CN105541601B - The method for separating and preparing of organic acid monomer and application in a kind of sunglo - Google Patents
The method for separating and preparing of organic acid monomer and application in a kind of sunglo Download PDFInfo
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- CN105541601B CN105541601B CN201510930024.1A CN201510930024A CN105541601B CN 105541601 B CN105541601 B CN 105541601B CN 201510930024 A CN201510930024 A CN 201510930024A CN 105541601 B CN105541601 B CN 105541601B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
- B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/91—Dibenzofurans; Hydrogenated dibenzofurans
Abstract
Method for separating and preparing and application the invention discloses organic acid monomer in a kind of sunglo, application of the NaOH in the organic acid monomer in preparing sunglo is separated using pH zone counter current chromatography.Petroleum ether ethyl acetate methanol aqueous solvent, adds trifluoroacetic acid that NaOH is added as preservative, in lower phase as eluant, eluent in the upper phase of the solvent system.1) the upper phase of trifluoroacetic acid will be added to pump into the splitter of adverse current chromatogram instrument in solvent system, as preservative；3) counter-current chromatograph is opened, sample solution is injected in adverse current chromatogram instrument；4) the lower phase of NaOH will be added in solvent system, in injection adverse current chromatogram instrument, as eluant, eluent；5) eluate is monitored, the chromatogram according to gained collects target components.The present invention carries out separation preparation to the organic acid monomer in sunglo, and good separating effect, fractional dose are big, the rate of recovery is high, organic acid monomer purity reaches more than 92%, and preparation cost is far below prior art.
Description
Technical field
The present invention relates to adverse current chromatogram method for separating and preparing, in particular to pH zone counter current chromatography from pine
The method that separation prepares organic acid monomer in trailing plants crude extract.
Technical background
Sunglo is Usneaceae plant sunglo long (Usnea longissima Ach.) and broken stem sunglo (Usnea
Diffracta Vain.) dry thallus, with clearing liver, resolving sputum, hemostasis, removing toxic substances the effects such as, can be used for treat headache, mesh
Red, coughing with a lot of sputum, malaria, scrofula, leukorrhea, uterine bleeding, traumatism and bleeding, carbuncle swells, venomous snake bite etc..Principle active component is organic acid
Class compound, including moss, barbatic acid, diffractaic acid and usnic acid.Wherein, usnic acid is its representative composition, is
Most gram-positive bacteriums are had powerful inhibitory action by a kind of broad-spectrum antibiotic, can also suppress the growth of tulase, are used for
Treatment suppuration sexual trauma, second-degree burn, local tuberculosis etc., additionally, the also effect such as anticancer, antiprotozoan and spasmolysis.
Chi Chunyan etc. exists《Food Science》(Vol.30, No.03,2009,127~129) deliver " preparative high-speed counter-current
Usnic acid in chromatographic separation and purification sunglo long ", research uses n-hexane-acetonitrile-ethyl acetate-water (8:7:5:0.8) two
The crude extract of (60-90 DEG C) backflow extraction of petroleum ether is prepared isolating and purifying for type high speed adverse current chromatogram by phase system, once
Sample introduction 25mg, can obtain usnic acid 11.2mg.Using high performance liquid chromatography detection, the sample purity reaches 99%.
Feng Jie etc. exists《CHINA JOURNAL OF CHINESE MATERIA MEDICA》(Vol.l34, No.6,2009,708~711) delivered " and sunglo chemistry long into
Divide research ", research isolates and purifies compound using silica gel column chromatography, Sephadex LH-20 column chromatographys and HPLC column chromatography,
Using IR, UV, MS and NMR data authenticating compound structure.Result is separated from sunglo and identifies 13 compounds, respectively red
Star clothing acetoacetic ester (1), suberone (2), β-amyrin (3), cupreol (4), 2,4- dihydroxy -3,6- mesitylenic acids
Methyl esters (5), barbatic acid (6), damp room terpene (7), ethyl orsellinate (8), 3 beta-hydroxies-viscous mould -5- alkene (9), oleanolic acid (10),
(+)-usnic acid (11), methyl orsellinate (12) and 4- methyl -2,6- 4-dihydroxy benzaldehydes (13).
Wang Xiao etc. exist《Journal of Chromatography A》(Vol.1103,2006,166~169) are sent out
Table " Preparative separation of cichoric acid from Echinacea Purpurea by pH-
Zone-refining counter-current chromatography ", research is extracted using EtOH Sonicate, reclaims ethanol,
Obtain Cichoric acid concentrate 2.3L.Preliminary purification is carried out with the chromatographic column of AB-8 macroporous absorbent resins, macropore is rinsed with water first and is inhaled
Attached resin column, without color, then with 30% ethanol elution target compound, obtains 14.9g sample crude extracts to eluent.Finally adopt
With t-butyl methyl ether-acetonitrile-water (4:1:5) solvent system, upper organic phase adds 10mmol/L trifluoroacetic acids as fixing phase,
Lower floor's water is added 10mmol/L ammoniacal liquor as mobile phase, and single injected sampling 3g, by 2 separation, obtains target compound 563mg,
Learn that gained Cichoric acid purity is 95.6% by HPLC analysis detections.
