CN112480191A

CN112480191A - Separation and purification method of monomer A and B of dysphagia

Info

Publication number: CN112480191A
Application number: CN202011373957.2A
Authority: CN
Inventors: 李俊; 张高荣; 周德雄; 黄锡山; 黄艳; 邓胜平
Original assignee: Guangxi Normal University
Current assignee: Guangxi Normal University
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2021-03-12

Abstract

The invention discloses a separation and purification method of a monomer A and a monomer B of dysphagia rice, which takes dysphagia rice leaves as a raw material, and the method comprises the steps of ethanol extraction with specific concentration, macroporous adsorption resin adsorption, silica gel chromatographic column chromatography, and high performance liquid phase preparation C₁₈Purifying by silica gel chromatography column to obtain two new monomer compounds. The method can effectively remove a large amount of impurities in the dysphagia miqueliana, and can obtain two dysphagia miqueliana glycosides at one time, thereby providing a material basis for the activity research of the dysphagia miqueliana glycosides and the development of new medicines with single component.

Description

Separation and purification method of monomer A and B of dysphagia

Technical Field

The invention relates to the technical field of separation and purification of active ingredients of traditional Chinese medicines, in particular to a separation and purification method of monomers A and B of dysphagia.

Background

Choking Miyayan (Streblus tonkinensis) belongs to Moraceae (Moraceae) and belongs to the genus Streblus (Streblus laurus). Belongs to the Moraceae (Moraceae) plant, and belongs to the genus Streblus (Streblus lour). Dysphagia due to the fact that the rice poplar is often used as a folk medicinal plant for treating diseases. Through research in the literature, no report on the chemical components and the biological activity of the miyangjie is found so far. Based on that the dysphagia miqueliana is taken as a medicinal plant commonly used by people, in order to better and deeply research components in the medicinal plant, develop and utilize the plant, a separation and purification process of dysphagia miqueliana glycoside A and B, which has the advantages of high extraction rate, low cost and easy industrial production, is found, and can lay a foundation for the research of the characteristic biological activity of dysphagia miqueliana and provide a premise for the research of structure-activity relationship.

Disclosure of Invention

Aiming at the problem that the separation and purification of the dysphagia-a monomer is not available at present, the invention provides a separation and purification method of dysphagia-a and-B monomers. The method can effectively remove a large amount of impurities in the dysphagia miqueliana, and can obtain two dysphagia miqueliana glycosides at one time, thereby providing a material basis for the activity research of the dysphagia miqueliana glycosides and the development of new medicines with single component.

The technical scheme for realizing the purpose of the invention is as follows:

a separation and purification method of monomer of dysphagia aspergilloside A and B comprises the following steps:

1) extracting dysphagia Miyana leaf with 10-15 mass ratios of 65-75% ethanol water solution, recovering ethanol, and diluting the remaining water with 10 times of distilled water to obtain dysphagia Miyana leaf crude extract water solution;

2) carrying out D101 type macroporous adsorption resin column chromatography on the crude extract aqueous solution obtained in the step 1), after full adsorption, washing with distilled water with 4-6 column volumes to remove unadsorbed impurities, then sequentially eluting with 20-35% ethanol aqueous solutions with 4-6 column volumes, discarding the eluent, then eluting with 38-45% ethanol aqueous solutions with 4-6 volume, and concentrating the eluent to obtain an eluted part rich in the icariside A and B;

3) dissolving the part of the sample eluted by 38% -45% ethanol water solution in the step 2) by using methanol, then carrying out silica gel chromatographic column chromatography, sequentially eluting by using 4-6 column volumes of mixed solution of 3% -10% of dichloromethane and 97% -90% of methanol in volume ratio as eluent, discarding 3% -5% of dichloromethane and 97% -95% of methanol mixed solution eluent, and collecting 10% of dichloromethane and 90% of methanol mixed solution eluent;

4) subjecting the eluate of 10% dichloromethane and 90% methanol mixture collected in step 3) to high performance liquid chromatography C₁₈Performing column chromatography to obtain the icaritin A and B, which are new compounds and have the structural formulas shown as the following formulas respectively:

the extract of the dysphagia miqueliana in the step 1) is prepared by the following method: crushing dysphagia Miyana leaves, adding 65-75% ethanol water solution with 10-15 times of weight of the leaves, heating and refluxing for 2-3 times, concentrating under reduced pressure, and drying to obtain dysphagia Miyana leaf extract.

