CN103694213B - A kind of extraction and isolation preparation method of Lignans in Schisandra chinensis monomer - Google Patents

A kind of extraction and isolation preparation method of Lignans in Schisandra chinensis monomer Download PDF

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CN103694213B
CN103694213B CN201310429192.3A CN201310429192A CN103694213B CN 103694213 B CN103694213 B CN 103694213B CN 201310429192 A CN201310429192 A CN 201310429192A CN 103694213 B CN103694213 B CN 103694213B
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CN103694213A (en
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楚楚
章诗迪
颜继忠
童胜强
李行诺
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Zhejiang University of Technology ZJUT
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/70—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with ring systems containing two or more relevant rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
    • C07C41/01—Preparation of ethers
    • C07C41/34—Separation; Purification; Stabilisation; Use of additives
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Abstract

The invention discloses a kind of extraction and isolation preparation method of Lignans in Schisandra chinensis monomer: schisandra chinensis medicinal material powder is through CO 2supercritical extraction, obtain crude extract and carry out high speed adverse current chromatogram separation, gradient elution, with normal hexane, ethyl acetate, methyl alcohol, water composition high-speed counter-current solvent system A, leave standstill after mixing fully, getting phase A is stationary phase A, and lower phase A is mobile phase A, and wash-out obtains schisandrin, Wuweizichun B, change solvent system, methyl alcohol, water volume ratio are become large, and taking off phase B is Mobile phase B, and wash-out obtains Wuweizi ester B; Change solvent system, methyl alcohol, water volume ratio are increased further, taking off phase C is moving phase C, and wash-out obtains deoxyschizandrin and Wuweizisu B.The method that the present invention adopts supercritical extraction to be separated in conjunction with high speed adverse current chromatogram, can be directly used in the separation and purification of follow-up monomer without the need to the sample preparation of complexity after extraction, only need flash liberation can obtain 5 lignanoid's monomers, simply efficiently.

Description

A kind of extraction and isolation preparation method of Lignans in Schisandra chinensis monomer
Technical field
The invention belongs to field of natural medicinal chemistry, relate to a kind of combined preparation process rapidly and efficiently extracting and developing purifying lignanoid monomer component from shizandra berry.Be specifically related to the monomer component prepared in conjunction with high-speed countercurrent chromatography extraction and isolation by super critical extraction in shizandra berry.
Background technology
Shizandra berry is the mature fruit of magnoliaceae schisandra Schisandra chinensis (Turcz.) Baill or schisandra chinensis Schisandra sphenanthera Rehd.et Wils..The former practises title " Schisandra chinensis ", and the latter practises title " kadsura longepedunculata ".Begin to be loaded in Shennong's Herbal, be classified as top grade.Shizandra berry is warm in nature, and taste is sweet, sour, returns lung, the heart, kidney channel.There is convergence astringent or styptic treatment for spontaneous sweating, supplementing QI for promoting the production of body fluid, effect of kidney calming.Can be used for that treatment chronic cough void is breathed heavily, spontaneous sweating, chronic diarrhea, Tianjin wound are thirsty, palpitaition, insomnia and dreamful sleep etc.Modern pharmacology research show shizandra berry have antitumor, anti-inflammatory, anti-oxidant, protect the liver, the multiple pharmacological effect such as neuroprotective.
Containing number of chemical compositions such as lignanoid, volatile oil, organic acid, polysaccharide, triterpene and sesquiterpenes in shizandra berry, wherein lignanoid is principle active component.What in lignan component, content was higher comprises schisandrin, Wuweizichun B, deoxyschizandrin, Wuweizisu B etc.Along with going deep into of research; several main lignan component is all proved and has good pharmacologically active above; as schisandrin have anti-oxidant, anti-ageing, the pharmacological action such as to protect the liver; deoxyschizandrin has pharmacologically actives such as protecting the liver; Wuweizisu B has and protects the effects such as liver, anticancer, antiviral, antiulcer agent, anti-oxidant, neuroprotective; therefore, develop a kind of fast, the preparation method of efficient, low cost is concerning most important the further research and development of this constituents.