Wang Xiao etc. exist《Journal of separation science》(Vol.30,2007,3214~3217)
" Large-scale separation of salvianolic acid B from the Chinese are delivered
medicinal plant Salvia miltiorrhiza by pH-zone-refining countercurrent
Chromatography ", research is extracted with 95% alcohol reflux, is concentrated under reduced pressure, and obtains concentrate；The concentrate amount of doubling water, uses
Na2CO3PH to 7.5 is adjusted, is extracted with ether；Water adjusts pH value to 3 with hydrochloric acid, is extracted with ether, reclaims ether, obtains red powder
Shape red sage root crude extract 57.19g.Using t-butyl methyl ether-water (1:1), upper organic phase adds trifluoroacetic acid (concentration 10mmol/L)
Used as fixing phase, lower floor's water is added ammoniacal liquor (concentration 10mmol/L) as mobile phase.Single injected sampling 2.0g, obtains tanshin polyphenolic acid B
524.2mg and a small amount of Rosmarinic acid, gained compound purity are all higher than 93%.
To be 201210595556.0 disclosure of the invention a kind of that usnic acid is extracted from sunglo long for Chinese Patent Application No.
Production technology, the method is：Raw material is put into after coarse crushing from cast hopper, extractor group main frame rotation, by material from unit
Front end is slowly advanced backward, while the feed tube of Extraction solvent slave group end enters in extractor, by tank rear end through movement
The flowing of material forward end, solid-liquid two-phase material is fully contacted in this reverse movement, so as to active ingredient in medicinal material be carried
Take out.The dregs of a decoction are pushed to slag notch and discharge through discharge conveyor pressure, and special Juice squeezer is extruded the dregs of a decoction, by medicine
Residual liquor extrusion medicinal material tissue, reduces residual liquor content in the dregs of a decoction in slag.Extraction efficiency is higher, extract more complete.This hair
Bright to be purified while extracting simultaneously binding silica gel column chromatography from continuous extraction tank group, separating effect is more preferably.Highest yield can be obtained is
1%, content is more than 95% usnic acid finished product.
A kind of Chinese Patent Application No. method for preparing usnic acid that has been 201010236842.9 disclosure of the invention.The method
Comprise the following steps：1) mixed with organic solvent after sunglo long is crushed, carry out refluxing extraction, obtain usnic acid crude extract；2)
Selection two phase solvent system, using high-speed countercurrent chromatography, by the step 1) the usnic acid crude extract that obtains separated, obtained
To usnic acid.The present invention carries out the separation of usnic acid using high speed adverse current chromatogram, with conventional laboratory preparation techniques means phase
Than with clear advantage：Single injected sampling amount is big, separating power is strong, disengaging time is short, sample loss is small, and separates and purify
Can synchronously complete.The method that the present invention is provided is suitable to the high-speed counter-current chromatograph of column capacity 325ml, and gained is prepared using the method
The high purity 99% of usnic acid.
The existing method isolated and purified on organic acid monomer in sunglo mainly prepares HPLC, repeatedly silicagel column color using half
Spectrum, Sephadex LH-20 gel column chromatographies, its limitation be waste time and energy, high cost, be also easy to produce Irreversible Adsorption, sample return
Yield is low, preparation efficiency is low；Have been reported that organic acid monomer in sunglo is isolated and purified with high-speed countercurrent chromatography, although
With sample size it is big, separating power is strong, disengaging time is short, sample loss is small the features such as, but relative to pH zone adverse current chromatogram
Speech, it is still suffered from, and sample size is small, experiment condition more difficult optimization the shortcomings of；Have been reported that using continuous extraction tank group binding silica gel post layer
The method of the organic acid monomer in analysis method purifying sunglo, its highest yield is 1%, and finished product is more than 95% usnic acid.It is above-mentioned
The separation that be directed to the main component usnic acid of sunglo research method carries out more is prepared and purified, as other organic acid monomers simultaneously
It is not directed to.Also there is document report that the organic acid monomer prepared in Chinese medicinal material extract is separated with pH zone countercurrent chromatography,
Selected solvent system has t-butyl methyl ether-water, t-butyl methyl ether-acetonitrile-water, t-butyl methyl ether-acetonitrile-n-butanol-water
With t-butyl methyl ether-tetrahydrofuran-water, using trifluoroacetic acid as preservative, ammoniacal liquor is used as eluant, eluent；Also be related to chloroform-
Methanol-water solvent systematic account, using ammoniacal liquor as preservative, trifluoroacetic acid is used as eluant, eluent；Have been reported that with petroleum ether-acetic acid
, used as solvent system, using hydrochloric acid as preservative, ammoniacal liquor is used as eluant, eluent for ethyl ester-methanol-water.
The content of the invention
Method for separating and preparing and application it is an object of the invention to provide organic acid monomer in a kind of sunglo, the method have
Low cost, it is simple to operate, rapidly and efficiently, a large amount of separation the advantage of organic acid monomer can be prepared from sunglo.
To achieve the above object, technical scheme is as follows：
Application of the NaOH in the organic acid monomer in preparing sunglo is separated using pH zone counter current chromatography.
The solvent system for preparing the organic acid monomer in sunglo is separated using pH zone counter current chromatography, is petroleum ether-second
Acetoacetic ester-methanol-water solvent system, adds trifluoroacetic acid as preservative in the upper phase of the solvent system, added in lower phase
NaOH is used as eluant, eluent.
