CN109485682B - Method for extracting kaempferol acetyl galactoside compound - Google Patents
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Abstract
The invention provides a method for extracting a kaempferol acetyl galactoside compound. The method comprises the steps of leaching and sampling the Nanshan tea cake to a macroporous resin chromatographic column, collecting eluent eluted by 15% ethanol water solution after gradient elution, and obtaining crude component liquid after post-treatment; loading to polyamide resin chromatographic column, gradient eluting to collect the eluent containing target compound, concentrating and freeze drying to obtain crude extract; performing DAC preparation by using a high performance liquid chromatograph and a C18HCE chromatographic column to obtain a crude isolate; performing isocratic elution by using a high performance liquid chromatograph and a C18ME chromatographic column and using an acetonitrile aqueous solution with the volume fraction of 20% as a mobile phase, collecting a flow fraction containing the target compound, concentrating and freeze-drying to obtain the target compound; the method provided by the application can effectively separate the target compound from the camellia plant and has high extraction purity.
Description
Technical Field
The invention relates to the field of compound extraction, and particularly relates to a method for extracting a kaempferol acetyl galactoside compound.
Background
Southern Camellia, also known as Camellia semiserrata (Chi.), belongs to the genus Camellia (Camellia L.) of the family Theaceae, and is a perennial tree or shrub. The Nanshan tea is mainly distributed in the southeast of Guangxi and the West of Guangdong in China. It is an important cultivated species of camellia oleifera. Through research and comparison, the Guangning safflower oil tea has relatively rich chemical components in various oil tea.
The camellia nandina and the cake thereof are particularly rich in flavonoids, saponins and other compounds with various biological activities. At present, researches on oil-tea camellia cakes mainly focus on optimization of various extraction processes by taking total flavonoids or saponins as indexes, and some researches focus on activities such as antioxidation and bacteriostasis of obtained crude extracts or preliminary purified products. However, the study of specific components in the extract is not sufficient.
Kaempferol and its derivatives are important compounds in Camellia oleifera, and have anticancer, diabetes and osteoporosis treating, and injured cell protecting effects.
The effective active substances in the camellia nankingense are analyzed, the active ingredients of the camellia nankingense are separated and extracted from complex and various ingredients, and a product with high purity is obtained, so that the method plays an important role in deep development of the camellia nankingense.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for extracting a kaempferol acetyl galactoside compound, which is used for extracting a target compound from Nanshan tea, and is quick, effective and high in purity.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a process for extracting kaempferol acetyl galactosides comprising the steps of:
A. leaching the south mountain tea cake to obtain a sample solution;
B. loading the sample liquid to a macroporous resin chromatographic column by a wet method, performing gradient elution by using ethanol aqueous solutions with volume fractions of 5%, 15%, 50% and 95% in sequence, collecting eluent eluted by using the 15% ethanol aqueous solution, and performing post-treatment to obtain a crude component liquid;
C. loading the crude component solution to a polyamide resin chromatographic column by a wet method, performing gradient elution by using distilled water and ethanol aqueous solution with volume fractions of 20%, 40% and 90%, performing chromatographic detection, collecting eluent containing a target compound, concentrating and freeze-drying to obtain a crude extract;
D. dissolving the crude extract with a first solvent, injecting a sample, performing DAC preparation by using a high performance liquid chromatograph and a C18HCE chromatographic column, performing isocratic elution by using a methanol aqueous solution as a mobile phase, collecting a fraction containing the target compound, concentrating and freeze-drying to obtain a crude isolate;
E. dissolving the crude isolate with a second solvent, injecting the solution, performing semi-preparation by using a high performance liquid chromatograph and a first C18ME chromatographic column, performing isocratic elution by using an acetonitrile aqueous solution with the volume fraction of 20% as a mobile phase, collecting a fluid fraction containing the target compound, concentrating and freeze-drying to obtain the target compound; the target compound has a structural formula:
according to the main components in the Nanshan tea leach liquor and the properties of the target compounds, the target compounds are effectively separated from the complex components of the Nanshan tea by adopting a method of leaching, extraction, macroporous resin column chromatography, C18ME chromatographic column elution, polyamide resin column chromatography, DAC (dynamic axial compression) preparation and C18ME chromatographic column high performance liquid chromatography preparation.
