CN105085498B - The method and system of extraction separation isovitexin from Desmodium styracifolium - Google Patents
The method and system of extraction separation isovitexin from Desmodium styracifolium Download PDFInfo
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Abstract
The present invention provides a kind of methods of extraction separation isovitexin, and this approach includes the following steps：Refluxing extraction is carried out to Desmodium styracifolium using ethyl alcohol, 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 using the first separate section of diol system pair, the second separate section is obtained, third chromatographic isolation is carried out using third alcohol the second separate section of system pair, to obtain the isovitexin.The present invention also provides a kind of systems for implementing the above method.Using method and/or system the separation and Extraction isovitexin from Desmodium styracifolium of the present invention, technological operation is simple, and the production time is short, and product yield is high, and purity is high, can obtain the isovitexin that purity reaches 99.7%.
Description
Technical field
The invention belongs to technical field of phytochemistry, and separation is extracted from Desmodium styracifolium specifically, the present invention relates to one kind
The method and system of isovitexin.
Background technology
Desmodium styracifolium is pulse family beggar-ticks plant Desmodium styracifolium (Osbeck) Merr., 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.
Isovitexin (isovitexin) is also referred to as Saponaretin or 6-C- glucose apiolins, is yellow powder；Point
Minor：C21H22O10；Molecular weight：432；Fig. 1 shows its chemical structural formula.
Isovitexin is present in Desmodium styracifolium medicinal material, differentiates the reference substance with assay as Desmodium styracifolium, with
And the quality control of Desmodium styracifolium and its relevant pharmaceutical formulations such as Desmodium styracifolium total flavone capsule is used.Existing extraction detaches different Vitex negundo var cannabifolia
The method and system of element urgently improves.
Invention content
The present invention is directed to solve at least one the problems of the prior art at least to a certain extent or provide a kind of quotient
Industry selects.For this purpose, the method for an object of the present invention is to provide a kind of from Desmodium styracifolium extraction separation isovitexin and
System, the isovitexin obtained using the extraction separation method and system, purity is high, differentiates and contains advantageous as Desmodium styracifolium
Measure the reference substance used surely and the quality control as Desmodium styracifolium and its relevant pharmaceutical formulations.
One side according to the present invention, the present invention provide it is a kind of extraction separation isovitexin method, this method include with
Lower step：Refluxing extraction is carried out to Desmodium styracifolium using ethyl alcohol, obtains Herba Desmodii Styracifolii extract；Using alcohol system to the extraction
Object carries out multistage chromatography separation, including carrying out the first chromatographic isolation to the extract using the first alcohol system, obtain the
One separate section carries out the second chromatographic isolation to first separate section using diol system, obtains the second separate section,
Third chromatographic isolation is carried out to second separate section using third alcohol system, to obtain the isovitexin.
This method of the present invention, is detached using multistage chromatography, including combination utilizes macroporous resin purification technology, middle compacting standby
And gel chromatography technology, using alcohol-water system, the separation and Extraction isovitexin from Herba Desmodii Styracifolii extract, technological operation letter
Single, the production time is short, and product yield is high, and purity is high, can obtain the isovitexin that purity reaches 99.7%, the different Vitex negundo var cannabifolia of acquisition
Element can be used as the reference substance of Desmodium styracifolium discriminating and assay.The Parameter Conditions of the processing step and each step are
Inventor considers, the combination and its respectively of chromatography and gel chromatography is pressed in multiple Adjustment Tests macroreticular resin chromatography, reverse phase
Elution requirement the influence for isolating and purifying effect of the isovitexin in Desmodium styracifolium is decided, the extraction purification mistake
Journey is not related to the relatively strong organic solvent of toxicity, extracts separation operation process and extract is all safe and reliable.
Fig. 2 shows the step flow chart of the method for compound shown in extraction separation Fig. 1 in one embodiment of the present of invention.