The macroporous resin in the step 2) is polystyrene resin type macroporous resin.

The model of the macroporous resin in the step 2) is D101, the preferred macroporous resin has large adsorption capacity, easy elution and good adsorption kinetics, and does not adsorb saccharides, proteins, inorganic acids, salts, alkalis and micromolecular hydrophilic organic matters, so that general glycoside substances can be separated from the substances, and a better separation effect can be obtained in a shorter time.

The silica gel chromatographic column in the step 3) is a normal phase silica gel chromatographic column.

The purification process in the step 3) is as follows: eluting with 38% -45% ethanol water solution in step 2), dissolving the sample rich in C and B with a small amount of methanol, performing silica gel column chromatography, and gradient eluting with 4-6 column volumes of mixed solution of 3% -10% dichloromethane and 97% -90% methanol as eluent.

The chromatographic column of the high performance liquid chromatography in the step 4) is a reversed-phase octadecylsilane chemically bonded silica chromatographic column, the specification is 20 x 250mm, the particle size of the filler is 5.0 mu m, the high performance liquid chromatography can be used at a higher temperature and under a lower pH condition, the stationary phase does not collapse, the column effect can be kept for a long time, and the service life of the column is prolonged.

The detection conditions of the high performance liquid chromatography are as follows:

stationary phase: chromatographic column with octadecylsilyl bonded silica gel as filler;

detection wavelength: 230 nm;

flow rate: 2.0-4.0 mL/min;

column temperature: 25-35 ℃;

mobile phase: the method comprises the following steps of (1) eluting with 85-80% of water as a mobile phase A and 15-20% of methanol as a mobile phase B, wherein the proportion of the mobile phase A to the mobile phase B is a volume ratio, and in the preparation process of high performance liquid chromatography, the selection of chromatographic conditions is very important and directly influences the retention time, the separation degree and the like of a substance chromatographic peak; the chromatographic conditions mainly comprise a chromatographic column (comprising filler, column length, column temperature and the like), a mobile phase (comprising components, flow rate and the like), a detector, a detection wavelength, column temperature and the like.

Performing high performance liquid chromatography C on the eluate of the mixture of 10% dichloromethane and 90% methanol collected in step 3) in step 4)₁₈The column chromatography purification process comprises: adding 10% dichloromethane and 90% methanol mixed solution eluate to reverse phase C₁₈Eluting with 15-20% methanol water solution in silica gel chromatographic column to obtain monomeric compounds of icaritin A and icaritin B.

In the technical scheme, the miyan dysphagia is a plant belonging to the genus magpie of the family Moraceae, and the medicinal material part is dry leaves.

The dysphagia oryzae contains not only dysphagia oryzae glycoside, but also flavonoid, alkaloids, saccharides, trace elements and other very complex components, so that separation of the dysphagia oryzae glycoside from other substances is difficult to realize, the content of the dysphagia oryzae glycoside is very low, the structure and the physicochemical properties are very similar, and a plurality of monomers of the dysphagia oryzae glycoside are difficult to obtain at one time by a simple separation method.

According to the method for separating and purifying the dysphagia miyagar monomer in the technical scheme, aiming at the physicochemical properties of each component in dysphagia miyagar, a large amount of impurities in the dysphagia miyagar can be effectively removed, the dysphagia miyagar monomer with higher purity can be obtained, the target preparation of the dysphagia miyagar can be realized, a plurality of dysphagia miyagar with known activity can be obtained at one time, and simultaneously, the trace glycosides are enriched and separated, so that the glycoside library can be enriched continuously, and a material basis is provided for the activity research of glycoside compounds and the development of new drugs with single components.

The method can effectively remove a large amount of impurities in the dysphagia miqueliana, and can obtain two dysphagia miqueliana glycosides at one time, thereby providing a material basis for the activity research of the dysphagia miqueliana glycosides and the development of new medicines with single component.

Drawings

FIG. 1 shows the reverse phases C of C and A of C in the examples₁₈Preparing chromatogram by silica gel column chromatography;

FIG. 2 shows dysphagia A of the example¹H NMR spectrum;

FIG. 3 shows dysphagia A of the example¹³C NMR spectrum;

FIG. 4 shows dysphagia B of the example¹H NMR spectrum;

FIG. 5 shows dysphagia B of the example¹³C NMR spectrum.

