CN103012118A - Lignans compounds and preparation method and application thereof - Google Patents
Lignans compounds and preparation method and application thereof Download PDFInfo
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- CN103012118A CN103012118A CN2013100141255A CN201310014125A CN103012118A CN 103012118 A CN103012118 A CN 103012118A CN 2013100141255 A CN2013100141255 A CN 2013100141255A CN 201310014125 A CN201310014125 A CN 201310014125A CN 103012118 A CN103012118 A CN 103012118A
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Abstract
The invention discloses lignans compounds and a preparation method and application thereof. According to the structural formulas of the compounds, R1 is -OH, R2 is -OCH3, a molecular formula is C20H24O8, and one compound is named as marphenol E; and -OCH2O- is between R1 and R2, a molecular formula is C20H22O8, and the other compound is named as marphenol F. According to the preparation method, the lignans compounds are prepared from the raw materials including dried branches, leaves and/or fruits of schisandra chinensis. The preparation method comprises the steps of extract extraction, organic solvent extraction, silica column chromatography and high pressure liquid chromatography separation of the raw materials. The invention further discloses the application of the lignans compounds to preparation of anti-AIDS medicines. According to cytotoxicity detection on C8166 host cells and inhibition tests on an HIV-1IIIB-induced cytopathic effect (CPE) of the C8166, the two fluorenone compounds, namely the marphenol E and the marphenol F, have good anti-HIV-1 activity, the EC50 values of the two fluorenone compounds are respectively 3.47 microgram/mL and 2.97 microgram/mL, and the therapeutic index (IT) values of the two fluorenone compounds are respectively 21.18 and 27.64. The fluorenone compounds have novel structures and high activity, and can serve as guiding compounds of the anti-AIDS medicines.
Description
Technical field
The invention belongs to effective ingredients in plant extractive technique field, be specifically related to a kind of Lignanoids compounds and its preparation method and application.
Background technology
Shizandra berry is Schisandraceae (Schisandraceae), dicotyledons, have Kadsura (
Kadsura) and Schizandra (
Schisandra)Two belong to, and totally 50 kinds, be distributed in East Asia, South East Asia and south, North America, China's two genus all produce it, approximately more than 30 plant, and originate in the west and south to the northeast, but main product ground are the west and south and the middle and south.Schizandra (
Schisandra)Approximately have 25 kinds, China has 19 kinds, is distributed in southwest to the southeast.
At present, from Schisandraceae Plant, separate the compound that obtains and be mainly two large classes: triterpene (Triterpenes) compounds and lignanoid (Lignans).Triterpene compound mostly is greatly lanostane-type and cyclic-ahltin type, and owing to its skeleton easily changes, and because the oxidisability difference makes this class triterpenoid complex structure unique.Lignanoid mostly is cyclohexyl biphenyl octene class.Modern pharmacology studies show that lignanoid and triterpene are the main active ingredient of this section plant, and the lignanoid that has in recent years report to be separated to from schisandra plant has certain potentiality aspect the anti-HIV.Therefore the continuation further investigation of this platymiscium seemed particularly important.
Summary of the invention
The first purpose of the present invention provides a kind of Lignanoids compounds; The second purpose is to provide the preparation method of above-mentioned Lignanoids compounds; The 3rd purpose is to provide the application of above-mentioned Lignanoids compounds and synthetic analogues thereof.
The first purpose of the present invention is to realize like this, described Lignanoids compounds is that shizandra berry branch, leaf and/or fruit take drying is as raw material, obtain through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, its structural formula is:
Described R
1For-OH, R
2For-OCH
3, molecular formula is C
20H
24O
8, this compound called after marphenol E.
Described R
1, R
2Between be-OCH
2O-, molecular formula is C
20H
22O
8, this compound called after marphenol F.
