CN106674313A - Method for simultaneously separating quercetin-3-O-gentian diglucoside and kaempferol-3-O-gentian diglucoside from folium sauropi - Google Patents
Method for simultaneously separating quercetin-3-O-gentian diglucoside and kaempferol-3-O-gentian diglucoside from folium sauropi Download PDFInfo
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- CN106674313A CN106674313A CN201611262101.1A CN201611262101A CN106674313A CN 106674313 A CN106674313 A CN 106674313A CN 201611262101 A CN201611262101 A CN 201611262101A CN 106674313 A CN106674313 A CN 106674313A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
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- C07H1/00—Processes for the preparation of sugar derivatives
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Abstract
The invention relates to a method for simultaneously separating quercetin-3-O-gentian diglucoside and kaempferol-3-O-gentian diglucoside from folium sauropi and belongs to the technical field of separation and purification of a compound monomer. According to the invention, the folium sauropi medicinal material is taken as a raw material, and the quercetin-3-O-gentian diglucoside and kaempferol-3-O-gentian diglucoside monomers are acquired according to the steps of ethanol extraction, concentration, macro-porous resin enrichment and elution, polyamide resin enrichment and elution, efficient preparation, liquid chromatography separation and product recycling. According to the invention, through mature processing steps and parameter conditions, the purities of the quercetin-3-O-gentian diglucoside and the kaempferol-3-O-gentian diglucoside in the product respectively reach above 99%; and meanwhile, the method is simple and convenient in operation and is high in separating efficiency, the raw materials are recyclable, and the quercetin-3-O-gentian diglucoside and kaempferol-3-O-gentian diglucoside monomers can be simultaneously separated and prepared in large batch and high purity.
Description
Technical field
The present invention relates to a kind of plant compound process for separation and purification, and in particular to Cortex querci dentatae is separated from Folium Sauropi simultaneously
The method of element -3-0- O-gentibiosides and Kaempferol -0- O-gentibiosides, belongs to technical field of Chinese medicines.
Background technology
Folium SauropiSauropus rostratusIt is Euphorbiaceae Sauropus plant, also known as leaf of dragon's tongue, imperial taste leaf, Flos Sauropi changiani
Leaf, the ears of an ox or cow leaf, are mainly distributed on the ground such as Guangdong, Guangxi, Fujian, originate in North Vietnam, and there is cultivation the Malay Peninsula.With heat clearing away
It is the effect of lung moistening, preventing phlegm from forming and stopping coughing, among the people for treating lung heat cough and asthma abundant expectoration, xerostomia, constipation and other diseases.The current report to Flos Sauropi changiani leaf
Road is only limitted to some Primary Studies, and the research to its chemical composition and its pharmacologically active is less.And Quercetin -3-O- in Folium Sauropi
O-gentibioside has most important effect in technical field of Chinese medicines with Kaempferol-O- O-gentibiosides.
Quercetin -3-O- O-gentibiosides（Quercetin 3-O-gentiobioside）, molecular formula is C27H30O17, point
Son amount is 626.52, and structural formula is as follows：
Kaempferol-O- O-gentibiosides（Kaempferol 3-O-diglucoside）, molecular formula is C27H30O16, molecule
Measure as 610.52, structural formula is as follows：
The at present main method for adopting silica gel column chromatography and crystalline phase to combine carry out Quercetin -3-O- O-gentibiosides, nimbecetin -
Refined, the long the production cycle of 3-O- O-gentibioside monomers, seriously, product yield is low for reagent waste.