Detailed Description

The invention will be further elucidated with reference to the drawings and examples, without however being limited thereto.

Example 1:

1) taking 10kg of dry miyan choke leaf, adding 10 times of 65% ethanol water solution (volume ratio) of raw materials by mass ratio, sequentially heating and refluxing for 3 times for 3 hours, 2 hours and 2 hours, combining filtrates, and recovering ethanol under reduced pressure to obtain extract;

2) diluting the extract obtained in the step 1) by 50 times with distilled water, adsorbing the extract by a D101 macroporous adsorption resin chromatographic column, washing and washing by distilled water with 4 column volumes to remove unadsorbed impurities, sequentially eluting by 10% ethanol water solution and 30% ethanol water solution with 4 column volumes, discarding the eluent, eluting by 40% ethanol water solution, and concentrating the eluent to obtain an eluted part rich in the icariin A and B;

3) taking the 40% ethanol eluate obtained in the step 2), performing silica gel column chromatography, sequentially eluting with 4 column volumes of 3% dichloromethane and 97% methanol mixed solution and 10% dichloromethane and 90% methanol mixed solution (volume ratio), discarding 3% dichloromethane and 97% methanol mixed solution (volume ratio) eluate, and collecting 10% dichloromethane and 90% methanol mixed solution eluate;

4) subjecting the eluate of the mixture of 10% dichloromethane and 90% methanol collected in step 3) to reversed phase C₁₈Performing silica gel column chromatography, eluting with water-methanol (80:20) to obtain first compound A (t)_R21.80min) and a second compound B (t)_R＝26.45min)。

In the step 4), the conditions for detecting and monitoring the high performance liquid chromatography are as follows:

a detector: an ultraviolet detector;

detection wavelength: 230 nm;

flow rate: 3.0 mL/min;

column temperature: 25 ℃;

mobile phase: water was 80% for mobile phase a and methanol was 20% for mobile phase B.

By detecting the obtained first monomer compound a and the second monomer compound B under the above-mentioned detection conditions, the purity of the first monomer compound a was 99.5%, and the purity of the second monomer compound B was 99.7%.

Example 2:

1) adding 12 times of 73% ethanol water solution (mass ratio) into 10kg of dried miyan dysphagia leaves, sequentially heating and reflux-extracting for 3 times (3 hr, 2 hr, and 2 hr), mixing filtrates, and recovering ethanol under reduced pressure to obtain extract;

2) suspending the extract in 50L water, adsorbing with D101 macroporous adsorbent resin chromatographic column, washing with water, and eluting with 20% and 50% ethanol to obtain 20% and 50% ethanol eluate respectively, wherein the 50% ethanol eluate is used as the extract of Choking leaf of Miyan that is rich in Chongxin A and Chongxin B;

3) collecting 625.0g of 50% ethanol eluate obtained in the step 2), performing silica gel column chromatography, sequentially eluting with 4 column volumes of 4% dichloromethane and 96% methanol mixed solution, 10% dichloromethane and 90% methanol mixed solution, and discarding 4% dichloromethane and 96% methanol mixed solution eluate, and collecting 10% dichloromethane and 90% methanol mixed solution eluate;

4) subjecting the eluate of the mixture of 10% dichloromethane and 90% methanol collected in step 3) to reversed phase C₁₈Performing silica gel column chromatography, eluting with water-methanol (80:20) to obtain first compound A (t)_R21.82min) and a second compound B (t)_R＝26.43min)。

In step 4), the conditions for detection and monitoring by high performance liquid chromatography were the same as in example 1 except that the flow rate was 3.5ml/min and the column temperature was 30 ℃.

By detecting the obtained first monomer compound a and the second monomer compound B under the above-mentioned detection conditions, the purity of the first monomer compound a was 99.2%, and the purity of the second monomer compound B was 99.4%.

Example 3:

1) taking 10kg of dry miyan choke leaf, adding 15 times of 75% ethanol water solution (mass ratio of raw materials), sequentially heating and refluxing for 3 times for 3 hours, 2 hours and 2 hours, combining filtrates, and recovering ethanol under reduced pressure to obtain extract;

2) suspending the extract obtained in the step 1) in 50L of water, adsorbing by a D101 macroporous adsorption resin chromatographic column, washing with water, and then eluting with 20% and 50% ethanol to obtain 20% and 50% ethanol eluate respectively, wherein the 50% ethanol eluate is used as the extract of the dysphagia miqueliana leaves rich in dysphagia glycoside A and B;

3) subjecting the 50% ethanol eluate obtained in step 2) to silica gel column chromatography, sequentially discarding the 5% dichloromethane and 95% methanol mixed solution eluate (volume ratio) and 4 column volumes of 5% dichloromethane and 95% methanol mixed solution eluate, 10% dichloromethane and 90% methanol mixed solution eluate, and collecting the 10% dichloromethane and 90% methanol mixed solution eluate;

4) subjecting the eluate of 10% dichloromethane and 90% methanol mixture collected in step 3) to reverse phase C₁₈Performing silica gel column chromatography, eluting with water-methanol (80:20) to obtain first compound A (t)_R21.81min) and a second compound B (t)_R＝26.44min)。

In step 4), the conditions for detection and monitoring by high performance liquid chromatography were the same as in example 1 except that the flow rate was 4.0ml/min and the column temperature was 35 ℃.

By detecting the obtained first monomer compound a and the second monomer compound B under the above-mentioned detection conditions, the purity of the first monomer compound a was 99.6%, and the purity of the second monomer compound B was 99.8%.

As shown in FIG. 1, FIG. 2, FIG. 3, FIG. 4 and FIG. 5, the two obtained monomer compounds are subjected to infrared, mass and nuclear magnetic resonance spectroscopy (R), (B), (C¹HNMR、¹³CNMR、DEPT、¹H-¹The spectrum techniques of HCOSY, HSQC, HMBC, ROESY), Mass Spectrum (MS) and optical rotation and the like identify the structures of the icariin A and B, which are new compounds, and the physicochemical data of the icariin A and B are shown as follows:

choxoside A [4- (3-hydroxy-but-1-enyl) -3,4-dimethyl-2- (3,4, 5-dihydroxy-6-hydroxymethy-l-tetrahydro-pyran-2-yloxymethyl) -cyclohex-2-enone, strigolycoside A]: colorless oil, dissolved in methanol. HRESIMS (positive) M/z 387.2012[ M + H ]]⁺,(calc.for C₁₉H₃₁O₈,387.2019)；

¹H-NMR(400MHz CD₃OD):δ_H2.55(2H,dd,J＝7.4,6.1Hz,H-5),1.92(2H,t,6.8,H-6),6.26(1H,dd,J＝15.9,5.1Hz,H-7),6.37(1H,dd,J＝15.9,1.2Hz,H-8),4.44(1H,m,H-9),1.36(3H,m,H-10),1.24(3H,s,H-11),1.24(3H,s,H-12),4.67(1H,d,J＝9.7Hz,H-13a),4.24(1H,d,J＝9.7Hz,H-13b),4.35(1H,d,J＝7.8Hz,H-1′),3.17(1H,t,8.3,H-2′),3.28(1H,m,H-3′),3.28(1H,m,H-4′),3.37(1H,m,H-5′),3.88(1H,dd,J＝11.9,2.2Hz,H-6′a),3.70(1H,dd,J＝11.9,5.3Hz,H-6′b)；¹³C-NMR(100MHz CD₃OD)δ_C 36.6(C-1),170.0(C-2),130.9(C-3),200.6(C-4,C＝O),35.0(C-5),37.6(C-6),143.9(C-7),124.1(C-8),68.8(C-9),23.5(C-10),27.4(C-11),27.4(C-12),64.4(C-13),104.1(C-1′),74.9(C-2′),77.8(C-3′),71.4(C-4′),78.0(C-5′),62.8(C-6′)。

Choxoside B [2- (4-p-Tolyl-pentyloxy) -tetrahydropyran-3, 4,5-triol, striglycoside B]: colorless needle crystals, dissolved in methanol. HRESIMS M/z 355.1766[ M + HCOO]^-(calcd For C₁₈H₂₇O₇,355.1780).

