A method for simultaneously separating, purifying and preparing multiple flavonoids using industrial chromatography technology
Ligumin and purify privetinoside and saccording from the extract of yam leaf through industrial chromatography. The reference product was solved, and the problems of difficult preparation, high cost and waste of resources in the prior art were solved, and high purity, environmental protection and The goal of simplifying the process.
Patent Information
- Application Number
- CN201911366454.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2019-12-26
- Publication Date
- 2024-12-31
- Estimated Expiration
- 2039-12-26
AI Technical Summary
It is difficult to efficiently separate and purify privetinoside and sequoside at the same time. The source of the reference product is limited and the separation process is complex, resulting in high costs, waste of resources and environmental pollution.
Ligumin and sequentially isolated and purified from the extract of yam leaf extract were used to prepare privetinoside and sequentialoside. The reference product was monitored in real time online, used less organic solvents, and simplified the process flow to achieve high-purity reference product preparation.
The preparation of high-purity (99.5% or more) privetinoside and sacranin control products has been achieved, which simplifies the process flow, reduces costs, increases the utilization rate of medicinal materials, and is process-friendly and environmentally friendly.
Smart Images
Figure CN113045614B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of separation and purification of active ingredients of traditional Chinese medicines, and particularly relates to a method for simultaneously separating, purifying and preparing a variety of flavonoid substances by utilizing industrial chromatography technology. Background Art
[0002] Ligustrum lucidum is a flavonoid glycoside compound, one of the main active ingredients of Ligustrum lucidum fruit and Rhizoma Citri Reticulatae leaves, which has the function of inhibiting influenza virus infection and replication and inhibiting neuraminidase. Rhus sutchuenensis is a flavonoid glycoside compound, one of the main active ingredients of Rhizoma Citri Reticulatae leaves and fruits of Rutaceae plants (such as Citrus aurantium), which has antioxidant, anti-tumor, anti-hypertensive and xanthine oxidase inhibitory effects.
[0003] At present, the control of medicinal material quality, screening of pharmacological efficacy, and development of new products all require the use of ligustrin and wild sumac glycoside reference materials. However, due to the limited sources of reference materials and the difficulty of separation and purification, the prices of ligustrin and wild sumac glycoside reference materials are relatively expensive, 500 yuan / vial for ligustrin and 1200 yuan / vial for sumac glycoside, which seriously limits the quality control of medicinal materials containing ligustrin and wild sumac glycoside and their related products. Therefore, the development of a simple, rapid and simultaneous separation and purification method for high-purity ligustrin and wild sumac glycoside components is the focus of current research.
[0004] The currently disclosed preparation methods for Ligustrum lucidum and Rhus chinensis glycosides are all for the preparation of single compounds. For example, CN101899078A discloses a method for extracting Ligustrum lucidum glycosides from Fructus Lucidi or Fructus Ligustri Lucidi, CN 102633850A discloses a method for extracting Rhus chinensis glycosides and its use for preparing medicines, CN 102286043A discloses a method for purifying Rhus chinensis glycosides, CN 103833810A discloses a new preparation technology for Rhus chinensis glycosides, and CN 106632546A discloses a method for simultaneously preparing two chemical reference substances, Rhus chinensis glycosides and Naringin. By comparison, the above methods have the disadvantages of small scale batch, long process steps, and environmental pollution, and cannot prepare reference substances in large quantities to meet the needs of current research, and separate single reference substances from medicinal materials, waste medicinal material resources, and are not conducive to environmental protection and sustainable utilization.
[0005] CN 102579522A discloses an ethanol percolation extract of total flavonoids from Psoralea corylifolia and a preparation method and use thereof, and CN 101862357A discloses an ethanol reflux extract of total flavonoids from Psoralea corylifolia and a preparation method and use thereof, wherein the extracts prepared mainly contain flavonoid compounds such as privet glycoside and scutellaria sutchuenensis. However, no prior art for simultaneously preparing privet glycoside and scutellaria sutchuenensis reference substances has been found. Summary of the invention
[0006] In order to overcome the shortcomings of the prior art, the present invention provides a method for simultaneously separating, purifying and preparing a variety of flavonoids by using industrial chromatography technology, specifically a method for simultaneously preparing glossy ligusticum glycoside and scutellaria baicalensis glycoside reference substances from a Scutellaria scabra leaf extract by using industrial chromatography preparation technology, which realizes real-time online monitoring of the separation process and a small amount of organic solvent; the preparation process is simple, the production cycle is short, it is easy to industrialize, and it is environmentally friendly, and it has high purity.