State Intellectual Property Office discloses a kind of Publication No. CN1844116 on October 11st, 2006, and patent name is
The patent of invention of " extracting and developing of flavonoid monomer, purification and identification in oriental blueberry melanin ", the patent is with Vaccinium bracteatum Thunb. Leaves
For raw material, Jing ethanol is extracted and obtains Vaccinium bracteatum Thunb. Leaves melanin solution, then Jing macroporous adsorbent resins to the flavonoid in melanin
Compound carries out crude separation, then purification is carried out to flavone compound monomer by polyamide column and HW-40 posts, then uses efficient liquid
Phase chromatography-mass spectroscopy, nuclear magnetic resonance technique carry out Structural Identification to purified components；7 kinds of flavone compound monomers are isolated therefrom,
Respectively A, B, C, D, E, F and G, and accurate Structural Identification has been carried out to B, C, D, E and F, it is determined that respectively Quercetin, Cortex Populi dividianae
Flavin, 4',5,7-trihydroxyflavone, kaempferol and luteolin, its relative amount are respectively：37.51%, 2.26%, 9.57%, 1.72% He
15.16%。
With Vaccinium bracteatum Thunb. Leaves as raw material, Jing ethanol is extracted and obtains Vaccinium bracteatum Thunb. Leaves melanin solution, then Jing macropores the patent
Adsorbent resin carries out crude separation to the flavone compound in melanin, then by polyamide column and HW-40 posts to flavonoid
Monomer adduct carries out purification.But the technical scheme process is complicated, relatively costly, involved solvent species are excessive, extracted Huang
In ketone compounds, Quercetin and nimbecetin content are low, and purity is not high；And involved Quercetin, chrysin, 4',5,7-trihydroxyflavone, mountain
How phenol and luteolin are flavone aglycone, without being mentioned to flavonoid glycoside compound, due to flavone aglycone and flavonoid glycoside
Both compounds polarity spectrum is very big, therefore the technical scheme is not suitable for Quercetin -3-0- O-gentibiosides and Kaempferol -0- is imperial
The extraction of gallbladder bioside is separated.
The content of the invention
Present invention seek to address that prior art problem, and propose from Folium Sauropi while separating meletin -3-0- Radix Gentianae
The method of bioside and Kaempferol -0- O-gentibiosides.The present invention makes product by ripe processing step and Parameter Conditions
Middle Quercetin -3-0- O-gentibiosides respectively reach more than 99% with Kaempferol -0- O-gentibioside purity；The method can be fast
Speed, high-purity, easy separation, Quercetin -3-0- O-gentibiosides and Kaempferol -0- gentiobioses in purification Folium Sauropi
Glycosides.
In order to realize above-mentioned technical purpose, following technical scheme is proposed：
The simultaneously method of separating meletin -3-0- O-gentibiosides and Kaempferol -0- O-gentibiosides from Folium Sauropi, including
Following steps：
（1）Extract
With Folium Sauropi medical material as raw material, the coarse powder of 1~4mm is ground into, then according to every kilogram of raw material adds 6 liters of ethanol solution,
Add 70% ethanol solution, reflux, extract, 3-4 time, each 2-3 hours, extracting solution to filter, collect, merge, obtain extracting solution I；
（2）Concentration
By step（1）After the extracting solution I of gained reclaims ethanol, the 15-25% of I volume of former extracting solution is concentrated into, is extracting solution II；
（3）Macroporous resin column is enriched with and eluting
By step（2）The extracting solution II of gained is crossed macroporous adsorptive resins and is adsorbed, and is cleaned with purified water to after colourless, then is used
60-70% ethanol solution eluted products sections, it is colourless to effluent, collect, merge eluent, then by eluent concentrating under reduced pressure,
Ethanol is reclaimed, extracting solution III is obtained；
（4）Polyamide resin column is enriched with and eluting
By step（3）The extracting solution III of gained is crossed polyamide resin column and is adsorbed, and first cleans polyamide resin with 10% ethanol solution
Fat post is to after colourless, then with 90% ethanol solution eluted products section, colourless to effluent, collects, merges eluent, then will wash
De- liquid concentrating under reduced pressure, after reclaiming ethanol, obtains the concentrated solution containing two kinds of target products, is extracting solution IV；
（5）High performance preparative liquid chromatography instrument is separated
Take step（4）The extracting solution IV of gained collects filtrate with 0.45 μm of organic membrane filter, and then filtrate sample introduction, carries out Cortex querci dentatae
The preparative separation of element -3-0- O-gentibiosides monomer and Kaempferol -0- O-gentibioside monomers, and specific aim collects Cortex querci dentatae
Element -3-0- O-gentibiosides prepare fraction solution with Kaempferol -0- two kinds of monomers of O-gentibioside；In the process, it is purple
External detector carries out on-line checking with wavelength 350nm；
（6）Product recycling
By step（5）In carry out the isolated Quercetin -3-0- O-gentibiosides of high performance preparative liquid chromatography and Kaempferol -
Two kinds of monomers of 0- O-gentibiosides prepare fraction solution, heating recovery methanol, and Quercetin -3-0- O-gentibiosides, nimbecetin -
3-0- O-gentibioside monomers are separated out in aqueous in a large number, place room temperature after, with funnel filter separate out crystal, cross filter solid in
It is dried 12 hours under the conditions of 65 DEG C, that is, obtains Quercetin -3-0- O-gentibiosides monomer and Kaempferol -0- O-gentibioside lists
Body target product.
