CN109265494B - Method for extracting kaempferol glucoside compounds from camellia reticulata - Google Patents
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Abstract
本发明提供了一种从滇山茶中提取山奈酚葡萄糖苷类化合物的方法。所述方法包括:将滇山茶茶饼用乙醇水溶液进行超声浸提，得到提取液；将提取液减压蒸馏至无醇得浓缩液；将浓缩液加入大孔树脂层析柱进行梯度洗脱，收集洗脱液得粗流份段，将粗流份段浓缩，得到粗流份段浓缩液；在粗流份段浓缩液中加入硅胶，搅拌均匀后烘干得到待处理样品；干法上样至硅胶层析柱中行梯度洗脱，收集洗脱液得准流份段；减压浓缩干燥得粗分离物；用C18色谱柱、甲醇‑甲酸水溶液体系作为流动相，将粗分离物用溶剂溶解后进样进行等度洗脱，收集含有目标化合物的流份段，浓缩冻干得到目标化合物。本申请提供的方法，首次从滇山茶中分离出目标化合物，提取纯度高。
The invention provides a method for extracting kaempferol glucoside compounds from Dianshan tea. The method includes: ultrasonically extracting the Dianshan tea cake with an aqueous ethanol solution to obtain an extract; distilling the extract under reduced pressure to obtain a concentrated solution without alcohol; adding the concentrated solution to a macroporous resin chromatography column for gradient elution, Collect the eluent to obtain a crude fraction, concentrate the crude fraction to obtain a concentrated liquid of the crude fraction; add silica gel to the concentrated liquid of the crude fraction, stir evenly, and then dry to obtain a sample to be treated; dry sample loading Carry out gradient elution in silica gel chromatography column, collect eluent to obtain quasi-fluid section; concentrate and dry under reduced pressure to obtain crude isolate; use C18 chromatographic column and methanol-formic acid aqueous system as mobile phase, and dissolve crude isolate with solvent After injection, isocratic elution was performed, and the fractions containing the target compound were collected, concentrated and lyophilized to obtain the target compound. The method provided in this application separates the target compound from Dianshan tea for the first time, and the extraction purity is high.
Description
Technical Field
The invention relates to the field of compound extraction, and particularly relates to a method for extracting kaempferol glucoside compounds from Yunnan camellia.
Background
Yunnan Camellia (Camellia reticulata Lindl), also known as Yunnan tea or Tengchonghua Camellia oleifera, and perennial evergreen trees in the genus Camellia (Camellia L.) of the family Theaceae are special species in China and secondary important protection plants in China. As the origin of the oil tea in China, more than 2300 years of cultivation history exists, Yunnan province is the big province of the oil tea in China, and the oil tea has rich oil tea resources.
Generally, the content of fatty acids such as oleic acid and linoleic acid in camellia seeds is an important index for evaluating the quality of camellia oleifera. Compared with the common camellia oleifera, the camellia reticulata has high oil content, bright oil body and the content of unsaturated fatty acid reaching 83.5 percent. The camellia reticulata contains rich bioactive substances, such as flavone, organic acid, saponin, triterpenoid compounds and the like, has certain nutrition and health care effects, can reduce cholesterol, prevent tumors and enhance the immunity of organisms, and has good biological utilization value.
Kaempferol and its derivatives are important compounds in Camellia oleifera, and have anticancer, diabetes and osteoporosis treating, and injured cell protecting effects.
