CN109694366A - A kind of method of separating-purifying Tamarix austro effective component - Google Patents
A kind of method of separating-purifying Tamarix austro effective component Download PDFInfo
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- CN109694366A CN109694366A CN201910025362.9A CN201910025362A CN109694366A CN 109694366 A CN109694366 A CN 109694366A CN 201910025362 A CN201910025362 A CN 201910025362A CN 109694366 A CN109694366 A CN 109694366A
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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- C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
- C07D307/10—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
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- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
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- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
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- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
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- C07H17/07—Benzo[b]pyran-4-ones
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- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
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Abstract
The invention discloses a kind of methods of separating-purifying Tamarix austro effective component, comprising the following steps: (1) is extracted using aerial part of the ethyl alcohol to Tamarix austro；(2) normal phase silica gel chromatography column on carries out gradient elution；(3) by normal phase silica gel column chromatography in extract A 2, gradient elution is carried out, sephadex chromatography column Sephadex LH-20 on eluent is collected, is eluted；(4) it by normal phase silica gel chromatography column in extract A 3, elutes, is concentrated by evaporation；(5) it by normal phase silica gel chromatography column in extract A 5, is eluted and is concentrated；(6) after extract A 6 being concentrated, n-butanol is added and is repeatedly extracted, upper reverse phase silica gel chromatographic column, is eluted using methanol aqueous solution after merging n-butanol phase and being concentrated, and is collected eluent, is obtained extract after being concentrated and dried.This method can individually purification comes out one by one by the active material in Tamarix austro, and the material purity after purification is high.
Description
Technical field
The present invention relates to chemical purification fields, and in particular to a kind of method of separating-purifying Tamarix austro effective component.
Background technique
Tamarix austro (Clematis filamentosa Dunn), alias eye Chinese drugs for snakebites, Ranunculaceae Clematis, Guangdong and Guangxi Provinces distribution,
It is used as medicine with base of leaf, it is sweet in flavor micro- cool.It is civil to see red headache etc. for treating, there is decompression, calm, hypnosis expands blood vessel and improves blood
The effect of circulation.It is considered as a kind of nontoxicity, the plant of highly-safe and medicine source treatment coronary heart disease and hypertension abundant
Medicine.Folk prescription Chinese patent drug Guanxinkang Granule piece made from it is clinically used for the treatment of coronary heart disease, hypertension etc..
There is document report to claim, found after being extracted to Tamarix austro, the main active in extract is flavones
Class compound.Flavone compound is a kind of chemical skeleton with important medical value, and such skeleton has various active function
Can, especially there is apparent therapeutic effect to myocardial ischemia.At present to the separating-purifying of ingredient in Tamarix austro usually first by sweet wood
After logical aerial part crushes, is impregnated and purified using ethyl alcohol.However this purification mode also rests on the degree of coarse extraction, slightly mentions
Taking object is the mixture of many kinds of substance, wherein the inactive impurity also containing there are many, is carried out further using conventional separation method
Found when separation, TLC(thin-layer chromatography) on point overlap, also there is overlap peak in liquid chromatogram, it is difficult to by active material
It is individually separated out one by one, so that the specific composition and structure of the various flavone compounds contained by it are still not clear, it can not
Learn its definite mechanism of action on curing myocardial ischemia.
Therefore, smart purification is carried out to Tamarix austro, obtains its pure active material, treat myocardial ischemia to it is researched and analysed
Mechanism have great significance, and obtained active material can be further used for being fabricated to drug after purifying, and be coronary heart disease
Treatment is brought with hypertensive patient.
Summary of the invention
The purpose of the present invention is to provide a kind of method of separating-purifying Tamarix austro effective component, this method can be by sweet wood
Individually purification comes out active material in logical one by one, and the material purity after purification is high.
The purpose of the present invention is achieved through the following technical solutions:
A kind of method of separating-purifying Tamarix austro effective component, comprising the following steps:
(1) it is extracted using aerial part of the ethyl alcohol to Tamarix austro, obtains ethanol extract；
(2) the use of volume ratio is successively 11:1~9:1,8:1~6:1,4 by normal phase silica gel chromatography column on the ethanol extract:
1~2:1, petroleum ether/acetone mixed solvent of 1.5:1~0.5:1, acetone and methanol carry out gradient elution, and collect respectively
Eluent simultaneously removes solvent, obtains extract A 1, A2, A3, A4, A5, A6；
(3) by normal phase silica gel column chromatography in extract A 2, the chlorine for the use of volume ratio being successively 100:1~80:1 and 60:1~40:1
Imitative/methanol mixed solvent carries out gradient elution, collects the eluent of the chloroform/methanol of 60:1~40:1 and evaporates solvent concentration
Afterwards, upper sephadex chromatography column Sephadex LH-20, the chloroform/methanol mixed solvent for being 2:1~0.5:1 with volume ratio are washed
It is de-, it stands eluent and powdered precipitating is precipitated, be separated by filtration, obtain salviarin (No. CAS: 19103-54-9) after dry；
(4) by normal phase silica gel chromatography column in extract A 3, using the chloroform/methanol mixed solvent that volume ratio is 40:1~20:1 into
Row elution, successively secondary collection obtains eluent t1 and t2 in two batches, obtains extract F1 and extract F2 after evaporating solvent concentration；
(4a) is by sephadex chromatography column Sephadex LH-20 on extract F1, the chlorine for being 2:1~0.5:1 with volume ratio
Imitative/methanol mixed solvent is eluted, and stands eluent and powdered precipitating is precipitated, be separated by filtration to obtain oleanolic acid (No. CAS:
508-02-1)；
(4b) is by sephadex chromatography column Sephadex LH-20 on extract F2, the chlorine for being 2:1~0.5:1 with volume ratio
Imitative/methanol mixed solvent is eluted, and is handled by HPLC preparative chromatography, is respectively obtained purification liquid t3 and t4, is removed
Solvent simultaneously respectively obtains lariciresinol (No. CAS: 27003-73-2) and two coniferyl alcohol of dihydro dehydrogenation after drying
(Dihydrodehydrodiconiferyl Alcohol, No. CAS: 126253-41-6)；
(5) it by normal phase silica gel chromatography column in extract A 5, is carried out with the chloroform/methanol mixed solvent that volume ratio is 25:1~5:1
Elution, successively secondary collection obtains eluent t5 and t6 in two batches, and extract F3 and F4 are obtained after concentration；
(5a) is by sephadex chromatography column Sephadex LH-20 on extract F3, the chlorine for being 2:1~0.5:1 with volume ratio
Imitative/methanol mixed solvent is eluted, and stands eluent and powdered precipitating is precipitated, be separated by solid-liquid separation, will be powdered with organic solvent
After precipitating dissolution, upper MCI GEL CHP20/P120 column is eluted with 60~80% methanol, collects eluent, concentration, recrystallization is simultaneously
Apiolin (No. CAS: 520-36-5) is obtained after drying；
(5b) elutes MCI GEL CHP20/P120 column on extract F4 with 70~90% methanol, collects eluent, is concentrated, weight
It crystallizes and obtains luteolin (No. CAS: 491-70-3) after drying；
(6) after extract A 6 being concentrated, be dissolved in water, be then added with plus water after the isometric n-butanol of total liquid volume carry out
Repeatedly extraction, upper reverse phase silica gel chromatographic column after merging n-butanol phase and being concentrated, successively using 10~20%, 20~35% and 35~
50% methanol aqueous solution is eluted, and collects eluent, extract F5, F6 and F7 are obtained after being concentrated and dried；
(6a) upper sephadex chromatography column Sephadex LH-20 after extract F5 is concentrated, with volume ratio be 2:1~
The chloroform/methanol mixed solvent of 0.5:1 is eluted, and upper normal phase silica gel chromatography column, uses volume after collecting eluent and being concentrated
Than being eluted for the chloroform/methanol mixed solvent of 9:1~7:1, eluent is collected, after being concentrated and dried, obtains caffeic acid
(No. CAS: 331-39-5)；
(6b) upper sephadex chromatography column Sephadex LH-20 after extract F6 is concentrated, with volume ratio be 2:1~
The chloroform/methanol mixed solvent of 0.5:1 is eluted, and upper normal phase silica gel chromatography column, uses volume after collecting eluent and being concentrated
Than being eluted for the chloroform/methanol mixed solvent of 7:1~5:1, eluent is collected, after being concentrated and dried, is obtained
No. Urolignoside(CAS: 131723-83-6)；
(6c) upper sephadex chromatography column Sephadex LH-20 after extract F7 is concentrated, with volume ratio be 2:1~
The chloroform/methanol mixed solvent of 0.5:1 is eluted, successively secondary collection eluent t7 and t8 in two batches, after concentrate drying respectively
Obtain astragalin (No. CAS: 480-10-4) and linarin (No. CAS: 480-36-4).
