CN114280201A - Efficient separation method for polyphenol components in dandelion - Google Patents
Efficient separation method for polyphenol components in dandelion Download PDFInfo
- Publication number
- CN114280201A CN114280201A CN202111676346.XA CN202111676346A CN114280201A CN 114280201 A CN114280201 A CN 114280201A CN 202111676346 A CN202111676346 A CN 202111676346A CN 114280201 A CN114280201 A CN 114280201A
- Authority
- CN
- China
- Prior art keywords
- phase
- sample
- solvent system
- separation
- dandelion
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a high-efficiency separation method of polyphenol components in dandelion, which adopts a mode of combining pH zone countercurrent chromatography (pH-ZRCCC) and high-speed countercurrent chromatography (HSCCC) to separate 6 polyphenol compounds from the dandelion. In the pH-ZRCCC separation, a solvent system of ethyl acetate-acetonitrile-water (4:1:5, v/v/v) is selected, trifluoroacetic acid (10mM) is added to the upper phase as a stationary phase, and ammonia water (10mM) is added to the lower phase as a mobile phase. Caffeic acid, hydroxycinnamic acid and mixture A are separated from the crude extract of dandelion ethyl acetate. Then further using HSCCC, selecting a solvent system of petroleum ether-ethyl acetate-methanol-water (1:4:1:4, v/v/v/v). 1-O-caffeoyl glycerol, 3, 4-dihydroxyphenylacetic acid, protocatechuic acid and p-hydroxyphenylacetic acid were isolated from 400mg of mixture A. The method has the advantages of large preparation amount and good reproducibility, and is suitable for separating and purifying polyphenol compounds of herba Taraxaci.
Description
Technical Field
The invention belongs to the technical field of chemical analysis, and particularly relates to a high-efficiency separation method of polyphenol components in dandelion.
Background
Herba Taraxaci (Taraxacum mongolicum hand. -Mazz.) is perennial herb of Compositae, and has effects of clearing away heat and toxic materials, resolving carbuncle and resolving hard mass. Researches show that the dandelion polyphenol substances have pharmacological activities in the aspects of oxidation resistance, cancer resistance, bacteriostasis, inflammation resistance, blood sugar reduction and the like. In order to further and deeply research the pharmacological activity of the dandelion polyphenol compound, more monomer compounds need to be separated and prepared. The components of the dandelion polyphenol substances are complex, the separation and preparation difficulty is high, the currently common separation and preparation method mainly adopts column chromatography, and the sample preparation efficiency is low and the time consumption is long. The countercurrent chromatography has the characteristics of large preparation amount, good separation effect, convenient operation and the like, and is widely applied to the separation and preparation of natural products. Relatively few research reports are reported for separating dandelion polyphenol by adopting counter-current chromatography at the present stage. The dandelion polyphenol is separated and purified by taking dandelion as a raw material and adopting pH zone countercurrent chromatography combined with high-speed countercurrent chromatography, and the chemical structure of the dandelion polyphenol is identified, so that technical support is provided for the research of pharmacological activity and quality standard of the dandelion.
Disclosure of Invention
In order to solve the technical problems, the invention provides a high-efficiency separation method of polyphenol components in dandelion. The invention adopts a mode of combining pH zone countercurrent chromatography (pH-ZRCCC) and high-speed countercurrent chromatography (HSCCC) to separate 6 polyphenol compounds from taraxacum. The method has the advantages of large preparation amount and good reproducibility, and is suitable for separating and purifying polyphenol compounds of herba Taraxaci.
The invention provides a high-efficiency separation method of polyphenol components in dandelion, which comprises the following steps: (1) extracting polyphenol of dandelion; pulverizing herba Taraxaci, extracting with solvent under reflux, and concentrating under reduced pressure to obtain concentrated solution; extracting the concentrated solution, eluting and concentrating after extraction to obtain a dandelion sample; (2) preparing a pH zone countercurrent chromatography solvent system and a high-speed countercurrent chromatography solvent system; (3) dissolving a dandelion sample in a pH zone countercurrent chromatography solvent system, carrying out pH zone countercurrent chromatography separation, and separating to obtain a compound I, a compound II and a mixture A; dissolving the mixture A in a high-speed counter-current chromatography solvent system, and performing high-speed counter-current chromatography separation to obtain a compound III, a compound IV and a compound V; (4) analyzing the dandelion sample and the compound obtained by separation and purification by using HPLC, and calculating the purity; the chemical structure was identified by ESI-MS and NMR analysis.
Further, the extracting of dandelion polyphenol specifically comprises: pulverizing appropriate amount of herba Taraxaci, extracting with 60% ethanol at a material-to-liquid ratio of 1:10(w/v) under reflux, filtering, and concentrating under reduced pressure to obtain concentrated solution; suspending the concentrated solution with water, extracting with petroleum ether, and extracting with ethyl acetate; concentrating ethyl acetate phase under reduced pressure to obtain paste, eluting with water and ethanol, and concentrating 30% of the eluate under reduced pressure to dry to obtain herba Taraxaci sample.
Furthermore, before preparing the pH zone countercurrent chromatography solvent system, the K value of the sample in the pH zone countercurrent chromatography is measured.
