CN109021046B - Method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori - Google Patents
Method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori Download PDFInfo
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Abstract
A method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of fructus Siraitiae Grosvenorii comprises the following steps: (1) sun drying fresh stem and leaf of fructus Siraitiae Grosvenorii, crushing, adding water, performing continuous countercurrent flash extraction, centrifuging, and filtering; (2) ultrafiltering, adsorbing with macroporous adsorbent resin column, washing with water, discarding water washing solution, eluting with organic solvent, collecting eluate, washing with water, collecting water washing solution, concentrating, and drying; (3) adding into alcohol solution, heating for dissolving, cooling, standing, filtering, concentrating, and drying; (4) dissolving with mobile phase, filtering, preparing liquid chromatography column, isocratic eluting, collecting target section eluate, concentrating, and drying to obtain quercetin and kaempferitrin. The purity of the quercetin product obtained by the method is more than or equal to 98.2 percent, the yield is more than or equal to 90.4 percent, the purity of the kaempferitrin product is more than or equal to 98.8 percent, and the yield is more than or equal to 91.3 percent; the method has simple and stable operation process, low energy consumption and cost and can realize continuous large-scale production.
Description
Technical Field
The invention relates to a method for extracting flavonoid glycoside substances, in particular to a method for simultaneously extracting quercetin and kaempferitrin from stems and leaves of momordica grosvenori.
Background
Momordica grosvenori is a perennial plant of the Cucurbitaceae family, and is mainly produced in Guangxi Guilin areas. The whole body of the momordica grosvenori is treasure: the fresh fructus momordicae contains rich components such as mogrosides, proteins, polysaccharides and the like, and particularly, the mogrosides serving as natural sweeteners have great market and economic values; the momordica grosvenori root tuber contains a large amount of starch and momordica grosvenori fruit acid, and the momordica grosvenori starch has high nutritional value; the momordica grosvenori seeds contain rich grease, and the main active component of the momordica grosvenori seeds is squalene, so that the momordica grosvenori seeds have the effects of resisting cancer, preventing cancer, preserving moisture and beautifying; the stem and leaf of momordica grosvenori contains more flavonoid glycoside substances, mainly quercetin and kaempferol, and the flavonoid glycoside substances have remarkable pharmacological and physiological activities, can expand blood vessels, increase blood circulation, inhibit thrombosis, and have the effects of resisting inflammation, resisting cancer, inhibiting bacteria, reducing blood sugar, reducing blood fat and the like.
At present, the early mature momordica glycoside is extracted from momordica grosvenori fruits, and after the fresh momordica grosvenori fruits are picked, stems, leaves and rattans of the momordica grosvenori fruits are usually directly thrown away, so that the environment is polluted, and huge resource waste is caused.
CN106668234A discloses an extraction and purification process of rose total flavonoids, which comprises the following steps: solvent extraction → macroporous resin purification → polyamide resin purification → C18 preparation and purification. Although the process can obtain the product with the total flavone purity of more than or equal to 90 percent, the whole process is complex, consumes more organic solvent, is difficult to recover, has low flavone recovery rate, and realizes large-scale industrial production with higher cost.
CN102138958A discloses a process for extracting and purifying flavonoid components of momordica grosvenori plants, which comprises the following steps: ethanol extraction → solvent recovery → macroporous resin impurity removal → elution → concentration and drying → alcohol soluble impurity removal → concentration and drying. Although the process can obtain the product with the purity of the flavonoid glycoside being more than or equal to 50 percent, and the process is simple and stable, the purity of the purified flavone is not high, and the recovery rate is low although the one-step alcohol-soluble impurity removal is simple and effective.
CN107698638A discloses an extraction and purification process, a detection method and application of total flavonoids of Chinese wolfberry, the process flow is as follows: drying → crushing → extraction → macroporous resin purification → polyamide resin purification. Although the total flavonoids of the Chinese wolfberry fruits obtained by the process are more than or equal to 40 percent, the extraction process uses 80 percent ethanol extraction and petroleum ether extraction, so that the consumption of a solvent is large, the requirement on production equipment is high, and the cost is high.
