CN110407906B - Method for separating and preparing triterpene compounds from fructus Hippophae extract - Google Patents
Method for separating and preparing triterpene compounds from fructus Hippophae extract Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention provides a chromatographic preparation method of a compound, which comprises the steps of preparing sea buckthorn into sea buckthorn extract, mixing the extract with silica gel, then carrying out column filling, and carrying out column flushing by taking petroleum ether and mixed liquid of the petroleum ether and ethyl acetate which are mixed in different volume proportions as a mobile phase; and (2) sequentially carrying out liquid chromatography analysis, normal phase chromatography preparation, liquid chromatography analysis and reverse phase chromatography preparation on the fraction obtained in the step (1), and finally carrying out liquid chromatography analysis on the purity. The compound is extracted and prepared from the seabuckthorn extract for the first time, has the alpha-glucosidase inhibition effect, and can be applied to preparation of products related to blood sugar reduction.
Description
Technical Field
The invention relates to the field of extraction of effective substances in sea buckthorn, in particular to a method for separating and preparing a triterpene compound from a sea buckthorn extract.
Background
Seabuckthorn (the name of Latin, Hippophae rhamnoides Linn.) is a deciduous shrub of the genus Hippophae of the family Elaeagnaceae, and is drought-resistant and sand-resistant, and can survive in saline-alkali lands, thus being widely used for water and soil conservation. Sea buckthorn is planted in great amount in northwest China for desert greening.
The sea-buckthorn fruit is rich in nutrition, and active substances such as various vitamins, fatty acids, trace elements, linolein, sea-buckthorn flavone, superoxide and the like and various amino acids required by a human body are determined to be contained in the sea-buckthorn fruit. Wherein the content of vitamin C is very high, and the content of vitamin C in every 100 g of fruit juice can reach 825-1100 mg, which is 2-3 times of that of kiwi fruit, and the vitamin has the beauty of the king of vitamin C. The method is widely applied to the production of sea-buckthorn juice and sea-buckthorn raw pulp, and in the production process, the skin of the sea-buckthorn needs to be peeled off, the peeled sea-buckthorn peel is usually discarded as waste, or the sea-buckthorn peel is produced into dry powder to be used as feed, so the price is very low.
It has been reported that seabuckthorn fruit peel contains two triterpenic acids, namely ursolic acid and oleanolic acid, has the effects of protecting liver, reducing blood sugar, resisting skin oxidation and the like, and more effective substances are expected to be detected in seabuckthorn, so that scientific development and utilization of seabuckthorn are facilitated.
Disclosure of Invention
The invention aims to provide a preparation method of a compound shown in a formula I,
the method comprises the following steps:
(1) and (2) applying the sea buckthorn extractum to a silica gel column, and eluting by taking petroleum ether and mixed liquid of the petroleum ether and ethyl acetate which are mixed in different volume ratios as a mobile phase, wherein the mixed ratio of the petroleum ether and the ethyl acetate is (9-1): (1-9) collecting a triterpene compound eluent;
(2) and (2) sequentially carrying out liquid phase normal phase chromatographic analysis, normal phase chromatographic preparation, reverse phase liquid chromatographic analysis and reverse phase chromatographic preparation on the eluent obtained in the step (1), and finally carrying out liquid chromatographic analysis on the purity to obtain the compound shown in the formula I.