In the prior art, the analysis of active substances contained in the camellia reticulata is not comprehensive, and a plurality of effective components need to be separated and extracted. The effective active substances in the camellia reticulata are separated and extracted from complex and various components to obtain a product with higher purity, and the method plays an important role in deep application of the camellia reticulata.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for extracting kaempferol glucoside compounds from Yunnan camellia, which firstly extracts target compounds from the Yunnan camellia, and has the advantages of rapidness, effectiveness and high purity.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for extracting kaempferol glucoside compounds from Yunnan camellia, which comprises the following steps:
A. ultrasonically leaching the Yunnan camellia tea cake with ethanol water solution to obtain an extracting solution; distilling the extracting solution under reduced pressure until no alcohol exists to obtain a concentrated solution;
B. adding the concentrated solution into a macroporous resin chromatographic column, performing gradient elution by sequentially using water, 20% ethanol, 50% ethanol and 80% ethanol, performing chromatographic detection, collecting eluent containing a target extract to obtain a crude fraction, and concentrating the crude fraction to obtain a crude fraction concentrated solution;
C. adding silica gel into the crude fraction concentrated solution, uniformly stirring, and drying to obtain a sample to be treated; loading the sample to be processed into a silica gel chromatographic column by using a dry method, carrying out gradient elution by using a dichloromethane-methanol solution system, carrying out chromatographic detection, and collecting an eluent containing a target extract to obtain a quasi-flow fraction; concentrating under reduced pressure, and drying to obtain crude extract;
D. dissolving the crude isolate with a solvent by using a C18 chromatographic column and using a methanol-formic acid aqueous solution system as a mobile phase, injecting a sample, performing isocratic elution, collecting a flow section containing a target compound, concentrating and freeze-drying to obtain the target compound, wherein the structural formula of the target compound is as follows:
the target compound can be effectively separated and extracted from a plurality of components of the Yunnan camellia by a preparation method of leaching, concentration, macroporous resin column chromatography, silica gel column chromatography and C18 chromatographic column.
Preferably, in the step B, the column volume of the macroporous resin chromatographic column is 2L; the eluent is collected once every 500mL and is sequentially labeled, and the crude fractions are combined from eluent A44-45.
The column volume and the time for collecting the eluent are limited so as to realize accurate separation and extraction. When the column volume and the collection unit volume are changed, the collection label is changed accordingly.
Preferably, the model of the macroporous resin in the macroporous resin chromatographic column is D101, the particle size is 60-16 meshes, and the specific surface area is more than or equal to 550m2The specific adsorption capacity (phenol/dry basis) is more than or equal to 20 mg/g; before use, the macroporous resin needs to be eluted by ethanol until the color is clear, and then is eluted by pure water until no alcohol smell exists.
The purpose of eluting with ethanol solution is to fully treat the macroporous resin and ensure the separation effect.
Further preferably, in the step C, the method for manufacturing the silica gel chromatographic column includes: and mixing silica gel with a dichloromethane solution, carrying out ultrasonic treatment to remove bubbles, slowly filling the mixture into a chromatographic column, and pressurizing the column by using a pressure pump to obtain the silica gel chromatographic column.
More preferably, in the step C, the dichloromethane-methanol solution system is: the mobile phase A is dichloromethane, and the mobile phase B is methanol; the proportion of gradient elution is as follows: the volume ratio of the mobile phase A to the mobile phase B is 7:1, 5:1, 3:1, 1:1 and 1:0 in sequence, and each elution gradient is eluted until colorless.
The mobile phase, the ratio thereof, and the elution gradient are determined based on parameters such as the polarity of the substance contained in the elution target, the separation time, and the degree of separation. The target compound can be quickly and effectively separated by proper mobile phase, proportion and elution gradient.
More preferably, in the step C, every 25-35g of the silica gel corresponds to 100mL of the crude stream fraction concentrated solution; the drying temperature is 45-55 ℃.
Due to the proper sample mixing method and the proper drying temperature, the effect of silica gel column chromatography can be effectively guaranteed.
Further preferably, in the step C, the column volume of the silica gel chromatographic column is 1.2L; the eluates were collected every 200mL and numbered sequentially, and the quasi-fractions were obtained from eluent No. C4.
Preferably, in the step D, the specification of the C18 chromatographic column is: venusil MP C18, size 10mm X250 mm, filler particle size 5 microns.
More preferably, the methanol-formic acid aqueous solution system is: the mobile phase C is methanol, and the mobile phase D is 0.1% of formic acid aqueous solution in volume fraction; the proportion of isocratic elution is as follows: the volume ratio of the mobile phase C to the mobile phase D is 25: 75.
optionally, the volume fraction of the ethanol aqueous solution is 50-60%; the leaching method comprises the following steps: the material-liquid ratio is 1kg, the Yunnan camellia tea cake corresponds to 3-4L of the ethanol water solution, ultrasonic leaching is carried out for 2-4 times under the condition of 30-40 ℃, and the extracting solution is obtained by filtering.
The extraction solvent and the extraction method are proper, so that the extraction yield can be ensured.
