CN110776541A - Preparation method and application of quercetin-3-gentiobioside - Google Patents
Preparation method and application of quercetin-3-gentiobioside Download PDFInfo
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Abstract
The invention discloses a preparation method of quercetin-3-gentiobioside, which comprises a crushing step, a reflux extraction step, a reduced pressure concentration step, a centrifugation step, a macroporous resin enrichment step, a preparation liquid chromatography enrichment step and a drying step.
Description
Technical Field
The invention relates to a preparation method and application of quercetin-3-gentiobioside.
Background
RHIZOMA PARIDIS (RHIZOMA PARIDIS) is a traditional Chinese medicinal material, and its base source is rhizome of plant of Paris of Liliaceae (liliaceac). Rhizoma paridis is also known as rhizoma paridis, herba Schefflerae Arboricolae, herba Typhonii gigantei, herba Anoectochili Roxburghii, rhizoma Helminthostachydis Zeylanicae, etc. There are 24 types of paris polyphylla in the world, the paris polyphylla is mainly distributed in tropical and temperate regions in the continental asia europe, and there are 19 types of paris polyphylla in China, and the paris polyphylla is mainly distributed in provinces or autonomous regions such as Yunnan, Guangxi, Sichuan, Guizhou, Shaanxi and the like.
The history of the drug use of Paris polyphylla is long, and the Paris polyphylla is recorded in the Shen nong's herbal Jing for the first time, and the paridis is the best choice in the Ben Cao gang mu of Li Shizhen; recorded in Tang Ben Cao (Tang materia Medica) as Paris rhizome. The 2010 version of the pharmacopoeia collects Parispolyphylla Smith var. yunnanensis (Franch.) hand-Mazz and Parispolyphylla Smith var. chinensis (Franch.) Hara as the basic source plants of the Chinese medicine Parispolyphylla.
The pharmacological activity research of modern pharmacology finds that the medicinal material has the effects of stopping bleeding, eliminating phlegm, inhibiting bacteria, relieving pain, calming, resisting cell toxicity and the like, and has more remarkable effects when being clinically applied to treating functional uterine bleeding, neurodermatitis, surgical inflammation and the like.
The extraction is carried out by taking the rhizoma paridis root as the raw material, which is common in the past researches, for example, the research of the rhizoma paridis is reviewed by Zhao Bao Sheng et al in the research progress of the traditional Chinese medicine rhizoma paridis. However, the rhizome of the paris polyphylla can be used as a medicine in 5 to 7 years, and with the increase of the demand of the paris polyphylla extract, the resource of the wild paris polyphylla is exhausted at present, and the search of the paris polyphylla root substitute is more and more important.
The rhizoma paridis shell is dry capsule shell of rhizoma paridis of Paris of Liliaceae. When a paris polyphylla grower collects paris polyphylla seeds, the mature capsules are picked, the seeds in the capsules are taken out, and the shells are discarded, so that paris polyphylla shells are single paris polyphylla plant parts which are easy to obtain. The invention can obtain the extract rich in the paris polyphylla total saponin and the total flavone after extracting and purifying the paris polyphylla fruit shell, and the quality of the batch is stable because a single part is used for extraction.
In the previous literature reports, the separation of quercetin-3-gentiobioside from the paris plant is not found, and the compound has certain antioxidant activity and can be used as an internal control substance for quality control of paris polyphylla shell extract.
Therefore, the method for separating the quercetin-3-gentiobioside, which is simple in process, is developed and the biological activity of the quercetin-3-gentiobioside is verified, so that the significance is great.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of quercetin-3-gentiobioside, which is used for separating and preparing the quercetin-3-gentiobioside from paris plants for the first time, wherein the purity is more than 99.0 percent.
The quercetin-3-gentiobioside obtained by the preparation method has stable quality, can be used as an antioxidant substance to be added into products for skin care, beauty treatment and the like, and can also be used as a reference substance to carry out quality control on a paris polyphylla shell extract.