Further, the step (2) comprises the following steps:
subjecting the fraction obtained in step (1) to normal phase liquid chromatography under chromatographic conditions comprising:
a chromatographic column: a hydrophilic HILIC chromatography column; preferably a 4.6mm 250mm,5 μm, more preferably a xamide4.6mm 250mm,5 μm, column;
mobile phase: a is ethanol, B is n-hexane; isocratic elution conditions: 2% -4% of A and 98% -96% of B;
preferably, at least one of the following conditions is also included:
detection wavelength: 203 +/-10 nm; preferably 203 +/-5 nm;
column temperature: 30 +/-5 ℃; preferably 30 +/-2 ℃;
flow rate: 1 plus or minus 0.5 mL/min; preferably 1 plus or minus 0.2 mL/min;
sample introduction amount: 10 +/-5 mu L; preferably 10 +/-2 mu L;
further, the normal phase chromatography preparation conditions comprise:
a chromatographic column: a HILIC chromatography column; the preferable specification is 20mm × 250mm,5 μm; more preferably a XAmide20mm x 250mm,5 μm chromatography column;
mobile phase: a is ethanol, B is n-hexane; isocratic elution conditions: 2% -4% of A and 98% -96% of B;
preferably, at least one of the following preparative chromatographic conditions is also included:
detection wavelength: 203 +/-10 nm; preferably 203. + -.5 nm
Column temperature: 30 +/-5 ℃; preferably 30. + -. 2 ℃ C
Flow rate: 19 plus or minus 2 mL/min; preferably 19 plus or minus 1 mL/min;
sample introduction amount: 1 plus or minus 0.5 mL; preferably 1 plus or minus 0.2 mL;
further, performing reverse phase liquid chromatography, wherein the chromatographic conditions of the reverse phase liquid chromatography comprise:
a chromatographic column: a C18 chromatography column, preferably of 4.6mm x 250mm,5 μm, more preferably a Kromasil C18 column (4.6mm x 250mm,5 μm);
mobile phase: a is water, B is acetonitrile, isocratic elution conditions are as follows: 20-30% of A and 80-70% of B;
preferably, at least one of the following chromatographic conditions is also included:
detection wavelength: 203 +/-10 nm;
column temperature: 30 +/-5 ℃;
flow rate: 1 plus or minus 0.5 mL/min;
further, the reverse phase preparative chromatography conditions include:
a chromatographic column: a C18 chromatographic column, preferably 21.2mm x 250mm,5 μm, Kromasil 100-5-C18 (21.2 x 250mm,5 μm);
column temperature: 30 ℃;
mobile phase: a is water, B is acetonitrile, isocratic elution conditions are as follows: 20-30% of A and 80-70% of B;
preferably, at least one of the following chromatographic preparation conditions is also included:
detection wavelength: 203 +/-10 nm;
column temperature: 30 +/-5 ℃;
flow rate: 21 plus or minus 2 mL/min.
Concentrating the prepared solution, performing liquid phase analysis under the same conditions, and judging the purity of the obtained component; if the purity is more than 98 percent, obtaining a target compound; if the purity is less than 98%, then the chromatographic preparation is carried out, preferably, the liquid chromatographic analysis comprises the following chromatographic conditions:
a chromatographic column: c18 chromatographic column, preferably 4.6mm × 250mm,5 μm;
mobile phase: a is water, B is acetonitrile, isocratic elution conditions are as follows: 20 to 30 percent of A and 80 to 70 percent of B.
In the above scheme of the invention, the column temperature, the detection wavelength, the sample injection amount, the flow rate and the like can be selected in a common range. Wherein the detection wavelength can be adjusted and selected within the ranges disclosed above by conventional means. When the optimal detection wavelength is searched, the method can be carried out by using a full-wave-band scanning method and the like which are matched with an ultraviolet spectrophotometry method and HPLC, and then the appropriate detection wavelength is found by adopting a conventional technology by matching with the detection effect of an HPLC detector (such as avoiding solvent interference). In one embodiment of the present invention, the detection wavelength is selected from 201 to 205nm, for example 203 nm.
Further, the mixing ratio of the petroleum ether and the ethyl acetate adopted in the step (1) in sequence is 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1: 9.
Preferably, the fraction of petroleum ether and ethyl acetate of 7:3 determined by thin layer chromatography during the elution in the step (1) is the fraction with the highest content of the triterpene compounds.
Further, after the preparation of the normal phase chromatography, collecting the corresponding components with the retention time of 14-19 min, and carrying out the next analysis.
