CN114773187A - Method for separating, preparing and screening antioxidant and anti-inflammatory active ingredients in bunge auriculate flower - Google Patents
Method for separating, preparing and screening antioxidant and anti-inflammatory active ingredients in bunge auriculate flower Download PDFInfo
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Abstract
The invention provides a separation preparation and screening method of antioxidant and anti-inflammatory active ingredients in cynanchum bungei flower, belonging to the technical field of biological medicines. The invention systematically screens and separates antioxidant and anti-inflammatory active sites in the white fleece-flower root Cynanchum auriculatum flower ex light flower based on an activity guide/spectrum effect separation technology, finally obtains 11 compounds with activity, and applies the compounds1H NMR、13The structure was determined by spectroscopic methods such as C NMR. The invention lays a foundation for establishing the quality standard of the bunge auriculate flower and comprehensively utilizing the plant resource of the bunge auriculate flower, and provides reference for the research of other natural products, thereby having good practical application value.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a method for separating, preparing and screening antioxidant and anti-inflammatory active ingredients in bunge auriculate flower.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The radix Cynanchi auriculati is mainly prepared from Cynanchum auriculatum Royle ex wight, Euphorbia pekinensis, Cynanchum auriculatum Bungei Decne, and Cynanchum auriculatum Wilfordii (Max)im.) hook.F dry root tuber, which is distributed in northeast, northwest, north China, east China, Heilongjiang, Liaoning, Gansu, Shanxi, Shaanxi, inner Mongolia, Hebei, Shandong, Jiansu, Hubei, Hunan, Henan, Anhui, etc., can be used as genuine medicinal materials in Shandong, Jiansu, Hunan, etc., and is food with nourishing and health-care functions in folk, has the effects of nourishing liver and kidney, strengthening bones and muscles, benefiting essence and blood, invigorating qi and strengthening spleen, and can be used for treating liver and kidney deficiency, soreness and weakness of waist and knees, insomnia, amnesia, dizziness and tinnitus, sore and carbuncle, gall, venomous snake bite, etc. Chemical research shows that the root of radix Cynanchi auriculati contains C21Steroid glycoside, benzophenones, flavonoids, phenolic acids and other traditional Chinese medicine components, and various nutrient components required by human body such as fatty acid, protein, vitamin, phospholipids, various trace elements and the like. Pharmacological research shows that the radix cynanchi bungei has the pharmacological effects of resisting tumor, resisting aging, regulating immunity, resisting oxidation, reducing blood fat, resisting anoxia and the like. The bunge auriculate root flower is a dried flower bud of bunge auriculate root, is used as scented tea in Hunan province, Jiangsu province and other places, is also developed into various places for sale, is used for treating hypertension, hyperglycemia, angiostenosis and aging, but has unknown chemical components and pharmacological activity, and needs to be deeply researched. The method can lay a foundation for formulating the quality standard of the bunge auriculate flower and comprehensively utilizing the bunge auriculate root which is a plant resource by separating and structurally identifying the chemical components of the bunge auriculate root flower and screening the active components in the bunge auriculate root flower.
An activity guide/spectrum effect separation technology is a method for analyzing the structure type of active ingredients of traditional Chinese medicine and guiding the separation of the active ingredients of the traditional Chinese medicine by comprehensively utilizing methods of spectroscopy and traditional Chinese medicine pharmacology. Firstly, preliminarily separating Chinese medicine extracts, determining active sites by measuring the in vitro activity of each separated site, rapidly separating compounds of the active sites by adopting an UPLC/MS method, speculating the structure of the compounds, establishing an optimal separation method according to the structural characteristics of the compounds, and accurately separating active ingredients. The method can improve the probability of finding active compounds, simplify the steps of separation operation, reduce the loss of compounds in separation and reduce the consumption of separation mobile phase and filler. However, the inventor finds that no report exists for separating, preparing and screening the antioxidant and anti-inflammatory active ingredients in the bunge auriculate flower by using an activity guiding/spectrum effect separation technology.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a method for separating, preparing and screening antioxidant and anti-inflammatory active ingredients in bunge auriculate flower. The invention carries out systematic screening and separation on the antioxidant and anti-inflammatory active sites in the flowers of the white fleece-flower root Cynanchum auriculatum flower ex light based on an activity guide/spectrum effect separation technology, and finally obtains 11 compounds with activity. The present invention has been completed based on the above results.
The specific technical scheme of the invention is as follows:
in a first aspect of the present invention, there is provided a method for separating, preparing and screening an antioxidant and anti-inflammatory active ingredient from flowers of bunge auriculate root, the method comprising:
s1, pulverizing radix Cynanchi auriculati, soaking in solvent, recovering solvent, concentrating, and drying to obtain extract;
s2, adding water into the extract for suspension, and sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butanol to obtain extracts of different parts;
s3, measuring the antioxidant and anti-inflammatory effects of the extracts of each part, and screening the ethyl acetate part with the best activity;
s4, determining the main compound structure of the ethyl acetate active site by using a UPLC-MS method;
and S5, separating the compound components at the ethyl acetate part, wherein the first separation adopts silica gel column chromatography, and the subsequent separation adopts gel column chromatography, polyamide column chromatography or preparative HPLC (separation filler is a reversed phase C18 column), and finally 11 active compounds are obtained.
By the separation method, 11 active compounds are obtained in total, which comprise: 2-trans-4-trans-abscisic acid (1), (2E,4E) -8-hydroxy-2,7-dimethyl-decadien- (2,4) -dissecure- (1,10) -dioic acid (2), quercetin (3), kaempferol (4), quercetin-3-O-beta-D-glucopyranose- (1 → 2) -beta-D-galactopyranoside (5), rutin (6), quercetin-3-O-beta-D-galactopyranoside (7), isoquercitrin (8), quercetin-3-O-beta-D-xyloside (9), quercetin-3-O-alpha-L-pyranoside (10), kaempferol-3-O-alpha-L-rhamnoside (11). The specific structural formula of the 11 compounds is shown in figure 7.
In a second aspect of the present invention, there is provided the use of the above separation, preparation and screening methods in the comprehensive utilization of cynanchum bunaei flower.
The beneficial technical effects of the technical scheme are as follows:
the technical scheme firstly uses the UPLC-MS method to research the chemical components of each extraction part of the bunge auriculate flower, speculates the structure of the bunge auriculate flower, and finds that the main active component of the ethyl acetate active part is a flavonoid compound. Aiming at the structural characteristics of the flavonoid compounds, a main separation scheme is determined; simultaneously, the antioxidant and anti-inflammatory active parts of the flowers of the bunge auriculate root are systematically separated for the first time, 11 compounds are separated from the active parts, and the application is carried out1H NMR、13The structure was confirmed by spectroscopic methods such as C NMR.
