CN103012118B - Lignans compounds and preparation method and application thereof - Google Patents
Lignans compounds and preparation method and application thereof Download PDFInfo
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- CN103012118B CN103012118B CN201310014125.5A CN201310014125A CN103012118B CN 103012118 B CN103012118 B CN 103012118B CN 201310014125 A CN201310014125 A CN 201310014125A CN 103012118 B CN103012118 B CN 103012118B
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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 mug/mL and 2.97 mug/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 two belongs to and all produces it, and approximately more than 30 plant, and originate in the west and south to northeast, but main product ground is 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 obtaining and be mainly two large classes: triterpene (Triterpenes) compounds and lignanoid (Lignans).Triterpene compound is mostly lanostane-type and cyclic-ahltin type, because its skeleton easily changes, and because oxidisability difference makes this class triterpenoid complex structure uniqueness.Lignanoid mostly is cyclohexyl biphenyl octene class.Modern pharmacology research shows the main active ingredient of lignanoid and triterpene Shi Gai section plant, and the lignanoid that has in recent years report to be separated to from schisandra plant has certain potentiality aspect anti-HIV.Therefore the continuation further investigation of this platymiscium is seemed to particularly important.
Summary of the invention
The first object of the present invention is to provide a kind of Lignanoids compounds; The second object is to provide the preparation method of above-mentioned Lignanoids compounds; The 3rd object is to provide the application of above-mentioned Lignanoids compounds and synthetic analogues thereof.
The first object of the present invention is to realize like this, described Lignanoids compounds be the shizandra berry branch, leaf and/or the fruit that are dried be 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 object of the present invention is achieved in that the shizandra berry branch, leaf and/or the fruit that are dried are raw material, obtains through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, and concrete steps are:
A, medicinal extract extract: by shizandra berry branch, leaf and/or fruit coarse reduction to 20 ~ 40 order, 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, filtering throw out, is condensed into medicinal extract a;
B, organic solvent extraction: in medicinal extract a, add the water of 1 ~ 2 times of amount of weight ratio, use and the isopyknic organic solvent extraction of water 3 ~ 5 times, merge organic solvent extraction phase, concentrating under reduced pressure becomes medicinal extract b;
C, silica gel column chromatography: the acetone solution by medicinal extract b by 1.5 ~ 3 times of amounts of weight ratio, then weigh 80 ~ 100 order silica gel silica gel mixed samples of 0.8 ~ 1.2 times with medicinal extract, then go up silica gel column chromatography, dress post silica gel is 160 ~ 200 orders, consumption is 6 ~ 8 times of amounts of medicinal extract b weight; The organic solvent solution gradient elution that is 1:0 ~ 0:1 by volume ratio, collects gradient eluent, concentrated, through TLC monitoring, merges identical part;
D, reversed phase column chromatography: by reversed phase column chromatography in the 9:1 part of C step elutriant, reversed-phase column is with reversed material C-18 dress post; The methanol aqueous solution that is 20 ~ 100% with volume content carries out gradient elution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part;
E, high performance liquid chromatography separate: 30% ~ 40% methanol aqueous solution wash-out part of D step elutriant, through high performance liquid chromatography separation and purification, is obtained to described Lignanoids compounds;
High performance liquid chromatography separation and purification described in F, E step is taking 25 ~ 45% methyl alcohol as 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, 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.Obtain described Lignanoids compounds marphenol E;
High performance liquid chromatography separation and purification described in G, E step is taking 25 ~ 45% methyl alcohol as 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, 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.Obtain described Lignanoids compounds marphenol F.
The 3rd object of the present invention is achieved in that described Lignanoids compounds is in the application of preparing in anti-AIDS drug.
Lignanoids compounds of the present invention is separated first, has determined for Lignanoids compounds, and characterized its concrete structure and be by nucleus magnetic resonance and measuring method of mass spectrum:
Its isomeric compound marphenol E, marphenol F can separate by method of the present invention.Taking marphenol E, marphenol F as raw material, through the cytotoxicity of C8166 host cell is detected, and inhibition test to HIV-1IIIB induction C8166 cytopathy (CPE), marphenol E, marphenol F all show good 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.