Further, the K value of pH band countercurrent chromatography was determined as: approximately 5mg of the sample was weighed into a 10mL tube. Preparing a solvent system, putting 5mL of each of the upper phase and the lower phase in a test tube containing a sample, dripping ammonia water until the pH value is about 10, shaking the test tube until the sample is completely dissolved, standing and layering; taking 10 μ L of each of the upper and lower phases, and detecting by High Performance Liquid Chromatography (HPLC) respectively; using the peak area of the upper phase (A)U) Peak area of phase (A)L) Calculating the partition coefficient K of the sample under alkaline conditionsbaseThe calculation formula is as follows: kbase＝AU/AL(ii) a Dripping trifluoroacetic acid into the test tube until the pH value is about 2, shaking up, standing for layering, respectively taking 10 μ L of upper and lower phases by the same method, and calculating the distribution coefficient K by HPLCacid。
Furthermore, before preparing the solvent system of the high-speed counter-current chromatography, the K value of the sample in the high-speed counter-current chromatography needs to be measured.
Further, the K value of the sample in high-speed counter-current chromatography was determined as: approximately 5mg of the sample was weighed into a 10mL tube. Preparing a solvent system, taking 5mL of upper phase and lower phase in a test tube containing a sample,shaking vigorously until the sample is completely dissolved, standing for layering, collecting upper and lower phase 10 μ L respectively, and detecting by HPLC to obtain upper phase peak area (A)1) Peak area of phase (A)2) Namely, the distribution coefficient K is calculated according to the following formula: k is A1/A2。
Further, the preparation of the pH zone countercurrent chromatography solvent system specifically comprises the following steps: selecting ethyl acetate-acetonitrile-water (4:1:5, v/v/v) as a two-phase solvent system, and separating by adopting a pH zone method; placing ethyl acetate, acetonitrile and water in a separating funnel according to a certain proportion, violently shaking, standing for layering, separating out an upper phase and a lower phase, adding trifluoroacetic acid into the upper phase, adding ammonia water into the lower phase, and respectively performing ultrasonic degassing; weighing a sample, taking an equivalent acidified upper phase and a lower phase without adding alkali to dissolve the sample, and using the sample for subsequent countercurrent chromatographic separation.
Further, the preparation of the high-speed counter-current chromatography solvent system comprises the following steps: selecting petroleum ether-ethyl acetate-methanol-water (1:4:1:4, v/v/v/v) as a solvent system, putting the solvent system into a separating funnel in proportion, shaking uniformly, standing for layering, separating an upper phase and a lower phase, and respectively performing ultrasonic degassing for later use; weighing the sample, taking the same amount of upper phase and lower phase, and dissolving the sample for later use.
Further, the pH zone countercurrent chromatography separation specifically comprises the following steps: pumping the upper phase into a high-speed counter-current chromatograph as a stationary phase, and injecting a sample solution into the separation column after the stationary phase is filled in the chromatographic spiral tube; starting a speed controller to enable the separation column to rotate clockwise, and simultaneously pumping a lower phase solution; starting an ultraviolet detector, adjusting the wavelength to 280nm, collecting chromatographic peak components according to a chromatogram, and detecting the pH value of the fraction by using a pH meter; and after the separation is finished, blowing out the residual liquid in the column by using a pressure pump, collecting the residual liquid in the measuring cylinder, and calculating the retention rate of the stationary phase by using the volume ratio of the stationary phase to the total volume of the tail blowing in the tail blowing.
Further, the high-speed counter-current chromatographic separation specifically comprises the following steps: pumping the upper phase as a stationary phase, starting a speed controller after the stationary phase is filled in the chromatographic spiral tube, enabling the separation column to rotate clockwise, and pumping the lower phase; when the system reaches the fluid dynamic equilibrium, injecting the mixture A into a separation column; starting an ultraviolet detector, and collecting chromatographic peak components according to a chromatogram map under the wavelength of 280 nm; after the separation is finished, blowing out the residual liquid in the column by using a pressure pump, collecting the residual liquid in a measuring cylinder, and calculating the retention rate of the stationary phase by using the volume ratio of the stationary phase in tail blowing to the total volume of the tail blowing; concentrating the separated components under reduced pressure to dry, sealing and storing in refrigerator.
Further, the total sample of dandelion and the fractions obtained by separation and purification were analyzed by HPLC. The chromatographic column is C18A chromatographic column (250 multiplied by 4.6mm, 5 mu m), wherein the mobile phase is methanol (A) -0.1% formic acid aqueous solution (B), 0-10 min, 20-24% A, 10-20 min, 24% A, 20-21 min, 24-34% A, 21-30 min, 34-40% A, 30-35 min, 40-100% A, the flow rate is 1.0mL/min, the detection wavelength is 280nm, and the sample injection amount is 10 mu L. And calculating the purity of the sample by adopting a normalization method.
The invention has the following beneficial effects:
the invention utilizes a method of combining pH-ZRCCC and HSCCC to separate dandelion polyphenol, and successfully separates 6 polyphenol compounds from dandelion. In the experimental process, the pH-ZRCCC is used for separating out the polyphenol components with high content in the dandelion and enriching the low-content components, and the HSCCC is used for separating out other polyphenol components with low content, so that the separation and preparation time is effectively shortened. The method is simple to operate, is rapid and efficient, provides a new efficient preparation method for separating polyphenol compounds, provides technical support for development and utilization of the dandelion, and has good application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure. Embodiments of the present disclosure are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: HPLC analysis of crude and pure fractions of taraxacum polyphenol.