The aerial part of Tamarix austro of the present invention includes the stem branch of Tamarix austro.
In order to overcome the problems, such as that conventional separation method can not separate effective component in Tamarix austro one by one, the present invention is through too long
After phase experiment, with the separation methods such as different types of chromatographic column combination HPLC and extraction, purified by substep, it is multiple in material composition
In miscellaneous Tamarix austro ethanol crude extract, will wherein have valuable effective component salviarin, oleanolic acid, fallen leaves pine resin
Alcohol, two coniferyl alcohol of dihydro dehydrogenation, apiolin, luteolin, caffeic acid, Urolignoside, astragalin and linarin height
Realize separating-purifying in effect ground, it is determined that the concrete composition of flavone compound in Tamarix austro, the material purity after purification is up to 95%
More than, all have great importance in scientific research and field of medicaments.
In the elution process of step (2) of the present invention, afforded with petroleum ether/acetone mixed solvent of 11:1~9:1
For extract A 1；Being afforded with petroleum ether/acetone mixed solvent of 8:1~6:1 is extract A 2；With the stone of 4:1~2:1
It is extract A 3 that oily ether/acetone mixed solvent, which affords,；With petroleum ether/acetone mixed solvent elution of 1.5:1~0.5:1
It obtains being extract A 4；Being afforded with acetone is extract A 5；Being afforded with methanol purification is extract A 6,
Middle extract A 1 and A4 are impurity component.Successive collection eluent secondary in two batches, is answered described in step (4), (5) and (6c)
It is interpreted as, is verified using TLC, the eluent of first eluate is collected and combined into as first batch, then by rear eluate
Eluent collect merge become second lot, with collect obtain two different extracts.
As one embodiment of the present invention, the process that step (1) extracts the aerial part of Tamarix austro is specific
Are as follows: the Tamarix austro aerial part after drying is crushed, is added in solid masses/liquid volume ratio 1:3~1:5 ratio
85% industrial alcohol extracts three times, merges supernatant, and vacuum concentration is simultaneously refrigerated under the conditions of -80 DEG C.
Preferably, step (2) successively using volume ratio be 10:1,7:1,3:1,1:1 petroleum ether/acetone mixed solvent,
Acetone and methanol carry out gradient elution.
Preferably, step (3) is successively 100:1 and 50:1 using volume ratio by normal phase silica gel column chromatography in extract A 2
Chloroform/methanol mixed solvent carry out gradient elution, after collecting the eluent of the chloroform/methanol of 50:1 and evaporating solvent concentration,
Upper sephadex chromatography column Sephadex LH-20 is eluted with the chloroform/methanol mixed solvent that volume ratio is 1:1.
Preferably, step (4) is eluted using the chloroform/methanol mixed solvent that volume ratio is 30:1.
Preferably, the chloroform/methanol mixed solvent that step (4a) is 1:1 with volume ratio is eluted.
Preferably, the chloroform/methanol mixed solvent that step (4b) is 1:1 with volume ratio is eluted.
Preferably, the chloroform/methanol mixed solvent that step (5) is 15:1 with volume ratio is eluted.
Preferably, sephadex chromatography column Sephadex LH-20 on extract F3 is by step (5a) with volume ratio
The chloroform/methanol mixed solvent of 1:1 is eluted, and stands eluent and powdered precipitating is precipitated, be separated by solid-liquid separation, will with organic solvent
After powdered precipitating dissolution, upper MCI GEL CHP20/P120 column is eluted with 75% methanol.
Preferably, step (5b) is eluted with 80% methanol.
Preferably, upper sephadex chromatography column Sephadex LH-20 after extract F5 is concentrated step (6a),
The chloroform/methanol mixed solvent for being 1:1 with volume ratio is eluted, upper normal phase silica gel chromatography column after collecting eluent and being concentrated,
It is eluted using the chloroform/methanol mixed solvent that volume ratio is 8:1.
Preferably, upper sephadex chromatography column Sephadex LH-20 after extract F6 is concentrated step (6b),
The chloroform/methanol mixed solvent for being 1:1 with volume ratio is eluted, upper normal phase silica gel chromatography column after collecting eluent and being concentrated,
It is eluted using the chloroform/methanol mixed solvent that volume ratio is 6:1.
Preferably, the chloroform/methanol mixed solvent that step (6c) is 1:1 with volume ratio is eluted.
The present invention recommends to use diameter 3.5cm, the silica gel chromatographic column of long 40cm glass column in the step (2)；Step
(3), (4), (5) middle use diameter 3.5cm, the silica gel chromatographic column of long 40cm glass column.
As one embodiment of the present invention:
After being separated by filtration powdered precipitating in the step (3), washed using methanol；
After being separated by filtration powdered precipitating in the step (4a), washed using acetone；
The step (4b) uses the methanol aqueous solution that during HPLC preparative chromatography, solvent is 30~50%；
The step (5a) organic solvent for dissolved powders shape precipitating is DMSO；
The step (5a), (5b) are recrystallized using methanol；
It is of the present invention dry using freeze-drying.