In a word, the technical scheme lays a foundation for establishing the quality standard of the bunge auriculate flower and comprehensively utilizing the plant resource of the bunge auriculate flower, and provides reference for the research of other natural products, so the method has good value of practical application.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 shows the results of in vitro antioxidant activity of each extraction site in example 1 of the present invention;
FIG. 2 shows the results of the safe concentration of RAW264.7 in vitro for each extraction moiety in example 1 of the present invention;
FIG. 3 shows the results of in vitro enhancement of the secretion of the cytokine IL-1. beta. from RAW264.7 at each extraction site in example 1 of the present invention;
FIG. 4 shows the results of promoting the secretion of IL-6, a cytokine, from RAW264.7, in vitro at each extraction site in example 1 of the present invention;
FIG. 5 shows the results of in vitro enhancement of the secretion of the cytokine IL-10 from RAW264.7 at each extraction site in example 1 of the present invention;
FIG. 6 is a BPC map with the ion molecular map of UPLC-MS of each active site in example 1 of the present invention;
FIG. 7 is a structural formula of compounds 1-11 prepared in example 1 of the present invention;
FIG. 8 is a drawing of Compound 11H NMR and13a C NMR spectrum;
FIG. 9 is a drawing of Compound 21H NMR and13a C NMR spectrum;
FIG. 10 is a drawing of Compound 31H NMR and13a C NMR spectrum;
FIG. 11 is a drawing of Compound 41H NMR and13a C NMR spectrum;
FIG. 12 is a drawing of Compound 51H NMR and13a C NMR spectrum;
FIG. 13 is a photograph of Compound 61H NMR and13a C NMR spectrum;
FIG. 14 is a drawing of Compound 71H NMR and13a C NMR spectrum;
FIG. 15 is of Compound 81H NMR and13a C NMR spectrum;
FIG. 16 is of Compound 91H NMR and13a C NMR spectrum;
FIG. 17 is a photograph of Compound 101H NMR and13a C NMR spectrum;
FIG. 18 is a photograph of Compound 111H NMR and13c NMR spectrum.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As described above, the flowers of bunge auriculate root are the dried buds of bunge auriculate root and are used for treating hypertension, hyperglycemia, angiostenosis and aging, but the chemical components and pharmacological activities thereof are not clear, and intensive research is urgently needed.
In view of the above, the present invention provides a method for separating, preparing and screening antioxidant and anti-inflammatory active ingredients from flowers of bunge auriculate root, which comprises: pulverizing radix Cynanchi auriculati into coarse powder, extracting with solvent, recovering solvent, lyophilizing or drying under reduced pressure to obtain dry extract, suspending the extract with water, sequentially extracting with different organic solvents, and recovering solvent to obtain different extraction parts. Measuring the free radical scavenging ability of the above extract part by DPPH method and ABTS method to determine antioxidant active site; and (3) measuring the capability of the extracted part on releasing cytokines and NO of RAW264.7 cells by an ELISA method, and determining the anti-inflammatory active part. The UPLC-MS technology is used for analyzing the active sites, the structural types of active compounds are deduced by combining documents, then according to the structural characteristics of the active compounds, an optimal separation scheme is screened and established from silica gel column chromatography, macroporous resin column chromatography, reversed phase column chromatography, preparative high performance liquid chromatography and other separation methods, and the active sites are rapidly enriched and accurately separated, so that the target compounds are obtained.
Specifically, in an exemplary embodiment of the present invention, a method for separating, preparing and screening an antioxidant and anti-inflammatory active ingredient from flowers of bunge auriculate root is provided, wherein the method comprises:
s1, pulverizing the flowers of bunge auriculate root, adding a solvent for soaking and extracting, recovering the solvent, concentrating and drying to obtain an extract;
s2, adding water into the extract for suspension, and sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol to obtain extracts of different parts;
s3, measuring the antioxidant and anti-inflammatory effects of the extracts of each part, and screening the ethyl acetate part with the best activity;
s4, determining the main compound structure of the ethyl acetate active site by using a UPLC-MS method;
s5, separating the compound components of the ethyl acetate part, wherein the first separation adopts silica gel column chromatography, and the subsequent separation adopts gel column chromatography, polyamide column chromatography or preparative HPLC (separation filler is a reversed phase C18 column), and finally obtaining 11 active compounds.
Wherein, in the step S1,
the solvent can be lower alcohol (such as methanol or ethanol) containing water 1-90% (volume fraction, v/v, the same below), or acetone containing water 1-90%, or water saturated ethyl acetate, water saturated dichloromethane, and water saturated n-butanol.
The soaking and extracting temperature can be room temperature (0-30 ℃) or heating condition (30 ℃ till the solvent is boiled) under normal pressure;
the concentration condition may be heating boiling (20 ℃ to solvent boiling) under reduced pressure (-0.1 to-0.09 MPa).
The drying can be freeze drying or decompression drying, the freeze drying condition is that the temperature is minus 80 to minus 20 ℃, the pressure is minus 0.1 to minus 0.09MPa, and the drying time is 5 to 7 days; the reduced pressure drying condition is room temperature (0-30 ℃) or heating boiling (30 ℃ to solvent boiling, generally not more than 45 ℃) under the reduced pressure state (-0.1 to-0.09 MPa).
In the step S2, in the above step,
the extraction solvent is water saturated petroleum ether (60-90 ℃), water saturated dichloromethane, water saturated ethyl acetate and water saturated n-butanol in sequence, the extraction times are 5-7 times for each extraction solvent, and the extraction temperature is room temperature (0-30 ℃).
In the step S3, in the above step,
the specific method for measuring the antioxidant and anti-inflammatory effects of the extracts of each part comprises but is not limited to measuring the antioxidant activity of the extracts of different parts of the cynanchum bunaei florum by adopting a DPPH method and an ABTS method; and selecting active extraction sites with anti-inflammatory effect by using RAW246.7 cells as target cells and measuring the influence of each extraction site on the release of cytokines and NO from the target cells.
Wherein, ELISA method can be adopted to determine the content of the cytokine released by RAW 246.7;
the content of NO released from RAW246.7 can be measured by Griess method.