CN102399252A discloses a preparation method of a cowherb seed flavonoid glycoside monomer, which comprises the following process flows: raw material extraction → macroporous resin enrichment → high performance liquid phase separation → macroporous resin re-enrichment → elution → concentration → drying. Although the cowherb seed flavonoid glycoside monomer obtained by the process is more than or equal to 99 percent, the process has complex steps, macroporous resin is used for enrichment for 2 times, the solvent consumption is large, and the recovery rate is low.
CN102050845A discloses a method for simultaneously preparing calycosin glycoside and formononetin chemical reference substances, which comprises the following process flows: enriching by silica gel column chromatography → removing impurities by resin column → refining by reverse high performance liquid chromatography → drying under reduced pressure. Although the process can simultaneously prepare the calycosin glycoside and the formononetin with the purity of more than or equal to 98 percent, the silica gel chromatographic enrichment method has the advantages of small treatment capacity, high consumption of organic solvent, high requirement on production environment and high large-scale production cost.
CN102503998A discloses a method for rapidly separating quercetin from albizia julibrissin, which comprises the following steps: raw material extraction → macroporous resin enrichment → concentrated crystallization → high-efficiency preparation liquid phase separation → cooling crystallization → drying. Although this process can obtain quercetin monomer, the recovery rate is low due to the 2-time crystallization.
CN104592327A discloses a method for preparing rutin and isoquercitrin in hops and detection of anti-allergic and antioxidant activities thereof, and the process flow is as follows: raw material extraction → extraction impurity removal → macroporous resin enrichment → semi-preparative high performance liquid separation. Although the process can obtain rutin and isoquercitrin monomers, the process is complex, the consumption of the solvent is large and the cost is high because petroleum ether, ethyl acetate and n-butanol are required to be used for extraction and impurity removal in sequence.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects in the prior art and providing the method for simultaneously extracting the quercetin and the kaempferitrin from the stem and the leaf of the momordica grosvenori, which has the advantages of high purity and yield of the obtained product, simple and stable operation process, low energy consumption and cost and capability of realizing continuous large-scale production.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of fructus Siraitiae Grosvenorii comprises the following steps:
(1) crushing and extracting: sun drying fresh stem and leaf of fructus Siraitiae Grosvenorii, crushing, adding water, performing continuous countercurrent flash extraction, centrifuging, and filtering to obtain extractive solution;
(2) ultrafiltration and macroporous resin adsorption: carrying out ultrafiltration on the extracting solution obtained in the step (1), adsorbing the extracting solution by using a macroporous adsorption resin column, washing with water until an effluent liquid is colorless, clear and transparent, discarding a washing liquid, eluting with an organic solvent, collecting an eluent, washing with water until no organic solvent exists, collecting the washing liquid, concentrating under reduced pressure, and drying in vacuum to obtain a crude product A of the flavonoid glycoside;
(3) alcohol dissolving and impurity removing: adding the crude flavonoid glycoside A obtained in the step (2) into an alcohol solution, heating for dissolving, cooling, standing, filtering, concentrating under reduced pressure, and drying in vacuum to obtain crude flavonoid glycoside B;
(4) preparing liquid phase chromatographic separation: and (4) dissolving the crude flavonoid glycoside B obtained in the step (3) by using a mobile phase, filtering, separating by using a preparative liquid chromatography column, performing isocratic elution by using the mobile phase, respectively collecting target section eluent, concentrating under reduced pressure, and drying in vacuum to respectively obtain the quercitrin and the kaempferitrin.
Preferably, in the step (1), the mass content of quercetin in the fresh stems and leaves of the momordica grosvenori is 0.04-0.08%, and the mass content of kaempferitrin is 0.5-1.0%.
Preferably, in the step (1), fresh stems and leaves of the momordica grosvenori are crushed to 20-40 meshes.
Preferably, in the step (1), the amount of the water is 4-20 times (more preferably 5-15 times) of the mass of the fresh stems and leaves of the momordica grosvenori. Because the momordica grosvenori flavonoid glycoside is very soluble in water, especially hot water, the cost is low by adopting water as a solvent, and the momordica grosvenori flavonoid glycoside is environment-friendly and safe.