FIG. 2: pH-ZRCCC separation diagram of herba Taraxaci crude extract.
FIG. 3: separating picture of recovered component HSCCC of herba Taraxaci.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.
Example 1
1 materials and methods
1.1 Main raw materials
The dandelion is purchased in the local medicinal material market of Shandong and is identified as Taraxacum mongolicum hand-Mazz by experts; in the experiment, reagents such as n-butyl alcohol, n-hexane, methanol, absolute ethyl alcohol, ethyl acetate, petroleum ether, ammonia water, trifluoroacetic acid and the like are analytically pure; the methanol used for HPLC analysis was chromatographically pure.
1.2 instrumentation
The high-speed countercurrent chromatography system is provided with an infusion pump, a low-temperature constant-temperature tank, an ultraviolet detector and a portable recorder; other devices are respectively a pH meter, a rotary evaporator, a high performance liquid chromatograph and a nuclear magnetic resonance spectrometer.
2 method of experiment
2.1 extraction of Dandelion polyphenols
Pulverizing 1kg herba Taraxaci Mongolici, extracting with 60% ethanol at a material-liquid ratio of 1:10(w/v) under reflux twice, each for 2 hr, filtering, mixing extractive solutions, and concentrating under reduced pressure. Suspending the concentrated solution with water, extracting with petroleum ether for three times to defat and remove impurities such as chlorophyll, and extracting with ethyl acetate for five times. Concentrating ethyl acetate phase under reduced pressure to obtain paste. And filling the sample into a 30-80-mesh polyamide atmospheric pressure column by adopting a dry loading mode. Eluting with water, eluting with 30% ethanol, and concentrating 30% of the eluate under reduced pressure to dry to obtain the final product. And (5) putting the sample into a refrigerator, and storing for later use.
2.2 determination of the K value of the target Compound in a two-phase solvent System
2.2.1 pH-band determination of K-value by countercurrent chromatography
Approximately 5mg of the sample was weighed into a 10mL tube. Preparing a solvent system, taking 5mL of upper phase and lower phase in a test tube containing a sample, dripping ammonia water until the pH value is about 10, and shaking the test tube to the sampleThe product is completely dissolved and kept stand for layering. The upper and lower phases were each 10. mu.L, and detected by High Performance Liquid Chromatography (HPLC), respectively. Using the peak area of the upper phase (A)U) Peak area of phase (A)L) Calculating the partition coefficient K of the sample under alkaline conditionsbaseThe calculation formula is as follows: kbase＝AU/AL. Dripping trifluoroacetic acid into the test tube until the pH value is about 2, shaking up, standing for layering, respectively taking 10 μ L of upper and lower phases by the same method, and calculating the distribution coefficient K by HPLCacid。
2.2.2 measurement of K value by high-speed countercurrent chromatography
Approximately 5mg of the sample was weighed into a 10mL tube. Preparing solvent system, placing 5mL of each of the upper and lower phases in a test tube containing sample, shaking vigorously until the sample is completely dissolved, standing for layering, respectively taking 10 μ L of each of the upper and lower phases, detecting by HPLC, and collecting peak area of upper phase (A)1) Peak area of phase (A)2) Namely, the distribution coefficient K is calculated according to the following formula: k is A1/A2。
2.3 preparation of the two-phase solvent System
2.3.1 preparation of pH zone countercurrent chromatography solvent System
Ethyl acetate-acetonitrile-water (4:1:5, v/v/v) was used as a two-phase solvent system, and the separation was carried out by a pH zone method. Placing ethyl acetate, acetonitrile and water in a separating funnel according to a certain proportion, violently shaking, standing for layering, separating out an upper phase and a lower phase, adding 10mM trifluoroacetic acid into the upper phase, adding 10mM ammonia water into the lower phase, and respectively carrying out ultrasonic degassing for 3min for later use.
Weighing a sample, taking an equivalent acidified upper phase and a lower phase without adding alkali to dissolve the sample, and using the sample for subsequent countercurrent chromatographic separation.
2.3.2 preparation of high-speed countercurrent chromatography solvent System
Selecting petroleum ether-ethyl acetate-methanol-water (1:4:1:4, v/v/v/v) as a solvent system, putting the solvent system into a separating funnel in proportion, shaking uniformly, standing for layering, separating an upper phase and a lower phase, and respectively performing ultrasonic degassing for later use.
Weighing the sample, taking the same amount of upper phase and lower phase, and dissolving the sample for later use.
2.4 countercurrent chromatography separation
2.4.1 pH zonal countercurrent chromatography
Pumping the upper phase into a high-speed counter-current chromatograph at a rate of 30mL/min to serve as a stationary phase, and injecting the sample solution into the separation column after the stationary phase is filled in the chromatographic spiral tube. The speed controller was turned on and the separation column was rotated clockwise at 800rpm while the lower phase solution was pumped in at 2 mL/min. Starting an ultraviolet detector, adjusting the wavelength to 280nm, collecting chromatographic peak components according to the chromatogram, and detecting the pH value of the fraction by using a pH meter. And after the separation is finished, blowing out the residual liquid in the column by using a pressure pump, collecting the residual liquid in the measuring cylinder, and calculating the retention rate of the stationary phase by using the volume ratio of the stationary phase to the total volume of the tail blowing in the tail blowing.
