CN103304537A - Biphenyl cyclooctene lignans compound as well as preparation method and application thereof - Google Patents
Biphenyl cyclooctene lignans compound as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a biphenyl cyclooctene lignans compound as well as a preparation method and application thereof. The biphenyl cyclooctene lignans compound is obtained by separating from branches, leaves and fruit of dry Yunnan-Tibet schisandra chinensis, and has the structure shown in the specification; the preparation method of the biphenyl cyclooctene lignans compound is as follows: performing extracting, silicagel column chromatography and high performance liquid chromatography separation on the branches, leaves and fruit of dry Yunnan-Tibet schisandra chinensis used as raw material; the method comprises the following specific steps of: crushing and extracting the branches, leaves and fruit of dry Yunnan-Tibet schisandra chinensis, filtering, reducing pressure and concentrating as an extract, extracting, performing the silicagel column chromatography on an extract phase, performing gradient eluting by using the organic solution in a volume proportion of (1:0)-(0:1), further performing the inverse-phase column chromatography and high performance liquid chromatography separation and purification on 9:1 part of the eluant to obtain the biphenyl cyclooctene lignans compound. According to the evidence, the biphenyl cyclooctene lignans compound disclosed by the invention is provided with good HIV-1 activity, the compound is novel in structure, good in activity and can be used as the pilot compound of anti-AIDS drugs.
Description
Technical field
The invention belongs to effective ingredients in plant extractive technique field, be specifically related to a kind of cyclohexyl biphenyl octene Lignanoids compounds and preparation method thereof and application.
Background technology
Schisandraceae (Schisandraceae) is dicotyledons, have only Kadsura (
Kadsura) and Schizandra (
Schisandra) two genus, totally 50 kinds, be distributed in East Asia, South East Asia and south, North America, China's two genus all produce it, and kind surplus in the of about 30 originates in the west and south to the northeast, but main product ground is the west and south and the middle and south.Schizandra (
Schisandra) there are 25 kinds approximately, China has 19 kinds, is distributed in southwest to the southeast.
At present, from the Schisandraceae medicinal plant, separate the compound that obtains and be mainly two big classes: lignanoid (Lignans) and triterpene (Triterpenes) compounds.Triterpene compound mostly is lanostane-type and cyclic-ahltin type greatly, and owing to its skeleton easily changes, and because the oxidisability difference makes this class triterpenoid complex structure uniqueness.Modern pharmacology studies show that lignanoid and triterpene are the main active ingredient of this section plant, and the Dibenzocyclooctene lignan that has report to be separated to from schisandra plant in recent years has certain potentiality aspect the anti-HIV.Therefore the continuation further investigation to this platymiscium seems particularly important.
Summary of the invention
First purpose of the present invention provides a kind of cyclohexyl biphenyl octene Lignanoids compounds; Second purpose is to provide the preparation method of described cyclohexyl biphenyl octene Lignanoids compounds; The 3rd purpose is to provide the application of described cyclohexyl biphenyl octene Lignanoids compounds in the medicine of the anti-AIDS of preparation.
First purpose of the present invention is achieved in that described cyclohexyl biphenyl octene Lignanoids compounds is to separate to obtain from Schisandra neglecat A. C. Smith. branch, leaf or fruit, called after neglignan B, and its molecular formula is C
23H
26O
9, have following structure:
Second purpose of the present invention is to realize like this, described cyclohexyl biphenyl octene Lignanoids compounds preparation method, it is characterized in that Schisandra neglecat A. C. Smith. branch, leaf or fruit with drying are raw material, through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high performance liquid chromatography separating step, be specially:
A, medicinal extract extract: Schisandra neglecat A. C. Smith. branch, leaf or fruit are crushed to 20 ~ 40 orders, and with organic solvent supersound extraction 2 ~ 4 times, each 30 ~ 60min, extracting solution merges, and filters, during concentrating under reduced pressure extracting solution to 1/4 ~ 1/2 volume, leave standstill back filtering throw out, be condensed into medicinal extract then;
B, organic solvent extraction: medicinal extract suspends in water, and uses and the isopyknic organic solvent extraction of water 3 ~ 5 times, will merge behind each organic solvent extraction phase concentrating under reduced pressure;
C, silica gel column chromatography: the medicinal extract after the extraction carries out silica gel column chromatography with 160-200 order silica gel dress post of 6 ~ 8 times of amounts of weight ratio; The organic solvent solution that with the volume proportion is 1:0 ~ 0:1 carries out gradient elution, collects the each several part elutriant and concentrates, and merges identical part;
D, reversed phase column chromatography: the 9:1 of C step elutriant part is further carried out chromatography with reversed material C-18 dress post, is that 20% ~ 100% methanol aqueous solution carries out gradient elution with volume proportion, collects each several part elutriant and concentrated, merges identical part;
E, high performance liquid chromatography separate: 40% ~ 60% methanol aqueous solution wash-out part of D step elutriant further namely gets described cyclohexyl biphenyl octene Lignanoids compounds with the high performance liquid chromatography separation and purification.