By studying for a long period of time, comparatively ripe to the extraction process of total lignans constituents in shizandra berry, but the preparation of lignanoid's monomer constituents is concentrated in the separation and purification of schisandrin, Wuweizichun B mostly, and less to the separation and purification report of deoxyschizandrin, Wuweizisu B, schisantherin the second grade monomer.Have in document and with methods such as solvent-extraction process, ultrasonic extraction, microwave loss mechanisms, supercritical fluid extraction, enzymolysis process auxiliary ultrasonic extraction method, ultrahigh-pressure extraction methods, Lignans in Schisandra chinensis constituents is extracted, with silica gel column chromatography, macroporous resin column chromatography, high-speed countercurrent chromatography, lignanoid's monomer is separated.The flow process of most extraction and isolation extracts with circumfluence method, adopts certain method to carry out enrichment removal of impurities to lignanoid, and then carry out separation and purification with column chromatography to it again.The patent of invention being 201210144871.1 as application number discloses " a kind of novel method of purifying schisandrin ".Adopt microwave counter current extraction, macroporous resin separation and purification and column chromatography to be further purified, finally obtain schisandrin monomer (content more than 98%).If application number is disclose in the patent of invention of 200910010631.0 " a kind of novel process preparing schisandrin and Wuweizichun B ", extraction process is by the extracting solution organic solvent extraction containing schisandrin and Wuweizichun B, extract is after column chromatography for separation repeatedly, collect the stream part containing schisandrin and Wuweizichun B, obtain purity and be all greater than 98% schisandrin and Wuweizichun B.For another example application number is disclose in the patent of invention of 201010510828.3 " a kind of technique extracting deoxyschizandrin from shizandra berry ", the method using ultrasonic wave added Petroleum ether extraction and alkali alcohol to strip prepares lignanoid's total extract, pass twice through silica gel aluminum oxide mixing column chromatography again, wash-out, recycling design, sherwood oil-acetone or hexanaphthene-ethyl alcohol recrystallization, dry, final acquisition deoxyschizandrin monomer.
Be not difficult to find out, the various drawbacks such as the process for extracting, separating and purifying of Lignans in Schisandra chinensis monomer exists complex steps, and preparation cycle is long, and complicated operation takes time and effort, and yield is low.The patent No. is disclose one " method for separating and preparing of shizandra berry monomer component " in the patent of invention of ZL200910242188.X, this patent is than front method, purification step adopts high speed adverse current chromatogram, makes purification cycle obtain shortening, can prepare multiple lignanoids monomer simultaneously.But sample pre-treatments is loaded down with trivial details, need, by multisteps such as extraction, extraction, silica gel column chromatographies, make whole preparation process still more complicated.
Supercritical fluid extraction is as a kind of environmental protection, fast and extraction means capable of being industrialized, be just widely used at present in the extraction of various natural product.Lignan component polarity in shizandra berry is less, to extract have the incomparable advantage of additive method by the method to it.If the patent No. is all have employed the total lignans constituents in supercritical fluid extraction extraction shizandra berry in the patent of invention of ZL200610041566.4, ZL201010119871.7.But there is not the report by supercritical extraction and high speed adverse current chromatogram separation and purification Lignans in Schisandra chinensis monomer so far.
Summary of the invention
The present invention is directed to the deficiency in existing technique, there is provided a kind of method of extracting and developing purifying deoxyschizandrin, Wuweizisu B, Wuweizi ester B, schisandrin, Wuweizichun B from schisandra chinensis medicinal material, the method simple process, efficiency is high, fast, product purity is greater than 85%.
The invention discloses a kind of with supercritical fluid extraction schisandra chinensis medicinal material powder, gained supercritical extract carries out separation and purification with high speed adverse current chromatogram, finally can obtain simultaneously purity higher than 85% deoxyschizandrin, Wuweizisu B, Wuweizi ester B, schisandrin, Wuweizichun B monomer lignans.Whole extraction and isolation process can complete the soonest in tens hours.The extraction separation and purification method that this method is more traditional is more quick, easy, efficient.