In still another embodiment of the present invention, the method for measuring antioxidant activity (DPPH method) comprises the steps of: 10mg of extracts of different extraction parts of the cynanchum bungei flower are respectively taken, absolute ethyl alcohol is used for preparing test solution with the mass concentration of 0.1, 0.5, 1.0, 1.5 and 2.0mg/mL, and deionized water is used for preparing vitamin C solution with the mass concentration of 0.02, 0.04, 0.06, 0.08 and 0.1 mg/mL. Respectively putting 2mL of each test solution into 10mL test tubes, adding 2mL of fully dissolved DPPH ethanol solution (0.1mmol/L) in portions, adding plugs, shaking uniformly, shielding and shading tin foil paper for 30min at room temperature, putting the test solution into a 1cm cuvette, putting the cuvette into a spectrophotometer, and measuring absorbance at 517nm to obtain the absorbance A of the test solutioniPurified water was used as a blank control group, and the absorbance was A0Absolute ethyl alcohol is used as a background group to replace DPPH ethanol solution, and the absorbance is Aj. Calculating Vc with different mass concentrations and the clearance rate of the sample solution to DPPH free radicals according to formulas (1) and (2), and obtaining IC through SPSS statistical analysis50。
The determination method (DPPH method) of the antioxidant activity has the calculation formula as follows:
vc on DPPH free radical clearance (%) (1-A)Vc/Ai)×100％ (1)；
The sample has DPPH free radical scavenging rate (%) [ A ]0-(Ai-Aj)]/A0×100％(2)
In still another embodiment of the present invention, the method for measuring antioxidant activity (ABTS method) comprises the steps of: respectively taking 10mg of extracts of different-polarity extraction parts of the bunge auriculate flower, preparing 0.05, 0.1, 0.15, 0.2 and 0.25mg/mL of the extracts by using absolute ethyl alcohol, and preparing 0.02, 0.04, 0.06, 0.08 and 0.1mg/mL of vitamin C solution by using water. And (3) placing the ABTS reagent in the kit in a test tube, screwing a tube cover, shaking up, and standing at room temperature for 12-16h to obtain an ABTS concentrated solution. Mixing ABTS solution (7.4mmol/L) and potassium persulfate solution (2.6mmol/L) at volume ratio of 1:1, standing at room temperature in dark place for 12 hr to obtain ABTS mother liquorThe solution was diluted 45 times with purified water as ABTS free radical working solution. Precisely measuring 2mL of ABTS free radical working solution and 1mL of each sample solution, fully shaking up, keeping out of the sun for 30min at room temperature, and then measuring absorbance at 734 nm. Measuring the absorbance of the test solution as AiPurified water was used as a blank control group, and the absorbance was A0ABTS working solution is used as a background group, and the absorbance is Aj. Calculating the clearance rate of the sample solution with different mass concentrations to DPPH free radical according to the formula (3) and obtaining IC through SPSS statistical analysis50. Researches find that the anti-oxidation activity of the bunge auriculate flower is an ethyl acetate part and an n-butyl alcohol part.
The measuring method (ABTS method) of the antioxidant activity has the following calculation formula:
ABTS free radical clearance (%) - [ A0-(Ai-Aj)]/A0×100％ (3)
In yet another embodiment of the present invention, the cell culture method for anti-inflammatory assays comprises the steps of: (1) cell recovery: taking out the frozen cells, quickly dissolving at 37 ℃, taking 15mL of centrifuge tube, adding 3mL of serum-containing culture medium, adding the liquid in the frozen tube into the centrifuge tube containing the culture medium, uniformly mixing by light blowing, centrifuging at the normal temperature of 1000-1500 r/min for 5-10 min, removing the supernatant, adding 3-5 mL of serum-containing culture medium, uniformly mixing by light blowing to obtain cell suspension, and adding the cell suspension into a culture bottle. (2) Culturing: placing the cells in a culture bottle containing complete culture medium for adherent culture under the culture conditions of 38 ℃ and 5% CO2When the cells grow over 80% of the culture flask, the cells can be passaged. (3) And (3) passage: discarding the culture solution in the culture bottle, adding 1-3 mL of trypsin, incubating and digesting for 2-5 min at 37 ℃, and immediately adding the culture solution containing the same volume of serum to stop digestion if cytoplasm retracts and intercellular space increases. And (4) blowing and beating the cells at the bottom of the bottle repeatedly and lightly in sequence to separate the adherent cells from the bottom of the bottle and suspend the adherent cells to form cell suspension. Inoculating into a new culture bottle according to a first transfer and a third transfer, and placing into an incubator for culture.
In still another embodiment of the present invention, the method for determining the safe concentration of an anti-inflammatory assay (MTT method) comprises the steps of: RAW246.7 cells were adjusted to a concentration of 1.0X 106Per mlAdded to a 96-well cell culture plate at 100. mu.L per well. Culturing for 12h, discarding culture solution, adding the five-concentration radix Cynanchi auriculati flower extract diluted with complete culture medium into cell culture plate at a concentration of 200 μ L per well, repeating for 4 wells, setting cell blank group (complete culture medium containing 0.1% DMSO only), DMSO control group (complete culture medium containing 0.1% DMSO), and placing at 37 deg.C and 5% CO2Culturing in an incubator. After culturing for 36h, adding 30 mu L of MTT into each well, continuing culturing for 4h, then discarding the supernatant, adding 150 mu L of DMSO into each well, placing on a micro-oscillator to shake and dissolve, then measuring the light absorption value at 570nm on an enzyme linked immunosorbent assay detector, and selecting the drug group A under the condition that the DMSO control group is not significantly different from the blank group570The concentration that did not significantly differ from the blank group was taken as the safe concentration of the bunge auriculate flower extract.
In another embodiment of the present invention, the method for measuring the cytokine content (ELISA method) in the anti-inflammatory assay comprises the following steps: (1) the kit is kept at the constant temperature to room temperature, and the standard substance and the working solution are prepared in advance. (2) Setting blank holes, standard holes and sample holes, adding sample diluent as blank holes, adding 100 μ L/hole of sample, standard or sample diluent, repeating each group for 3 times, sealing the ELISA plate with sealing plate film, and incubating at 37 deg.C for 90 min. (3) After incubation, removing liquid in the pores, spin-drying, adding 100 mu L/pore of antibody working solution, adding a membrane-covered sealing plate, and incubating for 60min at 37 ℃. (4) After incubation, removing liquid in the holes, adding the stock solution of the washing buffer solution with the same volume, soaking for 1min, removing waste liquid, washing the plate for 3 times repeatedly, and then drying by spinning. (5) Add ABC working solution 100. mu.L/well, add the sealing film, incubate for 30min at 37 ℃. (6) Pouring out residual liquid, adding washing buffer solution stock solution with the same volume, soaking for 1min, discarding waste liquid, repeatedly washing the plate for 3 times, and spin-drying. (7) Adding 90. mu.L of TMB color development liquid into each well, sealing the plate by a sealing membrane, protecting from light, and reacting for 20min at 37 ℃. (8) After the reaction, 100. mu.L of TMB stop solution was added to each well, and the absorbance was measured at 450 nm.