Preferably, in the step B, the preparation method of the macroporous resin chromatographic column comprises: soaking D101 macroporous resin in ethanol, loading into column, and washing with ethanol and water in sequence until no ethanol smell exists.
The macroporous resin is processed to prepare the chromatographic column, aiming at fully releasing the adsorption capacity of the macroporous resin, preventing impurities from generating adverse effect on the extraction process and ensuring the maximum column chromatography efficiency.
Further preferably, the column volume BV of the macroporous resin chromatography column1Is 10L, and the dosage of the 5 percent ethanol is 6 times of BV1The dosage of the 15 percent ethanol is 8 times of BV1The dosage of the 50 percent ethanol is 8 times of BV1The dosage of the 95 percent ethanol is 4 times of BV1。
Suitable eluent systems are based on the knowledge of the differences between the active ingredients and the target compounds in the leach solution. On one hand, the proper elution amount is used for ensuring that the target compound and other components can be separated as much as possible and the separation times are reduced; on the other hand, the extraction efficiency is ensured, and the extraction yield is improved as much as possible.
More preferably, the post-treatment comprises distilling the eluent under reduced pressure to remove ethanol, eluting with a second C18ME chromatographic column and methanol as a mobile phase to enrich DAC, concentrating the effluent under reduced pressure until no liquid is spun out, and dissolving with methanol and acetone at a volume ratio of 1:1 to obtain the crude component liquid, wherein the specification of the second C18ME chromatographic column is 100A, 50mm × 250mm and 10 μm.
The main purpose of treating the eluate with a chromatographic column is to separate the water. And after the methanol and the acetone are used for dissolution, the next separation is facilitated.
Preferably, in the step C, the column volume BV of the polyamide resin chromatographic column2Is 1L; the dosage of the distilled water, the ethanol with the volume fraction of 20 percent and the ethanol with the volume fraction of 40 percent are all 5 times BV2The dosage of the 90 percent ethanol is 2 times of BV2。
More preferably, during the gradient elution, every 500mL is a collection unit, the collection units are numbered sequentially, and the No. 5-18 eluents are collected.
The elution system, column volume and eluent amount of the polyurethane resin column chromatography are determined based on the nature of the substance to be separated, the eluent and the polyurethane chromatography column. The collection unit is set in order to better obtain a stream fraction containing the target compound.
Preferably, in the step D, the specification of the C18HCE chromatographic column is 100A, 50mm × 250mm and 10 μm, and the volume fraction of the methanol aqueous solution is 50%.
More preferably, the first solvent is methanol and dimethyl sulfoxide in a volume ratio of 1: 1.
The selection of the solvent, the specification of the column, the elution system, and the like is determined based on the polarity of the substance to be separated.
Preferably, in the step E, the first C18ME chromatographic column has the specification of 100A, 20 × 250mm and 10 μm, and the second solvent is an aqueous acetonitrile solution with the volume fraction of 20%.
Optionally, in the step a, the leaching treatment method includes: extracting with 50-60% ethanol water solution at a material-to-liquid ratio of 1kg of the Nanshan tea cake corresponding to 2-4L of the ethanol water solution, heating and reflux extracting at 60-70 deg.C for 2-4 times, filtering, and mixing to obtain extractive solution; then concentrating under reduced pressure, extracting with n-butanol to obtain extractive solution, and diluting with water 18-22 times to obtain the sample solution.
The ethanol water solution can well extract the components in the camellia nanensis, but due to different properties of the components, proper temperature and extraction time are needed; the choice of extractant depends on the nature of the target compound; when the high-concentration extract is directly subjected to column chromatography by using macroporous resin, the separation effect is poor, so that the extract needs to be diluted for use.
Compared with the prior art, the invention has the beneficial effects that:
(1) separating target compounds from the camellia plants for the first time;
(2) the extraction method provided by the application is simple and effective, and is suitable for popularization and application.
