CN108117572B - Method for preparing quercetin-3-O-2 ', 6' -dirhamnosylglucoside - Google Patents
Method for preparing quercetin-3-O-2 ', 6' -dirhamnosylglucoside Download PDFInfo
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Abstract
The invention provides a preparation method of quercetin-3-O-2 ', 6' -dirhamnosylglucoside, which adopts two-dimensional liquid chromatography for preparation, takes methanol (or containing 0.1% formic acid) -water, ethanol (or containing 0.1% formic acid) -water or acetonitrile (or containing 0.1% formic acid) -water as a mobile phase, takes a reversed phase chromatographic column as a one-dimensional preparation chromatographic column, cuts components of a ginkgo leaf extract, and collects a target component which is a crude product of quercetin-3-O-2 ', 6' -dirhamnosylglucoside. And then, taking the reversed phase chromatographic column as a two-dimensional preparation chromatographic column, and carrying out component cutting, collection and rotary evaporation concentration on the crude product of the quercetin-3-O-2 ', 6' -dirhamnosylglucoside under the guidance of a mass spectrum selected ion peak to obtain the high-purity quercetin-3-O-2 ', 6' -dirhamnosylglucoside, wherein the purity can reach over 75 percent. The preparation process has high repeatability and good operability, and simultaneously, the ginkgo leaf resource is rich and easy to obtain, thereby being suitable for the requirement of large-scale production.
Description
Technical Field
The title method relates to the technical field of medicine, in particular to a method for preparing quercetin-3-O-2 ', 6' -dirhamnosylglucoside from a ginkgo leaf extract. Furthermore, the invention relates to a method for preparing high-purity quercetin-3-O-2 ', 6' -dirhamnosylglucoside from ginkgo biloba extract by utilizing a two-dimensional liquid chromatography technology, wherein the purity of the quercetin-3-O-2 ', 6' -dirhamnosylglucoside can reach 75% or more.
Background
The quercetin-3-O-2 ', 6' -dirhamnosylglucoside belongs to a flavonoid compound, and the flavonoid compound has a good pharmaceutical effect, can reduce capillary permeability and fragility, promote cell proliferation, prevent hemagglutination and remove free radicals, and plays a very important role in treating cardiovascular and cerebrovascular diseases. Meanwhile, researches show that the quercetin-3-O-2 ', 6' -dirhamnosylglucoside also has an anti-tumor effect.
Quercetin-3-O-2 ', 6' -dirhamnosylglucoside is present in Phyllanthus urinaria, Dactylis asiatica, Vinca rosea, cortex Choerospondiatis, Cerbera Manghalensis, Elaeagnus longifolia, folium Ginkgo, etc. At present, for the extraction and separation of quercetin-3-O-2 ', 6' -dirhamnosylglucoside, mainly, a plant is subjected to alcohol extraction, then is subjected to separation and purification through macroporous resin or sephadex and other multiple column chromatography steps, the operation steps are complex, the process is complex, meanwhile, a large amount of organic solvents are consumed, and time and labor are consumed (Wuwei, loosestrife, the research on chemical components of anti-tumor effective parts of loosestrife, Chinese herbal medicines, 42 st volume 1: 38-41 in 2011; Zhaoyyi, Yinyi, the research on flavonoid glycosides chemical components in injection ginkgo leaf extracts, Chinese herbal medicines, 44 th volume 15: 2027 plus 2034 in 2013).
The two-dimensional liquid chromatography is a technique for separating a complex sample by using two chromatographic columns having the same or different properties or by adjusting the composition ratio of a mobile phase, and has been widely used in various fields such as medical and health, food science, environment, agricultural science, and the like. In recent years, with the development of two-dimensional liquid chromatography theory and application, two-dimensional preparative liquid chromatography has been gradually developed. Compared with single-dimensional preparative liquid chromatography, two-dimensional preparative liquid chromatography not only has higher peak capacity, but also has different selectivities in two dimensions. (Peak. use of two-dimensional liquid chromatography in drug analysis of impurities, vol.27, No. 4: 616-. So far, no report is found for preparing quercetin-3-O-2 ', 6' -dirhamnosylglucoside by adopting a two-dimensional liquid chromatography technology.