In another embodiment of the present invention, the method for determining the NO content (Griess method) in the anti-inflammatory assay comprises the following steps: (1) cell inoculation: adjusting the cell concentration of the adherent RAW264.7 cells to2.0×105one/mL, 100. mu.L/well in 96-well plates, and suspension cells were removed after 15h of culture. Blank groups were dosed with 100. mu.L PBS per well; LPS group LPS was added to the culture medium to give a final concentration of 10. mu.g/ml-1(ii) a The drug group is prepared by adding LPS into the drug, the LPS is added after 3h of treatment, the drugs are added for co-intervention for 36h, and 5 holes are made in parallel in each group. (2) Drawing a standard curve: preheating the microplate reader for more than 30min, and adjusting the wavelength to 550 nm. And placing the first reagent on ice for later use. Taking out the NO content detection kit and keeping the temperature to room temperature. The standards were diluted with distilled water to 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125. mu. mol/mL. Adding a Griess reagent I into a standard substance, carrying out vortex mixing, carrying out water bath at 37 ℃ for 60min, adding a reagent II into a sample, carrying out vortex mixing, standing at room temperature for 5min, centrifuging at 3500rpm for 10min, taking a supernatant, adding an isovolumetric developing solution into the supernatant, carrying out vortex mixing, standing at room temperature for 10min, measuring absorbance at 550nm, and calculating delta A according to a formula (4).
ΔAMeasurement of＝AMeasurement of-ABlank space(4)
In another embodiment of the present invention, the data processing method of the anti-inflammatory test comprises: data are expressed as Means ± SD and data analysis is performed using GraphPad prism 8.0.1 software. ". indicates the degree of significance of the difference.
In another embodiment of the present invention, in step S4,
the separation conditions used in the UPLC-MS method were: mobile phase: a: 0.1% aqueous formic acid; b: acetonitrile (0.1% formic acid); flow rate: 0.3 ml/min; mobile phase gradient setup: 0-2 min, 5% -5% of B; 2-22 min, 5% -95% of B; 22-26 min, 95% -95% B; 26-26.1 min, 95% -5% of B; 26.1-30 min, 5% -5% of B;
the mass spectrum equipment conditions used by the UPLC-MS method are as follows: mass spectrometer: waters Xevo G2-XS QT of systems (Waters, USA); an ion source: ESI (electrophoresis ion source); the mass spectrometry method comprises the following steps: spraying voltage: 3 KV; source offset: 80; ion source temperature: 115 ℃ of carbon dioxide; desolventizing temperature: at 450 ℃; taper hole airflow: 50L/h; desolventizing the gas stream: 600L/h; the data acquisition mode is as follows: MSe continuous; positive ions (negative ions); resolution mode data acquisition molecular weight range: 50-1200 Da; scanning speed: 1.5 s; gradient collision voltage: 20-40V.
In another embodiment of the present invention, in step S5,
the method for separating the ethyl acetate fraction comprises: subjecting ethyl acetate to silica gel column chromatography, gradient eluting with dichloromethane-methanol mixed solvent, developing thin layer, mixing the same eluates, subjecting to gel column chromatography or polyamide column chromatography, eluting with dichloromethane-methanol mixed solvent, subjecting to reversed phase C18 column, repeatedly eluting with methanol-water system and acetonitrile-water system, and repeatedly separating with preparative high performance liquid chromatography (p-HPLC).
In another embodiment of the present invention, the method for separating an ethyl acetate fraction comprises the steps of: dissolving the ethyl acetate part with methanol according to the ratio of 1:1 (g/mL), mixing with silica gel according to the ratio of 1:1(g/g), loading on a silica gel column, carrying out gradient elution by a dichloromethane-methanol system, detecting each flow by a thin layer chromatography, combining the same flow, and recovering the solvent to obtain Fr.1-Fr.8; fr.4 and methanol are dissolved according to the ratio of 1:1 (g/mL), the mixture and silica gel are mixed according to the ratio of 1:1(g/g), the mixture is put on a silica gel column, gradient elution is carried out by a dichloromethane-methanol system, each flow part is detected by a thin layer chromatography, and the same flow parts are combined to obtain flow parts Fr.4-1-Fr.4-6; fr.4-5 is put on a gel column, eluted by a dichloromethane-methanol system, each flow part is detected by a thin layer chromatography, the same flow parts are combined, and the solvent is recovered to obtain flow parts Fr.4-5-1 to Fr.4-5-12; Fr.4-5-3-Fr.4-5-6 and Fr.4-5-7-Fr.4-5-10, respectively, and separating with preparative HPLC, methanol-water system and acetonitrile-water system repeatedly to obtain compounds 1 and 2 and compounds 3 and 4; fr.6 and methanol are dissolved according to the ratio of 1:1 (g/mL), the mixture is put on a polyamide column, the gradient elution of a methanol-water system is carried out, the flow parts are detected by a thin layer chromatography, the same flow parts are combined, and the flow parts Fr.6-1-Fr.6-12 are obtained; Fr.6-2-Fr.6-4 and Fr.6-6-Fr.6-9 are respectively combined and subjected to preparative HPLC, repeated separation in a methanol-water system and an acetonitrile-water system to obtain a compound 5 and a compound 6-11;
in another embodiment of the present invention, in the method for separating an ethyl acetate fraction, the separation conditions of silica gel column chromatography used are as follows: silica gel for sample mixing is 100-200 meshes, the first separation silica gel is 100-200 meshes, and the rest is 200-300 meshes; the silica gel column elution solvent gradient was a dichloromethane-methanol system (100:1,50:1,20:1,10:1,4:1,1:1,0: 1); a developing agent for thin-layer detection is dichloromethane-methanol (20: 1-4: 1); the color developing agent is 5% sulfuric acid-ethanol solution and 1% ferric trichloride solution;
in another embodiment of the present invention, in the method for separating an ethyl acetate fraction, the separation conditions of the gel column used are: the separation filler is Sephadex LH-20, and the elution solvent is a dichloromethane-methanol system (2:1, 1:1,0: 1); a developing agent for thin layer detection is dichloromethane-methanol (20: 1-4: 1); the color developing agent is 5% sulfuric acid-ethanol solution and 1% ferric trichloride solution;
in another embodiment of the present invention, in the method for separating an ethyl acetate fraction, the separation conditions of the polyamide column used are: the elution solvent is a methanol-water system (0:1,1:9,3:7,1:1,7:3,9:1,1: 0); developing agent for thin layer detection is dichloromethane-methanol-water (65:35:10, lower layer) and n-butanol-acetic acid-water (4:5:1, upper layer); the color developing agent is 5% sulfuric acid-ethanol solution and 1% ferric trichloride solution.