In step（5）In, chromatographic condition is：Chromatographic column filler is C18 fillers, and column temperature is 30 DEG C, and flow velocity is 1.0mL/
Min, mobile phase are -0.1% phosphate aqueous solution of methanol, and both volume ratios are 46 to 54.
Before high performance preparative liquid chromatography instrument separation is carried out, can be conventional by liquid-mass chromatography or other the art
Method determines the peak shape of Quercetin -3-0- O-gentibiosides and Kaempferol -0- O-gentibioside monomers in high performance liquid chromatography.
By taking liquid-mass chromatography method as an example, can be identical with above-mentioned preparative separation chromatographic condition, using filler identical chromatographic column, composition phase
Mobile phase together, identical Detection wavelength etc., column temperature is room temperature.Take step（4）The extracting solution IV of gained is filtered, then sample introduction,
Carry out Quercetin -3-0- O-gentibiosides, the efficient liquid phase of Kaempferol -0- O-gentibioside monomers to isolate and purify, according to matter
Spectrum testing result, it may be determined that Quercetin -3-0- O-gentibiosides, Kaempferol -0- O-gentibioside monomers are in liquid chromatograph
Corresponding peak shape.
Quercetin -3-0- the O-gentibiosides, the purity of Kaempferol -0- O-gentibioside monomer products are rechecked and are adopted
Inverse analysis type liquid chromatograph, chromatographic condition is：With C18 as filler；With -0.1% phosphate aqueous solution of methanol as mobile phase, two
Person's volume ratio is 46 to 54；Flow velocity 1.0mL/min；Detection wavelength is 350nm.
Quercetin -3-0- O-gentibiosides respectively reach more than 99% with Kaempferol -0- O-gentibioside purity.
Using above-mentioned technical proposal, the Advantageous Effects for bringing are：
1. the present invention makes Quercetin -3-0- O-gentibiosides and Rhizoma Kaempferiae in product by ripe processing step and Parameter Conditions
Phenol -3-0- O-gentibioside purity respectively reaches more than 99%；Meanwhile, this method is easy to operate, separation efficiency is high, and raw material can be returned
Receive, can in a large number, high-purity and while separation prepares Quercetin -3-0- O-gentibiosides and Kaempferol -0- O-gentibioside lists
Body；
2., in the present invention, for the physicochemical property of various composition present in Folium Sauropi medical material, the technical program passes through step
Appropriate order arrange in pairs or groups, and appropriate parameter combination, effectively extracted Quercetin -3-O- O-gentibiosides and nimbecetin -
3-O- O-gentibioside compositions, and a large amount of impurity are eliminated, it is derived from entering preparative high performance liquid chromatography system
Sample solution, it is unlikely that very big impact is caused to highly effective liquid phase chromatographic system, extend its usage cycles as far as possible, saved and produced into
This；
3. in the present invention（1）In step, every kilogram of raw material adds 6 liters of ethanol solution, under the ratio, can effectively soak original
Material, can reduce ethanol usage amount, again so as to reduce the use cost of solvent；And 70% alcohol reflux is adopted, both can be by Cortex querci dentatae
Element -3-0- O-gentibiosides and Kaempferol -0- O-gentibiosides both flavones ingredients are effectively extracted, and make which again
His impurity component dissolution rate is relatively low, it is ensured that during preparation, impurity interference is few；
4. in the present invention（2）In step, extracting solution I reclaims ethanol, can reduce the ethanol content in concentrated solution；By concentrating, both
Recyclable ethanol reduces the volume of pregnant solution in subsequent step again；
5. in the present invention（3）In step, adsorbed by macroporous adsorptive resins, cleaned with purified water to colourless, by pure
Changing water guarantees to remove big polar impurity and some carbohydrate contents；And 60-70% ethanol solution is ensureing that it is same that product is eluted out
When, the less impurity of polarity is without being eluted；
6. in the present invention（4）In step, absorption of the eluent by polyamide resin column can effectively reach separating meletin -3-
0- O-gentibiosides and Kaempferol -0- O-gentibiosides, can play a part of to remove a part of impurity again；And pass through 10% second
Alcohol washes away the pigment and impurity of some big polarity, reduces impurity interference when preparing；90% ethanol quickly can be produced eluting
Product, can be reduced to minimal volumes preparing raw material during concentration again；
7. in the present invention（5）In step, using preparative high performance liquid chromatography system to Quercetin -3-O- O-gentibiosides, mountain
