WO2022237689A1 - 一种二氢槲皮素的制备方法 - Google Patents

一种二氢槲皮素的制备方法 Download PDF

Info

Publication number
WO2022237689A1
WO2022237689A1 PCT/CN2022/091499 CN2022091499W WO2022237689A1 WO 2022237689 A1 WO2022237689 A1 WO 2022237689A1 CN 2022091499 W CN2022091499 W CN 2022091499W WO 2022237689 A1 WO2022237689 A1 WO 2022237689A1
Authority
WO
WIPO (PCT)
Prior art keywords
dihydroquercetin
optionally
water
reaction
reduction reaction
Prior art date
Application number
PCT/CN2022/091499
Other languages
English (en)
French (fr)
Inventor
季俊虬
李孝常
李冰
李乔
苗青
刘丽梅
刘锦平
Original Assignee
合肥立方制药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 合肥立方制药股份有限公司 filed Critical 合肥立方制药股份有限公司
Priority to US18/008,044 priority Critical patent/US11753389B2/en
Publication of WO2022237689A1 publication Critical patent/WO2022237689A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic 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
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[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
    • C07D311/28—Benzo[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
    • C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic 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
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[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
    • C07D311/40—Separation, e.g. from natural material; Purification
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the disclosure relates to the field of drug synthesis, in particular, to a preparation method of dihydroquercetin.
  • dihydroquercetin 3,3',4',5,7-pentahydroxy-dihydroflavone, also known as Taxifolin, Taxifolin, and Taxifolin.
  • the structural formula is as follows:
  • dihydroquercetin Compared with quercetin, dihydroquercetin has superior solubility behavior in ethanol and water, so it has broader and more practical biological activities than quercetin, such as: antioxidant effect, or by regulating enzyme activity To affect fat metabolism, or inhibit the growth of malignant lymphocytes and leukemia cells, or inhibit bacteria (Staphylococcus aureus, Escherichia coli, Shigella and Salmonella typhi, etc.), and also have inhibitory effects on viral enzymes. Therefore, dihydroquercetin has a good prospect of popularization and application in the fields of health care products, functional foods, medicine, cosmetics, industry and agriculture.
  • the preparation methods of dihydroquercetin are mainly phytochemical methods and chemical synthesis methods. Due to the low content of dihydroquercetin in plants, and the complicated separation and purification process after extraction, moreover, the phytochemical method is more destructive to plant resources (such as Douglas fir tree roots) under large-scale production conditions.
  • Chemical synthesis methods can be divided into total synthesis and semi-synthesis. Although the raw materials of the former are easy to obtain and large-scale production is easy, but the synthesis steps are long, and there are toxicity of raw materials, reagents, and solvents, and the pollution of "three wastes" to varying degrees, etc., the industrial application prospect is not promising. On the other hand, unless asymmetric synthesis is used, it is difficult to obtain the target product consistent with the trans configuration of natural dihydroquercetin by total synthesis.
  • the present disclosure provides a preparation method of dihydroquercetin, comprising the following steps:
  • the concentrated crude product was recrystallized to obtain dihydroquercetin.
  • the mass ratio of the quercetin dihydrate to the reaction solvent water is 1:80-200.
  • the mass ratio of the quercetin dihydrate to the reaction solvent water is 1:100-180.
  • the mass ratio of the quercetin dihydrate to the reaction solvent water is 1:120-160.
  • the molar ratio of quercetin dihydrate to the alkalizing agent is 1:3.5-5.5.
  • the molar ratio of quercetin dihydrate to the alkalizing agent is 1:4.0-5.0.
  • the molar ratio of the quercetin dihydrate to the sulfite binary combination reducing agent is 1:3-8.
  • the molar ratio of quercetin dihydrate to the sulfite binary combination reducing agent is 1:4.5-7.5.
  • the molar ratio of the quercetin dihydrate to the sulfite binary combination reducing agent is 1:5.0-6.0.
  • the sulfite binary combination reducing agent includes a binary combination of sodium dithionite and sodium metabisulfite, or a binary combination of sodium dithionite and sodium bisulfite.
  • the sulfite binary combination reducing agent comprises a binary combination of sodium dithionite and sodium metabisulfite.
  • the molar ratio of sodium dithionite to sodium metabisulfite is 1:0.15-0.45. In some typical embodiments, the molar ratio of sodium dithionite to sodium metabisulfite is 1:0.2-0.40. In some more typical embodiments, the molar ratio of sodium dithionite to sodium metabisulfite is 1:0.25-0.35.
  • the reaction temperature of the reduction reaction is 60-120° C.
  • the reaction time is 55-115 minutes.
  • the reaction temperature of the reduction reaction is 80-100° C.
  • the reaction time is 70-100 min.
  • the mass ratio of the reaction solvent water to the dilution water after the reduction reaction is 1:0.1-0.6. In some embodiments, the mass ratio of the reaction solvent water to the dilution water after the reduction reaction is 1:0.2-0.5.
  • the acidification refers to dilute the terminal reduction reaction solution with water and then acidify it with dilute acid.
  • the acidification endpoint pH is 0.5-4.0. In some typical embodiments, the acidification terminal pH value is 1.5-3.0.
  • the temperature of the solution during the acidification is 0-35°C. In some typical implementations, the temperature of the solution during the acidification is 15-25°C;
  • the dilute acid includes dilute hydrochloric acid, dilute sulfuric acid, or dilute acetic acid. In some typical embodiments, the dilute acid is dilute sulfuric acid.
  • the mass concentration of the dilute sulfuric acid is 5%-15%. In some typical implementations, the mass concentration of the dilute sulfuric acid is 8%-12%.
  • the filter cake is washed and purified with hot water, and then recovered for reuse.
  • the hot water is water at 50°C-100°C. In some typical embodiments, the hot water is water at 65°C-85°C.
  • the washing time is 10-30 minutes. In some typical embodiments, the washing time is 15-25 minutes.
  • the extraction solvent used in the extraction is a hydrophobic organic solvent.
  • the hydrophobic organic solvent includes at least one of aliphatic ethers, alicyclic ethers, aliphatic esters, and hydrophobic aliphatic ketones.
  • the hydrophobic organic solvent includes at least one of isopropyl ether, tert-butyl ether, ethyl acetate, isopropyl acetate, and methyl isobutyl ketone.
  • the recrystallization process includes: performing primary crystallization on the concentrated crude product to obtain a primary crystal; performing secondary crystallization on the primary crystal to obtain a secondary crystal.
  • the solvent used for the recrystallization includes a mixed solvent of lower aliphatic alcohol and water.
  • the lower aliphatic alcohol includes at least one of methanol, ethanol, and isopropanol.
  • the lower aliphatic alcohols include methanol and ethanol.
  • the concentration of lower aliphatic alcohol in the solvent used in the recrystallization process is 20%-70%. In some typical embodiments, the concentration of lower aliphatic alcohol in the solvent used in the recrystallization process is 30%-60%.
  • the solid-liquid ratio of the concentrated crude product to the solvent used for the recrystallization is 1:3-10. In some typical embodiments, the solid-liquid ratio of the concentrated crude product to the solvent used for the recrystallization is 1:5-8.
  • the solid-liquid ratio of the primary crystallization product to the solvent used for the recrystallization is 1:5-10. In some typical embodiments, the solid-liquid ratio of the primary crystallized product to the solvent used for the recrystallization is 1:6-9.
  • the present disclosure also provides dihydroquercetin prepared by the preparation method.
  • Figure 1 is a liquid chromatogram of the semi-synthesized concentrated crude product of dihydroquercetin in Example 2 of the present disclosure
  • Figure 2 is a liquid chromatogram of a dihydroquercetin primary crystallization sample in Example 2 of the present disclosure
  • Example 3 is a liquid chromatogram of a secondary crystallization sample of dihydroquercetin in Example 2 of the present disclosure
  • Figure 4 is a liquid chromatogram of the semi-synthesized concentrated crude product of dihydroquercetin in comparative example 1 of the present disclosure
  • Fig. 5 is the 1 H NMR spectrum of the secondary crystallization sample of dihydroquercetin in Example 2 of the present disclosure.
  • the preparation route of semi-synthetic dihydroquercetin using quercetin as raw material not only has economic and environmental advantages, but also can obtain the target product consistent with the trans configuration of natural dihydroquercetin. This is a necessary condition for whether dihydroquercetin products can enter the field of medicine and food; however, whether the semi-synthetic method is feasible and economical depends on whether the acidity and alkalinity of the reaction solvent, reagent and reaction medium are compatible with quercetin. Matches the dominant dissolution behavior of primes.
