreticular resin with alcohol-water (50%, 90%),
Obtain flow point A-1 and A-2.Flow point A-1 and A-2 is further purified with incremental ethanol/water gradient elution by ODS, is flowed
Divide A-1-1, A-2-1, A-3-1.A-1-1, A-2-1 and A-3-1 obtain flow point A-1-1-1 extremely further across silica gel column chromatography
A-1-1-3, A-2-1-1 are to A-2-1-16 and A-3-1-1 to A-3-1-14.Wherein, by half preparation HPLC with acetonitrile-water (48:
52) it elutes, isolates compound Genkwanoid H (24mg, t from flow point A-2-1-4R 35.5min)、Genkwanoid J
(13mg,tR25.0min)；It is eluted with acetonitrile-water (40:60), separates compound Genkwanoid G from flow point A-2-1-8
(55mg,tR35.9min).Mobile phase is done with acetonitrile-water (43:57), purifies to obtain compound from flow point A-3-1-9
Genkwanoid I(24mg,tR24.5min)；Mobile phase is done with acetonitrile-water (46:54), flow point A-3-1-12 is purified, obtains
Compound Genkwanoid K (30mg, tR 41.2min)。
Compound in 2 present invention of experimental example is in vitro to by H2The neuroblastoma SH-SY5Y cell damage of O2 induction
The neuroprotection of wound measures
(1) cell culture.
Neuroblastoma SH-SY5Y cell line (is purchased from American Type Culture collection warehousing ATCC, Manassas, USA)
With the DMEM culture medium (be purchased from the U.S. Luo Genhai cron Hyclone, Logan, USA) containing 10%FBS, at 37 DEG C, 5%CO2's
It is cultivated in incubator, rear cell is adherent for 24 hours, discards old culture solution.
(2) cell is grouped
Blank group: being free of any drug, only uses the culture of DMEM complete culture solution.
Model group: 200 μM of H are added after DMEM complete culture solution culture 4h in cell2O2, continue culture 36 hours.
Positive drug group: cell is added 50 μM of Trolox and cultivates 1 hour, add after DMEM complete culture solution culture 4h
200μM H2O2, continue culture 36 hours.
1 group of compound: cell is added various concentration (12.5 μM, 25 μM, 50 μM) after DMEM complete culture solution culture 4h
Compound 1 is cultivated 1 hour, and 200 μM of H are added2O2, continue culture 36 hours.
2 groups of compound: cell is added various concentration (12.5 μM, 25 μM, 50 μM) after DMEM complete culture solution culture 4h
Compound 2 is cultivated 1 hour, and 200 μM of H are added2O2, continue culture 36 hours.
3 groups of compound: cell is added various concentration (12.5 μM, 25 μM, 50 μM) after DMEM complete culture solution culture 4h
Compound 3 is cultivated 1 hour, and 200 μM of H are added2O2, continue culture 36 hours.
4 groups of compound: cell is added various concentration (12.5 μM, 25 μM, 50 μM) after DMEM complete culture solution culture 4h
Compound 4 is cultivated 1 hour, and 200 μM of H are added2O2, continue culture 36 hours.
5 groups of compound: cell is added various concentration (12.5 μM, 25 μM, 50 μM) after DMEM complete culture solution culture 4h
Compound 5 is cultivated 1 hour, and 200 μM of H are added2O2, continue culture 36 hours.
MTT detection
20 hole μ L MTT solution (0.5mg/mL) is added, 37 DEG C are continued to be incubated for 4h.After discard waste liquid, 150 μ L/ of DMSO is added
Hole, vibrates 10min on constant temperature oscillator, and with microplate reader (Thermo Scientific Multiskan MK3, Shanghai,
China each hole absorbance value) is detected at 490nm.