One technical scheme for achieving the purpose is as follows: a preparation method of quercetin-3-gentiobioside comprises the following steps:
s1, a crushing step: crushing dried paris polyphylla husks to obtain paris polyphylla husk fine powder, wherein particles with the particle size of at least 95 percent of paris polyphylla husk fine powder pass through a 80-mesh sieve;
s2, reflux extraction step: refluxing and extracting the paris polyphylla shell fine powder by adopting an alcohol/water mixed solution to obtain an extracting solution, wherein the mass content of alcohol in the alcohol/water mixed solution is 70-80%, and the using weight of the alcohol/water mixed solution is 5-10 times of that of the paris polyphylla shell fine powder; the temperature of reflux extraction is 75-90 ℃;
s3, vacuum concentration step: concentrating the extracting solution at 45-65 ℃ under reduced pressure, and removing alcohol in the extracting solution to obtain a concentrated solution;
s4, a centrifugation step: centrifuging the concentrated solution at the rotating speed of 3000-16000 rpm, and taking out a supernatant;
s5, macroporous resin enrichment: adsorbing the supernatant by using macroporous resin, eluting by using an eluant to obtain an eluent, and concentrating the eluent to obtain a paris polyphylla shell extracting solution;
s6, preparation of liquid chromatography enrichment step: subjecting the rhizoma paridis fruit shell extractive solution to gradient elution enrichment by preparative liquid chromatography, wherein the preparative liquid chromatography column is Xbridge Prep; the inner diameter of the chromatographic column is 19mm, the length of the chromatographic column is 150mm, the filler of the chromatographic column is OBD C18, the particle size of the filler is 5 microns, and the column temperature of the chromatographic column is 25 ℃; the detection wavelength is 349nm, and the elution gradient of the mobile phase is shown in the table 1;
table 1. mobile phase elution gradient, where mobile phase a is water and mobile phase B is acetonitrile:
| time of day | Mobile phase A% | Mobile phase B% | Flow rate of flow |
| 0min | 82 | 18 | 10mL/min |
| 7min | 82 | 18 | 10mL/ |
| 9min | |||
| 0 | 100 | 10mL/ | |
| 12min | |||
| 0 | 100 | 10mL/min | |
| 13min | 82 | 18 | 10mL/min |
| 19min | 82 | 18 | 10mL/min |
S7, drying: and combining the mobile phases subjected to gradient elution and enrichment, and then concentrating and drying under reduced pressure to obtain the quercetin-3-gentiobioside.
In the above preparation method of quercetin-3-gentiobioside, in the pulverizing step, the rhizoma paridis is Yunnan rhizoma paridis, or rhizoma paridis, which are plants of Paris genus of Liliaceae family; the rhizoma paridis dry shell is the dry capsule shell of the rhizoma paridis capsule after seeds are removed.
In the step of reflux extraction, alcohol in the alcohol/water mixed solution is ethanol, and the alcohol/water mixed solution is adopted to carry out reflux extraction on the fine powder of the paris polyphylla husks for 1-3 times, wherein the time of each reflux extraction is 1-3 hours.
In the step of concentrating under reduced pressure, the concentration degree of the concentrated solution is equal to 0.5 to 1.5 grams of the fine powder of the paris polyphylla husks per milliliter of the concentrated solution.
In the centrifugation step, the concentrated solution is centrifuged at the rotating speed of 3000-16000 r/min, the centrifugal precipitate after the supernatant is taken out is redissolved by water, the redissolved solution is centrifuged again at the rotating speed of 3000-16000 r/min, the supernatant is taken out, the supernatant after the concentrate is centrifuged and the supernatant after the redissolved solution is centrifuged are combined, and the macroporous resin enrichment step is carried out.
In the step of enriching the macroporous resin, the macroporous resin is AB-8 type macroporous resin or D101 type macroporous resin, and the weight ratio of the macroporous resin to the paris polyphylla shell fine powder is 1: 1-1: 2; the eluent is prepared by mixing ethanol and water into 20% ethanol eluent, 40% ethanol eluent and 80% ethanol eluent.
In the above method for preparing quercetin-3-gentiobioside, the macroporous resin enrichment step specifically comprises the following steps:
s51, carrying out sample loading adsorption on the supernatant by adopting macroporous resin, wherein the sample loading speed is 0.5 column volume/hour, and standing for 2-12 hours after the adsorption of the supernatant is finished;
s52, eluting by water for 4-10 column volumes;
s53, eluting by using a 20% ethanol eluent for 4-8 column volumes;
s54, eluting 4-8 column volumes by using 40% ethanol eluent, and collecting eluent;
s55, eluting 4-8 column volumes by using an 80% ethanol eluent, and collecting eluent;
and S56, merging the eluates obtained in the steps S54 and S55, and concentrating to obtain the paris polyphylla shell extracting solution, wherein the concentration degree of the paris polyphylla shell extracting solution is 0.5-1.5 times of the total weight of the paris polyphylla shell extracting solution and the total weight of the paris polyphylla shell fine powder.