The technical solution used in the present invention is:
An extraction and isolation preparation method for Lignans in Schisandra chinensis monomer, described method is: 10 ~ 65 object schisandra chinensis medicinal material powder are through CO 2supercritical extraction, obtained shizandra berry crude extract, shizandra berry crude extract carries out high speed adverse current chromatogram separation, gradient elution, be first x1:x2:y1:(x1+x2-y1 with volume ratio) normal hexane, ethyl acetate, methyl alcohol, water composition high-speed counter-current solvent system A, 1≤x1<10, 1≤x2<10, 1≤y1<10, and x1+x2-y1>0, leave standstill after mixing fully, by upper and lower two-phase separately, getting phase A is stationary phase A, lower phase A is mobile phase A, get shizandra berry crude extract, with phase A, the mixed solvent of lower phase A volume ratio 1:1 dissolves, as need testing solution sample introduction, setting high-speed counter current chromatograph, rotating speed 500 ~ 1000r/min, flow velocity 0.1 ~ the 10ml/min of moving phase, UV-detector wavelength 180 ~ 400nm, collect the effluent liquid of each chromatographic peak on ultraviolet detection spectrogram respectively, concentrated, dry, obtained schisandrin and Wuweizichun B respectively, after collecting the effluent liquid containing Wuweizichun B, mobile phase A is transformed to Mobile phase B, collects the effluent liquid of chromatographic peak on ultraviolet detection spectrogram, concentrated, dry, obtained Wuweizi ester B, described Mobile phase B obtains as follows: take volume ratio as x1:x2:y2:(x1+x2-y2) normal hexane, ethyl acetate, methyl alcohol, water composition high-speed counter-current solvent system B, y1<y2<10, and x1+x2-y2>0, leave standstill after mixing fully, by upper and lower two-phase separately, taking off phase B is Mobile phase B, after collecting the effluent liquid containing Wuweizi ester B, Mobile phase B is transformed to moving phase C, collects the effluent liquid of chromatographic peak on ultraviolet detection spectrogram, concentrated, dry, obtained deoxyschizandrin and Wuweizisu B respectively respectively, described moving phase C obtains by the following method: take volume ratio as x1:x2:y3:(x1+x2-y3) normal hexane, ethyl acetate, methyl alcohol, water composition high-speed counter-current solvent system C, y2<y3<10, and x1+x2-y3>0, leave standstill after mixing fully, by upper and lower two-phase separately, taking off phase C is moving phase C.
Further, preferred the method for the invention is carried out according to the following steps:
(1) take after schisandra fruit is pulverized and cross 10 ~ 65 mesh sieves, obtain schisandra chinensis medicinal material powder, carry out CO 2supercritical extraction, Extracting temperature 35 ~ 60 DEG C, extracts pressure 14 ~ 30MPa, extraction time 0.5 ~ 6h, obtained shizandra berry crude extract;
(2) step (1) gained shizandra berry crude extract carries out high speed adverse current chromatogram separation, gradient elution, be first x1:x2:y1:(x1+x2-y1 with volume ratio) normal hexane, ethyl acetate, methyl alcohol, water composition high-speed counter-current solvent system A, 1≤x1<10, 1≤x2<10, 1≤y1<10, and x1+x2-y1>0, leave standstill after mixing fully, by upper and lower two-phase separately, getting phase A is stationary phase A, lower phase A is mobile phase A, get shizandra berry crude extract, with phase A, the mixed solvent of lower phase A volume ratio 1:1 dissolves, as need testing solution, stationary phase A is full of the multilayer coil separator column of high-speed counter-current chromatograph, setting high-speed counter current chromatograph, under 500 ~ 1000r/min rotating speed, mobile phase A is injected with the flow velocity of 0.1 ~ 10ml/min, detect with the UV-detector of wavelength 180 ~ 400nm, when obviously there being moving phase to flow out, get need testing solution sample introduction, according to UV-detector spectrogram, the corresponding effluent liquid of each chromatographic peak be separated is collected, detect through HPLC, will containing schisandrin, the effluent liquid of Wuweizichun B composition merges respectively, concentrated, dry, obtained schisandrin and Wuweizichun B respectively,
After collecting the effluent liquid containing Wuweizichun B, mobile phase A is transformed to Mobile phase B, according to UV-detector spectrogram, the effluent liquid of the chromatographic peak be separated is collected, detects through HPLC, the effluent liquid containing Wuweizi ester B composition is merged, concentrated, dry, obtained Wuweizi ester B; Described Mobile phase B obtains as follows: take volume ratio as x1:x2:y2:(x1+x2-y2) normal hexane, ethyl acetate, methyl alcohol, water composition high-speed counter-current solvent system B, y1<y2<10, and x1+x2-y2>0, leave standstill after mixing fully, by upper and lower two-phase separately, taking off phase B is Mobile phase B;
After collecting the effluent liquid containing Wuweizi ester B, Mobile phase B is converted moving phase C, according to UV-detector spectrogram, the corresponding effluent liquid of each chromatographic peak be separated is collected, detect through HPLC, effluent liquid containing deoxyschizandrin, Wuweizisu B is merged respectively, concentrated, dry, obtained deoxyschizandrin and Wuweizisu B respectively; Described moving phase C obtains by the following method: take volume ratio as x1:x2:y3:(x1+x2-y3) normal hexane, ethyl acetate, methyl alcohol, water composition high-speed counter-current solvent system C, y2<y3<10, and x1+x2-y3>0, leave standstill after mixing fully, by upper and lower two-phase separately, taking off phase C is moving phase C.
In described step (1), Extracting temperature preferably 35 ~ 55 DEG C.Extraction time is 1 ~ 4h preferably.
In described step (2), the volume ratio of the quality of the shizandra berry crude extract contained in described need testing solution and the multilayer coil separator column of high-speed counter-current chromatograph is generally 0.1 ~ 0.5:300g/ml.