In another embodiment of the present invention, in the method for separating an ethyl acetate fraction, the preparative HPLC used separation conditions are: the separation chromatographic column is a reversed phase C18 column; the separation solvent is a methanol-water system (20: 80-85: 15, v/v) and an acetonitrile-water system (15: 85-85: 15).
By the separation method, 11 active compounds are obtained in total, and the method comprises the following steps: 2-trans-4-trans-abscisic acid (1), (2E,4E) -8-hydroxy-2,7-dimethyl-decadien- (2,4) -distortion- (1,10) -dioic acid (2), quercetin (3), kaempferol (4), quercetin-3-O-beta-D-glucopyranosyl- (1 → 2) -beta-D-galactopyranoside (5), rutin (6), quercetin-3-O-beta-D-galactopyranoside (7), isoquercitrin (8), quercetin-3-O-beta-D-xyloside (9), quercetin-3-O-alpha-L-rhamnoside (10), Kaempferol-3-O-alpha-L-rhamnoside (11). The specific structural formulas of the 11 compounds are shown in figure 7.
The structural identification of the compound mainly comprises the following steps: ultraviolet (UV) and infrared (I) spectraR), Mass Spectrometry (MS), Nuclear magnetic resonance Hydrogen Spectrometry (M: (M)1H NMR and nuclear magnetic resonance carbon Spectroscopy: (13C NMR)。
In another embodiment of the present invention, the application of the above separation, preparation and screening methods in the comprehensive utilization of cynanchum bunaei flower is provided.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1: preparation of various extraction parts of bunge auriculate flower
Pulverizing 4kg of radix Cynanchi auriculati flower into fine powder, cold-soaking and extracting with 90% ethanol for 2 times, each time for 15 days and 10 days, cold-soaking and extracting with 80% ethanol for 1 time and 8 days, with the ethanol dosage being 32L, 24L and 24L respectively, filtering the medicinal liquid, mixing, concentrating under reduced pressure, and freeze-drying to obtain 1.2kg of extract. Adding 1200mL of water into the extract to prepare suspension, sequentially extracting with petroleum ether (60-90 ℃), dichloromethane, ethyl acetate and n-butanol with the same volume for 4-8 times, concentrating and drying the extract liquor of each part under reduced pressure, and freeze-drying to obtain a petroleum ether part (77.9g), a dichloromethane part (35.0g), an ethyl acetate part (36.5g), an n-butanol part (226.4g) and an extraction mother liquor part (728.0 g).
The antioxidant activity of each extraction part is measured by adopting a DPPH method, and the method comprises the following specific steps: 10mg of extracts of different-polarity extraction parts of the cynanchum bungei flowers are respectively taken, anhydrous ethanol is used for preparing test solution with the mass concentration of 0.1, 0.5, 1.0, 1.5 and 2.0mg/mL, and water is used for preparing vitamin C solution with the mass concentration of 0.02, 0.04, 0.06, 0.08 and 0.1 mg/mL. Respectively placing 2mL of each sample solution in 10mL test tubes, adding 2mL of sufficiently dissolved DPPH ethanol solution (0.1mmol/L) in portions, adding plugs, shaking, shielding and shading tin foil paper, shading at room temperature for 30min, placing in a 1cm cuvette, placing in a spectrophotometer, measuring absorbance at 517nm, and measuring the absorbance of the sample solution to be AiPurified water was used as a blank control group, and the absorbance was A0Absolute ethyl alcohol is used as a background group to replace DPPH ethanol solution, and the absorbance is Aj. Calculating Vc with different mass concentrations and the clearance rate of the sample solution to DPPH free radicals according to formulas (1) and (2)And obtaining IC by SPSS statistical analysis50。
The calculation formula of the DPPH free radical clearance rate of each extraction part is as follows:
vc on DPPH free radical clearance (%) (1-A)Vc/Ai)×100％ (1)；
The sample has DPPH free radical scavenging rate (%) [ A ]0-(Ai-Aj)]/A0×100％ (2)
The method for measuring the antioxidant activity of each extraction part by adopting an ABTS method comprises the following specific steps: respectively taking 10mg of extracts of different-polarity extraction parts of the bunge auriculate flower, preparing 0.05, 0.1, 0.15, 0.2 and 0.25mg/mL of the extracts by using absolute ethyl alcohol, and preparing 0.02, 0.04, 0.06, 0.08 and 0.1mg/mL of vitamin C solution by using water. And (3) placing the ABTS reagent in the kit in a test tube, screwing a tube cover, shaking up, and standing at room temperature for 12-16h to obtain an ABTS concentrated solution. Mixing the ABTS solution (7.4mmol/L) and the potassium persulfate solution (2.6mmol/L) in a volume ratio of 1:1, standing at room temperature in a dark place for 12h to obtain an ABTS mother liquor, and diluting the mother liquor by 45 times by using purified water to obtain the ABTS free radical working solution. Precisely measuring 2mL of ABTS free radical working solution and 1mL of each sample solution, fully shaking up, keeping out of the sun for 30min at room temperature, and then measuring absorbance at 734 nm. Measuring the absorbance of the test solution as AiPurified water was used as a blank control group, and the absorbance was A0ABTS working solution is used as a background group, and the absorbance is Aj. Calculating the clearance rate of the sample solutions with different mass concentrations to DPPH free radicals according to the formula (3) and obtaining IC through SPSS statistical analysis50。
Researches find that the anti-oxidation activity of the bunge auriculate flower is an ethyl acetate part and an n-butyl alcohol part.
The formula for calculating the ABTS free radical clearance rate of each extraction part is as follows:
ABTS free radical scavenging rate (%) - (A)0-(Ai-Aj)]/A0×100％ (3)
Selecting RAW246.7 cell as target cell, and screening active extraction site with anti-inflammatory effect by determining influence of each extraction site on target cell release cytokine and NO. The method for culturing RAW246.7 cells is as follows: (1) cell recoveryThreo: taking out the frozen cells, quickly dissolving at 37 ℃, taking 15mL of centrifuge tube, adding 3mL of serum-containing culture medium, adding the liquid in the frozen tube into the centrifuge tube containing the culture medium, uniformly mixing by light blowing, centrifuging at the normal temperature of 1000-1500 r/min for 5-10 min, removing the supernatant, adding 3-5 mL of serum-containing culture medium, uniformly mixing by light blowing to obtain cell suspension, and adding the cell suspension into a culture bottle. (2) Culturing: the cells are placed in a culture bottle containing complete culture medium to grow in an adherent way under the culture conditions of 38 ℃ and 5 percent CO2When the cells grow over 80% of the culture flask, the cells can be passed. (3) Passage: discarding the culture solution in the culture bottle, adding 1-3 mL of trypsin, incubating and digesting for 2-5 min at 37 ℃, if cytoplasm retracts and intercellular space increases, immediately adding the culture solution containing the same volume of serum, and stopping digestion. And (3) repeatedly and gently blowing the cells at the bottom of the bottle in sequence to separate the adherent cells from the bottom of the bottle to form cell suspension. Inoculating the strain into a new culture bottle according to the first transfer and the third transfer, and putting the new culture bottle into an incubator for culture.