Further, the seabuckthorn extract in the step (1) is obtained by extracting seabuckthorn with ethanol; further, ethanol with the volume concentration of 95% is adopted for extraction; further on; mixing the sea-buckthorn pomace and ethanol according to a material-liquid ratio of 1: (10-30) kg/L of the mixture is extracted at 70-80 ℃ for 1.5-2.5 h each time for 3-4 times, and the extracting solutions are combined to remove the solvent to obtain an extract.
The invention has the following beneficial effects:
the compound is prepared from the seabuckthorn extract for the first time by liquid chromatography analysis and a normal phase and reverse phase chromatography preparation method, has the activity inhibition effect of alpha-glucosidase, and can be used for preparing products related to blood sugar reduction.
Drawings
FIG. 1 is a normal phase liquid chromatography profile;
FIG. 2 is a normal phase chromatogram preparation map;
FIG. 3 is a reversed phase liquid chromatography profile;
FIG. 4 is a reverse phase chromatography preparation map;
FIG. 5 is a prepared liquid chromatography chromatogram.
FIG. 6 acarbose inhibition curves at different concentrations;
FIG. 7 inhibition curves for different concentrations of inhibitor.
Detailed Description
The present invention is further illustrated by the following specific embodiments. The following description is only exemplary of the present invention and is not intended to limit the scope of the present invention, which is defined by the claims and their equivalents, as well as any equivalents thereof, which may be directly or indirectly applied to other related arts.
In the following examples, Obtusol is a compound of formula I as described in the present invention
Example 1
1. Preparation of extract
Extracting 5kg of seabuckthorn fruit residues with 95% ethanol in a traditional Chinese medicine decocting machine for 2 hours each time, wherein the material-liquid ratio is 1:10kg/L, extracting for 3 times, combining 3 extracting solutions, and concentrating under reduced pressure to obtain about 500g of extract.
2. Preparation of the Compounds
Main instruments and reagents
The instrument comprises the following steps: agilent 1260 series HPLC is equipped with G1311C quaternary gradient pump, G1329B autosampler, G1316A column oven, G1315D detector.
Reagent: the analytical and preparative acetonitrile is from new blue view chemical industry of Yunnan, and the water for chromatography is Wahaha purified water.
2.1, mixing the extract with silica gel, washing the column with petroleum ether and ethyl acetate in different proportions, wherein the proportions of the petroleum ether and the ethyl acetate are respectively 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9, determining that the fraction part in the petroleum ether and the ethyl acetate 7:3 is the part with higher content of triterpenic acid through a silica gel column point plate, and carrying out normal phase chromatographic analysis and preparation on 4-13 parts (each 500mL of the fraction is collected into one bottle, and the fractions in the 4 th bottle to the 13 th bottle are collected) in the 7:3 fraction.
2.2 establishment of liquid chromatography
Fractions 4-13 of the 7:3 fraction were analyzed on a XAmide (4.6X 250mm,5 μm) analytical column under the following chromatographic conditions: mobile phase: a is ethanol, B is n-hexane; isocratic elution conditions: 0-45 min, 4% of A and 96% of B; flow rate: 1 mL/min; column temperature: 30 ℃; detection wavelength: 203nm, and the sample size is 10 μ L.
2.3 preparation by normal phase chromatography
The fraction 7:3 was subjected to normal phase chromatography on a XAmide (20 mm. times.250 mm,5 μm) column for 4 to 13 portions. The preparation time is 1mL, flow rate: 19mL/min, otherwise identical to the XAmide analytical column assay conditions. Collecting components: f-1 in 3-3.8 min; f-2 in 3.9-4.6 min; f is between 6.2 and 8min and is between 3 and F; f-4 in 8.5-10 min; f-5 in 10-14 min; f-6 in 14-19 min; f-7 in 20-23 min; f-8 in 23-25 min; f-9 in 25-30 min; f-10 in 30-35 min; f-11 in 35-37 min.