Naphthol -3-O- O-gentibiosides monomer carries out separating, purification, has reached good separating effect, high performance preparative liquid chromatography phase
For other conventional separation methods（Such as silicagel column elution is separated）Can be to product sharp separation, and the yield to product
Improve a lot, cost is substantially reduced.And by online ultraviolet detection, targetedly collect Quercetin -3-O- Radix Gentianae two
Glucosides, Kaempferol-O- O-gentibioside monomers, it is with clearly defined objective, it is to avoid the wasting of resources that conventional column chromatography etc. is caused, and
It is easy to control product quality, product purity is up to more than 99%；
8. during high performance liquid chromatography separation, selection of each chromatographic condition and combinations thereof is particularly important, because its direct shadow
Ring appearance time, peak shape of material etc.；Chromatographic condition mainly includes chromatographic column（Including filler, column length, column temperature etc.）, mobile phase
（Including composition, flow velocity etc.）, detector and Detection wavelength etc..The present invention by substantial amounts of experimental study and relative analyses, it is determined that
Above-described chromatographic condition, so that the appearance time of material, peak shape, separating effect etc. reach optimization, realizes Mongolian oak
The high efficiency separation of Pi Su -3-O- O-gentibiosides, Kaempferol-O- O-gentibioside monomers；
9. the recyclable recycling of organic reagent in the present invention, in each processing step, solvent-oil ratio are few, cost-effective.
Description of the drawings
Fig. 1 is the HPLC collection of illustrative plates of Quercetin -3-O- O-gentibioside monomer products in the present invention
Fig. 2 is the HPLC collection of illustrative plates of Kaempferol-O- O-gentibioside monomer products in the present invention.
Specific embodiment
Below by being clearly and completely described to the technical scheme in the embodiment of the present invention, it is clear that described reality
Apply a part of embodiment that example is only the present invention, rather than the embodiment of whole.Based on the embodiment in the present invention, this area is general
All other embodiment that logical technical staff is obtained under the premise of creative work is not made, belongs to present invention protection
Scope.
Embodiment 1
As shown in Figure 1 and Figure 2：While separating meletin -3-0- O-gentibiosides and Kaempferol -0- Radix Gentianae two from Folium Sauropi
The method of glucosides, comprises the technical steps that：
（1）Extract
Folium Sauropi medical material is weighed into 1Kg, the coarse powder of 1~4mm is ground into, 70% ethanol solution 6L is subsequently adding, reflux, extract, 3 times,
2 hours/time, filter, merging filtrate, and common 30L is extracting solution I；
（2）Concentration
By step（1）After the extracting solution I of gained reclaims ethanol, small size is concentrated into, gained concentrated solution is 5L, is extracting solution II；
（3）Macroporous resin column is enriched with and eluting
By step（2）The extracting solution II of gained is adsorbed with macroporous resin, and resin is cleaned with 10L purified water to after colourless, then
It is with the 60% ethanol solution eluted products section of 15L, colourless to effluent, collect, merge eluent, eluent concentrating under reduced pressure is returned
Ethanol is received, 5L extracting solution III is obtained；
（4）Polyamide resin column is enriched with and eluting
By step（3）The extracting solution III of gained is crossed polyamide resin column and is adsorbed, and first cleans tree with 10% ethanol solution of 10L
Fat is to after colourless, then with the 90% ethanol solution eluted products of 16L, colourless to effluent, collects, merges eluent, by eluting
Liquid concentrating under reduced pressure, after reclaiming ethanol, obtains concentrated solutions of the 1L containing product, is extracting solution IV；
（5）High performance preparative liquid chromatography is separated
Chromatographic condition is as follows：Chromatographic column of the filler for C18；
Mobile phase is consisted of：- 0.1% phosphate aqueous solution of methanol（V/V）, the two volume ratio is 46 to 54；
Detection wavelength is 350nm；Take step（4）The extracting solution IV of gained is collected filtrate, is then filtered with 0.45 μm of organic membrane filter
Liquid sample introduction, carries out the preparative separation of Quercetin -3-0- O-gentibiosides monomer and Kaempferol -0- O-gentibioside monomers, and
Specific aim collection Quercetin -3-0- O-gentibiosides prepare fraction solution with Kaempferol -0- two kinds of monomers of O-gentibioside,