Compared with the prior art, the invention has the following beneficial effects:
1. the present invention is during separating-purifying, after carrying out gradient crude separation using normal phase silicagel column, using sephadex
Chromatography or the mode for combining high performance liquid chromatography separation, it is de- to realize salviarin, oleanolic acid, lariciresinol and dihydro
The rapidly and efficiently separation of two coniferyl alcohol of hydrogen, purity is up to 95% or more；
2. the present invention using sephadex chromatography combined with MCI chromatography or MCI chromatographic column is single separate by the way of, realization
The separation of flavone compound apigenin and luteolin rapidly and efficiently, purity can reach 95% or more, overcome efficient liquid
The difficult technical problem of phase chromatography component peak separation；
3. the present invention using sephadex chromatography or combine the constant elution of normal phase silicagel column after, realize caffeic acid,
The rapidly and efficiently separation of Urolignoside, astragalin and linarin, purity overcome high-efficient liquid phase color up to 95% or more
Compose into the difficult technical problem of swarming separation；
4. the present invention effectively separates the active material realization in Tamarix austro one by one, purification obtains pure compound, helps
In scientific research, with determine its treat Cardiovascular material base, the chemical structure for identifying monomer, bright analysis its act on machine
Reason, purifying technique can also can be amplified, and the active constituent after purification is used to be made the medicine for reducing chronic disease occurrence and development
Object, application field is wide, and market potential is huge, realizes that upgrading and quality control are realized in the secondary development of Tamarix austro, this is to promotion
The development of Tamarix austro resource, pushes the sustainable development of Tamarix austro industry with important at the economic benefit for improving Tamarix austro industry
Meaning.
Detailed description of the invention
Below by way of attached drawing, the present invention is further illustrated.
3 separating-purifying flow diagram of Fig. 1 embodiment.
The NMR hydrogen spectrogram of Fig. 2 compound C1.
The NMR carbon spectrogram of Fig. 3 compound C1.
The molecular structural formula of Fig. 4 compound C1.
The NMR hydrogen spectrogram of Fig. 5 compound C2.
The NMR carbon spectrogram of Fig. 6 compound C2.
The molecular structural formula of Fig. 7 compound C2.
The NMR hydrogen spectrogram of Fig. 8 compound C3.
The NMR carbon spectrogram of Fig. 9 compound C3.
The molecular structural formula of Figure 10 compound C3.
The NMR hydrogen spectrogram of Figure 11 compound C4.
The NMR carbon spectrogram of Figure 12 compound C4.
The molecular structural formula of Figure 13 compound C4.
The NMR hydrogen spectrogram of Figure 14 compound C5.
The NMR carbon spectrogram of Figure 15 compound C5.
The molecular structural formula of Figure 16 compound C5.
The NMR hydrogen spectrogram of Figure 17 compound C6.
The NMR carbon spectrogram of Figure 18 compound C6.
The molecular structural formula of Figure 19 compound C6.
The NMR hydrogen spectrogram of Figure 20 compound C7.
The molecular structural formula of Figure 21 compound C7.
The NMR hydrogen spectrogram of Figure 22 compound C8.
The NMR carbon spectrogram of Figure 23 compound C8.
The molecular structural formula of Figure 24 compound C8.
The NMR hydrogen spectrogram of Figure 25 compound C9.
The NMR carbon spectrogram of Figure 26 compound C9.
The molecular structural formula of Figure 27 compound C9.
The NMR hydrogen spectrogram of Figure 28 compound C10.
The molecular structural formula of Figure 29 compound C10.
Specific embodiment
Below by way of specific embodiment, the present invention is further illustrated.
Embodiment 1
(1) Tamarix austro aerial part of the 10kg after dry is crushed, is added in solid masses/liquid volume ratio 1:5 ratio
Entering 85% industrial alcohol, soak extraction 12h is extracted three times, merges supernatant, and vacuum concentration is simultaneously refrigerated under the conditions of -80 DEG C,
Obtain ethanol extract；
It (2) the use of volume ratio is successively 11:1,8:1,4:1,1.5:1 by normal phase silica gel chromatography column on the ethanol extract
Petroleum ether/acetone mixed solvent, acetone and methanol carry out gradient elution, and collect eluent respectively and remove solvent, obtain
Extract A 1, A2, A3, A4, A5, A6；
(3) it by normal phase silica gel column chromatography in extract A 2, is successively mixed using the chloroform/methanol that volume ratio is 90:1 and 60:1 molten
Agent carries out gradient elution, after collecting the eluent of the chloroform/methanol of 60:1 and evaporating solvent concentration, upper sephadex chromatography column
Sephadex LH-20 is eluted with the chloroform/methanol mixed solvent that volume ratio is 2:1, stands eluent and powdered precipitating is precipitated,
It is separated by filtration, is washed using methanol, obtain compound C1 after dry；
(4) it by normal phase silica gel chromatography column in extract A 3, is washed using the chloroform/methanol mixed solvent that volume ratio is 40:1
De-, successively secondary collection obtains eluent t1 and t2 in two batches, obtains extract F1 and extract F2 after evaporating solvent concentration；
(4a) by sephadex chromatography column Sephadex LH-20 on extract F1, the chloroform/methanol for being 2:1 with volume ratio is mixed
Bonding solvent is eluted, and stands eluent and powdered precipitating is precipitated, washed using acetone, be separated by filtration to obtain compound
C2；
(4b) by sephadex chromatography column Sephadex LH-20 on extract F2, the chloroform/methanol for being 2:1 with volume ratio is mixed
Bonding solvent is eluted, and is handled by HPLC preparative chromatography, the methanol aqueous solution that solvent is 30%；, respectively obtain purification
Liquid t3 and t4 remove solvent and respectively obtain compound C3 and compound C4 after drying；
(5) by normal phase silica gel chromatography column in extract A 5, the chloroform/methanol mixed solvent for being 25:1 with volume ratio is eluted,
Successively secondary collection obtains eluent t5 and t6 in two batches, and extract F3 and F4 are obtained after concentration；
(5a) by sephadex chromatography column Sephadex LH-20 on extract F3, the chloroform/methanol for being 2:1 with volume ratio is mixed
Bonding solvent is eluted, and stands eluent and powdered precipitating is precipitated, be separated by solid-liquid separation, after powdered precipitating is dissolved with DMSO, on
MCI GEL CHP20/P120 column is eluted with 60% methanol, collects eluent, and concentration obtains after recrystallizing and dry in methyl alcohol
C5；
(5b) elutes MCI GEL CHP20/P120 column on extract F4 with 70% methanol, collects eluent, concentration, in methanol
It is middle to recrystallize and obtain C6 after drying；
(6) after extract A 6 being concentrated, be dissolved in water, be then added with plus water after the isometric n-butanol of total liquid volume carry out
Repeatedly extraction, upper reverse phase silica gel chromatographic column, successively water-soluble using 10%, 20% and 35% methanol after merging n-butanol phase and being concentrated
Liquid is eluted, and collects eluent, extract F5, F6 and F7 are obtained after being concentrated and dried；
(6a) upper sephadex chromatography column Sephadex LH-20 after extract F5 is concentrated, is 2:1's with volume ratio
Chloroform/methanol mixed solvent is eluted, and upper normal phase silica gel chromatography column after collecting eluent and being concentrated, is 9:1 using volume ratio
Chloroform/methanol mixed solvent eluted, collect eluent, after being concentrated and dried, obtain compound C7；
(6b) upper sephadex chromatography column Sephadex LH-20 after extract F6 is concentrated, is 2:1's with volume ratio
Chloroform/methanol mixed solvent is eluted, and upper normal phase silica gel chromatography column after collecting eluent and being concentrated, is 7:1 using volume ratio
Chloroform/methanol mixed solvent eluted, collect eluent, after being concentrated and dried, obtain compound C8；
(6c) upper sephadex chromatography column Sephadex LH-20 after extract F7 is concentrated, is 2:1's with volume ratio
Chloroform/methanol mixed solvent is eluted, successively secondary collection eluent t7 and t8 in two batches, respectively obtains chemical combination after concentrate drying
Object C9 and compound C10.