[0007] The invention provides a method for preparing a flavonoid reference substance by utilizing industrial preparation chromatography purification technology, comprising the following steps: injecting a sample solution of a safflower leaf extract into a high-pressure preparation chromatography according to a certain injection volume, eluting with a solvent system 1, detecting separation conditions with an ultraviolet online detector, collecting eluates of prepared privet glycoside and wild sumac glycoside monomer compounds respectively according to the elution time and chromatographic peak height of privet glycoside and wild sumac glycoside, wherein the collection start and end times are 175-200 minutes and 220-250 minutes respectively, and concentrating and drying the eluates respectively;
[0008] The flavonoid reference substances include privet glycoside and wild sumac glycoside;
[0009] The preparation of the sample solution of the extract of the leaves of the safflower tree is as follows: the extract of the leaves of the safflower tree is taken, and an appropriate amount of the solvent system 2 is added to dissolve it and prepare a sample solution of a certain concentration, and the solution is filtered for later use.
[0010] Preferably, the content of privet glycoside in the extract of the leaves of the Coptis chinensis is 1.0%-9.0% (W / W), preferably 2.0%-8.0% (W / W); the content of flaxseed glycoside is 0.5%-7.0% (W / W), preferably 1.0%-6.0% (W / W); and / or, the solvent system 2 is 5.0-15.0% (V / V) methanol aqueous solution; preferably 8.0%-12.0% (V / V) methanol aqueous solution.
[0011] Preferably, the sample solution concentration is 50-100 mg / mL; preferably 70-90 mg / mL.
[0012] Preferably, the industrially prepared chromatographic filler is a polystyrene-divinylbenzene polymer filler; preferably CG161M or MCI GEL CHP20P.
[0013] Preferably, the solvent system 1 is one or more of methanol, water, and trifluoroacetic acid; preferably a mixed solution of methanol, water, and trifluoroacetic acid; more preferably, the volume ratio of methanol, water, and trifluoroacetic acid is 30-50:70-50:0.5, such as 40:60:0.5, 43:57:0.5, and 45:55:0.5.
[0014] Preferably, the elution flow rate is 2.5-7.5CV / h; preferably 2.5-5.0CV / h;
[0015] Preferably, the injection volume is 120-150L.
[0016] Preferably, the detection wavelength of the ultraviolet online detector is 336 nm.
[0017] Preferably, the concentration is reduced-pressure concentration; and / or the concentration temperature is 50-60°C.
[0018] Preferably, the drying is vacuum drying; and / or the drying temperature is 40-50°C.
[0019] Preferably, the method for extracting the Cyperus rotundus leaf is as follows: take an appropriate amount of Cyperus rotundus leaf, add ethanol to reflux and extract several times (such as 1, 2, 3 times, etc.), each reflux extraction takes 1-3 hours, and filter; combine the filtrates, recover ethanol from the filtrate, adsorb the aqueous solution with a non-polar macroporous adsorption resin, elute with ethanol below 20% to remove impurities, then elute with 25-90% ethanol, collect the eluate, recover ethanol, concentrate, and dry.
[0020] Beneficial technical effects of the present invention:
[0021] 1. The present invention directly separates and purifies the extract of the leaves of the Rhizoma Cibotii by industrial preparation chromatography, and simultaneously obtains two reference compounds (ligustrin and scutellaria baicalensis glycosides) with a purity of more than 99.5%, thereby solving the problems of difficult preparation and high price of reference compounds.
[0022] 2. The present invention utilizes industrial chromatography preparation technology to simultaneously prepare a reference substance, thereby achieving real-time online monitoring of the separation process; it has the characteristics of small amount of organic solvent used, simple preparation process, short production cycle, easy industrialization, and environmental friendliness.