Compared with the prior art, the invention has the beneficial effects that:
(1) separating a target extract from the Yunnan camellia 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 an HPLC chromatogram of the preparation of the crude isolate of example 1;
FIG. 2 is a graph showing the purity measurement of the objective compound prepared in example 1;
FIG. 3 is a mass spectrum of a target compound prepared according to the present application;
FIG. 4 is a chart of the target compound prepared in the present application as H;
fig. 5 is a spectrum C of the 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 Yunnan camellia tea fruit in the sun, removing shell, removing oil by cold pressing method, drying in the air and pulverizing properly to obtain Yunnan camellia tea cake. Performing ultrasonic extraction with 50% ethanol water solution at 40 deg.C for 3 times (each time for 6 hr) at a material-to-liquid ratio of 1:4(m/v), filtering, and mixing to obtain extractive solution. Concentrating the extractive solution under reduced pressure until no alcohol is removed, and concentrating to obtain concentrated 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 with the model number of D101: the particle diameter is 60-16 meshes, and the specific surface area is more than or equal to 550m2The specific adsorption capacity (phenol/dry basis) is more than or equal to 20 mg/g. Mixing 2L macroporous resin with appropriate amount of water, loading into 2L chromatographic column, decolorizing with 5L 100% ethanol until the color is clear, and washing with pure water until no alcohol smell is detected. Taking 2L of concentrated solution, slowly adding the concentrated solution into a macroporous resin chromatographic column, sequentially eluting with water, 20% ethanol, 50% ethanol and 80% ethanol until the concentrated solution is colorless, collecting the eluent by using a clean glass bottle, wherein each bottle is 500mL, and the bottles are sequentially marked with A1, A2, A3 and … …, detecting the marked eluent by using a high performance liquid chromatography analyzer, collecting the eluent A44-45 containing a target compound to obtain a crude fraction section, and concentrating the crude fraction section to obtain a crude fraction section concentrated solution.
Mixing 200-mesh silica gel with 300 meshes with a dichloromethane solution, slowly filling the mixture into a chromatographic column with the column volume of 1.2L after removing bubbles through ultrasonic treatment, and pressurizing the column by using a pressure pump to obtain a silica gel chromatographic column; adding 60g of silica gel into 200mL of the crude fraction concentrated solution, uniformly stirring, and drying at 50 ℃ to obtain a sample to be treated; loading a sample to be treated into a silica gel chromatographic column by using a dry method, and carrying out gradient elution by using a dichloromethane-methanol solution system: the mobile phase A is dichloromethane, and the mobile phase B is methanol; the proportion of gradient elution is as follows: the volume ratio of the mobile phase A to the mobile phase B is 7:1, 5:1, 3:1, 1:1 and 1:0 in sequence, and each elution gradient is eluted until colorless. In the elution process, collecting the eluent by using a clean glass bottle, numbering C1, C2 and C3 … … according to the receiving sequence of 200mL of each bottle, performing high performance liquid chromatography on the collected eluent, merging the eluents according to the detection result, and taking the C4 eluent to obtain a quasi-flow fraction; and (4) concentrating the quasi-flow sections under reduced pressure, drying by using a nitrogen blowing instrument, and freeze-drying to obtain a crude isolate.
The crude isolate was purified by a volume ratio of 25: dissolving 75% methanol-0.1% formic acid water solution with concentration of 20 mg/mL; performing isocratic elution with LC3000 high performance liquid chromatograph and C18 chromatographic column by using methanol-formic acid aqueous solution system as mobile phase, collecting fraction containing target compound, concentrating, and lyophilizing to obtain target compound. Wherein, the specification of the C18 chromatographic column is as follows: venusil MP C18, size 10mm × 250mm, filler particle size 5 micron; the methanol-formic acid aqueous solution system is as follows: the mobile phase C is methanol, and the mobile phase D is 0.1% of formic acid aqueous solution in volume fraction; the proportion of isocratic elution is as follows: the volume ratio of the mobile phase C to the mobile phase D is 25: 75.
the HPLC profile of the crude isolate prepared using a Venusil MP C18 column is shown in fig. 1, where the xii peak corresponds to the target compound.