2.4.2 high-speed countercurrent chromatography
Pumping the upper phase as the stationary phase at a speed of 30mL/min, starting a speed controller after the stationary phase is filled in the chromatographic spiral tube, enabling the separation column to rotate at a speed of 800rpm in a clockwise direction, and pumping the lower phase at a speed of 2 mL/min. When the system reaches hydrodynamic equilibrium, the sample is injected into the separation column. Starting an ultraviolet detector, and collecting chromatographic peak components according to the chromatogram map under the wavelength of 280 nm. And after the separation is finished, blowing out the residual liquid in the column by using a pressure pump, collecting the residual liquid in the measuring cylinder, and calculating the retention rate of the stationary phase by using the volume ratio of the stationary phase to the total volume of the tail blowing in the tail blowing.
Concentrating the separated components under reduced pressure to dry, sealing and storing in refrigerator.
2.5 HPLC analysis and structural identification
The dandelion total sample and the fractions obtained by separation and purification were analyzed by HPLC. The chromatographic column is C18A chromatographic column (250 multiplied by 4.6mm, 5 mu m), wherein the mobile phase is methanol (A) -0.1% formic acid aqueous solution (B), 0-10 min, 20-24% A, 10-20 min, 24% A, 20-21 min, 24-34% A, 21-30 min, 34-40% A, 30-35 min, 40-100% A, the flow rate is 1.0mL/min, the detection wavelength is 280nm, and the sample injection amount is 10 mu L. And calculating the purity of the sample by adopting a normalization method. The separated and purified components are dissolved in DMSO, and the chemical structures of the components are identified through ESI-MS and NMR analysis.
3 results and analysis
3.1 HPLC analysis results
FIG. 1 is a HPLC analysis chart of crude extract and prepared pure product of taraxacum polyphenol. The peak area ratios of the target compounds I to VI in the total sample were 4.50% (compound V), 1.34% (compound IV), 8.37% (compound VI), 40.01% (compound I), 6.68% (compound III), and 9.07% (compound II), respectively, as calculated using peak area normalization at 280 nm.
3.2 pH zonal countercurrent chromatography separation
The pH-ZRCCC is prepared in large quantities and is suitable for components with a concentration of more than 0.1mM, preferably more than 1 mM. Therefore, the pH zone countercurrent chromatography is firstly selected to separate polyphenol components with higher content in the dandelion, and simultaneously, trace components are enriched.
The proper partition coefficient (K) is the key to the successful separation of pH zone countercurrent chromatography, and when separating acidic substances, the K of a solvent system is requiredbase1 and Kacid> 1, while satisfying the requirement that the sample has good solubility in the solvent system. Selecting a solvent system of ethyl acetate-n-butanol-water (4:1:5, v/v/v) according to literature reports and experience of the laboratory, and measuring a distribution coefficient K of the target compound in the solvent systemacidAnd KbaseThe results are shown in Table 1. The results showed that the solvent system was suitable for the separation of the sample, and therefore 1.6g of crude dandelion extract was separated using ethyl acetate-n-butanol-water (4:1:5, v/v/v) as the solvent system, trifluoroacetic acid (10mM) as the stationary phase added to the upper phase and ammonia water (10mM) as the mobile phase added to the lower phase, and the target compound was eluted as irregular rectangular peaks as shown in fig. 2, with a retention of the stationary phase of 61.1%. Compound I, II was successfully isolated at 10h by HPLC with 98.1% and 98.8% purity, respectively, whereas the other compounds were less separated and were combined as mixture a. After freeze-drying of each component sample, 60.2mg of compound I, 6.3mg of compound II and 590mg of mixture A were obtained.
TABLE 1 partition coefficient K of the object compounds I to VI in solvent systemsacidAnd Kbase
3.3 separation by high-speed countercurrent chromatography
The mixture A can not achieve the separation effect after being separated by the pH zone countercurrent chromatography, and the pH value is presumed to be possibly similar or the component content is less, so the method is suitable for separating the mixture A again by applying the high-speed countercurrent chromatography.
The key point of the high-speed counter-current chromatography separation is the selection of a solvent system, and the K value of a target compound in the solvent system is 0.5-2.0. First, a common solvent system of ethyl acetate-acetonitrile-water (4:1:5, v/v/v) is selected, and the K value of the target compound in the solvent system is found to be far greater than 2.0. It is therefore speculated that the target compound may be less polar, and petroleum ether is added to the solvent system to adjust the polarity of the solvent system. Finally, petroleum ether-ethyl acetate-acetonitrile-water (1:4:1:4, V/V/V/V) is selected as a solvent system, and K values of the compounds III, IV, V and VI are respectively 1.03, 0.97, 0.86 and 0.78 which are determined to be in accordance with the selection range of the solvent system. Therefore, 400mg of mixture A was separated by using petroleum ether-ethyl acetate-acetonitrile-water (1:4:1:4, v/v/v/v) as a solvent system for HSCCC separation, and as a result, as shown in FIG. 3, the mixture A was separated within 6.5h, and the stationary phase retention rate was 69.4%. HPLC analysis showed that compound III (7.7mg, 82.2% purity), compound IV (5.4mg, 24.8% purity), compound V (6.2mg, 95.3% purity), compound VI (3.4mg, 89.0% purity). The purity of the product is up to more than 90% by purification of semi-preparative high performance liquid chromatography, and the product is enriched for subsequent structural identification for many times.