Disclosure of Invention
The invention provides a method for preparing quercetin-3-O-2 ', 6' -dirhamnosylglucoside by using a ginkgo leaf extract as a raw material and utilizing a two-dimensional chromatographic separation technology, which comprises the following operation steps:
1. dissolving folium Ginkgo extract in 20-80% methanol-water solution to obtain folium Ginkgo extract solution, wherein the concentration of folium Ginkgo extract is 10-2000 mg/mL, the best folium Ginkgo extract is dissolved in 40-60% methanol-water solution, and the best concentration of folium Ginkgo extract is 250-550 mg/mL;
2. the chromatographic columns prepared in one dimension by liquid chromatography are reversed phase C18, reversed phase C8, Click PN. The columns prepared two-dimensionally by liquid chromatography were reversed phase C18, reversed phase C8, Click PN. The optimal two-dimensional chromatographic column combination is that one dimension is reverse phase C18, and the two dimensions are Click PN;
3. the one-dimensional liquid chromatography adopts methanol as a mobile phase, ethanol or acetonitrile (an organic phase) is mixed with water (a water phase), and elution conditions are carried out according to the gradient that the organic phase is 5-60% isocratic or is increased from 5% to 80% in 0-60 minutes. The optimal mobile phase for one-dimensional preparation is acetonitrile (or containing 0.1 percent of formic acid) and water (or containing 0.1 percent of formic acid), and the organic phase proportion is increased to 30-40 percent from 10 percent in 20-30 minutes by adopting a gradient mode;
4. collecting the component with main ingredient of quercetin-3-O-2 ', 6' -dirhamnosylglucoside after retention time of 5-20 min, and rotary evaporating to dryness to obtain crude product of quercetin-3-O-2 ', 6' -dirhamnosylglucoside as one-dimensional preparation. Dissolving the one-dimensionally prepared quercetin-3-O-2 ', 6' -dirhamnosylglucoside crude product with methanol-water to a concentration of 20-200 mg/mL. One-dimensional preparation with optimal collection time of 12-18 min, which is mainly composed of quercetin-3-O-2 ", 6" -dirhamnosylglucoside, and concentration to concentration of 50-100 mg/mL;
5. and (4) carrying out two-dimensional liquid chromatography preparation on the one-dimensional collected and concentrated sample. The adopted mobile phase is methanol, ethanol or acetonitrile (organic phase) and water (water phase) to be mixed, and the elution condition is carried out according to the gradient that the organic phase is 5-15% isocratic or is increased from 5% to 30% after 0-30 minutes. The optimal two-dimensional preparation mobile phase is acetonitrile (or containing 0.1% formic acid) and water (or containing 0.1% formic acid), and the concentration of the acetonitrile (or containing 0.1% formic acid) is 8-15% by adopting isocratic conditions; two-dimensional preparation of mass spectrum, selecting ions with m/z 303, and collecting fractions with m/z 303;
6. the flow rate of the mobile phase is 20-120 mL/min during preparation, the column temperature is room temperature or 25-40 ℃ during preparation, the sample injection amount is 200-; the optimal flow rate is 80-100 mL/min, the column temperature is room temperature, the sample injection amount is 2000 and 2500 mu L/needle, and the ultraviolet detector is 360 nm;
7. and (3) rotationally evaporating and concentrating the quercetin-3-O-2 ', 6' -dirhamnosylglucoside fraction collected by the two-dimensional liquid chromatography to dryness to obtain the high-purity quercetin-3-O-2 ', 6' -dirhamnosylglucoside compound.
It should be understood by those skilled in the art that the technical method of the present invention is not limited to the preparation of quercetin-3-O-2 ", 6" -dirhamnosylglucoside from Ginkgo biloba extract, but can also be applied to the extraction of other medicinal materials quercetin-3-O-2 ", 6" -dirhamnosylglucoside.
The invention has the advantages that:
compared with the prior art, the invention has the following characteristics:
1. quercetin-3-O-2 ', 6' -dirhamnosylglucoside has high purity: because the technical method used by the invention directly prepares the 6 '-dirhamnosylglucoside aiming at the quercetin-3-O-2' contained in the plant without heating and changing acid and alkali, the quercetin-3-O-2 ', 6' -dirhamnosylglucoside is prepared in the existing state in the medicinal materials, and no hydrolysate is doped, the purity of the obtained quercetin-3-O-2 ', 6' -dirhamnosylglucoside is high and reaches more than 75 percent.