  • quercetin Since quercetin is insoluble in water, it is slightly soluble in lower aliphatic alcohols, aliphatic ketones and fatty acid ester solvents at room temperature, and its solubility is less than 1% even in boiling glacial acetic acid. Therefore, in terms of economy and practicability , neutral or acidic water and most neutral and/or acidic organic solvents are not suitable as a semi-synthetic reaction solvent; therefore, the inventor hopes to be able to implement and complete the reduction reaction of quercetin in an alkaline aqueous solution, however, It is not easy to find a reduction reagent that can successfully reduce the C ring double bond in the molecular structure of flavonols in alkaline aqueous solution.
  • the disclosure provides a binary combination reducing agent of sulfite (oxysulfite) with sodium dithionite as the main component, and dihydrate A new method for the reduction of quercetin (or anhydrous quercetin) to dihydroquercetin.
  • Some embodiments of the present disclosure provide a method for preparing dihydroquercetin, which includes the following steps:
  • Dihydroquercetin was obtained by recrystallizing the concentrated crude product from a mixed solvent of alcohol-water.
  • the present disclosure in order to fully improve the utilization rate of starting materials and reduce production costs, the present disclosure will acidify and filter the collected filter cake with hot water (such as purified water or deionized water or distilled water) After washing and purification, it is recycled and reused. That is, when the liquid phase purity of the purified quercetin dihydrate after washing with hot water is ⁇ 98%, it can be used as a starting material for the preparation method of dihydroquercetin described in the present disclosure; optionally, the Hot water is water at 50°C-100°C. In some typical embodiments, the hot water is water at 65°C-85°C. In some typical embodiments, the washing time is 10-30 minutes. In some typical embodiments, the washing time is 15-25 minutes.
  • hot water such as purified water or deionized water or distilled water
  • the molar ratio of quercetin dihydrate to the alkalizing agent is 1:3.5-5.5, such as 1:3.5, 1:3.8, 1:4.0, 1:4.5 , 1:5.0 or 1:5.5. In some typical embodiments, the molar ratio of quercetin dihydrate to the alkalizing agent is 1:4.0-5.0.
  • the reaction solvent water is non-salt water, including purified water, deionized water or distilled water. In some optional embodiments of the present disclosure, the reaction solvent water is purified water.
  • the mass ratio of quercetin dihydrate to reaction solvent water is 1:80-200, such as 1:80, 1:100, 1:120, 1:140, 1 :160, 1:180, 1:200; As some embodiments of the present disclosure, the mass ratio of quercetin dihydrate to the reaction solvent water can be 1:100-180, more can be 1:120-160.
  • the mass ratio of the reaction solvent water to the dilution water for diluting the final reduction reaction solution is 1:0.1-0.6, such as 1:0.1, 1:0.2, 1:0.3, 1: 0.4, 1:0.5, 1:0.6;
  • the mass ratio of the reaction solvent water to the dilution water for diluting the terminal reduction reaction solution is 1:0.2-0.5.
  • the molar ratio of quercetin dihydrate to the sulfite binary combination reducing agent is 1:3-8, such as 1:3, 1:4, 1:3 5. 1:6, 1:7 or 1:8; In some embodiments of the present disclosure, the molar ratio of quercetin dihydrate to the sulfite binary combination reducing agent is 1:4.5-7.5 , more can be 1:5.0-6.0.
  • the sulfite binary combination reducing agent includes a binary combination of sodium dithionite and sodium metabisulfite, or a binary combination of sodium dithionite and sodium bisulfite; in this disclosure, the sulfite binary combination reducing agent includes a binary combination of sodium dithionite and sodium pyrosulfite; when the sulfite binary combination reducing agent is a binary combination of sodium dithionite and sodium pyrosulfite When combined, the mol ratio of the sodium dithionite to sodium metabisulfite is 1:0.15-0.45, such as 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4 or 1:0.45 .
  • the molar ratio of sodium dithionite to sodium metabisulfite is 1:0.2-0.40. In some more typical embodiments, the molar ratio of sodium dithionite to sodium metabisulfite is 1:0.25-0.35.
  • the disclosure uses sodium dithionite in combination with sodium pyrosulfite or sodium bisulfite, compared to using sodium dithionite alone, can effectively inhibit the generation of by-products during the reduction reaction; although the reducing ability of sodium dithionite is higher than that of all Sulfates are the strongest, but they also have certain oxidizing properties under certain conditions. This tendency is especially obvious when sodium dithionite is used alone as a reducing agent. Under the conditions of strong alkalinity (such as pH ⁇ 10.0) and high reaction temperature (temperature such as ⁇ 90 ° C), its oxidative properties will be more obvious, and more undesirable by-products will be produced; And when the alkalinity is weak or the reaction temperature is low, the reaction yield will obviously decrease again. Therefore, in order to minimize the adverse effect of the oxidation tendency of sodium dithionite, the present disclosure uses sodium dithionite in combination with sodium metabisulfite or sodium bisulfite in an appropriate ratio.
  • the reaction temperature of the reduction reaction is 60-120°C, such as 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C °C, 110°C or 120°C. In some typical embodiments, the reaction temperature of the reduction reaction is 80-100°C.
  • the reaction time of the reduction reaction is 55-115min, such as 55min, 60min, 70min, 80min, 90min, 100min, 110min or 115min. In some typical embodiments, the reaction time of the reduction reaction is 70-100 min.
  • the extraction solvent is a hydrophobic organic solvent.
  • the hydrophobic organic solvent includes at least one of aliphatic ethers, alicyclic ethers, aliphatic esters and hydrophobic aliphatic ketones.
  • the hydrophobic organic solvent includes at least one of aliphatic ethers, aliphatic esters and hydrophobic aliphatic ketones. More optionally, the hydrophobic organic solvent includes at least one of isopropyl ether, tert-butyl ether, ethyl acetate, isopropyl acetate, and methyl isobutyl ketone.
  • the mass ratio of the acidic filtrate after the reduction reaction to the above-mentioned hydrophobic organic solvent is 1:0.2-0.6, and the extraction is divided into three times according to conventional methods.
  • the extracts are combined, washed with purified water and aqueous sodium chloride solution in order to desalinate and separate and remove possible emulsified layers, that is, first use purified water (corresponding to In the quercetin dihydrate batch charging amount of 20.0g, water consumption here is 300-500ml) wash 1 time, then with 15% sodium chloride aqueous solution (corresponding to the quercetin dihydrate batch charging amount of 20.0g, this The consumption of sodium chloride aqueous solution is 200-300ml) washes 1 time.
  • purified water corresponding to In the quercetin dihydrate batch charging amount of 20.0g, water consumption here is 300-500ml
  • 15% sodium chloride aqueous solution corresponding to the quercetin dihydrate batch charging amount of 20.0g, this
  • the consumption of sodium chloride aqueous solution is 200-300ml
  • the desiccant can be anhydrous sodium sulfate, It can also be anhydrous magnesium sulfate, anhydrous copper sulfate, anhydrous calcium chloride, or molecular sieve.
  • the dehydrated extract is concentrated in vacuum, the temperature of the concentration may be 50-70° C., and the degree of vacuum may be 650-750 mmHg.
  • the concentrated crude product is obtained after the above-mentioned extraction, washing, drying and vacuum concentration; at this time, the liquid phase purity of the concentrated crude product under normal reaction conditions is about 80% (at least ⁇ 75%) .
  • the present disclosure puts the above crude product into a mixed solvent of lower aliphatic alcohol and purified water for recrystallization.
  • the present disclosure puts the semi-synthesized crude product of dihydroquercetin into a round bottom bottle, adds recrystallization solvent, heats on a water bath and stirs until completely dissolved, adds activated carbon to reflux for decolorization for about 10 minutes, and filters while hot; Transfer the collected filtrate to a crystallization bottle, cool down to 10-5°C with slow stirring, and let it stand for crystallization, then vacuum filter, wash the filter cake with a cold solvent of the same concentration for 2-3 times, and then vacuum-dry to dryness , to obtain the primary crystallization of dihydroquercetin; on the basis of the primary recrystallization conditions, the solvent concentration and the proportioning ratio are moderately adjusted, and then the primary crystallization product of dihydroquercetin is subjected to secondary crystallization to obtain the secondary crystallization of dihydroquercetin Crystals, that is, dihydroquercetin with liquid phase purity ⁇ 99.0%.
  • the recrystallization solvent may be a mixed solvent of methanol, ethanol, isopropanol and other lower aliphatic alcohols and water.
  • the lower aliphatic alcohols are methanol and ethanol.
  • the concentration of lower fatty alcohol in the recrystallization solvent is 20%-70 (v/v, the same below)%.
  • the concentration of lower aliphatic alcohol in the recrystallization solvent is 30%-60%, such as 30%, 40%, 50% or 60%.
  • the solid-liquid ratio of the concentrated crude product to the recrystallization solvent is 1:3-10. Typically, the solid-liquid ratio of the concentrated crude product to the recrystallization solvent is 1:5-8.
  • the solid-liquid ratio of the primary crystal of dihydroquercetin to the solvent used for the recrystallization is 1:5-10.
