CN104974204B - The method and system of separating flavone oxygen glycosides compound monomer is extracted from Desmodium styracifolium - Google Patents
The method and system of separating flavone oxygen glycosides compound monomer is extracted from Desmodium styracifolium Download PDFInfo
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Abstract
The invention provides a kind of method that extraction separating genistein 7 O β D furans celery glycosyl (1 → 6) O β D glucopyranosides are compound shown in formula 1, this method comprises the following steps：Refluxing extraction is carried out to Desmodium styracifolium using ethanol, obtains Herba Desmodii Styracifolii extract；Multistage chromatography separation is carried out to extract using alcohol system, including, the first chromatographic isolation is carried out to extract using the first alcohol system, obtain the first separate section, the second chromatographic isolation is carried out to the first separate section using diol system, the second separate section is obtained, the 3rd chromatographic isolation is carried out to the second separate section using triol system, to obtain compound shown in the formula 1.Using the method for the present invention from Desmodium styracifolium compound shown in separation and Extraction formula 1, technique is simple, and the production time is short, high income, and purity is high, can obtain compound shown in the formula 1 of purity more than 95%.
Description
Technical field
The invention belongs to technical field of phytochemistry, is related to the extraction separating flavone oxygen glycosides compound list from Desmodium styracifolium
The method and system of body, specifically, extracting separating flavone oxygen glycosides compound monomer from Desmodium styracifolium the present invention relates to one kind
The method and system of genistein -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides.
Background technology
Desmodium styracifolium is pulse family beggar-ticks plant Desmodium styracifolium (Osbeck) Merr., and its is medicinal
Position is dry aerial parts, and main chemical compositions are the compounds such as flavones, saponin(e, polysaccharide, alkaloid.With removing damp-heat,
The effect of inducing diuresis for treating strangurtia.For heat gonorrhea, Sha Lin, urolithiasis, difficulty and pain in micturition, oedema oliguria, jaundice, lithangiuria.
Genistein -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides, its chemical structural formula is such as
Shown in formula 1, it is yellow powder, molecular formula：C26H28O14；Molecular weight：564；Compound shown in formula 1 is as follows：
R1=Glc (6 → 1) Api.
The compound is present in Desmodium styracifolium medicinal material, is one of effective substance important in Desmodium styracifolium, and content phase
To higher.Meanwhile compound shown in formula 1 can also differentiate as Desmodium styracifolium and the reference substance of assay, and for wide
The quality control of desmodium and its relevant pharmaceutical formulations such as Desmodium styracifolium total flavone capsule is used.Existing extraction separates the compound
Method and system urgently improves.
The content of the invention
It is contemplated that at least solve at least one technical problem of the prior art or offer one to a certain extent
Kind business selects.Therefore, it is an object of the present invention to propose that one kind extracts separating genistein -7-O- from Desmodium styracifolium
The method and system of β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides, utilizes the extraction separation method and system
Compound shown in obtained formula 1, purity is high, differentiates reference substance, the Yi Jizuo with assay advantageous as Desmodium styracifolium
For the quality control of Desmodium styracifolium and its relevant pharmaceutical formulations.
According to an aspect of of the present present invention, the present invention provides a kind of method for extracting compound shown in separate type 1, this method bag
Include following steps：Refluxing extraction is carried out to Desmodium styracifolium using ethanol, obtains Herba Desmodii Styracifolii extract；Using alcohol system to described
Extract carry out multistage chromatography separation, including, using the first alcohol system to the extract carry out the first chromatographic isolation, obtain
The first separate section is obtained, the second chromatographic isolation is carried out to first separate section using diol system, obtains the second separation
Part, the 3rd chromatographic isolation is carried out to second separate section using triol system, to obtain chemical combination shown in the formula 1
Thing.