2. The method is simple and easy to implement, and the operation is simple and convenient: the method for preparing quercetin-3-O-2 ', 6' -dirhamnosylglucoside, which is related by the invention, has the advantages of high instrument automation degree, convenient and simple operation, capability of being carried out at normal temperature and normal pressure, high repeatability and suitability for the requirement of large-scale production.
3. The medicinal materials have wide sources, are easy to obtain and have low price: ginkgo biloba is a unique precious tree species in China, accounts for 70 percent of the world resources, and isThe total leaf yield is 2X 10 per year7Over kg, the resource is very rich.
Detailed Description
The present invention will be described in detail with reference to examples.
Example 1: weighing folium Ginkgo extract 500g, dissolving in 1L methanol-water solution with volume concentration of 50% to obtain folium Ginkgo extract solution with concentration of 500mg/mL, filtering with 0.45 μm microporous membrane, and performing one-dimensional liquid chromatography on the liquid permeating the membrane. The one-dimensional liquid chromatographic column adopts Unitry C18(50 multiplied by 250mm, 10 mu, Wasie New science and technology Co., Ltd.), the mobile phase adopts acetonitrile (containing formic acid with the mass concentration of 0.1%) as an organic phase, water (containing formic acid with the mass concentration of 0.1%) as a water phase, and the linear gradient elution mode is as follows: the volume concentration of the organic phase increased from 5% to 30% over 60 minutes. Selecting absorption wavelength by adopting a DAD ultraviolet detector at 360nm, preparing at room temperature, sampling at 3000 mu L/needle, and flowing at a flow rate of 90mL/min for a mobile phase, collecting fractions for 20-25 min, and performing rotary evaporation and concentration to dryness to obtain a crude product of quercetin-3-O-2 ', 6' -dirhamnosylglucoside in one dimension. Dissolving a crude product of quercetin-3-O-2 ', 6' -dirhamnosylglucoside by using a methanol-water solution with the volume concentration of 40%, wherein the concentration is 10mg/mL, filtering the solution by using a microporous filter membrane, and preparing the solution penetrating through the membrane by using a two-dimensional liquid chromatography, wherein a chromatographic column is prepared by selecting methanol (containing 0.1% by mass of formic acid) as an organic phase and water (containing 0.1% by mass of formic acid) as a water phase for a Click PN (50X 150mm, 5 mu, Wasabun New technology Co., Ltd.) mobile phase and adopting the organic phase with the volume concentration of 10% for isocratic elution. Selecting an absorption wavelength by adopting a DAD ultraviolet detector at 360nm, selecting ions m/z as 303 by mass spectrum, preparing at room temperature, sampling volume of 1000 mu L/needle, flow velocity of a mobile phase of 100mL/min, collecting fractions with m/z of 303, rotating and evaporating to dryness to obtain quercetin-3-O-2 ', 6' -dirhamnosylglucoside compound, analyzing by liquid chromatography to obtain the purity of 90%, and preparing quercetin-3-O-2 ', 6' -dirhamnosylglucoside crude product with the mass content of quercetin-3-O-2 ', 6' -dirhamnosylglucoside of 35%.
Example 2: weighing folium Ginkgo extract 1000g, dissolving in 2L methanol-water solution with volume concentration of 50% to obtain folium Ginkgo extract solution with concentration of 500mg/mL, filtering with 0.45 μm microporous membrane, and performing one-dimensional liquid chromatography. The one-dimensional liquid chromatography adopts Unitry C8(50 x 250mm, 10 mu, Wasienna New science and technology Co., Ltd.), ethanol (containing 0.1% formic acid) is adopted as an organic phase as a mobile phase, water is adopted as a water phase, the volume concentration of the organic phase is 60% isocratic, a DAD ultraviolet detector is adopted to select an absorption wavelength at 360nm, the preparation temperature is room temperature, the sample injection amount is 500 mu L/needle, the flow rate of the mobile phase is 60mL/min, fractions of 5-10 minutes are collected and are subjected to rotary evaporation and concentration to dryness, and the one-dimensional preparation of crude quercetin-3-O-2 ', 6' -dirhamnosylglucoside is carried out. Dissolving quercetin-3-O-2 ', 6' -dirhamnosylglucoside crude product with a methanol-water solution with a volume concentration of 20%, wherein the concentration is 500mg/mL, filtering the solution through a microporous filter membrane, and performing two-dimensional liquid chromatography, wherein a chromatographic column is XUnion C18 (50X 150mm, 5 mu, Wasabh spectral New technology Co., Ltd.) mobile phase, acetonitrile (containing 0.1% by mass of formic acid) is selected as an organic phase, water (containing 0.1% by mass of formic acid) is selected as an aqueous phase, and the volume concentration of the organic phase is 30% isocratic. Selecting an absorption wavelength by adopting a DAD ultraviolet detector at 360nm, selecting ions m/z as 303 by mass spectrum, preparing at 40 ℃, sampling at 200 mu L/needle, collecting fractions with m/z of 303, rotating and evaporating to dryness to obtain quercetin-3-O-2 ', 6' -dirhamnosylglucoside compound, analyzing by liquid chromatography to obtain the purity of 70%, and preparing quercetin-3-O-2 ', 6' -dirhamnosylglucoside crude product with the mass content of quercetin-3-O-2 ', 6' -dirhamnosylglucoside of 20% by one dimension.