Cell survival rate/%=(experimental group A value/blank control group A value) × 100%
(3) experimental result
Experimental result is as shown in figure 16, compared to the blank group, H2O2The cell survival rate of processing reduces.It is compareed with positive drug
Group is compared, and 50 μM of compounds 3 and compound 4 are to H2O2The SH-SY5Y cellular damage protective effect of induction is stronger.Experimental example
The bis- dye method detection compounds 1 of 7Annexin V-FITC/PI, compound 2, compound 3, compound 4, compound 5 are to H2O2Induction
Human neuroblastoma SH-SY5Y Apoptosis influence.
(4) cell culture
Step is the same as experimental example 2
(5) cell is grouped
Specific steps are the same as embodiment 2, wherein the concentration of compound 1, compound 2, compound 3, compound 4, compound 5
It is 12.5 μM.
Apoptosis rate is detected using Annexin V-FITC and PI apoptosis detection kit.It first uses at room temperature
Annexin V-FITC dyeing, then dyed 15 minutes with PI.Using flow cytometer (Becton Dickinson, Franklin
Lakes, USA) to carry out apoptosis rate quantitative, pass through FACScan flow cytometry (BD Biosciences, NJ, USA) immediately after
Analyze the cell of dyeing.
(6) experimental result
Experimental result is as shown in figure 17, compared with the control group, 200 μM of H is applied alone2O2When processing, apoptosis rate is obviously risen
It is high.And after being pre-processed with 12.5 μM of compound 3 and compound 4, H2O2The SH-SY5Y apoptosis rate of induction reduces.The above knot
Fruit shows compound 3 and compound 4 to H2O2The SH-SY5Y Apoptosis of induction is inhibited.
Claims (10)
1. such as the guainane type sesquiterpenoid and its pharmaceutically acceptable salt, isomers of flowering structure:
2. guainane type sesquiterpenoid according to claim 1, it is characterised in that: the guainane type times
Half terpenoid be extracted from root of lilac daphne it is isolated.
3. guainane type sesquiterpenoid according to claim 1 and its pharmaceutically acceptable salt, isomers
Preparation method, which comprises the steps of:
(1) take dry root of lilac daphne with 70-80% alcohol reflux extraction 3 times, combined extract is concentrated to get medicinal extract, gained medicinal extract
Petroleum ether is successively used, ethyl acetate, extracting n-butyl alcohol, ethyl acetate portion is through silica gel column chromatography, with methylene chloride/tri- chloromethanes
Alkane-methanol system carries out gradient elution, is collected into 4 fraction A-D altogether；
(2) fraction A is through HP20 column chromatography, with ethanol water gradient elution, is collected into 2 1-A2 of fraction A altogether；
(3) fraction A 1, A2 is through ODS column chromatography, with ethanol water gradient elution, is collected into 3 fraction As 1-1, A2-1, A3-1 altogether；
(4) fraction A 1-1, A2-1 and A3-1 through silica gel column chromatography, collect fraction and obtain A-1-1-1-A-1-1-3, A-2- respectively
1-1-A-2-1-16 and A-3-1-1-A-3-1-14；
(5) fraction A -2-1-4, A-2-1-8, A-3-1-9, A-3-1-12 distinguish inverted HPLC, with CH3CN-H2O elution, obtains
Compound 1-5.
4. preparation method as claimed in claim 3, which is characterized in that the concentration of ethanol water in step (2) are as follows: 50%-90%.
5. preparation method as claimed in claim 3, which is characterized in that the concentration of ethanol water in step (3) are as follows: 30-90%.
6. preparation method as claimed in claim 3, which is characterized in that the condition of silica gel column chromatography in step (4) are as follows: petroleum
Ether-ethyl acetate 50:1-1:1 gradient elution.
7. preparation method as claimed in claim 3, which is characterized in that in step (5), fraction A -2-1-4, A-2-1-8, A-3-
The inverted HPLC of 1-9, A-3-1-12, mobile phase are as follows: the CH of 40%-50%3CN-H2O。
8. pharmaceutical composition includes guainane type sesquiterpenoid described in claim 1 and its pharmaceutically acceptable
Salt, isomers and pharmaceutically acceptable carrier or excipient.
9. guainane type sesqui described in claim 1 and its pharmaceutically acceptable salt, isomers are in preparation mind
Through the purposes in protection drug.
10. purposes according to claim 9, which is characterized in that the guainane type sesquiterpenoid and its medicine
Acceptable salt, isomers protect SH-SY5Y cell by reverse both apoptosis on.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111072456A (en) * | 2019-12-26 | 2020-04-28 | 沈阳药科大学 | Sesquiterpene compound in corn stigma and application thereof |
| CN113683507A (en) * | 2021-08-24 | 2021-11-23 | 沈阳药科大学 | Guaiane sesquiterpene derivative in regachia and application thereof |
| CN113788840A (en) * | 2021-09-15 | 2021-12-14 | 中国科学院昆明植物研究所 | Guaiane sesquiterpene polymer and its medicine, preparation method and application |
| CN113912482A (en) * | 2020-07-09 | 2022-01-11 | 沈阳药科大学 | Guaiane type sesquiterpenoids, preparation and application thereof |
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| CN115304466A (en) * | 2022-07-13 | 2022-11-08 | 沈阳药科大学 | Guaiane type sesquiterpenoids separated from lilac daphne root as well as preparation method and application thereof |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101279964A (en) * | 2008-02-29 | 2008-10-08 | 中国药科大学 | Guaiane type sesquiterpenes, preparation and medical use thereof |
| CN101531644A (en) * | 2009-02-18 | 2009-09-16 | 沈阳药科大学 | New daphnane diterpene compounds in Daphne genkwa as well as preparation method and application of same |
| CN102260234A (en) * | 2010-05-26 | 2011-11-30 | 福建医科大学 | Preparation method and application of guaiane-type sesquiterpene |
| CN103242275A (en) * | 2013-05-13 | 2013-08-14 | 沈阳药科大学 | Sesquiterpenoids in zedoary as well as preparation method and application of sesquiterpenoids |
-
2019
- 2019-04-11 CN CN201910288681.9A patent/CN110305093B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101279964A (en) * | 2008-02-29 | 2008-10-08 | 中国药科大学 | Guaiane type sesquiterpenes, preparation and medical use thereof |
| CN101531644A (en) * | 2009-02-18 | 2009-09-16 | 沈阳药科大学 | New daphnane diterpene compounds in Daphne genkwa as well as preparation method and application of same |
| CN102260234A (en) * | 2010-05-26 | 2011-11-30 | 福建医科大学 | Preparation method and application of guaiane-type sesquiterpene |
| CN103242275A (en) * | 2013-05-13 | 2013-08-14 | 沈阳药科大学 | Sesquiterpenoids in zedoary as well as preparation method and application of sesquiterpenoids |
Non-Patent Citations (3)
| Title |
|---|
| HEINRICH E.BODE等: "Electrolyte-assisted stereoselection and control of cyclization vs saturation in electroreductive cyclizations", 《TETRAHEDRON LETTERS》 * |
| QING-YUN MA等: "Two New Guaiane Sesquiterpenoids from Daphne holosericea(Diels) Hamaya", 《MOLECULES》 * |
| 李玲芝等: "芫花的化学成分及药理作用研究进展", 《沈阳药科大学学报》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111072456B (en) * | 2019-12-26 | 2022-07-12 | 沈阳药科大学 | Sesquiterpene compound in corn stigma and application thereof |
| CN111072456A (en) * | 2019-12-26 | 2020-04-28 | 沈阳药科大学 | Sesquiterpene compound in corn stigma and application thereof |
| CN113912482B (en) * | 2020-07-09 | 2023-10-27 | 沈阳药科大学 | Guaiane sesquiterpene compound and preparation and application thereof |
| CN113912482A (en) * | 2020-07-09 | 2022-01-11 | 沈阳药科大学 | Guaiane type sesquiterpenoids, preparation and application thereof |
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