This method of the present invention, is separated using multistage chromatography, including combines the macroporous resin purification technology that utilizes, and middle compacting is standby
And gel chromatography technology, using alcohol-water system, the compound shown in separation and Extraction formula 1 from Herba Desmodii Styracifolii extract, technique behaviour
To make simple, the production time is short, and product yield is high, and purity is high, can obtain purity and reach compound shown in more than 95% formula 1,
Compound shown in the formula 1 of acquisition can be used as assay chemical reference substance.The parameter bar of the processing step and each step
Part, be inventor consider, multiple Adjustment Tests macroreticular resin chromatogram, it is anti-phase it is middle pressure chromatogram and gel chromatography built-up sequence
And its respective elution testing conditions determine down to the influence for isolating and purifying effect of compound shown in the formula 1 in Desmodium styracifolium
Come, the extraction purification process is not related to the relatively strong organic solvent of toxicity, extracts separation operation process and extract is all safe
Reliably.
Fig. 1 be one embodiment of the present of invention in extraction separate type 1 shown in compound method step flow chart.Root
According to embodiments of the invention, compound extraction separation method shown in the formula 1 of the invention described above one side, there can also be following skill
At least one art feature：
According to one embodiment of present invention, it is described that refluxing extraction is carried out to Desmodium styracifolium using ethanol, obtain wide money
Careless extract, including：Refluxing extraction twice is carried out to the Desmodium styracifolium using concentration 50-95% ethanol, merging is returned twice
The extract solution of stream extraction gained, obtains and merges extract solution, removes the ethanol merged in extract solution, and/or remove the conjunction
And ethanol and ion in extract solution, and optional, it is dried, to obtain the extract.Concentration is 50-95% twice
Alcohol reflux extraction obtains extract, is that inventor gropes to determine by test of many times, raw material can be made full use of also to save
Save time, prepared beneficial to large-scale production；Remove extract solution in ethanol and/or ion, beneficial to exclude they to later separation
The interference of purifying.
According to one embodiment of present invention, the refluxing extraction twice, wherein ethanol and the weight of Desmodium styracifolium once
Amount is than being 12:1, the time of refluxing extraction is 1.5h-3h, optional for 2h, the ethanol of another time and the weight ratio of Desmodium styracifolium
For 10:1, the time of refluxing extraction is 1h-2h, optional for 1.5h.During backflow, concentration is 50-95% ethanol and Desmodium styracifolium
Ratio and return time, be inventor is determined by test of many times detection, and raw material can be made to obtain high extraction.
According to some embodiments of the present invention, the first alcohol system is ethanol-water system, in the ethanol-water system
The concentration of ethanol is 60-65%, preferably, the concentration of the ethanol in the ethanol-water system is 60%, first chromatogram is
Macroreticular resin.The concentration of ethanol is that inventor combines follow-up chromatographic separation condition, test of many times is examined in the alcohol-water eluent system
Compound ratio shown in the formula 1 surveyed in the peak eluted, determined by multiple adjusting and optimizing.
According to some embodiments of the present invention, the diol system is ethanol-water system, in the ethanol-water system
The concentration of ethanol is 20-40%, and second chromatogram is reverse chromatograms, preferably, the reverse chromatograms of selection are reversely middle pressure color
Spectrum, more preferably, C18 posts are selected, its post pressure is 1-10bar, and elution flow rate is 5~30ml/min.In the alcohol-water eluent system
Concentration of alcohol be ratio that inventor combines target component in the first separate section, and ethanol in the first chromatographic isolation-
Water elution system, test of many times optimization determines, beneficial in simple operations and short time, obtains the high formula 1 of high income, purity
Shown compound.
According to one embodiment of present invention, it is described that the second chromatogram point is carried out to the first separate section using diol system
From, the second separate section is obtained, including：First separate section is carried out using the ethanol-water system that concentration of alcohol is a etc.
Degree elution 28-35min, then use the ethanol-water system that concentration of alcohol is [a, b] instead and gradient is carried out to first separate section
180-220min is eluted, is collected to obtain second separate section, wherein, a span is 20-28%, b value model
Enclose for 35-40%.First with the ethanol-water system isocratic elution of a concentration, then carry out gradient elution, it is possible to increase separation carries
Take efficiency；The concentration of alcohol or concentration range of isocratic elution and gradient elution, and elution time, be inventor consider yield,
Ratio of target component etc. in time, the complexity of the separate section reclaimed, separate section, what test of many times determined.
As shown in Fig. 2 according to another embodiment of the invention, it is described that the first separate section is entered using diol system
The chromatographic isolation of row second, the second separate section is obtained, including：Using the ethanol-water system that concentration of alcohol is c to described first point
From part carry out isocratic elution 30min, then use instead concentration of alcohol be [c, d] ethanol-water system to first separation unit
Divide and carry out gradient elution 200min, collect and obtain primary second separate section；Utilize the ethanol-water system pair that concentration of alcohol is e
Primary second separate section carries out isocratic elution 30min, then uses the ethanol-water system pair that concentration of alcohol is [e, f] instead
Primary second separate section carries out gradient elution 200min, collects and obtains second separate section, wherein, c and e's takes
Value scope is that 20-28%, d and f span are 35-40%, e>C, d>f.It is front and rear twice, all first with the second of a concentration
Alcohol-water system isocratic elution, then carry out gradient elution, it is possible to increase the efficiency of compound shown in separation and Extraction formula 1；It is front and rear twice
Isocratic elution and the concentration of alcohol or concentration range of gradient elution, respective elution time, speed, be that inventor takes into consideration
Target component in the condition of isocratic twice-gradient elution, yield, time, the complexity of separate section of recovery, separate section
Ratio etc., test of many times optimization determines, beneficial in simple operations and short time, obtains shown in the high formula 1 of high income, purity
Compound.In the preferred embodiment of the present invention, c=20%, d=40%, e=28%, f=35%, it can make final
The purity of the extraction purification product of acquisition is up to more than 95%.
According to another embodiment of the invention, the triol system is pure methanol, and the 3rd chromatogram is gel color
Spectrum.In one embodiment of the invention, selected gel chromatography is sephadex chromatography Sephadex LH-20.It is beneficial into one
Step improves the purity of target compound.
According to another aspect of the present invention, the present invention provides a kind of system for extracting compound shown in separate type 1, the system
Can be to implement compound extraction separation method shown in formula 1 of the invention described above on the one hand or in any embodiment, this is
System includes：Refluxing extraction device, to carry out refluxing extraction to Desmodium styracifolium using ethanol, obtain Herba Desmodii Styracifolii extract；Color
Separator is composed, for carrying out multistage chromatography separation to the extract using alcohol system, it includes, the first chromatographic isolation list
Member, it is connected with the refluxing extraction device, for carrying out the first chromatographic isolation to the extract using the first alcohol system, obtains
First separate section, the second chromatographic isolation unit, it is connected with first chromatographic isolation unit, for utilizing diol system pair
First separate section carries out the second chromatographic isolation, obtains the second separate section, the 3rd chromatographic isolation unit, with described second
Chromatographic isolation unit is connected, for carrying out the 3rd chromatographic isolation to second separate section using triol system, to obtain
Compound shown in the formula 1.Fig. 3 shows the structure of the extraction piece-rate system 1000 in one embodiment of the present of invention, including returns
Extraction element 100 and chromatographic separation device 200 are flowed, chromatographic separation device includes the first chromatographic isolation unit 202, the second chromatogram point
From the chromatographic isolation unit 206 of unit 204 and the 3rd.Compound extraction separation method shown in the above-mentioned formula 1 to one aspect of the present invention
Technical characteristic and advantage description, the system of equally applicable this aspect of the present invention, will not be repeated here.People in the art
Member is it is appreciated that the system can also include other devices, sub-device or functional unit to implement above-mentioned corresponding implementation
Example.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination accompanying drawings below to embodiment
Substantially and it is readily appreciated that, wherein：
Fig. 1 show extraction separating genistein -7-O- β-D- furans celerys glycosyl in one embodiment of the present of invention-(1 →
6) the step flow of the method for-O- β-D- glucopyranosides；
Fig. 2 show extraction separating genistein -7-O- β-D- furans celerys glycosyl in one embodiment of the present of invention-(1 →
6) the step flow of the method for-O- β-D- glucopyranosides；
Fig. 3 show extraction separating genistein -7-O- β-D- furans celerys glycosyl in one embodiment of the present of invention-(1 →
6) structural representation of the system of-O- β-D- glucopyranosides；
Fig. 4 show extraction separating genistein -7-O- β-D- furans celerys glycosyl in one embodiment of the present of invention-(1 →
6) flow chart of the method for-O- β-D- glucopyranosides；
Fig. 5 shows the ESI-MS mass spectrograms of the extraction purification product in one embodiment of the invention；
Fig. 6 shows the H of the extraction purification product in one embodiment of the invention1- NMR spectra；And
Fig. 7 shows the C of the extraction purification product in one embodiment of the invention13- NMR spectra.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, is only used for explaining the present invention, and is not considered as limiting the invention.
It should be noted that term " first ", " second " are only used for describing purpose, and it is not intended that instruction or hint phase
To importance or the implicit quantity for indicating indicated technical characteristic.Thus, define " first ", the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the invention, unless otherwise saying
Bright, " multiple " are meant that two or more.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carried out according to the technology described by document in the art or condition or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, being can be by the conventional products of acquisition purchased in market.
Embodiment 1
Fig. 4 shows the general flow of the inventive method.
1. raw material：From legume Desmodium styracifolium 45000-50000 parts by weight, 5-10cm segments are cut into, drinking water is clear
Wash silt off, drain, feed intake.
2. ethanol extracts：80% alcohol reflux extracts twice, for the first time 12 times of amounts 2h, second of 10 times of amount 1.5h；Merge
Alcohol extract twice, recovery ethanol is to without alcohol taste.
3. it is concentrated under reduced pressure：Extraction concentrate is diluted with water to 5 times of volumes of medicinal material weight；200 mesh filter-cloth filterings；Obtain upper prop
Liquid；The net product resin crossed in advance with 95% Ethanol Treatment is filled into post, purified water is replaced；Upper prop liquid is squeezed into post with pump, adsorbed,
0.5-1 times of BV/h of coutroi velocity, after absorption, stand 2h；Rinsed with purified water, elution flow rate 3-4BV/h, elution amount
10BV；Again with 60% ethanol elution, elution flow rate 2-3BV/h, elution amount 5BV；60% eluent merges, and squeezes into concentration tank, returns
Ethanol is received to without alcohol taste, the 50-70 DEG C of drying into vacuum drying chamber, obtains extensively golden bulk drug 300-400 parts by weight.
4. multistage chromatography separates
(1) the wide golden bulk drug 300-400 parts by weight AB-8 macroreticular resin chromatographic isolations of general, i.e. chromatographic isolation I, specifically
For：With ethanol-water mixed solvent 20：80,30:70,40:60,60:40 gradient elutions, each gradient are flushed to no color, close
And each fraction obtains 20%, 30%, 40%, 60% totally four positions.
(2) the anti-phase middle pressure chromatographic isolations of 60% position ODS obtained chromatographic isolation I, i.e. chromatographic isolation II, specifically
For：With alcohol-water, condition is 20% isocratic 30min, 20-40% gradients 200min elutions, according to chromatogram peak sequence, is divided
Part is collected, and collects portion per 100-150 parts by volume, altogether 40-50 fraction of collection, and high performance liquid chromatography inspection merges phase cocurrent flow after knowing
Part.
(3) the 36-50 fractions for obtaining chromatographic isolation II merge, further with the anti-phase middle pressure chromatographic isolations of ODS, i.e. chromatogram
Separating III, it is specially：With alcohol-water, condition is 28% isocratic 30min, and 28-35% gradients 200min is eluted, according to chromatogram
Peak sequence, portioning are collected, and portion is collected per 100-150 parts by volume, collect 35-45 fraction altogether, and high performance liquid chromatography inspection is known
After merge identical fraction.
(4) the 30-35 fractions for obtaining chromatographic isolation III merge, and are concentrated under reduced pressure to give the crude product of compound shown in formula 1
(purity 90%).Gel chromatography separation is finally used, i.e. chromatographic isolation IV, concrete operations are：By compound shown in formula 1 add to
Methanol-water (volume ratio 2:1) in, ultrasonic dissolution.Selected gel chromatography is sephadex chromatography Sephadex LH-20, is entered
Sample volume is 10 milliliters, rushes post with pure methanol, portioning is collected, and every 5 parts by volume collects portion, collects 20-30 fraction altogether, efficiently
Liquid chromatogram inspection merges identical fraction after knowing；The 15-25 fractions that chromatographic isolation IV obtains merge, and recycling design obtains yellow powder
End.
Wherein, (2) and (3) are that chromatographic isolation II and chromatographic isolation III are to utilize the anti-phase middle pressure chromatograms of ODS, in correspondence
State the second alleged chromatographic isolation.
5. Structural Identification
Through electrospray ionization mass spectrometry (ESI-MS) and spectroscopic methodology (H1- NMR and C13- NMR) reflected to obtaining yellow powder
It is fixed, with reference to ESI-MS, H1- NMR and C13The molecular formula of-NMR spectra information inference the compound is C26H28O14, it is determined that of the invention
The yellow powder isolated that extracted from Desmodium styracifolium is genistein -7-O- β-D- furans celerys glycosyls-(1 → 6)-O- β -
D- glucopyranosides (compound shown in formula 1), through HPLC purity checks, purity reaches more than 95%, and it is as Desmodium styracifolium
Differentiate the reference substance with assay, and the matter of Desmodium styracifolium and its relevant pharmaceutical formulations such as Desmodium styracifolium total flavone capsule
Amount control is used.
Fig. 5 is the mass spectrogram that compound shown in formula 1 carries out electrospray ionization mass spectrometry, shows point of each composition in yellow powder
Son amount, principal component molecular weight is ESI-MS m/z:565[M+H]+。
The H of compound shown in Fig. 6 display types 11- NMR spectra, H1- NMR (DMSO, 600HZ) δ：12.92(5-OH),8.39
(1H, s, H-3), 7.41 (2H, d, J=8.4Hz, H-2 ', 6 '), 6.84 (2H, d, J=8.4Hz, H-3,5), 6.73 (1H, d, J
=1.8Hz, H-8), 6.47 (1H, d, J=1.9Hz, H-6).
The C of compound shown in Fig. 7 display types 113- NMR spectra, C13- NMR (DMSO, 125HZ) δ:154.6(C-2),
122.5(C-3),180.5(C-4),161.6(C-5),99.6(C-6),162.9(C-7),94.6(C-8),157.3(C-9),
109.4(C-10),121.0(C-1’),130.2(C-2’6’),115.1(C-3’5’),157.5(C-4’),94.6(C-1”of
Glc), 73.0 (C-2 " of Glc), 76.4 (C-3 ' of Glc), 69.9 (C-4 " of Glc), 75.6 (C-5 " of Glc), 67.7
(C-6 " of Glc), 99.8 (C-1 " ' of Api), 75.9 (C-2 " ' ofApi), 78.7 (C-3 " ' of Api), 73.3 (C-4 " '
Of Api), 63.2 (C-5 " ' of Api).Wherein described Api represents celery sugar, and Glc represents glucose.
Embodiment 2
1st, the step 1-3 of embodiment 1 is carried out.