According to an embodiment of the invention, the isovitexin extraction separation method of aforementioned present invention one side can also have following technology
At least one feature：
According to one embodiment of present invention, described that refluxing extraction is carried out to Desmodium styracifolium using ethyl alcohol, obtain wide money
Careless extract, including：Refluxing extraction twice is carried out to the Desmodium styracifolium using the ethyl alcohol of concentration 50-95%, merging is returned twice
The extracting solution of stream extraction gained, obtains and merges extracting solution, removes the ethyl alcohol merged in extracting solution, and/or remove the conjunction
And the ethyl alcohol in extracting solution and ion, and optional, it is dried, to obtain the extract.A concentration of 50-95% twice
Alcohol reflux extraction obtains extract, is that inventor gropes determination by test of many times, can make full use of raw material that can also save
It saves time, is conducive to large-scale production and preparation；The ethyl alcohol and/or ion in extracting solution are removed, is conducive to exclude them to later separation
The interference of purifying.
According to one embodiment of present invention, the refluxing extraction twice, wherein the weight of primary ethyl alcohol and Desmodium styracifolium
Amount is than being 12:1, the time of refluxing extraction is 1.5h-3h, is optionally 2h, the weight ratio of another ethyl alcohol and Desmodium styracifolium
It is 10:1, the time of refluxing extraction is 1h-2h, is optionally 1.5h.When reflux, a concentration of 50-95% ethyl alcohol and Desmodium styracifolium
Ratio and return time, be inventor determining by test of many times detection, 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
A concentration of 50-65% of ethyl alcohol, preferably, a concentration of 60% of ethyl alcohol in the ethanol-water system, first chromatography is
Macroreticular resin.The concentration of ethyl alcohol is that inventor combines follow-up chromatographic separation condition, test of many times to examine in the alcohol-water eluent system
The isovitexin ratio in the peak eluted is surveyed, is 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
A concentration of 18-40% of ethyl alcohol, preferably, being 20-40%.Second chromatography is reverse chromatograms, preferably, selection is reversed
Chromatography is reversed middle pressure chromatography, and more preferably, it is 1-10bar to select C18 columns, column pressure, and elution flow rate is 5~30ml/min.It should
Concentration of alcohol in alcohol-water eluent system is the ratio and first that inventor combines target component in the first separate section
Alcohol-water eluent system in chromatographic isolation, test of many times optimization determination, it is conducive in simple operations and short time, obtains
The high isovitexin of high income, purity.
According to one embodiment of present invention, described to carry out the second chromatography point using the first separate section of diol system pair
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 25-40min then uses the ethanol-water system that concentration of alcohol is [a, b] instead and carries out gradient to first separate section
180-250min is eluted, is collected to obtain second separate section, wherein the value range of a is 18-28%, the value model of b
It encloses for 35-40%.First with the ethanol-water system isocratic elution of a concentration, then gradient elution is carried out, separation can be improved and carried
Take efficiency；The concentration of alcohol or concentration range and elution time of isocratic elution and gradient elution, be inventor consider yield,
The ratio etc. of target component in time, the complexity of separate section of recycling, separate section, what test of many times determined.
As shown in figure 3, according to another embodiment of the invention, it is described using the first separate section of diol system pair into
The second chromatographic isolation of row obtains the second separate section, 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, collects 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's and e takes
The value range for being worth ranging from 18-28%, d and f is 35-40%, e>C, d>f.It is front and back twice, all first with the second of a concentration
Alcohol-water system isocratic elution, then gradient elution is carried out, the efficiency of separation and Extraction isovitexin can be improved；It is front and back twice etc.
The concentration of alcohol or concentration range, respective elution time, speed of degree elution and gradient elution, are that inventor takes into consideration twice
The ratio of target component in the condition of isocratic-gradient elution, yield, time, the complexity of separate section of recycling, separate section
Deng, test of many times optimization determination, it is conducive in simple operations and short time, obtains the high isovitexin of high income, purity.?
In the preferred embodiment of the present invention, c=20%, e=25%, d=40%, f=35% can make the product finally obtained
Purity reaches 99.7%.
According to another embodiment of the invention, the third alcohol system is pure methanol, and the third chromatography is gel color
Spectrum.In one embodiment of the invention, selected gel chromatography is sephadex chromatography Sephadex LH-20.Conducive into one
Step improves the purity of target compound.