The MTT method is adopted to determine the safe concentration of each extraction part on RAW246.7 cells, and the specific method is as follows: RAW246.7 cells were adjusted to a concentration of 1.0X 106One/ml, added to a 96 well cell culture plate at 100. mu.L per well. Culturing for 12 hr, discarding culture solution, adding the extract of flos Cynanchi auriculati diluted with complete culture medium into cell culture plate at a concentration of five times, respectively, 200 μ L per well, repeating for 4 wells at each concentration, setting cell blank group (adding complete culture medium only), DMSO control group (containing complete culture medium of 0.1% DMSO), and placing at 37 deg.C and 5% CO2Culturing in an incubator. Culturing for 36h, adding MTT30 μ L into each well, culturing for 4h, discarding supernatant, adding 150 μ L of MSO into each well, shaking and dissolving on a micro-oscillator, measuring light absorption value at 570nm with enzyme linked immunosorbent assay, and selecting drug group A under the condition that DMSO control group and blank group have no significant difference570The concentration that did not significantly differ from the blank group was taken as the safe concentration of the bunge auriculate flower extract.
The content of the cytokine released by RAW246.7 is measured by ELISA method, which comprises the following steps: (1) the kit is kept at the constant temperature to room temperature, and the standard substance and the working solution are prepared in advance. (2) Setting blank holes, standard holes and sample holes, wherein only the sample diluent is added as the blank holes, the sample, the standard product or the sample diluent is respectively added into each 100 mu L/hole, 3 holes are set in each group, the enzyme label plate is sealed by a sealing plate film, and incubation is carried out for 90min at 37 ℃. (3) And (3) removing liquid in the holes after incubation is finished, spin-drying, adding 100 mu L of antibody working solution into each hole, adding a film-covered sealing plate, and incubating for 60min at 37 ℃. (4) After incubation, removing liquid in the holes, adding the stock solution of the washing buffer solution with the same volume, soaking for 1min, removing waste liquid, repeatedly washing the plate for 3 times, and then spin-drying. (5) Adding 100 mu L/hole of ABC working solution, adding a sealing film, and incubating for 30min at 37 ℃. (6) After incubation, removing the liquid in the pores, adding the stock solution of the washing buffer solution with the same volume, soaking for 1min, removing the waste liquid, washing the plate for 3 times repeatedly, and spin-drying. (7) Adding 90. mu.L of TMB color development liquid/hole, sealing with a sealing plate film, keeping out of the light, and reacting for 20min at 37 ℃. (8) After the reaction, 100. mu.L of TMB stop solution was added to each well, and the absorbance was measured at 450 nm.
The method for measuring the NO content (Griess method) of the anti-inflammatory experiment comprises the following steps: (1) cell inoculation: cultured RAW264.7 cells were adjusted to a cell concentration of 2.0X 105one/mL, 100. mu.L/well in 96-well plates, and suspension cells were removed after 15h of culture. Blank groups were dosed with 100. mu.L per well; LPS group LPS was added to the culture medium to give a final concentration of 10. mu.g/ml-1(ii) a The drug group is prepared by adding LPS into the drug, the LPS is added after 3h of treatment, the drug is added for co-intervention for 36h, and each group is repeatedly subjected to 5-hole treatment. (2) Drawing a standard curve: preheating the microplate reader for more than 30min, and adjusting the wavelength to 550 nm. And placing the reagent I on ice for later use. Taking out the NO content detection kit and keeping the temperature constant to room temperature. The standards were diluted with distilled water to 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125. mu. mol/mL. Adding a Griess reagent I into a standard substance, carrying out vortex mixing, carrying out water bath at 37 ℃ for 60min, adding a reagent II into a sample, carrying out vortex mixing, standing at room temperature for 5min, centrifuging at 3500rpm for 10min, taking a supernatant, adding an isovolumetric developing solution into the supernatant, carrying out vortex mixing, standing at room temperature for 10min, and measuring absorbance at 550 nm. Calculating Delta AMeasurement of＝AMeasurement of-ABlank space。
The data processing method of the anti-inflammatory experiment comprises the following steps: data are expressed as Means ± SD and data analysis is performed using GraphPad prism 8.0.1 software. ". indicates the degree of significance of the difference.
The results show that the anti-oxidation active parts of the bunge auriculate flower are ethyl acetate parts and n-butyl alcohol parts, and the most active part is the ethyl acetate part. Taken together, the chemical composition of the ethyl acetate sites was further investigated.
The method for determining the structure of the active site by using the UPLC-MS method comprises the following steps: (1) preparation of a sample: 0.2g of each extraction site is weighed into a 50mL centrifuge tube, 20mL of 70% methanol is added, and ultrasonic treatment is carried out for 2h, 2X 104g, centrifuging for 10min, taking the supernatant, transferring the supernatant into a volumetric flask, metering the volume of 70% methanol to a scale, and injecting a sample. The UPLC-MS method uses the following instruments and separation chromatographic columns: the liquid phase system comprises the following instruments: waters ACQUITY UPLC system (Waters, USA); the types of chromatographic columns are: ACQUITY BEH C18column (1.7 μm, 2.1X 100mm, Waters, USA); column temperature: 35 ℃ is carried out. The separation conditions used in the above UPLC-MS method were: mobile phase: a: 0.1% formic acid water; b: acetonitrile (0.1% formic acid); flow rate: 0.3 ml/min; mobile phase gradient setting: 0-2 min, 5% -5% of B; 2-22 min, 5% -95% of B; 22-26 min, 95% -95% B; 26-26.1 min, 95% -5% of B; 26.1-30 min, 5% -5% of B. The mass spectrum equipment conditions used by the UPLC-MS method are as follows: mass spectrometer: waters Xevo G2-XS QT of systems (Waters, USA); an ion source: ESI (electrophoresis ion source); the mass spectrometry method comprises the following steps: spraying voltage: 3 KV; source offset: 80; ion source temperature: 115 ℃ of carbon dioxide; desolvation temperature: at 450 ℃; taper hole airflow: 50L/h; desolventizing the gas stream: 600L/h; the data acquisition mode comprises the following steps: MSe continuous; positive ions (negative ions); resolution mode data acquisition molecular weight range: 50-1200 Da; scanning speed: 1.5 s; gradient collision voltage: 20-40V.