Structure with the cyclohexyl biphenyl octene Lignanoids compounds of method for preparing is measured out by the following method:
The compounds of this invention neglignan B is yellow jelly; Optical value
+ 23.8 (solvent is methyl alcohol c 0.20); UV spectrum (solvent is methyl alcohol),
λ Max(log
ε): 210 (4.58), 250 (3.64), 320 (0.68) nm; CD (
c0.05, MeOH)
λ MaxNm (Δ
ε) 250 (38.6), 238 (15.3), 222 (+8.14), 215 (3.05); Infrared spectra (pressing potassium bromide troche)
ν Max3401,3092,2922,2855,1687,1628,1559,1466,1323,1235,1168,1070,973,862 cm
-1HRESIMS shows the The compounds of this invention quasi-molecular ion peak
M/z[469.1470 M+Na]
+(calculated value is 469.1475), in conjunction with
13C and
1H NMR spectrum (figure-1 and figure-2, carbon spectrum attribution data sees Table-1) provides its molecular formula C
23H
26O
9 1H NMR and
13C NMR (C
5D
5N, 500 MHz, 125 MHz) data, see Table 1.
Compound neglignan B's
1H and
13C NMR spectrogram shows 26 hydrogen and 23 carbon signals respectively, corresponding to two phenyl ring (
δ C105.2-154.1) a fragrant hydrogen (
δ H6.62), dioxy methylene radical (
d C 101.3 t
; d H 5.99,5.92, each s), two methynes (
δ C40.3,36.6), oxidized methyne (
δ C80.7), carboxyl (
δ C175.0,
δ H9.28) and three methoxyl groups (
δ C60.3,60.0,60.8).Ultraviolet absorption spectrum has maximum absorption at 210 and 248 nm,
1H-
1H COSY spectrum shows H-6/H-7/H-8/H-9, and H-7/H-17 is relevant with H-8/H-18, HMBC spectrum demonstration H-11 (
δ H6.62 s) with C-9 (
δ C80.7 d), C-10 (
δ C132.0 s) and C-15 (
δ C117.9 s) relevant, also have H-9 (
δ H4.70, d,
J=8.5 Hz) with C-10 (
δ C132.0 s), C-11 (
δ C105.2 d), and C-15 (
δ C117.9 s) relevant, and H-6 (
δ H1.79,2.46) and C-4 (
δ C106.9 s), C-5 (
δ C133.1 s), and C-16 (
δ C119.0 s) relevant, more than these data show that all compound neglignan B should be a cyclohexyl biphenyl octene lignanoid.Carboxyl hydrogen in the HMBC spectrum (
δ H9.28) with the C-4 of phenyl ring (
δ C106.9 s) relevant, illustrate that carboxyl substituted is in the C-4 position.Two hydrogen of dioxy methylene radical (
d H 5.99,5.92, each s) and C-12 (
δ C149.1 s) and C-13 (
δ C137.9 s) relevant, illustrate that the dioxy methylene radical is substituted in C-12 and C-13.Three methoxyl group hydrogen on phenyl ring (
δ H3.79,3.93,3.82 s) respectively with C-1 (
δ C154.1 s), C-2 (
δ C142.0 s), and C-14 (
δ C142.5 s) have HMBC relevant, the position of three methoxyl groups be described respectively at C-1, C-2 and C-14 position.Oxidized methyne signal instruction has a hydroxyl to be substituted in the C-9 position.Also have an aromatic ring hydroxyl (
δ H10.04) and C-3 (
δ C148.7), C-4 (
δ C106.9) and C-2 (
δ C142.0) HMBC relevant, show that hydroxyl is in the C-3 position.The CD spectrum of this compound is presented at 250nm negative absorption, just has at 222nm to absorb, and illustrates that it is
S-biphenyl configuration.The ROESY spectrum shows H-19 (carboxyl proton)/CH
3-17 is relevant with H-11/H-8, illustrates that this compound is the cyclohexyl biphenyl octene lignanoid of turning round ship chair configuration.The ROESY related description OH-9 of H-11/H-9 is
a-orientation.The ROESY spectrum shows that also H-7 is relevant with H-9 respectively with H-8, and Me-17 is relevant with Me-18 and H-19, illustrates that Me-17 and Me-18 are
R-orientation.So far the structure of this compound is determined, and called after neglignan B.