2.4 reverse phase chromatography preparation
After F-6 (fraction 6 obtained by subjecting 4-13 parts of the fraction 7:3 to normal phase chromatography) was analyzed on a Kromasil 100-5-C18 (4.6X 250mm,5 μm) analytical column, reverse phase chromatography was performed on a Kromasil 100-5-C18 (21.2X 250mm,5 μm) preparative column, and then analysis was performed on a Kromasil 100-5-C18 (4.6X 250mm,5 μm) analytical column under the same liquid phase conditions.
The chromatographic analysis conditions were: mobile phase: a: water, B: acetonitrile; isocratic elution conditions: 0-40 min, 20% of A and 80% of B; analyzing the flow rate: 1 mL/min; detection wavelength: 203 nm; column temperature: at 30 ℃. The analysis condition is also the liquid chromatogram condition for the qualitative and quantitative detection of the compound.
The chromatographic preparation conditions are as follows: mobile phase: a: water, B: acetonitrile; isocratic elution conditions: 0-40 min, 20% of A and 80% of B; flow rate: 21 mL/min; detection wavelength: 203 nm; column temperature: 30 ℃;
the prepared fractions were subjected to liquid chromatography, and the results are shown in FIG. 5, with a chromatographic peak purity of 96.8%. NMR identification of the obtained compound:
the NMR information for this compound is as follows: obtusol compound of formula: c30H50O2The amount of the white powder,1H NMR(600MHz,MeOD)δ5.14(1H,t,J＝3.6Hz,H～12)，3.56,3.03(2H,(a)d,J＝11.0Hz；(b)d,J＝11.0Hz,H～27)，3.16(1H,dd,J＝11.4,4.8Hz,H～3)，1.95(1H,m,H～18)，1.94(2H,m,H～16)，1.91(2H,m,H～11)，1.62(2H,m,H～21)，1.61(1H,m,H～19)，1.57(2H,m,H～22)，1.55(2H,m,H～1)，1.41(2H,m,H～7)，1.39(2H,m,H～6)，1.32(1H,m,H～9)，1.25(2H,m,H～2)，1.23(2H,m,H～15)，1.12(3H,s,H～28)，1.03(3H,s,H～26)，1.0(1H,m,H～20)，0.98(3H,s,H～23)，0.97(3H,s,H～24)，0.93(3H,d,J＝6.6Hz,H～30)，0.81(3H,d,J＝6.6Hz,H～29)，0.78(3H,s,H～25)，0.76(2H,dd,J＝12.0,1.2Hz,H～5).
13C NMR(151MHz,MeOD)δ140.21(s,C～13),126.32(s,C～12),79.68(s,C～3),70.20(s,C～27),56.68(s,C～18),55.64(s,C～5),49.57(s,C～9),49.00,43.18(s,C～17),41.29(s,C～8),40.86(s,C～19),40.74(s,C～4),40.14(s,C～1),39.86(s,C～14),39.17(s,C～20),37.99(s,C～10),36.61(s,C～22),34.04(s,C～7),31.82(s,C～15),28.73(s,C～2),27.92(s,C～23),27.11(s,C～21),24.45(s,C～11),24.13(s,C～30),23.86(s,C～16),21.78(s,C～29),19.46(s,C～6),17.93(s,C～28),17.34(s,C～24),16.37(s,C～25),16.22(s,C～26).
determiningThe compound has the molecular formula C30H50O2
Molecular weight 442.3811
Structural formula is
Experiment 1: alpha-glucosidase inhibition assay for the Compounds of the invention
Instrument and reagent
NaH2PO4·2H2O (metallocene chemical reagent works, Tianjin);
Na2HPO4·12H2o (metallocene chemical reagent works, Tianjin);
anhydrous Na2CO3(red rock reagent factory of eastern river, Tianjin);
absolute ethanol (Kanton Chemicals, Inc., Tianjin);
yeast alpha-glucosidase (sigma, 100UN, usa);
pNPG (alatin reagent (shanghai) ltd);
acarbose (Shanghai-derived leaf science and technology Co., Ltd.);
enzyme-linked immunosorbent assay (BioTek company, model: EPOCH)2)；
A constant temperature oscillator (THOMO SHAKER, model: BE-9010).