Respectively Quercetin -3-0- O-gentibiosides prepare solution 5L, Kaempferol -0- O-gentibiosides and prepare solution 6L；Here mistake
Cheng Zhong, UV-detector on-line checking；
（6）Product recycling
By step（5）In carry out the isolated Quercetin -3-0- O-gentibiosides of high performance preparative liquid chromatography, Kaempferol -
Two kinds of monomers of 0- O-gentibiosides prepare fraction solution, heating recovery methanol, and Quercetin -3-0- O-gentibiosides, nimbecetin -
3-0- O-gentibioside monomers are separated out in aqueous in a large number, place room temperature after with funnel filter separate out crystal, cross filter solid in
It is dried 12 hours under the conditions of 65 DEG C, that is, obtains Quercetin -3-0- O-gentibioside solid 1.1g, Kaempferol -0- gentiobioses
Glycosides solid 1.3g.
Quercetin -3-0- the O-gentibiosides, the purity of Kaempferol -0- O-gentibioside monomer products are rechecked and are adopted
Inverse analysis type liquid chromatograph（RP-HPLC）Method, chromatographic condition are as follows：With C18 as filler；It is water-soluble with -0.1% phosphoric acid of methanol
Liquid is mobile phase, and both volume ratios are 46 to 54）；Flow velocity 1.0mL/min；Detection wavelength is 350nm.
Whole about 4 days production procedure used times.
Calculating product yield is：
Quercetin -3-O- O-gentibiosides：（1.1/1000）×100%=0.11%；
Kaempferol-O- O-gentibiosides：（1.3/1000）×100%=0.13%.
By changing mobile phase composition, using inverse analysis type liquid chromatograph（RP-HPLC）Product purity is rechecked, knot is measured
It is really Quercetin -3-O- O-gentibiosides 99.15%, Kaempferol-O- O-gentibiosides 99.35%.
Embodiment 2
The simultaneously method of separating meletin -3-0- O-gentibiosides and Kaempferol -0- O-gentibiosides from Folium Sauropi, including
Following processing step：
（1）Extract
Folium Sauropi medical material is weighed into 10Kg, the coarse powder of 1~4mm is ground into, is added 60L volumetric concentrations to be 70% ethanol solution, is extracted
3 times, 3 hours/time are filtered, merging filtrate, and common 90L is extracting solution I；
（2）Concentration
By step（1）After the extracting solution I of gained reclaims ethanol, small size is concentrated into, gained concentrated solution is 15L, is extracting solution II；
（3）Macroporous resin enrichment and parsing
By step（2）The extracting solution II of gained is adsorbed with macroporous resin, and resin is cleaned with 80L purified water to after colourless, then
It is with the 65% ethanol solution eluted products section of 120L, colourless to effluent, collect, merge eluent, by eluent concentrating under reduced pressure,
Ethanol is reclaimed, 18L extracting solution III is obtained；
（4）Polyamide resin column is enriched with and eluting
By step（3）The extracting solution III of gained is crossed polyamide resin column and is adsorbed, and first cleans tree with 10% ethanol solution of 100L
Fat is to after colourless, then with the 90% ethanol solution eluted products of 180L, colourless to effluent, collects, merges eluent, by eluting
Liquid concentrating under reduced pressure, after reclaiming ethanol, obtains concentrated solutions of the 20L containing product, is extracting solution IV；
（5）High performance preparative liquid chromatography is separated
Chromatographic condition is as follows：Chromatographic column of the filler for C18；
Mobile phase is consisted of：- 0.1% phosphate aqueous solution of methanol（V/V）, the two volume ratio is 46 to 54；
Detection wavelength is 350nm；Take step（4）The extracting solution IV of gained is collected filtrate, is then filtered with 0.45 μm of organic membrane filter
Liquid sample introduction, carries out the preparative separation of Quercetin -3-0- O-gentibiosides monomer and Kaempferol -0- O-gentibioside monomers, and
Specific aim collection Quercetin -3-0- O-gentibiosides prepare fraction solution with Kaempferol -0- two kinds of monomers of O-gentibioside,
Respectively Quercetin -3-0- O-gentibiosides prepare solution 60L, Kaempferol -0- O-gentibiosides and prepare solution 75L；Here
During, UV-detector on-line checking；
（6）Product recycling
By step（5）In carry out the isolated Quercetin -3-0- O-gentibiosides of high performance preparative liquid chromatography, Kaempferol -
Two kinds of monomers of 0- O-gentibiosides prepare fraction solution, heating recovery methanol, and Quercetin -3-0- O-gentibiosides, nimbecetin -
3-0- O-gentibioside monomers are separated out in aqueous in a large number, place room temperature after with funnel filter separate out crystal, cross filter solid in
It is dried 12 hours under the conditions of 65 DEG C, that is, obtains Quercetin -3-0- O-gentibiosides solid 12, Kaempferol -0- O-gentibiosides
Solid 14g.