The second purpose of the present invention is achieved in that shizandra berry branch, leaf and/or fruit take drying as raw material, separates obtaining through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography, and concrete steps are:
A, medicinal extract extract: with shizandra berry branch, leaf and/or fruit coarse reduction to 20 ~ 40 orders, use organic solvent supersound extraction 2 ~ 5 times, and each 30 ~ 60min, extracting solution merges; Extracting liquid filtering is evaporated to 1/4 ~ 1/2 of volume; Leave standstill, the filtering throw out is condensed into medicinal extract a;
B, organic solvent extraction: add the water of 1 ~ 2 times of amount of weight ratio among the medicinal extract a, use and the isopyknic organic solvent extraction of water 3 ~ 5 times, merge the organic solvent extraction phase, concentrating under reduced pressure becomes medicinal extract b;
C, silica gel column chromatography: with the acetone solution of medicinal extract b with 1.5 ~ 3 times of amounts of weight ratio, then weigh 0.8 ~ 1.2 times 80 ~ 100 order silica gel silica gel mixed samples with medicinal extract, then go up silica gel column chromatography, dress post silica gel is 160 ~ 200 orders, and consumption is 6 ~ 8 times of amounts of medicinal extract b weight; Be the organic solvent solution gradient elution of 1:0 ~ 0:1 with volume ratio, collect gradient eluent, concentrated, through the TLC monitoring, merge identical part;
D, reversed phase column chromatography: the 9:1 of C step elutriant is partly gone up reversed phase column chromatography, and reversed-phase column is with reversed material C-18 dress post; Be that 20 ~ 100% methanol aqueous solution carries out gradient elution with volume content, collect each several part elutriant and concentrated, through the TLC monitoring, merge identical part;
E, high performance liquid chromatography separate: 30% ~ 40% methanol aqueous solution wash-out part of D step elutriant through the high performance liquid chromatography separation and purification, is namely got described Lignanoids compounds;
F, the described high performance liquid chromatography separation and purification of E step are as moving phase take 25 ~ 45% methyl alcohol, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 10 ~ 25min, repeatedly cumulative rear evaporate to dryness.Namely get described Lignanoids compounds marphenol E;
G, the described high performance liquid chromatography separation and purification of E step are as moving phase take 25 ~ 45% methyl alcohol, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 25 ~ 40min, repeatedly cumulative rear evaporate to dryness.Namely get described Lignanoids compounds marphenol F.
The 3rd purpose of the present invention is achieved in that the application of described Lignanoids compounds in the preparation anti-AIDS drug.
Lignanoids compounds of the present invention is separated first, has determined for Lignanoids compounds by nucleus magnetic resonance and measuring method of mass spectrum, and has characterized its concrete structure and be:
Its isomeric compound marphenol E, marphenol F can separate by method of the present invention.Take marphenol E, marphenol F as raw material, detect through the cytotoxicity to the C8166 host cell, and the inhibition test of HIV-1IIIB being induced C8166 cytopathy (CPE), marphenol E, marphenol F all show preferably anti-HIV-1 activity, EC
50Value is respectively 3.47 μ g/mL, 2.97 μ g/mL, and therapeutic index (TI) is respectively 21.18,27.64.The compounds of this invention novel structure, active good has good application prospect in the medicine of the anti-AIDS of preparation, can be used as the guiding compound of anti-AIDS drug.
Description of drawings
Fig. 1 be compound marlphenol E carbon-13 nmr spectra (
13C NMR);
Fig. 2 be compound marlphenol E proton nmr spectra (
1H NMR);
Fig. 3 be compound marlphenol F carbon-13 nmr spectra (
13C NMR);
Fig. 4 be compound marlphenol F proton nmr spectra (
1H NMR);
Fig. 5 is the main HMBC(of compound marlphenol F
→) 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.
Lignanoids compounds of the present invention be shizandra berry branch, leaf and/or fruit take drying as raw material, separate to obtain through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography, its structural formula is:
Described R
1For-OH, R
2For-OCH
3, molecular formula is C
20H
24O
8, this compound called after marphenol E.
Described R
1, R
2Between be-OCH
2O-, molecular formula is C
20H
22O
8, this compound called after marphenol F.
R
1, R
2Can select-H ,-OH or-OCH
3, perhaps R
1, R
2Between formation-OCH
2O-, marphenol E, marphenol F are two kinds of preferred compounds.