(3) The extraction purity of the target compound is high.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a spectrum of DAC preparation of example 1;
FIG. 2 is a spectrum of a high performance liquid chromatography preparation of example 1;
FIG. 3 is a graph showing the measurement of the purity of the objective compound prepared in example 1;
FIG. 4 is a mass spectrum of a target compound prepared according to the present application;
FIG. 5 is a graph of the spectrum H of a target compound prepared according to the present application;
FIG. 6 is a C spectrum of a target compound prepared according to the present application;
FIG. 7 is a chart of the H-H COSY of the target compound prepared in the present application;
FIG. 8 is a HSQC spectrum of a target compound prepared according to the present application;
fig. 9 is a HMBC spectrum of a target compound prepared in the present application.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
Drying the tea fruit of the camellia nanensis in the sun, removing the shell, drying the cake generated after removing the grease by a physical squeezing method, and properly crushing to obtain the camellia nanensis cake. Ultrasonic extracting with 50% ethanol water solution at 60 deg.C for 3 times (2 hr each time) at a material-to-liquid ratio of 1:4(m/v), filtering, and mixing to obtain extractive solution. Then concentrating the extracting solution, extracting with n-butanol of the same volume to obtain an extract, and diluting with water by 20 times to obtain a sample solution.
The shelling and the grease removal are carried out to ensure the extraction yield to the maximum extent and reduce the interference in the separation process. Suitable methods of feedstock treatment are also advantageous in avoiding loss of the target compound.
Selecting macroporous resin D101 type, soaking the macroporous resin D101 in ethanol, and packing column (column volume BV)110L), washing with ethanol and water in sequence until no ethanol smell exists, detecting with an alcohol meter, and reading less than 5. Slowly adding the sample solution into a macroporous resin chromatographic column, and sequentially using 6 times of BV1The volume fraction of the ethanol is 5 percent ethanol and 8 times of BV1The volume fraction of the ethanol is 15 percent ethanol and 8 times of BV1The volume fraction of the ethanol is 50 percent and 4 times of BV1Is eluted with 95% ethanol, chromatographically detected, collected with 1Distilling the eluent eluted by 5% ethanol under reduced pressure to remove ethanol, eluting with C18ME chromatographic column (100A, 50mm × 250mm, 10 μm) and methanol as mobile phase (DAC enrichment) at flow rate of 80mL/min for 10min, concentrating the eluate under reduced pressure until no liquid is spun out, and dissolving with methanol and acetone (volume ratio 1:1) to obtain crude component liquid.
Washing polyamide resin chromatographic column with pure water until effluent is colorless, and wet loading the crude component solution to polyamide resin chromatographic column (BV)21L) is added, distilled water, ethanol with volume fraction of 20 percent, ethanol with volume fraction of 40 percent and ethanol with volume fraction of 90 percent are sequentially used for gradient elution, and the dosage of each eluent is 5 times BV2Performing chromatographic detection, wherein each 500mL is a collection unit, numbering is performed in sequence, and the No. 5-18 eluents are collected, concentrated and freeze-dried to obtain a crude extract.
DAC preparation is carried out by using a C18HCE chromatographic column (100A, 50mm × 250mm, 10 mu m) and using a methanol water solution with the volume fraction of 50% as a mobile phase, dissolving the crude extract by using methanol and dimethyl sulfoxide with the volume ratio of 1:1, then injecting, controlling the flow rate to be 70mL/min and the sample injection amount to be 16mL, carrying out isocratic elution, collecting a flow part section containing a target compound (shown as a peak No. 5 in figure 1), concentrating and freeze-drying to obtain a crude isolate.
The crude isolate was dissolved in a 20% aqueous acetonitrile solution using a C18ME column (100A, 20 × 250mm, 10 μm) using a 19% volume aqueous acetonitrile solution as a mobile phase, then the sample was taken, the flow rate was controlled at 20mL/min, the amount of the sample was controlled at 2mL, isocratic elution was performed, the fractions containing the target compound were collected, concentrated and lyophilized to obtain the target compound (see peak No. 3, FIG. 2).
The purity of the objective compound was measured by using acetonitrile-0.1% formic acid aqueous solution (20: 80 in volume ratio) as a mobile phase, and using an LC3000 high performance liquid chromatograph, a C18HCE column (4.6 × 250mm, 10 μm), a detector, UV254nm, at a flow rate of 1mL/min, to measure the purity of the objective compound as 99.48% (see FIG. 3), and the extraction yield as 32 mg/kg.