3.4 structural characterization of Compounds
A compound I: ESI-MS, M/z 179.0332[ M-H ]]-。1H-NMR(400MHz,DMSO)δ: 8.43(s,1H),7.33(d,J＝15.8Hz,1H),7.02(s,1H),6.90(d,J＝8.1Hz,1H), 6.75(d,J＝8.1Hz,1H),6.18(d,J＝15.8Hz,2H).13C-NMR (100MHz, DMSO). delta. 169.35(s),148.64(s),146.30(s),143.50(s),126.40(s),121.16(s),117.61(s), 116.38(s), 115.06(s). And the literature [ inhibitors of bioassay-regulated isolation of anti-glycation components from Taraxacum coreanum and simultaneous quantification.Molecules,2018,23(9):2148-2161]The reported caffeic acid data are consistent, and compound i was identified as caffeic acid.
Compound II: ESI-MS, M/z 163.0288[ M-H ]]-。1H-NMR(400MHz,DMSO)δ: 7.49(t,J＝11.7Hz,3H),6.79(d,J＝8.5Hz,2H),6.29(d,J＝15.9Hz,1H),1.23 (s,1H).13C NMR (100MHz, DMSO). delta. 159.88(s),144.27(s),130.48(s), 116.20(s). And the literature [ Defuscin, a new phenolic ester from Dendrobium fuscescens: transformation of shikimic acid. phytochemistry,1989,28(1):290-]The reported data of p-hydroxycinnamic acid are consistent, so that the compound II is identified as p-hydroxycinnamic acid.
Compound III: ESI-MS, M/z 253.0653[ M-H ]]-。1H NMR(400MHz,DMSO)δ 7.49(d,J＝15.9Hz,1H),7.05(s,1H),7.00(d,J＝8.2Hz,1H),6.77(d,J＝8.1 Hz,1H),6.26(d,J＝15.9Hz,1H),4.15(dd,J＝11.2,4.0Hz,1H),4.00(dd,J＝ 11.1,6.5Hz,1H),3.75–3.66(m,1H),1.24(s,2H).13C NMR (100MHz, DMSO) δ 167.09(s),148.92(s),146.08(s),145.55(s),125.96(s),121.80(s),116.24(s), 115.23(s),114.46(s),69.90(s),66.08(s), 63.17(s). And the literature [ the research of the chemical components of phenolic acids in the Senecio chuanensis, Henan Chinese medicine, 2014,34(9):1847-1849]The reported data for 1-O-caffeoyl glycerol are consistent, so compound III was identified as 1-O-caffeoyl glycerol.
Compound IV: ESI-MS, M/z 167.0277[ M-H ]]-。1H NMR(600MHz,DMSO)δ 8.84(s,1H),6.64(d,J＝2.6Hz,2H),6.63(s,1H),6.48(dd,J＝8.0,2.1Hz,1H), 3.32(s,3H).13C NMR (150MHz, DMSO). delta. 173.77(s),145.43(s),144.43(s), 126.32(s),120.46(s),117.13(s), 115.79(s). And documents [ cortex Fraxini phenolic component and its analgesic activity, Chinese patent medicine, 2019,41(7): 1582-1586-]The reported data for 3, 4-dihydroxybenzoic acid are consistent, so that compound IV is identified as 3, 4-dihydroxybenzoic acid.
Compound V: ESI-MS, M/z 153.0089[ M-H ]]-。1H NMR(400MHz,DMSO)δ 9.46(s,2H),7.33(s,1H),7.31–7.25(m,1H),6.78(d,J＝8.2Hz,1H).13C NMR (100MHz,DMSO)δ167.77(s) 150.45(s),145.34(s),122.34(s),122.14(s), 117.01(s), 115.60(s). And the literature [ two novel phenolic acid compounds in drynaria, pharmaceutical science, 2010,45(7):874-878]The protocatechuic acid data reported were consistent, so compound v was identified as protocatechuic acid.
Compound VI: ESI-MS, M/z 151.0288[ M-H ]]-。1H NMR(400MHz,DMSO)δ 12.15(s,1H),9.27(s,1H),7.03(d,J＝8.3Hz,1H),6.69(d,J＝8.3Hz,1H).13C NMR (100MHz, DMSO). delta. 173.59(s),156.50(s),130.71(s),125.60(s), 115.48(s). And the literature [ phenolic acid component in Fufang Sanzi Yangqin Tang, Shenyang pharmaceutical university, 2007(11): 679-]The reported data for p-hydroxyphenylacetic acid are consistent, and therefore, compound VI is identified as p-hydroxyphenylacetic acid.