Example 2
Drying Yunnan camellia tea fruit in the sun, removing shell, removing oil by cold pressing method, drying in the air and pulverizing properly to obtain Yunnan camellia tea cake. Ultrasonic extracting with 60% ethanol water solution at 30 deg.C for 4 times (6 hr each time) at a material-to-liquid ratio of 1:3(m/v), filtering, and mixing to obtain extractive solution. Concentrating the extractive solution under reduced pressure until no alcohol is removed, and concentrating to obtain concentrated solution.
Selecting macroporous resin with the model number of D101: the particle diameter is 60-16 meshes, and the specific surface area is more than or equal to 550m2The specific adsorption capacity (phenol/dry basis) is more than or equal to 20 mg/g. Mixing 2L macroporous resin with appropriate amount of water, loading into 2L chromatographic column, decolorizing with 4L 100% ethanol until the color is clear, and washing with pure water until no alcohol smell is detected. Taking 2L of the concentrated solution, slowly adding the concentrated solution into a macroporous resin chromatographic column, then sequentially eluting with water, 20% ethanol, 50% ethanol and 80% ethanol until the concentrated solution is colorless, collecting the eluent by using a clean glass bottle, wherein each bottle is 500mL, and the bottles are sequentially marked with A1, A2, A3 and … …, detecting the marked eluent by using a high performance liquid chromatography analyzer, and collecting the A44-45 eluent containing the target compound to obtain a crude fraction section.
Mixing 200-mesh silica gel with 300 meshes with a dichloromethane solution, slowly filling the mixture into a chromatographic column with the column volume of 1.2L after removing bubbles through ultrasonic treatment, and pressurizing the column by using a pressure pump to obtain a silica gel chromatographic column; adding 50g of silica gel into 200mL of the crude fraction concentrated solution, uniformly stirring, and drying at 45 ℃ to obtain a sample to be treated; loading a sample to be treated into a silica gel chromatographic column by using a dry method, and carrying out gradient elution by using a dichloromethane-methanol solution system: the mobile phase A is dichloromethane, and the mobile phase B is methanol; the proportion of gradient elution is as follows: the volume ratio of the mobile phase A to the mobile phase B is 7:1, 5:1, 3:1, 1:1 and 1:0 in sequence, and each elution gradient is eluted until colorless. In the elution process, collecting the eluent by using a clean glass bottle, numbering C1, C2 and C3 … … according to the receiving sequence of 200mL of each bottle, performing high performance liquid chromatography on the collected eluent, merging the eluents according to the detection result, and taking the C4 eluent to obtain a quasi-flow fraction; and (4) concentrating the quasi-flow sections under reduced pressure, drying by using a nitrogen blowing instrument, and freeze-drying to obtain a crude isolate.
The crude isolate was purified by a volume ratio of 25: dissolving 75% methanol-0.1% formic acid water solution with concentration of 20 mg/mL; performing isocratic elution with LC3000 high performance liquid chromatograph and C18 chromatographic column by using methanol-formic acid aqueous solution system as mobile phase, collecting fraction containing target compound, concentrating, and lyophilizing to obtain target compound. Wherein, the specification of the C18 chromatographic column is as follows: venusil MP C18, size 10mm × 250mm, filler particle size 5 micron; the methanol-formic acid aqueous solution system is as follows: the mobile phase C is methanol, and the mobile phase D is 0.1% of formic acid aqueous solution in volume fraction; the proportion of isocratic elution is as follows: the volume ratio of the mobile phase C to the mobile phase D is 25: 75.
example 3
Drying Yunnan camellia tea fruit in the sun, removing shell, removing oil by cold pressing method, drying in the air and pulverizing properly to obtain Yunnan camellia tea cake. Performing ultrasonic extraction with 55% ethanol water solution at 35 deg.C for 2 times (6 hr each time) at a material-to-liquid ratio of 1:3.5(m/v), filtering, and mixing to obtain extractive solution. Concentrating the extractive solution under reduced pressure until no alcohol is removed, and concentrating to obtain concentrated solution.