The method of Lignanoids compounds of the present invention, be shizandra berry branch, leaf and/or fruit take drying as raw material, separate to obtain through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography, concrete steps are:
A, medicinal extract extract: with shizandra berry branch, leaf and/or fruit coarse reduction to 20 ~ 40 orders, use organic solvent supersound extraction 2 ~ 5 times, and each 30 ~ 60min, extracting solution merges; Extracting liquid filtering is evaporated to 1/4 ~ 1/2 of volume; Leave standstill, the filtering throw out is condensed into medicinal extract a;
B, organic solvent extraction: add the water of 1 ~ 2 times of amount of weight ratio among the medicinal extract a, use and the isopyknic organic solvent extraction of water 3 ~ 5 times, merge the organic solvent extraction phase, concentrating under reduced pressure becomes medicinal extract b;
C, silica gel column chromatography: with the acetone solution of medicinal extract b with 1.5 ~ 3 times of amounts of weight ratio, then weigh 0.8 ~ 1.2 times 80 ~ 100 order silica gel silica gel mixed samples with medicinal extract, then go up silica gel column chromatography, dress post silica gel is 160 ~ 200 orders, and consumption is 6 ~ 8 times of amounts of medicinal extract b weight; Be the organic solvent solution gradient elution of 1:0 ~ 0:1 with volume ratio, collect gradient eluent, concentrated, through the TLC monitoring, merge identical part;
D, reversed phase column chromatography: the 9:1 of C step elutriant is partly gone up reversed phase column chromatography, and reversed-phase column is with reversed material C-18 dress post; Be that 20 ~ 100% methanol aqueous solution carries out gradient elution with volume content, collect each several part elutriant and concentrated, through the TLC monitoring, merge identical part;
E, high performance liquid chromatography separate: 30% ~ 40% methanol aqueous solution wash-out part of D step elutriant through the high performance liquid chromatography separation and purification, is namely got described Lignanoids compounds;
F, the described high performance liquid chromatography separation and purification of E step are as moving phase take 25 ~ 45% methyl alcohol, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 10 ~ 25min, repeatedly cumulative rear evaporate to dryness.Namely get described Lignanoids compounds marphenol E;
G, the described high performance liquid chromatography separation and purification of E step are as moving phase take 25 ~ 45% methyl alcohol, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 25 ~ 40min, repeatedly cumulative rear evaporate to dryness.Namely get described Lignanoids compounds marphenol F.
Described shizandra berry is the Heqing shizandra berry.The present invention is take shizandra berry as raw material, and the restriction that is not subjected to the area and plants all can realize the present invention, preferred Heqing shizandra berry.Heqing shizandra berry (Schisandra wilsoniana A. C. Smith) originates in the jungle of 1800 ~ 2600 meters of northwestern Yunnan Province, height above sea level or the small stream limes marginis.
The described organic solvent of A step is a kind of in 70 ~ 100% acetone, ethanol or the methyl alcohol.
The described organic solvent of B step is a kind of in ethyl acetate, chloroform, ether, sherwood oil or the benzene.
The described organic solvent solution of C step is a kind of in normal hexane-acetone, chloroform-acetone, chloroform-methanol, sherwood oil-acetone or the petroleum ether-ethyl acetate, and the volume proportion of organic solvent solution is 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1.
The described high performance liquid chromatography separation and purification of E step is as moving phase take 25 ~ 45% methyl alcohol, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 10 ~ 40min, repeatedly cumulative rear evaporate to dryness.
The application of Lignanoids compounds of the present invention in the preparation anti-AIDS drug.The compound synthetic take Lignanoids compounds of the present invention as template can be realized purpose of the present invention equally.
Embodiment 1
Get branch, leaf and/or the fruit 4.5kg of Heqing shizandra berry, 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 523g medicinal extract a; Add 784.5g water in medicinal extract a, use and the isopyknic ethyl acetate extraction of water 5 times, merge extraction phase, concentrating under reduced pressure becomes 385g medicinal extract b; With 200 order silica gel 2310g dress post, in medicinal extract b, add the acetone solution of 577.5g, then add 100 order silica gel 385g and mix sample, mix upper prop behind the sample; Be respectively the chloroform-methanol organic solvent solution 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, concentrated, monitor through TLC, merge identical part, obtain 6 parts, the elutriant c of the chloroform-methanol organic solvent solution of volume ratio 9:1 is 63g; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c carries out gradient elution take the methanol aqueous solution of volume content as 20 ~ 100%, collects each several part elutriant and concentrated, through the TLC monitoring, merges identical part; Get the elutriant that obtains with volume content 30 ~ 40% methanol aqueous solution wash-outs, methyl alcohol take 35% 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 58 mL, collect the chromatographic peak of 15min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol E; Collect the chromatographic peak of 30min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol F.