Example 2
Drying the tea fruit of the camellia nanensis in the sun, removing the shell, drying the cake generated after removing the grease by a physical squeezing method, and properly crushing to obtain the camellia nanensis cake. Ultrasonic extracting with 60% ethanol water solution at 70 deg.C for 2 times (each time for 2 hr) at a material-to-liquid ratio of 1:2(m/v), filtering, and mixing to obtain extractive solution. Then concentrating the extracting solution, extracting with n-butanol of the same volume to obtain an extract, and diluting with water for 22 times to obtain a sample solution.
Selecting macroporous resin D101 type, soaking the macroporous resin D101 in ethanol, and packing column (column volume BV)110L), washing with ethanol and water in sequence until no ethanol smell exists, detecting with an alcohol meter, and reading less than 5. Slowly adding the sample solution into a macroporous resin chromatographic column, and sequentially using 6 times of BV1The volume fraction of the ethanol is 5 percent ethanol and 8 times of BV1The volume fraction of the ethanol is 15 percent ethanol and 8 times of BV1The volume fraction of the ethanol is 50 percent and 4 times of BV1The volume fraction of (1) is 95% ethanol elution, chromatographic detection is carried out, an eluent eluted by 15% ethanol is collected, reduced pressure distillation is carried out to remove the ethanol, then a C18ME chromatographic column (100A, 50mm × 250mm, 10 mu m) is used for elution by taking methanol as a mobile phase (DAC enrichment), the flow rate is 80mL/min, elution is carried out for 10min, the effluent is received and decompressed and concentrated until no liquid is spun out, and the crude component liquid is obtained by dissolving the eluent by methanol and acetone (volume ratio is 1: 1).
Washing polyamide resin chromatographic column with pure water until effluent is colorless, and wet loading the crude component solution to polyamide resin chromatographic column (BV)21L) is added, distilled water, ethanol with volume fraction of 20 percent, ethanol with volume fraction of 40 percent and ethanol with volume fraction of 90 percent are sequentially used for gradient elution, and the dosage of each eluent is 5 times BV2Performing chromatographic detection, wherein each 500mL is a collection unit, numbering is performed in sequence, and the No. 5-18 eluents are collected, concentrated and freeze-dried to obtain a crude extract.
Using a C18HCE chromatographic column (100A, 50mm × 250mm, 10 μm) and using a methanol aqueous solution with the volume fraction of 50% as a mobile phase to carry out DAC preparation, dissolving the crude extract by using methanol and dimethyl sulfoxide with the volume ratio of 1:1, then injecting a sample, controlling the flow rate to be 70mL/min and the sample injection amount to be 16mL, carrying out isocratic elution, collecting a flow part section containing the target compound, concentrating and freeze-drying to obtain the crude extract.
The crude isolate was dissolved in a 20% acetonitrile aqueous solution using a C18ME column (100A, 20 × 250mm, 10 μm) and a 19% volume fraction acetonitrile aqueous solution as a mobile phase, and then the sample was introduced, and the flow rate was controlled at 20mL/min and the amount of the sample was controlled at 2mL, followed by isocratic elution, and the fractions containing the objective compound were collected, concentrated and lyophilized to obtain the objective compound.
Example 3
Drying the tea fruit of the camellia nanensis in the sun, removing the shell, drying the cake generated after removing the grease by a physical squeezing method, and properly crushing to obtain the camellia nanensis cake. Ultrasonic extracting with 55% ethanol water solution at 65 deg.C for 2 hr for 4 times at a ratio of 1:4(m/v), filtering, and mixing to obtain extractive solution. Then concentrating the extracting solution, extracting with n-butanol of the same volume to obtain an extract, and diluting with water by 18 times to obtain a sample solution.