Second, Experimental methods
1. Preparation of reagents
(1) Preparation of liquid A: NaH2PO4·2H2Weighing 15.603g, metering to 500mL, storing in a brown bottle at 4 ℃ for later use.
(2) Preparing liquid B: na (Na)2HPO4·12H2Weighing 35.822g, metering to 500mL, storing in a brown bottle at 4 ℃ for later use.
(3) Preparation of 0.1M phosphate buffer: 51mL of the solution A and 49mL of the solution B are measured, 100mL of water is added, and the mixture is mixed uniformly to obtain a phosphate buffer solution with the pH value of 6.8, and the phosphate buffer solution is stored in a brown bottle at 4 ℃ for later use.
(4) Yeast α -glucosidase: the enzyme solution was diluted to 20U/mL with a phosphate buffer (pH 6.8) and frozen for use, and was diluted to 1U/mL with a phosphate buffer (pH 6.8) before use.
(5) Substrate pNPG preparation: 0.3766g of pNPG is precisely weighed, added into a proper amount of sodium phosphate buffer solution for dissolution, and then the volume is determined to 50mL to prepare 25mmol/L mother liquor, and the mother liquor is prepared into 0.5mmol/L with the sodium phosphate buffer solution before use for standby.
(6) Preparing an acarbose inhibitor: 13.9mg of acarbose is precisely weighed, and the volume is adjusted to 1mL by DMSO to prepare 13.9mg/mL for later use.
(7)0.1mol/L of Na2CO3Preparation: weighing 1.06g of Na2CO3Adding a proper amount of distilled water into a beaker for dissolving, fixing the volume to 100mL, and storing at 4 ℃ for later use.
2. Preparation of inhibitors
Precisely weighing 2.5mg of Obtusol into a centrifuge tube, dissolving the Obtusol in 1mL of absolute ethyl alcohol to obtain a mother solution, and diluting the mother solution to a proper concentration when the mother solution is required to be used, thus obtaining a series of inhibitors with different concentrations of Obtusol.
3. Obtusol activity inhibition effect test on alpha-glucosidase
The principle is as follows: p-nitrophenol-alpha-D-glucoside (pNPG) can be hydrolyzed by alpha-glucosidase to generate p-nitrophenol, which is specifically absorbed at 405nm, so that the activity of the alpha-glucosidase can be detected by detecting the generation amount of the p-nitrophenol.
The experiment is divided into a blank group, a control group, a sample blank group and a sample group, and the preparation method comprises the following steps: adding samples of each reactant in a 96-well plate according to the dosage in the table 1, wherein each group comprises 3 parallel samples, uniformly mixing an inhibitor, ethanol, a buffer solution and an enzyme solution, preserving the temperature in a constant temperature oscillator at 37 ℃ for 10min, taking out after the completion, adding 50 mu L of 0.5mmol/L pNPG solution, fully and uniformly mixing, carrying out water bath reaction at 37 ℃ for 20min, adding 50 mu L of 0.1mol/L Na after the completion2CO3And stopping the reaction of the solution to obtain each test group (blank group, control group, sample blank group and sample group).
Because PNPG can be hydrolyzed to generate glucose and PNP under the action of alpha-glucosidase, PNP has maximum absorption at 405nm, the absorbance of PNP is measured by an enzyme-labeling instrument, and the inhibition rate and IC50 value of the alpha-glucosidase of each sample can be calculated according to a formula.
The formula:
wherein Ac is blank absorbance, ABAs is the absorbance of the control group, As is the absorbance of the sample group, ASBThe absorbance values are for sample blanks.