Preferably, in the step (1), the temperature of the continuous countercurrent flash extraction is 50-90 ℃, the extraction frequency is more than or equal to 2 times, and the extraction time is 60-200 s (more preferably 100-180 s) each time. Due to the special property of easy oxidation of flavonoids, if the extraction temperature is too high or the extraction time is too long, the extraction recovery rate is reduced, so that continuous and large-scale production can be realized by adopting continuous countercurrent flash extraction, the extraction time is shortened, and the recovery rate can be improved.
Preferably, in the step (1), the rotation speed of the centrifugation is 10000-80000 r/min (more preferably 40000-60000 r/min).
Preferably, in the step (1), the centrifugal filtration is performed by firstly horizontal spiral centrifugal filtration and then butterfly centrifugal filtration.
Preferably, in the step (2), the ultrafiltration membrane used for ultrafiltration is a ceramic membrane, and the pore diameter of the ceramic membrane is 0.2-2.0 μm (more preferably 0.3-1.0 μm).
Preferably, in the step (2), the flow rate of the upper column is 1.0-4.0 BV/h. Since the flavonoid glycoside is a substance with larger polarity, the flavonoid glycoside is easy to be absorbed by macroporous absorption resin, thereby achieving the purpose of purification and separation.
Preferably, in the step (2), the type of the macroporous adsorption resin is one or more of D-101 type, AB-8 type, DM-130 type, X-7 type or XDA-7 type and the like.
In the method, before the macroporous adsorption resin is used, 1-3 BV of ethanol with the volume fraction of 90-99% is soaked for 20-30 h, then the macroporous adsorption resin is washed with 90-99% of ethanol until effluent liquid is colorless and odorless, the macroporous adsorption resin is washed with water until the effluent liquid is free of alcohol smell, then the macroporous adsorption resin is washed with alkali by 3-5 BV of sodium hydroxide solution with the mass concentration of 4-6%, then the macroporous adsorption resin is washed with water until the effluent liquid is neutral, finally the macroporous adsorption resin is washed with hydrochloric acid solution with the mass concentration of 3-5 BV of 4-6%, and then the macroporous adsorption resin is washed with water.
Preferably, in the step (2), the volume-to-mass ratio (L/kg) of the macroporous adsorption resin to the fresh stems and leaves of the momordica grosvenori is 1: 1-20 (more preferably 1: 5-15).
Preferably, in the step (2), the diameter-height ratio of the macroporous adsorption resin column is 1: 2-8 (more preferably 1: 4-6).
Preferably, in the step (2), the flow rate of the water washing is 1.0-4.0 BV/h. The purpose of water washing is mainly to remove impurities with strong water solubility, such as monosaccharide, polysaccharide, pectin and the like.
Preferably, in the step (2), the amount of the organic solvent is 1-4 BV.
Preferably, in the step (2), the flow rate of the organic solvent elution is 0.25 to 2.00BV/h (more preferably 0.5 to 1.0 BV/h). The purpose of the organic solvent elution is to elute off the flavonoid glycosides adsorbed by the resin.
Preferably, in the step (2), the volume fraction of the organic solvent is 50-90%.
Preferably, in step (2), the organic solvent is food grade ethanol.
Preferably, in the step (2), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%.
Preferably, in the step (2), the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the time is 4-12 h.
Preferably, in the step (3), the mass-to-volume ratio (kg/L) of the crude flavonoid glycoside A to the alcohol solution is 1: 8-20 (more preferably 1: 9-15). The alcohol-soluble impurity removal mainly removes impurities with good water solubility and poor alcohol solubility, such as protein and the like.
Preferably, in the step (3), the heating temperature is 50-80 ℃.
Preferably, in the step (3), the alcohol solution is food-grade anhydrous methanol or food-grade ethanol with a volume fraction of 90-99%.
Preferably, in step (3), the cooling is to room temperature. Plate cooling is preferred.