Embodiment 2
(1) Tamarix austro aerial part of the 15kg after dry is crushed, is added in solid masses/liquid volume ratio 1:4 ratio
Entering 85% industrial alcohol, soak extraction 12h is extracted three times, merges supernatant, and vacuum concentration is simultaneously refrigerated under the conditions of -80 DEG C,
Obtain ethanol extract；
It (2) is successively the stone of 9:1,6:1,2:1,0.5:1 using volume ratio by normal phase silica gel chromatography column on the ethanol extract
Oily ether/acetone mixed solvent, acetone and methanol carry out gradient elution, and collect eluent respectively and remove solvent, are mentioned
Take object A1, A2, A3, A4, A5, A6；
(3) it by normal phase silica gel column chromatography in extract A 2, is successively mixed using the chloroform/methanol that volume ratio is 80:1 and 40:1 molten
Agent carries out gradient elution, after collecting the eluent of the chloroform/methanol of 40:1 and evaporating solvent concentration, upper sephadex chromatography column
Sephadex LH-20 is eluted with the chloroform/methanol mixed solvent that volume ratio is 0.5:1, stands eluent and powdered sink is precipitated
It forms sediment, is separated by filtration, is washed using methanol, obtain compound C1 after dry；
(4) it by normal phase silica gel chromatography column in extract A 3, is washed using the chloroform/methanol mixed solvent that volume ratio is 20:1
De-, successively secondary collection obtains eluent t1 and t2 in two batches, obtains extract F1 and extract F2 after evaporating solvent concentration；
(4a) is by sephadex chromatography column Sephadex LH-20 on extract F1, the chloroform/methanol for being 0.5:1 with volume ratio
Mixed solvent is eluted, and stands eluent and powdered precipitating is precipitated, washed using acetone, be separated by filtration to obtain compound
C2；
(4b) is by sephadex chromatography column Sephadex LH-20 on extract F2, the chloroform/methanol for being 0.5:1 with volume ratio
Mixed solvent is eluted, and is handled by HPLC preparative chromatography, the methanol aqueous solution that solvent is 50%；, respectively obtain and mention
Pure liquid t3 and t4 removes solvent and respectively obtains C3 and C4 after drying；
(5) by normal phase silica gel chromatography column in extract A 5, the chloroform/methanol mixed solvent for being 5:1 with volume ratio is eluted, first
Secondary collection obtains eluent t5 and t6 in two batches afterwards, and extract F3 and F4 are obtained after concentration；
(5a) is by sephadex chromatography column Sephadex LH-20 on extract F3, the chloroform/methanol for being 0.5:1 with volume ratio
Mixed solvent is eluted, and stands eluent and powdered precipitating is precipitated, be separated by solid-liquid separation, after powdered precipitating is dissolved with DMSO,
Upper MCI GEL CHP20/P120 column is eluted with 80% methanol, collects eluent, and concentration obtains after recrystallizing and dry in methyl alcohol
To compound C5；
(5b) elutes MCI GEL CHP20/P120 column on extract F4 with 90% methanol, collects eluent, concentration, in methanol
It is middle to recrystallize and obtain compound C6 after drying；
(6) after extract A 6 being concentrated, be dissolved in water, be then added with plus water after the isometric n-butanol of total liquid volume carry out
Repeatedly extraction, upper reverse phase silica gel chromatographic column, successively water-soluble using 20%, 35% and 50% methanol after merging n-butanol phase and being concentrated
Liquid is eluted, and collects eluent, extract F5, F6 and F7 are obtained after being concentrated and dried；
(6a) upper sephadex chromatography column Sephadex LH-20 after extract F5 is concentrated, is 0.5:1 with volume ratio
Chloroform/methanol mixed solvent eluted, upper normal phase silica gel chromatography column after collecting eluent and being concentrated, is 7 using volume ratio:
1 chloroform/methanol mixed solvent is eluted, and is collected eluent, after being concentrated and dried, is obtained compound C7；
(6b) upper sephadex chromatography column Sephadex LH-20 after extract F6 is concentrated, is 0.5:1 with volume ratio
Chloroform/methanol mixed solvent eluted, upper normal phase silica gel chromatography column after collecting eluent and being concentrated, is 5 using volume ratio:
1 chloroform/methanol mixed solvent is eluted, and is collected eluent, after being concentrated and dried, is obtained compound C8；
(6c) upper sephadex chromatography column Sephadex LH-20 after extract F7 is concentrated, is 0.5:1 with volume ratio
Chloroform/methanol mixed solvent eluted, successively secondary collection eluent t7 and t8 in two batches, respectively obtaining after concentrate drying
Close object C9 and compound C10.