Get branch, leaf and/or the fruit 5kg of Heqing shizandra berry, 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 600g medicinal extract a; The water that adds 600g in medicinal extract a is used and the isopyknic chloroform extraction of water 3 times, merges extraction phase, and concentrating under reduced pressure becomes 428g medicinal extract b; With 160 order silica gel 3.42Kg dress post, in medicinal extract b, add the acetone solution of 1284g, then add 80 order silica gel 342.4g and mix sample, mix upper prop behind the sample; Be respectively the normal hexane of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1-acetone organic solvent solution gradient elution with volume ratio, collect gradient eluent, concentrated, through the TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c carries out gradient elution take the methanol aqueous solution of volume content as 20 ~ 100%, collects each several part elutriant and concentrated, through the TLC monitoring, merges identical part; Get the elutriant that obtains with volume content 30 ~ 40% methanol aqueous solution wash-outs, methyl alcohol take 25% 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 25min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol E; Collect the chromatographic peak of 40min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol F.
Get branch, leaf and/or the fruit 6kg of Heqing shizandra berry, 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 635g medicinal extract a; The water that adds 1270g 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 500g medicinal extract b; With 180 order silica gel 3.5Kg dress post, in medicinal extract b, add the acetone solution of 1000g, then add 90 order silica gel 600g and mix sample, mix upper prop behind the sample; Be respectively the chloroform of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1-acetone organic solvent solution gradient elution with volume ratio, collect gradient eluent, concentrated, through the TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c carries out gradient elution take the methanol aqueous solution of volume content as 20 ~ 100%, collects each several part elutriant and concentrated, through the TLC monitoring, merges identical part; Get the elutriant that obtains with volume content 30 ~ 40% methanol aqueous solution wash-outs, methyl alcohol take 45% 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 10min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol E; Collect the chromatographic peak of 25min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol F.
Get branch, leaf and/or the fruit 5.5kg of Heqing shizandra berry, 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 612g medicinal extract a; The water that adds 918g 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 496g medicinal extract b; With 160 order silica gel 2976g dress post, in medicinal extract b, add the acetone solution of 744g, then add 80 order silica gel 496g and mix sample, mix upper prop behind the sample; Be respectively the sherwood oil of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1-acetone organic solvent solution gradient elution with volume ratio, collect gradient eluent, concentrated, through the TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c carries out gradient elution take the methanol aqueous solution of volume content as 20 ~ 100%, collects each several part elutriant and concentrated, through the TLC monitoring, merges identical part; Get the elutriant that obtains with volume content 30 ~ 40% methanol aqueous solution wash-outs, methyl alcohol take 35% 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 20min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol E; Collect the chromatographic peak of 35min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol F.
Embodiment 5
Get branch, leaf and/or the fruit 5kg of Heqing shizandra berry, 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 590g medicinal extract a; The water that adds 1180g in medicinal extract a is used and the isopyknic benzene extraction of water 5 times, merges extraction phase, and concentrating under reduced pressure becomes 475g medicinal extract b; With 200 order silica gel 3325g dress post, in medicinal extract b, add the acetone solution of 1425g, then add 100 order silica gel 380g and mix sample, mix upper prop behind the sample; Be respectively the petroleum ether-ethyl acetate organic solvent solution 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, concentrated, through the TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c carries out gradient elution take the methanol aqueous solution of volume content as 20 ~ 100%, collects each several part elutriant and concentrated, through the TLC monitoring, merges identical part; Get the elutriant that obtains with volume content 30 ~ 40% methanol aqueous solution wash-outs, methyl alcohol take 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 50mL, collect the chromatographic peak of 13min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol E; Collect the chromatographic peak of 36min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol F.
Get the compound marphenol E of embodiment 1 preparation, be yellow jelly; Optical value [a] 24.8 D+19.6 (solvent is methyl alcohol c 0.18); 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
e): 205(4.92), 282(4.18), 326 (2.25) nm;
2) infrared spectra (pressing potassium bromide troche),
n Max3480,2968,2947,2885,2843,1622,1592,1517,1458,1368,1255,1262,1152,1022,977,824 cm
-1
3) high resolution mass spectrum (HRESIMS) shows the compounds of this invention quasi-molecular ion peak
M/z[415.1362 M+H]
+(calculated value is 415.1369), in conjunction with
13C and
1H NMR spectrum (figure-1 and figure-2, attribution data sees Table-1) provides its molecular formula C
20H
24O
8, degree of unsaturation is 9.
1H?NMR（C
5D
5N,?500?MHz）?