In the method for preparing quercetin-3-gentiobioside, the purity of quercetin-3-gentiobioside in the quercetin-3-gentiobioside prepared by the method for preparing quercetin-3-gentiobioside is more than 99.0 percent; IC50 was less than 0.05mg/mL for DPPH clearance.
The invention also provides an application of the quercetin-3-gentiobioside, the quercetin-3-gentiobioside is prepared by adopting the preparation method of the quercetin-3-gentiobioside, and the quercetin-3-gentiobioside is added into a formula of a skin care product as an antioxidant active ingredient.
The invention also provides the application of another quercetin-3-gentiobioside, the quercetin-3-gentiobioside is prepared by adopting the preparation method of the quercetin-3-gentiobioside, and the quercetin-3-gentiobioside is used as a reference substance for carrying out quality control on the extract of the Chinese paris fruit shell.
By adopting the technical scheme of the preparation method of the quercetin-3-gentiobioside, the positive progress effects are as follows:
(1) according to the invention, the paris polyphylla husks are independently used as raw materials for the first time, waste materials are changed into valuable materials, and enrichment and purification are carried out after the paris polyphylla husks are extracted to obtain the active monomer quercetin-3-gentiobioside;
(2) the invention separates the compound quercetin-3-gentiobioside from the paris plant for the first time;
(3) the quercetin-3-gentiobioside obtained by the preparation method has stronger antioxidant activity, can be used as an active substance for skin care products and medical auxiliary products, and can be used as an active marker for carrying out quality control on the extract of the Chinese paris rhizome.
Drawings
FIG. 1 is a flow chart of the method of preparing quercetin-3-gentiobioside according to the present invention;
FIG. 2 is a chromatogram of a preparative liquid chromatography enrichment step in the preparation method of the present invention;
FIG. 3 is an HPLC chromatogram of quercetin-3-gentiobioside prepared by the preparation method of the present invention;
FIG. 4 is a high resolution mass spectrum of quercetin-3-gentiobioside prepared by the preparation method of the present invention;
FIG. 5 is a nuclear magnetic hydrogen spectrum of quercetin-3-gentiobioside prepared by the preparation method of the present invention;
FIG. 6 is a nuclear magnetic carbon spectrum of quercetin-3-gentiobioside prepared by the preparation method of the present invention;
FIG. 7 is a graph showing the DPPH radical scavenging effect of quercetin-3-gentiobioside prepared by the preparation method of the present invention.
Detailed Description
In order that those skilled in the art will better understand the technical solution of the present invention, the following detailed description is given with reference to the accompanying drawings:
referring to the implementation of FIG. 1, the solvent formulations in the following examples are mass ratios, and the reagents and starting materials used are commercially available. Rhizoma paridis is selected from rhizoma paridis Yunnanensis, rhizoma paridis, or rhizoma paridis of Paris of Liliaceae; the rhizoma paridis dry shell is the dry capsule shell of rhizoma paridis capsule after seeds are removed. The eluent is mixed by ethanol and water, and the mass content of the ethanol eluent is 20 percent of ethanol eluent, 40 percent of ethanol eluent and 80 percent of ethanol eluent.
Example 1:
a preparation method of quercetin-3-gentiobioside comprises the following steps:
s1, a crushing step: pulverizing 200g of rhizoma paridis dry shell to obtain 80-mesh rhizoma paridis shell fine powder;
s2, reflux extraction step: filling the fine powder of the paris polyphylla husks into an extraction tank, performing 3 times of reflux extraction by adopting 70% ethanol/water mixed solution to obtain an extracting solution, wherein the dosage of the ethanol/water mixed solution is 1Kg, the reflux extraction temperature is 75-90 ℃, the reflux extraction time of each time is 2 hours, and combining the extracting solutions obtained by the 3 times of reflux extraction;
s3, vacuum concentration step: concentrating the extractive solution at 60 deg.C under reduced pressure, and removing alcohol to obtain 100g concentrated solution;
s4, a centrifugation step: centrifuging the concentrated solution at the rotating speed of 3000 r/min, taking out supernatant, re-dissolving the centrifugal precipitate after taking out the supernatant with 100g of water (the addition amount of the water is the weight of the concentrated solution), centrifuging the re-dissolved solution at the rotating speed of 3000 r/min again, taking out the supernatant, and combining the supernatant obtained after centrifuging the concentrated solution and the supernatant obtained after centrifuging the re-dissolved solution;
s5, a macroporous resin enrichment step, which specifically comprises the following steps:
s51, loading 220g of AB-8 type macroporous resin into a column, eluting with clear water until no alcohol smell exists, loading the supernatant by using the macroporous resin for adsorption, wherein the loading speed is 0.5 column volume/hour, and standing for 3 hours after the supernatant is completely adsorbed;
s52, eluting 5 column volumes with water;
s53, eluting 5 column volumes with 20% ethanol eluent;
s54, eluting 5 column volumes by using 40% ethanol eluent, and collecting eluent;
s55, eluting 5 column volumes by using 80% ethanol eluent, and collecting eluent;
s56, mixing the eluates obtained in the steps S54 and S55, concentrating and drying to obtain 200g of paris polyphylla fruit shell extract;
s6, preparation of liquid chromatography enrichment step: referring to fig. 2, the paris polyphylla shell extract is subjected to gradient elution enrichment by adopting preparative liquid chromatography, wherein a chromatographic column of the preparative liquid chromatography is XBridge Prep; the inner diameter of the chromatographic column is 19mm, the length of the chromatographic column is 150mm, the filler of the chromatographic column is OBD C18, the particle size of the filler is 5 microns, and the column temperature of the chromatographic column is 25 ℃; the detection wavelength is 349nm, and the elution gradient of the mobile phase is shown in table 1, wherein the mobile phase A is water, and the mobile phase B is acetonitrile:
| time of day | Mobile phase A% | Mobile phase B% | Flow rate of flow |
| 0min | 82 | 18 | 10mL/min |
| 7min | 82 | 18 | 10mL/ |
| 9min | |||
| 0 | 100 | 10mL/ | |
| 12min | |||
| 0 | 100 | 10mL/min | |
| 13min | 82 | 18 | 10mL/min |
| 19min | 82 | 18 | 10mL/min |
Table 1S7, drying step: mixing the mobile phases after gradient elution and enrichment, concentrating under reduced pressure, and drying to obtain quercetin-3-gentiobioside, with reference to FIG. 3, with purity of 99.6% by HPLC.
Example 2:
a preparation method of quercetin-3-gentiobioside comprises the following steps:
s1, a crushing step: pulverizing 205g of rhizoma paridis dry fruit shell to obtain 80-mesh rhizoma paridis fruit shell fine powder;
s2, reflux extraction step: filling the fine powder of the paris polyphylla husks into an extraction tank, performing reflux extraction for 3 times by adopting 1.6Kg of 80% ethanol/water mixed solution to obtain an extracting solution, wherein the reflux extraction temperature is 75-90 ℃, the reflux extraction time of each reflux extraction is 2 hours, and combining the extracting solutions obtained by the reflux extraction for 3 times;
s3, vacuum concentration step: concentrating the extractive solution at 60 deg.C under reduced pressure, and removing alcohol to obtain 120g concentrated solution;
s4, a centrifugation step: centrifuging the concentrated solution at the rotating speed of 3000 r/min, taking out supernatant, re-dissolving the centrifugal precipitate after taking out the supernatant with 120g of water (the addition amount of the water is the weight of the concentrated solution), centrifuging the re-dissolved solution at the rotating speed of 3000 r/min again, taking out the supernatant, and combining the supernatant obtained after centrifuging the concentrated solution and the supernatant obtained after centrifuging the re-dissolved solution;
s5, a macroporous resin enrichment step, which specifically comprises the following steps:
s51, loading 240g of D101 type macroporous resin into a column, eluting with clear water until no alcohol smell exists, loading the supernatant by using the macroporous resin for adsorption, wherein the loading speed is 0.5 column volume/hour, and standing for 8 hours after the supernatant is completely adsorbed;
s52, eluting 5 column volumes with water;
s53, eluting 5 column volumes with 20% ethanol eluent;
s54, eluting 6 column volumes by using 40% ethanol eluent, and collecting eluent;
s55, eluting 8 column volumes by using 80% ethanol eluent, and collecting eluent;
s56, mixing the eluates obtained in the steps S54 and S55, concentrating and drying to obtain 210g of paris polyphylla fruit shell extract;
s6, preparation of liquid chromatography enrichment step: referring to fig. 2, the paris polyphylla shell extract is subjected to gradient elution enrichment by adopting preparative liquid chromatography, wherein a chromatographic column of the preparative liquid chromatography is XBridge Prep; the inner diameter of the chromatographic column is 19mm, the length of the chromatographic column is 150mm, the filler of the chromatographic column is OBD C18, the particle size of the filler is 5 microns, and the column temperature of the chromatographic column is 25 ℃; the detection wavelength is 349nm, and the elution gradient of the mobile phase is shown in table 1, wherein the mobile phase A is water, and the mobile phase B is acetonitrile;
s7, drying: mixing the mobile phases after gradient elution and enrichment, concentrating under reduced pressure, and drying to obtain quercetin-3-gentiobioside with purity of 99.3% by HPLC.
Example 3:
a preparation method of quercetin-3-gentiobioside comprises the following steps:
s1, a crushing step: 197g of dried paris polyphylla husks are crushed to obtain 80-mesh paris polyphylla husk fine powder;
s2, reflux extraction step: filling the fine powder of the paris polyphylla husks into an extraction tank, performing 3 times of reflux extraction by adopting 80% ethanol/water mixed solution to obtain an extracting solution, wherein the dosage of the ethanol/water mixed solution is 1.6Kg, the reflux extraction temperature is 75-90 ℃, the reflux extraction time of each time is 2 hours, and combining the extracting solutions obtained by the 3 times of reflux extraction;
s3, vacuum concentration step: concentrating the extractive solution at 60 deg.C under reduced pressure, and removing alcohol to obtain 101g concentrated solution;
s4, a centrifugation step: centrifuging the concentrated solution at the rotating speed of 3000 r/min, taking out supernatant, re-dissolving the centrifugal precipitate after taking out the supernatant with 101g of water (the addition amount of the water is the weight of the concentrated solution), centrifuging the re-dissolved solution at the rotating speed of 3000 r/min again, taking out the supernatant, and combining the supernatant obtained after centrifuging the concentrated solution and the supernatant obtained after centrifuging the re-dissolved solution;
s5, a macroporous resin enrichment step, which specifically comprises the following steps:
s51, loading 225g of AB-8 type macroporous resin into a column, eluting with clear water until no alcohol smell exists, loading the supernatant by using the macroporous resin for adsorption, wherein the loading speed is 0.5 column volume/hour, and standing for 12 hours after the supernatant is completely adsorbed;
s52, eluting 5 column volumes with water;
s53, eluting 6 column volumes with 20% ethanol eluent;
s54, eluting 8 column volumes by using 40% ethanol eluent, and collecting eluent;
s55, eluting 8 column volumes by using 80% ethanol eluent, and collecting eluent;
s56, combining the eluates obtained in the steps S54 and S55, concentrating and drying to obtain 207g of paris polyphylla nutshell extracting solution;
s6, preparation of liquid chromatography enrichment step: referring to fig. 2, the paris polyphylla shell extract is subjected to gradient elution enrichment by adopting preparative liquid chromatography, wherein a chromatographic column of the preparative liquid chromatography is XBridge Prep; the inner diameter of the chromatographic column is 19mm, the length of the chromatographic column is 150mm, the filler of the chromatographic column is OBD C18, the particle size of the filler is 5 microns, and the column temperature of the chromatographic column is 25 ℃; the detection wavelength is 349nm, and the elution gradient of the mobile phase is shown in table 1, wherein the mobile phase A is water, and the mobile phase B is acetonitrile;
s7, drying: mixing the mobile phases after gradient elution and enrichment, concentrating under reduced pressure, and drying to obtain quercetin-3-gentiobioside, with reference to FIG. 3, with purity of 99.6% by HPLC.
Measuring the content of quercetin-3-gentiobioside in the paris polyphylla shell extract:
referring to fig. 3 to 6, the quercetin-3-gentiobioside prepared by the preparation method of the present invention has a purity of more than 99.0%, and can be used as a reference substance for quality control of the extract of the nut shell of the paris polyphylla.
Specifically, the quercetin-3-gentiobioside prepared in example 1 was used as a control, the paris polyphylla nutshell extract obtained in the macroporous resin enrichment step was further concentrated and dried to obtain a paris polyphylla nutshell extract, and the content of quercetin-3-gentiobioside in the paris polyphylla nutshell extract was measured.
Taking quercetin-3-gentiobioside prepared in example 1, and preparing into control solutions with the concentrations of 0.26400mg/mL, 0.13200mg/mL, 0.06600mg/mL, 0.03300mg/mL, 0.01650mg/mL and 0.00825 mg/mL; and preparing the paris polyphylla shell extract into a to-be-detected sample solution of 2.88 mg/mL. Respectively analyzing the reference substance and the sample to be detected by using an LC-MS method, determining quercetin-3-gentiobioside by using MS and retention time, and drawing a standard curve by using an integral area and the concentration of the quercetin-3-gentiobioside:
Y＝0.0003X+0.0002,R
2＝0.9997
wherein Y is concentration, X is integral area, R
2Is a data correlation coefficient.
Through calculation, the content of quercetin-3-gentiobioside in the paris polyphylla shell extract to be detected is 3.277%.
DPPH free radical scavenging effect test of quercetin-3-gentiobioside:
DPPH radical scavenging action of quercetin-3-gentiobioside in example 1 was measured with vitamin C as a control.
Preparing standard vitamin C solution with concentration of 0.414mg/mL, 0.207mg/mL, 0.103mg/mL, 0.052mg/mL, 0.026mg/mL, 0.013mg/mL, each 1mL for use; taking the quercetin-3-gentiobioside in the embodiment 1 to respectively prepare to-be-detected sample solutions with the concentration of 0.264mg/mL, and taking 1mL for later use; after DPPH is taken and prepared into 0.51mg/mL DPPH solution, 1mL DPPH solution is added into a sample to be detected and a control sample respectively, after reaction for 30 minutes, the absorbance is measured at the wavelength of 405nm, and a curve is drawn according to the absorbance and the concentration of the sample. The results of the DPPH radical scavenging assay are shown in FIG. 7.
The results show that under the conditions, vitamin C has 0.024mg/mL of DPPH free radical scavenging effect IC50, quercetin-3-gentiobioside in example 1 has 0.034mg/mL of DPPH free radical scavenging effect IC50, and quercetin-3-gentiobioside has excellent oxidation resistance and can be added into products for skin care, cosmetology and the like as an antioxidant substance.
In conclusion, the raw material for preparing the quercetin-3-gentiobioside is the waste fruit shell in the process of collecting the paris polyphylla seeds, the source of the raw material is stable, the effective components of the raw material are extracted and purified to obtain an extract with stable quality, and the quercetin-3-gentiobioside can be obtained by preparing liquid phase and purifying. The compound has strong oxidation resistance, can be used as an active ingredient of skin care products and medical auxiliary products, and has the effects of resisting oxidation, removing free radicals and the like; the quercetin-3-gentiobioside prepared by the preparation method has high purity, and can be used as a reference substance for controlling the quality of the paris polyphylla extract.
It should be understood by those skilled in the art that the above embodiments are only for illustrating the present invention and are not to be used as a limitation of the present invention, and that changes and modifications to the above described embodiments are within the scope of the claims of the present invention as long as they are within the spirit and scope of the present invention.
Claims (10)
1. A preparation method of quercetin-3-gentiobioside is characterized by comprising the following steps:
s1, a crushing step: crushing dried paris polyphylla husks to obtain paris polyphylla husk fine powder, wherein particles with the particle size of at least 95 percent of paris polyphylla husk fine powder pass through a 80-mesh sieve;
s2, reflux extraction step: refluxing and extracting the paris polyphylla shell fine powder by adopting an alcohol/water mixed solution to obtain an extracting solution, wherein the mass content of alcohol in the alcohol/water mixed solution is 70-80%, and the using weight of the alcohol/water mixed solution is 5-10 times of that of the paris polyphylla shell fine powder; the temperature of reflux extraction is 75-90 ℃;
s3, vacuum concentration step: concentrating the extracting solution at 45-65 ℃ under reduced pressure, and removing alcohol in the extracting solution to obtain a concentrated solution;
s4, a centrifugation step: centrifuging the concentrated solution at the rotating speed of 3000-16000 rpm, and taking out a supernatant;
s5, macroporous resin enrichment: adsorbing the supernatant by using macroporous resin, eluting by using an eluant to obtain an eluent, and concentrating the eluent to obtain a paris polyphylla shell extracting solution;
s6, preparation of liquid chromatography enrichment step: subjecting the rhizoma paridis fruit shell extractive solution to gradient elution enrichment by preparative liquid chromatography, wherein the preparative liquid chromatography column is Xbridge Prep; the inner diameter of the chromatographic column is 19mm, the length of the chromatographic column is 150mm, the filler of the chromatographic column is OBD C18, the particle size of the filler is 5 microns, and the column temperature of the chromatographic column is 25 ℃; the detection wavelength is 349nm, and the elution gradient of the mobile phase is shown in the table 1;
table 1. mobile phase elution gradient, where mobile phase a is water and mobile phase B is acetonitrile:
S7, drying: and combining the mobile phases subjected to gradient elution and enrichment, and then concentrating and drying under reduced pressure to obtain the quercetin-3-gentiobioside.
2. The method of claim 1, wherein in the step of pulverizing, the rhizoma paridis is selected from the group consisting of Yunnan rhizoma paridis, Hua rhizoma paridis, and Paris polyphylla Smith; the rhizoma paridis dry shell is the dry capsule shell of the rhizoma paridis capsule after seeds are removed.
3. The method of claim 1, wherein in the step of reflux extraction, ethanol is used as the alcohol in the alcohol/water mixture, and the alcohol/water mixture is used to carry out reflux extraction on the fine powder of the hull of the paris polyphylla for 1-3 times, wherein the time for each reflux extraction is 1-3 hours.
4. The method of claim 1, wherein in the step of concentrating under reduced pressure, the concentration of the concentrated solution is equal to 0.5 g to 1.5 g of the fine powder of the nut shell of paris polyphylla per ml of the concentrated solution.
5. The method according to claim 1, wherein the centrifugation step comprises centrifuging the concentrated solution at 3000-16000 rpm, re-dissolving the supernatant in water, centrifuging the re-dissolved solution at 3000-16000 rpm, collecting the supernatant, combining the centrifuged supernatant with the centrifuged supernatant, and subjecting the combined supernatant to the macroporous resin enrichment step.
6. The method of claim 1, wherein in the macroporous resin enrichment step, the macroporous resin is AB-8 type macroporous resin or D101 type macroporous resin, and the weight ratio of the macroporous resin to the fine powder of the paris polyphylla husks is 1: 1-1: 2; the eluent is prepared by mixing ethanol and water into 20% ethanol eluent, 40% ethanol eluent and 80% ethanol eluent.
7. The method of claim 6, wherein the macroporous resin enrichment step comprises the steps of:
s51, carrying out sample loading adsorption on the supernatant by adopting macroporous resin, wherein the sample loading speed is 0.5 column volume/hour, and standing for 2-12 hours after the adsorption of the supernatant is finished;
s52, eluting by water for 4-10 column volumes;
s53, eluting by using a 20% ethanol eluent for 4-8 column volumes;
s54, eluting 4-8 column volumes by using 40% ethanol eluent, and collecting eluent;
s55, eluting 4-8 column volumes by using an 80% ethanol eluent, and collecting eluent;
and S56, merging the eluates obtained in the steps S54 and S55, and concentrating to obtain the paris polyphylla shell extracting solution, wherein the concentration degree of the paris polyphylla shell extracting solution is 0.5-1.5 times of the total weight of the paris polyphylla shell extracting solution and the total weight of the paris polyphylla shell fine powder.
8. The method of claim 1, wherein the purity of quercetin-3-gentiobioside in quercetin-3-gentiobioside prepared by the method of claim 1 is greater than 99.0%; IC50 was less than 0.05mg/mL for DPPH clearance.
9. The use of quercetin-3-gentiobioside, wherein the quercetin-3-gentiobioside is prepared by the method for preparing quercetin-3-gentiobioside according to claim 1, and the quercetin-3-gentiobioside is added into a skin care product formula as an antioxidant active ingredient.
10. The use of quercetin-3-gentiobioside, wherein the quercetin-3-gentiobioside is prepared by the method for preparing quercetin-3-gentiobioside according to claim 1, and the quercetin-3-gentiobioside is used as a reference substance for quality control of a paris polyphylla shell extract.
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