Another aspect according to the present invention, the present invention provide a kind of system of extraction separation isovitexin, which can
To implement isovitexin extraction separation method of the aforementioned present invention on the one hand or in any embodiment, which includes：It returns
Extraction element is flowed, to carry out refluxing extraction to Desmodium styracifolium using ethyl alcohol, obtains Herba Desmodii Styracifolii extract；Chromatographic isolation fills
Set, for using alcohol system to extract progress multistage chromatography separation comprising, the first chromatographic isolation unit is and described
Refluxing extraction device is connected, and for carrying out the first chromatographic isolation to the extract using the first alcohol system, obtains the first separation
Part, the second chromatographic isolation unit are connected with first chromatographic isolation unit, for utilizing diol system to described first
Separate section carries out the second chromatographic isolation, obtains the second separate section, third chromatographic isolation unit, with second chromatographic isolation
Unit is connected, described different male to obtain for carrying out third chromatographic isolation to second separate section using third alcohol system
Jing Su.Fig. 4 shows the structure of the extraction piece-rate system 1000 in one embodiment of the present of invention, including refluxing extraction device 100
With chromatographic separation device 200, chromatographic separation device includes the first chromatographic isolation unit 202, the second chromatographic isolation unit 204 and the
Three chromatographic isolation units 206.The technical characteristic of the above-mentioned isovitexin extraction separation method to one aspect of the present invention and advantage
Description, the system of equally applicable this aspect of the present invention, details are not described herein.It will be understood by those skilled in the art that the system
It can also implement above-mentioned corresponding embodiment including other devices, sub-device or functional unit.
According to the present invention in another aspect, the present invention also provides it is a kind of can extract simultaneously separation Desmodium styracifolium in it is a variety of
The method of compound monomer, alleged compound monomer further include Vicenin-1, Vicenin-2, Xia Fo except including isovitexin
At least one of tower glycosides and isovitexin.This approach includes the following steps：Refluxing extraction is carried out to Desmodium styracifolium using ethyl alcohol,
Obtain Herba Desmodii Styracifolii extract；Multistage chromatography separation is carried out to the extract using alcohol system, including utilizing different the
One alcohol system carries out the first chromatographic isolation to the extract, obtains multiple first separate sections, is distinguished using diol system
Second chromatographic isolation is carried out to first separate section, corresponding second separate section is obtained, using third alcohol system to institute
It states the second separate section and carries out third chromatographic isolation, to obtain corresponding compound monomer respectively.
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
Obviously, or practice through the invention is recognized.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination following accompanying drawings to embodiment
Obviously and it is readily appreciated that, wherein：
Fig. 1 shows the chemical structural formula of isovitexin；
Fig. 2 shows the step flow of the method for compound shown in extraction separation Fig. 1 in one embodiment of the present of invention；
Fig. 3 shows the step flow of the method for compound shown in extraction separation Fig. 1 in one embodiment of the present of invention；
Fig. 4 shows the structural schematic diagram of the system of compound shown in extraction separation Fig. 1 in one embodiment of the present of invention；
Fig. 5 shows the flow chart of the method for compound shown in extraction separation Fig. 1 in one embodiment of the present of invention；
Fig. 6 shows the ESI-MS mass spectrograms of the extraction purification product in one embodiment of the invention；
Fig. 7 shows the H of the extraction purification product in one embodiment of the invention1NMR spectra；And
Fig. 8 shows the C of the extraction purification product in one embodiment of the invention13NMR spectra.
Specific implementation mode
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, and 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 used for description purposes only, it is not understood to indicate or imply phase
To importance or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the present invention, unless otherwise saying
Bright, the meaning of " plurality " is 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.Particular technique or item are not specified in embodiment
Part, it is carried out according to technology or condition described in document in the art or according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Embodiment 1
Fig. 5 shows the general flow of the method for the present invention, including following：
1, feedstock processing：Legume Desmodium styracifolium 45000-50000 parts by weight are selected, 5-10cm segments is cut into, drinks
Water washes silt, drains, and feeds intake.