δ?7.11（1H,?s,?H-2）,?6.80?-?6.84?（1H,?overlap,?H-5）,?6.91?-6.95（1H,?overlap,?H-6）,?3.60（1H,?d?
J?=?11.5?Hz,?H-7）,?3.24（1H,?m,?H-8）,?7.08（1H,?s,?H-2￠）,?6.80?-?6.84（1H,?overlap,?H-5￠）,?6.91?-6.95（1H,?overlap,?H-6￠）,?4.27?-?4.34（2?H,?overlap,?H-7￠）,?2.35（1H,?m,?H-8￠）,?4.27-4.34（2?H,?overlap,?H-7￠）,?11.13（1H,?brs,?-COOH）,?10.54?（2H,?brs,?Ar-OH）,?3.77,?3.77（3H?s,?2?×?-OMe）。
13C NMR data see Table 1.
Compound marphenol E's
1H and
13C NMR spectrogram shows respectively 24 hydrogen and 20 carbon signals, corresponding to 6 fragrant hydrogen of 2 phenyl ring, and 2 oxidized methylene radical, 3 methynes, 1 carboxyl and 2 methoxyl groups.The carbon relevant (C-2, C-6, C-2', and C-6') of H-7 and 2 phenyl ring illustrates that 2 phenyl ring pass through (C-7) continuous in the HMBC spectrum.H-7/H-8/H-8'/H-9' and H-8'/H-7''s
1H-
1H COSY coherent signal, and H-7 and C-8 in the HMBC spectrum, C-9 is relevant with C-8', H-7' and C-8', C-9' is relevant with C-8, and there are OH-CH in carboxyl hydrogen and C-8 related description in this compound
2-CH(OH-CH
2)-CH(COOH)-CH structural unit.On phenyl ring two methoxyl groups respectively with the position of C-4 and these two methoxyl groups of C-4' related description in C-4 and C-4' position.So far the structure of this compound is determined.
Get the compound marphenol F of embodiment 1 preparation, be yellow jelly; Optical value [a] 24.8 D+26.8 (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
e): 208(4.82), 282(4.22), 325 (2.18) nm;
2) infrared spectra (pressing potassium bromide troche),
n Max3468,2977,2942,2887,2849,2816,1627,1593,1514,1455,1363,1254,1265,1153,1031,1018,987,806 cm
-1
3) high resolution mass spectrum (HRESIMS) shows the compounds of this invention quasi-molecular ion peak
M/z[413.1217 M+H]
+(calculated value is 413.1212), in conjunction with
13C and
1H NMR spectrum (figure-3 and figure-4, attribution data sees Table-1) provides its molecular formula C
20H
22O
8, degree of unsaturation is 10.
1H?NMR（C
5D
5N,?500?MHz）?
δ7.10（1H,?s,?H-2）,?6.76?-?6.79（1H,?overlap,?H-5）,?6.99?-7.01（1H,?overlap,?H-6）,?3.61（1H,?d?
J?=?11.9?Hz,?H-7）,?3.14（1H,?m,?H-8）,?7.37（1H,?s,?H-2￠）,?6.76?-?6.79（1H,?overlap,?H-5￠）,?6.99?-7.01（1H,?overlap,?H-6￠）,?4.36?-?4.50（2?H,?overlap,?H-7￠）,?2.35（1H,?m,?H-8￠）,?4.36-4.50（2?H,?overlap,?H-7￠）,?11.13（1H,?brs,?-COOH）,?10.69（1H,?brs,?Ar-OH）,?5.82,?5.87（1H,?s,?-OCH
2O-）,?3.75（3H?s,?-OMe）。
13C NMR data see Table 1.
Compound marphenol F's
1H and
13C NMR and marphenol E's is closely similar.Find by contrast, the difference of these two compounds is to have lacked 1 methoxyl group among the marphenol F, 1 two Oxymethylene occurred.The HMBC coherent signal of two Oxymethylenes and C-3' and C-4' illustrates that its position of substitution is in C-3' and C-4' position.1 methoxyl group and its position of substitution of C-3 related description are in the C-3 position.So far the structure of this compound is determined.