Selecting macroporous resin with the model number of D101: the particle diameter is 60-16 meshes, and the specific surface area is more than or equal to 550m2The specific adsorption capacity (phenol/dry basis) is more than or equal to 20 mg/g. Mixing 2L macroporous resin with appropriate amount of water, loading into 2L chromatographic column, and purifying with 4-5L macroporous resinDecolorizing with 100% ethanol until the color is clear, and washing with pure water until no alcohol smell is obtained. Taking 2L of the concentrated solution, slowly adding the concentrated solution into a macroporous resin chromatographic column, then sequentially eluting with water, 20% ethanol, 50% ethanol and 80% ethanol until the concentrated solution is colorless, collecting the eluent by using a clean glass bottle, wherein each bottle is 500mL, and the bottles are sequentially marked with A1, A2, A3 and … …, detecting the marked eluent by using a high performance liquid chromatography analyzer, and collecting the A44-45 eluent containing the target compound to obtain a crude fraction section.
Mixing 200-mesh silica gel with 300 meshes with a dichloromethane solution, slowly filling the mixture into a chromatographic column with the column volume of 1.2L after removing bubbles through ultrasonic treatment, and pressurizing the column by using a pressure pump to obtain a silica gel chromatographic column; adding 70g of silica gel into 200mL of the crude fraction concentrated solution, uniformly stirring, and drying at 55 ℃ to obtain a sample to be treated; loading a sample to be treated into a silica gel chromatographic column by using a dry method, and carrying out gradient elution by using a dichloromethane-methanol solution system: the mobile phase A is dichloromethane, and the mobile phase B is methanol; the proportion of gradient elution is as follows: the volume ratio of the mobile phase A to the mobile phase B is 7:1, 5:1, 3:1, 1:1 and 1:0 in sequence, and each elution gradient is eluted until colorless. In the elution process, collecting the eluent by using a clean glass bottle, numbering C1, C2 and C3 … … according to the receiving sequence of 200mL of each bottle, performing high performance liquid chromatography on the collected eluent, merging the eluents according to the detection result, and taking the C4 eluent to obtain a quasi-flow fraction; and (4) concentrating the quasi-flow sections under reduced pressure, drying by using a nitrogen blowing instrument, and freeze-drying to obtain a crude isolate.
The crude isolate was purified by a volume ratio of 25: dissolving 75% methanol-0.1% formic acid water solution with concentration of 20 mg/mL; performing isocratic elution with LC3000 high performance liquid chromatograph and C18 chromatographic column by using methanol-formic acid aqueous solution system as mobile phase, collecting fraction containing target compound, concentrating, and lyophilizing to obtain target compound. Wherein, the specification of the C18 chromatographic column is as follows: venusil MP C18, size 10mm × 250mm, filler particle size 5 micron; the methanol-formic acid aqueous solution system is as follows: the mobile phase C is methanol, and the mobile phase D is 0.1% of formic acid aqueous solution in volume fraction; the proportion of isocratic elution is as follows: the volume ratio of the mobile phase C to the mobile phase D is 25: 75.
the purity of the monomer compound obtained in examples 1 to 3 was measured under the following conditions: using LC3000 high performance liquid chromatograph, Venusil MP C18(4.6mm × 250mm, 5 μm) chromatographic column, mobile phase A is methanol, and mobile phase B is 0.1% formic acid water; controlling the flow rate to be 1mL/min, the detector UV205nm and the sample injection amount to be 2 mu L, and carrying out isocratic elution, wherein the elution ratio is as follows: the volume ratio of mobile phase a to mobile phase B was 25: 75. wherein the purity of the product obtained in example 1 is 97.8%, and the extraction yield is 22.6 mg/kg. FIG. 2 shows the purity measurement chart. The purity and yield of the target extract obtained in examples 2 to 3 were both equal to or higher than the above values.
The compound monomers obtained in examples 1 to 3 were subjected to LC-MS measurement and nuclear magnetic resonance detection.