Preferably, in the step (3), the standing time is 2-4 h. The purpose of standing is to fully remove impurities, easily separate the impurities and facilitate subsequent filtration.
Preferably, in the step (3), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%.
Preferably, in the step (3), the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the time is 4-12 h.
Preferably, in the step (4), the mass-to-volume ratio (kg/L) of the crude flavonoid glycoside B to the mobile phase is 0.08-0.30: 1. The separation and purification effect is better under the proportion.
Preferably, in the step (4), the mobile phase is an organic solvent-water solution with a volume ratio of 15-40: 95-60. The separation and purification effect is better and the recovery rate is higher under the proportion.
Preferably, in the step (4), the organic solvent is one or more of ethanol, methanol, acetonitrile, or the like. More preferably, ethanol. The solvent has good separation effect, is convenient to recover and has lower cost.
Preferably, in the step (4), the filter membrane used for filtration is an organic phase filter membrane, and the pore diameter is 0.20-0.45 μm. The main purpose of filtration is to remove a small amount of suspended impurities and prevent the prepared liquid phase from blocking the column and affecting the separation effect.
Preferably, in the step (4), the flow rate of the sample injection is 0.25-1.00 BV/h.
Preferably, in the step (4), the preparative liquid chromatography column is an ODS column, and the packing is C-18 reverse phase bonded silica gel. The inventor researches and discovers that the column type and the filler have more obvious separation and purification effects, higher separation degree and higher recovery rate.
Preferably, in the step (4), the flow rate of the elution is 0.5-2.0 BV/h. The inventor finds that the separation effect is better under the flow rate, and the recovery rate of the target component is higher.
Preferably, in the step (4), after the flow elution, eluents of the target products of quercitrin and kaempferitrin are respectively collected according to the retention time of two independent peaks appearing on a signal display screen of a prepared liquid-phase UV detector.
Preferably, in the step (4), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%.
Preferably, in the step (4), the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the time is 4-12 h.
In the steps (3) and (4), the filtration is preferably plate-frame filtration or plate-frame pressure filtration.
The method has the following beneficial effects:
(1) the quercetin product and the kaempferitrin product obtained by the method are yellow, the purity of the quercetin in the quercetin product is more than or equal to 98.2 percent, the yield is more than or equal to 90.4 percent, the purity of the kaempferitrin in the kaempferitrin product is more than or equal to 98.8 percent, and the yield is more than or equal to 91.3 percent;
(2) because the solvent extracted by the method is water, compared with the traditional organic solvent extraction, the method is more environment-friendly, safer and low in cost; continuous countercurrent flash extraction is adopted, and compared with the traditional reflux extraction, the method can ensure continuous and large-scale production, and has short extraction time and high recovery rate;
(3) the preparation liquid phase of the method adopts the organic solvent-water mobile phase, and is eluted at equal degree, compared with other organic mixed solvents, the method has the advantages of lower cost, simple and convenient operation, and capability of recycling the mobile phase, and is suitable for large-scale industrial production;
(4) the method combines macroporous resin adsorption and purification → alcohol soluble impurity removal → preparation of liquid chromatography separation, has simple and stable process and ensures the high purity of the product.
Detailed Description
The present invention will be further described with reference to the following examples.
The stem and leaf of the momordica grosvenori used in the embodiment of the invention are purchased from Guangxi Guilin, and the HPLC detection shows that the mass content of the quercitrin is 0.06 percent, and the mass content of the kaempferitrin is 0.73 percent; the ceramic membranes with the aperture of 0.3 mu m and the aperture of 0.5 mu m used in the embodiment of the invention are all purchased from Jiangsu Jiu Anyuan environmental protection science and technology Limited company; the D-101 type and AB-8 type macroporous adsorption resins used in the embodiment of the invention are purchased from New science and technology materials, Inc. of Xian blue and Xiao; the X-5 type macroporous adsorption resin used in the embodiment of the invention is purchased from Tianjin Nankai and science and technology Limited; organic phase filter membranes with the aperture of 0.22 mu m and the aperture of 0.45 mu m used in the embodiment of the invention are purchased from Shanghai Xinya purification device factories; the ODS column and the C-18 reverse phase bonding silica gel used in the embodiment of the invention are purchased from Jiangsu Hanbang science and technology Co., Ltd; the raw materials and chemicals used in the examples of the present invention were obtained from conventional commercial sources unless otherwise specified.