Preferably, petroleum ether, ethyl acetate, the volume ratio of first alcohol and water are 5:5:(2-3):(8-7), preferably 5:5:3:
7。
Preferably, the concentration of the trifluoroacetic acid in upper phase is 10-15mmol/L, preferably 10mmol/L；Hydrogen in lower phase
The concentration of sodium oxide molybdena is 10-20mmol/L, preferably preceding 1.5 hours 10mmol/L, and 20mmol/L is converted to afterwards.
The preparation process of the solvent system, comprises the following steps：By component A, B component, component C and D components by above-mentioned
Proportioning is mixed, and is stood after shaking up, and after being layered, upper and lower two-phase is separated；Trifluoroacetic acid is added in upper phase, in lower phase
Add NaOH.
Above-mentioned solvent system is separating the application in preparation sunglo in organic acid monomer using pH zone counter current chromatography.
The method that organic acid monomer separation in sunglo is carried out using the solvent system, is comprised the following steps：
The sample solution that above-mentioned solvent system is pumped into counter-current chromatograph to the organic acid crude extract of sunglo is separated,
Monitoring eluate, the chromatogram according to gained collects target components.
Comprise the following steps that：
1) the upper phase of trifluoroacetic acid will be added to pump into the splitter of adverse current chromatogram instrument in solvent system, as preservative；
2) take equivalent add the upper phase of trifluoroacetic acid and be not hydrogenated with sodium oxide molybdena mix after dissolving sunglo organic acid
Crude extract, obtains sample solution；Sample is set to keep free state and can be completely dissolved.
3) counter-current chromatograph is opened, sample solution is injected in adverse current chromatogram instrument；
4) the lower phase of NaOH will be added in solvent system, in injection adverse current chromatogram instrument, as eluant, eluent；
5) eluate is monitored, the chromatogram according to gained collects target components.
Preferably, step 1) in, the speed that the upper phase of solvent system pumps into the splitter of adverse current chromatogram instrument is 10-
20ml/min。
Preferably, step 2) in, the preparation method of the organic acids extract of sunglo comprises the following steps；Using organic molten
Agent is extracted to sunglo, by extract solution concentration, after freeze-drying, the organic acids extract of sunglo is obtained.
It is further preferred that step 2) in, in the sample solution, every gram of organic acids extract phase and 5- on 5-10ml
The mixture of phase is dissolved under 10ml.
Preferably, step 3) in, when in sample solution injection adverse current chromatogram instrument, the rotating speed of counter-current chromatograph is 750-
1000rpm。
Preferably, step 4) in, the speed of the lower phase injection adverse current chromatogram instrument of solvent system is 2ml/min；Further
The concentration of the NaOH in lower phase in preferred first 1.5 hours is the concentration of NaOH after 10mmol/L, 1.5 hours
Be converted to 20mmol/L, it is therefore an objective to realizing reducing the time used by separation process on the basis of compound is successfully separated.
Preferably, the high-efficient liquid phase chromatogram condition of analysis organic acid crude extract is：YMC-Pack ODS-A C18column
(250mm × 4.6mm), ultraviolet detection wavelength：300nm, column temperature：25 DEG C, flow velocity：1.0ml/min, sample size：10 μ l, mobile phase
Using methanol-water, gradient elution program is：0-40min, 50%-100% methyl alcohol；40-55min, 100%-100% methyl alcohol.Point
The purpose for analysing organic acids extract is to analyze the number and separating degree of composition in crude extract, detection pH zone adverse current chromatogram cuts
Purity and belong to compound in each cut using retention time.
The authentication method at pH zone adverse current chromatogram rectangles peak, using Agilent 5973N mass spectrographs and Bruker 400MHz
Nuclear magnetic resonance chemical analyser carries out MS respectively,1H-NMR and13The measure of C-NMR spectrums.
The application separated in preparing of above method organic acid monomer in sunglo.
The optimization of pH zone adverse current chromatogram optimum experimental condition of the invention experienced following process：
According to " the Preparative separation of cichoric acid from that Wang Xiao etc. are delivered
Echinacea Purpurea by pH-zone-refining counter-current chromatography ", from uncle
Butyl methyl ether-acetonitrile-water (4:1:5) it is dicyandiamide solution, upper addition 10mmol/L trifluoroacetic acids, lower addition 10mmol/L ammoniacal liquor,
PH zone is not formed, is detected through HPLC, it is impossible to which realization is successfully separated；According to " the Preparative that Cheng Chunru etc. are delivered
isolation of triterpenoids from Ganoderma lucidum by counter-current
Chromatography combined with pH-zone-refining " select chloroform-methanol-water (13:7:4) it is solvent
System, upper addition ammoniacal liquor 10mmol/L, lower addition trifluoroacetic acid 10mmol/L, does not form pH zone, it is impossible to realize successfully yet
Separate.