In described step (2), the flow velocity of described mobile phase A is preferably 1 ~ 3mL/min, more preferably 1.8mL/min.
The flow velocity of described Mobile phase B is preferably 1 ~ 3mL/min, more preferably 1.8mL/min.
The flow velocity of described moving phase C is preferably 1 ~ 3mL/min, more preferably 1.8mL/min.
In described step (2), the preferred 800r/min of described rotating speed.
In described step (2), the preferred 210-280nm of determined wavelength of described UV-detector, more preferably 254nm.
In described step (2), high speed adverse current chromatogram is separated and usually carries out at normal temperatures, preferred constant temperature 25 DEG C.
Described high-speed counter-current solvent system A, B, in C, normal hexane, ethyl acetate, methyl alcohol, the volume ratio of water is respectively x1:x2:y1:(x1+x2-y1), x1:x2:y2:(x1+x2-y2), x1:x2:y3:(x1+x2-y3), wherein normal hexane, the volume ratio of ethyl acetate is x1:x2, x1, the numerical value of x2 is identical, represent high-speed counter-current solvent system A, B, in C, normal hexane, the volume ratio of ethyl acetate is identical all the time to remain unchanged, and methyl alcohol, in the volume ratio of water, y1<y2<y3<10, and x1+x2-y1>0, x1+x2-y2>0, x1+x2-y3>0, represent methyl alcohol, the volume ratio of water is at high-speed counter-current solvent system A, B, increase gradually in C.
Comparatively preferred, in described high-speed counter-current solvent system A, the volume ratio of normal hexane, ethyl acetate, methyl alcohol, water is x1:x2:y1:(x1+x2-y1), wherein 5≤x1≤9,1≤x2≤5,4≤y1≤9.
Preferred, in described step (2) high speed counter current solvent system A, the volume ratio of normal hexane, ethyl acetate, methyl alcohol, water is 6:4:5:5,7:3:5:5 or 8:2:5:5.In high-speed counter-current solvent system B, keep the volume ratio of the normal hexane in high-speed counter-current solvent system A, ethyl acetate constant, the volume ratio of methyl alcohol, water is greater than the volume ratio of methyl alcohol, water in high-speed counter-current solvent system A; In high-speed counter-current solvent system C, keep the volume ratio of the normal hexane in high-speed counter-current solvent system B, ethyl acetate constant, the volume ratio of methyl alcohol, water is greater than the volume ratio of methyl alcohol, water in high-speed counter-current solvent system B.
After the effluent liquid collected containing Wuweizichun B of the present invention, mobile phase A is transformed to Mobile phase B, refers to according to UV-detector spectrogram, after the chromatographic peak being collected into Wuweizichun B terminates, mobile phase A is transformed to Mobile phase B.But consider the column volume of multilayer coil separator column itself, after changing the mobile phase A that separator column ingress is injected into Mobile phase B, the moving phase in separator column becomes Mobile phase B completely needs the longer time.In order to save disengaging time, can before the chromatographic peak of Wuweizichun B terminate, reasonable time changes the mobile phase A that separator column ingress is injected into Mobile phase B in advance, is convenient to become Mobile phase B completely by separator column as early as possible.Those skilled in the art can be transformed to the time of Mobile phase B according to substantial sepn situation from Row sum-equal matrix mobile phase A.
Same, described in collect containing Wuweizi ester B effluent liquid after, Mobile phase B is converted moving phase C, refers to according to UV-detector spectrogram, after the chromatographic peak being collected into Wuweizi ester B terminates, Mobile phase B is transformed to moving phase C.But consider the column volume of multilayer coil separator column itself, after changing the Mobile phase B that separator column ingress is injected into moving phase C, the moving phase in separator column becomes moving phase C completely needs the longer time.In order to save disengaging time, can before the chromatographic peak of Wuweizi ester B terminate, the reasonable time Mobile phase B that just separator column ingress is injected changes moving phase C in advance, is convenient to become moving phase C completely by separator column as early as possible.Those skilled in the art can be transformed to the time of moving phase C according to substantial sepn situation from Row sum-equal matrix Mobile phase B.
Highly preferred, gradient elution program of the present invention is one of following:
(1) high-speed counter-current solvent system A: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 6:4:5:5, leave standstill after mixing fully, by upper and lower two-phase separately, getting phase A is stationary phase A, lower phase A is mobile phase A, and wash-out obtains schisandrin and Wuweizichun B, treats that the complete wash-out of Wuweizichun B is complete, mobile phase A is become Mobile phase B, and wash-out obtains Wuweizi ester B; Described Mobile phase B obtains as follows: high-speed counter-current solvent system B: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 6:4:6:4, leaves standstill after mixing fully, and by upper and lower two-phase separately, taking off phase B is Mobile phase B; Treat that the complete wash-out of Wuweizi ester B is complete, Mobile phase B is become moving phase C, and wash-out obtains deoxyschizandrin and Wuweizisu B; Described moving phase C obtains as follows: high-speed counter-current solvent system C: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 6:4:8:2, leaves standstill after mixing fully, and by upper and lower two-phase separately, taking off phase C is moving phase C;
(2) high-speed counter-current solvent system A: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 6:4:5:5, leave standstill after mixing fully, by upper and lower two-phase separately, getting phase A is stationary phase A, lower phase A is mobile phase A, and wash-out obtains schisandrin and Wuweizichun B, treats that the complete wash-out of Wuweizichun B is complete, mobile phase A is become Mobile phase B, and wash-out obtains Wuweizi ester B; Described Mobile phase B obtains as follows: high-speed counter-current solvent system B: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 6:4:6:4, leaves standstill after mixing fully, and by upper and lower two-phase separately, taking off phase B is Mobile phase B; Treat that the complete wash-out of Wuweizi ester B is complete, Mobile phase B is become moving phase C, and wash-out obtains deoxyschizandrin and Wuweizisu B; Described moving phase C obtains as follows: high-speed counter-current solvent system C: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 6:4:9:1, leaves standstill after mixing fully, and by upper and lower two-phase separately, taking off phase C is moving phase C;
(3) high-speed counter-current solvent system A: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 6:4:5:5, leave standstill after mixing fully, by upper and lower two-phase separately, getting phase A is stationary phase A, lower phase A is mobile phase A, and wash-out obtains schisandrin and Wuweizichun B, treats that the complete wash-out of Wuweizichun B is complete, mobile phase A is become Mobile phase B, and wash-out obtains Wuweizi ester B; Described Mobile phase B obtains as follows: high-speed counter-current solvent system B: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 6:4:6:4, leaves standstill after mixing fully, and by upper and lower two-phase separately, taking off phase B is Mobile phase B; Treat that the complete wash-out of Wuweizi ester B is complete, Mobile phase B is become moving phase C, and wash-out obtains deoxyschizandrin and Wuweizisu B; Described moving phase C obtains as follows: high-speed counter-current solvent system C: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 6:4:7:3, leaves standstill after mixing fully, and by upper and lower two-phase separately, taking off phase C is moving phase C;
(4) high-speed counter-current solvent system A: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 7:3:5:5, leave standstill after mixing fully, by upper and lower two-phase separately, getting phase A is stationary phase A, lower phase A is mobile phase A, and wash-out obtains schisandrin and Wuweizichun B, treats that the complete wash-out of Wuweizichun B is complete, mobile phase A is become Mobile phase B, and wash-out obtains Wuweizi ester B; Described Mobile phase B obtains as follows: high-speed counter-current solvent system B: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 7:3:6:4, leaves standstill after mixing fully, and by upper and lower two-phase separately, taking off phase B is Mobile phase B; Treat that the complete wash-out of Wuweizi ester B is complete, Mobile phase B is become moving phase C, and wash-out obtains deoxyschizandrin and Wuweizisu B; Described moving phase C obtains as follows: high-speed counter-current solvent system C: normal hexane, ethyl acetate, methyl alcohol, water volume ratio are 7:3:7:3, leaves standstill after mixing fully, and by upper and lower two-phase separately, taking off phase C is moving phase C.
Beneficial effect of the present invention is:
1, supercritical fluid technology is adopted to carry out extracting and developing and purifying in conjunction with high-speed countercurrent chromatography to kind of the main lignan component of five in shizandra berry (comprising schisandrin, Wuweizichun B, Wuweizi ester B, deoxyschizandrin, Wuweizisu B) first.Wherein, high speed adverse current chromatogram separation and purification have employed the pattern of gradient elution.
2, the supercritical liquid extraction technique not only environmental protection adopted, fast, meanwhile, to Lignans in Schisandra chinensis selective extraction effect, the separation and purification that lignanoid's monomer is follow-up is conducive to.Only need once to extract the separation and purification that can be directly used in follow-up monomer, extraction step is simply efficient, simplifies the extraction step of pre-treatment than prior art.
3, adopt high-speed countercurrent chromatography to carry out separation and purification, only need flash liberation can obtain 5 lignanoid's monomers, simple to operate, product purity is higher.
4, quick, efficient, easy, the low cost of the method, environmental protection, compared with prior art, extraction step is simple, time is short, separation and purification efficiency is high, achieves technological innovation and lifting, serves important reference function to the development research of Lignans in Schisandra chinensis constituents.
Accompanying drawing explanation
Fig. 1 is the process flow diagram of the inventive method.
Fig. 2 is the high speed adverse current chromatogram figure of embodiment 6, and in figure, peak 1 is schisandrin, and peak 2 is Wuweizichun B, and peak 3 is Wuweizi ester B, and peak 4 is deoxyschizandrin, and peak 5 is Wuweizisu B.
Fig. 3 is the HPLC color atlas of the schisandrin that embodiment 6 obtains.
Fig. 4 is the HPLC color atlas of the Wuweizichun B that embodiment 6 obtains.
Fig. 5 is the HPLC color atlas of the Wuweizi ester B that embodiment 6 obtains.
Fig. 6 is the HPLC color atlas of the deoxyschizandrin that embodiment 6 obtains.
Fig. 7 is the HPLC color atlas of the Wuweizisu B that embodiment 6 obtains.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this.
Embodiment 1: supercritical extraction
Get Schisandra chinensis fruit, pulverize, obtain schisandra chinensis medicinal material powder, particle diameter is 50 ~ 65 orders, gets schisandra chinensis medicinal material powder 2g, puts into supercritical fluid extraction tank, Extracting temperature 40 DEG C, extracts pressure 25MPa, CO 2flow is 2L/min, and extraction time is 1h, obtains shizandra berry crude extract.Be representative with schisandrin, calculating its extraction yield is 0.5294%.
Embodiment 2: supercritical extraction
Get Schisandra chinensis fruit, pulverize, obtain schisandra chinensis medicinal material powder, particle diameter is 10 ~ 20 orders, gets schisandra chinensis medicinal material powder 3g, puts into supercritical fluid extraction tank, Extracting temperature 35 DEG C, extracts pressure 14MPa, CO 2flow is 2L/min, and extraction time is 2h, obtains shizandra berry crude extract.Be representative with schisandrin, calculating its extraction yield is 0.3926%.
Embodiment 3: supercritical extraction
Get Schisandra chinensis fruit, pulverize, obtain schisandra chinensis medicinal material powder, particle diameter is 50 ~ 65 orders, gets schisandra chinensis medicinal material powder 3g, puts into supercritical fluid extraction tank, Extracting temperature 35 DEG C, extracts pressure 30MPa, CO 2flow is 2L/min, and extraction time is 2h, obtains shizandra berry crude extract.Be representative with schisandrin, calculating its extraction yield is 0.6001%.
Embodiment 4: supercritical extraction
Get Schisandra chinensis fruit, pulverize, obtain schisandra chinensis medicinal material powder, particle diameter is 50 ~ 65 orders, gets schisandra chinensis medicinal material powder 3g, puts into supercritical fluid extraction tank, Extracting temperature 55 DEG C, extracts pressure 25MPa, CO 2flow is 2L/min, and extraction time is 2h, obtains shizandra berry crude extract.Be representative with schisandrin, calculating its extraction yield is 0.5650%.
Embodiment 5: supercritical extraction technique
Get Schisandra chinensis fruit, pulverize, obtain schisandra chinensis medicinal material powder, particle diameter is 20 ~ 40 orders, gets schisandra chinensis medicinal material powder 3kg, puts into supercritical fluid extraction tank, Extracting temperature 36 DEG C, extracts pressure 15MPa, CO 2flow is 40 ± 5kg/h, and extraction time is 4h, obtains shizandra berry crude extract.Be representative with schisandrin, calculating its extraction yield is 0.2806%.