Quercetin -3-0- the O-gentibiosides, the purity of Kaempferol -0- O-gentibioside monomer products are rechecked and are adopted
Inverse analysis type liquid chromatograph（RP-HPLC）Method, chromatographic condition are as follows：With C18 as filler；It is water-soluble with -0.1% phosphoric acid of methanol
Liquid is mobile phase, and both volume ratios are 46 to 54；Flow velocity 1.0mL/min；Detection wavelength is 350 nm.
Whole about 6 days production procedure used times.
Calculating product yield is：
Quercetin -3-O- O-gentibiosides：（12/1000）×100%=0.12%；
Kaempferol-O- O-gentibiosides：（14/1000）×100%=0.14%.
By changing mobile phase composition, using inverse analysis type liquid chromatograph（RP-HPLC）Product purity is rechecked, knot is measured
It is really Quercetin -3-O- O-gentibiosides 99.25%, Kaempferol-O- O-gentibiosides 99.45%.
Embodiment 3
The simultaneously method of separating meletin -3-0- O-gentibiosides and Kaempferol -0- O-gentibiosides from Folium Sauropi, including
Following processing step：
（1）Extract
Folium Sauropi medical material is weighed into 20Kg, the coarse powder of 1~4mm is ground into, 70% ethanol solution of 120L is subsequently adding, 4 are extracted
Secondary, 3 hours/time are filtered, merging filtrate, and common 185L is extracting solution I；
（2）Concentration
By step（1）After the extracting solution I of gained reclaims ethanol, small size is concentrated into, gained concentrated solution is 30L, is extracting solution II；
（3）Macroporous resin enrichment and parsing
By step（2）The extracting solution II of gained is adsorbed with macroporous resin, and resin is cleaned with 150L purified water to after colourless, then
It is with the 70% ethanol solution eluted products section of 200L, colourless to effluent, collect, merge eluent, by eluent concentrating under reduced pressure,
Ethanol is reclaimed, 25L extracting solution III is obtained；
（4）Polyamide resin column is enriched with and eluting
By step（3）The extracting solution III of gained is crossed polyamide resin column and is adsorbed, and first cleans tree with 10% ethanol solution of 100L
Fat is to after colourless, then with the 90% ethanol solution eluted products of 200L, colourless to effluent, collects, merges eluent, by eluting
Liquid concentrating under reduced pressure, after reclaiming ethanol, obtains concentrated solutions of the 21L containing product, is extracting solution IV；
（5）High performance preparative liquid chromatography is separated
Chromatographic condition is as follows：Chromatographic column of the filler for C18；
Mobile phase is consisted of：- 0.1% phosphate aqueous solution of methanol（V/V）, the two volume ratio is 46 to 54；
Detection wavelength is 350nm；
Take step（4）The extracting solution IV of gained collects filtrate with 0.45 μm of organic membrane filter, and then filtrate sample introduction, carries out Cortex querci dentatae
The preparative separation of element -3-0- O-gentibiosides monomer and Kaempferol -0- O-gentibioside monomers, and specific aim collects Cortex querci dentatae
Element -3-0- O-gentibiosides and Kaempferol -0- two kinds of monomers of O-gentibioside prepare fraction solution, respectively Quercetin -
3-0- O-gentibiosides prepare solution 80L, Kaempferol -0- O-gentibiosides and prepare solution 95L；In the process, ultraviolet inspection
Survey device on-line checking；
（6）Product recycling
By step（5）In carry out the isolated Quercetin -3-0- O-gentibiosides of high performance preparative liquid chromatography, Kaempferol -
Two kinds of monomers of 0- O-gentibiosides prepare fraction solution, heating recovery methanol, and Quercetin -3-0- O-gentibiosides, nimbecetin -
3-0- O-gentibioside monomers are separated out in aqueous in a large number, place room temperature after with funnel filter separate out crystal, cross filter solid in
It is dried 12 hours under the conditions of 65 DEG C, that is, obtains Quercetin -3-0- O-gentibioside solid 25g, Kaempferol -0- O-gentibiosides
Solid 27g.