Reference example 1
Before the macroporous adsorption resin is used, 2BV of ethanol with volume fraction of 95% is soaked for 24 hours, then the macroporous adsorption resin is washed by the ethanol with volume fraction of 95% until effluent liquid is colorless and odorless, and is washed by water until no alcohol odor exists, then sodium hydroxide solution with mass concentration of 4BV and 5% is used for alkali washing, then the macroporous adsorption resin is washed by the water until the effluent liquid is neutral, finally hydrochloric acid solution with mass concentration of 4BV and 5% is used for acid washing, and then the macroporous adsorption resin is washed by the water until the effluent liquid is neutral for later use.
Example 1
(1) Crushing and extracting: sun drying 10t fresh stem and leaf of fructus Siraitiae Grosvenorii, crushing to 20 mesh, adding 100t water, performing continuous countercurrent flash extraction at 60 deg.C for 2 times, 150s each time, centrifuging at 60000r/min with horizontal spiral centrifuge, and centrifuging with butterfly centrifuge to obtain 95t extractive solution;
(2) ultrafiltration and macroporous resin adsorption: performing ultrafiltration on 95t of the extract obtained in the step (1) by using a 0.5-micron ceramic membrane, adsorbing an ultrafiltration permeate by using an AB-8 type macroporous adsorption resin column (the volume of the AB-8 type macroporous adsorption resin is 1000L, and the diameter-height ratio is 1: 4) at the flow rate of 3BV/h, washing with water at the flow rate of 2BV/h until an effluent is colorless, clear and bright, discarding a water washing solution, eluting with food grade ethanol with the volume fraction of 80% by using 2BV at the flow rate of 0.5BV/h, collecting the eluent, washing with water until no ethanol exists, collecting a water washing solution, concentrating the collected eluent and the water washing solution under reduced pressure at the temperature of 50 ℃ and under the pressure of 0.1MPa until the solid content is 45%, and performing vacuum drying at the temperature of 40 ℃ and the pressure of-0.1 MPa for 6h to obtain 131.29kg of a crude flavonoid glycoside product A;
(3) alcohol dissolving and impurity removing: 131.29kg of crude flavonoid glycoside A obtained in the step (2) is added into 1500L of food grade ethanol solution with the volume fraction of 95%, the solution is heated and dissolved at the temperature of 60 ℃, the solution is cooled to the room temperature in a plate type, the solution is kept stand for 2h, the filtrate is filtered by a plate frame, the filtrate is decompressed and concentrated at the temperature of 40 ℃ and the pressure of-0.1 MPa until the solid content is 50%, and the filtrate is dried in vacuum at the temperature of 40 ℃ and the pressure of-0.1 MPa for 8h to obtain 93.23kg of crude flavonoid glycoside B;
(4) preparing liquid phase chromatographic separation: 93.23kg of crude flavonoid glycoside B obtained in the step (3) is dissolved by 1100L of mobile phase ethanol-water solution (volume ratio is 30: 70), the dissolved solution is filtered by an organic phase filter membrane plate frame with the aperture of 0.22 mu m, then, a liquid chromatography column (ODS column, packing C-18 reverse phase bonded silica gel) was prepared at a flow rate of 0.25BV/h for separation, and then isocratic elution was carried out with a mobile phase ethanol-water solution (volume ratio 30: 70) at a flow rate of 1.0BV/h, collecting 10-25 BV of eluent of a quercitrin target section and 30-40 BV of eluent of a kaempferitrin target section respectively according to the retention time of two independent peaks appearing on a signal display screen of a prepared liquid-phase UV detector, concentrating under reduced pressure at 40 deg.C and-0.1 MPa to solid content of 55%, and vacuum drying at 40 deg.C and-0.1 MPa for 6 hr to obtain quercetin 5.58kg and kaempferitrin 67.89kg, respectively.