As shown in FIG. 3, M/z951.2856[ M + H ]]+. The molecular formula is presumed to be C43H50O24。
FIG. 4(1H-NMR), delta 6.20(H, s) and delta 6.37(H, s) are characteristic of the hydrogen at the meta position on the benzene ring, and are inferred to belong to Kaempferol A ring H-6 and H-8. δ 8.01(2H, d, J ═ 8.5Hz) and δ 6.89(2H, d, J ═ 8.5Hz), the two sets of signals, which by integration of the areas, contain 2 protons, should be of symmetrical structure, with chemical shifts, splits and coupling constants matching the characteristics of the ortho hydrogens on the phenyl ring, the results being in agreement with the kaempferol B ring structure, and therefore are inferred to belong to kaempferol B rings H-2', 6' and H-3', 5'. δ 5.47(H, d, J ═ 7.3Hz) is the proton signal at the end of the glucosyl group, which can be inferred from the coupling constants (J ═ 7.3Hz > 7.0Hz) to be in the β configuration; δ 4.59(H, s), 4.67(H, s) are rhamnosyl end-group proton signals; δ 2.15(3H, s), 2.11(3H, s), 2.04(3H, s), 1.95(3H, s), 1.94(3H, s) are acetyl proton signals; δ 0.78(3H, d, J ═ 6.2Hz), 1.12(3H, d, J ═ 6.2Hz) are rhamnosylmethyl proton signals; the delta 4.0-3.0 section is glycosyl other proton signal.
FIG. 5(13C-NMR), δ 103.13, 75.85, 76.58, 71.97, 78.00, 67.11 is one group of glucose-based signals, δ 99.92, 70.91, 75.33, 73.76, 67.50, 17.62 and δ 99.92, 70.91, 75.33, 73.76, 67.50, 17.62 are two groupsThe signals for the five groups of acetyl carbons are the signals for the rhamnosyl groups, δ 172.16, 171.81, 171.76, 171.70, 171.47 and δ 20.94, 20.74, 20.72, 20.67, 20.52. The rest signals are highly consistent with characteristic signals of kaempferol.
Attribution of peaks of the H and C spectra:
1H-NMR：8.01(2H,d,J＝8.5Hz,H-2’,6’)、6.89(2H,d,J＝8.5Hz,H-3’,5’)、6.37(H,s,H-8)、6.20(H,s,H-6)、5.47(H,d,J＝7.3Hz,H-1”)、4.59(H,s,H-1’”)4.67(H,s,H-1””)、2.15(3H,s,AcO-3””)、2.11(3H,s,AcO-2”’)、2.04(3H,s,AcO-4””)、1.95(3H,s,AcO-4”’)、1.94(3H,s,AcO-2””)、1.12(3H,d,J＝6.2Hz,H-6””)、0.78(3H,d,J＝6.2Hz,H-6”’)。
13C-NMR：158.82(C-2)、134.85(C-3),、179.40(C-4)、163.15(C-5)、98.90(C-6)、165.94(C-7)、94.74(C-8)、158.50(C-9)、105.67(C-10)、122.78(C-1’)、132.20(C-2’,6’)、116.18(C-3’,5’)、161.49(C-6’)、103.13(C-1”)、75.85(C-2”)、76.58(C-3”)、71.97(C-4”)、78.00(C-5”)、67.11(C-6”)、99.92(C-1”’)、70.91(C-2”’)、75.33(C-3”’)、73.76(C-4”’)、67.50(C-5”’)、17.62(C-6”’)、99.98(C-1””)、70.00(C-2””)、71.07(C-3””)、72.55(C-4””)、68.35(C-5””)、17.19(C-6””)、171.76,20.52(AcO-C-2”’)、172.16,20.67(AcO-C-4””)、171.70,20.72(AcO-C-2””)、171.81,20.94(AcO-C-3””)、171.47,20.74(AcO-C-4””)。
and (3) integrating all nuclear magnetic spectrum and mass spectrum results, identifying the compound as kaempferol-3-O- [ 2', 3', 4 ' -triacetyl-alpha-L-rhamnose- (1 → 3) -2', 4 ' -diacetyl-alpha-L-rhamnose (1 → 6) ] -beta-D-glucoside, wherein the structural formula is as follows:
the method for extracting the kaempferol glucoside compounds from the Yunnan camellia, provided by the application, separates and extracts the target compounds from the Yunnan camellia 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 Yunnan camellia.