According to " countercurrent chromatography isolates and purifies the usnic acid in sunglo long " delivered such as gorgeous of slow spring, from just
Hexane-ethylacetate-acetonitrile-water (8:5:7:0.8) it is dicyandiamide solution, in upper addition trifluoroacetic acid 10mmol/L, in lower addition
Ammoniacal liquor 10mmol/L, it is unrealized to be successfully separated；According to " the Accelerated solvent that Liu Yuqin are delivered
extraction of monacolin K from red yeast rice and purification by high-speed
Counter-current chromatography ", from petroleum ether-ethyl acetate-methanol-water (5:5:4:6) it is solvent body
System, upper addition hydrochloric acid 10mmol/L, lower addition ammoniacal liquor 10mmol/L form two obvious zone, are detected through HPLC, obtain tongue
Color acid and 4- methoxyl group moss, purity are respectively 92.5% and 91.9%, and required time is about 3.5 hours, and each composition has
The trend for separating；Continue to optimize on this basis to separate preparation condition, dicyandiamide solution is constant, changes hydrochloric acid into trifluoro second
Acid, causes reserve capability to weaken so that moss and 4- methoxyl groups moss are eluted out simultaneously, only obtain evinic acid
And barbatic acid, purity is respectively 94.5% and 96.9%, and required time is about 4 hours, and other organic acid monomers have
The trend for separating.
In order to realize the separation of moss and 4- methoxyl group moss, the content of methyl alcohol in dicyandiamide solution, solvent are reduced
System is changed into petroleum ether-ethyl acetate-methanol-water (5:5:2:8) three pH zone, are ultimately formed, is detected through HPLC, obtain tongue
Color acid, 4- methoxyl groups moss and evinic acid, purity are respectively 94.5%, 96.9% and 95.8%, and required time is about 5
Hour, and other organic acids have the trend for separating；In order to realize successfully separation, ammoniacal liquor is changed into NaOH to strengthen
The eluting power of lower phase, finally give moss, 4- methoxyl groups moss, evinic acid, barbatic acid, diffractaic acid and
Usnic acid, analyzes through HPLC, and purity is respectively 92.5%, 96.9%, 93.5%, 95.1%, 93.0% and 94.2%, is taken
Between about 11 hours, realize and be successfully separated.
Due to completing the overlong time required for separating, the present invention has been carried out further to pH zone adverse current chromatogram conditions
Optimization, it is therefore an objective to the shortening of disengaging time is realized on the basis of being successfully separated.According to separating experience, dicyandiamide solution is changed into stone
Oily ether-acetate-methanol-water (5:5:3:7), equally realize and be successfully separated, form 5 pH zone and post is tolerant, obtain
Moss, 4- methoxyl groups moss, evinic acid, barbatic acid, diffractaic acid and usnic acid, analyze, purity through HPLC
Respectively 93.2%, 95.4%, 94.1%, 95.7%, 92.7% and 96.8%, required time are about 9.5 hours, save
1.5 hours；Continue Optimal Experimental condition, dicyandiamide solution is petroleum ether-ethyl acetate-methanol-water (5:5:3:7) it is constant, on
Trifluoroacetic acid 10mmol/L is added, lower phase is divided into two parts, and a part adds 10mmol/L NaOH, another part to add
20mmol/L NaOH, wherein first 1.5 hours are eluted with the lower phase containing 10mmol/L NaOH, switches to afterwards
The lower phase of 20mmol/L NaOH, as a result also achieves and is successfully separated, formed 5 pH zone and post it is tolerant, obtain moss,
4- methoxyl groups moss, evinic acid, barbatic acid, diffractaic acid and usnic acid, analyze through HPLC, and purity is respectively
92.7%th, 97.7%, 93.8%, 94.8%, 92.2% and 95.7%, required time is 8 hours, has saved 1.5 hours, relatively
3 hours have been saved altogether in initial disengaging time, have realized the target of the shortening disengaging time of initial setup.
It can be seen that, experiment condition of the invention is the basis that inventor uses for reference according to the experience of itself and to former achievements
On, by repeatedly grope what is gradually built up, the process gradually groped needs inventor to pay substantial amounts of creative work
On the basis of could finally determine, so, the technical scheme is that non-obvious.
Advantageous Effects of the invention are：The present invention is entered using pH zone adverse current chromatogram to the organic acid monomer in sunglo
Row is separated and prepared, good separating effect, fractional dose be big, simple to operate, rapidly and efficiently, the rate of recovery is high, organic acid monomer purity reaches
To more than 92%, the preparation cost of organic acid monomer is far below prior art, is a kind of dividing from sunglo medicinal material for economical and efficient
Method from organic acid monomer is prepared.
Brief description of the drawings
The chromatogram that Fig. 1 for the pH zone adverse current chromatogram of the sunglo crude extract of embodiment 5 separate, 1, moss, 2,4- methoxies
Base moss, 3, evinic acid, 4, barbatic acid, 5, diffractaic acid, 6, usnic acid；
Fig. 2 is the high-efficient liquid phase chromatogram of crude extract；
Fig. 3 is the high-efficient liquid phase chromatogram of the isolated organic acid monomer from sunglo.
Specific embodiment
With reference to specific embodiment and accompanying drawing, the invention will be further described.
Embodiment 1
1st, air-dried sunglo thallus 1.5kg is taken, 40 mesh sieves is crossed after crushing, flowed back with 4 times of ethyl acetate solutions of amount and carried
Take 3 times, 1.5 hours every time, filtration, merging filtrate was concentrated under reduced pressure, and freeze-drying obtains 119g crude extracts, is stored in 4 DEG C of refrigerators
Prepared for further separating；
2nd, the organic acid monomer prepared in sunglo crude extract is separated with pH zone countercurrent chromatography
Solvent system is petroleum ether (60-90 DEG C)-acetate-methanol-water (5:5:2:8), add in upper phase fixing phase
Enter 10mmol/L trifluoroacetic acids as preservative, 10mmol/L NaOH is added in lower phase mobile phase as eluant, eluent, it is high
The column volume of fast adverse current chromatogram instrument is 300ml, sample size 1.5g, rotating speed 800rpm, flow velocity 2.0ml/min, and fixing phase retains
Rate 41%, Detection wavelength 254nm.