The invention adopts a mode of combining pH zone countercurrent chromatography (pH-ZRCCC) and high-speed countercurrent chromatography (HSCCC) to separate 6 polyphenol compounds from taraxacum. In the pH-ZRCCC separation, a solvent system of ethyl acetate-acetonitrile-water (4:1:5, v/v/v) is selected, trifluoroacetic acid (10mM) is added to the upper phase as a stationary phase, and ammonia water (10mM) is added to the lower phase as a mobile phase. Caffeic acid (60.2mg, 98.1% purity) and hydroxycinnamic acid (6.3mg, 98.8% purity) and 590mg of mixture A were isolated from 1.6g of the crude ethyl acetate extract of dandelion. Then further using HSCCC, selecting a solvent system of petroleum ether-ethyl acetate-methanol-water (1:4:1:4, v/v/v/v). 1-O-caffeoyl glycerol (4.0mg, purity 99.7%), 3, 4-dihydroxyphenylacetic acid (0.4mg, purity 93.3%), protocatechuic acid (6.2mg, purity 95.3%) and p-hydroxyphenylacetic acid (2.1mg, purity 94.3%) were isolated from 400mg of mixture A. The method has the advantages of large preparation amount and good reproducibility, and is suitable for separating and purifying polyphenol compounds of herba Taraxaci.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for efficiently separating polyphenol components in dandelion is characterized by comprising the following steps:
(1) extracting polyphenol of dandelion; pulverizing herba Taraxaci, extracting with solvent under reflux, and concentrating under reduced pressure to obtain concentrated solution; extracting the concentrated solution, eluting and concentrating after extraction to obtain a dandelion sample;
(2) preparing a pH zone countercurrent chromatography solvent system and a high-speed countercurrent chromatography solvent system;
(3) dissolving a dandelion sample in a pH zone countercurrent chromatography solvent system, carrying out pH zone countercurrent chromatography separation, and separating to obtain a compound I, a compound II and a mixture A; dissolving the mixture A in a high-speed counter-current chromatography solvent system, and performing high-speed counter-current chromatography separation to obtain a compound III, a compound IV and a compound V;
(4) analyzing the dandelion sample and the compound obtained by separation and purification by using HPLC, and calculating the purity; ESI-MS and NMR analysis are adopted to identify the chemical structure of the compound obtained by separation and purification.
2. The method according to claim 1, characterized in that step (1) is in particular: pulverizing appropriate amount of herba Taraxaci, extracting with 60% ethanol at a material-to-liquid ratio of 1:10(w/v) under reflux, filtering, and concentrating under reduced pressure to obtain concentrated solution; suspending the concentrated solution with water, extracting with petroleum ether, and extracting with ethyl acetate; concentrating ethyl acetate phase under reduced pressure to obtain paste, eluting with water and ethanol, and concentrating 30% of the eluate under reduced pressure to dry to obtain herba Taraxaci sample.
3. The method as claimed in claim 1, wherein the K value of the sample in the pH zone countercurrent chromatography and the high speed countercurrent chromatography is determined before the pH zone countercurrent chromatography and the high speed countercurrent chromatography solvent system are prepared.
4. The method of claim 3, wherein the method is performed in a batch processCharacterized in that the K value of the pH zone countercurrent chromatography is determined by the following method: weighing a sample in a test tube; preparing a solvent system, taking the upper phase and the lower phase in a test tube filled with a sample, dripping ammonia water until the pH value is about 10, shaking the test tube until the sample is completely dissolved, standing and layering; detecting the upper and lower phases by High Performance Liquid Chromatography (HPLC); using the peak area of the upper phase (A)U) Peak area of phase (A)L) Calculating the partition coefficient K of the sample under alkaline conditionsbaseThe calculation formula is as follows: kbase＝AU/AL(ii) a Dripping trifluoroacetic acid into the test tube until the pH value is about 2, shaking up, standing for layering, taking the upper phase and the lower phase respectively according to the same method, and calculating the distribution coefficient K by using HPLCacid。
5. The method of claim 3, wherein the K value of the sample is determined by high-speed countercurrent chromatography as follows: weighing a sample in a test tube; preparing solvent system, placing upper and lower phases in test tube containing sample, shaking vigorously until the sample is completely dissolved, standing for layering, respectively taking upper and lower phases, detecting by HPLC, and collecting peak area of upper phase (A)1) Peak area of phase (A)2) Namely, the distribution coefficient K is calculated according to the following formula: k is A1/A2。
6. The method according to claim 1, wherein the pH-zonal countercurrent chromatography solvent system is formulated as: selecting ethyl acetate-acetonitrile-water (4:1:5, v/v/v) as a two-phase solvent system, and separating by adopting a pH zone method; placing ethyl acetate, acetonitrile and water in a separating funnel according to a certain proportion, violently shaking, standing for layering, separating out an upper phase and a lower phase, adding trifluoroacetic acid into the upper phase, adding ammonia water into the lower phase, and respectively performing ultrasonic degassing; weighing a sample, taking an equivalent acidified upper phase and a lower phase without adding alkali to dissolve the sample, and using the sample for subsequent countercurrent chromatographic separation.
7. The method according to claim 1, wherein the solvent system for high-speed countercurrent chromatography is specifically formulated as: selecting petroleum ether-ethyl acetate-methanol-water (1:4:1:4, v/v/v/v) as a solvent system, putting the solvent system into a separating funnel in proportion, shaking uniformly, standing for layering, separating an upper phase and a lower phase, and respectively performing ultrasonic degassing for later use; weighing the sample, taking the same amount of upper phase and lower phase, and dissolving the sample for later use.
8. The method according to claim 1, characterized in that the pH-zone countercurrent chromatography separation is in particular: pumping the upper phase into a high-speed counter-current chromatograph as a stationary phase, and injecting a sample solution into the separation column after the stationary phase is filled in the chromatographic spiral tube; starting a speed controller to enable the separation column to rotate clockwise, and simultaneously pumping a lower phase solution; starting an ultraviolet detector, adjusting the wavelength to 280nm, collecting chromatographic peak components according to a chromatogram, and detecting the pH value of the fraction by using a pH meter; and after the separation is finished, blowing out the residual liquid in the column by using a pressure pump, collecting the residual liquid in the measuring cylinder, and calculating the retention rate of the stationary phase by using the volume ratio of the stationary phase to the total volume of the tail blowing in the tail blowing.