Example 3: weighing folium Ginkgo extract 2000g, dissolving in 2L methanol-water solution with volume concentration of 30% to obtain folium Ginkgo extract solution with concentration of 1000mg/mL, filtering with 0.45 μm microporous membrane, and performing one-dimensional liquid chromatography. One-dimensional liquid chromatography adopts XUnion C8 (20X 250mm, 10 mu, Wasaburra Innovation science and technology Co., Ltd.), the mobile phase adopts methanol as the organic phase, water as the water phase, and the elution is carried out in an isocratic mode by adopting the organic phase with the volume concentration of 5 percent. Selecting absorption wavelength by 360nm of DAD ultraviolet detector, preparing at 30 deg.C, sampling amount of 200 μ L/needle, flow rate of mobile phase of 20mL/min, collecting 15-25 min fraction, rotary evaporating for concentrating to dryness to obtain crude product of quercetin-3-O-2 ", 6" -dirhamnosylglucoside. Dissolving quercetin-3-O-2 ', 6' -dirhamnosylglucoside crude product with 70% methanol-water solution, the concentration is 500mg/mL, filtering with microporous membrane, and performing two-dimensional liquid chromatography with XTerra C18(50 × 150mm, 5 μ, Waters) as chromatographic column, methanol as organic phase and water as water phase as mobile phase, and increasing the volume concentration of the organic phase from 5% to 35% in 30 min by linear gradient. The absorption wavelength is selected by adopting a DAD ultraviolet detector at 360nm, the ion m/z is selected to be 303 by mass spectrum, the preparation temperature is 30 ℃, the sample injection amount is 200 mu L/needle, the flow velocity of a mobile phase is 120mL/min, and the fraction with the m/z of 303 is collected. Rotating to evaporate to dryness to obtain quercetin-3-O-2 ', 6' -dirhamnosylglucoside compound, analyzing by liquid chromatography to obtain a product with purity of 50%, and one-dimensionally preparing crude product of quercetin-3-O-2 ', 6' -dirhamnosylglucoside with quercetin-3-O-2 ', 6' -dirhamnosylglucoside content of 15%.
Example 4: weighing 100g folium Ginkgo extract, dissolving in 500mL 55% methanol-water solution with volume concentration of 200mg/mL to obtain folium Ginkgo extract solution, filtering with 0.45 μm microporous membrane, and performing one-dimensional liquid chromatography. The one-dimensional liquid chromatography adopts Click PN (50 multiplied by 100mm, 5 mu, Wasabun New science and technology Co., Ltd.), the mobile phase adopts methanol (containing formic acid with mass concentration of 0.1%) as an organic phase, water (containing formic acid with mass concentration of 0.1%) as a water phase, and the gradient is adopted: the volume concentration of the organic phase is increased from 5 percent to 70 percent after 50 minutes, a DAD ultraviolet detector is adopted to select the absorption wavelength at 360nm, the preparation temperature is 25 ℃, the sample injection amount is 3000 mu L/needle, the flow rate of the mobile phase is 120mL/min, the fraction for 20-25 minutes is collected and is subjected to rotary evaporation concentration to dryness, and the crude product of quercetin-3-O-2 ', 6' -dirhamnosylglucoside is prepared in one dimension. Dissolving quercetin-3-O-2 ', 6' -dirhamnosylglucoside crude product with concentration of 80mg/mL with 60% methanol-water solution, filtering with microporous membrane, and performing two-dimensional liquid chromatography with chromatographic column of Unitry C8(50 × 100mm, 10 μ, Wasabre New technology Co., Ltd.), mobile phase of ethanol (containing 0.1% formic acid by mass concentration) as organic phase, water (containing 0.1% formic acid by mass concentration) as water phase, and isocratic elution with organic phase concentration of 15% by volume concentration. Selecting an absorption wavelength by adopting a DAD ultraviolet detector at 360nm, selecting ions m/z as 303 by mass spectrum, preparing at 25 ℃, sampling at 200 mu L/needle, collecting fractions with m/z of 303, rotating and evaporating to dryness to obtain quercetin-3-O-2 ', 6' -dirhamnosylglucoside compound, analyzing by liquid chromatography to obtain the purity of 80%, and preparing quercetin-3-O-2 ', 6' -dirhamnosylglucoside crude product with the content of quercetin-3-O-2 ', 6' -dirhamnosylglucoside of 35% in one-dimensional way.