[0023] 3. The present invention adopts one medicinal material to prepare two reference compounds at the same time, thereby improving the utilization rate of the medicinal material and saving costs. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 : HPLC chromatogram of the extract of Rhizoma Cyperi leaves;
[0025] Figure 2 :Process flow chart for preparing privet glycosides and wild sumac glycosides;
[0026] Figure 3 : HPLC chromatogram of Ligustrum lucidum glycosides;
[0027] Figure 4 : HPLC chromatogram of wild sumac glycoside; DETAILED DESCRIPTION
[0028] In order to better understand the present invention, the technical scheme of the present invention is further described and illustrated by the following examples.
[0029] Example 1: Preparation of the extract of Rhizoma Cyperi
[0030] Take an appropriate amount of the leaves of the Rhizoma Cyrthospermi, add 10 times the volume of 70% ethanol, reflux and extract twice, each reflux extraction for 1 hour, filter; combine the filtrates, recover ethanol from the filtrate, adsorb the aqueous solution with a non-polar macroporous adsorption resin, elute with 20% or less ethanol (such as 18%) to remove impurities, then elute with 70% ethanol, collect the eluate, recover ethanol, concentrate, and dry to obtain the Rhizoma Cyrthospermi leaf extract, wherein the content of privet glycosides is about 8.0% (W / W), and the content of wild sumac glycosides is about 6.0% (W / W). Figure 1 .
[0031] Example 2: Preparation of the extract of Rhizoma Cyperi
[0032] Take an appropriate amount of Scutellaria scabra leaves, add 5 times the volume of 30% ethanol, reflux and extract once, each reflux extraction is 1 hour, and filter; combine the filtrates, recover ethanol from the filtrate, adsorb the aqueous solution with a non-polar macroporous adsorption resin, elute with 20% or less ethanol (such as 15%) to remove impurities, then elute with 25% ethanol, collect the eluate, recover ethanol, concentrate, and dry to obtain a Scutellaria scabra leaf extract, in which the content of privet glycoside is about 2.0% (W / W) and the content of wild lacquercoside is about 1.0% (W / W).
[0033] Example 3: Preparation of the extract of Rhizoma Cyperi
[0034] Take an appropriate amount of Scutellaria scabra leaves, add 15 times the volume of 90% ethanol, reflux and extract twice, each time for 2 hours, and filter; combine the filtrates, recover ethanol from the filtrate, adsorb the aqueous solution with a non-polar macroporous adsorption resin, elute with 20% or less ethanol (such as 10%) to remove impurities, then elute with 90% ethanol, collect the eluate, recover ethanol, concentrate, and dry to obtain a Scutellaria scabra leaf extract, in which the content of privet glycoside is about 6.0% (W / W) and the content of flaxseed glycoside is about 3.0% (W / W).
[0035] Example 4: Preparation of Ligustrum lucidum and Rhus chinensis glycosides
[0036] A method for simultaneously preparing privet glycosides and wild sumac glycoside reference substances from the extract of the leaves of the safflower using industrial chromatography preparation technology, the process flow chart is as follows Figure 2 As shown, the steps are as follows:
[0037] (1) 10800 g of the extract of the leaves of the scutellaria baicalensis of Example 1 was dissolved by adding 12.0% (V / V) methanol aqueous solution to prepare a sample solution with a concentration of 90 mg / mL. The sample solution was filtered and set aside.
[0038] (2) 120L of the sample solution from step (1) was injected into an industrial preparation chromatography, with CG161M as the purification filler, methanol: water: trifluoroacetic acid solution (40:60:0.5, V / V / V) as the eluent, the elution flow rate of 5.0CV / h, and the UV detection wavelength of 336nn. The prepared eluates containing ligustrin and wild sumac glycoside were collected according to the elution time of the ligustrin and wild sumac glycoside components and the height of the chromatographic peaks, with the collection start and end times being 175-200min and 220-250min, respectively. The eluates containing the target component with high purification were concentrated to dryness under reduced pressure at 55°C and then vacuum dried at 45°C for 24h to obtain 691.21g of ligustrin with a purity of more than 99.5% (see Figure 3 ), wild sumac glycoside 518.45g, purity above 99.5% (see Figure 4 ).