Selecting macroporous resin D101 type, soaking the macroporous resin D101 in ethanol, and packing column (column volume BV)110L), washing with ethanol and water in sequence until no ethanol smell exists, detecting with an alcohol meter, and reading less than 5. Slowly adding the sample solution into a macroporous resin chromatographic column, and sequentially using 6 times of BV1The volume fraction of the ethanol is 5 percent ethanol and 8 times of BV1The volume fraction of the ethanol is 15 percent ethanol and 8 times of BV1The volume fraction of the ethanol is 50 percent and 4 times of BV1The volume fraction of (1) is 95% ethanol elution, chromatographic detection is carried out, an eluent eluted by 15% ethanol is collected, reduced pressure distillation is carried out to remove the ethanol, then a C18ME chromatographic column (100A, 50mm × 250mm, 10 mu m) is used for elution by taking methanol as a mobile phase (DAC enrichment), the flow rate is 80mL/min, elution is carried out for 10min, the effluent is received and decompressed and concentrated until no liquid is spun out, and the crude component liquid is obtained by dissolving the eluent by methanol and acetone (volume ratio is 1: 1).
Washing polyamide resin chromatographic column with pure water until effluent is colorless, and wet loading the crude component solution to polyamide resin chromatographic column (BV)21L) is added, distilled water, ethanol with volume fraction of 20 percent, ethanol with volume fraction of 40 percent and ethanol with volume fraction of 90 percent are sequentially used for gradient elution, and the dosage of each eluent is 5 times BV2Performing chromatographic detection, wherein each 500mL is a collection unit, numbering in sequence, and collectingConcentrating the eluent of No. 5-18, and lyophilizing to obtain crude extract.
Using a C18HCE chromatographic column (100A, 50mm × 250mm, 10 μm) and using a methanol aqueous solution with the volume fraction of 50% as a mobile phase to carry out DAC preparation, dissolving the crude extract by using methanol and dimethyl sulfoxide with the volume ratio of 1:1, then injecting a sample, controlling the flow rate to be 70mL/min and the sample injection amount to be 16mL, carrying out isocratic elution, collecting a flow part section containing the target compound, concentrating and freeze-drying to obtain the crude extract.
The crude isolate was dissolved in a 20% acetonitrile aqueous solution using a C18ME column (100A, 20 × 250mm, 10 μm) and a 19% volume fraction acetonitrile aqueous solution as a mobile phase, and then the sample was introduced, and the flow rate was controlled at 20mL/min and the amount of the sample was controlled at 2mL, followed by isocratic elution, and the fractions containing the objective compound were collected, concentrated and lyophilized to obtain the objective compound.
The compound monomers obtained in examples 1 to 3 were subjected to LC-MS measurement and nuclear magnetic resonance detection.
As shown in FIG. 4, the mass spectrum data M/z783.2256[ M + H [ ]]+Proton fragment M/z637.1692[ (M + H) -146 [ ]]+，449.1029[(M+H)-(146+42)]+，287.0515[(M+H)-(146+42)-162]+. According to ion fragmentation results, and binding1H-NMR and13C-NMR data presume that the molecular formula is C35H42O20It contains a kaempferol nucleus, a group of rhamnosides, a group of acetyl rhamnosides and a group of galactosides.
1In H-NMR (see fig. 5), δ 6.19(H, d, J ═ 1.9Hz) and δ 6.39(H, d, J ═ 1.9Hz), which are characterized by the hydrogen in the meta position on the phenyl ring, are deduced to belong to kaempferol a ring H-6 and H-8, δ 7.95(2H, d, J ═ 8.7Hz) and δ 6.88(2H, d, J ═ 8.7Hz), by integrating the area, both groups of signals contain 2 protons, whose chemical shifts, splits and coupling constants correspond to the hydrogen in the ortho position on the phenyl ring, which result corresponds to kaempferol B ring structure, and thus are deduced to belong to kaempferol B ring H-2', 6' and H-3', 5', δ 5.39(H, d, J ═ 7.4Hz) as terminal proton signals, by which coupling constants (J ═ 7.5 > 7.0, 7.5 ═ H, 25.35H ═ 5, 25.52H ═ 5, 25.5H ═ 25.52H ═ 5H, h.5 ═ 25.5 h.5H, d, J ═ 52 h.5H ═ 5 h.5 h.Plum glycoside terminal proton signal; δ 1.03(3H, d, J ═ 6.2Hz) and 0.72(3H, d, J ═ 6.2Hz) belong to two groups of rhamnoside methyl proton signals; the delta 3.00-4.00(m) section signal is other glycoside proton signal; δ 1.91(3H, s) is the acetyl proton signal.