Brief description of the 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
Below in conjunction with accompanying drawing, the present invention is further illustrated, but never in any form the present invention is limited, and any conversion or the improvement done based on training centre of the present invention, all fall into protection scope of the present invention.
Lignanoids compounds of the present invention be the shizandra berry branch, leaf and/or the fruit that are dried be raw material, separate and 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, or 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 the fruit being dried be raw material, separate and to obtain through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography, concrete steps are:
A, medicinal extract extract: by shizandra berry branch, leaf and/or fruit coarse reduction to 20 ~ 40 order, 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, filtering throw out, is condensed into medicinal extract a;
B, organic solvent extraction: in medicinal extract a, add the water of 1 ~ 2 times of amount of weight ratio, use and the isopyknic organic solvent extraction of water 3 ~ 5 times, merge organic solvent extraction phase, concentrating under reduced pressure becomes medicinal extract b;
C, silica gel column chromatography: the acetone solution by medicinal extract b by 1.5 ~ 3 times of amounts of weight ratio, then weigh 80 ~ 100 order silica gel silica gel mixed samples of 0.8 ~ 1.2 times with medicinal extract, then go up silica gel column chromatography, dress post silica gel is 160 ~ 200 orders, consumption is 6 ~ 8 times of amounts of medicinal extract b weight; The organic solvent solution gradient elution that is 1:0 ~ 0:1 by volume ratio, collects gradient eluent, concentrated, through TLC monitoring, merges identical part;
D, reversed phase column chromatography: by reversed phase column chromatography in the 9:1 part of C step elutriant, reversed-phase column is with reversed material C-18 dress post; The methanol aqueous solution that is 20 ~ 100% with volume content carries out gradient elution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part;
E, high performance liquid chromatography separate: 30% ~ 40% methanol aqueous solution wash-out part of D step elutriant, through high performance liquid chromatography separation and purification, is obtained to described Lignanoids compounds;
High performance liquid chromatography separation and purification described in F, E step is taking 25 ~ 45% methyl alcohol as 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, 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.Obtain described Lignanoids compounds marphenol E;
High performance liquid chromatography separation and purification described in G, E step is taking 25 ~ 45% methyl alcohol as 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, 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.Obtain described Lignanoids compounds marphenol F.
Described shizandra berry is Heqing shizandra berry.The present invention is taking shizandra berry as raw material, and the restriction that is not subject to area and plants, all can realize the present invention, preferably 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 small stream limes marginis.
Organic solvent described in A step is the one in 70 ~ 100% acetone, ethanol or methyl alcohol.
Organic solvent described in B step is the one in ethyl acetate, chloroform, ether, sherwood oil or benzene.
Organic solvent solution described in C step is the one in normal hexane-acetone, chloroform-acetone, chloroform-methanol, sherwood oil-acetone or 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.
High performance liquid chromatography separation and purification described in E step is taking 25 ~ 45% methyl alcohol as 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, 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.
Lignanoids compounds of the present invention is in the application of preparing in anti-AIDS drug.Can realize equally object of the present invention taking Lignanoids compounds of the present invention as the synthetic compound of template.
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, filtering throw out, is condensed into 523g medicinal extract a; In medicinal extract a, add 784.5g water, 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 posts, in medicinal extract b, add the acetone solution of 577.5g, then add 100 order silica gel 385g to mix sample, mix upper prop after 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 by 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 taking volume content as 20 ~ 100% methanol aqueous solution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part; Get the elutriant obtaining with volume content 30 ~ 40% methanol aqueous solution wash-outs, methyl alcohol taking 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, 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, obtains described Lignanoids compounds marphenol E; Collect the chromatographic peak of 30min, repeatedly cumulative rear evaporate to dryness, obtains described Lignanoids compounds marphenol F.