Table 1 compound
13(solvent is C to C NMR data
5D
5N)
| Atom | marlphenol E | marlphenol F | Atom | marlphenol E | marlphenol F |
| 1 | 136.3 s | 134.2 s | 3￠ | 148.9 s | 147.9 |
| 2 | 110.9 d | 112.4 |
4￠ | 145.0 s | 145.9 |
| 3 | 148.8 s | 149.6 s | 5￠ | 114.7 d | 108.8 |
| 4 | 144.9 s | 146.0 s | 6￠ | 119.8 d | 120.5 d |
| 5 | 114.7 d | 116.5 |
7￠ | 70.0 t | 70.4 |
| 6 | 119.7 d | 112.4 d | 8￠ | 49.8 d | 46.7 |
| 7 | 58.8 d | 58.4 |
9￠ | 70.0 t | 70.4 t |
| 8 | 49.8 d | 49.5 d | OMe-3 | 55.9 s | 55.8 |
| 9 | 178.6 s | 179.7 s | OMe-4 | ? | ? |
| 1￠ | 136.8 s | 133.3 s | OMe-3￠ | 55.9 s | ? |
| 2￠ | 111.0 d | 108.3 d | -OCH2O- | ? | 101.6 t |
Embodiment 8
The compound marphenol E, the marphenol F that get embodiment 2 preparations carry out structure determination by the method among the embodiment 6,7 respectively, and the result is: its structure is with embodiment 6,7, and molecular formula is respectively C
20H
24O
8And C
20H
22O
8
The compound marphenol E, the marphenol F that get embodiment 3 preparations carry out structure determination by the method among the embodiment 6,7 respectively, and the result is: its structure is with embodiment 6,7, and molecular formula is respectively C
20H
24O
8And C
20H
22O
8
The compound marphenol E, the marphenol F that get embodiment 4 preparations carry out structure determination by the method among the embodiment 6,7 respectively, and the result is: its structure is with embodiment 6,7, and molecular formula is respectively C
20H
24O
8And C
20H
22O
8
The compound marphenol E, the marphenol F that get embodiment 5 preparations carry out structure determination by the method among the embodiment 6,7 respectively, and the result is: its structure is with embodiment 6,7, and molecular formula is respectively C
20H
24O
8And C
20H
22O
8
Embodiment 12
The Lignanoids compounds (marphenol E) of getting embodiment 1 preparation carries out the anti-HIV-1 activity test, and is as follows:
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 the degerming of μ m membrane filtration ,-20 ℃ of preservations after the packing.
HEPES(N-2(2-Hydroxyothyl) piperazine-N'-(2-ethanesufonic acid), MTT (3-(4 reagent:, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), DMF(N, N '-Dimethyl formamine), penicillin (Penicillin), Vetstrep (Streptomycin sulfate), glutamine (Glutamine) is all available from Sigma company; DMSO, 2 mercapto ethanol (2-Me, 2-Mercaptoethanol) are Bio-Rad company product; RPMI-1640 and newborn calf serum are Gibco company product.
Substratum: the RPMI-1640 perfect medium, contain 10 % newborn calf serums, 2 mM L-glutaminate, 10 mM HEPES, 50 μ M 2 mercapto ethanols, 100,000 IU penicillin, 100 μ g/mL Streptomycin sulphates.
Cell and virus: the human T lymphocyte is C8166, and MT4 and HIV-1 experiment strain HIV-1IIIB all come from Britain Medical Research Council, AIDS Reagent Project.All cells and virus are all cultivated with the RPMI-1640 perfect medium that contains 10% calf serum.Prepare according to a conventional method HIV-1IIIB, titration also calculates viral TCID
50After the packing of virus stock solution, put-70 ℃ of preservations.Cell and virus is freezing and thawing according to a conventional method.
The HIV-1 infectious titration: carry out titration by the described method improvement of Johnson﹠Byington, 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 the C8166 cell, and 50 μ L(4 * 105/mL), every hole final volume is 200 μ L, 37 ℃, and 5% CO
2Cultivate.Added fresh RPMI-1640 perfect medium 100 μ L on the 3rd day, under inverted microscope, observed cytopathic effect (the Cytopathic Effect that HIV-1 induces in every hole on the 7th day, CPE), whether there is the formation of synplasm (Syncytium) to determine with every hole; Press the TCID that the Reed﹠Muench method is calculated virus
50(50% Tissue Culture Infection Dose).
Sample detects the cytotoxicity of C8166 host cell: 4 * 105/mL C8166 cell suspension, 100 μ L and different testing compound solution mixing, establish three repeating holes.The control wells that does not contain compound, 37 ℃, 5% CO are set simultaneously
2Cultivated three days, and adopted MTT colorimetric determination 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 when namely 50% normal T lymphocyte series C8166 being produced toxicity.