Quercetin -3-0- the O-gentibiosides, the purity of Kaempferol -0- O-gentibioside monomer products are rechecked and are adopted
Inverse analysis type liquid chromatograph（RP-HPLC）Method, chromatographic condition are as follows：With C18 as filler；It is water-soluble with -0.1% phosphoric acid of methanol
Liquid is mobile phase, and both volume ratios are 4 to 54；Flow velocity 1.0mL/min；Detection wavelength is 350nm.
Whole about 7 days production procedure used times.
Calculating product yield is：
Quercetin -3-O- O-gentibiosides：（25/2000）×100%=0.125%；
Kaempferol-O- O-gentibiosides：（27/2000）×100%=0.135%.
By changing mobile phase composition, using inverse analysis type liquid chromatograph（RP-HPLC）Product purity is rechecked, knot is measured
It is really Quercetin -3-O- O-gentibiosides 99.45%, Kaempferol-O- O-gentibiosides 99.35%.
Embodiment 4
On the basis of embodiment 1-3, mobile phase can also be -0.1% phosphate aqueous solution of acetonitrile, and both volume ratios are 25 to 75；
Before high performance liquid chromatography separation is carried out, determine that Quercetin -3-0- is imperial in high performance liquid chromatography by liquid-mass chromatography method
The peak shape of gallbladder bioside, Kaempferol -0- O-gentibioside monomers, i.e. take step（5）Middle gained mixed liquor sample introduction, carries out Mongolian oak
The preparative separation of Pi Su -3-0- O-gentibiosides, Kaempferol -0- O-gentibioside monomers, according to Mass Spectrometer Method result, it is determined that
Quercetin -3-0- O-gentibiosides and Kaempferol -0- O-gentibioside monomers corresponding peak shape in liquid chromatograph.
Claims (4)
1. the simultaneously method of separating meletin -3-0- O-gentibiosides and Kaempferol -0- O-gentibiosides from Folium Sauropi, its
It is characterised by, comprises the steps：
（1）Extract
With Folium Sauropi medical material as raw material, the coarse powder of 1~4mm is ground into, then according to every kilogram of raw material adds 6 liters of ethanol solution,
Add 70% ethanol solution, reflux, extract, 3-4 time, each 2-3 hours, extracting solution to filter, collect, merge, obtain extracting solution I；
（2）Concentration
By step（1）After the extracting solution I of gained reclaims ethanol, the 15-25% of I volume of former extracting solution is concentrated into, is extracting solution II；
（3）Macroporous resin column is enriched with and eluting
By step（2）The extracting solution II of gained is crossed macroporous resin column and is adsorbed, and is cleaned with purified water to after colourless, then uses 60-
70% ethanol solution eluted products section, it is colourless to effluent, collect, merge eluent, then by eluent concentrating under reduced pressure, reclaim
Ethanol, obtains extracting solution III；
（4）Polyamide resin column is enriched with and eluting
By step（3）The extracting solution III of gained is crossed polyamide resin column and is adsorbed, and first cleans polyamide resin with 10% ethanol solution
Fat post is to after colourless, then with 90% ethanol solution eluted products section, colourless to effluent, collects, merges eluent, then will wash
De- liquid concentrating under reduced pressure, after reclaiming ethanol, obtains the concentrated solution containing two kinds of target products, is extracting solution IV；
（5）High performance preparative liquid chromatography instrument is separated
Take step（4）The extracting solution IV of gained collects filtrate with 0.45 μm of organic membrane filter, and then filtrate sample introduction, carries out Cortex querci dentatae
The preparative separation of element -3-0- O-gentibiosides monomer and Kaempferol -0- O-gentibioside monomers, and specific aim collects Cortex querci dentatae
Element -3-0- O-gentibiosides prepare fraction solution with Kaempferol -0- two kinds of monomers of O-gentibioside；In the process, it is purple
External detector carries out on-line checking with wavelength 350nm；
（6）Product recycling
By step（5）In carry out the isolated Quercetin -3-0- O-gentibiosides of high performance preparative liquid chromatography and Kaempferol -
Two kinds of monomers of 0- O-gentibiosides prepare fraction solution, heating recovery methanol, and Quercetin -3-0- O-gentibiosides, nimbecetin -
3-0- O-gentibioside monomers are separated out in aqueous in a large number, place room temperature after, with funnel filter separate out crystal, cross filter solid in
It is dried 12 hours under the conditions of 65 DEG C, that is, obtains Quercetin -3-0- O-gentibiosides monomer and Kaempferol -0- O-gentibioside lists
Body target product.