Specifically operating procedure is：Solvent system is prepared by above-mentioned solvent ratios, is placed in separatory funnel, stood after shaking up
Layering, ready to balance for a period of time afterwards separates upper and lower two-phase, and 10mmol/L trifluoroacetic acids are added in upper phase fixing phase as guarantor
Agent is stayed, adds 10mmol/L NaOH as eluant, eluent in lower phase mobile phase, taken 1.5g sunglo crude extracts and be dissolved in 10ml
Add trifluoroacetic acid upper phase and 10ml be not hydrogenated with it is stand-by in the lower phase mixture of sodium oxide molybdena.Had with field biotechnology using Shanghai
Limit company develop semi-preparative high-speed counter-current chromatograph device, it be by plunger displacement pump, sampling valve, UV-detector, recorder and
Chromatography column (spiral tube formed by polyfluortetraethylene pipe multi-lay winding, capacity is 300ml) etc. is constituted, and sample introduction is made first
Valve is in sample introduction state, and fixing phase pump is filled into chromatography column, termination of pumping with high flow rate (20ml/min).The sample that will have been dissolved
Product are injected in the liquid storage tube of adverse current chromatogram instrument sampling valve with syringe, and rotation sampling valve enables sample to enter to connect column state
Enter chromatography column.Opening speed controller, rotates forward the chromatography column of high-speed counter-current chromatograph device, and rotating speed reaches 800rpm
When, setting flow rate of mobile phase is 2.0ml/min, starts pump mobile phase.Then according to detector ultraviolet spectrogram receive target into
Point, moss, 4- methoxyl groups moss, evinic acid, barbatic acid, diffractaic acid and usnic acid are obtained, analyzed through HPLC,
Purity is respectively 92.5%, 96.9%, 93.5%, 95.1%, 93.0% and 94.2%, and required time is about 11 hours.
Using efficient liquid phase chromatographic analysis separator, liquid-phase condition：YMC-Pack ODS-A C18column(250mm×
4.6mm), ultraviolet detection wavelength 300nm, column temperature：25 DEG C, flow velocity：1.0ml/min, sample size：10 μ l, mobile phase using methyl alcohol-
Water (0-40min, 50%-100% methyl alcohol；40-55min, 100%-100% methyl alcohol).
Embodiment 2
1st, air-dried sunglo thallus 1.5kg is taken, 40 mesh sieves is crossed after crushing, flowed back with 4 times of ethyl acetate solutions of amount and carried
Take 3 times, 1.5 hours every time, filtration, merging filtrate was concentrated under reduced pressure, and freeze-drying obtains 121g crude extracts, is stored in 4 DEG C of refrigerators
Prepared for further separating；
2nd, the organic acid monomer prepared in sunglo crude extract is separated with pH zone countercurrent chromatography
Solvent system is petroleum ether (60-90 DEG C)-acetate-methanol-water (5:5:2.5:7.5), in upper phase fixing phase
Middle addition 15mmol/L trifluoroacetic acids add 15mmol/L NaOH as wash-out as preservative in lower phase mobile phase
Agent, the column volume of high-speed counter-current chromatograph device is 300ml, sample size 1.5g, rotating speed 800rpm, flow velocity 2.0ml/min, fixing phase
Retention rate 40%, Detection wavelength 254nm.
Specifically operating procedure is：Solvent system is prepared by above-mentioned solvent ratios, is placed in separatory funnel, stood after shaking up
Layering, ready to balance for a period of time afterwards separates upper and lower two-phase, and 10mmol/L trifluoroacetic acids are added in upper phase fixing phase as guarantor
Agent is stayed, adds 10mmol/L NaOH as eluant, eluent in lower phase mobile phase, taken 1.5g sunglo crude extracts and be dissolved in 10ml
Add trifluoroacetic acid upper phase and 10ml be not hydrogenated with it is stand-by in the lower phase mixture of sodium oxide molybdena.Had with field biotechnology using Shanghai
Limit company develop semi-preparative high-speed counter-current chromatograph device, it be by plunger displacement pump, sampling valve, UV-detector, recorder and
Chromatography column (spiral tube formed by polyfluortetraethylene pipe multi-lay winding, capacity is 300ml) etc. is constituted, and sample introduction is made first
Valve is in sample introduction state, and fixing phase pump is filled into chromatography column, termination of pumping with high flow rate (10ml/min).The sample that will have been dissolved
Product are injected in the liquid storage tube of adverse current chromatogram instrument sampling valve with syringe, and rotation sampling valve enables sample to enter to connect column state
Enter chromatography column.Opening speed controller, rotates forward the chromatography column of high-speed counter-current chromatograph device, and rotating speed reaches 800rpm
When, setting flow rate of mobile phase is 2.0ml/min, starts pump mobile phase.Then according to detector ultraviolet spectrogram receive target into
Point, moss, 4- methoxyl groups moss, evinic acid, barbatic acid, diffractaic acid and usnic acid are obtained, analyzed through HPLC,
Purity is respectively 92.3%, 94.9%, 95.5%, 95.8%, 93.4% and 93.2%, and required time is about 10.5 hours.