The quercetin product and the kaempferitrin product obtained in the embodiment of the invention are yellow, and the purity of the quercetin in the quercetin product is 98.2 percent, the yield is 91.3 percent, the purity of the kaempferitrin is 99.1 percent and the yield is 92.2 percent through HPLC detection.
Example 2
(1) Crushing and extracting: sun drying 10t fresh stem and leaf of fructus Siraitiae Grosvenorii, pulverizing to 40 mesh, adding 150t water, performing continuous countercurrent flash extraction at 80 deg.C for 3 times, each time for 100s, centrifuging at 40000r/min with horizontal spiral centrifuge, and centrifuging with butterfly centrifuge to obtain 145t extractive solution;
(2) ultrafiltration and macroporous resin adsorption: performing ultrafiltration on 145t of the extract obtained in the step (1) by using a 0.3 mu m ceramic membrane, adsorbing an ultrafiltration permeating liquid by using an X-5 type macroporous adsorption resin column (the volume of the X-5 type macroporous adsorption resin is 1500L, and the diameter-height ratio is 1: 6) at the flow rate of 4BV/h, washing with water at the flow rate of 4BV/h until an effluent liquid is colorless, clear and bright, discarding a water washing liquid, eluting with food grade ethanol with the volume fraction of 60% at the flow rate of 1.0BV/h by using 4BV, collecting the eluent, washing with water until no ethanol exists, collecting the water washing liquid, concentrating the collected eluent and the water washing liquid under reduced pressure at 70 ℃ and under 0.08MPa until the solid content is 50%, and performing vacuum drying at 60 ℃ and under-0.08 MPa for 8h to obtain 125.6kg of a crude flavonoid glycoside product;
(3) alcohol dissolving and impurity removing: adding 125.6kg of crude flavonoid glycoside A obtained in the step (2) into 1200L of food-grade anhydrous methanol solution, heating and dissolving at 80 ℃, cooling in a plate-type manner to room temperature, standing for 4h, filtering with a plate frame, concentrating the filtrate at 60 ℃ and-0.08 MPa under reduced pressure until the solid content is 40%, and drying in vacuum at 60 ℃ and-0.08 MPa for 6h to obtain 91.8kg of crude flavonoid glycoside B;
(4) preparing liquid phase chromatographic separation: dissolving 91.8kg of crude flavonoid glycoside B obtained in step (3) with 800L of mobile phase methanol-water solution (volume ratio of 20: 80), press-filtering the dissolved solution with organic phase filter membrane plate frame with pore diameter of 0.45 μm, then, a preparative liquid chromatography column (ODS column, packing material C-18 reverse phase bonded silica gel) was separated at a flow rate of 0.75BV/h, and isocratic elution was carried out with a mobile phase methanol-water solution (volume ratio 20: 80) at a flow rate of 2.0BV/h, collecting 10-25 BV of eluent of a quercitrin target section and 30-40 BV of eluent of a kaempferitrin target section respectively according to the retention time of two independent peaks appearing on a signal display screen of a prepared liquid-phase UV detector, concentrating under reduced pressure at 60 deg.C and-0.08 MPa to solid content of 60%, and vacuum drying at 60 deg.C and-0.08 MPa for 10 hr to obtain quercetin 5.5kg and kaempferitrin 66.8kg, respectively.
The quercetin product and the kaempferitrin product obtained in the embodiment of the invention are yellow, and the purity of the quercetin in the quercetin product is 98.6 percent, the yield is 90.4 percent, the purity of the kaempferitrin is 99.8 percent and the yield is 91.3 percent through HPLC detection.
Example 3
(1) Crushing and extracting: sun drying 10t fresh stem and leaf of fructus Siraitiae Grosvenorii, crushing to 30 mesh, adding 120t water, performing continuous countercurrent flash extraction at 70 deg.C for 2 times (120 s each time), centrifuging at 50000r/min with horizontal spiral centrifuge, and centrifuging with butterfly centrifuge to obtain 95t extractive solution;
(2) ultrafiltration and macroporous resin adsorption: performing ultrafiltration on 95t of the extract obtained in the step (1) by using a 0.5-micron ceramic membrane, adsorbing an ultrafiltration permeate by using a D-101 type macroporous adsorption resin column (the volume of the D-101 type macroporous adsorption resin is 1200L, and the diameter-height ratio is 1: 5) at the flow rate of 2BV/h, washing with water at the flow rate of 3BV/h until an effluent is colorless, clear and bright, discarding a water washing solution, eluting with food grade ethanol with the volume fraction of 70% at the flow rate of 0.75BV/h by using 3BV, collecting the eluent, washing with water until no ethanol exists, collecting the water washing solution, concentrating the collected eluent and the water washing solution under reduced pressure at the temperature of 60 ℃ and the pressure of 0.09MPa until the solid content is 48%, and performing vacuum drying at the temperature of 50 ℃ and the pressure of-0.09 MPa for 11h to obtain 140.3kg of a crude flavonoid glycoside product A;
(3) alcohol dissolving and impurity removing: adding 140.3kg of crude flavonoid glycoside A obtained in the step (2) into 1500L of 92% food grade ethanol solution by volume, heating and dissolving at 70 ℃, cooling in a plate-type manner to room temperature, standing for 3h, filtering with a plate frame, concentrating the filtrate at 50 ℃ and-0.09 MPa under reduced pressure until the solid content is 47%, and vacuum-drying at 50 ℃ and-0.09 MPa for 10h to obtain 89.5kg of crude flavonoid glycoside B;
(4) preparing liquid phase chromatographic separation: dissolving 89.5kg of crude flavonoid glycoside B obtained in step (3) with 1000L of mobile phase acetonitrile-water solution (volume ratio of 26: 74), press-filtering the dissolved solution with an organic phase filter membrane plate frame with the aperture of 0.22 μm, then, the mixture was subjected to separation by preparative liquid chromatography (ODS column packed with C-18 reverse phase bonded silica gel) at a flow rate of 0.5BV/h, followed by isocratic elution with a mobile phase acetonitrile-water solution (volume ratio: 26: 74) at a flow rate of 1.5BV/h, collecting eluents of 5-15 BV of quercetin target section and 20-30 BV of kaempferitrin target section respectively according to the retention time of two independent peaks appearing on a signal display screen of a prepared liquid-phase UV detector, concentrating under reduced pressure at 50 deg.C and-0.09 MPa to solid content of 56%, and vacuum drying at 50 deg.C and-0.09 MPa for 8 hr to obtain quercetin 5.75kg and kaempferitrin 70.5kg, respectively.
The quercetin product and the kaempferitrin product obtained in the embodiment of the invention are yellow, and the purity of the quercetin in the quercetin product is 98.4%, the yield is 94.3%, the purity of the kaempferitrin is 98.8% and the yield is 95.4% through HPLC detection.
Claims (12)
1. A method for simultaneously extracting quercetin and kaempferitrin from stem and leaves of momordica grosvenori is characterized by comprising the following steps:
(1) crushing and extracting: sun drying fresh stem and leaf of fructus Siraitiae Grosvenorii, crushing, adding water, performing continuous countercurrent flash extraction, centrifuging, and filtering to obtain extractive solution;
(2) ultrafiltration and macroporous resin adsorption: carrying out ultrafiltration on the extracting solution obtained in the step (1), adsorbing the extracting solution by using a macroporous adsorption resin column, washing with water until an effluent liquid is colorless, clear and transparent, discarding a washing liquid, eluting with an organic solvent, collecting an eluent, washing with water until no organic solvent exists, collecting the washing liquid, concentrating under reduced pressure, and drying in vacuum to obtain a crude product A of the flavonoid glycoside;
(3) alcohol dissolving and impurity removing: adding the crude flavonoid glycoside A obtained in the step (2) into an alcohol solution, heating for dissolving, cooling, standing, filtering, concentrating under reduced pressure, and drying in vacuum to obtain crude flavonoid glycoside B;