Embodiment 2
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, filtering throw out, is condensed into 600g medicinal extract a; In medicinal extract a, add the water of 600g, use and the isopyknic chloroform extraction of water 3 times, merge extraction phase, concentrating under reduced pressure becomes 428g medicinal extract b; With 160 order silica gel 3.42Kg dress posts, in medicinal extract b, add the acetone solution of 1284g, then add 80 order silica gel 342.4g to mix sample, mix upper prop after sample; Be respectively normal hexane-acetone organic solvent solution gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 by volume ratio, collect gradient eluent, concentrated, through TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c, carries out gradient elution taking volume content as 20 ~ 100% methanol aqueous solution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part; Get the elutriant obtaining with volume content 30 ~ 40% methanol aqueous solution wash-outs, methyl alcohol taking 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, 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, obtains described Lignanoids compounds marphenol E; Collect the chromatographic peak of 40min, repeatedly cumulative rear evaporate to dryness, obtains described Lignanoids compounds marphenol F.
Embodiment 3
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, filtering throw out, is condensed into 635g medicinal extract a; In medicinal extract a, add the water of 1270g, use and the isopyknic extracted with diethyl ether of water 4 times, merge extraction phase, concentrating under reduced pressure becomes 500g medicinal extract b; With 180 order silica gel 3.5Kg dress posts, in medicinal extract b, add the acetone solution of 1000g, then add 90 order silica gel 600g to mix sample, mix upper prop after sample; Be respectively chloroform-acetone organic solvent solution gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 by volume ratio, collect gradient eluent, concentrated, through TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c, carries out gradient elution taking volume content as 20 ~ 100% methanol aqueous solution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part; Get the elutriant obtaining with volume content 30 ~ 40% methanol aqueous solution wash-outs, methyl alcohol taking 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, it is 254nm that UV-detector detects wavelength, each sample introduction 60mL, collect the chromatographic peak of 10min, repeatedly cumulative rear evaporate to dryness, obtains described Lignanoids compounds marphenol E; Collect the chromatographic peak of 25min, repeatedly cumulative rear evaporate to dryness, obtains described Lignanoids compounds marphenol F.
Embodiment 4
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, filtering throw out, is condensed into 612g medicinal extract a; In medicinal extract a, add the water of 918g, use and the isopyknic petroleum ether extraction of water 4 times, merge extraction phase, concentrating under reduced pressure becomes 496g medicinal extract b; With 160 order silica gel 2976g dress posts, in medicinal extract b, add the acetone solution of 744g, then add 80 order silica gel 496g to mix sample, mix upper prop after sample; Be respectively sherwood oil-acetone organic solvent solution gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 by volume ratio, collect gradient eluent, concentrated, through TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c, carries out gradient elution taking volume content as 20 ~ 100% methanol aqueous solution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part; Get the elutriant obtaining with volume content 30 ~ 40% methanol aqueous solution wash-outs, methyl alcohol taking 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, it is 254nm that UV-detector detects wavelength, each sample introduction 55mL, collect the chromatographic peak of 20min, repeatedly cumulative rear evaporate to dryness, obtains described Lignanoids compounds marphenol E; Collect the chromatographic peak of 35min, repeatedly cumulative rear evaporate to dryness, obtains 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, filtering throw out, is condensed into 590g medicinal extract a; In medicinal extract a, add the water of 1180g, use and the isopyknic benzene extraction of water 5 times, merge extraction phase, concentrating under reduced pressure becomes 475g medicinal extract b; With 200 order silica gel 3325g dress posts, in medicinal extract b, add the acetone solution of 1425g, then add 100 order silica gel 380g to mix sample, mix upper prop after 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 by volume ratio, collect gradient eluent, concentrated, through TLC monitoring, merge identical part; With reversed material C-18 dress post, the upper reversed-phase column of elutriant c, carries out gradient elution taking volume content as 20 ~ 100% methanol aqueous solution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part; Get the elutriant obtaining with volume content 30 ~ 40% methanol aqueous solution wash-outs, methyl alcohol taking 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, it is 254nm that UV-detector detects wavelength, each sample introduction 50mL, collect the chromatographic peak of 13min, repeatedly cumulative rear evaporate to dryness, obtains described Lignanoids compounds marphenol E; Collect the chromatographic peak of 36min, repeatedly cumulative rear evaporate to dryness, obtains described Lignanoids compounds marphenol F.