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 (9)
1. A method for extracting kaempferol glucoside compounds from Yunnan camellia, which is characterized by comprising the following steps:
A. ultrasonically leaching the Yunnan camellia tea cake with ethanol water solution to obtain an extracting solution; distilling the extracting solution under reduced pressure until no alcohol exists to obtain a concentrated solution;
B. adding the concentrated solution into a macroporous resin chromatographic column, performing gradient elution by sequentially using water, 20% ethanol, 50% ethanol and 80% ethanol, performing chromatographic detection, collecting eluent containing a target extract to obtain a crude fraction, and concentrating the crude fraction to obtain a crude fraction concentrated solution;
C. adding silica gel into the crude fraction concentrated solution, uniformly stirring, and drying to obtain a sample to be treated; loading the sample to be processed into a silica gel chromatographic column by using a dry method, carrying out gradient elution by using a dichloromethane-methanol solution system, carrying out chromatographic detection, and collecting an eluent containing a target extract to obtain a quasi-flow fraction; concentrating under reduced pressure, and drying to obtain crude extract;
D. dissolving the crude isolate with a solvent by using a C18 chromatographic column and using a methanol-formic acid aqueous solution system as a mobile phase, injecting a sample, performing isocratic elution, collecting a flow section containing a target compound, concentrating and freeze-drying to obtain the target compound, wherein the structural formula of the target compound is as follows:
wherein in the step B, theThe column volume of the macroporous resin chromatographic column is 2L; collecting eluent once per 500mL, and sequentially labeling, wherein the crude fractions are obtained by combining eluent A44-45; the model of the macroporous resin in the macroporous resin chromatographic column is D101, the particle size of the macroporous resin is 60-16 meshes, and the specific surface area is more than or equal to 550m2The specific adsorption capacity of phenol/dry basis is more than or equal to 20 mg/g.
2. The method as claimed in claim 1, wherein the macroporous resin is eluted with ethanol until its color is clear and then eluted with pure water until no alcohol smell is produced before use.
3. The method according to claim 1, wherein in the step C, the silica gel chromatographic column is prepared by: and mixing silica gel with a dichloromethane solution, carrying out ultrasonic treatment to remove bubbles, slowly filling the mixture into a chromatographic column, and pressurizing the column by using a pressure pump to obtain the silica gel chromatographic column.
4. The method according to claim 3, wherein in the step C, the dichloromethane-methanol solution system is: the mobile phase A is dichloromethane, and the mobile phase B is methanol; the proportion of gradient elution is as follows: the volume ratio of the mobile phase A to the mobile phase B is 7:1, 5:1, 3:1, 1:1 and 1:0 in sequence, and each elution gradient is eluted until colorless.
5. The method as claimed in claim 4, wherein in step C, every 25-35g of the silica gel corresponds to 100mL of the crude fraction concentrate; the drying temperature is 45-55 ℃.
6. The method according to claim 5, wherein in the step C, the column volume of the silica gel chromatographic column is 1.2L; the eluates were collected every 200mL and numbered sequentially, and the quasi-fractions were obtained from eluent No. C4.
7. The method of claim 1, wherein in the step D, the specification of the C18 chromatographic column is: venusil MP C18, size 10mm X250 mm, filler particle size 5 microns.
8. The method of claim 7, wherein the aqueous methanol-formic acid system is: the mobile phase C is methanol, and the mobile phase D is 0.1% of formic acid aqueous solution in volume fraction; the proportion of isocratic elution is as follows: the volume ratio of the mobile phase C to the mobile phase D is 25: 75.
9. the process according to any one of claims 1 to 8, wherein the volume fraction of the aqueous ethanol solution is from 50 to 60%; the leaching method comprises the following steps: the material-liquid ratio is 1kg, the Yunnan camellia tea cake corresponds to 3-4L of the ethanol water solution, ultrasonic leaching is carried out for 2-4 times under the condition of 30-40 ℃, and the extracting solution is obtained by filtering.
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| CN101270139A (en) * | 2008-04-17 | 2008-09-24 | 大连大学 | Camellia flavonoid glycoside A with estrogen activity and its preparation method and use |
| CN105924419A (en) * | 2016-04-29 | 2016-09-07 | 中南林业科技大学 | Method for extracting kaempferol and derivative thereof from camellia oleifera leaves |
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| CN101270139A (en) * | 2008-04-17 | 2008-09-24 | 大连大学 | Camellia flavonoid glycoside A with estrogen activity and its preparation method and use |
| CN105924419A (en) * | 2016-04-29 | 2016-09-07 | 中南林业科技大学 | Method for extracting kaempferol and derivative thereof from camellia oleifera leaves |
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