Embodiment 3
(1) as shown in Figure 1, Tamarix austro aerial part of the 5kg after dry is crushed, by solid masses/liquid volume ratio 1:3
Ratio 85% industrial alcohol is added, soak extraction 12h is extracted three times, merge supernatant, vacuum concentration simultaneously under the conditions of -80 DEG C into
Row refrigeration, obtains ethanol extract；
It (2) is successively the stone of 10:1,7:1,3:1,1:1 using volume ratio by normal phase silica gel chromatography column on the ethanol extract
Oily ether/acetone mixed solvent, acetone and methanol carry out gradient elution, and collect eluent respectively and remove solvent, are mentioned
Take object A1, A2, A3, A4, A5, A6；
(3) it by normal phase silica gel column chromatography in extract A 2, is successively mixed using the chloroform/methanol that volume ratio is 100:1 and 50:1
Solvent carries out gradient elution, after collecting the eluent of the chloroform/methanol of 50:1 and evaporating solvent concentration, upper sephadex chromatography
Column Sephadex LH-20 is eluted with the chloroform/methanol mixed solvent that volume ratio is 1:1, stands eluent and powdered sink is precipitated
It forms sediment, is separated by filtration, is washed using methanol, obtain compound C1 after dry；
(4) it by normal phase silica gel chromatography column in extract A 3, is washed using the chloroform/methanol mixed solvent that volume ratio is 30:1
De-, successively secondary collection obtains eluent t1 and t2 in two batches, obtains extract F1 and extract F2 after evaporating solvent concentration；
(4a) by sephadex chromatography column Sephadex LH-20 on extract F1, the chloroform/methanol for being 1:1 with volume ratio is mixed
Bonding solvent is eluted, and stands eluent and powdered precipitating is precipitated, washed using acetone, be separated by filtration to obtain compound
C2；
(4b) by sephadex chromatography column Sephadex LH-20 on extract F2, the chloroform/methanol for being 1:1 with volume ratio is mixed
Bonding solvent is eluted, and is handled by HPLC preparative chromatography, the methanol aqueous solution that solvent is 40%；, respectively obtain purification
Liquid t3 and t4 remove solvent and respectively obtain compound C3 and compound C4 after drying；
(5) by normal phase silica gel chromatography column in extract A 5, the chloroform/methanol mixed solvent for being 15:1 with volume ratio is eluted,
Successively secondary collection obtains eluent t5 and t6 in two batches, and extract F3 and F4 are obtained after concentration；
(5a) by sephadex chromatography column Sephadex LH-20 on extract F3, the chloroform/methanol for being 1:1 with volume ratio is mixed
Bonding solvent is eluted, and stands eluent and powdered precipitating is precipitated, be separated by solid-liquid separation, after powdered precipitating is dissolved with DMSO, on
MCI GEL CHP20/P120 column is eluted with 75% methanol, collects eluent, and concentration obtains after recrystallizing and dry in methyl alcohol
Compound C5；
(5b) elutes MCI GEL CHP20/P120 column on extract F4 with 80% methanol, collects eluent, concentration, in methanol
It is middle to recrystallize and obtain compound C6 after drying；
(6) after extract A 6 being concentrated, be dissolved in water, be then added with plus water after the isometric n-butanol of total liquid volume carry out
Repeatedly extraction, upper reverse phase silica gel chromatographic column, successively water-soluble using 15%, 30% and 40% methanol after merging n-butanol phase and being concentrated
Liquid is eluted, and collects eluent, extract F5, F6 and F7 are obtained after being concentrated and dried；
(6a) upper sephadex chromatography column Sephadex LH-20 after extract F5 is concentrated, is 1:1's with volume ratio
Chloroform/methanol mixed solvent is eluted, and upper normal phase silica gel chromatography column after collecting eluent and being concentrated, is 8:1 using volume ratio
Chloroform/methanol mixed solvent eluted, collect eluent, after being concentrated and dried, obtain compound C7；
(6b) upper sephadex chromatography column Sephadex LH-20 after extract F6 is concentrated, is 1:1's with volume ratio
Chloroform/methanol mixed solvent is eluted, and upper normal phase silica gel chromatography column after collecting eluent and being concentrated, is 6:1 using volume ratio
Chloroform/methanol mixed solvent eluted, collect eluent, after being concentrated and dried, obtain compound C8；
(6c) upper sephadex chromatography column Sephadex LH-20 after extract F7 is concentrated, is 1:1's with volume ratio
Chloroform/methanol mixed solvent is eluted, successively secondary collection eluent t7 and t8 in two batches, respectively obtains chemical combination after concentrate drying
Object C9 and compound C10.
After purification, using DMSO as solvent, the NMR that frequency of use is 500MHz compound C1 resulting to purification~
C10 carries out the detection and analysis of H map and C map, and testing result is as follows:
1) as shown in Fig. 2, being the NMR hydrogen spectrogram of compound C1, hydrogen modal data specifically: 8.07 (Hz of 2H, d, J=9.0,
H-2′, 6′), 7.12 (2H, d, J=9.0 Hz, H-3′, 5′), 6.94 (1H, s, H-8), 6.96 (1H, s,
H-3), 12.87 (1H, s, OH-5), 3.12 (1H, s, OCH3), 3.18 (1H, s, OCH3), 3.13 (1H,
s, OCH3)；
As shown in figure 3, being the NMR carbon spectrogram of compound C1, carbon modal data specifically: 163.6 (C-2), 103.3 (C-
3), 182.2 (C-4), 152.5 (C-5), 131.9 (C-6), 158.1 (C-7), 91.6 (C-8), 152.8 (C-
5), 105.2 (C-10), 122.7(C-1’), 128 (C-2’, 6’), 114.6 (C-3’, 5’), 162.4 (C-
4’), 60.2 (-OCH3), 56.4 (-OCH3), 55.6 (-OCH3)。
After carrying out comprehensive analysis to above-mentioned data, show that the molecular structure of compound C1 is as shown in Figure 4, it was demonstrated that compound
C1 is salviarin (No. CAS: 19103-54-9).
2) as shown in figure 5, being the NMR hydrogen spectrogram of compound C2, hydrogen modal data specifically: 12.04 (s, 1H,
COOH), 5.16 ( t, J = 3.7, 3.3 Hz, 1H, H-12), 2.99 (dd, J = 10.6, 5.1 Hz, 2H,
H-3), 2.75 ( dd, J = 13.9, 4.0Hz, 1H, H-18), 1.91 ( td, J = 13.6, 4.0 Hz, 1H,
H-16), 1.81 (dd, J=8.8,3.3 Hz, 2H, H-11), 1.58-1.67 (m, 3H, H-2, H-15, H-
19), 1.38-1.52 (m, 8H, H-1, H-2, H-6, H-7, H-9, H-16, H -21), 1.07 (d, J=
5.9 Hz, 3H, H-6), 1.28-1.34 ( m, 2H, H-19, H-21), 1.23 ( td, J = 9.9, 3.3 Hz,
1H, H-5), 1.13 ( m, 1H, H-19), 1.03-1.07 ( m,1H, H-21) , 0.99 ( td, J = 11.0,
3.3 Hz, 1H, H-15), 1.09, 0.89, 0.87, 0.87, 0.85, 0.72, 0.67 (s, 3H)；
As shown in fig. 6, being the NMR carbon spectrogram of compound C2, carbon modal data specifically: 178.6 (C-28), 143.8 (C-
13), 121.5 (C-12), 76.8 (C-3), 54.8 (C-5), 47.1 (C-9), 45.7 (C-19), 45.4 (C-
17), 41.3 (C-18), 40.8 (C-14), 38.9 (C-8), 38.4 (C-1), 36.6 (C-10), 33.3 (C-
21), 32.8 (C-29), 32.4 (C-7), 32.1 (C-22), 30.4 (C-20), 28.2 (C-23), 27.2 (C-
15), 26.9 (C-27), 25.6 (C-2), 23.4 (C-30), 22.9 (C-11), 22.6 (C-16), 18.0 (C-
6), 16.8 (C-26), 16.0 (C-24), 15.1 (C-25)。
After carrying out comprehensive analysis to above-mentioned data, show that the molecular structure of compound C2 is as shown in Figure 7, it was demonstrated that compound
C2 is oleanolic acid (No. CAS: 508-02-1).