  • the solid-liquid ratio of the primary crystal of dihydroquercetin to the solvent used for the recrystallization is 1:6-9.
  • the acidification refers to dilute the terminal reduction reaction solution with water and acidify it with a dilute acid; the dilute acid can be dilute hydrochloric acid, dilute sulfuric acid, or dilute acid. Acetic acid, etc.; when the present disclosure is illustrated in the following embodiments, dilute sulfuric acid with a mass concentration of 5%-15% is used; of course, the concentration can also be 7%, 8%, 9%, 10%, 11% , 12%, 13%, etc. any concentration within the range of 5%-15%.
  • the pH value at the end point of the acidification is 0.5-4.0, typically, the pH value is 1.5-3.0.
  • the solution temperature in the acidification process is 0-35°C, typically, the temperature is 15-25°C.
  • the preparation method of dihydroquercetin described in the present disclosure mainly comprises the following steps:
  • Step 1 After adjusting the reaction solvent water to be alkaline with an alkalizing reagent, an alkaline aqueous solution is obtained; dissolving quercetin dihydrate in the alkaline aqueous solution, and adding a sulfite binary combination reducing agent to carry out a reduction reaction to obtain terminal reduction reaction solution;
  • Step 2 Dilute the terminal reduction reaction liquid with water, acidify, age, and filter to obtain a filtrate and a filter cake;
  • Step 3 extracting, washing, drying and vacuum concentrating the filtrate to obtain a concentrated crude product; recrystallizing the concentrated crude product to obtain dihydroquercetin.
  • the reaction temperature of the reduction reaction is 60-120°C, or 80-100°C; the reaction time of the reduction reaction is 55-115min, or 70-100min.
  • the dilute acid used in the acidification can be dilute hydrochloric acid, dilute sulfuric acid, dilute acetic acid, etc.; when the present disclosure is illustrated in the following examples, the mass concentration 5%-15% dilute sulfuric acid; of course, the mass concentration can also be 8%-12%.
  • the pH value at the end of the acidification is 0.5-4.0, or 1.5-3.0.
  • the temperature of the reaction solution during the acidification process should be kept within 0-35°C, optionally 15-25°C.
  • the extraction refers to: the acidified filtrate is shaken and extracted three times with a hydrophobic organic solvent, wherein the mass ratio of the acidified filtrate to the hydrophobic organic solvent is 1:0.1-1:1.0.
  • the mixing mass ratio of the acidified filtrate to the hydrophobic organic solvent can also be 1:0.2-1:0.6.
  • the hydrophobic organic solvent is exemplified below, and the hydrophobic organic solvent can be aliphatic ether, aliphatic ester and/or hydrophobic aliphatic ketone Any one of them; such as isopropyl ether, tert-butyl ether, ethyl acetate, isopropyl acetate, methyl isobutyl ketone, etc.
  • the desiccant can be anhydrous sodium sulfate, anhydrous magnesium sulfate, or It can be anhydrous copper sulfate, anhydrous calcium chloride, or molecular sieve.
  • Concentrate the dehydrated extract, the concentration temperature may be 50-70° C., and the vacuum degree may be 650-750 mmHg.
  • the semi-synthesized crude product of dihydroquercetin can be obtained.
  • the liquid phase purity of the obtained concentrated crude product is about 80%.
  • the inventors conducted extensive experiments. Research, respectively design the technical scheme of carrying out two recrystallizations with lower aliphatic alcohol-purified water mixed solvent as described below.
  • the present disclosure puts the crude semi-synthesized dihydroquercetin into a round bottom bottle, adds recrystallization solvent, heats on a water bath and stirs until fully dissolved, adds activated carbon, refluxes for decolorization, and filters while hot; Transfer the collected filtrate to a crystallization bottle, cool down to 8-2°C with slow stirring and let it stand for crystallization for a specified period of time, then vacuum filter, wash the filter cake with a cold solvent of the same concentration for 2-3 times, and then dry it in vacuum To dryness, obtain the dihydroquercetin primary crystal (liquid phase purity ⁇ 95.0%); on the basis of the primary recrystallization conditions, adjust the solvent concentration and the proportioning ratio appropriately, and carry out the secondary crystallization of the dihydroquercetin primary crystal product Crystallization to obtain secondary crystals of dihydroquercetin, that is, dihydroquercetin with liquid phase purity ⁇ 99.0%.
  • the disclosure of this application also provides an additional step, which is to recycle the filter cake obtained in step 2.
  • the specific steps are as follows:
  • step 2 Collect the filter cake (uncompletely reacted quercetin dihydrate) obtained in step 2 separately or collectively, put it in a round bottom flask, and stir and wash it in hot water at 60-100°C for 10-30min, so as to remove the trapped
  • the dihydroquercetin and other water-soluble components in it are fully extracted or removed from the filter cake, and then filtered and dried to increase the purity of dihydroquercetin to more than 98%, which can be prepared as described in the present disclosure.
  • the starting materials of the method are recovered and reused.
  • the main difference between the present disclosure and the prior art lies in the differences in the reducing agent, the reaction solvent, the pH environment of the reaction medium and the recrystallization method. Among them, combining the reducing agent sodium dithionite and sodium metabisulfite in a certain proportion is one of the main invention points of the present disclosure.
  • a special advantage of the dihydroquercetin preparation method (semi-synthetic method) described in the present disclosure is that it can obtain nearly 100% dihydroquercetin with the same trans configuration of natural quercetin.
  • the reducing agent used in this disclosure is a sulfite binary combination reducing agent based on sodium dithionite, such as a binary combination of sodium dithionite and sodium metabisulfite or sodium dithionite and sodium bisulfite, or sodium dithionite and other A binary composition of sulfates.
  • the inventors have found through research that, under the premise of other reduction reaction conditions remaining unchanged, when a single sodium dithionite is used instead of the binary combination reducing agent described in the present disclosure as a reducing agent, compared with the latter, the reduction obtained by the former
  • the liquid phase purity of the target product dihydroquercetin in the crude product is very low (see comparative example 1 of Table 4), and the by-products are many and miscellaneous, thus there is no further research and industrial practical value; wherein, the structure of the main by-products is as follows:
  • the molar ratio of the reducing agent can also be 1:4.5-7.5, more preferably 1:5.0-6.0.
  • the combination ratio of each component in the binary combination reducing agent is exemplified below:
  • the sulfite binary combination reducing agent is sodium dithionite and coke
  • the molar ratio of the two is 1:0.15-0.45, it can also be 1:0.2-0.4, and it can be 1:0.25-0.35.
  • This disclosure uses cheap alkaline aqueous solution as the solvent for the reduction reaction of quercetin, and limits the amount of water used, that is: the mass ratio of quercetin dihydrate to water is 1:80-200; optionally , the mass ratio of quercetin dihydrate to water is 1:100-180; more alternatively, the mass ratio of quercetin dihydrate to water is 1:120-160; the terminal reduction reaction solution When diluting with water, the mass ratio of the reaction solvent water to the dilution water is 1:0.1-0.6, more alternatively, the mass ratio of the reaction solvent water to the dilution water is 1:0.2-0.5.
  • the present disclosure uses water as the base solvent, and after being adjusted to basicity with an alkalizing agent, implements and completes the semi-reduction of quercetin with the sulfite binary combination reducing agent described in the present disclosure.
  • Synthetic reaction, and the consumption of basifying reagent is limited, promptly the mol ratio of quercetin dihydrate and basifying reagent described in step 1 is 1:3.5-5.5, can be selected as 1:4.0-5.0;
  • the pH range of the reaction medium from the beginning to the end of the reduction reaction is 7.0-13.0, optionally 7.5-12.5.
  • the terminal reduction reaction solution is acidified with dilute acid to acidify, and then the unfinished quercetin-chalcone is re-closed (regenerated quercetin), and is rapidly fully precipitated from the aqueous phase system, and aged for a certain period of time.
  • the most convenient way of filtration liquid-solid separation is used to fully separate it from the main product of the reaction (ie, dihydroquercetin, dissolved in an acidic aqueous solution).
  • the above-mentioned separation method is based on quercetin (or dihydrate) being dissolved in alkaline aqueous solution (opening ring), almost insoluble in acidic aqueous solution (closed ring), and dihydroquercetin is both in alkaline and acidic aqueous solution. Soluble properties are different, so it is designed to dilute the end-point reduction reaction solution with a proportioned amount of dilution water and cool it to room temperature, and then directly acidify it to the specified pH value with dilute acid to make the unfinished quercetin dihydrate (starting The starting material) is fully precipitated from the mixed system, while the product dihydroquercetin remains in the aqueous phase. After the acidified product is aged (standstill) for a certain period of time, the sediment is filtered out (retained) with double-layer filter paper or No. 2 sand core filter, so as to easily realize the sufficient separation of the product and the non-acted product.