2. simultaneously separating meletin -3-0- O-gentibiosides and Kaempferol -0- from Folium Sauropi according to claim 1
The method of O-gentibioside, it is characterised in that in step（5）In, chromatographic condition is：Chromatographic column filler is C18 fillers；Column temperature is
Room temperature；Flow velocity is 1.0mL/min；Mobile phase is -0.1% phosphate aqueous solution of methanol, and both volume ratios are 46 to 54.
3. simultaneously separating meletin -3-0- O-gentibiosides and Kaempferol -0- from Folium Sauropi according to claim 1
The method of O-gentibioside, it is characterised in that the Quercetin -3-0- O-gentibiosides, Kaempferol -0- O-gentibioside lists
The purity of body product is rechecked using inverse analysis type liquid chromatograph, and chromatographic condition is：With C18 as filler；With -0.1% phosphorus of methanol
Aqueous acid is mobile phase, and both volume ratios are 46 to 54；Flow velocity 1.0mL/min；Detection wavelength is 350nm.
4. simultaneously separating meletin -3-0- O-gentibiosides and Kaempferol -0- from Folium Sauropi according to claim 1
The method of O-gentibioside, it is characterised in that Quercetin -3-0- O-gentibiosides and Kaempferol -0- O-gentibioside purity
Respectively reach more than 99%.
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| CN110251552A (en) * | 2019-06-19 | 2019-09-20 | 苏州大学 | A kind of preparation method of Folium Sauropi general flavone |
| CN110441444A (en) * | 2019-09-20 | 2019-11-12 | 广东一方制药有限公司 | A kind of Folium Sauropi medicinal material UPLC characteristic spectrum construction method and its discrimination method |
| CN110776541A (en) * | 2019-11-19 | 2020-02-11 | 云南贝泰妮生物科技集团股份有限公司 | Preparation method and application of quercetin-3-gentiobioside |
| CN111481583A (en) * | 2020-03-31 | 2020-08-04 | 右江民族医学院 | Method for efficiently extracting total flavonoids from sauropus spatulifolius |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110251552A (en) * | 2019-06-19 | 2019-09-20 | 苏州大学 | A kind of preparation method of Folium Sauropi general flavone |
| CN110251552B (en) * | 2019-06-19 | 2021-06-15 | 苏州大学 | Preparation method of total flavonoids of sauropus spatulifolius |
| CN110441444A (en) * | 2019-09-20 | 2019-11-12 | 广东一方制药有限公司 | A kind of Folium Sauropi medicinal material UPLC characteristic spectrum construction method and its discrimination method |
| CN110441444B (en) * | 2019-09-20 | 2022-03-29 | 广东一方制药有限公司 | Method for constructing UPLC characteristic spectrum of sauropus miq and identification method thereof |
| CN110776541A (en) * | 2019-11-19 | 2020-02-11 | 云南贝泰妮生物科技集团股份有限公司 | Preparation method and application of quercetin-3-gentiobioside |
| CN110776541B (en) * | 2019-11-19 | 2021-08-13 | 云南贝泰妮生物科技集团股份有限公司 | Preparation method and application of quercetin-3-gentiobioside |
| CN111481583A (en) * | 2020-03-31 | 2020-08-04 | 右江民族医学院 | Method for efficiently extracting total flavonoids from sauropus spatulifolius |
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