¹H-NMR(400MHz CD₃OD):δ_H 7.07(2H,m,H-2,6),7.06(2H,m,H-3,5),3.75(1H,dt,9.2,6.8,H-10a),3.49(1H,m,H-10b),2.63(1H,dt,12.0,7.2,H-7),2.28(3H,s,C-12),1.63(2H,m,H-8),1.48(2H,m,H-9),1.23(3H,d,7.2,H-11),4.14(1H,d,J＝7.6Hz,H-1′),3.14(1H,m,H-2′),3.48(1H,m,H-3′),3.46(1H,m,H-4′),3.82(2H,dd,11.6,5.6,H-5′)；¹³C-NMR(100MHz CD₃OD)δ_C 145.5(C-1),127.9(C-2,6),129.9(C-3,5),136.3(C-4),40.6(C-7),35.7(C-8),29.0(C-9),70.9(C-10),23.0(C-11),21.4(C-12),105.1(C-1′),74.9(C-2′),77.8(C-3′),71.2(C-4′),66.9(C-5′)。

Claims

1. a method for separation and purification of A and B monomers, is characterized in that, comprises the steps:

1) extracting the 65-75% ethanol aqueous solution of 10-15 mass ratios of the leaves of Populus militaris, after reclaiming the ethanol of the extract, the remaining water part is diluted with 10 times of distilled water to obtain the aqueous solution of crude extracts of the leaves of Populus militaris;

2) The crude extract aqueous solution obtained in step 1) is subjected to D101 type macroporous adsorption resin column chromatography, and after sufficient adsorption, rinse with 4-6 column volumes of distilled water to remove unadsorbed impurities, and then use each 4- 6 column volumes of 20%-35% ethanol aqueous solution to elute, discard the eluent, then eluate with 4-6 column volumes of 38-45% ethanol aqueous solution, and concentrate the eluent to obtain a eluted fractions of glycosides A and B;

3) Dissolve with methanol step 2) 38%-45% ethanol aqueous solution to elute part of the sample, and then perform silica gel column chromatography, sequentially use 4-6 column volumes of 3%-10% dichloromethane and 97% -90% methanol mixture as eluent for elution, discard 3%-5% dichloromethane and 97%-95% methanol mixture eluent, collect 10% dichloromethane and 90% The methanol mixture eluate;

4) The 10% dichloromethane and 90% methanol mixed solution eluate collected in step 3) is subjected to high performance liquid chromatography C ₁₈ column chromatography to obtain miloside A and B, both of which are new Compounds, their structural formulas are shown as follows:

2. the method for separating and purifying A and B monomers according to claim 1, is characterized in that, the extract of the leaf of poplar choking described in step 1) is obtained by the following method: the leaf of poplar choking is pulverized, Add 10-15 times the mass of the leaves with an ethanol aqueous solution with a concentration of 65-75%, extract under reflux for 2-3 times, concentrate under reduced pressure, and dry to obtain the extract of Populus oryzae leaves.

3. the method for separating and purifying A and B monomer according to claim 1, is characterized in that, the macroporous resin described in step 2) is polystyrene resin type macroporous resin.

4. the method for separation and purification of A and B monomer according to claim 3, is characterized in that, the model of macroporous resin described in step 2) is D101.

5. the method for separating and purifying A and B monomer according to claim 1, is characterized in that, the silica gel chromatographic column described in step 3) is normal phase silica gel chromatographic column.

6. the method for separation and purification of A and B monomer according to claim 1, is characterized in that, the purification process described in step 3) is: the ethanol of 38%-45% in step 2) is used Aqueous solution elution, the samples rich in miloside A and B were dissolved with a small amount of methanol, and then subjected to silica gel column chromatography, followed by 4-6 column volumes of 3%-10% dichloromethane and 97%-90% dichloromethane. % methanol mixture was used as eluent for gradient elution.

7. the method for separating and purifying A and B monomer according to claim 1, is characterized in that, the chromatographic column of the high performance liquid chromatography described in step 4) is reversed-phase octadecyl silane bond A silica gel chromatographic column with a size of 20 × 250 mm and a particle size of the filler of 5.0 μm.

8. the method for separating and purifying A and B monomers according to claim 1, is characterized in that, described in step 4), the 10% dichloromethane and 90% dichloromethane collected in step 3) are collected. The eluate of the methanol mixture was subjected to high-performance liquid chromatography C ₁₈ column chromatography purification process as follows: adding 10% dichloromethane and 90% methanol mixture eluate to a reverse-phase C ₁₈ silica gel column, using the volume Elution with a concentration of 15-20% methanol aqueous solution, respectively, to obtain the monoclonal compounds A and B.