  • the applicant has innovatively developed the filter cake (quercetin dihydrate precipitate) obtained through filtration described in the present disclosure into a beaker, adding a specified amount and preheating to Purified water at a specified temperature is continuously stirred and washed for an appropriate time under heat preservation conditions, and then the washing solvent is filtered off.
  • the liquid phase purity of the obtained filter cake is ⁇ 99%.
  • the recovered by-products of this purity fully meet the quality requirements for recycling.
  • the lower aliphatic alcohol includes at least one of methanol, ethanol and isopropanol, optionally, the lower aliphatic alcohol includes methanol and ethanol; considering the use safety of methanol, the present disclosure optionally uses ethanol.
  • the concentration range of the lower fatty alcohol is 20%-70%, optionally 30%-60%.
  • liquid phase chromatographic conditions in the method for measuring the liquid phase purity of the primary and secondary crystallization products described in the reduction reaction crude product and the two recrystallization processes described in the present disclosure are:
  • the concentration of the test product 0.5mg/ml.
  • the present disclosure is based on the molar ratio of quercetin dihydrate (abbreviated as starting material in Table 3) and binary combination reducing agent (abbreviated as reducing agent in Table 3), sodium dithionite in binary combination reducing agent (referred to in Table 3 as Abbreviated as A) and sodium pyrosulfite (abbreviated as B in table 3) or sodium bisulfite (abbreviated as C in table 3), the reaction temperature of reduction reaction and the reaction time are variable parameters, and in table 3 Corresponding positions of each embodiment and comparative example are arranged in pairs.
  • Example 2 in the above table is an example of Example 1 enlarged by 5 times (mass ratio).
  • Step 2 Acidification, Aging, Filtration
  • step 1 After the terminal reduction reaction solution obtained in step 1 was cooled to room temperature, 10% dilute sulfuric acid was evenly added dropwise under stirring condition to acidify to pH 2.0-2.5, left to age for 2.5h, and filtered to obtain a clear dilute The acidic filtrate of hydrogen quercetin, stand for later use;
  • Step 3 Extraction, washing, drying, concentration
  • the acidic filtrate containing dihydroquercetin obtained in step 2 is divided into 3 times (500+350+300ml) with 1150ml isopropyl acetate for shaking extraction, the combined extracts are washed once with 350ml purified water, and then Wash once with 250ml of 15% sodium chloride aqueous solution; separate the clean water layer and the possible emulsified layer, add 35g of anhydrous sodium sulfate, stir and wash for 5min, then let it stand for dehydration for 2.5h, filter out the sodium sulfate to obtain the dehydrated Extract: Concentrate the extract in a water bath with a water bath temperature of 50-70°C and a vacuum of 650-750mmHg until no liquid drips out, then dry pump for 30 minutes to obtain a light amber honeycomb-like semi-synthetic dihydroquercetin concentrate The crude product was collected and sampled for liquid phase detection.
  • the molar mass of quercetin dihydrate is 338.27, content: ⁇ 95.0%, and the molar mass of dihydroquercetin is 304.25;
  • Comparative examples 1-3 are two kinds of binary combination reducing agents of A+B and A+C in Example 1 and Example 11 replaced by A, B, C single reducing agent respectively, the experiment of 3 comparative examples The result clearly shows that: compared with the results of Examples 1 and 11, the experimental results using a single reducing agent have a large gap.
  • the reducing agent used in Comparative Example 1 is A component, and its comparison with Example 1 (A+B) shows that: only when the reducing agent A component forms a binary combination with B or C components, will it appear as in Table 4 Example 1.
  • the superiority of the disclosed scheme is just printed from the reverse side. Obviously, both the reduction reaction yield and the liquid phase purity of the concentrated crude product in Examples 1-11 of the present disclosure are significantly higher than those in Comparative Examples 1-3.
  • Examples 7-10 are the experimental results after cross-change of reaction temperature and time, and the data show that: compared with Examples 3-4 (molar ratio changes), the gap between Examples 7-10 and Example 1 is significantly narrowed . This shows that among the many process parameters of the reduction reaction, whether the setting of the molar ratio parameter between the starting material and the combined reducing agent and the internal A and B components of the combined reducing agent is appropriate is the first and most important factor affecting the reaction result. Second element.
  • Example 1 and Example 11 in Table 4 show that when other parameters are fixed, when component B is replaced with reducing agent component C (that is, the binary combination of A+C), compared to the implementation Example 11, the reduction reaction of Example 1 shows a significant improvement in the conversion yield level of the liquid phase, indicating that in the sulfite binary combination reducing agent, compared with the binary combination of A+C, the binary combination of A+B The meta-combination can obtain higher reduction reaction yield and crude liquid phase purity results.
  • Table 5 A recrystallization experiment result of some representative examples in Table 4
  • the recrystallization test results of each embodiment also show that the parameters of alcohol concentration and liquid-solid ratio in the two alcohol-water mixed recrystallization solvents set by the present disclosure are not only suitable, but also very effective. Compared with the conventional polyamide column chromatography or macroporous resin adsorption separation method, the efficiency is greatly improved.
  • Fig. 1 is a liquid chromatogram of the semi-synthesized concentrated crude product of dihydroquercetin in Example 2 of the present disclosure
  • Fig. 2 is a primary crystal of dihydroquercetin in Example 2 of the present disclosure (crystal sample)
  • Fig. 3 is the liquid chromatogram of the dihydroquercetin secondary crystallization (crystal sample) of embodiment 2 of the present disclosure
  • Fig. 4 is the semi-synthesis of dihydroquercetin of comparative example 1 of the present disclosure The liquid chromatogram of the concentrated crude product
  • FIG. 5 is the 1 H NMR spectrum of the dihydroquercetin secondary crystal (crystal sample) in Example 2 of the present disclosure.
  • test sample The elemental analysis result of the secondary crystallization thing (hereinafter referred to as test sample) of embodiment 2 in table 6 is as shown in table 7:
  • the 2 and 3 hydrogens of this compound are trans (1:1 mixture of 2R, 3R and 2S, 3S isomers). According to the coupling constant of these two H, it is 11.3Hz. If the two H are in cis, the coupling constant should be 2-3Hz.
  • the content of the cis-isomer in the sample is extremely small.
  • the H-2 of the cis isomer should show a signal around ⁇ 5.34, accounting for only about 1% of this spectrum.