9. The method according to claim 1, wherein the high-speed counter-current chromatography is specifically: pumping the upper phase as a stationary phase, starting a speed controller after the stationary phase is filled in the chromatographic spiral tube, enabling the separation column to rotate clockwise, and pumping the lower phase; when the system reaches the fluid dynamic equilibrium, injecting the mixture A into a separation column; starting an ultraviolet detector, and collecting chromatographic peak components according to a chromatogram map under the wavelength of 280 nm; after the separation is finished, blowing out the residual liquid in the column by using a pressure pump, collecting the residual liquid in a measuring cylinder, and calculating the retention rate of the stationary phase by using the volume ratio of the stationary phase in tail blowing to the total volume of the tail blowing; concentrating the separated components under reduced pressure to dry, sealing and storing in refrigerator.
10. The method according to claim 1, wherein the dandelion sample and the isolated and purified compound are analyzed by HPLC; wherein the chromatographic column is C18A chromatographic column (250 multiplied by 4.6mm, 5 mu m), a mobile phase of methanol (A) -0.1 percent formic acid aqueous solution (B), 0 to 10min, 20 to 24 percent A, 10 to 20min, 24 percent A, 20 to 21min, 24 to 34 percent A, 21 to 30min, 34 percent to E40% A, 30-35 min, 40-100% A, flow rate of 1.0mL/min, detection wavelength of 280nm, and sample injection amount of 10 muL; and calculating the purity of the sample by adopting a normalization method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111676346.XA CN114280201B (en) | 2021-12-31 | 2021-12-31 | Efficient separation method of polyphenol components in dandelion |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111676346.XA CN114280201B (en) | 2021-12-31 | 2021-12-31 | Efficient separation method of polyphenol components in dandelion |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114280201A true CN114280201A (en) | 2022-04-05 |
| CN114280201B CN114280201B (en) | 2023-06-13 |
Family
ID=80879758
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111676346.XA Active CN114280201B (en) | 2021-12-31 | 2021-12-31 | Efficient separation method of polyphenol components in dandelion |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114280201B (en) |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5332504A (en) * | 1993-03-05 | 1994-07-26 | The United States Of America As Represented By The Department Of Health And Human Services | pH-zone-refining countercurrent chromatography |
| CN1506680A (en) * | 2002-12-10 | 2004-06-23 | 中国农业科学院茶叶研究所 | Countercurrent chromatographic separation process for reducing fixed phase |
| JP2004256481A (en) * | 2003-02-27 | 2004-09-16 | Asahi Breweries Ltd | Method for separating polyphenols |
| US20070054868A1 (en) * | 2005-06-20 | 2007-03-08 | The Trustees Of Columbia University In The City Of New York | Synergistic polyphenol compounds, compositions thereof, and uses thereof |
| CN105223300A (en) * | 2015-08-03 | 2016-01-06 | 广东药学院 | The method of quality control of lichee polyphenol and products thereof |
| RU2597661C1 (en) * | 2015-07-06 | 2016-09-20 | федеральное государственное бюджетное образовательное учреждение высшего образования "Воронежский государственный университет" (ФГБОУ ВО "ВГУ") | Method of determining polyphenolic compounds by stepped elution in thin layer of sorbent |
| CN106866602A (en) * | 2017-03-27 | 2017-06-20 | 浙江工业大学 | A kind of method that application high speed adverse current chromatogram separates flavone compound in Hericium erinaceus |
| CN108743471A (en) * | 2018-06-25 | 2018-11-06 | 佛山市聚成生化技术研发有限公司 | A kind of antibacterial anti-inflammatory face cream and preparation method thereof |
| CN109503373A (en) * | 2019-01-10 | 2019-03-22 | 辽宁大学 | A kind of method of polyphenolic substance in fast separating and purifying serviceberry |
| CN110746302A (en) * | 2019-10-16 | 2020-02-04 | 山东省分析测试中心 | Method for separating and preparing phenolic acid compounds in echinacea purpurea |
| CN112110886A (en) * | 2020-10-10 | 2020-12-22 | 长春师范大学 | Method for separating polyphenol compounds in phellinus igniarius by utilizing high-efficiency countercurrent chromatography |
| CN113185406A (en) * | 2021-04-16 | 2021-07-30 | 山东省分析测试中心 | Efficient preparation method of phenolic acid active ingredients in salvia yunnanensis |
| CN113402574A (en) * | 2021-04-07 | 2021-09-17 | 江西省科学院应用化学研究所 | Method for separating and purifying flavonoids compounds in ethyl acetate phase of polygonum multiflorum by high-speed counter-current chromatography |
| CN113484434A (en) * | 2021-07-01 | 2021-10-08 | 苏州求是本草健康科技有限公司 | Detection method of rose polyphenol |
-
2021
- 2021-12-31 CN CN202111676346.XA patent/CN114280201B/en active Active
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5332504A (en) * | 1993-03-05 | 1994-07-26 | The United States Of America As Represented By The Department Of Health And Human Services | pH-zone-refining countercurrent chromatography |
| CN1506680A (en) * | 2002-12-10 | 2004-06-23 | 中国农业科学院茶叶研究所 | Countercurrent chromatographic separation process for reducing fixed phase |