The 3rd purpose of the present invention is achieved in that and is about to the preparation that described cyclohexyl biphenyl octene Lignanoids compounds is applied to anti-AIDS medicine.
The compound of table-1. 1H NMR and
13(solvent is C to C NMR data
5D
5N)
Cyclohexyl biphenyl octene Lignanoids compounds of the present invention is separated first, is defined as cyclohexyl biphenyl octene Lignanoids compounds by nucleus magnetic resonance and other spectroscopic measurement methods, and has characterized its concrete structure.Through right
C8166The cytotoxicity of host cell detects, and right
HIV-1IIIBInduce
C8166Cytopathy (
CPE) inhibition test, such cyclohexyl biphenyl octene lignanoid has anti-HIV-1 activity preferably, EC
50Value is 1.4 ± 0.14
μG/mL, therapeutic index (TI) values is 55.3.Above result has disclosed compound of the present invention has good prospects for application in the medicine of the anti-AIDS of preparation.The compounds of this invention novel structure activity is good, can be used as the guiding compound of anti-AIDS medicine.
Table 2. the Anti-HIV activity of compound
Description of drawings
Fig. 1 be compound neglignan B carbon-13 nmr spectra (
13C NMR);
Fig. 2 be compound neglignan B proton nmr spectra (
1H NMR);
The main HMBC of neglignan B of Fig. 3 compound (
→) and
1H-
1H COSY (
–) relevant.
Embodiment
The present invention is further illustrated below in conjunction with accompanying drawing, but never in any form the present invention is limited, and any conversion or improvement based on training centre of the present invention is done all fall into protection scope of the present invention.
Cyclohexyl biphenyl octene Lignanoids compounds of the present invention is to separate to obtain from Schisandra neglecat A. C. Smith. branch, leaf or the fruit of drying, called after neglignan B, and its molecular formula is C
23H
26O
9, have following structure:
Cyclohexyl biphenyl octene Lignanoids compounds preparation method of the present invention, it is characterized in that Schisandra neglecat A. C. Smith. branch, leaf or fruit with drying are raw material, through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separating step, be specially:
A, medicinal extract extract: Schisandra neglecat A. C. Smith. branch, leaf or fruit are crushed to 20 ~ 40 orders, and with organic solvent supersound extraction 2 ~ 4 times, each 30 ~ 60min, extracting solution merges, and filters, during concentrating under reduced pressure extracting solution to 1/4 ~ 1/2 volume, leave standstill back filtering throw out, be condensed into medicinal extract then;
B, organic solvent extraction: medicinal extract suspends in water, and uses and the isopyknic organic solvent extraction of water 3 ~ 5 times, will merge behind each organic solvent extraction phase concentrating under reduced pressure;
C, silica gel column chromatography: the medicinal extract after the extraction carries out silica gel column chromatography with 160-200 order silica gel dress post of 6 ~ 8 times of amounts of weight ratio; The organic solvent solution that with the volume proportion is 1:0 ~ 0:1 carries out gradient elution, collects the each several part elutriant and concentrates, and merges identical part;
D, reversed phase column chromatography: the 9:1 of C step elutriant part is further carried out chromatography with reversed material C-18 dress post, is that 20% ~ 100% methanol aqueous solution carries out gradient elution with volume proportion, collects each several part elutriant and concentrated, merges identical part;
E, high performance liquid chromatography separate: 40% ~ 60% methanol aqueous solution wash-out part of D step elutriant further namely gets described cyclohexyl biphenyl octene Lignanoids compounds with the high performance liquid chromatography separation and purification.
The preparation method of described cyclohexyl biphenyl octene Lignanoids compounds is characterized in that solvent in the described A step can adopt any one in the acetone, ethanol, methyl alcohol of 70%-100%.
The preparation method of described cyclohexyl biphenyl octene Lignanoids compounds is characterized in that the organic solvent that extracts in the described B step can adopt any one in ethyl acetate, chloroform, ether, sherwood oil, the benzene.
The preparation method of described cyclohexyl biphenyl octene Lignanoids compounds is characterized in that medicinal extract weighs 0.8 ~ 1.2 times 80 ~ 100 order silica gel silica gel mixed samples with medicinal extract before the silica gel column chromatography rough segmentation behind the acetone solution with 1.5 ~ 3 times of amounts of weight ratio in the described C step.
The preparation method of described cyclohexyl biphenyl octene Lignanoids compounds, when it is characterized in that carrying out silica gel column chromatography in the described C step, carry out the used organic solvent solution of gradient elution and can adopt arbitrary system in normal hexane-acetone, chloroform-acetone, chloroform-methanol, sherwood oil-acetone, the petroleum ether-ethyl acetate.
The preparation method of described cyclohexyl biphenyl octene Lignanoids compounds is characterized in that organic solution system volume proportion is 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 in the described D step.
The preparation method of described cyclohexyl biphenyl octene Lignanoids compounds, it is characterized in that high performance liquid chromatography separation and purification in the described E step is that to adopt 40 ~ 60% methyl alcohol be moving phase, flow velocity 10 ~ 14mL/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5 mm is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60 mL, collect the chromatographic peak of 20 ~ 40 min, the back evaporate to dryness repeatedly adds up.
The application of cyclohexyl biphenyl octene Lignanoids compounds of the present invention in the preparation anti-AIDS drug.
Shizandra berry of the present invention is not limited by area and kind, all can realize the present invention.
Be that the synthetic compound of template can be realized purpose of the present invention equally with Lignanoids compounds of the present invention.
Get branch, leaf and/or the fruit 4.5kg of Schisandra neglecat A. C. Smith., coarse reduction to 40 order, the acetone supersound extraction with 70% 5 times, each 60min, extracting solution merges; Extracting liquid filtering is evaporated to 1/4 of volume; Leave standstill, the filtering throw out is condensed into 510g medicinal extract a; Add 765g water in medicinal extract a, use and water equal volume of ethyl acetate 5 times, merge extraction phase, concentrating under reduced pressure becomes 370g medicinal extract b; With 200 order silica gel 2220g dress post, in medicinal extract b, add the acetone solution of 555g, add 100 order silica gel 370g then and mix sample, mix upper prop behind the sample; Be respectively the chloroform-methanol mixed organic solvents gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 with volume ratio, collect gradient eluent, concentrate, monitor through TLC, merge identical part, obtain 6 parts, the elutriant c of the chloroform-methanol mixed organic solvents of volume ratio 9:1 is 70g; With reversed material C-18 dress post, elutriant c goes up reversed-phase column, is that 20 ~ 100% methanol aqueous solution carries out gradient elution with volume content, collects the each several part elutriant and concentrates, and through the TLC monitoring, merges identical part; Get the elutriant that obtains with volume content 40 ~ 60% methanol aqueous solution wash-outs, methyl alcohol with 45% is moving phase, flow velocity 10ml/min, 21.2 ' 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 50mL, collect the chromatographic peak of 35min, the back evaporate to dryness that repeatedly adds up namely gets described Lignanoids compounds neglignan B.
Embodiment 2
Get branch, leaf and/or the fruit 5kg of Schisandra neglecat A. C. Smith., coarse reduction to 20 order, the ethanol ultrasonic extraction with 100% 2 times, each 50min, extracting solution merges; Extracting liquid filtering is evaporated to 1/3 of volume; Leave standstill, the filtering throw out is condensed into 580g medicinal extract a; The water that adds 580g in medicinal extract a is used and the isopyknic chloroform extraction of water 3 times, merges extraction phase, and concentrating under reduced pressure becomes 350g medicinal extract b; With 160 order silica gel 2796g dress post, in medicinal extract b, add the acetone solution of 1284g, add 80 order silica gel 342g then and mix sample, mix upper prop behind the sample; Be respectively normal hexane-acetone mixed organic solvents gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 with volume ratio, collect gradient eluent, concentrate, through the TLC monitoring, merge identical part; With reversed material C-18 dress post, elutriant c goes up reversed-phase column, is that 20 ~ 100% methanol aqueous solution carries out gradient elution with volume content, collects the each several part elutriant and concentrates, and through the TLC monitoring, merges identical part; Get the elutriant that obtains with volume content 40 ~ 55% methanol aqueous solution wash-outs, methyl alcohol with 50% is moving phase, flow velocity 14ml/min, 21.2 ' 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 50mL, collect the chromatographic peak of 30min, the back evaporate to dryness that repeatedly adds up namely gets described Lignanoids compounds neglignan B.
Embodiment 3
Get branch, leaf and/or the fruit 6kg of Schisandra neglecat A. C. Smith., coarse reduction to 30 order, the methyl alcohol supersound extraction with 80% 4 times, each 30min, extracting solution merges; Extracting liquid filtering is evaporated to 1/2 of volume; Leave standstill, the filtering throw out is condensed into 535g medicinal extract a; The water that adds 1070g in medicinal extract a is used and the isopyknic extracted with diethyl ether of water 4 times, merges extraction phase, and concentrating under reduced pressure becomes 420g medicinal extract b; With 180 order silica gel 2940g dress post, in medicinal extract b, add the acetone solution of 1000g, add 90 order silica gel 490g then and mix sample, mix upper prop behind the sample; Be respectively chloroform-acetone mixed organic solvents gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 with volume ratio, collect gradient eluent, concentrate, through the TLC monitoring, merge identical part; With reversed material C-18 dress post, elutriant c goes up reversed-phase column, is that 20 ~ 100% methanol aqueous solution carries out gradient elution with volume content, collects the each several part elutriant and concentrates, and through the TLC monitoring, merges identical part; Get the elutriant that obtains with volume content 45 ~ 50% methanol aqueous solution wash-outs, methyl alcohol with 40% is moving phase, flow velocity 12ml/min, 21.2 ' 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, and it is 254nm that UV-detector detects wavelength, each sample introduction 60mL, collect the chromatographic peak of 40min, the back evaporate to dryness that repeatedly adds up namely gets described Lignanoids compounds neglignan B.
Embodiment 4
Get branch, leaf and/or the fruit 5.5kg of Schisandra neglecat A. C. Smith., coarse reduction to 40 order, the ethanol ultrasonic extraction with 90% 3 times, each 45min, extracting solution merges; Extracting liquid filtering is evaporated to 1/4 of volume; Leave standstill, the filtering throw out is condensed into 468g medicinal extract a; The water that adds 838g in medicinal extract a is used and the isopyknic petroleum ether extraction of water 4 times, merges extraction phase, and concentrating under reduced pressure becomes 326g medicinal extract b; With 160 order silica gel 1948g dress post, in medicinal extract b, add the acetone solution of 652g, add 80 order silica gel 350g then and mix sample, mix upper prop behind the sample; Be respectively sherwood oil-acetone mixed organic solvents gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 with volume ratio, collect gradient eluent, concentrate, through the TLC monitoring, merge identical part; With reversed material C-18 dress post, elutriant c goes up reversed-phase column, is that 20 ~ 100% methanol aqueous solution carries out gradient elution with volume content, collects the each several part elutriant and concentrates, and through the TLC monitoring, merges identical part; Get the elutriant that obtains with volume content 45 ~ 55% methanol aqueous solution wash-outs, methyl alcohol with 55% is moving phase, flow velocity 10ml/min, 21.2 ' 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, and it is 254nm that UV-detector detects wavelength, each sample introduction 55mL, collect the chromatographic peak of 26min, the back evaporate to dryness that repeatedly adds up namely gets described Lignanoids compounds neglignan B.
Embodiment 5
Get branch, leaf and/or the fruit 5kg of Schisandra neglecat A. C. Smith., coarse reduction to 20 order, the methyl alcohol supersound extraction with 70% 5 times, each 35min, extracting solution merges; Extracting liquid filtering is evaporated to 1/2 of volume; Leave standstill, the filtering throw out is condensed into 490g medicinal extract a; The water that adds 980g in medicinal extract a is used and the isopyknic benzene extraction of water 5 times, merges extraction phase, and concentrating under reduced pressure becomes 355g medicinal extract b; With 200 order silica gel 2538g dress post, in medicinal extract b, add the acetone solution of 995g, add 100 order silica gel 300 then
gMix sample, mix upper prop behind the sample; Be respectively the petroleum ether-ethyl acetate mixed organic solvents gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 with volume ratio, collect gradient eluent, concentrate, through the TLC monitoring, merge identical part; With reversed material C-18 dress post, elutriant c goes up reversed-phase column, is that 20 ~ 100% methanol aqueous solution carries out gradient elution with volume content, collects the each several part elutriant and concentrates, and through the TLC monitoring, merges identical part; Get the elutriant that obtains with volume content 40 ~ 60% methanol aqueous solution wash-outs, methyl alcohol with 60% is moving phase, flow velocity 12ml/min, 21.2 ' 250mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, and it is 254nm that UV-detector detects wavelength, each sample introduction 50mL, collect the chromatographic peak of 20min, the back evaporate to dryness that repeatedly adds up namely gets described Lignanoids compounds neglignan B.
Embodiment 6
Get the compound neglignan B of embodiment 1 preparation, be yellow jelly; Optical value
+ 23.8 (solvent is methyl alcohol c 0.20); Measuring method is: use nucleus magnetic resonance, identify structure in conjunction with other spectroscopic technique.
1) UV spectrum (solvent is methyl alcohol),
λ Max(log
ε): 210 (4.58), 250 (3.64), 320 (0.68) nm;
2) CD spectrum (
c0.05 solvent is methyl alcohol)
λ MaxNm (Δ
ε) 250 (38.6), 238 (15.3), 222 (+8.14), 215 (3.05);
3) infrared spectra (pressing potassium bromide troche),
ν Max3401,3092,2922,2855,1687,1628,1559,1466,1323,1235,1168,1070,973,862 cm
-1
4) HRESIMS shows the The compounds of this invention quasi-molecular ion peak
M/z[469.1470 M+Na]
+(calculated value is 469.1475), in conjunction with
13C and
1H NMR spectrum (figure-1 and figure-2) provides its molecular formula C
23H
26O
9, degree of unsaturation is 11.
1H NMR and
13C NMR (C
5D
5N, 500 MHz, 125 MHz) data, see Table 1.
Compound neglignan B's
1H and
13C NMR spectrogram shows 26 hydrogen and 23 carbon signals respectively, corresponding to two phenyl ring (
δ C105.2-154.1) a fragrant hydrogen (
δ H6.62), dioxy methylene radical (
d C 101.3 t
; d H 5.99,5.92, each s), two methynes (
δ C40.3,36.6), oxidized methyne (
δ C80.7), carboxyl (
δ C175.0,
δ H9.28) and three methoxyl groups (
δ C60.3,60.0,60.8).Ultraviolet absorption spectrum has maximum absorption at 210 and 248 nm,
1H-
1H COSY spectrum shows H-6/H-7/H-8/H-9, and H-7/H-17 is relevant with H-8/H-18, HMBC spectrum demonstration H-11 (
δ H6.62 s) with C-9 (
δ C80.7 d), C-10 (
δ C132.0 s) and C-15 (
δ C117.9 s) relevant, also have H-9 (
δ H4.70, d,
J=8.5 Hz) with C-10 (
δ C132.0 s), C-11 (
δ C105.2 d), and C-15 (
δ C117.9 s) relevant, and H-6 (
δ H1.79,2.46) and C-4 (
δ C106.9 s), C-5 (
δ C133.1 s), and C-16 (
δ C119.0 s) relevant, more than these data show that all compound neglignan B should be a cyclohexyl biphenyl octene lignanoid.Carboxyl hydrogen in the HMBC spectrum (
δ H9.28) with the C-4 of phenyl ring (
δ C106.9 s) relevant, illustrate that carboxyl substituted is in the C-4 position.Two hydrogen of dioxy methylene radical (
d H 5.99,5.92, each s) and C-12 (
δ C149.1 s) and C-13 (
δ C137.9 s) relevant, illustrate that the dioxy methylene radical is substituted in C-12 and C-13.Three methoxyl group hydrogen on phenyl ring (
δ H3.79,3.93,3.82 s) respectively with C-1 (
δ C154.1 s), C-2 (
δ C142.0 s), and C-14 (
δ C142.5 s) have HMBC relevant, the position of three methoxyl groups be described respectively at C-1, C-2 and C-14 position.Oxidized methyne signal instruction has a hydroxyl to be substituted in the C-9 position.Also have an aromatic ring hydroxyl (
δ H10.04) and C-3 (
δ C148.7), C-4 (
δ C106.9) and C-2 (
δ C142.0) HMBC relevant, show that hydroxyl is in the C-3 position.The CD spectrum of this compound is presented at 250nm negative absorption, just has at 222nm to absorb, and illustrates that it is
S-biphenyl configuration.The ROESY spectrum shows H-19 (carboxyl proton)/CH
3-17 is relevant with H-11/H-8, illustrates that this compound is the cyclohexyl biphenyl octene lignanoid of turning round ship chair configuration.The ROESY related description OH-9 of H-11/H-9 is
a-orientation.The ROESY spectrum shows that also H-7 is relevant with H-9 respectively with H-8, and Me-17 is relevant with Me-18 and H-19, illustrates that Me-17 and Me-18 are
R-orientation.So far the structure of this compound is determined, and called after neglignan B.
Embodiment 7
Get the compound of embodiment 2 preparations, be yellow jelly; Optical value
+ 23.8 (solvent is methyl alcohol c 0.20); Carry out structure determination by the method among the embodiment 6, the result is: its structure is with embodiment 6, and molecular formula is C
23H
26O
9The compound of confirming embodiment 2 preparations is described cyclohexyl biphenyl octene Lignanoids compounds neglignan B.
Embodiment 8
Get the compound of embodiment 3 preparations, be yellow jelly; Optical value
+ 23.8 (solvent is methyl alcohol c 0.20); Carry out structure determination by the method among the embodiment 6, the result is: its structure is with embodiment 6, and molecular formula is C
23H
26O
9The compound of confirming embodiment 3 preparations is described cyclohexyl biphenyl octene Lignanoids compounds neglignan B.
Embodiment 9
Get the compound of embodiment 4 preparations, be yellow jelly; Optical value
+ 23.8 (solvent is methyl alcohol c 0.20); Carry out structure determination by the method among the embodiment 6, the result is: its structure is with embodiment 6, and molecular formula is C
23H
26O
9The compound of confirming embodiment 4 preparations is described cyclohexyl biphenyl octene Lignanoids compounds neglignan B.
Get the compound of embodiment 5 preparations, be yellow jelly; Optical value
+ 23.8 (solvent is methyl alcohol c 0.20); Carry out structure determination by the method among the embodiment 6, the result is: its structure is with embodiment 6, and molecular formula is C
23H
26O
9The compound of confirming embodiment 5 preparations is described cyclohexyl biphenyl octene Lignanoids compounds neglignan B.
Embodiment 11
The cyclohexyl biphenyl octene Lignanoids compounds of getting embodiment 1 ~ 5 preparation carries out the anti-HIV-1 activity test, and test situation is as follows:
(1)Measure medicine and compound
Testing sample dissolves with DMSO, positive control compound Zidovodine (AZT, 3 '-Azido-3 '-deoxythymidine) available from Sigma company, be dissolved in the RPMI-1640 perfect medium 0.22
μThe degerming of m membrane filtration ,-20 ℃ of preservations after the packing.
(2)Reagent
(N-2 (2-Hydroxyothyl) piperazine-N'-(2-ethanesufonic acid), (3-(4 for MTT for HEPES, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), DMF (N, N '-Dimethyl formamine), penicillin (Penicillin), Vetstrep (Streptomycin sulfate), glutamine (Glutamine) are all available from Sigma company; (2-Me 2-Mercaptoethanol) is Bio-Rad company product for DMSO, 2 mercapto ethanol.RPMI-1640 and newborn calf serum are Gibco company product.
(3)Substratum
The RPMI-1640 perfect medium contains 10 % newborn calf serums, 2 mM L-glutaminate, 10 mM HEPES, 50
μThe M 2 mercapto ethanol, 100,000 IU penicillin, 100
μThe g/mL Streptomycin sulphate.
(4)Cell and virus
The human T lymphocyte is C8166, MT
4And HIV-1 experiment strain HIV-1IIIB is by Britain Medical Research Council, and AIDS Reagent Project is so kind as to give.All cells and virus are all cultivated with the RPMI-1640 perfect medium that contains 10% calf serum.Prepare HIV-1IIIB according to a conventional method, titration also calculates viral TCID
50After the packing of virus stock solution, put-70 ℃ of preservations.Cell and virus is frozen and recovery according to a conventional method.
(5) HIV-1Infectious titration
Carry out titration by the described method improvement of Johnson ﹠ Byington, be summarized as follows: the HIV-1IIIB stock solution is done 4 times of dilutions at 96 orifice plates, 10 gradients, 6 repeating holes of every gradient arrange control wells 6 holes simultaneously.Every hole adds C8166 cell 50
μL (4 * 10
5/ mL), every hole final volume is 200
μL.37 ℃, 5% CO
2Cultivate.Added fresh RPMI-1640 perfect medium 100 on the 3rd day
μL, (whether Cytopathic Effect CPE), has the formation of synplasm (Syncytium) to determine with every hole to the cytopathic effect that HIV-1 induced in the every hole of observation under inverted microscope in the 7th day; Press the TCID that the Reed﹠Muench method is calculated virus
50(50% Tissue Culture Infection Dose).
(6)Sample is right
C8166The cytotoxicity of host cell detects
4 * 10
5/ mL C8166 cell suspension 100
μL mixes with different testing compound solution, establishes three repeating holes.The control wells that does not contain compound, 37 ℃, 5% CO are set simultaneously
2Cultivated three days, and adopted the MTT colorimetry to detect cytotoxicity.ELx800 ELISA instrument is measured the OD value, and measuring wavelength is 595 nm, and reference wavelength is 630 nm.Calculate CC
50Value (50% Cytotoxic Concentration), the compound concentration during namely to 50% normal T lymphocyte series C8166 toxigenicity.
(7)Sample is right
HIV-1IIIBInduce
C8166Cytopathy (
CPE) inhibition test
With 8 * 10
5/ mL C8166 cell 50
μThe L/ hole is inoculated into contains 100
μOn the 96 porocyte culture plates of L/ hole doubling dilution compound, add 50 then
μThe HIV-1IIIB dilution supernatant (M.O.I.0.0016) of L.If three repeating holes.The normal cell control wells that does not contain compound, 37 ℃, 5% CO are set simultaneously
2Cultivated three days, (100 *) count plasmodial formation under the inverted microscope.EC
50(50% Effective Concentration) forms 50% o'clock compound concentration for suppressing synplasm.
(8)Calculation formula
Draw dose response curve according to experimental result, calculate the 50% effective concentration (EC that compound suppresses virus by the Reed﹠Muench method
50), 50% cell growth inhibiting concentration (CC
50) and the therapeutic index TI value (Therapeutic index) of anti-HIV-1 activity be: TI=CC
50/ EC
50
(9)Test-results
Through right
C8166The cytotoxicity of host cell detects, and right
HIV-1IIIBInduce
C8166Cytopathy (
CPE) inhibition test, such cyclohexyl biphenyl octene lignanoid has anti-HIV-1 activity preferably, EC
50Value is 1.4 ± 0.14
μG/mL, therapeutic index (TI) values is 55.3.Above result has disclosed compound of the present invention has good prospects for application in the medicine of the anti-AIDS of preparation.The compounds of this invention novel structure activity is good, can be used as the guiding compound of anti-AIDS medicine.