  • the present disclosure provides a method for preparing dihydroquercetin, which has the advantages of short reaction steps, inexpensive reaction solvent (non-toxic) and reagents, simple crude product purification process, less pollutants, and low production cost and, after the reduction reaction, the low-purity starting material (filter cake) has not been fully recovered and reused after the simple and efficient purification treatment described in the present disclosure, which further reduces the cost of semi-synthetic raw materials; especially the semi-synthetic
  • the proportion of trans isomer in the target product obtained by the method is about 99%, which is consistent with the trans configuration of natural quercetin. Therefore, it has good popularization and application value, and is especially suitable for large-scale industrial production.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Pyrane Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

本公开提供了一种二氢槲皮素的制备方法，属于药物合成领域。所述方法包括以下步骤：将反应溶剂水用碱化试剂调节至碱性后，得碱性水溶液；将二水槲皮素溶解于碱性水溶液中，并加入亚硫酸盐二元组合还原剂进行还原反应，得到终点还原反应液；将所述终点还原反应液用水稀释后经酸化、陈化、过滤，得到滤液和滤饼；将所述滤液作萃取、洗涤、干燥和真空浓缩后得到浓缩粗品；将浓缩粗品经重结晶后，得到二氢槲皮素。本公开所述制备方法所用原料易得、工艺简便、生产成本低、尤其适用于工业化生产。

Description

一种二氢槲皮素的制备方法
相关申请的交叉引用
本公开要求于2021年5月10日提交中国专利局的申请号为“CN 202110519620.6”名称为“一种二氢槲皮素的制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及药物合成领域，具体而言，涉及一种二氢槲皮素的制备方法。
背景技术
二氢槲皮素化学名为3,3',4',5,7-五羟基-二氢黄酮，也叫花旗松素、黄杉叶素、黄杉素，结构式如下：
Figure PCTCN2022091499-appb-000001
与槲皮素相比较，二氢槲皮素在乙醇和水中的溶解行为更为优越，因而比槲皮素具有更宽广和更实用的生物活性，如：抗氧化作用，或通过调节酶的活性来影响脂肪代谢，或抑制恶性淋巴细胞和白血病细胞生长，或抑菌作用(金黄色葡萄球菌、大肠杆菌、痢疾杆菌和伤寒杆菌等)，对病毒酶也有抑制作用等。因此，二氢槲皮素在保健品、功能食品、医药、化妆品和工、农业等领域具有良好的推广应用前景。
截止目前，二氢槲皮素的制备方法主要为植化方法和化学合成法。由于二氢槲皮素在植物中的含量较低，且提取后的分离、纯化过程繁复，况且，植化方法在规模化生产条件下对植物资源(如花旗松树根)的破坏性较大。化学合成方法又可分为全合成法和半合成法。前者虽原料易得，且易于实现规模化生产，但合成步骤较长，且存在原料、试剂、溶媒的毒害性和不同程度地“三废”污染问题等，工业应用前景并不看好。另一方面，除非采用不对称合成法，否则，全合成法难以获得与天然二氢槲皮素反式构型一致的目标产物。
发明内容
本公开提供一种二氢槲皮素的制备方法，包括以下步骤：
将反应溶剂水用碱化试剂调节至碱性后，得碱性水溶液；
将二水槲皮素溶解于碱性水溶液中，并加入亚硫酸盐二元组合还原剂进行还原反应，得到终点还原反应液；
将所述终点还原反应液用水稀释后经酸化、陈化、过滤，得到滤液和滤饼；
将所述滤液作萃取、洗涤、干燥和真空浓缩后得到浓缩粗品；
将浓缩粗品经重结晶后，得到二氢槲皮素。
在一些实施方式中，所述二水槲皮素与反应溶剂水的质量比为1:80-200。
在一些实施方式中，所述二水槲皮素与反应溶剂水的质量比为1:100-180。
在一些实施方式中，所述二水槲皮素与反应溶剂水的质量比为1:120-160。
在一些实施方式中，所述二水槲皮素与所述碱化试剂的摩尔比例为1：3.5-5.5。
在一些实施方式中，所述二水槲皮素与所述碱化试剂的摩尔比例为1：4.0-5.0。
在一些实施方式中，所述二水槲皮素与所述亚硫酸盐二元组合还原剂的摩尔比为1:3-8。
在一些实施方式中，所述二水槲皮素与所述亚硫酸盐二元组合还原剂的摩尔比为1:4.5-7.5。
在一些实施方式中，所述二水槲皮素与所述亚硫酸盐二元组合还原剂的摩尔比为1:5.0-6.0。
在一些实施方式中，所述亚硫酸盐二元组合还原剂包括连二亚硫酸钠与焦亚硫酸钠的二元组合，或连二亚硫酸钠与亚硫酸氢钠的二元组合。
在一些实施方式中，所述亚硫酸盐二元组合还原剂包括连二亚硫酸钠与焦亚硫酸钠的二元组合。
在一些实施方式中，所述连二亚硫酸钠与焦亚硫酸钠的摩尔比为1:0.15-0.45。在一些典型的实施方式中，所述连二亚硫酸钠与焦亚硫酸钠的摩尔比为1:0.2-0.40。在一些更典型的实施方式中，所述连二亚硫酸钠与焦亚硫酸钠的摩尔比为1:0.25-0.35。
在一些实施方式中，所述还原反应的反应温度为60-120℃，反应时间为55-115min。
在一些实施方式中，所述还原反应的反应温度为80-100℃，反应时间为70-100min。
在一些实施方式中，所述反应溶剂水与还原反应后的稀释用水的质量比为1：0.1-0.6。在一些实施方式中，所述反应溶剂水与还原反应后的稀释水的质量比为1:0.2-0.5。
在一些实施方式中，所述酸化是指将所述终点还原反应液用水稀释后用稀酸酸化。
在一些实施方式中，所述酸化终点pH值为0.5-4.0。在一些典型的实施方式中，所述酸化终点pH值为1.5-3.0。
在一些实施方式中，所述酸化过程中的溶液温度为0-35℃。在一些典型的实施方式中，所述酸化过程中的溶液温度为15-25℃；
在一些实施方式中，所述稀酸包括稀盐酸、稀硫酸或稀醋酸。在一些典型的实施方式中，所述稀酸为稀硫酸。
在一些实施方式中，所述稀硫酸的质量浓度为5％-15％。在一些典型的实施方式中，所述稀硫酸的质量浓度为8％-12％。
在一些实施方式中，将所述滤饼经用热水洗涤纯化后回收再利用。
在一些实施方式中，所述热水为50℃-100℃的水。在一些典型的实施方式中，所述热水为65℃-85℃的水。
在一些实施方式中，所述洗涤时间为10-30min。在一些典型的实施方式中，所述洗涤时间为15-25min。
在一些实施方式中，所述萃取时所用的萃取溶剂为憎水性有机溶剂。
在一些实施方式中，所述憎水性有机溶剂包括脂肪族醚、脂环族醚、脂肪族酯和憎水性脂族酮中的至少一种。
在一些实施方式中，所述憎水性有机溶剂包括异丙基醚、叔丁基醚、乙酸乙酯、乙酸异丙酯、甲基异丁基酮中的至少一种。
在一些实施方式中，所述重结晶过程包括：将浓缩粗品经一次结晶后得到一次结晶物；将一次结晶物进行二次结晶后得到二次结晶物。
在一些实施方式中，所述重结晶所用溶剂包括低级脂肪醇和水的混合溶剂。在一些典型的实施方式中，所述低级脂肪醇包括甲醇、乙醇、异丙醇中的至少一种。在一些典型的实施方式中，所述低级脂肪醇包括甲醇和乙醇。
在一些实施方式中，所述重结晶过程所用溶剂中低级脂肪醇的浓度为20％-70％。在一些典型的实施方式中，所述重结晶过程所用溶剂中低级脂肪醇的浓度为30％-60％。
在一些实施方式中，所述浓缩粗品与所述重结晶所用溶剂的料液比为1:3-10。在一些典型的实施方式中，所述浓缩粗品与所述重结晶所用溶剂的料液比为1:5-8。