3) as shown in figure 8, being the NMR hydrogen spectrogram of compound C3, hydrogen modal data specifically: 6.81 (4H, m, H-5,
2′, 5′, 6′), 6.63 (2H, m, H-2, 6), 4.72 (1H, d, J = 7.2 Hz, H-7′), 4.00
(1H, dd, J = 8.0, 7.2 Hz, H-9α), 3.89 (1H, m, H-9′α), 3.70 (2H, m, H-9β, 9′
β), 2.92 (1H, dd, J = 12.6, 4.8 Hz, H-7α), 2.69 (1H, m, H-8), 2.48 (1H, dd, J
= 12.6, 10.6 Hz, H-7β), 2.39 (1H, m, H-8′), 3.88 (3H, s, 3′-OCH3), 3.82 (3H,
s, 3-OCH3)；
As shown in figure 9, being the NMR carbon spectrogram of compound C3, carbon modal data specifically: 147.3 (C-3 '), 146.9 (C-
3), 144.9 (C-4′), 144.0 (C-4), 135.3 (C-1′), 132.6 (C-1), 121.7 (C-6), 119.2
(C-6′), 114.7 (C-5), 114.6 (C-5′), 111.8 (C-2), 108.9 (C-2′), 83.0 (C-7′),
73.1 (C-9), 60.9 (C-9′), 56.1 (3, 3′-OCH3), 52.9 (C-8′), 42.9 (C-8), 33.8 (C-
7)。
After carrying out comprehensive analysis to above-mentioned data, show that the molecular structure of compound C3 is as shown in Figure 10, it was demonstrated that chemical combination
Object C3 is lariciresinol (No. CAS: 27003-73-2).
It 4) as shown in figure 11, is the NMR hydrogen spectrogram of compound C4, hydrogen modal data is specially (C5D5N): 7.33 (1H,
d, J = 1.8 Hz, H-2), 7.20 (1H, d, J = 1.8 Hz, H-5), 7.25 (1H, dd, J = 1.8,
8.1 Hz, H-6), 6.06 (1H, d, J = 6.8 Hz, H-7), 3.97 (1H, m, H-8), 4.21 (1H, m,
H-9a), 4.27 (1H, m, H-9b), 6.92 (1H, brs, H-2′), 7.06 (1H, brs, H-6′), 2.87
(2H, t, J = 7.6 Hz, H-7′), 2.09 (2H, m, H-8′), 3.92 (2H, t, J = 6.4 Hz, H-
9′), 3.63 (3H, s, 3-OCH3), 3.84 (3H, s, 3′-OCH3)；
It as shown in figure 12, is the NMR carbon spectrogram of compound C4, carbon modal data is specially (C5D5N): 134.4 (C-1),
111.4 (C-2), 148.6 (C-3), 147.9 (C-4), 117.0 (C-5), 120.3 (C-6), 88.9 (C-7),
55.6 (C-8), 64.9 (C-9), 130.7 (C-1′), 114.2 (C-2′), 145.2 (C-3′), 147.9 (C-
4′), 136.7 (C-5′), 118.1 (C-6′), 33.2 (C-7′), 36.5 (C-8′), 62.0 (C-9′), 56.3
(3-OCH3), 56.8 (3′-OCH3)。
After carrying out comprehensive analysis to above-mentioned data, show that the molecular structure of compound C4 is as shown in figure 13, it was demonstrated that chemical combination
Object C4 is two coniferyl alcohol of dihydro dehydrogenation (Dihydrodehydrodiconiferyl Alcohol, No. CAS: 126253-41-6).
It 5) as shown in figure 14, is the NMR hydrogen spectrogram of compound C5, hydrogen modal data specifically: 6.78 (1H, s), 6.
49 (1H, d, J = 1.8 Hz), 6. 19 (1H, d, J = 1. 8 Hz), 6.94 (2H, dd, J=9.0, 1.2
Hz), 7.94 (2H, dd, J = 9.0, 1. 2 Hz)；
It as shown in figure 15, is the NMR carbon spectrogram of compound C5, carbon modal data specifically: 182.0 (C-4), 164.2 (C-
2), 163.9 (C-7), 161.3 (C-5), 161.1 (C-4’), 156.9 (C-9), 128.6 (C-2′, 6′),
121.0 (C-1 '), 116.0 (C-3 ', 5 '), 105.3 (C-10), 103.1 (C-3), 99.9 (C-6), 94.8 (C-
8)。
After carrying out comprehensive analysis to above-mentioned data, show that the molecular structure of compound C5 is as shown in figure 16, it was demonstrated that chemical combination
Object C5 is apiolin (No. CAS: 520-36-5).
It 6) as shown in figure 17, is the NMR hydrogen spectrogram of compound C6, hydrogen modal data specifically: 6.67 (1H, s),
6.45 (Hz of 1H, d, J=1.8), 6.19 (Hz of 1H, d, J=1.8), 7.43 (1H, dd, J=8.4,1.2 Hz),
7.43 (Hz of 1H, d, J=2.4), 6.90 (Hz of 1H, d, J=8.4)；
It as shown in figure 18, is the NMR carbon spectrogram of compound C6, carbon modal data specifically: 181.6 (C-4), 164.1 (C-
2), 163.9 (C-7), 161.4 (C-5), 157.3 (C-9), 149.7 (C-4 '), 145.7 (C-3 '), 121.5 (C-
1 '), 118.9 (C-6 '), 116.0 (C-5 '), 113.4 (C-2), 103.7 (C-l '), 102.8 (C-3), 98.8 (C-
6), 93.8 (C-8).
After carrying out comprehensive analysis to above-mentioned data, show that the molecular structure of compound C6 is as shown in figure 19, it was demonstrated that chemical combination
Object C6 is luteolin (No. CAS: 491-70-3).
It 7) as shown in figure 20, is the NMR hydrogen spectrogram of compound C7, hydrogen modal data specifically: 7.53 (1H, d, J=
16.0 Hz, H-3), 6.22 (1H, d, J =15.6 Hz, H-2), 7.02 (1H, d, J =2.0 Hz, H-2′),
6.93 (1H, dd, J =8.0 Hz, H-6′), 6.77 (1H, d, J =8.0 Hz, H-5′)。
After carrying out comprehensive analysis to above-mentioned data, show that the molecular structure of compound C7 is as shown in figure 21, it was demonstrated that chemical combination
Object C7 is caffeic acid (No. CAS: 331-39-5).