Comparative example 1
The operation process is the same as that of example 1, except that: the one-dimensional chromatographic column adopts XTerra C18(50 × 250mm, 5 μ, Waters) to obtain quercetin-3-O-2 ", 6" -dirhamnosylglucoside compound, and liquid chromatography analysis shows that the purity is 30%, and the one-dimensional preparation of quercetin-3-O-2 ", 6" -dirhamnosylglucoside crude product contains quercetin-3-O-2 ", 6" -dirhamnosylglucoside 5%.
Comparative example 2
The operation process is the same as that of example 1, except that: the one-dimensional chromatographic column adopts XAmide (50 × 250mm, 10 μ, Wasp. New science and technology Co., Ltd.) to prepare quercetin-3-O-2 ″, the content of quercetin-3-O-2 ″,6 ″ -dirhamnosylglucoside in crude product of 6 ″ -dirhamnosylglucoside is 0%.
Comparative example 3
The operation process is the same as that of example 1, except that: the two-dimensional chromatographic column adopts XAmide (50 × 150mm, 10 μ, Wasabsu New science and technology, Ltd.), and the obtained quercetin-3-O-2 ', 6' -dirhamnosylglucoside compound content is 0%.
The invention provides a preparation method of quercetin-3-O-2 ', 6' -dirhamnosylglucoside, which adopts two-dimensional liquid chromatography for preparation, takes methanol (or containing 0.1% formic acid) -water, ethanol (or containing 0.1% formic acid) -water or acetonitrile (or containing 0.1% formic acid) -water as a mobile phase, takes a reversed phase chromatographic column as a one-dimensional preparation chromatographic column, cuts components of a ginkgo leaf extract, and collects a target component which is a crude product of quercetin-3-O-2 ', 6' -dirhamnosylglucoside. And then, taking the reversed phase chromatographic column as a two-dimensional preparation chromatographic column, and carrying out component cutting, collection and rotary evaporation concentration on the crude product of the quercetin-3-O-2 ', 6' -dirhamnosylglucoside under the guidance of a mass spectrum selected ion peak to obtain the high-purity quercetin-3-O-2 ', 6' -dirhamnosylglucoside, wherein the purity can reach over 75 percent. The preparation process has high repeatability and good operability, and simultaneously, the ginkgo leaf resource is rich and easy to obtain, thereby being suitable for the requirement of large-scale production.
The applicant states that the present invention is illustrated by the above examples to show the detailed process flow of the present invention, and the present invention is not limited to the above detailed process flow, i.e. it does not mean that the present invention must rely on the above detailed process flow to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitution of each raw material and addition of auxiliary components to the product of the present invention, selection of specific modes, etc., are within the scope of protection and disclosure of the present invention.