[0039] Example 5: Preparation of Ligustrum lucidum and Rhus chinensis glycosides
[0040] A method for simultaneously preparing privet glycosides and wild sumac glycoside reference substances from the extract of the leaves of the safflower using industrial chromatography preparation technology, the process flow chart is as follows Figure 2 As shown, the steps are as follows:
[0041] 8.0% (V / V) methanol aqueous solution was added to dissolve 10500 g of the extract of the leaves of the Codonopsis pilosula in Example 2 and prepare a sample solution with a concentration of 70 mg / mL. The sample solution was filtered and set aside.
[0042] (2) Separation, purification and drying: 150 L of the sample solution from step (1) was injected into an industrial preparation chromatography, with MCI GEL CHP20P as the purification filler, methanol: water: trifluoroacetic acid solution (45:55:0.5, V / V / V) as the eluent, the elution flow rate being 2.5 CV / h, and the ultraviolet detection wavelength being 336 nm. The eluates containing privet glycoside and wild sumac glycoside were collected according to the elution time of privet glycoside and wild sumac glycoside components and the height of the chromatographic peaks, with the collection start and end times being 175-200 min and 220-250 min, respectively. The eluates containing the target component with high purity were concentrated to dryness under reduced pressure at 55° C. and then dried in vacuo at 45° C. for 24 h to obtain 178.36 g of privet glycoside with a purity of more than 99.5% and 89.25 g of wild sumac glycoside with a purity of more than 99.5%.
[0043] The above preferred embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Those skilled in the art should understand that any modification or equivalent replacement of the technical solution of the present invention without departing from the purpose and scope of the technical solution of the present invention should be included in the scope of protection of the present invention.
Claims
1. A method for simultaneously separating, purifying and preparing a plurality of flavonoids using industrial chromatography technology, comprising the following steps: The sample solution of the extract of the leaves of the Codonopsis pilosula is injected into a high-pressure industrial preparation chromatography according to a certain injection volume, and eluted with solvent system 1. The separation is detected by an ultraviolet online detector. According to the elution time and chromatographic peak height of privet glycoside and wild sumac glycoside, the eluates of the prepared privet glycoside and wild sumac glycoside monomer compounds are collected respectively, and the collection start and end times are 175-200min and 220-250min respectively, and the eluates are concentrated and dried respectively; the injection volume is 120-150L; the industrial chromatography filler is CG161M or MCI GEL CHP20P polystyrene-divinylbenzene polymer; the solvent system 1 is a mixed solution of methanol, water and trifluoroacetic acid, wherein the volume ratio of methanol, water and trifluoroacetic acid is 30-50:70-50:0.5; the elution flow rate is 2.5-5.0CV / h; the detection wavelength of the ultraviolet online detector is 336nm; the concentration is reduced pressure concentration, and the concentration temperature is 50-60°C; the drying is vacuum drying, and the drying temperature is 40-50°C; Preparation of the sample solution of the extract of the leaves of the safflower: take the extract of the leaves of the safflower, add an appropriate amount of solvent system 2 to dissolve it and prepare a sample solution of a certain concentration, filter it for later use; the solvent system 2 is 8.0%-12.0% V / V methanol aqueous solution; the concentration of the sample solution is 70-90 mg / mL; The extraction method of the leaves of the mountain osmanthus is as follows: taking an appropriate amount of the leaves of the mountain osmanthus, adding ethanol to reflux and extracting once or twice, each reflux extraction takes 1-2 hours, and filtering; combining the filtrates, recovering ethanol from the filtrates, adsorbing the aqueous solution with a non-polar macroporous adsorption resin, eluting with ethanol below 20% to remove impurities, and then eluting with 25-90% ethanol, collecting the eluate, recovering ethanol, concentrating, and drying.
2. The method according to claim 1, wherein the content of privet glycoside in the extract of the leaves of the Rhizoma Cyperi is 1.0%-9.0% W / W; the content of scutellaria baicalensis glycoside is 0.5%-7.0% W / W.
3. The method according to claim 2, wherein the content of privet glycoside in the extract of the leaves of the Rhizoma Cyperi is 2.0%-8.0% W / W; the content of scutellaria baicalensis glycoside is 1.0%-6.0% W / W.
4. The method according to claim 1, wherein the solvent system 1 is a mixed solution of methanol, water and trifluoroacetic acid, wherein the volume ratio of methanol, water and trifluoroacetic acid is 40:60:0.5 or 45:55:0.5.