It 8) as shown in figure 22, is the NMR hydrogen spectrogram of compound C8, hydrogen modal data is specially (CD3OD): 6.71 (1H,
s, H-6), 6.73 (1H, s, H-6), 2.62 (2H, t, J=7.2Hz, H-7), 1.80 (2H, m, H-8),
3.81 (2H, t, J=6.5 Hz, H-9), 7.02 (1H, d, J=1.8 Hz, H-2′), 7.12 (1H, d, J=8.5
Hz, H-5′), 6.92 (1H, dd, J=8.5, 1.8 Hz, H-6′), 5.54 (1H, d, J=5.8 Hz, H-7′),
3.44 (1H, m, H-8′), 3.81 (2H, t, J=6.5, H-9), 4.88 (1H, d, J=8.0 Hz, Glc H-
1), 3.85 (3H, s, 3-OCH3), 3.83 (3H, s, 3'-OCH3)；
It as shown in figure 23, is the NMR carbon spectrogram of compound C8, carbon modal data is specially (CD3OD): 137.1 (C-1),
114.1 (C-2), 145.2 (C-3), 147.6 (C-4), 129.5 (C-5), 117.9 (C-6), 32.9 (C-7),
35.8 (C-8), 62.1 (C-9), 138.3 (C-1′), 111.0 (C-2′), 150.9 (C-3′), 147.4 (C-
4′), 117.8 (C-5′), 119.3 (C-6′), 88.5 (C-7′), 55.7 (C-8′), 65.0 (C-9′), 56.7
(3-OCH3), 56.6 (3′-OCH3), 102.7 (Glc H-1), 74.9 (Glc H-2), 78.2(Glc H-3),
71.3 (Glc H-4), 77.8 (Glc H-5), 62.4 (Glc H-6)。
After carrying out comprehensive analysis to above-mentioned data, show that the molecular structure of compound C8 is as shown in figure 24, it was demonstrated that chemical combination
Object C8 is No. Urolignoside(CAS: 131723-83-6).
9) as shown in figure 25, it is the NMR hydrogen spectrogram of compound C9, hydrogen modal data is specially (DMSO): 5.45 (1 H,
d, J=7.5 Hz, H-1"), 6.21 (1H, d, J=2.1Hz, H-6), 6.43 (1H, d, J=2.1 Hz, H-8),
6.88 (2H, d, J=8. 9 Hz, H-3, 5′), 8.04 (2H, d, J=8.9 Hz, H-2′, 6′), 12. 61
(1H, s, 5-OH)；
It as shown in figure 26, is the NMR carbon spectrogram of compound C9, carbon modal data specifically: 61. 0 (C-6 "), 70. 0 (C-
4"), 74. 4 (C-2"), 76. 6 (C-3"), 77. 6 (C-5"), 93. 8 (C-8), 98. 8 (C-6), 101.
0 (C-1"), 104. 2 (C-10), 115. 2 (C-3′, 5′), 121.1 (C-1′), 131. 0 (C-2′, 6′),
133. 4 (C-3), 156. 4 (C-2), 156. 5 (C-9), 160. 1 (C-4′), 161. 4 (C-5), 164. 3
(C-7), 177. 6 (C-4)。
After carrying out comprehensive analysis to above-mentioned data, show that the molecular structure of compound C9 is as shown in figure 27, it was demonstrated that chemical combination
Object C9 is astragalin (No. CAS: 480-10-4).
It 10) as shown in figure 28, is the NMR hydrogen spectrogram of compound C9, hydrogen modal data specifically: 12.96 (1H, s, 5-
OH), 8.06 (2H, d, J=8.6Hz, H-2′, 6′), 7.16 (2H, d, J=8.6Hz, H-3, 5′), 6.96
(1H, s, H-3), 6.80 (1H, br s, H-8), 6.46 (1H, br s, H-6), 5.07 (1H, d, J=
6.7Hz, H-1"), 4.55 (1H, br s, C-1"′), 3.86 (3H, s, 4′-OCH3), 1.07 (3H, d, J=
5.5Hz, H-6"′)。
After carrying out comprehensive analysis to above-mentioned data, show that the molecular structure of compound C10 is as shown in figure 29, it was demonstrated that chemical combination
Object C10 is linarin (No. CAS: 480-36-4).
From above result as it can be seen that method of the present invention successfully separates each effective component in Tamarix austro one by one
Purification, and from the peak type of hydrogen spectrogram as it can be seen that the material purity height obtained after separating-purifying, is suitable for further studying or being used for
It is fabricated to drug.
It should be pointed out that above-described embodiment be only to further explanation of the invention, rather than limit, art technology
Any adjustment or change of the personnel in the comparable meaning and scope with technical solution of the present invention are all considered as being included in this
In the protection scope of invention.
Claims (10)
1. a kind of method of separating-purifying Tamarix austro effective component, which comprises the following steps:
(1) it is extracted using aerial part of the ethyl alcohol to Tamarix austro, obtains ethanol extract；
(2) the use of volume ratio is successively 11:1~9:1,8:1~6:1,4 by normal phase silica gel chromatography column on the ethanol extract:
1~2:1, petroleum ether/acetone mixed solvent of 1.5:1~0.5:1, acetone and methanol carry out gradient elution, and collect respectively
Eluent simultaneously removes solvent, obtains extract A 1, A2, A3, A4, A5, A6；
(3) by normal phase silica gel column chromatography in extract A 2, the chlorine for the use of volume ratio being successively 100:1~80:1 and 60:1~40:1
Imitative/methanol mixed solvent carries out gradient elution, collects the eluent of the chloroform/methanol of 60:1~40:1 and evaporates solvent concentration
Afterwards, upper sephadex chromatography column Sephadex LH-20, the chloroform/methanol mixed solvent for being 2:1~0.5:1 with volume ratio are washed
It is de-, it stands eluent and powdered precipitating is precipitated, be separated by filtration, obtain salviarin after dry；
(4) by normal phase silica gel chromatography column in extract A 3, using the chloroform/methanol mixed solvent that volume ratio is 40:1~20:1 into
Row elution, successively secondary collection obtains eluent t1 and t2 in two batches, obtains extract F1 and extract F2 after evaporating solvent concentration；
(4a) is by sephadex chromatography column Sephadex LH-20 on extract F1, the chlorine for being 2:1~0.5:1 with volume ratio
Imitative/methanol mixed solvent is eluted, and stands eluent and powdered precipitating is precipitated, be separated by filtration to obtain oleanolic acid；
(4b) is by sephadex chromatography column Sephadex LH-20 on extract F2, the chlorine for being 2:1~0.5:1 with volume ratio
Imitative/methanol mixed solvent is eluted, and is handled by HPLC preparative chromatography, is respectively obtained purification liquid t3 and t4, is removed
Solvent simultaneously respectively obtains two coniferyl alcohol of lariciresinol and dihydro dehydrogenation after drying；
(5) it by normal phase silica gel chromatography column in extract A 5, is carried out with the chloroform/methanol mixed solvent that volume ratio is 25:1~5:1
Elution, successively secondary collection obtains eluent t5 and t6 in two batches, and extract F3 and F4 are obtained after concentration；
(5a) is by sephadex chromatography column Sephadex LH-20 on extract F3, the chlorine for being 2:1~0.5:1 with volume ratio
Imitative/methanol mixed solvent is eluted, and stands eluent and powdered precipitating is precipitated, be separated by solid-liquid separation, will be powdered with organic solvent
After precipitating dissolution, upper MCI GEL CHP20/P120 column is eluted with 60~80% methanol, collects eluent, concentration, recrystallization is simultaneously
Apiolin is obtained after drying；
(5b) elutes MCI GEL CHP20/P120 column on extract F4 with 70~90% methanol, collects eluent, is concentrated, weight
It crystallizes and obtains luteolin after drying；