The positive ion diagram result shows that the ethyl acetate part of the bunge auriculate root comprises the following main components: the table for positive ion mode detection is as follows:
the anion result shows that the ethyl acetate part of the bunge auriculate root comprises the following main components: the table for negative ion pattern detection is as follows:
the method for separating the ethyl acetate part comprises the following steps: dissolving the ethyl acetate part with methanol according to a ratio of 1:1 (g/mL), mixing with silica gel according to a ratio of 1:1(g/g), loading on a silica gel column, carrying out gradient elution by a dichloromethane-methanol system, detecting each flow by a thin layer chromatography, combining the same flow, and recovering the solvent to obtain Fr.1-Fr.8. Fr.4 and methanol are dissolved according to the ratio of 1:1 (g/mL), the mixture is mixed with silica gel according to the ratio of 1:1(g/g), the mixture is loaded on a silica gel column, the dichloromethane-methanol system is used for gradient elution, each flow part is detected by a thin layer chromatography, and the same flow parts are combined to obtain flow parts Fr.4-1-Fr.4-6; fr.4-5 is put on a gel column, eluted by a dichloromethane-methanol system, and each flow part is detected by thin layer chromatography, the same flow parts are combined, and the solvent is recovered to obtain flow parts Fr.4-5-1 to Fr.4-5-12. Fr.4-5-3-Fr.4-5-6 and Fr.4-5-7-Fr.4-5-10, and separating with preparative HPLC, methanol-water system and acetonitrile-water system to obtain compounds 1 and 2 and compounds 3 and 4. Fr.6 and methanol are dissolved according to the ratio of 1:1 (g/mL), the mixture is put on a polyamide column, the gradient elution is carried out on a methanol-water system, the fractions are detected by a thin layer chromatography, the same fractions are combined, and the fractions Fr.6-1 to Fr.6-12 are obtained; Fr.6-2-Fr.6-4 and Fr.6-6-Fr.6-9 are respectively combined and repeatedly separated by preparative HPLC, a methanol-water system and an acetonitrile-water system to obtain a compound 5 and a compound 6-11.
By passing1H NMR and13the structures of the compounds 1-11 are determined by the spectrum methods such as C NMR and the like and by comparison with literature reports as follows: 2-trans-4-trans-abscisic acid (1), (2E,4E) -8-hydroxy-2,7-dimethyl-decadien- (2,4) -distortion- (1,10) -dioic acid (2), quercetin (3), kaempferol (4), quercetin-3-O-beta-D-glucopyranosyl- (1 → 2) -beta-D-galactopyranoside (5), rutin (6), quercetin-3-O-beta-D-galactopyranoside (7), isoquercitrin (8), quercetin-3-O-beta-D-xyloside (9), quercetin-3-O-alpha-L-rhamnoside (10), Kaempferol-3-O-alpha-L-rhamnoside (11).
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. A method for separating, preparing and screening an antioxidant and anti-inflammatory active ingredient in cynanchum bungei flower is characterized by comprising the following steps of:
s1, pulverizing the flowers of bunge auriculate root, adding a solvent for soaking and extracting, recovering the solvent, concentrating and drying to obtain an extract;
s2, adding water into the extract for suspension, and sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol to obtain extracts of different parts;
s3, measuring the antioxidant and anti-inflammatory effects of the extracts of each part, and screening the ethyl acetate part with the best activity;
s4, determining the main compound structure of the ethyl acetate active site by using a UPLC-MS method;
and S5, separating the compound components at the ethyl acetate part, wherein the first separation adopts silica gel column chromatography, and the subsequent separation adopts gel column chromatography, polyamide column chromatography or preparative HPLC.
2. The method according to claim 1, wherein in step S1,
the solvent is lower alcohol (including methanol or ethanol) containing water 1-90% (volume fraction, v/v), or acetone containing water 1-90%, or water saturated ethyl acetate, water saturated dichloromethane, and water saturated n-butanol;
the soaking and extracting temperature is room temperature (0-30 ℃) or heating (30 ℃ to boiling of the solvent) under normal pressure;
the concentration condition is that the mixture is heated and boiled (20 ℃ to the boiling of the solvent) under the reduced pressure state (-0.1 to-0.09 MPa);
the drying is freeze drying or drying under reduced pressure.
3. The method of claim 2, wherein the freeze-drying conditions are a temperature of-80 to-20 ℃, a pressure of-0.1 to-0.09 MPa, and a drying time of 5 to 7 days; the reduced pressure drying condition is room temperature (-0.1 to-0.09 MPa) at room temperature (0-30 deg.C) or boiling under heating (30 deg.C to boiling of the solvent, preferably not more than 45 deg.C) in a reduced pressure state.
4. The method according to claim 1, wherein in step S2,
the extraction solvent comprises water saturated petroleum ether, water saturated dichloromethane, water saturated ethyl acetate and water saturated n-butyl alcohol in sequence, the extraction times are 5-7 times for each extraction solvent, and the extraction temperature is room temperature (0-30 ℃).
5. The method of claim 1, wherein the step S3, the specific method for measuring the antioxidant and anti-inflammatory effects of the extracts of different parts comprises measuring the antioxidant activity of the extracts of different parts of the flowers of cynanchum bungei by DPPH method and ABTS method; and screening active extraction sites with anti-inflammatory effect by using RAW246.7 cells as target cells and measuring the influence of each extraction site on the release of cytokines and NO by the target cells.
6. The method of claim 5, wherein determining the amount of RAW246.7 released cytokine comprises ELISA;
the content of NO released by RAW246.7 is measured by Griess method.
7. The method according to claim 1, wherein in step S4,
the separation conditions used in the UPLC-MS method were: mobile phase: a: 0.1% aqueous formic acid; b: acetonitrile (0.1% formic acid); flow rate: 0.3 ml/min; mobile phase gradient setting: 0-2 min, 5% -5% of B; 2-22 min, 5% -95% B; 22-26 min, 95% -95% B; 26-26.1 min, 95% -5% of B; 26.1-30 min, 5% -5% of B;
the mass spectrum equipment conditions used by the UPLC-MS method are as follows: an ion source: ESI; the mass spectrometry method comprises the following steps: spraying voltage: 3 KV; source offset: 80; ion source temperature: 115 ℃ of carbon dioxide; desolvation temperature: at 450 ℃; taper hole airflow: 50L/h; desolventizing the gas stream: 600L/h; the data acquisition mode comprises the following steps: MSe Continum; positive ions (negative ions); resolution mode data acquisition molecular weight range: 50-1200 Da; scanning speed: 1.5 s; gradient collision voltage: 20-40V.