2nd, multistage chromatography separates
(1) the wide golden bulk drug 300-400 parts by weight AB-8 macroreticular resin chromatographic isolations of general, i.e. chromatographic isolation I, ethanol-
Water mixed solvent 25:75,35:65,45:55,65:35 gradient elutions, each gradient are flushed to no color, merge each fraction and obtain
25%, 35%, 45%, 65% totally four positions；
(2) the anti-phase middle pressure chromatographic isolations of 65% position ODS obtained chromatographic isolation I, i.e. chromatographic isolation II, use second
Alcohol-water, condition are 20% isocratic 30min, and 20-40% gradients 200min elutions, according to chromatogram peak sequence, portioning is collected,
Portion is collected per 100-150 parts by volume, collects 40-50 fraction altogether, high performance liquid chromatography inspection merges identical fraction after knowing；
(3) the 36-50 fractions for obtaining chromatographic isolation II merge, further with the anti-phase middle pressure chromatographic isolations of ODS, i.e. chromatogram
Separating III, with alcohol-water, condition is 25% isocratic 30min, and 25-35% gradients 200min elutions are suitable according to chromatogram appearance
Sequence, portioning are collected, and portion is collected per 100-150 parts by volume, collect 35-45 fraction altogether, and high performance liquid chromatography inspection merges after knowing
Identical fraction；
(4) the 30-35 fractions for obtaining chromatographic isolation III merge, and are recycled to solvent and obtain crude compound shown in formula 1
(purity 85%).Gel chromatography separation is finally used, i.e. chromatographic isolation IV, concrete operations are：By compound shown in formula 1 add to
Methanol-water (volume ratio 1:1) in, ultrasonic dissolution.Selected gel chromatography is Sephadex G-50, and sampling volume is 10 milliliters,
Post is rushed with pure methanol, portioning is collected, and every 5 parts by volume collects portion, 20-30 fraction is collected altogether, after high performance liquid chromatography inspection is known
Merge identical fraction；The 15-25 fractions that chromatographic isolation IV obtains merge, and recycling design obtains the powder of yellow, utilizes EFI
Mist MALDI-MS (ESI-MS) and spectroscopic methodology (H1- NMR and C13- NMR) identify obtaining yellow powder, identify yellow powder
For compound shown in formula 1, purity 89%.
Compound shown in the yellow powder formula 1 that the process conditions of embodiment 2 are produced, purity are divided significantly lower than embodiment 1
From compound shown in the formula 1 of extraction.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of departing from the principle and objective of the present invention a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The scope of invention is limited by claim and its equivalent.
Claims (10)
1. the side of one kind extraction separating genistein -7-O- β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides
Method, it is characterised in that comprise the following steps：
Refluxing extraction is carried out to Desmodium styracifolium using ethanol, obtains Herba Desmodii Styracifolii extract；
Multistage chromatography separation is carried out to the extract using alcohol system, including,
The first chromatographic isolation is carried out to the extract using the first alcohol system, obtains the first separate section,
The second chromatographic isolation is carried out to first separate section using diol system, obtains the second separate section,
The 3rd chromatographic isolation is carried out to second separate section using triol system, to obtain the genistein -7-O-
β-D- furans celerys glycosyl-(1 → 6)-O- β-D- glucopyranosides,
The concentration of alcohol is 50-95%；
The first alcohol system is ethanol-water system, and the concentration of the ethanol in the ethanol-water system is 60-65%；
First chromatogram is macroreticular resin；
The diol system is ethanol-water system, and the concentration of the ethanol in the ethanol-water system is 20-40%；
Second chromatogram is C18 posts reversely middle pressure chromatogram；
The triol system is pure methanol, and the 3rd chromatogram is gel chromatography.
2. according to the method for claim 1, it is characterised in that it is described that refluxing extraction is carried out to Desmodium styracifolium using ethanol,
Herba Desmodii Styracifolii extract is obtained, including：
Refluxing extraction twice is carried out to the Desmodium styracifolium using ethanol, merges the extract solution obtained by refluxing extraction twice, obtains
Merge extract solution,
The ethanol in the merging extract solution is removed, and/or removes the ethanol and ion merged in extract solution, to obtain
State extract.
3. according to the method for claim 2, it is characterised in that further comprise：It will remove in the merging extract solution
Extract solution obtained by ethanol, and/or the removing ethanol and ion merged in extract solution is dried, to be carried described in acquisition
Take thing.
4. according to the method for claim 2, it is characterised in that the refluxing extraction twice, wherein
Ethanol and the weight ratio of Desmodium styracifolium once is 12:1, the time of refluxing extraction is 1.5h-3h,
The ethanol of another time and the weight ratio of Desmodium styracifolium are 10:1, the time of refluxing extraction is 1h-2h.
5. according to the method for claim 4, it is characterised in that the time of the refluxing extraction once is 2h.
6. according to the method for claim 4, it is characterised in that the time of the refluxing extraction of described another time is 1.5h.
7. according to the method for claim 1, it is characterised in that the concentration of the ethanol in first ethanol-water system is
60%.
8. according to the method for claim 1, it is characterised in that the post pressure of the reversely middle pressure chromatogram is 1-10bar, elution
Flow velocity is 5~30ml/min.
9. according to the method for claim 1, it is characterised in that described that the first separate section is carried out using diol system
Second chromatographic isolation, the second separate section is obtained, including：
Isocratic elution 28-35min is carried out to first separate section using the ethanol-water system that concentration of alcohol is 20-28%,
Then use the ethanol-water system that concentration of alcohol is 20~40% instead and gradient elution 180- is carried out to first separate section
220min, collect to obtain second separate section.
10. according to the method for claim 1, it is characterised in that described that the first separate section is entered using diol system
The chromatographic isolation of row second, the second separate section is obtained, including：
Isocratic elution 30min is carried out to first separate section using the ethanol-water system that concentration of alcohol is 20-28%, connect
The ethanol-water system for using concentration of alcohol instead as 20-40% and gradient elution 200min is carried out to first separate section, collect
Primary second separate section is obtained,
Isocratic elution is carried out to primary second separate section using the ethanol-water system that concentration of alcohol is 20-28%
30min, then use the ethanol-water system that concentration of alcohol is 20-40% instead and the primary second separate section progress gradient is washed
De- 200min, collects and obtains second separate section, wherein,
The concentration of alcohol that isocratic elution is carried out to first separate section is less than and primary second separate section carried out etc.
Spend the concentration of alcohol of elution；
The maximum that the span of the concentration of alcohol of gradient elution is carried out to first separate section is more than to the primary
Second separate section carries out the maximum of the span of the concentration of alcohol of gradient elution, and ladder is carried out to first separate section
The minimum value for spending the span of the concentration of alcohol of elution is less than the second that gradient elution is carried out to primary second separate section
The minimum value of the span of determining alcohol.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL205635B1 (en) * | 2001-04-09 | 2010-05-31 | Inst Farmaceutyczny | Novel derivatives of genistein and pharmacological agents containing such derivatives |
| EP2202237A1 (en) * | 2008-12-23 | 2010-06-30 | Libragen | Hydrosoluble [6)-O-alpha-d-glcp-(1->]n-6-o-bêta-d-glcp-(1->-phenllic derivatives with dermocosmetic, nutritional and therapeutic applications, and compositions containing said water soluble compounds |
| CN102329312A (en) * | 2011-07-26 | 2012-01-25 | 苏州宝泽堂医药科技有限公司 | Method for purifying schaftoside |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL205635B1 (en) * | 2001-04-09 | 2010-05-31 | Inst Farmaceutyczny | Novel derivatives of genistein and pharmacological agents containing such derivatives |
| EP2202237A1 (en) * | 2008-12-23 | 2010-06-30 | Libragen | Hydrosoluble [6)-O-alpha-d-glcp-(1->]n-6-o-bêta-d-glcp-(1->-phenllic derivatives with dermocosmetic, nutritional and therapeutic applications, and compositions containing said water soluble compounds |
| CN102329312A (en) * | 2011-07-26 | 2012-01-25 | 苏州宝泽堂医药科技有限公司 | Method for purifying schaftoside |
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| Biochanin A triglycoside from Andira inermis;B.P. da Silva，等;《Fitoterapia》;20001231;第71卷;第664页 * |
| 通脉方化学成分;王付荣，等;《中国实验方剂学杂志》;20111031;第17卷(第20期);第63-64页 * |
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