Embodiment 6: high speed adverse current chromatogram separation and purification
Get shizandra berry crude extract (preparing extract obtained with extraction process in embodiment 5) 300mg, solvent system n-hexane-ethyl acetate-methanol-water volume ratio is 6:4:5:5, leave standstill after mixing fully, by upper and lower two-phase separately, shizandra berry crude extract 300mg mutually each 7mL dissolving up and down, as need testing solution.Upper as stationary phase, lower to moving phase using solvent system.Stationary phase is full of the multilayer coil separator column of high-speed counter-current chromatograph, the column volume of separator column is 300ml, setting high-speed counter current chromatograph, flow rate of mobile phase 1.8mL/min, centrifugation instrument rotating speed 800rpm, ultraviolet detection wavelength 254nm, 25 DEG C, constant temperature circulating water tank.When obviously there being moving phase to flow out, get need testing solution sample introduction.Gradient elution program is: 1. solvent system n-hexane-ethyl acetate-methanol-water volume ratio is 6:4:5:5, elution time 0-260min, and elution time is the actual elution time recorded according to the Wuweizichun B wash-out complete time; 2. solvent system n-hexane-ethyl acetate-methanol-water volume ratio is 6:4:6:4, leave standstill after mixing fully, by upper and lower two-phase separately, taking off is moving phase mutually, elution time 260-400min, elution time is the actual elution time recorded according to the Wuweizi ester B wash-out complete time; 3. solvent system n-hexane-ethyl acetate-methanol-water volume ratio is 6:4:8:2, leaves standstill after mixing fully, and by upper and lower two-phase separately, taking off is moving phase mutually, elution time 400-670min.According to UV-detector spectrogram, as shown in Figure 2, peak 1 is schisandrin, and peak 2 is Wuweizichun B, and peak 3 is Wuweizi ester B, and peak 4 is deoxyschizandrin, and peak 5 is Wuweizisu B.The corresponding effluent liquid of each chromatographic peak be separated is collected, detects with HPLC, merge the effluent liquid of identical component, concentrating under reduced pressure, lyophilize, obtains schisandrin 12.5mg respectively, Wuweizichun B 7.1mg, Wuweizi ester B 1.8mg, deoxyschizandrin 4.4mg, Wuweizisu B 6.8mg, detect three kinds of separated products with high performance liquid chromatography, purity is respectively 98.0%, 98.1%, 93.3%, 92.9%, 89.1%, as shown in figure 3 to figure 7.The testing conditions of high performance liquid chromatography is: acetonitrile-water system, gradient elution (0 ~ 10min, 45% ~ 58% acetonitrile; 10 ~ 15min, 58% ~ 60% acetonitrile; 15 ~ 25min, 60% ~ 70% acetonitrile; 25 ~ 35min, 70% ~ 75% acetonitrile; 35 ~ 45min, 75% ~ 95% acetonitrile; 45 ~ 50min, 95% ~ 100% acetonitrile), sample size is 10 μ L, and flow rate of mobile phase is 1mL/min, and column temperature is 30 DEG C, and determined wavelength is 225nm, and elution time is 68min.Chromatographic column is: phenomenex Gemini C18,250 × 4.6mm, 5 μm.
Embodiment 7: high speed adverse current chromatogram separation and purification
Get shizandra berry crude extract (preparing extract obtained with extraction process in embodiment 5) 300mg, with the mutually each 7mL dissolving up and down of solvent system n-hexane-ethyl acetate-methanol-water (6:4:5:5, v/v).Gradient elution program is: 1. solvent system n-hexane-ethyl acetate-methanol-water (6:4:5:5, v/v), elution time 0-280min; 2. solvent system n-hexane-ethyl acetate-methanol-water (6:4:6:4, v/v), elution time 280-410min; 3. solvent system n-hexane-ethyl acetate-methanol-water (6:4:9:1, v/v), elution time 410-700min; Other operational conditions are with embodiment 6.Obtain schisandrin, Wuweizichun B, Wuweizi ester B, deoxyschizandrin, Wuweizisu B component, concentrating under reduced pressure, lyophilize, obtain schisandrin 15.6mg, Wuweizichun B 6.6mg, Wuweizi ester B 1.4mg, deoxyschizandrin 2.3mg, Wuweizisu B 3.2mg, purity is respectively 98.5%, 98.5%, 92.7%, 85.3%, 85.1%.
Embodiment 8: high speed adverse current chromatogram separation and purification
Get shizandra berry crude extract (preparing extract obtained with extraction process in embodiment 5) 300mg, with the mutually each 7mL dissolving up and down of solvent system n-hexane-ethyl acetate-methanol-water (6:4:5:5, v/v).Gradient elution program is: 1. solvent system n-hexane-ethyl acetate-methanol-water (6:4:5:5, v/v), elution time 0-300min; 2. solvent system n-hexane-ethyl acetate-methanol-water (6:4:6:4, v/v), elution time 300-460min; 3. solvent system n-hexane-ethyl acetate-methanol-water (6:4:7:3, v/v), elution time 460-850min; Other operational conditions are with embodiment 6.Obtain schisandrin, Wuweizichun B, Wuweizi ester B, deoxyschizandrin, Wuweizisu B component, concentrating under reduced pressure, lyophilize, obtain schisandrin 14.3mg, Wuweizichun B 7.3mg, Wuweizi ester B 1.9mg, deoxyschizandrin 5.4mg, Wuweizisu B 6.5mg, purity is respectively 98.3%, 99.1%, 93.7%, 91.9%, 90.7%.