13In C-NMR (see FIG. 6), δ 101.24, 76.44, 75.25, 69.76, 74.18, 65.78 is a set of galactoside signals, δ 100.64, 70.33, 70.71, 72.25, 68.58, 17.71 and δ 102.38, 71.89, 69.55, 76.09, 67.16, 16.93 are two sets of rhamnoside signals, δ 169.72, 20.47 are acetyl signals, the remaining signals are highly consistent with characteristic kaempferol signals, which is consistent with the above inference.
In H-H COSY (shown in figure 7), delta 6.19(H, d, J ═ 1.9Hz) and 6.39(H, d, J ═ 1.9Hz) are mutually coupled, are spin-coupled systems of protons in meta positions on a benzene ring, and conform to a kaempferol A ring structure; δ 7.95(2H, d, J ═ 8.7Hz) and δ 6.88(2H, d, J ═ 8.7Hz) were coupled to each other to form a typical AA 'BB' spin-coupled system of para-substituted benzenes, conforming to the kaempferol B ring structure;
δ 5.39(H, d, J ═ 7.4Hz) was coupled to δ 3.00-4.00(m), and from the chemical shift values, it was concluded that δ 5.39(H, d, J ═ 7.4Hz) was assigned to the galactoside end group. δ 5.35(H, d, J ═ 3.7Hz), 4.52(H, s) and δ 3.00-4.00(m) are coupled to each other, and δ 1.03(3H, d, J ═ 6.2Hz), 0.72(3H, d, J ═ 6.2Hz) and δ 3.00-4.00(m) are coupled to each other, and meet the rhamnoside methyl proton signal characteristics.
In HSQC (see fig. 8), δ 98.87 corresponds to δ 6.19(H, d, J ═ 1.9Hz), δ 93.72 corresponds to δ 6.39(H, d, J ═ 1.9Hz), δ 130.77 corresponds to δ 7.95(2H, d, J ═ 8.7Hz), δ 115.09 corresponds to 6.88(2H, d, J ═ 8.7Hz), δ 101.24 corresponds to δ 5.39(H, d, J ═ 7.4Hz), δ 100.64 corresponds to δ 4.52(H, s), δ 102.38 corresponds to δ 5.35(H, d, J ═ 3.7Hz), δ 17.71 corresponds to δ 1.03(3H, d, J ═ 6.2Hz), δ 16.93 corresponds to δ 0.72(3H, d, J ═ 6.2Hz), δ 3.47 (3H, d, J ═ 3.47H, J ═ 4H, J ═ 9.75 Hz), δ 75(H, d, J ═ 8.7 Hz).
In conjunction with HSQC, the ligation sites for the different fragments were determined by HMBC (see FIG. 9). In HMBC, δ 6.19(H, d, J ═ 1.9Hz) is associated with δ 161.22, 164.73, δ 6.39(H, d, J ═ 1.9Hz) is associated with δ 164.73, 156.49, δ 7.95(2H, d, J ═ 8.7Hz) is associated with δ 120.82, 115.09, 159.97, δ 6.88(2H, d, J ═ 8.7Hz) is associated with δ 130.77, 159.97, 120.82, and its characteristics are fully consistent with kaempferol. δ 5.39(H, d, J ═ 7.4Hz) correlates with δ 133.06, indicating that the galactoside is attached to kaempferol C-3; δ 4.36(H, d, J ═ 1.0Hz) correlates with δ 76.44, suggesting that the acetyl rhamnoside is linked to Gal-C-2; δ 4.75(H, t, J ═ 9.9Hz) correlates with δ 72.25, knowing that acetyl is attached to the acetyl rhamnoside C-4; δ 5.35(H, d, J ═ 3.7Hz) correlates with δ 65.78, indicating that another rhamnoside is linked to Glu-C-6.