| JP2004256481A (en) * | 2003-02-27 | 2004-09-16 | Asahi Breweries Ltd | Method for separating polyphenols |
| US20070054868A1 (en) * | 2005-06-20 | 2007-03-08 | The Trustees Of Columbia University In The City Of New York | Synergistic polyphenol compounds, compositions thereof, and uses thereof |
| RU2597661C1 (en) * | 2015-07-06 | 2016-09-20 | федеральное государственное бюджетное образовательное учреждение высшего образования "Воронежский государственный университет" (ФГБОУ ВО "ВГУ") | Method of determining polyphenolic compounds by stepped elution in thin layer of sorbent |
| CN105223300A (en) * | 2015-08-03 | 2016-01-06 | 广东药学院 | The method of quality control of lichee polyphenol and products thereof |
| CN106866602A (en) * | 2017-03-27 | 2017-06-20 | 浙江工业大学 | A kind of method that application high speed adverse current chromatogram separates flavone compound in Hericium erinaceus |
| CN108743471A (en) * | 2018-06-25 | 2018-11-06 | 佛山市聚成生化技术研发有限公司 | A kind of antibacterial anti-inflammatory face cream and preparation method thereof |
| CN109503373A (en) * | 2019-01-10 | 2019-03-22 | 辽宁大学 | A kind of method of polyphenolic substance in fast separating and purifying serviceberry |
| CN110746302A (en) * | 2019-10-16 | 2020-02-04 | 山东省分析测试中心 | Method for separating and preparing phenolic acid compounds in echinacea purpurea |
| CN112110886A (en) * | 2020-10-10 | 2020-12-22 | 长春师范大学 | Method for separating polyphenol compounds in phellinus igniarius by utilizing high-efficiency countercurrent chromatography |
| CN113402574A (en) * | 2021-04-07 | 2021-09-17 | 江西省科学院应用化学研究所 | Method for separating and purifying flavonoids compounds in ethyl acetate phase of polygonum multiflorum by high-speed counter-current chromatography |
| CN113185406A (en) * | 2021-04-16 | 2021-07-30 | 山东省分析测试中心 | Efficient preparation method of phenolic acid active ingredients in salvia yunnanensis |
| CN113484434A (en) * | 2021-07-01 | 2021-10-08 | 苏州求是本草健康科技有限公司 | Detection method of rose polyphenol |
Non-Patent Citations (2)
| Title |
|---|
| LIHUA XU ET AL.: "Preparative separation of seven polyphenols from Perillae Folium via pH-zone-refining countercurrent chromatography combined with highspeed counter-current chromatography", ANAL. METHODS, pages 1232 - 1237 * |
| 吕迎春 等: "逆流色谱技术在国内茶叶天然产物中的研究及应用", 云南化工, vol. 35, no. 4, pages 50 - 53 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114280201B (en) | 2023-06-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106866602B (en) | A method of using flavone compound in high speed adverse current chromatogram separation Hericium erinaceus | |
| Lu et al. | Preparative high-speed counter-current chromatography for purification of shikonin from the Chinese medicinal plant Lithospermum erythrorhizon | |
| CN103145677B (en) | Method for separating active ingredients from aquilaria sinensis lamina by utilizing high-speed countercurrent chromatography | |
| CN103421077B (en) | Method for separating and purifying limonin compounds from pomelo fruits | |
| CN110294673B (en) | Caffeoylquinic acid butyl ester isomer and preparation method and application thereof | |
| Zhang et al. | Application of accelerated solvent extraction coupled with counter-current chromatography to extraction and online isolation of saponins with a broad range of polarity from Panax notoginseng | |
| Lu et al. | Preparative separation of gallic acid from Chinese traditional medicine by high-speed counter-current chromatography and followed by preparative liquid chromatography | |
| Wang et al. | Application of high-speed counter-current chromatography for preparative separation of cyclic peptides from Vaccaria segetalis | |
| CN102020691A (en) | High performance liquid chromatographic separation method of four monomer components of aescine | |
| CN106957310B (en) | The high efficiency preparation method of flavonoids monomer in a kind of leaves of Hawthorn | |
| CN110746302B (en) | Method for separating and preparing phenolic acid compounds in echinacea purpurea | |
| CN101805352B (en) | Method for preparing eriocalyxin B | |
| CN114989152B (en) | Method for separating and preparing two apigenin glycosides from dendrobium candidum leaves | |
| CN114280201A (en) | Efficient separation method for polyphenol components in dandelion | |
| CN109053433B (en) | Combined preparation method of ginkgolic acid | |
| CN100500689C (en) | Method of separating and preparing wild jujube seed saponin | |
| CN106957309B (en) | The preparation method of lignanoids monomer in a kind of justicia | |
| CN109369382B (en) | A method for preparing ginkgolic acids | |
| CN106831892B (en) | Preparation method of flavone monomer in hawthorn leaves | |
| CN102532147B (en) | Preparation method of high purity dictamnine monomer | |
| CN100526329C (en) | Production of high-purity peiminine and fritimine | |
| CN108440612A (en) | The isolation and purification method of three kinds of iridoid constituents in a kind of radix scrophulariae | |
| CN111718318B (en) | Method for separating flavone monomer in spina gleditsiae based on countercurrent chromatography | |
| CN109053417B (en) | Preparation method of high-purity ginkgolic acid | |
| CN101070335A (en) | Process for preparing high-purity gentiopicrin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |