CN111423404A - Method for separating rutin and quercetin from black tartary buckwheat - Google Patents

Method for separating rutin and quercetin from black tartary buckwheat Download PDF

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CN111423404A
CN111423404A CN202010354451.0A CN202010354451A CN111423404A CN 111423404 A CN111423404 A CN 111423404A CN 202010354451 A CN202010354451 A CN 202010354451A CN 111423404 A CN111423404 A CN 111423404A
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tartary buckwheat
quercetin
rutin
aqueous solution
ethanol
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戴富才
李政
陈梦佳
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Langfang Normal University
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Langfang Normal University
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D311/00Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D311/00Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/40Separation, e.g. from natural material; Purification
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00Processes for the preparation of sugar derivatives
    • C07H1/06Separation; Purification
    • C07H1/08Separation; Purification from natural products
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06Benzopyran radicals
    • C07H17/065Benzo[b]pyrans
    • C07H17/07Benzo[b]pyran-4-ones

Abstract

The invention discloses a method for separating rutin and quercetin from black tartary buckwheat. (1) Providing a black tartary buckwheat primary product; (2) dispersing the black tartary buckwheat primary product into a methanol solution to form a primary product adsorption solution, adding the primary product adsorption solution into a polyamide resin adsorption column, and preliminarily eluting the adsorption column with water; then, carrying out primary elution by using a first ethanol water solution; collecting the eluent obtained when the lower color band is eluted as a primary eluent; finally, secondary elution is carried out by using a second ethanol aqueous solution, and the eluent obtained when the upper color band is eluted is collected and used as secondary eluent; (3) concentrating and crystallizing the primary eluent to obtain a tartary buckwheat rutin product; concentrating and crystallizing the secondary eluent to obtain a tartary buckwheat quercetin product; wherein the first ethanol aqueous solution contains 40-65 vol% of ethanol; the second aqueous solution of ethanol contains 70 to 90 vol% of ethanol. The method has good reproducibility and is suitable for industrial production.

Description

Method for separating rutin and quercetin from black tartary buckwheat
Technical Field
The invention relates to a method for separating and purifying rutin and quercetin in black tartary buckwheat.
Background
The Rutin (Rutin) is also used for the auxiliary treatment of hemorrhagic diseases, also used for treating hypertension, senile bronchitis and the like, has certain radioactivity resistance, has strong absorption effect on ultraviolet rays and X rays, participates in vivo oxido-reductase function, has strong medicinal value and health-preserving efficacy, has the functions of eliminating phlegm, relieving cough, reducing blood pressure, reducing capillary fragility and the like, and the structure of the Quercetin determines that the Quercetin can be used as a metal chelate, can be used as an antioxidant of ascorbic acid or oil, can be used for producing free group receptors in oxidation processes of oil and fat and the like, and can be used for producing Rutin in a certain amount in plant leaves and other industrial plants, and can also be used for producing antioxidant of Rutin in addition to industrial pigments.
At present, rutin is mainly derived from sophora flower buds and is extracted by methods such as solvent extraction, alkali dissolution and acid precipitation, ultrasonic and the like. The problems of the method include single source, low purity of the obtained rutin and quercetin products, and the like. Therefore, people attract attention to explore other raw materials and methods to obtain high-purity rutin and quercetin products. The black tartary buckwheat is one of tartary buckwheat, and flavonoid substances, particularly rutin and quercetin, in the black tartary buckwheat are relatively rich. The research and report on the hydrolysis of rutin into quercetin are relatively mature, but the research on how to separate the hydrolyzed rutin from the quercetin and purify the rutin is relatively less.
CN103340938A discloses a preparation method of tartary buckwheat standard extract FT83 and high-purity quercetin, which takes tartary buckwheat bran as a raw material, and obtains the high-content quercetin through the steps of lower alcohol soaking or ultrasonic extraction, reduced pressure concentration, spray drying, column chromatography purification and the like. However, the method has long production period and large equipment investment, only one active ingredient quercetin in the tartary buckwheat is obtained, substances harmful to human bodies are used in the production process, and the method is not suitable for industrial production. CN1385428A discloses a process for extracting tartary buckwheat bioflavonoid, which comprises the steps of taking tartary buckwheat powder or tartary buckwheat leaves as raw materials, taking ethanol as a solvent, and carrying out continuous countercurrent extraction, filtration, reduced pressure concentration, vacuum drying and the like to obtain the tartary buckwheat bioflavonoid. However, the method has no separation and purification process, and the obtained product is a flavone mixture. CN104069187A discloses a process for extracting and purifying buckwheat flavonoids from buckwheat, which comprises the steps of taking the buckwheat as a raw material, and obtaining the buckwheat flavonoids with the content of 86.5 percent through the steps of ethanol reflux extraction, centrifugation, ethanol recovery, water addition precipitation, precipitation separation and the like. The method also has no separation and purification process. CN1258999C discloses a process for extracting flavone from tartary buckwheat, which comprises the steps of squeezing and peeling tartary buckwheat, carrying out microwave treatment on tartary buckwheat peel powder, adding ethanol for microwave extraction, carrying out reduced pressure concentration, adding acid for precipitation, carrying out vacuum drying and the like to obtain a rutin finished product. The obtained product has low rutin content.
Disclosure of Invention
The invention aims to provide a method for separating rutin and quercetin from black tartary buckwheat. The method can ensure the separation of rutin and quercetin. The technical scheme adopted by the application achieves the aim.
The invention provides a method for separating rutin and quercetin from black tartary buckwheat, which comprises the following steps:
(1) providing a black tartary buckwheat primary product containing rutin and quercetin;
(2) dispersing the black tartary buckwheat primary product containing rutin and quercetin in a methanol solution to form a primary product adsorption solution, adding the primary product adsorption solution into a polyamide resin adsorption column, and preliminarily eluting the adsorption column with water; then, eluting once by using a first ethanol aqueous solution, wherein the polyamide resin adsorption column is obviously layered and comprises an upper color band and a lower color band; collecting the eluent obtained when the lower color band is eluted as a primary eluent; finally, secondary elution is carried out by using a second ethanol aqueous solution, and the eluent obtained when the upper color band is eluted is collected and used as secondary eluent; wherein the first ethanol aqueous solution contains 40-65 vol% of ethanol, and the second ethanol aqueous solution contains 70-90 vol% of ethanol.
(3) Concentrating and crystallizing the primary eluent to obtain a tartary buckwheat rutin product; and concentrating and crystallizing the secondary eluent to obtain the tartary buckwheat quercetin product.
According to the method, the length-diameter ratio of the polyamide resin adsorption column is preferably 5-15: 1; the addition amount of the primary product adsorption solution is 0.5-1 BV; the using amount of the distilled water is 2-5 BV; wherein BV represents the volume of the column.
According to the method of the present invention, preferably, the method further comprises a step of pretreating the polyamide resin of the polyamide resin adsorption column:
soaking polyamide resin in a third ethanol aqueous solution with the ethanol concentration of 90-98 vol% for 12-36 hours, and then washing with water to be neutral; soaking the mixture for 6 to 15 hours by using 3 to 8 weight percent of sodium hydroxide aqueous solution, and then washing the mixture to be neutral by using water; then soaking the mixture for 6 to 15 hours by using 3 to 8 weight percent acetic acid aqueous solution, and then washing the mixture to be neutral by using water.
According to the method, the flow rate of the first ethanol aqueous solution and the flow rate of the second ethanol aqueous solution are both 1.5-2.5 BV/h; wherein BV represents the volume of the column.
According to the method of the invention, preferably, the black tartary buckwheat primary product containing rutin and quercetin is prepared by the following steps:
extracting black tartary buckwheat in methanol water solution in a refluxing manner, filtering, collecting filtrate to obtain a crude black tartary buckwheat extract, and concentrating, precipitating and drying the obtained crude black tartary buckwheat extract to obtain a black tartary buckwheat primary product containing rutin and quercetin.
According to the method, the black tartary buckwheat is preferably used in the form of black tartary buckwheat powder, and the particle size of the black tartary buckwheat powder is 10-40 meshes.
According to the method, the mass ratio of the methanol water solution to the black tartary buckwheat is preferably 10-20: 1.
According to the method, preferably, the content of methanol in the methanol aqueous solution is 40-80 vol%; the reflux extraction times are 1-5 times; the single reflux extraction time is 0.5-1.5 hours.
According to the method, preferably, the black tartary buckwheat is used in the form of black tartary buckwheat powder, and the particle size of the black tartary buckwheat powder is 20-40 meshes; the content of methanol in the methanol aqueous solution is 55-65 vol%; the reflux extraction time is 1.0-1.2 hours; the reflux extraction times are 3-5; evaporating and concentrating the crude extract of the black tartary buckwheat on a rotary evaporator; the drying temperature is 55-65 ℃, and the drying time is 6-10 hours.
According to the method, preferably, the first eluent and the second eluent are subjected to rotary evaporation concentration by using a rotary evaporator respectively, and then are kept stand for 16-32 hours to obtain a tartary buckwheat rutin product and a tartary buckwheat quercetin product.
The invention carries out reflux extraction on the black tartary buckwheat powder after being crushed and sieved by a methanol water solution, crystallizing a primary product, separating by a polyamide resin column and crystallizing the product to obtain a tartary buckwheat rutin product and a tartary buckwheat quercetin product. Thus, rutin and quercetin can be sufficiently separated. The method has good separation degree and high purity of the obtained rutin and quercetin. In addition, the method of the invention has good reproducibility and is suitable for industrial production.
Drawings
FIG. 1 is a high performance liquid chromatography spectrum of the rutin standard substance in the invention.
FIG. 2 is a high performance liquid chromatography spectrum of the quercetin standard substance of the present invention.
FIG. 3 is a high performance liquid analysis spectrogram of the crude extract of Tartary buckwheat of the present invention.
FIG. 4 is a high performance liquid chromatography analysis chart of the black tartary buckwheat primary product in the invention.
FIG. 5 is a high performance liquid analysis spectrogram of the buckwheat rutin product of the present invention.
FIG. 6 is a high performance liquid chromatography analysis spectrogram of the tartary buckwheat quercetin product of the present invention.
FIG. 7 is a nuclear magnetic resonance hydrogen spectrum of the buckwheat rutin product of the invention.
FIG. 8 is a nuclear magnetic resonance hydrogen spectrum of the tartary buckwheat quercetin product of the present invention.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.
In the present invention, "vol%" represents a volume percentage, and "wt%" represents a weight percentage.
In the present invention, the "quantitative analysis" is an external standard curve quantitative analysis unless otherwise specified.
The invention provides a method for separating rutin and quercetin from black tartary buckwheat. The present inventors have unexpectedly found that rutin and quercetin have different adsorption forces on a resin column and need to be eluted with different elution reagents, thereby completing the present invention. The method comprises the following steps of (1) preparing a primary product; (2) separating by an adsorption column; (3) and (4) crystallizing the product. Optionally, a polyamide resin pretreatment step may also be included. As described in detail below.
< preparation of Primary product >
The preparation step of the initial product is to provide the black tartary buckwheat initial product containing rutin and quercetin. The black tartary buckwheat primary product contains rutin and quercetin to be separated. Preferably, the black tartary buckwheat primary product is obtained by extracting with an alcohol solution. The alcohol solution used is preferably an aqueous methanol solution.
In some embodiments, the black tartary buckwheat is extracted in methanol water solution in a reflux manner, then is filtered, filtrate is collected to obtain a crude extract of the black tartary buckwheat containing rutin, and the obtained crude extract of the black tartary buckwheat is concentrated, precipitated and dried to obtain a primary product of the black tartary buckwheat containing the rutin and the quercetin.
The black tartary buckwheat of the invention is preferably black tartary buckwheat with shell produced in great summer mountains in Sichuan. Preferably, the black tartary buckwheat of the present invention is used in the form of black tartary buckwheat powder. The black tartary buckwheat powder can be prepared by crushing black tartary buckwheat. For example, the dried black tartary buckwheat is put into an XFB-400 high-speed traditional Chinese medicine grinder to be ground for 1-5 minutes, preferably 2-4 minutes, and more preferably 2-3 minutes to obtain black tartary buckwheat powder. Thus being helpful to fully extract the rutin and the quercetin in the black tartary buckwheat. The particle size of the obtained black tartary buckwheat powder is 10-40 meshes; preferably 20-40 meshes; more preferably 30 to 40 mesh.
In the methanol aqueous solution of the present invention, the content of methanol may be 40 to 80 vol%, preferably 50 to 70 vol%, and more preferably 55 to 65 vol%. The mass ratio of the methanol water solution to the black tartary buckwheat is 10-20: 1, preferably 12-18: 1, and more preferably 14-16: 1. This facilitates removal of other impurity components, thereby improving the purity of the extract.
In the present invention, the number of reflux extractions may be 1 to 5, preferably 2 to 5, and more preferably 3 to 5. The single reflux extraction time can be 0.5-1.5 hours, preferably 0.8-1.4 hours, and more preferably 1.0-1.2 hours. The volume of aqueous methanol solution used for each reflux extraction may be the same. Therefore, more rutin and quercetin can be extracted, and the purity of the rutin and the quercetin in the crude extract can be improved.
In the present invention, in order to improve efficiency, filtration may be performed after reflux extraction by suction filtration. And combining the filtrates obtained by the three times of filtration to obtain the coarse extract of the black tartary buckwheat.
The content of rutin in the crude extract of the black tartary buckwheat is high, and the content of quercetin is little. Concentrating, precipitating and drying the crude extract of black tartary buckwheat to obtain a primary product containing rutin and quercetin. After the rutin in the crude extract is subjected to rotary evaporation and concentration, a part of the rutin is hydrolyzed into quercetin.
In the invention, the crude extract of black tartary buckwheat can be concentrated under reduced pressure, preferably by a rotary evaporator. According to one embodiment of the invention, the crude extract is concentrated on a rotary evaporator, and the rotary evaporation is stopped when a small amount of yellow precipitate appears; then, the concentrated solution is placed in a beaker for standing for sufficient precipitation, and the standing time can be 16-32 hours, preferably 18-30 hours, and more preferably 22-28 hours. Standing to obtain a large amount of yellow precipitate, and drying the precipitate to obtain a primary product containing rutin and quercetin. The drying temperature can be 45-70 ℃, preferably 50-65 ℃, and more preferably 55-65 ℃. The drying time is generally 4 to 10 hours, preferably 6 to 10 hours, and more preferably 8 to 10 hours. The precipitate may be filtered before drying to improve drying efficiency. Preferably, the filtration is carried out by suction filtration. The initial product obtained by this treatment contains fewer impurities.
< separation by adsorption column >
Dispersing the black tartary buckwheat primary product containing rutin and quercetin in a methanol solution to form a primary product adsorption solution. In the present invention, the methanol solution for dispersing the primary product containing rutin and quercetin is an analytically pure methanol solution, and the amount thereof is not particularly limited as long as the primary product containing rutin and quercetin can be completely dissolved.
And after the primary product adsorption liquid is formed, adding the primary product adsorption liquid into a polyamide resin adsorption column. And adding 0.5-2 BV of the adsorption solution of the primary product into the adsorption column, preferably, adding 0.5-1 BV of the adsorption solution of the primary product into the adsorption column. Too large an amount of the initial product added results in poor separation efficiency, and too small an amount results in lowering of separation efficiency. The adsorption rate is 1.5-2.5 BV/h, preferably 1.5-2 BV/h, and more preferably 1.5-1.8 BV/h. By controlling the adsorption rate, the purity of rutin and quercetin can be improved.
First, the adsorption column was preliminarily eluted with water. The preliminary elution may be performed using distilled water, deionized water, or ultrapure water. And (3) adding the primary product adsorption solution into the adsorption column, and then carrying out primary elution by using 2-5 BV of water. Thus, impurities with polarity higher than that of rutin in the initial product can be removed, and methanol for dissolving the initial product can be eluted out, so that the purity of the product is improved.
Then, the first ethanol water solution is used for elution, and the polyamide resin adsorption column has obvious layering, including an upper color band and a lower color band. The color band is light yellow or yellow. Collecting eluate obtained during eluting lower layer color band as primary eluate, wherein rutin is contained. The concentration of the first ethanol aqueous solution is 40-65 vol%; preferably, the concentration is 45 to 60 vol%, more preferably, the concentration is 50 to 55 vol%. In this way, rutin can be eluted, but quercetin is kept on the adsorption column, so that separation and purification of the rutin and the quercetin are realized. The process has good reproducibility and is suitable for industrial production.
In the invention, the flow rate of the first ethanol aqueous solution is 1.5-2.5 BV/h, preferably 1.5-2 BV/h, and more preferably 1.5-1.8 BV/h. By controlling the elution rate, elution of quercetin can be avoided.
And finally, carrying out secondary elution by using a second ethanol aqueous solution, and collecting the eluent obtained when the upper color band is eluted as a secondary eluent, wherein the secondary eluent contains quercetin. The concentration of ethanol in the second ethanol aqueous solution is 70-90 vol%; preferably, the concentration is 75-90 vol%; more preferably, the concentration is 80 vol%. This allows elution of quercetin. The process has good reproducibility and is suitable for industrial production.
In the invention, the flow rate of the second ethanol aqueous solution is 1.5-2.5 BV/h, preferably 1.5-2 BV/h, and more preferably 1.5-1.8 BV/h. By controlling the elution rate, quercetin can be sufficiently eluted.
< crystallization of product >
And concentrating and crystallizing the primary eluent to obtain a tartary buckwheat rutin product. The concentration can be performed by rotary evaporation. For example, the eluate is subjected to rotary evaporation and concentration by using a RE-52A rotary evaporator until a large number of crystals are precipitated, the rotary evaporation is stopped, and then the eluate is kept stand for 16-32 hours, preferably 18-30 hours, and more preferably 22-28 hours, so that a tartary buckwheat rutin product is obtained.
And concentrating and crystallizing the secondary eluent to obtain the tartary buckwheat quercetin product. The concentration can be performed by rotary evaporation. For example, the eluate is subjected to rotary evaporation and concentration by using a RE-52A rotary evaporator until a large number of crystals are precipitated, the rotary evaporation is stopped, and then the eluate is kept stand for 16-32 hours, preferably 18-30 hours, and more preferably 22-28 hours, so that the tartary buckwheat quercetin product is obtained.
< pretreatment of Polyamide resin >
The polyamide resin needs to be pretreated before being filled into the column. The pretreatment step of the polyamide resin adsorption column comprises the following steps:
soaking polyamide resin in a third ethanol aqueous solution, and then washing the polyamide resin with water to be neutral; soaking with sodium hydroxide aqueous solution, and washing with water to neutrality; then soaking the mixture in acetic acid aqueous solution, and then washing the mixture with water to be neutral.
The ethanol concentration of the third ethanol aqueous solution is 90 to 98 vol%, preferably 91 to 96 vol%, and more preferably 93 to 95 vol%. The third ethanol aqueous solution is soaked for 12 to 36 hours, preferably 15 to 30 hours, and more preferably 24 to 28 hours. After the third ethanol aqueous solution is soaked, deionized water or distilled water and the like can be adopted for washing.
The concentration of the sodium hydroxide aqueous solution is 3 to 8 wt%, preferably 3 to 7 wt%, and more preferably 5 to 6 wt%. The soaking time of the sodium hydroxide aqueous solution is 6 to 15 hours, preferably 8 to 13 hours, and more preferably 9 to 12 hours. After the soaking with the aqueous solution of sodium hydroxide, deionized water or distilled water may be used for washing.
The concentration of the acetic acid aqueous solution is 5-10 wt%, preferably 6-10 wt%, and more preferably 8-10 wt%. The soaking time of the acetic acid aqueous solution is 6-15 hours, preferably 8-13 hours, and more preferably 9-12 hours. After the acetic acid aqueous solution is soaked, deionized water or distilled water and the like can be adopted for washing. After the treatment, the adsorption force of the polyamide resin on the quercetin is greater than that of the rutin in an alcohol-water system, so that the separation of the two is facilitated.
According to one embodiment of the invention, the polyamide resin is soaked in a third ethanol aqueous solution with the ethanol concentration of 90-98 vol% for 12-36 hours and then washed to be neutral by water; soaking the mixture for 6 to 15 hours by using 3 to 8 weight percent of sodium hydroxide aqueous solution, and then washing the mixture to be neutral by using water; then soaking the mixture for 6 to 15 hours by using 3 to 8 weight percent acetic acid aqueous solution, and then washing the mixture to be neutral by using water. According to one embodiment of the present invention, the polyamide resin is soaked with a 95 vol% third aqueous solution of ethanol for 24 hours, then washed to neutrality with deionized water, then soaked with a 5 wt% aqueous solution of sodium hydroxide for 12 hours, finally washed to neutrality with deionized water, then soaked with an 8 wt% aqueous solution of acetic acid for 12 hours, and then washed to neutrality with deionized water.
And loading the pretreated polyamide resin into a glass chromatographic column to obtain the polyamide resin adsorption column. The aspect ratio (column length/column diameter) of the adsorption column may be 10 to 20:1, preferably 15 to 20: 1. Thus, the adsorption and separation effect can be ensured, and the product content in the extract can be improved.
According to one embodiment of the invention, the primary product containing rutin and quercetin is dispersed in analytically pure methanol to form a primary product adsorption solution, and the primary product adsorption solution is added into a pretreated polyamide resin adsorption column, wherein the length-diameter ratio of the adsorption column is 10: 1. The adsorption column is preliminarily eluted by distilled water, and the using amount of the distilled water is 2-5 BV. After the primary elution is finished, performing primary elution by using a first ethanol aqueous solution with the ethanol concentration of 50 vol%, wherein the polyamide resin adsorption column is obviously layered and comprises an upper color band and a lower color band; collecting the eluate obtained when the lower layer color band is eluted as a primary eluate, and concentrating and crystallizing the obtained primary eluate to obtain a tartary buckwheat rutin product; and after the primary elution is finished, performing secondary elution by using a second ethanol water solution with the ethanol concentration of 80 vol%, collecting the eluent obtained when the upper color band is eluted as a secondary eluent, and concentrating and crystallizing the obtained secondary eluent to obtain the tartary buckwheat quercetin product. The flow rates of the first ethanol aqueous solution and the second ethanol aqueous solution are both 2 BV/h.
The starting materials used in the following examples are illustrated below:
0.4 wt% phosphoric acid solution: the high-grade pure phosphoric acid is purchased from chemical reagent supply and sale company of Tianjin and prepared into 0.4 wt% phosphoric acid aqueous solution;
rutin and quercetin standards: purchased from Wuhan Tian Biotechnology GmbH;
methanol (chromatographically pure): purchased from chemical reagents supply and distribution company of Tianjin;
polyamide resin powder (100-200 mesh): from chemical reagents of national drug group, Ltd
< description of test and analytical methods >
1) High performance liquid chromatography analysis of buckwheat rutin and buckwheat quercetin:
the rutin standard substance and the quercetin standard substance are accurately weighed to be 5.0mg, the rutin standard substance and the quercetin standard substance are respectively dissolved by chromatographic pure methanol and then are subjected to constant volume to be prepared into a rutin standard solution with the concentration of 0.2mg/m L and a quercetin standard solution with the concentration of 0.2mg/m L, the rutin standard solution (0.2mg/m L) and the quercetin standard solution (0.2mg/m L) with the concentrations of 0.5m L0, 1.0m L, 1.5m L, 2.0m L, 2.5m L and 3.0m L are respectively measured into a volumetric flask with the concentration of 10m L, the chromatographic pure methanol is used for constant volume, the peak area of the rutin standard substance and the quercetin standard substance is measured by high performance liquid chromatography, and the standard curve is drawn by taking the concentration of the rutin standard substance or the concentration of the quercetin standard substance as a horizontal coordinate and the peak area as a vertical coordinate.
Accurately weighing each 5mg of the tartary buckwheat rutin product and the tartary buckwheat quercetin product, and respectively dissolving in a 25m L volumetric flask by using chromatographic pure methanol to obtain a tartary buckwheat rutin product solution and a tartary buckwheat quercetin product solution, wherein the concentrations are both 0.2mg/m L.
Filtering rutin standard solution, quercetin standard solution, black radix Et rhizoma Fagopyri Tatarici crude extract, primary product adsorption solution, radix Et rhizoma Fagopyri Tatarici rutin product solution, and radix Et rhizoma Fagopyri Tatarici quercetin product solution with 0.45 μm filter membrane, and analyzing with high performance liquid chromatography.
The measurement conditions for the high performance liquid chromatography were set as follows:
the chromatographic column was SB-C18(250 × 4.6.6 mm, 5 μm), the mobile phase was chromatographically pure methanol-0.4 wt% phosphoric acid aqueous solution, the flow rate was set at 1.0m L/min, the column temperature was set at 25 deg.C, the amount of sample was set at 10 μ L, and the detection wavelength was set at 358 nm.
2) Calculating the purity of the tartary buckwheat rutin product and the tartary buckwheat quercetin product:
performing high performance liquid chromatography analysis on a tartary buckwheat rutin (tartary buckwheat quercetin) product solution of 0.2mg/m L and a rutin (quercetin) standard substance solution of 0.2mg/m L under the same liquid chromatography condition, recording peak areas, and substituting the peak areas into the following formula to calculate the product purity;
Figure BDA0002472992180000111
wherein, P is the quantitative purity (%) of the tartary buckwheat rutin (tartary buckwheat quercetin) product, A is the liquid chromatogram peak area (mAU & s) of the tartary buckwheat rutin (tartary buckwheat quercetin) product, and As is the liquid chromatogram peak area (mAU & s) of the rutin (quercetin) standard product.
3) Calculating the yield of the tartary buckwheat rutin and tartary buckwheat quercetin products:
accurately weighing the dried extract, calculating, and calculating the product yield by substituting the following formula:
Figure BDA0002472992180000121
wherein Y is the yield of the tartary buckwheat rutin (tartary buckwheat quercetin) product percent; m the quality (g) of a tartary buckwheat rutin (tartary buckwheat quercetin) product; g is the mass (G) of the black tartary buckwheat powder.
4) Nuclear magnetic resonance hydrogen spectrum analysis of rutin extract and quercetin extract:
taking a proper amount of rutin extract and a proper amount of quercetin extract, respectively dissolving the rutin extract and the quercetin extract by using deuterated dimethyl sulfoxide as a solvent, and carrying out nuclear magnetic resonance hydrogen spectrum determination.
Example 1
The method comprises the steps of putting dried black tartary buckwheat into an XFB-400 high-speed traditional Chinese medicine grinder to be ground for 3 minutes to obtain black tartary buckwheat powder, carrying out reflux extraction on 40g of black tartary buckwheat powder for 1 hour by using a 60 vol% methanol water solution with the concentration of 600m L, carrying out suction filtration after the reflux extraction, collecting obtained filtrate, repeating the reflux extraction process for three times, and combining the filtrate obtained by the three times of filtration to obtain the crude extract of the black tartary buckwheat.
And (3) evaporating and concentrating the crude black tartary buckwheat extract on a rotary evaporator, and stopping rotary evaporation when a small amount of yellow precipitate appears. The concentrate was allowed to stand for 24 hours to sufficiently precipitate to obtain a large amount of yellow precipitate. And (3) carrying out suction filtration on the concentrated solution with the precipitate, and drying the precipitate at 60 ℃ for 6 hours to obtain a black tartary buckwheat primary product containing rutin and quercetin.
Soaking polyamide resin in a third ethanol aqueous solution with the ethanol concentration of 95 vol% for 24h, then washing with deionized water to be neutral, then soaking with a 5 wt% sodium hydroxide aqueous solution for 12h, finally washing with deionized water to be neutral, then soaking with an 8 wt% acetic acid aqueous solution for 12h, and then washing with deionized water to be neutral.
And (3) loading the treated polyamide resin into a glass chromatographic column to obtain a pretreated polyamide resin adsorption column with the column height of 10cm and the length-diameter ratio of 10: 1. Dispersing the black tartary buckwheat primary product containing rutin and quercetin in a small amount of analytically pure methanol to form a primary product adsorption solution.