在一些实施方式中，所述一次结晶品与所述重结晶所用溶剂的料液比为1:5-10。在一些典型的实施方式中，所述一次结晶品与所述重结晶所用溶剂的料液比为1:6-9。
本公开还提供了由所述制备方法制备获得的二氢槲皮素。
附图说明
为了更清楚地说明本公开实施方式和实施例的技术方案，下面将对实施方式和实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施方式和实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本公开实施例2的二氢槲皮素半合成浓缩粗品的液相色谱图；
图2为本公开实施例2的二氢槲皮素一次结晶样品的液相色谱图；
图3为本公开实施例2的二氢槲皮素二次结晶样品的液相色谱图；
图4为本公开对比例1的二氢槲皮素半合成浓缩粗品的液相色谱图；
图5为本公开实施例2的二氢槲皮素二次结晶样品的 1H NMR谱图。
具体实施方式
下面将结合实施方式和实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施方式和实施例仅用于说明本公开，而不应视为限制本公开的范围。实施方式和实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以槲皮素为原料的半合成二氢槲皮素的制备途径不仅具有经济优势和环境优势，尤其能够得到与天然二氢槲皮素反式构型一致的目标产品。此乃二氢槲皮素产品能否进入药品和食品领域的必要条件；然而，半合成法是否具有可行性和经济性，则取决于反应溶剂、试剂以及反应介质的酸碱性是否与槲皮素优势溶解行为相匹配。
由于槲皮素不溶于水，常温下微溶于低级脂族醇、脂族酮和脂肪酸酯类溶剂，即使在沸腾的冰乙酸中溶解度也不足1％，因此，就经济性和实用性而言，中性或酸性水以及绝大多数中性和/或酸性有机溶剂均不适合作为半合成反应溶剂；因此，发明人希望能够在碱性水溶液中实施并完成槲皮素的还原反应，然而，欲寻找一种能够在碱性水溶液中顺利地还原黄酮醇分子结构中C环双键的还原反应试剂实属不易。
经过发明人精心设计和大量的实验研究，本公开提供了一种以连二亚硫酸钠为主要成分的亚硫酸盐(含氧亚硫酸盐)二元组合还原剂，并在碱性水溶液中将二水槲皮素(或无水槲皮素)还原为二氢槲皮素的制备新方法。
本公开一些实施方式提供了一种二氢槲皮素的制备方法，其包括以下步骤：
将反应溶剂水用碱化试剂调节至碱性后，得碱性水溶液；
将二水槲皮素溶解于碱性水溶液中，并加入亚硫酸盐(也可以称为含氧亚硫酸盐)二元组合还原剂进行还原反应，得到终点还原反应液；
将所述终点还原反应液用水稀释后经酸化、陈化、过滤，得到滤液和滤饼；
将所述滤液作萃取、洗涤、干燥和真空浓缩后得到浓缩粗品；
将浓缩粗品经用醇-水混合溶剂重结晶后，得到二氢槲皮素。
在本公开一些可选的实施方式中，为充分提高起始原料利用率，降低生产成本，本公开将酸化并过滤后收集的滤饼经用热水(如纯化水或去离子水或蒸馏水)洗涤、纯化后回收再利用。即经过用热水洗涤纯化后的二水槲皮素的液相纯度≥98％时，即可再作为本公开所述二氢槲皮素制备方法的起始原料使用；可选地，所述热水为50℃-100℃的水。在一些典型的实施方式中，热水为65℃-85℃的水。在一些典型的实施方式中，所述洗涤时间为10-30min。在一些典型的实施方式中，洗涤时间为为15-25min。
在本公开一些可选的实施方式中，所述二水槲皮素与所述碱化试剂的摩尔比例为1：3.5-5.5，例如1：3.5、1：3.8、1：4.0、1：4.5、1：5.0或1：5.5。在一些典型的实施方式中，二水槲皮素与所述碱化试剂的摩尔比例为为1：4.0-5.0。
在本公开一些可选的实施方式中，反应溶剂水为无盐水，包括纯化水、去离子水或蒸馏水。在本公开一些可选的实施方式中，反应溶剂水为纯化水。
在本公开一些可选的实施方式中，所述二水槲皮素与反应溶剂水的质量比为1:80-200，例如1:80、1:100、1:120、1:140、1:160、1:180、1:200；作为本公开的一些实施方式，所述二水槲皮素与反应溶剂水的质量比可以为1:100-180，更可以为1:120-160。
在本公开一些可选的实施方式中，所述反应溶剂水与稀释终点还原反应液的稀释水的质量比为1:0.1-0.6，诸如1:0.1、1:0.2、1:0.3、1:0.4、1:0.5、1:0.6；可选地，所述反应溶剂水与稀释终点还原反应液的稀释水的质量比为1:0.2-0.5。
在本公开一些可选的实施方式中，所述二水槲皮素与所述亚硫酸盐二元组合还原剂的摩尔比为1:3-8，例如1:3、1:4、1:5、1:6、1:7或1:8；在本公开的一些实施方式中，所述二水槲皮素与所述亚硫酸盐二元组合还原剂的摩尔比为1:4.5-7.5，更可以为1:5.0-6.0。
在本公开一些可选的实施方式中，所述亚硫酸盐二元组合还原剂包括连二亚硫酸钠与焦亚硫酸钠的二元组合，或连二亚硫酸钠与亚硫酸氢钠的二元组合；在本公开一些可选的实施方式中，所述亚硫酸盐二元组合还原剂包括连二亚硫酸钠与焦亚硫酸钠的二元组合；当所述亚硫酸盐二元组合还原剂为连二亚硫酸钠与焦亚硫酸钠的二元组合时，所述连二亚硫酸钠与焦亚硫酸钠的摩尔比为1:0.15-0.45，例如1:0.15、1:0.2、1:0.25、1:0.3、1:0.35、1:0.4或1:0.45。在一些典型的实施方式中，连二亚硫酸钠与焦亚硫酸钠的摩尔比为1:0.2-0.40。在一些更典型的实施方式中，连二亚硫酸钠与焦亚硫酸钠的摩尔比为1:0.25-0.35。
本公开将连二亚硫酸钠与焦亚硫酸钠或亚硫酸氢钠组合使用，相较于单独使用连二亚硫酸钠，在还原反应过程中能够有力地抑制副产物的产生；虽然连二亚硫酸钠的还原能力在所有亚硫酸盐类是最强的，但它在一定条件下也具有一定的氧化性，当单独使用连二亚硫酸钠作为还原剂时，此倾向尤为明显。在碱性偏强(诸如pH≥10.0)且反应温度较高(温度诸如≥90℃)的条件下，它的氧化性会较明显的显现出来，进而产生较多的不希望出现的副产物；而当碱性偏弱或反应温度偏低时，反应收率又会明显降低。因此，为了最大程度地减少连二亚硫酸钠的氧化倾向的不利影响，本公开将连二亚硫酸钠与焦亚硫酸钠或亚硫酸氢钠以恰当的比例复配(组合)使用。
在本公开一些可选的实施方式中，所述还原反应的反应温度为60-120℃，例如60℃、65℃、70℃、75℃、80℃、85℃、90℃、95℃、100℃、110℃或120℃。在一些典型的实施方式中，还原反应的反应温度为80-100℃。所述还原反应的反应时间为55-115min，例如55min、60min、70min、80min、90min、100min、110min或115min。在一些典型的实施方式中，还原反应的反应时间为70-100min。
在本公开一些可选的实施方式中，所述的萃取溶剂为憎水性有机溶剂。可选地，所述憎水性有机溶剂包括脂肪族醚、脂环族醚、脂族酯和憎水性脂族酮中的至少一种。可选地，憎水性有机溶剂包括脂肪族醚、脂族酯和憎水性脂族酮中的至少一种。更可选地，所述憎水性有机溶剂包括异丙基醚、叔丁基醚、乙酸乙酯、乙酸异丙酯、甲基异丁基酮中的至少一种。在本公开一些可选的实施方式中，还原反应后的酸性滤液与上述憎水性有机溶剂的质量比为1：0.2-0.6，并按常规法分作3次萃取。
在本公开一些可选的实施方式中，在完成3次萃取后，合并萃取液，并依次用纯化水和氯化钠水溶液洗涤除盐及分离除去可能的乳化层，即先用纯化水(对应于20.0g的二水槲皮素批投料量，此处用水量为300-500ml)洗涤1次，再用15％氯化钠水溶液(对应于20.0g的二水槲皮素批投料量，此处氯化钠水溶液的用量为200-300ml)洗涤1次。在洗涤结束并分尽水层后，加入干燥剂于有机相中干燥脱水2-3小时)，滤除吸水后的 干燥剂后得到干燥的萃取液；所述干燥剂可以是无水硫酸钠、也可以是无水水硫酸镁、也可以是无水硫酸铜、也可以是无水氯化钙、也可以是分子筛等。将脱水后的萃取液进行真空浓缩，所述浓缩的温度可以是50-70℃，真空度可以是650-750mmHg。
在本公开一些可选的实施方式中，经过上述萃取、洗涤、干燥和真空浓缩后得到浓缩粗品；此时，正常反应条件下的浓缩粗品的液相纯度约为80％(至少≥75％)。为了大幅度地提高其液相纯度，本公开将上述粗品置于低级脂肪醇与纯化水的混合溶剂中重结晶。在实际操作中，本公开将所述半合成二氢槲皮素粗品投入圆底瓶中，加入重结晶溶剂后于水浴上加热并搅拌至全溶，加入活性炭回流脱色约10min，趁热过滤；将收集的滤液转移至结晶瓶中，在缓慢搅拌中降温至10-5℃之间静置结晶后作真空过滤、滤饼经用同浓度的冷溶剂洗涤2-3次，再真空干燥至干，得到二氢槲皮素一次结晶物；在一次重结晶条件基础上适度调整溶剂浓度和配比率后将所述二氢槲皮素一次结晶产物进行二次结晶，得到二氢槲皮素二次结晶物，即液相纯度≥99.0％的二氢槲皮素。
为了便于本领域技术人员重复本公开所述方法，下面对重结晶溶剂的用量以及组成进行举例说明：
(1)所述重结晶溶剂可以是甲醇、乙醇、异丙醇等低级脂族醇和水的混合溶剂。典型地，低级脂肪醇为甲醇和乙醇。所述重结晶溶剂中低级脂肪醇的浓度为20％-70(v/v,下同)％。典型地，重结晶溶剂中低级脂肪醇的浓度为30％-60％，如30％、40％、50％或60％等。
(2)所述浓缩粗品与所述重结晶溶剂的料液比为1:3-10。典型地，浓缩粗品与所述重结晶溶剂的料液比为1:5-8。
(3)所述二氢槲皮素一次结晶物与所述重结晶所用溶剂的料液比为1:5-10。典型地，二氢槲皮素一次结晶物与所述重结晶所用溶剂的料液比为1:6-9。
在本公开一些可选的实施方式中，所述酸化是指将所述终点还原反应液用水稀释后用稀酸酸化；所述稀酸可以是稀盐酸、也可以是稀硫酸、还可以是稀醋酸等；本公开在下面实施方式中举例说明时，采用了质量浓度为5％-15％的稀硫酸；当然，所述浓度也可以为7％、8％、9％、10％、11％、12％、13％等在5％-15％范围内的任意浓度。所述酸化终点的pH值为0.5-4.0，典型地，pH值为1.5-3.0。所述酸化过程中的溶液温度为0-35℃，典型地，温度为15-25℃。
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限制本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下针对本公开所述二氢槲皮素的制备方法进行说明：
本公开所述的二氢槲皮素的制备方法，主要包括以下步骤：
步骤1：将反应溶剂水用碱化试剂调节至碱性后，得碱性水溶液；将二水槲皮素溶解于碱性水溶液中，并加入亚硫酸盐二元组合还原剂进行还原反应，得到终点还原反应 液；
步骤2：将所述终点还原反应液用水稀释后经酸化、陈化、过滤，得到滤液和滤饼；
步骤3：将所述滤液作萃取、洗涤、干燥和真空浓缩后得到浓缩粗品；将浓缩粗品经重结晶后，得到二氢槲皮素。
在上述步骤1中，所述还原反应的反应温度为60-120℃，也可以为80-100℃；所述还原反应的反应时间为55-115min，也可以为70-100min。
在上述步骤2中，所述酸化(关环)所用的稀酸可以是稀盐酸、也可以是稀硫酸、还可以是稀醋酸等；本公开在下面实施例中举例说明时，采用了质量浓度为5％-15％的稀硫酸；当然，所述质量浓度也可以为8％-12％。所述酸化终点的pH值为0.5-4.0，也可以是1.5-3.0。所述酸化过程中反应液的温度应保持在0-35℃内，可选为15-25℃。
在上述步骤3中，萃取是指：采用憎水性有机溶剂对酸化滤液分作3次振摇萃取，其中，酸化滤液与憎水性有机溶剂质量比为1:0.1-1:1.0。当然所述酸化滤液与憎水性有机溶剂的混合质量比也可以为1:0.2-1:0.6。为了便于本领域技术人员更好的重复本公开所述方法，下面对憎水性有机溶剂进行举例说明，所述憎水性有机溶剂可以是脂肪族醚、脂肪族酯和/或憎水性脂族酮中的任一一种；如异丙基醚、叔丁基醚、乙酸乙酯、乙酸异丙酯、甲基异丁基酮等。
在上述萃取3次后，合并萃取液后进行洗涤：
即先用300-500ml纯化水洗涤1次，再用200-300ml的15％氯化钠水溶液洗涤1次。在洗涤结束后，加入干燥剂于有机相中干燥脱水，滤除吸水后的干燥剂后得到脱水萃取液；所述干燥剂可以是无水硫酸钠、也可以是无水水硫酸镁、也可以是无水硫酸铜、也可以是无水氯化钙、也可以是分子筛等。将脱水后的萃取液进行浓缩，所述浓缩的温度可以是50-70℃，真空度可以是650-750mmHg。
经过上述萃取、洗涤、干燥和浓缩后，可以得到二氢槲皮素半合成的粗品，此时，在如附表3所示的还原反应条件及按后续步骤1-步骤3所规定的试验条件下所得浓缩粗品的液相纯度约为80％，在此基础上，为了快速、简便地大幅度将此等粗品的液相纯度提高至95％或99％以上水平，本发明人经过深入地实验研究，分别设计出如下文所述的用低级脂族醇-纯化水混合溶剂进行两次重结晶的技术方案。在实际操作中，本公开将所述半合成二氢槲皮素粗品投入圆底瓶中，加入重结晶溶剂后于水浴上加热并搅拌至全溶，加入活性炭后回流脱色，并趁热过滤；将收集的滤液转移至结晶瓶中，在缓慢搅拌下降温至8-2℃之间静置结晶规定时间后作真空过滤、滤饼经用同浓度的冷溶剂洗涤2-3次，再真空干燥至干，得到二氢槲皮素一次结晶物(液相纯度≥95.0％)；在一次重结晶条件基础上适度调整溶剂浓度和配比率后将所述二氢槲皮素一次结晶产物进行二次结晶，得到二氢槲皮素二次结晶物，即液相纯度≥99.0％的二氢槲皮素。
此外，为了提高原料利用率，本申请公开还提供一个附加步骤，即将步骤2中所得滤饼回收再利用，具体步骤如下所示：
将步骤2所得到的滤饼(未反应完全的二水槲皮素)单独或集中收集后置于圆底烧 瓶中，并于60-100℃的热水中搅拌洗涤10-30min，以便将裹挟在其中的二氢槲皮素及其他水溶性成分自滤饼中充分萃取或除去，然后再过滤、干燥，使二水槲皮素的纯度提高至98％以上，即可作为本公开所述制备方法的起始原料回收再利用。
为了便于本领域技术人员重复本公开所述方法，下面对重结晶溶剂的用量以及组成进行举例说明：(1)所述重结晶溶剂可以是甲醇、乙醇或异丙醇等低级脂族醇和纯化水的混合溶剂；所述低级脂肪醇可选为甲醇和乙醇；所述重结晶溶剂中低级脂肪醇的浓度为20％-70％，可选为30％-60％，如30％、40％、50％或60％等。(2)所述浓缩粗品与所述重结晶溶剂的料液比为1:3-10，可选为1:5-8；(3)所述二氢槲皮素一次结晶物与所述重结晶所用溶剂的料液比为1:5-10，可选为1:6-9。
本公开所述二水槲皮素半合成路线如下：
Figure PCTCN2022091499-appb-000002
本公开与现有技术主要不同之处在于还原剂、反应溶剂、反应介质pH环境和重结晶方法的不同。其中，将还原剂连二亚硫酸钠与焦亚硫酸钠按一定比例组合使用是本公开的主要发明点之一。
本公开所述的二氢槲皮素制备方法(半合成方法)的一个特殊优势是：它可以近乎100％地得到天然槲皮素的反式构型一致的二氢槲皮素。
本公开所用还原剂为以连二亚硫酸钠为主的亚硫酸盐二元组合还原剂，如连二亚硫酸钠与焦亚硫酸钠或连二亚硫酸钠与亚硫酸氢钠的二元组合，或连二亚硫酸钠与其他亚硫酸盐的二元组合物。发明人经研究发现，在其他还原反应条件不变的前提下，当以单一连二亚硫酸钠代替本公开所述二元组合还原剂作为还原剂使用时，相较于后者，前者所获得的还原粗品中目标产物二氢槲皮素的液相纯度很低(见表4的对比例1)，副产物多而杂，因而没有进一步研究和工业实用价值；其中，主要副产物的结构如下：
Figure PCTCN2022091499-appb-000003
此副产物的极性与目标产物十分接近(液相图谱显示二者的保留时间仅相差约1min)，不易分离，且在分离过程中会造成较多的产品损失。
基于上述原因，为了最大程度抑制此副产物生成，发明人经过深入的实验研究发现，将焦亚硫酸钠或亚硫酸氢钠与连二亚硫酸钠按一定的比例组合使用时，使得目标产物的 纯度得到大幅度提升(见表4的实施例1、实施例2和实施例11)，从而使得本公开方案具有较大的开发应用价值。
如上所述的亚硫酸盐二元组合还原剂在本公开所述方法中的摩尔比例为二水槲皮素：组合还原剂＝1:3.0-8.0，当然，所述二水槲皮素与组合还原剂的摩尔比也可以为1:4.5-7.5，更可以是1:5.0-6.0。
为了便于本领域技术人员重复本公开所述方法，下面对二元组合还原剂中各组分的组合比例进行举例说明：如当所述亚硫酸盐二元组合还原剂为连二亚硫酸钠和焦亚硫酸钠的二元组合时，二者的摩尔比为1:0.15-0.45，也可以为1:0.2-0.4，更可以为1:0.25-0.35。
本公开以廉价的碱性水溶液作为槲皮素的还原反应溶剂，并对水的用量进行了限定，即：所述二水槲皮素与水的质量比为1:80-200；可选地，所述二水槲皮素与水的质量比为1:100-180；更可选地，所述二水槲皮素与水的质量比为1:120-160；所述终点还原反应液用水稀释时，反应溶剂水与稀释水的质量比为1：0.1-0.6，更可选地，所述反应溶剂水与稀释水的质量比为1：0.2-0.5。
相较于中性和酸性有机溶剂，本公开以水作为基础溶剂，经用碱化剂调节至碱性后用本公开所述的亚硫酸盐二元组合还原剂实施并完成槲皮素的半合成反应，并对碱化试剂的用量进行了限定，即在步骤1中所述二水槲皮素与碱化试剂的摩尔比为1:3.5-5.5，可选为1:4.0-5.0；所述从还原反应开始至结束时的反应介质的pH值范围为7.0-13.0，可选为7.5-12.5。