Claims (6)
1. A method for preparing quercetin-3-O-2 ', 6' -dirhamnosylglucoside comprises the following steps:
(1) dissolving the Chinese medicinal extract in methanol-water, ethanol-water or acetonitrile-water to obtain Chinese medicinal extract solution;
(2) preparing quercetin-3-O-2 '', 6 '' -dirhamnosylglucoside from the Chinese medicinal extract by two-dimensional liquid chromatography separation and purification technology; the chromatographic column prepared in one dimension by adopting liquid chromatography is reversed phase C18, reversed phase C8 or Click PN; the chromatographic column prepared in two dimensions by adopting liquid chromatography is reversed phase C18, reversed phase C8 or Click PN; the column for Click PN was 50 x 150mm, 5 μ,
the traditional Chinese medicine extract is a ginkgo leaf extract;
dissolving folium Ginkgo extract in 20-80 vol% methanol-water, ethanol-water or acetonitrile-water solution to obtain folium Ginkgo extract solution with folium Ginkgo extract concentration of 10-2000 mg/mL;
the preparation of the one-dimensional liquid chromatography adopts a mobile phase of methanol, ethanol or acetonitrile and an organic phase, wherein the organic phase does not contain or contains 0.1 percent of formic acid in mass concentration and is mixed with water of water phase, and the elution condition is carried out according to the linear gradient that the organic phase volume concentration is increased from 5 percent to 80 percent in 5-60 percent isocratic or from 0 to 15-60 minutes; collecting the component with main ingredient of quercetin-3-O-2 '', 6 '' -dirhamnosylglucoside with retention time of 5-25 min, rotary evaporating to dryness, and one-dimensionally preparing crude product of quercetin-3-O-2 '', 6 '' -dirhamnosylglucoside; dissolving the crude product of quercetin-3-O-2 ', 6' -dirhamnosylglucoside prepared in one dimension with methanol-water solution with volume concentration of 20-70% until the concentration is 10-500 mg/mL;
performing two-dimensional liquid chromatography on the one-dimensional collected and concentrated sample; the adopted mobile phase is methanol ethanol or acetonitrile of an organic phase without containing or containing 0.1 percent of formic acid by mass concentration and water of a water phase are mixed, elution is carried out according to the 5-30 percent isocratic degree of the organic phase or the gradient that the volume concentration of the organic phase is increased from 5 percent to 30 percent after 0-15-60 minutes, and the quercetin-3-O-2 ', 6' -dirhamnosylglucoside component is collected;
the specification of the chromatographic column is 20-50 cm of inner diameter; the flow rate of the mobile phase is 20-120 mL/min during preparation, the column temperature is room temperature or 25-40 ℃ during preparation, and the sample injection amount is 200-; the separation and purification technology is to combine two-dimensional liquid chromatography preparation with mass spectrometry coupling technology to prepare folium Ginkgo extract quercetin-3-O-2 '', 6 '' -dirhamnosylglucoside, wherein the detector is a DAD ultraviolet detector during one-dimensional preparation and a mass spectrometry detector during two-dimensional preparation.
2. The method of claim 1, wherein: the ginkgo biloba extract is dissolved in 40-60% methanol-water, ethanol-water or acetonitrile-water solution, and the concentration of the ginkgo biloba extract is 250-550 mg/mL.
3. The method of claim 1, wherein: the optimal two-dimensional chromatographic column combination is one-dimensional reverse phase C18 and two-dimensional reverse phase Click PN.
4. The method of claim 3, wherein: the optimal mobile phase for one-dimensional preparation is acetonitrile containing no or 0.1% by mass of formic acid and water containing no or 0.1% by mass of formic acid, and the volume proportion of the acetonitrile phase is increased from 10% to 30-40% in 20-30 minutes by adopting a linear gradient mode.
5. The method of claim 4, wherein: one-dimensional preparation and collection are carried out for 12-18 minutes, wherein the component mainly contains quercetin-3-O-2 ', 6' -dirhamnosylglucoside, and one-dimensional preparation of a crude product of quercetin-3-O-2 ', 6' -dirhamnosylglucoside is prepared into a crude solution of quercetin-3-O-2 ', 6' -dirhamnosylglucoside with the concentration of 20-200 mg/mL by using a methanol-water solution with the volume concentration of 40-60%; the optimal mobile phase for two-dimensional preparation is acetonitrile containing no or 0.1% by mass of formic acid and water containing no or 0.1% by mass of formic acid, and the volume proportion of the acetonitrile phase is increased from 5% to 15% -25% in 25-35 minutes by adopting a linear gradient condition.
6. The method of claim 3, wherein: the optimal flow rate of the mobile phase during preparation is 80-100 mL/min, the column temperature during preparation is room temperature, the sample injection amount during preparation is 2000-; performing rotary evaporation and concentration on quercetin-3-O-2 '', 6 '' -dirhamnosylglucoside fractions collected by two-dimensional liquid chromatography to dry to obtain a quercetin-3-O-2 '', 6 '' -dirhamnosylglucoside compound.
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