The 0.5BV of the adsorption solution of the initial product was added to a pretreated polyamide resin adsorption column. The adsorption column was first eluted with 3BV of distilled water. After the primary elution is finished, performing primary elution by using a first ethanol aqueous solution (50 vol% ethanol aqueous solution), wherein the flow rate of the first ethanol aqueous solution is 2 BV/h. The pretreated polyamide resin adsorption column has obvious layering, including an upper color band and a lower color band. Collecting the eluate obtained when the lower layer color band is eluted as a primary eluate, and concentrating and crystallizing to obtain the tartary buckwheat rutin product. And after the first elution is finished, performing secondary elution on the adsorption column by using a second ethanol aqueous solution (80 vol% ethanol aqueous solution), wherein the flow rate of the second ethanol aqueous solution is 2BV/h, collecting eluent obtained when an upper color band is eluted as secondary eluent, and concentrating and crystallizing to obtain the tartary buckwheat quercetin product.
The high performance liquid chromatography analysis spectrogram of each stage and the standard product is shown in figures 1-6, and the nuclear magnetic resonance hydrogen spectrum of the tartary buckwheat rutin product and the tartary buckwheat quercetin product is shown in figures 7 and 8.
As can be seen from FIGS. 1 to 6, the retention time of the rutin standard substance is 9.261 min; the retention time of quercetin standard substance is 26.025 min. The crude extract of black tartary buckwheat mainly contains rutin, the content of quercetin is little, and impurities exist (figure 3). After rotary evaporation and concentration, the initial product contains rutin and quercetin hydrolyzed from rutin (figure 4), and the impurity content is greatly reduced. After chromatographic separation and product crystallization, a high-purity tartary buckwheat rutin product (figure 5) and a tartary buckwheat quercetin product (figure 6) are obtained.
As can be seen from fig. 7, the nuclear magnetic hydrogen spectrum data of the tartary buckwheat rutin product is as follows: h12.6 (1H's' 5-OH), H (10.85, 9.68, 9.19) (3H 3 '-OH 4' -OH 7-OH), H7.54 (2H'd' J ═ 8.0Hz H-2 'H-6'), H6.84 (1H'd' J ═ 8.0Hz H-5 '), H6.39 (1H'd 'J ═ 2.0Hz H-8), H6.19 (1H'd 'J ═ 1.6Hz H-6), H5.35 (1H'd 'J ═ 7.2 Hz' Glu 'H-1 ″), H4.38 (1H' brs 'Rha' H-1 "'), H0.99 (3H'd 'J ═ 6.0 Hz' Rha-Me).
As can be seen from fig. 8, the nuclear magnetic hydrogen spectrum data of the tartary buckwheat quercetin product is as follows: h12.5 (1H's ' 5-OH), H (10.80, 9.62, 9.39) (3H 3 ' -OH ' 4 ' -OH ' 7-OH), H7.69 (1H'd ' J ═ 6.0Hz H-2 '), H7.55 (1H ' dd ' J ═ 8.8,7.2Hz H-6 '), H6.89 (1H'd ' J ═ 8.0Hz H-5 '), H6.42 (1H'd ' J ═ 6.0Hz H-8), H6.20 (1H'd ' J ═ 6.0Hz H-6).
The procedure of example 1 was repeated twice, as denoted by S2 and S3, and the purity was quantitatively analyzed. The analysis results are shown in table 1.
TABLE 1
Numbering Example 1 S2 S3 Mean value of
Yield of rutin/%) 1.71 1.68 1.74 1.71
Quercetin yield/%) 2.45 2.49 2.35 2.43
Purity of rutin/%) 98.5 97.8 98.7 98.3
Purity/% of quercetin 99.4 97.4 98.6 98.5
As can be seen from the table, the method of the present invention has good reproducibility and is suitable for industrial production.
The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.

Claims (10)

1. A method for separating rutin and quercetin from black tartary buckwheat is characterized by comprising the following steps:
(1) providing a black tartary buckwheat primary product containing rutin and quercetin;
(2) dispersing the black tartary buckwheat primary product containing rutin and quercetin in a methanol solution to form a primary product adsorption solution, adding the primary product adsorption solution into a polyamide resin adsorption column, and preliminarily eluting the adsorption column with water; then, eluting once by using a first ethanol aqueous solution, wherein the polyamide resin adsorption column is obviously layered and comprises an upper color band and a lower color band; collecting the eluent obtained when the lower color band is eluted as a primary eluent; finally, secondary elution is carried out by using a second ethanol aqueous solution, and the eluent obtained when the upper color band is eluted is collected and used as secondary eluent; wherein the first ethanol aqueous solution contains 40-65 vol% of ethanol, and the second ethanol aqueous solution contains 70-90 vol% of ethanol;
(3) concentrating and crystallizing the primary eluent to obtain a tartary buckwheat rutin product; and concentrating and crystallizing the secondary eluent to obtain the tartary buckwheat quercetin product.