TABLE 1 metering and sequence of addition of the reactants (. mu.L)
The inhibition rates of the inhibitors at different concentrations of acarbose and different concentrations of Obtusol are shown in tables 2 and 3, and fitting curves are respectively drawn for the two sets of data, as shown in fig. 1 and 2, the inhibition rates are plotted on the abscissa, the inhibition rates are plotted on the ordinate, the inhibition curves of acarbose at different concentrations are shown in fig. 6, and the inhibition curves of the inhibitors at different concentrations of Obtusol are shown in fig. 7. And obtaining a curve equation from the fitted curve:
the equation for the acarbose inhibition curves at different concentrations is: y 0.3639x +0.0767, R2＝0.9926。
The inhibition curve equation for different concentrations of Obtusol inhibitor is: 19.107x +0.0546, R2＝0.9963。
The concentrations of acarbose and Obtusol at 50% inhibition rate of alpha-glucosidase can be respectively obtained by the two curve equations, and the half-inhibition concentration IC50 value of acarbose to alpha-glucosidase is 1.1632mg/mL, and the half-inhibition concentration IC50 value of Obtusol to alpha-glucosidase is 0.0233 mg/mL.
Table 2 inhibition of acarbose at different concentrations (n ═ 3)
| Concentration (mg/mL) | Inhibition ratio (%) | RSD |
| 0.07 | 9.82 | 14.32％ |
| 0.139 | 13.22 | 18.68％ |
| 0.278 | 19.11 | 8.53％ |
| 0.556 | 25.79 | 7.79％ |
| 1.112 | 48.82 | 2.72％ |
IC50 value of 1.1632mg/mL
Table 3 inhibition of different concentrations of Obtusol inhibitor (n ═ 3)
| Concentration (mg/mL) | Inhibition ratio (%) | RSD |
| 0.05 | 100.54％ | 1.67％ |
| 0.025 | 55.29％ | 7.99％ |
| 0.0125 | 27.59％ | 12.82％ |
| 0.00625 | 15.01％ | 7.23％ |
| 0.003125 | 13.97％ | 5.81％ |
IC50 value of 0.0233mg/mL
As can be seen, the compound of the present invention has a significant inhibitory effect on the activity of alpha-glucosidase, and the IC thereof50Much lower than acarbose.
Claims (7)
1. A preparation method of a compound shown as a formula I is characterized by comprising the following steps:
1) loading the sea-buckthorn extractum on a silica gel column, sequentially eluting with petroleum ether and ethyl acetate according to the mixing ratio of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9 as mobile phases, determining the fraction with the petroleum ether and the ethyl acetate of 7:3 as the fraction with the highest content of the triterpene compound by thin-layer chromatography in the elution process, and collecting eluent;
(2) sequentially carrying out liquid phase normal phase liquid chromatography analysis, normal phase chromatography preparation, reverse phase liquid chromatography analysis and reverse phase chromatography preparation on the eluent obtained in the step (1), and finally carrying out liquid chromatography analysis on purity to obtain the compound shown in the formula I;
in the step (1), the seabuckthorn extract is obtained by extracting seabuckthorn with 95% ethanol;
in the step (2), the normal phase liquid chromatography conditions are as follows:
a chromatographic column: a hydrophilic HILIC chromatography column; mobile phase: a is ethanol, B is n-hexane; isocratic elution conditions: 2% -4% of A and 98% -96% of B; detection wavelength: 203 +/-10 nm; column temperature: 30 +/-5 ℃;
the preparation conditions of the normal phase chromatography are as follows:
a chromatographic column: a HILIC chromatography column; mobile phase: a is ethanol, B is n-hexane; isocratic elution conditions: 2% -4% of A, 98% -96% of B; detection wavelength: 203 +/-10 nm; column temperature: 30 +/-5 ℃;
after the normal phase chromatography is prepared, collecting components with the retention time of 14-19 min, and performing the following reverse phase liquid chromatography analysis, wherein the chromatographic conditions are as follows:
a chromatographic column: c18 column, mobile phase: a is water, B is acetonitrile, isocratic elution conditions are as follows: 20% -30% of A and 80% -70% of B; detection wavelength: 203 +/-10 nm; column temperature: 30 +/-5 ℃;
the reverse phase preparative chromatography conditions were:
a chromatographic column: c18 column, mobile phase: a is water, B is acetonitrile, isocratic elution conditions are as follows: 20% -30% of A and 80% -70% of B; detection wavelength: 203 +/-10 nm; column temperature: 30 +/-5 ℃;
concentrating the prepared solution for liquid phase analysis, and obtaining a target compound if the purity is more than 98%; if the purity is less than 98%, performing chromatographic preparation, wherein the chromatographic conditions of the liquid chromatographic analysis are as follows: a chromatographic column: a C18 chromatography column; mobile phase: a is water, B is acetonitrile, isocratic elution conditions are as follows: 20% -30% of A and 80% -70% of B.