(4) preparing liquid phase chromatographic separation: dissolving the flavonoid glycoside crude product B obtained in the step (3) by using a mobile phase, filtering, carrying out separation by using a preparative liquid chromatography column, carrying out isocratic elution by using the mobile phase, respectively collecting target section eluent, carrying out reduced pressure concentration, and carrying out vacuum drying to respectively obtain quercetin glycoside and kaempferitrin;
in the step (1), crushing fresh stems and leaves of momordica grosvenori to 20-40 meshes; the amount of the water is 4-20 times of the mass of the fresh stems and leaves of the momordica grosvenori; the temperature of the continuous countercurrent flash extraction is 50-90 ℃, the extraction frequency is more than or equal to 2 times, and the extraction time is 60-200 s each time;
in the step (2), the ultrafiltration membrane used for ultrafiltration is a ceramic membrane, and the aperture of the ceramic membrane is 0.2-2.0 μm;
in the step (2), the model of the macroporous adsorption resin is one or more of D-101 type, AB-8 type, DM-130 type, X-7 type or XDA-7 type; the volume-mass ratio of the macroporous adsorption resin to fresh stems and leaves of the momordica grosvenori is 1: 1-20;
in the step (2), the flow rate of the water washing is 1.0-4.0 BV/h; the dosage of the organic solvent is 1-4 BV; the flow rate of the organic solvent elution is 0.25-2.00 BV/h; the volume fraction of the organic solvent is 50-90%; the organic solvent is food grade ethanol;
in the step (3), the mass-to-volume ratio of the crude flavonoid glycoside A to the alcoholic solution is 1: 8-20; the heating temperature is 50-80 ℃; the alcoholic solution is food-grade anhydrous methanol or food-grade ethanol with the volume fraction of 90-99%; the standing time is 2-4 h;
in the step (4), the mass-to-volume ratio of the crude flavonoid glycoside B to the mobile phase is 0.08-0.30: 1; the mobile phase is an organic solvent-water solution with a volume ratio of 15-40: 95-60; the organic solvent is one or more of ethanol, methanol or acetonitrile; the filter membrane used for filtering is an organic phase filter membrane, and the aperture is 0.20-0.45 mu m; the flow rate of the sample introduction is 0.25-1.00 BV/h; the preparative liquid chromatography column is an ODS column, and the filler is C-18 reverse phase bonded silica gel.
2. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 1, wherein: in the step (1), the rotating speed of the centrifugation is 10000-80000 r/min; the centrifugal filtration mode is horizontal spiral centrifugal filtration and then butterfly centrifugal filtration.
3. The method for simultaneously extracting quercetin and kaempferitrin from momordica grosvenori stem and leaf according to claim 1 or 2, wherein: in the step (2), the flow rate of the upper column is 1.0-4.0 BV/h; the diameter-height ratio of the macroporous adsorption resin column is 1: 2-8.
4. The method for simultaneously extracting quercetin and kaempferitrin from momordica grosvenori stem and leaf according to claim 1 or 2, wherein: in the step (2), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.
5. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 3, wherein: in the step (2), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.
6. The method for simultaneously extracting quercetin and kaempferitrin from momordica grosvenori stem and leaf according to claim 1 or 2, wherein: in the step (3), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.
7. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 3, wherein: in the step (3), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.
8. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 4, wherein: in the step (3), the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.
9. The method for simultaneously extracting quercetin and kaempferitrin from momordica grosvenori stem and leaf according to claim 1 or 2, wherein: in the step (4), the flow rate of elution is 0.5-2.0 BV/h; the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.
10. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 3, wherein: in the step (4), the flow rate of elution is 0.5-2.0 BV/h; the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.
11. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 4, wherein: in the step (4), the flow rate of elution is 0.5-2.0 BV/h; the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.
12. The method for simultaneously extracting quercetin and kaempferitrin from stem and leaf of momordica grosvenori according to claim 6, wherein: in the step (4), the flow rate of elution is 0.5-2.0 BV/h; the temperature of the reduced pressure concentration is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the concentration is carried out until the solid content is 30-60%; the temperature of the vacuum drying is 40-70 ℃, the pressure is-0.1 to-0.07 MPa, and the time is 4-12 h.
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