Attribution of peaks of the H and C spectra:
1H-NMR:7.95(2H,d,J＝8.7Hz,H-2’,6’),6.88(2H,d,J＝8.7Hz,H-3’,5’),6.39(H,d,J＝1.9Hz,H-8),6.19(H,d,J＝1.9Hz,H-6),5.39(H,d,J＝7.4Hz,H-1”),5.35(H,d,J＝1.7Hz,H-1””),4.75(H,t,J＝9.9Hz,H-4”’),4.52(H,s,H-1”’),1.91(3H,s,AcO-C-4”’),1.03(3H,d,J＝6.2Hz,H-6”’),0.72(3H,d,J＝6.2Hz,H-6””)。
13C-NMR:156.67(C-2),133.06(C-3),177.34(C-4),161.22(C-5),98.87(C-6),164.73(C-7),93.72(C-8),156.49(C-9),103.77(C-10),120.82(C-1’),130.77(C-2’,6’),115.09(C-3’,5’),159.97(C-4’),101.24(C-1”),76.44(C-2”),75.25(C-3”),69.76(C-4”),74.18(C-5”),65.78(C-6”),100.64(C-1”’),70.33(C-2”’),70.71(C-3”’),72.25(C-4”’),68.58(C-5”’),17.71(C-6”’),102.38(C-1””),71.89(C-2””),69.55(C-3””),76.09(C-4””),67.16(C-5””),16.93(C-6””),169.72,20.47(AcO-C-4”’)。
the compound is identified as kaempferol-3-O- [ 4' -acetyl- α -L-rhamnose (1 → 2) - [ α -L-rhamnose (1 → 6) ] - β -D-galactoside by integrating all nuclear magnetic spectrum and mass spectrum results, and the structural formula is as follows:
the method for extracting the kaempferol acetyl galactoside compound from the Nanshan tea, provided by the application, separates and extracts the target compound from the camellia plant for the first time, is simple and practical, is suitable for large-scale application, has high product purity, and has positive significance for deep development and utilization of the Nanshan tea.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (6)
1. A method for extracting kaempferol acetyl galactoside compounds is characterized by comprising the following steps:
A. leaching the south mountain tea cake to obtain a sample solution; the leaching treatment method comprises the following steps: extracting with 50-60% ethanol water solution at a material-to-liquid ratio of 1kg of the Nanshan tea cake corresponding to 2-4L of the ethanol water solution, heating and reflux extracting at 60-70 deg.C for 2-4 times, filtering, and mixing to obtain extractive solution; then concentrating under reduced pressure, extracting with n-butanol to obtain extract, and diluting with water by 18-22 times to obtain the sample solution;
B. loading the sample liquid to a macroporous resin chromatographic column by a wet method, performing gradient elution by using ethanol aqueous solutions with volume fractions of 5%, 15%, 50% and 95% in sequence, collecting eluent eluted by using the 15% ethanol aqueous solution, and performing post-treatment to obtain a crude component liquid;
C. loading the crude component solution to a polyamide resin chromatographic column by a wet method, performing gradient elution by using distilled water and ethanol aqueous solution with volume fractions of 20%, 40% and 90%, performing chromatographic detection, collecting eluent containing a target compound, concentrating and freeze-drying to obtain a crude extract;
D. dissolving the crude extract with a first solvent, injecting the dissolved crude extract, performing DAC preparation by using a high performance liquid chromatograph and a C18HCE chromatographic column, performing isocratic elution by using a methanol aqueous solution as a mobile phase, collecting a flow fraction containing the target compound, concentrating and freeze-drying to obtain a crude extract, wherein the specification of the C18HCE chromatographic column is 100A, 50mm × 250mm and 10 mu m, the volume fraction of the methanol aqueous solution is 50%, and the first solvent is methanol and dimethyl sulfoxide with the volume ratio of 1: 1;
E. dissolving the crude isolate with a second solvent, injecting, performing semi-preparation by using a high performance liquid chromatograph and a first C18ME chromatographic column, performing isocratic elution by using an acetonitrile aqueous solution with the volume fraction of 20% as a mobile phase, collecting a fluid section containing the target compound, concentrating and freeze-drying to obtain the target compound, wherein the specification of the first C18ME chromatographic column is 100A, 20 × 250mm and 10 mu m, the second solvent is an acetonitrile aqueous solution with the volume fraction of 20%, and the structural formula of the target compound is as follows:
2. the method according to claim 1, wherein in the step B, the macroporous resin chromatographic column is prepared by the following steps: soaking D101 macroporous resin in ethanol, loading into column, and washing with ethanol and water in sequence until no ethanol smell exists.