Embodiment 6
Getting compound marphenol E prepared by embodiment 1, is yellow jelly; Optical value [a] 24.8 D+19.6 (solvent is methyl alcohol c 0.18); Measuring method is: with nucleus magnetic resonance, in conjunction with other spectroscopic technique qualification structure.
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 is in 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 are in 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.H-7 relevant to the carbon of 2 phenyl ring (C-2, C-6, C-2', and C-6') in HMBC spectrum, illustrates that 2 phenyl ring are connected by (C-7).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 HMBC spectrum, C-9 is relevant with C-8', H-7' and C-8', C-9' is relevant with C-8, and in this compound, there is OH-CH in carboxyl hydrogen and C-8 related description
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.
Embodiment 7
Getting compound marphenol F prepared by embodiment 1, is yellow jelly; Optical value [a] 24.8 D+26.8 (solvent is methyl alcohol c 0.20); Measuring method is: with nucleus magnetic resonance, in conjunction with other spectroscopic technique qualification structure.
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 is in 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 are in 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 in marphenol F, has occurred 1 two Oxymethylene.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 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 s |
| 2 | 110.9 d | 112.4 d | 4￠ | 145.0 s | 145.9 s |
| 3 | 148.8 s | 149.6 s | 5￠ | 114.7 d | 108.8 d |
| 4 | 144.9 s | 146.0 s | 6￠ | 119.8 d | 120.5 d |
| 5 | 114.7 d | 116.5 d | 7￠ | 70.0 t | 70.4 t |
| 6 | 119.7 d | 112.4 d | 8￠ | 49.8 d | 46.7 d |
| 7 | 58.8 d | 58.4 d | 9￠ | 70.0 t | 70.4 t |
| 8 | 49.8 d | 49.5 d | OMe-3 | 55.9 s | 55.8 s |
| 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
Get compound marphenol E, marphenol F prepared by embodiment 2 and carry out structure determination by the method in embodiment 6,7 respectively, result is: its structure is with embodiment 6,7, and molecular formula is respectively C
20h
24o
8and C
20h
22o
8.
Embodiment 9
Get compound marphenol E, marphenol F prepared by embodiment 3 and carry out structure determination by the method in embodiment 6,7 respectively, result is: its structure is with embodiment 6,7, and molecular formula is respectively C
20h
24o
8and C
20h
22o
8.
Embodiment 10
Get compound marphenol E, marphenol F prepared by embodiment 4 and carry out structure determination by the method in embodiment 6,7 respectively, result is: its structure is with embodiment 6,7, and molecular formula is respectively C
20h
24o
8and C
20h
22o
8.
Embodiment 11
Get compound marphenol E, marphenol F prepared by embodiment 5 and carry out structure determination by the method in embodiment 6,7 respectively, result is: its structure is with embodiment 6,7, and molecular formula is respectively C
20h
24o
8and C
20h
22o
8.
Embodiment 12
Get Lignanoids compounds (marphenol E) prepared by embodiment 1 and carry out anti-HIV-1 activity test, as follows:
Measure medicine and compound: testing sample dissolves with DMSO, positive control compound Zidovodine (AZT, 3 '-Azido-3 '-deoxythymidine) purchased from Sigma company, be dissolved in RPMI-1640 perfect medium, 0.22 μ m membrane filtration degerming ,-20 DEG C of preservations after packing.