Using efficient liquid phase chromatographic analysis separator, liquid-phase condition：YMC-Pack ODS-A C18column(250mm×
4.6mm), ultraviolet detection wavelength 300nm, column temperature：25 DEG C, flow velocity：1.0ml/min, sample size：10 μ l, mobile phase using methyl alcohol-
Water (0-40min, 50%-100% methyl alcohol；40-55min, 100%-100% methyl alcohol).
Embodiment 3
1st, air-dried sunglo thallus 1.5kg is taken, 40 mesh sieves is crossed after crushing, flowed back with 4 times of ethyl acetate solutions of amount and carried
Take 3 times, 1.5 hours every time, filtration, merging filtrate was concentrated under reduced pressure, and freeze-drying obtains 122g crude extracts, is stored in 4 DEG C of refrigerators
Prepared for further separating；
2nd, the organic acid monomer prepared in sunglo crude extract is separated with pH zone countercurrent chromatography
Solvent system is petroleum ether (60-90 DEG C)-acetate-methanol-water (5:5:3:7), add in upper phase fixing phase
Enter 10mmol/L trifluoroacetic acids as preservative, 10mmol/L NaOH is added in lower phase mobile phase as eluant, eluent, it is high
The column volume of fast adverse current chromatogram instrument is 300ml, sample size 1.5g, rotating speed 800rpm, flow velocity 2.0ml/min, and fixing phase retains
Rate 43%, Detection wavelength 254nm.
Specifically operating procedure is：Solvent system is prepared by above-mentioned solvent ratios, is placed in separatory funnel, stood after shaking up
Layering, ready to balance for a period of time afterwards separates upper and lower two-phase, and 15mmol/L trifluoroacetic acids are added in upper phase fixing phase as guarantor
Agent is stayed, adds 20mmol/L NaOH as eluant, eluent in lower phase mobile phase, taken 1.5g sunglo crude extracts and be dissolved in 10ml
Add trifluoroacetic acid upper phase and 10ml be not hydrogenated with it is stand-by in the mixture of sodium oxide molybdena.Using Shanghai with the limited public affairs of field biotechnology
The semi-preparative high-speed counter-current chromatograph device developed is taken charge of, it is by plunger displacement pump, sampling valve, UV-detector, recorder and chromatogram
The compositions such as splitter (having the spiral tube that polyfluortetraethylene pipe multi-lay winding is formed, capacity is 300ml), make at sampling valve first
In sample introduction state, fixing phase pump is filled into chromatography column, termination of pumping with high flow rate.The sample that will have been dissolved is injected with syringe
In the liquid storage tube of adverse current chromatogram instrument sampling valve, rotation sampling valve enables sample to enter chromatography column to connect column state.Open
Speed control is opened, the chromatography column of high-speed counter-current chromatograph device is rotated forward, when rotating speed reaches 800rpm, mobile phase stream is set
Speed is 2.0ml/min, starts pump mobile phase.Then target component is received according to detector ultraviolet pipe spectrogram, obtains moss, 4- first
Epoxide moss, evinic acid, barbatic acid, diffractaic acid and usnic acid, analyze through HPLC, and purity is respectively
92.2%th, 93.4%, 94.3%, 93.7%, 92.9% and 95.8%, required time is about 9.5 hours.
Using efficient liquid phase chromatographic analysis separator, liquid-phase condition：YMC-Pack ODS-A C18column(250mm×
4.6mm), ultraviolet detection wavelength 300nm, column temperature：25 DEG C, flow velocity：1.0ml/min, sample size：10 μ l, mobile phase using methyl alcohol-
Water (0-40min, 50%-100% methyl alcohol；40-55min, 100%-100% methyl alcohol).
Embodiment 4
1st, air-dried sunglo thallus 1.5kg is taken, 40 mesh sieves is crossed after crushing, flowed back with 4 times of ethyl acetate solutions of amount and carried
Take 3 times, 1.5 hours every time, filtration, merging filtrate was concentrated under reduced pressure, and freeze-drying obtains 123g crude extracts, is stored in 4 DEG C of refrigerators
Prepared for further separating；
2nd, the organic acid monomer prepared in sunglo crude extract is separated with pH zone countercurrent chromatography
Solvent system is petroleum ether (60-90 DEG C)-acetate-methanol-water (5:5:3:7), add in upper phase fixing phase
Enter 10mmol/L trifluoroacetic acids as preservative, 15mmol/L NaOH is added in lower phase mobile phase as eluant, eluent, it is high
The column volume of fast adverse current chromatogram instrument is 300ml, sample size 1.5g, rotating speed 800rpm, flow velocity 2.0ml/min, and fixing phase retains
Rate 42%, Detection wavelength 254nm.