Sample is induced the inhibition test of C8166 cytopathy (CPE) to HIV-1IIIB: 8 * 105/mL C8166 cell, 50 μ L/ holes are inoculated on the 96 porocyte culture plates that contain 100 μ L/ hole doubling dilution compounds, then add the HIV-1IIIB dilution supernatant (M.O.I.0.0016) of 50 μ 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.Compound concentration during EC50(50% Effective Concentration) for inhibition Syncytium formation 50%.
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 Active be: TI=CC
50/ EC
50
Test-results: detect through the cytotoxicity to the C8166 host cell, and the inhibition test of HIV-1IIIB being induced C8166 cytopathy (CPE), Lignanoids compounds (marphenol E) has preferably anti-HIV-1 activity, EC
50Value is 3.47 μ g/mL, and therapeutic index (TI) sees Table 2 for 21.18().It is good that above result has disclosed marphenol E novel structure activity, in the medicine of the anti-AIDS of preparation good application prospect arranged, and can be used as the guiding compound of anti-AIDS drug.
The Anti-HIV of table 2 marphenol E and marphenol F is active
Embodiment 13
The Lignanoids compounds (marphenol F) of getting embodiment 1 preparation carries out the anti-HIV-1 activity test by the method for embodiment 12, the result is as follows: detect through the cytotoxicity to the C8166 host cell, and the inhibition test of HIV-1IIIB being induced C8166 cytopathy (CPE), Lignanoids compounds (marphenol E) has preferably anti-HIV-1 activity, EC
50Value is 2.97 μ g/mL, and therapeutic index (TI) sees Table 2 for 27.64().It is good that above result has disclosed marphenol F novel structure activity, in the medicine of the anti-AIDS of preparation good application prospect arranged, and can be used as the guiding compound of anti-AIDS drug.
Claims (9)
1. Lignanoids compounds, it is characterized in that: described Lignanoids compounds is that shizandra berry branch, leaf and/or fruit take drying is as raw material, obtain through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, its structural formula is:
2. Lignanoids compounds according to claim 1 is characterized in that: described R
1For-OH, R
2For-OCH
3, molecular formula is C
20H
24O
8, this compound called after marphenol E.
3. Lignanoids compounds according to claim 1 is characterized in that: described R
1, R
2Between be-OCH
2O-, molecular formula is C
20H
22O
8, this compound called after marphenol F.
4. method for preparing Lignanoids compounds claimed in claim 1, it is characterized in that shizandra berry branch, leaf and/or fruit take drying is as raw material, obtain through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, concrete steps are:
A, medicinal extract extract: with shizandra berry branch, leaf and/or fruit coarse reduction to 20 ~ 40 orders, use organic solvent supersound extraction 2 ~ 5 times, and each 30 ~ 60min, extracting solution merges; Extracting liquid filtering is evaporated to 1/4 ~ 1/2 of volume; Leave standstill, the filtering throw out is condensed into medicinal extract a;
B, organic solvent extraction: add the water of 1 ~ 2 times of amount of weight ratio among the medicinal extract a, use and the isopyknic organic solvent extraction of water 3 ~ 5 times, merge the organic solvent extraction phase, concentrating under reduced pressure becomes medicinal extract b;
C, silica gel column chromatography: with the acetone solution of medicinal extract b with 1.5 ~ 3 times of amounts of weight ratio, then weigh 0.8 ~ 1.2 times 80 ~ 100 order silica gel silica gel mixed samples with medicinal extract, then go up silica gel column chromatography, dress post silica gel is 160 ~ 200 orders, and consumption is 6 ~ 8 times of amounts of medicinal extract b weight; Be the organic solvent solution gradient elution of 1:0 ~ 0:1 with volume ratio, collect gradient eluent, concentrated, through the TLC monitoring, merge identical part;
D, reversed phase column chromatography: the 9:1 of C step elutriant is partly gone up reversed phase column chromatography, and reversed-phase column is with reversed material C-18 dress post; Be that 20 ~ 100% methanol aqueous solution carries out gradient elution with volume content, collect each several part elutriant and concentrated, through the TLC monitoring, merge identical part;
E, high performance liquid chromatography separate: with 30% ~ 40% methanol aqueous solution wash-out part of D step elutriant through the high performance liquid chromatography separation and purification, methyl alcohol take 25 ~ 45% is moving phase, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, 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 50 ~ 60mL, collect the chromatographic peak of 10 ~ 40min, repeatedly cumulative rear evaporate to dryness.