Reagent: HEPES(N-2(2-Hydroxyothyl) piperazine-N'-(2-ethanesufonic acid), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), DMF(N, N '-Dimethyl formamine), penicillin (Penicillin), Vetstrep (Streptomycin sulfate), glutamine (Glutamine) is all purchased 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: RPMI-1640 perfect medium, contains 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: human T lymphocyte is C8166, 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 containing 10% calf serum.Prepare according to a conventional method HIV-1IIIB, titration also calculates viral TCID
50.After the packing of virus stock solution, put-70 DEG C of preservations.Cell and virus freezing and thawing according to a conventional method.
HIV-1 infectious titration: carry out titration by method improvement described in Johnson & Byington, as follows: HIV-1IIIB stock solution is done on 96 orifice plates to 4 times of dilutions, 10 gradients, 6 repeating holes of every gradient arrange control wells 6 holes simultaneously.Every hole adds C8166 cell 50 μ L(4 × 105/mL), every hole final volume is 200 μ L, 37 DEG C, 5% CO
2cultivate.Within the 3rd day, add fresh RPMI-1640 perfect medium 100 μ L, within the 7th day, under inverted microscope, observe cytopathic effect (the Cytopathic Effect of HIV-1 induction in every hole, CPE), whether there is the formation of synplasm (Syncytium) to determine with every hole; Calculate viral TCID by Reed & Muench method
50(50% Tissue Culture Infection Dose).
Sample detects the cytotoxicity of C8166 host cell: 4 × 105/mL C8166 cell suspension, 100 μ L mix from different testing compound solutions, establish three repeating holes.The not control wells containing compound is set simultaneously, 37 DEG C, 5% CO
2cultivate three days, adopt MTT colorimetric determination cytotoxicity.ELx800 ELISA instrument is measured OD value, and measuring wavelength is 595 nm, and reference wavelength is 630 nm.Calculate CC
50value (50% Cytotoxic Concentration), the compound concentration while 50% normal T lymphocyte series C8166 being produced to toxicity.
The inhibition test of sample to HIV-1IIIB induction C8166 cytopathy (CPE): 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 not normal cell control wells containing compound is set simultaneously, 37 DEG C, 5% CO
2cultivate three days, under inverted microscope, (100 ×) count plasmodial formation.EC50(50% Effective Concentration) be the compound concentration while suppressing Syncytium formation 50%.
Calculation formula: draw dose response curve according to experimental result, calculate by Reed & Muench method the 50% effective concentration (EC that compound suppresses virus
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: through the cytotoxicity of C8166 host cell is detected, and HIV-1IIIB is induced to the inhibition test of C8166 cytopathy (CPE), Lignanoids compounds (marphenol E) has good anti-HIV-1 activity, EC
50value is 3.47 μ g/mL, and therapeutic index (TI) is for 21.18(is in table 2).It is good that above result has disclosed marphenol E novel structure activity, in the medicine of the anti-AIDS of preparation, has good application prospect, can be used as the guiding compound of anti-AIDS drug.
The Anti-HIV activity of table 2 marphenol E and marphenol F
Embodiment 13
Get Lignanoids compounds (marphenol F) prepared by embodiment 1 and carry out anti-HIV-1 activity test by the method for embodiment 12, result is as follows: through the cytotoxicity of C8166 host cell is detected, and inhibition test to HIV-1IIIB induction C8166 cytopathy (CPE), Lignanoids compounds (marphenol E) has good anti-HIV-1 activity, EC
50value is 2.97 μ g/mL, and therapeutic index (TI) is for 27.64(is in table 2).It is good that above result has disclosed marphenol F novel structure activity, in the medicine of the anti-AIDS of preparation, has good application prospect, can be used as the guiding compound of anti-AIDS drug.
Claims (4)
1. a Lignanoids compounds, it is characterized in that: described Lignanoids compounds be the shizandra berry branch, leaf and/or the fruit that are dried be raw material, obtain through medicinal extract extraction, organic solvent extraction, silica gel column chromatography, high pressure liquid chromatography separation, its structural formula is:
; R
1for-OH, R
2for-OCH
3, molecular formula is C
20h
24o
8, this compound called after marphenol E; Or R
1, R
2common composition group-OCH
2o-, molecular formula is C
20h
22o
8, this compound called after marphenol F.