Specifically operating procedure is：Solvent system is prepared by above-mentioned solvent ratios, is placed in separatory funnel, stood after shaking up
Layering, ready to balance for a period of time afterwards separates upper and lower two-phase, and 10mmol/L trifluoroacetic acids are added in upper phase fixing phase as guarantor
Agent is stayed, adds 15mmol/L NaOH as eluant, eluent in lower phase mobile phase, taken 1.5g sunglo crude extracts and be dissolved in 10ml
Add trifluoroacetic acid upper phase and 10ml be not hydrogenated with it is stand-by in the mixture of sodium oxide molybdena.Using Shanghai with the limited public affairs of field biotechnology
The semi-preparative high-speed counter-current chromatograph device developed is taken charge of, it is by plunger displacement pump, sampling valve, UV-detector, recorder and chromatogram
The compositions such as splitter (having the spiral tube that polyfluortetraethylene pipe multi-lay winding is formed, capacity is 300ml), make at sampling valve first
In sample introduction state, fixing phase pump is filled into chromatography column, termination of pumping with high flow rate.The sample that will have been dissolved is injected with syringe
In the liquid storage tube of adverse current chromatogram instrument sampling valve, rotation sampling valve enables sample to enter chromatography column to connect column state.Open
Speed control is opened, the chromatography column of high-speed counter-current chromatograph device is rotated forward, when rotating speed reaches 800rpm, mobile phase stream is set
Speed is 2.0ml/min, starts pump mobile phase.Then target component is received according to detector ultraviolet pipe spectrogram, obtains moss, 4- first
Epoxide moss, evinic acid, barbatic acid, diffractaic acid and usnic acid, analyze through HPLC, and purity is respectively
89.2%th, 87.4%, 92.3%, 93.5%, 94.9% and 93.8%, required time is about 8.5 hours.
Using efficient liquid phase chromatographic analysis separator, liquid-phase condition：YMC-Pack ODS-A C18column(250mm×
4.6mm), ultraviolet detection wavelength 300nm, column temperature：25 DEG C, flow velocity：1.0ml/min, sample size：10 μ l, mobile phase using methyl alcohol-
Water (0-40min, 50%-100% methyl alcohol；40-55min, 100%-100% methyl alcohol).
Embodiment 5
1st, air-dried sunglo thallus 1.5kg is taken, 40 mesh sieves is crossed after crushing, flowed back with 4 times of ethyl acetate solutions of amount and carried
Take 3 times, 1.5 hours every time, filtration, merging filtrate was concentrated under reduced pressure, and freeze-drying obtains 120g crude extracts, is stored in 4 DEG C of refrigerators
Prepared for further separating；
2nd, the organic acid monomer prepared in sunglo crude extract is separated with pH zone countercurrent chromatography
Solvent system is petroleum ether (60-90 DEG C)-acetate-methanol-water (5:5:3:7), add in upper phase fixing phase
Enter 10mmol/L trifluoroacetic acids as preservative, lower phase mobile phase is divided into two parts, a portion adds 10mmol/L hydrogen
Sodium oxide molybdena；Another part adds 20mmol/L NaOH as eluant, eluent, is converted at about 1.5 hours.High-speed counter-current
The column volume of chromatographic apparatus be 300ml, sample size 1.5g, rotating speed 800rpm, flow velocity 2.0ml/min, fixing phase retention rate 42%,
Detection wavelength 254nm.
Specifically operating procedure is：Solvent system is prepared by above-mentioned solvent ratios, is placed in separatory funnel, stood after shaking up
Layering, ready to balance for a period of time afterwards separates upper and lower two-phase, and 10mmol/L trifluoroacetic acids are added in upper phase fixing phase as guarantor
Agent is stayed, lower phase is divided into two parts, add 10mmol/L NaOH, another part to add in phase mobile phase under a part
20mmol/L NaOH takes 1.5g sunglo crude extracts and is dissolved in upper phase and 10ml that 10ml adds trifluoroacetic acid as eluant, eluent
It is not hydrogenated with stand-by in the mixture of sodium oxide molybdena.The semi-preparative high-speed counter-current developed using Shanghai Tongtian Biotechnology Co., Ltd.
Chromatographic apparatus, it is that (have polyfluortetraethylene pipe multilayer by plunger displacement pump, sampling valve, UV-detector, recorder and chromatography column
The spiral tube being wound, capacity is 300ml) etc. composition, make first sampling valve be in sample introduction state, by fixing phase pump with
High flow rate fills chromatography column, termination of pumping.The sample that will have been dissolved injects the reservoir of adverse current chromatogram instrument sampling valve with syringe
Guan Zhong, rotation sampling valve enables sample to enter chromatography column to connect column state.Opening speed controller, makes high-speed counter-current
The chromatography column of chromatographic apparatus is rotated forward, and when rotating speed reaches 800rpm, setting flow rate of mobile phase is 2.0ml/min, starts to pump into
Added 10mmol/L NaOH mobile phases, after about 1.5 hours, start to pump into added 20mmol/L NaOH flow
Phase.Then target component is received according to detector ultraviolet pipe spectrogram (Fig. 1), obtains moss, 4- methoxyl groups moss, goes first ring trailing plants
Acid, barbatic acid, diffractaic acid and usnic acid, analyze through HPLC, purity is respectively 92.7%, 97.7%, 93.8%,
94.8%th, 92.2% and 95.7%, required time is 8 hours.
Using efficient liquid phase chromatographic analysis separator, liquid-phase condition：YMC-Pack ODS-A C18column(250mm×
4.6mm), ultraviolet detection wavelength 300nm, column temperature：25 DEG C, flow velocity：1.0ml/min, sample size：10 μ l, mobile phase using methyl alcohol-
Water (0-40min, 50%-100% methyl alcohol；40-55min, 100%-100% methyl alcohol).