2, ethyl alcohol 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, recycling ethyl alcohol to no 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% alcohol treatment is filled into column, purified water is replaced；Upper prop liquid pump is squeezed into column, is adsorbed,
0.5-1 times of BV/h of coutroi velocity after absorption, stands 2h；It is 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
Ethyl alcohol is received to no alcohol taste, until 50-70 DEG C of drying in vacuum drying chamber, obtains extensively golden bulk pharmaceutical chemicals 300-400 parts by weight.
4, three-level chromatographic isolation
(1) by wide golden bulk pharmaceutical chemicals 300-400 parts by weight AB-8 macroreticular resin chromatographic isolations, i.e. chromatographic isolation I：Ethyl alcohol-
Water mixed solvent 20：80,30:70,40:60,60:40 gradient elutions, each gradient are flushed to no color, merge each fraction and obtain
20%, 30%, 40%, 60% totally four positions.
(2) 60% position that chromatography I is obtained is with pressing chromatographic isolation, i.e. chromatographic isolation II in ODS reverse phases：With alcohol-water, item
Part is 20% isocratic 30min, and the 200min elutions of 20-40% gradients, according to chromatogram peak sequence, portioning is collected, per 100-150
Parts by volume collects portion, collects 40-50 fraction altogether, and high performance liquid chromatography inspection merges identical fraction after knowing；Chromatographic isolation II
The 25-35 fractions that arrive merge, further with pressing chromatographic isolation, i.e. chromatographic isolation III in ODS reverse phases：With alcohol-water, condition is
25% isocratic 30min, the 200min elutions of 25-35% gradients, according to chromatogram peak sequence, portioning is collected, per 100-150 volumes
Part collects portion, collects 35-45 fraction altogether, merges identical fraction after high performance liquid chromatography inspection knowledge.
(3) the 20-35 fractions that chromatographic isolation III obtains merge, and are concentrated to dryness, obtain isovitexin crude product.Finally
(Sephadex LH-20) is detached with sephadex chromatography, i.e. chromatographic isolation IV：Isovitexin is added to methanol-water (body
Product is than being 1:1) in, ultrasonic dissolution, sampling volume is 10 milliliters, rushes column with pure methanol, portioning is collected, and every 5 parts by volume collects one
Part, 20-30 fraction is collected altogether, and high performance liquid chromatography inspection merges identical fraction after knowing；The 10-20 streams that chromatographic isolation IV obtains
Part merges, and recycling design obtains yellow powder.
5, it identifies
Through electrospray ionization mass spectrometry (ESI-MS) and spectroscopic methodology (H1- NMR and C13- NMR) to obtained yellow powder substance
It is identified, in conjunction with ESI-MS, H1- NMR and C13The molecular formula of-NMR information inferences the compound is C21H20O10, determine above-mentioned
It is isovitexin that the yellow powder isolated is extracted from Desmodium styracifolium, and through HPLC purity checks, purity reaches 99.7%, can make
Differentiate for Desmodium styracifolium and the reference substance used of assay and as the quality control of Desmodium styracifolium and its relevant pharmaceutical formulations
With.
Fig. 6 is the mass spectrogram that electrospray ionization mass spectrometry is carried out to extraction separation product isovitexin, is shown in yellow powder
The molecular weight of the molecular weight of each ingredient, principal component is ESI-MS m/z:433[M+H]+。
The H of Fig. 7 display extraction separation product isovitexins1NMR spectra, H1- NMR (DMSO, 400HZ) δ：13.56(5-
OH), 7.93 (2H, d, J=8.4Hz, H-2 ', 6 '), 6.92 (2H, d, J=8.4Hz, H-2 ', 6 '), 6.50 (1H, s, H-3).