2. The method as claimed in claim 1, wherein the normal phase liquid chromatography conditions in step (2) are as follows:
the specification of the chromatographic column is 4.6mm x 250mm,5 μm; flow rate: 1 plus or minus 0.5 mL/min; sample introduction amount:
。
3. the method according to claim 1, wherein the normal phase chromatography preparation conditions are:
the specification of the chromatographic column is 20mm x 250mm,5 μm; flow rate: 19 plus or minus 2 mL/min; sample introduction amount: 1. + -. 0.5 mL.
4. The method of claim 1, wherein the chromatographic conditions of the reverse phase liquid chromatography after the normal phase chromatography preparation are as follows:
the specification of the chromatographic column is 4.6mm x 250mm,5 μm; flow rate: 1 plus or minus 0.5 mL/min.
5. The method of claim 1, wherein the reverse phase preparative chromatography conditions are:
the specification of the chromatographic column is 21.2mm x 250mm,5 μm; flow rate: 21 plus or minus 2 mL/min.
6. The method of claim 1, wherein the prepared solution is concentrated for liquid phase analysis using a column size of 4.6mm x 250mm,5 μm.
7. The method according to any one of claims 1 to 6, wherein the extraction operation of the seabuckthorn extract in the step (1) comprises the following steps: mixing the sea-buckthorn pomace and ethanol according to a material-liquid ratio of 1: mixing 10-30 kg/L, extracting at 70-80 ℃ for 1.5-2.5 h each time for 3-4 times, combining the extracting solutions, and removing the solvent to obtain an extract.
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| CN111732617B (en) * | 2020-06-29 | 2024-02-27 | 中国科学院西北高原生物研究所 | Extraction method of neoflavonoid Hyd |
| CN111732621B (en) * | 2020-06-29 | 2024-02-27 | 中国科学院西北高原生物研究所 | Extraction method of new flavonoid compound Hip A |
| CN111732620B (en) * | 2020-06-29 | 2024-01-30 | 中国科学院西北高原生物研究所 | Extraction method of new flavonoid compounds Hip B and Hip C |
| CN111789873B (en) * | 2020-08-04 | 2022-05-27 | 中国科学院西北高原生物研究所 | Method for extracting high-content seabuckthorn triterpenic acid extract |
| CN111773257B (en) * | 2020-08-04 | 2021-12-03 | 中国科学院西北高原生物研究所 | Hippophae rhamnoides extract and its use |
| CN111939183A (en) * | 2020-08-05 | 2020-11-17 | 中国科学院西北高原生物研究所 | Ultrasonic-extracted sea buckthorn extract and extraction method and application thereof |
| CN113336819B (en) * | 2021-05-13 | 2023-04-21 | 中国科学院西北高原生物研究所 | Method for separating and preparing three triterpenic acid compounds from sea buckthorn pomace |
| CN113336820B (en) * | 2021-05-13 | 2023-10-13 | 中国科学院西北高原生物研究所 | Method for separating and preparing multiple isomer compounds from sea buckthorn pomace |
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