5. namely get described Lignanoids compounds marphenol E and marphenol F;
F, the described high performance liquid chromatography separation and purification of E step are as moving phase take 25 ~ 45% methyl alcohol, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 10 ~ 25min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol E;
G, the described high performance liquid chromatography separation and purification of E step are as moving phase take 25 ~ 45% methyl alcohol, flow velocity 10 ~ 14ml/min, 21.2 ' 250 mm, the anti-phase preparative column of Zorbax PrepHT GF of 5mm is stationary phase, it is 254 nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, the chromatographic peak of collection 25 ~ 40min, repeatedly cumulative rear evaporate to dryness namely gets described Lignanoids compounds marphenol F;
The preparation method of Lignanoids compounds according to claim 4 is characterized in that: described shizandra berry is the Heqing shizandra berry.
6. the preparation method of Lignanoids compounds according to claim 4 is characterized in that: the described organic solvent of A step is a kind of in 70 ~ 100% acetone, ethanol or the methyl alcohol.
7. the preparation method of Lignanoids compounds according to claim 4 is characterized in that: the described organic solvent of B step is a kind of in ethyl acetate, chloroform, ether, sherwood oil or the benzene.
8. the preparation method of Lignanoids compounds according to claim 4, it is characterized in that: the described organic solvent solution of C step is a kind of in normal hexane-acetone, chloroform-acetone, chloroform-methanol, sherwood oil-acetone or the petroleum ether-ethyl acetate, and the volume proportion of organic solvent solution is 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1.
9. a claim 1, the application of 2 or 3 described Lignanoids compounds in the preparation anti-AIDS drug.
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| CN103756321A (en) * | 2014-01-03 | 2014-04-30 | 中山市点石塑胶有限公司 | High-thermal-conductivity polymer composite and preparation method thereof |
| CN107595838A (en) * | 2017-09-13 | 2018-01-19 | 北京理工大学 | A kind of application of Lignanoids compounds in anti-liver fiber medicine is prepared |
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| CN101982467A (en) * | 2010-09-26 | 2011-03-02 | 云南烟草科学研究院 | Lignans compounds contained in Schisandra chinensis, and preparation method and application thereof |
| CN102093749A (en) * | 2011-01-11 | 2011-06-15 | 烟台大学 | Preparation technology of schisandra chinensis natural pigment |
| US20120052142A1 (en) * | 2010-08-26 | 2012-03-01 | Dr. Chang Naturals, LLC | BIOLOGICALLY ACTIVE COMPOSITIONS FROM Schisandra chinensis FOR TREATING COLORECTAL CANCER |
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| US20120052142A1 (en) * | 2010-08-26 | 2012-03-01 | Dr. Chang Naturals, LLC | BIOLOGICALLY ACTIVE COMPOSITIONS FROM Schisandra chinensis FOR TREATING COLORECTAL CANCER |
| CN101982467A (en) * | 2010-09-26 | 2011-03-02 | 云南烟草科学研究院 | Lignans compounds contained in Schisandra chinensis, and preparation method and application thereof |
| CN102093749A (en) * | 2011-01-11 | 2011-06-15 | 烟台大学 | Preparation technology of schisandra chinensis natural pigment |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103756321A (en) * | 2014-01-03 | 2014-04-30 | 中山市点石塑胶有限公司 | High-thermal-conductivity polymer composite and preparation method thereof |
| CN103756321B (en) * | 2014-01-03 | 2016-05-04 | 中山市点石塑胶有限公司 | High heat-conductivity polymer composite and preparation method thereof |
| CN107595838A (en) * | 2017-09-13 | 2018-01-19 | 北京理工大学 | A kind of application of Lignanoids compounds in anti-liver fiber medicine is prepared |
| CN110585193A (en) * | 2017-09-13 | 2019-12-20 | 北京理工大学 | Application of diaryl butyrolactone lignan compound in preparation of anti-hepatic fibrosis medicine |
| CN110585193B (en) * | 2017-09-13 | 2022-08-02 | 北京理工大学 | Application of diaryl butyrolactone lignan compound in preparation of anti-hepatic fibrosis medicine |
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