The C of Fig. 8 display extraction separation product isovitexins13NMR spectra, C13- NMR (DMSO, 100HZ) δ:163.5(C-
2),102.7(C-3),181.9(C-4),160.7(C-5),108.9(C-6),163.2(C-7),93.6(C-8),156.2(C-
9),103.3(C-10),121.1(C-1’),128.5(C-2’6’),116.0(C-3’5’),161.2(C-4’),73.1(C-1”
Of Glc), 70.6 (C-2 " of Glc), 79.0 (C-3 " of Glc), 70.2 (C-4 " of Glc), 81.5 (C-5 " of Glc),
61.5 (C-6 " of Glc), wherein Glc represents glucose.
Embodiment 2
Legume Desmodium styracifolium 45000-50000 parts by weight are selected, are cut into 5-10cm segments, drinking water washes mud
Sand drains, and feeds intake.80% alcohol reflux extracts twice, for the first time 12 times of amounts 2h, second of 10 times of amount 1.5h；Merge alcohol twice
Extract, recycling ethyl alcohol to no alcohol taste.Extraction concentrate is diluted with water to 5 times of volumes of medicinal material weight；200 mesh filter-cloth filterings；?
Upper prop liquid；The net product resin crossed in advance with 95% alcohol treatment is filled into column, purified water is replaced；Upper prop liquid pump is squeezed into column,
Absorption, 0.5-1 times of BV/h of coutroi velocity after absorption, stand 2h；It is rinsed, elution flow rate 3-4BV/h, is eluted with purified water
Measure 10BV；Again with 60% ethanol elution, elution flow rate 2-3BV/h, elution amount 5BV；60% eluent merges, and squeezes into concentration tank,
Ethyl alcohol is recycled to no alcohol taste, until 50-70 DEG C of drying in vacuum drying chamber, obtains extensively golden bulk pharmaceutical chemicals 300-400 parts by weight.
It will extensively golden bulk pharmaceutical chemicals 300-400 parts by weight AB-8 macroreticular resin chromatographic isolations, i.e. chromatographic isolation I, alcohol-water mix
Bonding 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；65% position that chromatography I is obtained is with pressing chromatographic isolation, i.e. chromatography in ODS reverse phases
Separation II, with alcohol-water, condition is 18% isocratic 300min, and the 200min elutions of 18-40% gradients are suitable according to chromatogram appearance
Sequence, portioning are collected, and portion is collected per 100-150 parts by volume, collect 40-50 fraction altogether, and high performance liquid chromatography inspection merges after knowing
Identical fraction；25-40 fractions that chromatographic isolation II obtains merge, further with pressing chromatographic isolation, i.e. chromatographic isolation in ODS reverse phases
III, with alcohol-water, condition is 22% isocratic 30min, and the 200min elutions of 22-35% gradients divide according to chromatogram peak sequence
Part is collected, and collects portion per 100-150 parts by volume, altogether 35-45 fraction of collection, and high performance liquid chromatography inspection merges phase cocurrent flow after knowing
Part；The 20-40 fractions that chromatographic isolation III obtains merge, and are recycled to solvent and obtain isovitexin crude product.It is finally solidifying with glucan
Glue chromatographic isolation (Sephadex G-75), i.e. chromatographic isolation IV, specially：Isovitexin is added to methanol-water (volume ratio
It is 2:1) in, ultrasonic dissolution, sampling volume is 10 milliliters, and column is rushed with pure methanol, and portioning is collected, and every 5 parts by volume collects portion, altogether
20-30 fraction is collected, high performance liquid chromatography inspection merges identical fraction after knowing；The 10-26 fractions that chromatographic isolation IV obtains are closed
And recycling design obtains the powder of yellow.Utilize electrospray ionization mass spectrometry (ESI-MS) and spectroscopic methodology (H1-NMR and C13-NMR)
It identifies obtaining yellow powder, is identified as isovitexin, purity 92%.
Embodiment 3
Legume Desmodium styracifolium 45000-50000 parts by weight are selected, are cut into 5-10cm segments, drinking water washes mud
Sand drains, and feeds intake.80% alcohol reflux extracts twice, for the first time 12 times of amounts 2h, second of 10 times of amount 1.5h；Merge alcohol twice
Extract, recycling ethyl alcohol to no alcohol taste.Extraction concentrate is diluted with water to 5 times of volumes of medicinal material weight；200 mesh filter-cloth filterings；?
Upper prop liquid；The net product resin crossed in advance with 95% alcohol treatment is filled into column, purified water is replaced；Upper prop liquid pump is squeezed into column,
Absorption, 0.5-1 times of BV/h of coutroi velocity after absorption, stand 2h；It is rinsed, elution flow rate 3-4BV/h, is eluted with purified water
Measure 10BV；Again with 60% ethanol elution, elution flow rate 2-3BV/h, elution amount 5BV；60% eluent merges, and squeezes into concentration tank,
Ethyl alcohol is recycled to no alcohol taste, until 50-70 DEG C of drying in vacuum drying chamber, obtains extensively golden bulk pharmaceutical chemicals 300-400 parts by weight.
It will extensively golden bulk pharmaceutical chemicals 300-400 parts by weight AB-8 macroreticular resin chromatographic isolations, i.e. chromatographic isolation I, alcohol-water mix
Bonding solvent 20：80,30:70,40:60,50:50 gradient elutions, each gradient are flushed to no color, merge each fraction and obtain
20%, 30%, 40%, 60% totally four positions；50% position that chromatography I is obtained is with pressing chromatographic isolation, i.e. chromatography in ODS reverse phases
Separation II, with alcohol-water, condition is 22% isocratic 30min, and the 200min elutions of 22-40% gradients are suitable according to chromatogram appearance
Sequence, portioning are collected, and portion is collected per 100-150 parts by volume, collect 40-50 fraction altogether, and high performance liquid chromatography inspection merges after knowing
Identical fraction；25-38 fractions that chromatographic isolation II obtains merge, further with pressing chromatographic isolation, i.e. chromatographic isolation in ODS reverse phases
III, with alcohol-water, condition is 28% isocratic 30min, and the 200min elutions of 28-35% gradients divide according to chromatogram peak sequence
Part is collected, and collects portion per 100-150 parts by volume, altogether 35-45 fraction of collection, and high performance liquid chromatography inspection merges phase cocurrent flow after knowing
Part；The 20-40 fractions that chromatographic isolation III obtains merge, and are recycled to solvent and obtain isovitexin crude product.It is finally solidifying with glucan
Glue chromatographic isolation (Sephadex G-75), i.e. chromatographic isolation IV, specially：Isovitexin is added to methanol-water (volume ratio
It is 2:1) in, ultrasonic dissolution, sampling volume is 10 milliliters, and column is rushed with pure methanol, and portioning is collected, and every 5 parts by volume collects portion, altogether
20-30 fraction is collected, high performance liquid chromatography inspection merges identical fraction after knowing；The 10-25 fractions that chromatographic isolation IV obtains are closed
And recycling design obtains the powder of yellow.Through utilizing electrospray ionization mass spectrometry (ESI-MS) and spectroscopic methodology (H1-NMR and C13-
NMR it) identifies obtaining yellow powder, be identified as isovitexin, purity is less than 90.5%.
The yellow powder isovitexin that the process conditions of embodiment 2 and 3 are produced, purity are substantially less than the separation of embodiment 1
The isovitexin 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 spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiments or example in can be combined in any suitable 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 being detached from the principle of the present invention and objective a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The range of invention is limited by claim and its equivalent.
Claims (9)
1. a kind of method of extraction separation isovitexin, which is characterized in that include the following steps：
Refluxing extraction is carried out to Desmodium styracifolium using ethyl alcohol, 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,
Third chromatographic isolation is carried out to second separate section using third alcohol system, to obtain the isovitexin,
First chromatography is macroreticular resin；
Second chromatography is reverse-phase chromatography；
The third chromatography is gel chromatography,
Wherein,
The first alcohol system is ethanol-water system, a concentration of 50-65% of the ethyl alcohol in the ethanol-water system,
The diol system is ethanol-water system, a concentration of 18-40% of the ethyl alcohol in second ethanol-water system,
The third alcohol system is pure methanol,
The reverse-phase chromatography is C18 columns.
2. according to the method described in claim 1, it is characterized in that, it is described using ethyl alcohol to Desmodium styracifolium carry out refluxing extraction,
Herba Desmodii Styracifolii extract is obtained, including：
Refluxing extraction twice is carried out to the Desmodium styracifolium using the ethyl alcohol of concentration 50-95%, is merged obtained by refluxing extraction twice
Extracting solution, obtain merge extracting solution,
The ethyl alcohol in the merging extracting solution is removed, and/or removes the ethyl alcohol and ion merged in extracting solution, to obtain
State extract.
3. according to the method described in claim 1, it is characterized in that, further comprising：Remove the second in the merging extracting solution
Alcohol, and/or the ethyl alcohol and ion merged in extracting solution is removed, it is dried, to obtain the extract.
4. according to the method described in claim 2, it is characterized in that, the refluxing extraction twice, wherein
The weight ratio of primary ethyl alcohol and Desmodium styracifolium is 12:1, the time of refluxing extraction is 1.5h-3h,
Another ethyl alcohol and the weight ratio of Desmodium styracifolium are 10:1, the time of refluxing extraction is 1h-2h.
5. according to the method described in claim 4, it is characterized in that, the time of a refluxing extraction be 2h, it is described another
The time of secondary refluxing extraction is 1.5h.
6. according to the method described in claim 1, it is characterized in that, ethyl alcohol in first ethanol-water system it is a concentration of
60%.
7. according to the method described in claim 1, the column pressure of the C18 columns is 1-10bar, elution flow rate is 5~30ml/min.
8. according to the method described in claim 1, it is characterized in that, described carried out using the first separate section of diol system pair
Second chromatographic isolation obtains the second separate section, including：
Isocratic elution 25-40min is carried out to first separate section using the ethanol-water system that concentration of alcohol is a, is then changed
With concentration of alcohol be [a, b] ethanol-water system to first separate section carry out gradient elution 180-250min, collect with
Obtain second separate section, wherein
The value range of a is 18-28%, and the value range of b is 35-40%.
9. according to the method described in claim 1, it is characterized in that, described carried out using the first separate section of diol system pair
Second chromatographic isolation obtains the second separate section, including：
Isocratic elution 30min is carried out to first separate section using the ethanol-water system that concentration of alcohol is c, is then used instead
Concentration of alcohol is that the ethanol-water system of [c, d] carries out gradient elution 200min to first separate section, collects and obtains primary
Second separate section,
Isocratic elution 30min is carried out to primary second separate section using the ethanol-water system that concentration of alcohol is e, then
It uses the ethanol-water system that concentration of alcohol is [e, f] instead and gradient elution 200min is carried out to primary second separate section, collect
Obtain second separate section, wherein
The value range of c and e is that the value range of 18-28%, d and f are 35-40%, e>C, d>f.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103599123A (en) * | 2013-12-03 | 2014-02-26 | 广东世信药业有限公司 | Drug for treating purine metabolic disorder disease |
| CN103893246A (en) * | 2013-12-05 | 2014-07-02 | 人福医药集团股份公司 | General flavanone capsule of desmodium styracifolium and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103599123A (en) * | 2013-12-03 | 2014-02-26 | 广东世信药业有限公司 | Drug for treating purine metabolic disorder disease |
| CN103893246A (en) * | 2013-12-05 | 2014-07-02 | 人福医药集团股份公司 | General flavanone capsule of desmodium styracifolium and preparation method and application thereof |
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| Title |
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| FLAVONOID COMPOUNDS FROM Desmodium styracifolium OF VIETNAMESE ORIGIN;Minh Giang Phan,等;《Chemistry of Natural Compounds》;20101231;第46卷(第5期);第797-768页 * |
| 广金钱草化学成分的研究;王植柔，等;《广西医科大学学报》;19980930;第15卷(第3期);第10-14页 * |
| 广金钱草的生物活性成分研究;刘茁;《沈阳药科大学硕士学位论文》;20070115;第10-15页 * |
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