2. The method according to claim 1, wherein the length-diameter ratio of the polyamide resin adsorption column is 5-15: 1; the addition amount of the primary product adsorption solution is 0.5-1 BV; the using amount of water is 2-5 BV; wherein BV represents the volume of the column.
3. The method according to claim 1, further comprising a step of pretreating the polyamide resin of the polyamide resin adsorption column:
soaking polyamide resin in a third ethanol aqueous solution with the ethanol concentration of 90-98 vol% for 12-36 hours, and then washing with water to be neutral; soaking the mixture for 6 to 15 hours by using 3 to 8 weight percent of sodium hydroxide aqueous solution, and then washing the mixture to be neutral by using water; then soaking the mixture for 6 to 15 hours by using 3 to 8 weight percent acetic acid aqueous solution, and then washing the mixture to be neutral by using water.
4. The method according to claim 1, wherein the flow rate of the first aqueous ethanol solution and the flow rate of the second aqueous ethanol solution are both 1.5-2.5 BV/h; wherein BV represents the volume of the column.
5. The method according to claim 1, wherein the primary product of tartary buckwheat containing rutin and quercetin is prepared by the following steps:
extracting black tartary buckwheat in methanol water solution in a refluxing manner, filtering, collecting filtrate to obtain a crude black tartary buckwheat extract, and concentrating, precipitating and drying the obtained crude black tartary buckwheat extract to obtain a black tartary buckwheat primary product containing rutin and quercetin.
6. The method according to claim 5, wherein the black tartary buckwheat is used in the form of black tartary buckwheat powder, and the particle size of the black tartary buckwheat powder is 10-40 meshes.
7. The method according to claim 5, wherein the mass ratio of the methanol aqueous solution to the black tartary buckwheat is 10-20: 1.
8. The method according to claim 5, wherein the content of methanol in the methanol aqueous solution is 40 to 80 vol%; the reflux extraction times are 1-5 times; the single reflux extraction time is 0.5-1.5 hours.
9. The method of claim 5, wherein:
the black tartary buckwheat is used in the form of black tartary buckwheat powder, and the particle size of the black tartary buckwheat powder is 20-40 meshes;
the content of methanol in the methanol aqueous solution is 55-65 vol%;
the reflux extraction time is 1.0-1.2 hours; the reflux extraction times are 3-5;
evaporating and concentrating the crude extract of the black tartary buckwheat on a rotary evaporator;
the drying temperature is 55-65 ℃, and the drying time is 6-10 hours.
10. The method according to any one of claims 1 to 9, wherein the first eluent and the second eluent are respectively concentrated by rotary evaporation using a rotary evaporator, and then left for 16 to 32 hours to obtain a tartary buckwheat rutin product and a tartary buckwheat quercetin product.
CN202010354451.0A 2020-04-29 2020-04-29 Method for separating rutin and quercetin from black tartary buckwheat Pending CN111423404A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115777935A (en) * 2022-12-28 2023-03-14 西昌航飞苦荞科技发展有限公司 Method for preparing buckwheat rutin from buckwheat flavone extract
CN115974826A (en) * 2022-12-28 2023-04-18 西昌航飞苦荞科技发展有限公司 Method for preparing tartary buckwheat quercetin from tartary buckwheat flavone extract

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102731593A (en) * 2011-04-02 2012-10-17 中国科学院兰州化学物理研究所 Method for extracting rutin from tartary buckwheat
CN109369733A (en) * 2018-10-30 2019-02-22 湖南华诚生物资源股份有限公司 A method of extracting a variety of flavone compounds simultaneously from leaf of Radix Et Rhizoma Fagopyri Tatarici

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102731593A (en) * 2011-04-02 2012-10-17 中国科学院兰州化学物理研究所 Method for extracting rutin from tartary buckwheat
CN109369733A (en) * 2018-10-30 2019-02-22 湖南华诚生物资源股份有限公司 A method of extracting a variety of flavone compounds simultaneously from leaf of Radix Et Rhizoma Fagopyri Tatarici

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
于智峰等: "大孔吸附树脂对苦荞黄酮吸附分离特性研究", 《食品研究与开发》 *
刘金玉等: "不同大孔吸附树脂对苦荞芦丁分离纯化效果的研究", 《食品研究与开发》 *
郑峰等: "苦荞籽粒的化学成分研究", 《西北农林科技大学学报(自然科学版)》 *
黄莎等: "大孔树脂分离纯化苦荞麦中总黄酮的工艺", 《江苏农业科学》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115777935A (en) * 2022-12-28 2023-03-14 西昌航飞苦荞科技发展有限公司 Method for preparing buckwheat rutin from buckwheat flavone extract
CN115974826A (en) * 2022-12-28 2023-04-18 西昌航飞苦荞科技发展有限公司 Method for preparing tartary buckwheat quercetin from tartary buckwheat flavone extract

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