3. The method according to claim 2, wherein the macroporous resin chromatography column has a column volume BV1Is 10L, and the dosage of the 5 percent ethanol is 6 times of BV1The dosage of the 15 percent ethanol is 8 times of BV1The dosage of the 50 percent ethanol is 8 times of BV1The dosage of the 95 percent ethanol is 4 times of BV1。
4. The method as claimed in claim 3, wherein the post-treatment comprises distilling the eluate under reduced pressure to remove ethanol, subjecting the eluate to DAC enrichment by using a second C18ME chromatographic column and methanol as a mobile phase, concentrating the eluate under reduced pressure until no liquid is spun out, and dissolving the eluate with methanol and acetone at a volume ratio of 1:1 to obtain the crude component, wherein the second C18ME chromatographic column has a specification of 100A, 50mm × 250mm and 10 μm.
5. The method of claim 1, wherein in step C, the polyimide isColumn volume BV of amine resin chromatography column2Is 1L; the dosage of the distilled water, the ethanol with the volume fraction of 20 percent and the ethanol with the volume fraction of 40 percent are all 5 times BV2The dosage of the 90 percent ethanol is 2 times of BV2。
6. The method according to claim 5, wherein during the gradient elution, each 500mL is taken as a collection unit, the collection units are numbered sequentially, and the No. 5-18 eluents are collected.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270139A (en) * | 2008-04-17 | 2008-09-24 | 大连大学 | Common camellia flavone glycosides A with estrogen liveness, preparation method and application thereof |
| CN101899070A (en) * | 2010-07-19 | 2010-12-01 | 中国林业科学研究院林产化学工业研究所 | The preparation method of flavonoid glycoside in the compression leg sharp separation cake of camellia oleifera seeds in a kind of |
| CN104592784A (en) * | 2014-12-30 | 2015-05-06 | 苏秀珍 | Preparation method of water-soluble tawny natural pigments in camellia cleifera seed meal |
| CN105432673A (en) * | 2015-11-24 | 2016-03-30 | 仲恺农业工程学院 | Method for extracting bacteriostatic substances from oil tea by high-speed countercurrent chromatography and application of bacteriostatic substances |
| CN105440092A (en) * | 2016-01-18 | 2016-03-30 | 信阳师范学院华锐学院 | Method for quickly preparing flavonoid glycoside from oil-tea meal |
-
2018
- 2018-11-30 CN CN201811457238.1A patent/CN109485682B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270139A (en) * | 2008-04-17 | 2008-09-24 | 大连大学 | Common camellia flavone glycosides A with estrogen liveness, preparation method and application thereof |
| CN101899070A (en) * | 2010-07-19 | 2010-12-01 | 中国林业科学研究院林产化学工业研究所 | The preparation method of flavonoid glycoside in the compression leg sharp separation cake of camellia oleifera seeds in a kind of |
| CN104592784A (en) * | 2014-12-30 | 2015-05-06 | 苏秀珍 | Preparation method of water-soluble tawny natural pigments in camellia cleifera seed meal |
| CN105432673A (en) * | 2015-11-24 | 2016-03-30 | 仲恺农业工程学院 | Method for extracting bacteriostatic substances from oil tea by high-speed countercurrent chromatography and application of bacteriostatic substances |
| CN105440092A (en) * | 2016-01-18 | 2016-03-30 | 信阳师范学院华锐学院 | Method for quickly preparing flavonoid glycoside from oil-tea meal |
Non-Patent Citations (3)
| Title |
|---|
| "Acetylated and non-acetylated Flavonol Triglycosides from Galega Officinalis";Yves Champavier 等;《Chem. Pharm. Bull.》;20000229;第48卷(第2期);第281-282页 * |
| "Flavonoids with acetylated branched glycans and bioactivity of Tipuana tipu (Benth.) Kuntze leaf extract";Manal S.Afifia 等;《Natural Product Research》;20131021;第28卷(第4期);第257-264页 * |
| "广宁红花油茶饼黄酮类化合物的分离及结构鉴定";包莉圆;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20180115(第01期);第B024-320页 * |
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