Embodiment 9: high speed adverse current chromatogram separation and purification
Get shizandra berry crude extract (preparing extract obtained with extraction process in embodiment 5) 300mg, with the mutually each 7mL dissolving up and down of solvent system n-hexane-ethyl acetate-methanol-water (7:3:5:5, v/v).Gradient elution program is: 1. solvent system n-hexane-ethyl acetate-methanol-water (7:3:5:5, v/v), elution time 0-350min; 2. solvent system n-hexane-ethyl acetate-methanol-water (7:3:6:4, v/v), elution time 350-560min; 3. solvent system n-hexane-ethyl acetate-methanol-water (7:3:7:3, v/v), elution time 560-980min; Other operational conditions are with embodiment 6.Obtain schisandrin, Wuweizichun B, Wuweizi ester B, deoxyschizandrin, Wuweizisu B component, concentrating under reduced pressure, lyophilize, obtain schisandrin 13.2mg, Wuweizichun B 8.5mg, Wuweizi ester B 1.7mg, deoxyschizandrin 4.9mg, Wuweizisu B 5.7mg, purity is respectively 97.6%, 96.1%, 91.9%, 90.4%, 89.2%.

Claims (4)

1. an extraction and isolation preparation method for Lignans in Schisandra chinensis monomer, is characterized in that said method comprising the steps of:
(1) take after schisandra fruit is pulverized and cross 10 ~ 65 mesh sieves, obtain schisandra chinensis medicinal material powder, carry out CO 2supercritical extraction, Extracting temperature 35 ~ 60 DEG C, extracts pressure 14 ~ 30MPa, extraction time 0.5 ~ 6h, obtained shizandra berry crude extract;
(2) step (1) gained shizandra berry crude extract carries out high speed adverse current chromatogram separation, gradient elution, be first x1:x2:y1:(x1+x2-y1 with volume ratio) normal hexane, ethyl acetate, methyl alcohol, water composition high-speed counter-current solvent system A, 5≤x1≤9, 1≤x2≤5, 4≤y1≤9, and x1+x2-y1>0, leave standstill after mixing fully, by upper and lower two-phase separately, getting phase A is stationary phase A, lower phase A is mobile phase A, get shizandra berry crude extract, with phase A, the mixed solvent of lower phase A volume ratio 1:1 dissolves, as need testing solution, stationary phase A is full of the multilayer coil separator column of high-speed counter-current chromatograph, setting high-speed counter current chromatograph, under 500 ~ 1000r/min rotating speed, mobile phase A is injected with the flow velocity of 0.1 ~ 10ml/min, detect with the UV-detector of wavelength 180 ~ 400nm, when obviously there being moving phase to flow out, get need testing solution sample introduction, according to UV-detector spectrogram, the corresponding effluent liquid of each chromatographic peak be separated is collected, detect through HPLC, will containing schisandrin, the effluent liquid of Wuweizichun B composition merges respectively, concentrated, dry, obtained schisandrin and Wuweizichun B respectively,
After collecting the effluent liquid containing Wuweizichun B, mobile phase A is transformed to Mobile phase B, according to UV-detector spectrogram, the effluent liquid of the chromatographic peak be separated is collected, detects through HPLC, the effluent liquid containing Wuweizi ester B composition is merged, concentrated, dry, obtained Wuweizi ester B; Described Mobile phase B obtains as follows: take volume ratio as x1:x2:y2:(x1+x2-y2) normal hexane, ethyl acetate, methyl alcohol, water composition high-speed counter-current solvent system B, y1<y2<10, and x1+x2-y2>0, leave standstill after mixing fully, by upper and lower two-phase separately, taking off phase B is Mobile phase B;
After collecting the effluent liquid containing Wuweizi ester B, Mobile phase B is converted moving phase C, according to UV-detector spectrogram, the corresponding effluent liquid of each chromatographic peak be separated is collected, detect through HPLC, effluent liquid containing deoxyschizandrin, Wuweizisu B is merged respectively, concentrated, dry, obtained deoxyschizandrin and Wuweizisu B respectively; Described moving phase C obtains by the following method: take volume ratio as x1:x2:y3:(x1+x2-y3) normal hexane, ethyl acetate, methyl alcohol, water composition high-speed counter-current solvent system C, y2<y3<10, and x1+x2-y3>0, leave standstill after mixing fully, by upper and lower two-phase separately, taking off phase C is moving phase C.
2. the method for claim 1, is characterized in that in described step (1), Extracting temperature 35 ~ 55 DEG C, extraction time 1 ~ 4h.
3. the method for claim 1, it is characterized in that in described step (2), the volume ratio of the quality of the shizandra berry crude extract contained in described need testing solution and the multilayer coil separator column of high-speed counter-current chromatograph is 0.1 ~ 0.5:300g/ml.
4. the method for claim 1, is characterized in that the determined wavelength of described UV-detector is 210-280nm.
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