Fig. 2 is the HPLC analyses to sunglo crude extract, it is therefore an objective to understands the number and separating degree of organic acid in crude extract, obtains
It is that the purity detecting and ownership of pH each cuts of zone adverse current chromatogram determine analysis condition to the retention time of each composition；Fig. 3 is right
The HPLC detections of pH zone adverse current chromatogram cuts, it is therefore an objective to obtain the purity of each composition and according to each in retention time and crude extract
The retention time of composition is compared, and realizes the ownership of each cut.
Although above-mentioned be described with reference to accompanying drawing to specific embodiment of the invention, not to invention protection domain
Limitation, one of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art are not required to
The various modifications or deformation made by paying creative work are still within the scope of the present invention.
Claims (8)
1. the method that organic acid monomer separation preparation in sunglo is carried out using solvent system, it is characterised in that：Comprise the following steps：
The sample solution that solvent system is pumped into counter-current chromatograph to the organic acid crude extract of sunglo is separated, monitoring is washed out
Liquid, the chromatogram according to gained collects target components；The solvent system is petroleum ether-ethyl acetate-methanol-water solvent body
System, adds trifluoroacetic acid that NaOH is added as preservative, in lower phase as eluant, eluent in the upper phase of solvent system；
The speed that the upper phase of solvent system pumps into the splitter of adverse current chromatogram instrument is 10-20ml/min, the lower phase of solvent system
The speed for injecting adverse current chromatogram instrument is 2ml/min, in the organic acid crude extract sample solution injection adverse current chromatogram instrument of sunglo
When, the rotating speed of counter-current chromatograph is 750-1000rpm；
The preparation method of the sample solution of the organic acid crude extract of the sunglo, comprises the following steps；Using ethyl acetate to pine
Trailing plants is extracted, and by extract solution concentration, after freeze-drying, the organic acid crude extract of sunglo is obtained, and every gram of organic acid crude extract is used
5-10ml adds the upper phase and 5-10ml of trifluoroacetic acid not to add the mixture of the lower phase of NaOH to be dissolved, and obtains final product.
2. method according to claim 1, it is characterised in that：In the solvent system, petroleum ether, ethyl acetate, methyl alcohol
It is 5 with the volume ratio of water:5:(2-3):(8-7)；The concentration of the trifluoroacetic acid in upper phase is 10-15mmol/L；Hydrogen in lower phase
The concentration of sodium oxide molybdena is 10-20mmol/L.
3. method according to claim 2, it is characterised in that：In the solvent system, petroleum ether, ethyl acetate, methyl alcohol
It is 5 with the volume ratio of water:5:3:7.
4. method according to claim 2, it is characterised in that：The preparation method of the solvent system comprises the following steps：By stone
Oily ether, ethyl acetate, first alcohol and water are mixed by the proportioning of each component in claim 2, are stood after shaking up, after being layered,
Upper and lower two-phase is separated；Trifluoroacetic acid is added in upper phase, NaOH is added in lower phase.
5. method according to claim 2, it is characterised in that：The concentration of the NaOH in lower phase in first 1.5 hours
It is 10mmol/L, the concentration of later NaOH is converted to 20mmol/L within 1.5 hours.
6. according to any described methods of claim 1-5, it is characterised in that：Analyze the high performance liquid chromatography of organic acid crude extract
Condition is：YMC-Pack ODS-A C18Column, 250mm × 4.6mm, ultraviolet detection wavelength 300nm, column temperature：25 DEG C, flow velocity：
1.0ml/min, sample size：10 μ l, mobile phase uses the methanol-water, gradient elution program to be：0-40min, 50%-100% first
Alcohol；40-55min, 100%-100% methyl alcohol.
7. according to any described methods of claim 1-5, it is characterised in that：The identification side at pH zone adverse current chromatogram rectangles peak
Method, MS is carried out using Agilent 5973N mass spectrographs and Bruker 400MHz nuclear magnetic resonance chemical analysers respectively,1H-NMR and13C-
The measure of H NMR spectroscopy.
8. any described methods of claim 1-5 organic acid monomer in sunglo separates the application in preparing.
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| CN101050208A (en) * | 2007-05-10 | 2007-10-10 | 南京农业大学 | Technique for producing usnic acid |
| CN103086967A (en) * | 2013-02-05 | 2013-05-08 | 中国科学院新疆理化技术研究所 | Alkaloid having type 1 skeleton in Nigellaglandulifera Freyn grass seeds, and its preparation method |
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| CN1995027A (en) * | 2006-12-30 | 2007-07-11 | 山东省分析测试中心 | Separating preparation process of salvianolic acid B |
| CN101050208A (en) * | 2007-05-10 | 2007-10-10 | 南京农业大学 | Technique for producing usnic acid |
| CN103086967A (en) * | 2013-02-05 | 2013-05-08 | 中国科学院新疆理化技术研究所 | Alkaloid having type 1 skeleton in Nigellaglandulifera Freyn grass seeds, and its preparation method |
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Application publication date: 20160504 Assignee: Beijing Emilion Technology Co., Ltd. Assignor: Shandong Analysis Test Center Contract record no.: X2019370000027 Denomination of invention: Separation and preparation method of organic acid monomers in Chinese usnea and application thereof Granted publication date: 20170704 License type: Exclusive License Record date: 20191213 |
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