2. prepare the method for Lignanoids compounds claimed in claim 1 for one kind, shizandra berry branch, leaf and/or the fruit that it is characterized in that being dried is 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: by shizandra berry branch, leaf and/or fruit coarse reduction to 20 ~ 40 order, 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, filtering throw out, is condensed into medicinal extract a; Organic solvent described in this step is the one in 70 ~ 100% acetone, ethanol or methyl alcohol;
B, organic solvent extraction: in medicinal extract a, add the water of 1 ~ 2 times of amount of weight ratio, use and the isopyknic organic solvent extraction of water 3 ~ 5 times, merge organic solvent extraction phase, concentrating under reduced pressure becomes medicinal extract b; Organic solvent described in this step is the one in ethyl acetate, chloroform, ether, sherwood oil or benzene;
C, silica gel column chromatography: the acetone solution by medicinal extract b by 1.5 ~ 3 times of amounts of weight ratio, then weigh 80 ~ 100 order silica gel mixed samples of 0.8 ~ 1.2 times with medicinal extract, then go up silica gel column chromatography, dress post silica gel is 160 ~ 200 orders, consumption is 6 ~ 8 times of amounts of medicinal extract b weight; Be the organic solvent solution gradient elution of 1:0,20:1,9:1,8:2,3:2,1:1,1:2,0:1 by volume ratio, collect gradient eluent, concentrated, through TLC monitoring, merge identical part; Organic solvent solution described in this step is the one in normal hexane-acetone, chloroform-acetone, chloroform-methanol, sherwood oil-acetone or petroleum ether-ethyl acetate;
D, reversed phase column chromatography: by reversed phase column chromatography in the 9:1 part of C step elutriant, reversed-phase column is with reversed material C-18 dress post; The methanol aqueous solution that is 20 ~ 100% with volume content carries out gradient elution, collects each several part elutriant concentrated, through TLC monitoring, merges identical part;
E, high performance liquid chromatography separate: by 30% ~ 40% methanol aqueous solution wash-out part of D step elutriant through high performance liquid chromatography separation and purification, methyl alcohol taking 25 ~ 45% is moving phase, flow velocity 10 ~ 14ml/min, the anti-phase preparative column of Zorbax PrepHT GF of 21.2 × 250mm, 5 μ m is stationary phase, it is 254nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, collect the chromatographic peak of 10 ~ 40min, repeatedly cumulative rear evaporate to dryness, obtains described Lignanoids compounds marphenol E and marphenol F;
High performance liquid chromatography separation and purification described in F, E step is taking 25 ~ 45% methyl alcohol as moving phase, flow velocity 10 ~ 14ml/min, the anti-phase preparative column of Zorbax PrepHT GF of 21.2 × 250mm, 5 μ m is stationary phase, it is 254nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, collect the chromatographic peak of 10 ~ 25min, repeatedly cumulative rear evaporate to dryness, obtains described Lignanoids compounds marphenol E;
High performance liquid chromatography separation and purification described in G, E step is taking 25 ~ 45% methyl alcohol as moving phase, flow velocity 10 ~ 14ml/min, the anti-phase preparative column of Zorbax PrepHT GF of 21.2 × 250mm, 5 μ m is stationary phase, it is 254nm that UV-detector detects wavelength, each sample introduction 50 ~ 60mL, collect the chromatographic peak of 25 ~ 40min, repeatedly cumulative rear evaporate to dryness, obtains described Lignanoids compounds marphenol F.
3. the preparation method of Lignanoids compounds according to claim 2, is characterized in that: described shizandra berry is Heqing shizandra berry.
4. a Lignanoids compounds claimed in claim 1 is in the application of preparing in anti-AIDS drug.
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| 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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| 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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