8. The method according to claim 1, wherein in step S5,
the method for separating the ethyl acetate fraction comprises: subjecting ethyl acetate to silica gel column chromatography, gradient eluting with dichloromethane-methanol mixed solvent, developing thin layer, mixing the same eluates, subjecting to gel column chromatography or polyamide column chromatography, respectively eluting with dichloromethane-methanol mixed solvent, subjecting to reversed phase C18 column, repeatedly eluting with methanol-water system and acetonitrile-water system, and repeatedly separating with preparative high performance liquid chromatograph to obtain the final product;
preferably, the method for separating an ethyl acetate fraction comprises the following steps: dissolving the ethyl acetate part with methanol according to the ratio of 1:1 (g/mL), mixing with silica gel according to the ratio of 1:1(g/g), loading on a silica gel column, carrying out gradient elution by a dichloromethane-methanol system, detecting each flow by a thin layer chromatography, combining the same flow, and recovering the solvent to obtain Fr.1-Fr.8; fr.4 and methanol are dissolved according to the ratio of 1:1 (g/mL), the mixture is mixed with silica gel according to the ratio of 1:1(g/g), the mixture is loaded on a silica gel column, the dichloromethane-methanol system is used for gradient elution, each flow part is detected by a thin layer chromatography, and the same flow parts are combined to obtain flow parts Fr.4-1-Fr.4-6; fr.4-5 is put on a gel column, eluted by a dichloromethane-methanol system, each flow is detected by a thin layer chromatography, the same flow is combined, and the solvent is recovered to obtain the flow Fr.4-5-1 to Fr.4-5-12; Fr.4-5-3-Fr.4-5-6 and Fr.4-5-7-Fr.4-5-10, respectively, and separating with preparative HPLC, methanol-water system and acetonitrile-water system repeatedly to obtain compounds 1 and 2 and compounds 3 and 4; fr.6 and methanol are dissolved according to the ratio of 1:1 (g/mL), the mixture is put on a polyamide column, the gradient elution of a methanol-water system is carried out, the flow parts are detected by a thin layer chromatography, the same flow parts are combined, and the flow parts Fr.6-1-Fr.6-12 are obtained; Fr.6-2-Fr.6-4 and Fr.6-6-Fr.6-9 are respectively combined and subjected to preparative HPLC, repeated separation in a methanol-water system and an acetonitrile-water system to obtain a compound 5 and a compound 6-11;
further preferably, the separation conditions of the silica gel column chromatography are as follows: silica gel for sample mixing is 100-200 meshes, the first separation silica gel is 100-200 meshes, and the rest is 200-300 meshes; the silica gel column elution solvent gradient was a dichloromethane-methanol system (100:1,50:1,20:1,10:1,4:1,1:1,0: 1); a developing agent for thin-layer detection is dichloromethane-methanol (20: 1-4: 1); the color developing agent is 5% sulfuric acid-ethanol solution and 1% ferric trichloride solution;
further preferably, the separation conditions of the gel column used are: the separation filler is Sephadex LH-20, and the elution solvent is a dichloromethane-methanol system (2:1, 1:1,0: 1); a developing agent for thin-layer detection is dichloromethane-methanol (20: 1-4: 1); the color developing agent is 5% sulfuric acid-ethanol solution and 1% ferric trichloride solution;
further preferably, the separation conditions of the polyamide column used are: the elution solvent is a methanol-water system (0:1,1:9,3:7,1:1,7:3,9:1,1: 0); developing agent for thin layer detection is dichloromethane-methanol-water (65:35:10, lower layer) and n-butanol-acetic acid-water (4:5:1, upper layer); the color developing agent is 5% sulfuric acid-ethanol solution and 1% ferric trichloride solution;
further preferably, the preparative HPLC used has separation conditions of: the separation chromatographic column is a reversed phase C18 column; the separation solvent is a methanol-water system (20: 80-85: 15, v/v) and an acetonitrile-water system (15: 85-85: 15).
9. The method of claim 1, wherein obtaining 11 active compounds comprises: 2-trans-4-trans-abscisic acid (1), (2E,4E) -8-hydroxy-2,7-dimethyl-decadien- (2,4) -dissecure- (1,10) -dioic acid (2), quercetin (3), kaempferol (4), quercetin-3-O-beta-D-glucopyranose- (1 → 2) -beta-D-galactopyranoside (5), rutin (6), quercetin-3-O-beta-D-galactopyranoside (7), isoquercitrin (8), quercetin-3-O-beta-D-xyloside (9), quercetin-3-O-alpha-L-pyranoside (10), Kaempferol-3-O-alpha-L-rhamnoside (11).
10. The use of the method of any one of claims 1 to 9 for the comprehensive utilization of flowers of Cynanchum bungei.
Priority Applications (1)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115737664A (en) * | 2023-01-05 | 2023-03-07 | 中央民族大学 | Acer truncatum leaf extract with anti-inflammatory activity and preparation method thereof |
Citations (3)
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| JPS6426512A (en) * | 1987-07-20 | 1989-01-27 | Lion Corp | Composition for oral cavity application |
| CN104585573A (en) * | 2013-11-03 | 2015-05-06 | 郑方 | A pollen essence of polygoni multiflori flowers |
| CN107712210A (en) * | 2017-11-24 | 2018-02-23 | 卢世旺 | A kind of radix cynanchi bungei jasmine tea |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6426512A (en) * | 1987-07-20 | 1989-01-27 | Lion Corp | Composition for oral cavity application |
| CN104585573A (en) * | 2013-11-03 | 2015-05-06 | 郑方 | A pollen essence of polygoni multiflori flowers |
| CN107712210A (en) * | 2017-11-24 | 2018-02-23 | 卢世旺 | A kind of radix cynanchi bungei jasmine tea |
Non-Patent Citations (3)
| Title |
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| XIAOXUE DING, ET AL.: "Chemical constituents from the flowers of Cynanchum auriculatum Royle ex Wight" * |
| 刘泽鑫等: "白首乌花多糖对小鼠酒精性肝损伤的保护作用" * |
| 张明等: "白首乌的化学成分、药理作用及栽培技术综述" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115737664A (en) * | 2023-01-05 | 2023-03-07 | 中央民族大学 | Acer truncatum leaf extract with anti-inflammatory activity and preparation method thereof |
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