Claims (9)
1. a cyclohexyl biphenyl octene Lignanoids compounds is characterized in that described cyclohexyl biphenyl octene Lignanoids compounds is to separate to obtain from Schisandra neglecat A. C. Smith. branch, leaf or the fruit of drying, called after neglignan B, and its molecular formula is C
23H
26O
9, have following structure:
2. the described cyclohexyl biphenyl octene of claim 1 Lignanoids compounds preparation method, it is characterized in that it being that Schisandra neglecat A. C. Smith. branch, leaf or fruit with drying is raw material, through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separating step, be specially:
A, medicinal extract extract: Schisandra neglecat A. C. Smith. branch, leaf or fruit are crushed to 20 ~ 40 orders, and with organic solvent supersound extraction 2 ~ 4 times, each 30 ~ 60min, extracting solution merges, and filters, during concentrating under reduced pressure extracting solution to 1/4 ~ 1/2 volume, leave standstill back filtering throw out, be condensed into medicinal extract then;
B, organic solvent extraction: medicinal extract suspends in water, and uses and the isopyknic organic solvent extraction of water 3 ~ 5 times, will merge behind each organic solvent extraction phase concentrating under reduced pressure;
C, silica gel column chromatography: the medicinal extract after the extraction carries out silica gel column chromatography with 160-200 order silica gel dress post of 6 ~ 8 times of amounts of weight ratio; The organic solvent solution that with the volume proportion is 1:0 ~ 0:1 carries out gradient elution, collects the each several part elutriant and concentrates, and merges identical part;
D, reversed phase column chromatography: the 9:1 of C step elutriant part is further carried out chromatography with reversed material C-18 dress post, is that 20% ~ 100% methanol aqueous solution carries out gradient elution with volume proportion, collects each several part elutriant and concentrated, merges identical part;
E, high performance liquid chromatography separate: 40% ~ 60% methanol aqueous solution wash-out part of D step elutriant further namely gets described cyclohexyl biphenyl octene Lignanoids compounds with the high performance liquid chromatography separation and purification.
3. the preparation method of cyclohexyl biphenyl octene Lignanoids compounds according to claim 2 is characterized in that solvent in the described A step can adopt a kind of in the acetone, ethanol, methyl alcohol of 70%-100%.
4. the preparation method of cyclohexyl biphenyl octene Lignanoids compounds according to claim 2 is characterized in that the organic solvent that extracts in the described B step can adopt a kind of in ethyl acetate, chloroform, ether, sherwood oil, the benzene.
5. the preparation method of cyclohexyl biphenyl octene Lignanoids compounds according to claim 2, it is characterized in that medicinal extract weighs 0.8 ~ 1.2 times 80 ~ 100 order silica gel silica gel mixed samples with medicinal extract before the silica gel column chromatography rough segmentation behind the acetone solution with 1.5 ~ 3 times of amounts of weight ratio in the described C step.
6. the preparation method of cyclohexyl biphenyl octene Lignanoids compounds according to claim 2, when it is characterized in that carrying out silica gel column chromatography in the described C step, carry out the used organic solvent solution of gradient elution and can adopt arbitrary system in normal hexane-acetone, chloroform-acetone, chloroform-methanol, sherwood oil-acetone, the petroleum ether-ethyl acetate.
7. the preparation method of cyclohexyl biphenyl octene Lignanoids compounds according to claim 2 is characterized in that organic solution system volume proportion is 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 in the described D step.
8. the preparation method of cyclohexyl biphenyl octene Lignanoids compounds according to claim 2, it is characterized in that high performance liquid chromatography separation and purification in the described E step is that to adopt 40 ~ 60% methyl alcohol be moving phase, flow velocity 10 ~ 14mL/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5 mm is stationary phase, and it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60 mL, collect the chromatographic peak of 20 ~ 40 min, the back evaporate to dryness repeatedly adds up.
9. the application of the described cyclohexyl biphenyl octene of claim 1 Lignanoids compounds in the preparation anti-AIDS drug.
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2013
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Non-Patent Citations (1)
| Title |
|---|
| XUE-MEI GAO,等: "Bioactive Dibenzocyclooctadiene Lignans from the Stems of Schisandra neglecta", 《JOURNAL OF NATURAL PRODUCTS》 * |
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Application publication date: 20130918 |