(6) after extract A 6 being concentrated, be dissolved in water, be then added with plus water after the isometric n-butanol of total liquid volume carry out
Repeatedly extraction, upper reverse phase silica gel chromatographic column after merging n-butanol phase and being concentrated, successively using 10~20%, 20~35% and 35~
50% methanol aqueous solution is eluted, and collects eluent, extract F5, F6 and F7 are obtained after being concentrated and dried；
(6a) upper sephadex chromatography column Sephadex LH-20 after extract F5 is concentrated, with volume ratio be 2:1~
The chloroform/methanol mixed solvent of 0.5:1 is eluted, and upper normal phase silica gel chromatography column, uses volume after collecting eluent and being concentrated
Than being eluted for the chloroform/methanol mixed solvent of 9:1~7:1, eluent is collected, after being concentrated and dried, obtains caffeic acid；
(6b) upper sephadex chromatography column Sephadex LH-20 after extract F6 is concentrated, with volume ratio be 2:1~
The chloroform/methanol mixed solvent of 0.5:1 is eluted, and upper normal phase silica gel chromatography column, uses volume after collecting eluent and being concentrated
Than being eluted for the chloroform/methanol mixed solvent of 7:1~5:1, eluent is collected, after being concentrated and dried, is obtained
Urolignoside；
(6c) upper sephadex chromatography column Sephadex LH-20 after extract F7 is concentrated, with volume ratio be 2:1~
The chloroform/methanol mixed solvent of 0.5:1 is eluted, successively secondary collection eluent t7 and t8 in two batches, after concentrate drying respectively
Obtain astragalin and linarin.
2. the method for separating-purifying Tamarix austro effective component according to claim 1, which is characterized in that step (1) is to sweet
The process that the aerial part of caulis akebiae extracts specifically: crush the Tamarix austro aerial part after drying, by solid matter
85% industrial alcohol is added in amount/liquid volume ratio 1:3~1:5 ratio, extracts three times, merges supernatant, vacuum concentration simultaneously -80
It is refrigerated under the conditions of DEG C.
3. the method for separating-purifying Tamarix austro effective component according to claim 1 or 2, which is characterized in that step (2) according to
It is secondary to carry out gradient elution using petroleum ether/acetone mixed solvent, acetone and the methanol that volume ratio is 10:1,7:1,3:1,1:1.
4. the method for separating-purifying Tamarix austro effective component according to claim 3, which is characterized in that step (3) will mention
Normal phase silica gel column chromatography on object A2 is taken, successively carries out gradient using the chloroform/methanol mixed solvent that volume ratio is 100:1 and 50:1
Elution, after collecting the eluent of the chloroform/methanol of 50:1 and evaporating solvent concentration, upper sephadex chromatography column Sephadex
LH-20 is eluted with the chloroform/methanol mixed solvent that volume ratio is 1:1.
5. the method for separating-purifying Tamarix austro effective component according to claim 4, which is characterized in that step (4) uses
Volume ratio is that the chloroform/methanol mixed solvent of 30:1 is eluted；Step (4a) is mixed with the chloroform/methanol that volume ratio is 1:1
Solvent is eluted；The chloroform/methanol mixed solvent that step (4b) is 1:1 with volume ratio is eluted；Step (5) uses volume ratio
It is eluted for the chloroform/methanol mixed solvent of 15:1.
6. the method for separating-purifying Tamarix austro effective component according to claim 5, which is characterized in that step (5a) will mention
Sephadex chromatography column Sephadex LH-20 on object F3 is taken, the chloroform/methanol mixed solvent for being 1:1 with volume ratio is washed
It is de-, it stands eluent and powdered precipitating is precipitated, be separated by solid-liquid separation, after powdered precipitating is dissolved with organic solvent, upper MCI GEL
CHP20/P120 column is eluted with 75% methanol；Step (5b) is eluted with 80% methanol.
7. the method for separating-purifying Tamarix austro effective component according to claim 6, which is characterized in that step (6a) will mention
Upper sephadex chromatography column Sephadex LH-20 after taking object F5 to be concentrated, is mixed with the chloroform/methanol that volume ratio is 1:1
Solvent is eluted, upper normal phase silica gel chromatography column after collecting eluent and being concentrated, mixed using the chloroform/methanol that volume ratio is 8:1
Bonding solvent is eluted；Step (6b) upper sephadex chromatography column Sephadex LH-20 after extract F6 is concentrated,
The chloroform/methanol mixed solvent for being 1:1 with volume ratio is eluted, upper normal phase silica gel chromatography column after collecting eluent and being concentrated,
It is eluted using the chloroform/methanol mixed solvent that volume ratio is 6:1；Step (6c) is mixed with the chloroform/methanol that volume ratio is 1:1
Bonding solvent is eluted.
8. the method for separating-purifying Tamarix austro effective component according to claim 1 or claim 7, which is characterized in that in step (3)
After powdered precipitating is separated by filtration, washed using methanol；After being separated by filtration powdered precipitating in step (4a), use
Acetone is washed.
9. the method for separating-purifying Tamarix austro effective component according to claim 8, which is characterized in that step (4b) uses
During HPLC preparative chromatography, solvent be 30~50% methanol aqueous solution.
10. the method for separating-purifying Tamarix austro effective component according to claim 9, which is characterized in that step (5a) institute
Stating for the organic solvent of dissolved powders shape precipitating is DMSO；Step (5a), (5b) are recrystallized using methanol.
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| CN114651685A (en) * | 2022-04-28 | 2022-06-24 | 西藏自治区农牧科学院农业研究所 | Lodging-resistant stable-yield cultivation method for wheat |
| CN115340906A (en) * | 2022-09-16 | 2022-11-15 | 广西中烟工业有限责任公司 | Spice for increasing cocoa aroma characteristics of cigarette and preparation method thereof |
| CN115430174A (en) * | 2022-09-16 | 2022-12-06 | 广西中烟工业有限责任公司 | Fructus momordicae extract with characteristic flavor and preparation method thereof |
| CN115340906B (en) * | 2022-09-16 | 2023-10-20 | 广西中烟工业有限责任公司 | Spice for increasing cocoa aroma characteristics of cigarettes and preparation method thereof |
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