本公开将终点还原反应液用稀酸酸化成酸性后使未作用完的槲皮素查尔酮重新关环(再生槲皮素)，并迅速自水相体系中充分沉淀析出，陈化一定时间后用最简便的过滤(液固分离)方式使之与反应主产物(即二氢槲皮素，溶于酸性水溶液中)实现充分分离。上述分离方法是基于槲皮素(或二水物)在碱性水溶液中溶解(开环)，在酸性水溶液中几乎不溶解(闭环)，而二氢槲皮素在碱性和酸性水溶液中均易溶的性质差别，从而设计出将终点还原反应液先用配比量的稀释水稀释并冷却至室温后直接用稀酸酸化至规定pH值后使未作用完的二水槲皮素(起始原料)自混合体系中充分沉淀析出，而生成物二氢槲皮素则留在水相中。酸化物经陈化(静置)一定时间后经用双层滤纸或2号砂芯滤器滤去(截留)沉淀物，从而轻松实现生成物与未作用物的充分分离。
为了充分再生利用这种可贵的废弃资源，申请人创新开发了本公开所述的将上述经过滤得到的滤饼(二水槲皮素沉淀物)倒入烧杯中，加入规定量并预热至规定温度的纯化水中，保温条件下持续搅拌洗涤适当时间后滤除洗涤溶剂，所得滤饼的液相纯度≥99％，这种纯度的回收副产品完全满足回收再利用的质量要求。
已知自生物黄酮类混合物中分离单一黄酮成分的常规方法是柱层析分离或大孔树脂吸附分离法，虽然分离效果好，但分离周期长，尤其是使用过的聚酰胺或大孔树脂均需要作相对漫长的再生处理过程，作为一种工业生产方法，显然缺乏经济性，且难以实现规模化生产。着眼于未来的工业生产，本申请人推出的这种既经济又方便、省时的低级脂族醇与纯化水混合溶剂两次重结晶方法，能够将液相纯度约80％的半合成二氢槲皮 素粗品纯度快速提升至99.5％的高纯级二氢槲皮素产品。作为当前市场需求量更大的液相纯度90-95％的二氢槲皮素工业级产品，则只需一次重结晶过程即可轻松得到。
所述低级脂族醇包括甲醇、乙醇和异丙醇中的至少一种，可选地，所述低级脂族醇包括甲醇和乙醇；考虑到甲醇的使用安全性问题，本公开可选地使用乙醇。所述重结晶溶剂中，所述低级脂肪醇的浓度范围为20％-70％，可选为30％-60％。
在本公开所述的还原反应粗品和两次重结晶过程所述的一次和二次结晶产品的液相纯度测定方法中的液相色谱条件为：
色谱柱：Intertsustain C18，250*4.6，5μm，CN-6020-07346，S/N5JR98073；
流动相：A泵0.1％磷酸水溶液，B泵-甲醇；
梯度法：流速1.5ml/min，进样量10μL，柱温40℃，检测波长290nm；
供试品浓度：0.5mg/ml。
以下结合实施例对本公开所述二氢槲皮素的制备方法作进一步的详细描述。
下述所有实施例和对比例中所用主要仪器信息如表1所示，所用原料、试剂和溶剂信息如表2所示：
表1：
仪器名称 型号/规格 仪器编号 制造厂商
电子天平 B3002 YHA009 上海良平仪器仪表公司
PH-温度测量系统 M400 / 梅特勒-托利多仪器(上海)有限公司
恒速电动搅拌机 JJ-2Q / 常州国宇仪器制造有限公司
恒温真空干燥箱 DZF-6050 B61016 上海博迅实验有限公司
真空过滤器 1000mlx500ml / 上海玻璃仪器有限公司
表2：
名称 规格 纯度/含量 制造厂商
二水槲皮素 药用级 含量≥95％ 陕西嘉禾生物科技股份公司
连二亚硫酸钠 分析纯 含量≥88.0％ 麦克林公司
焦亚硫酸钠 分析纯 含量≥99.0％ 麦克林公司
亚硫酸氢钠 分析纯 含量≥99.0％ 麦克林公司
无水碳酸钠 分析纯 含量≥99.0％ 国药集团上海化学试剂有限公司
纯化水 符合GMP标准 / 企业自制
乙醇 药用级 含量≥95％ 徐州香醅
乙酸异丙酯 工业优级品 含量≥99％ 国药集团上海化学试剂有限公司
硫酸 试剂级 含量95-98％ 扬州沪宝化学试剂有限公司
氯化钠 试剂级 含量≥99.0％ 国药集团上海化学试剂有限公司
活性炭 药用级 含量：99.9％ 上海活性炭有限公司
本公开以二水槲皮素(表3中简称为起始原料)与二元组合还原剂(表3中简称为还原剂)的摩尔比、二元组合还原剂中连二亚硫酸钠(表3中简称为A)与焦亚硫酸钠(表3中简称为B)或亚硫酸氢钠(表3中简称为C)的摩尔比、还原反应的反应温度 以及反应时间为可变参数，并在表3中各实施例和对比例的相应位置对号排列。
表3：
Figure PCTCN2022091499-appb-000004
备注：上表中实施例2为实施例1放大5倍(质量比)的实施例。
表3中各实施例和对比例中二氢槲皮素制备过程中的固定工艺条件、参数和制备程序如下所示：
步骤1：还原反应
向反应瓶中投加3000ml纯化水和24.5g的碳酸钠，持续搅拌至pH稳定后，水浴加热至50-55℃，加入20g二水槲皮素；继续加热至各实施例和对比例所规定的反应温度后，加入各实施例规定量的组合(或单一)还原剂进行还原反应，持续搅拌反应至规定的结束时间后，迅速加入750ml预冷至5℃以下的纯化水对反应液进行稀释，以便终止反应，得终点还原反应液。
步骤2：酸化、陈化、过滤
将步骤1所获得的终点还原反应液降温至室温后，于搅拌条件下均匀滴加10％的稀硫酸酸化至pH值2.0-2.5时，静置陈化2.5h，过滤，得到澄清的含二氢槲皮素的酸性滤液，静置备用；
附：滤饼的洗涤纯化
将各批回收的滤饼合并于烧杯中，加入10倍量80℃热纯化水后于80-85℃水浴上搅拌洗涤20min后趁热过滤，再用5倍量的热纯化水分2次洗涤滤饼，抽干后送入鼓风干燥箱中鼓风干燥至干。取样检测其液相纯度，当其纯度≥98％时，即可作为本半合成反应起始原料回收再利用。
步骤3：萃取、洗涤、干燥、浓缩
将步骤2所获得的含二氢槲皮素的酸性滤液用1150ml乙酸异丙酯分作3次(500+350+300ml)振摇萃取，合并萃取液，用350ml纯化水洗涤1次，再用250ml,浓度为15％氯化钠水溶液洗涤1次；分净水层和可能的乳化层后加入35g的无水硫酸钠搅拌洗涤5min后静置脱水2.5h，滤除硫酸钠后得到脱水后的萃取液；将此萃取液在水浴温度50-70℃水浴、真空度650-750mmHg条件下浓缩至无液滴滴出后再干抽30min后得到淡琥珀色蜂窝状半合成二氢槲皮素浓缩粗品，收料并取样做液相检测。
步骤4：重结晶
1)一次重结晶
向带有冷凝器的圆底瓶中加入由步骤3所获得的规定量的二氢槲皮素半合成浓缩粗品和6.5倍质量、乙醇浓度为40％的乙醇-水混合溶剂，再置于水浴上加热并搅拌至全溶，加入相当于粗品质量0.5％的活性炭并回流脱色10min，趁热过滤，并用相同浓度的热溶媒15ml分2次(10ml+5ml)洗涤滤饼，合并滤、洗液，并转移至结晶瓶中，在缓慢搅拌中降温至8℃以下，并于8-3℃之间静置结晶2h后真空过滤，滤饼经用预冷至5℃以下的冷溶媒洗涤2次(10ml+7ml)，滤干后于60℃以下真空干燥至干，得到二氢槲皮素一次结晶产物；
2)二次结晶
将规定量的一次结晶物置于圆底瓶中，加入相对于一次结晶物质量比8.0倍、乙醇浓度为50％的乙醇-水混合溶剂后依照一次结晶的方法依次作溶解、脱色、过滤、冷却结晶、过滤和真空干燥，得到液相纯度≥99％的二氢槲皮素二次结晶品。
表4：表3各实施例和对比例还原反应条件下的实验结果
Figure PCTCN2022091499-appb-000005
备注：二水槲皮素的摩尔质量为338.27，含量：≥95.0％，二氢槲皮素的摩尔质为304.25；
通过表3和表4中的条件与结果的对应关系可以看出：
1)对比例1-3分别是以A、B、C单一还原剂替代实施例1和实施例11中的A+B和A+C的两种二元组合还原剂，3个对比例的实验结果清楚地表明：使用单一还原剂的实验结果与实施例1和11的结果相比较，差距甚大。对比例1使用的还原剂是A成分，其与实施例1(A+B)的比较结果表明：还原剂A成分只有与B或C成分形成二元组合时，才会出现如表4实施例1、实施例2-实施例11所示的理想或较好或可接受的效果。若单独使用单一成分A,或B，或C时，结果必然糟糕。恰好从反面印正了本公开方案的优越性。显然，本公开实施例1-11的还原反应收率和浓缩粗品液相纯度均显著高于对比例1-3。
2)在反应温度和反应时间相同的条件下，由于实施例1、实施例2(实施例1的放大验证例，下同)、实施例3和实施例4中的起始原料与还原剂的摩尔配比不同，使得实施例1和2的还原反应收率和目标物纯度(相对应的指标是最大单杂、总杂面积百分比，下同)明显高于实施例3和4。在实施例5和6中，适度改变实施例1组合还原剂(A+B)中A与B成分之间的摩尔配比，可以看出，相比于实施例5和6，实施例1还原反应收率和目标物纯度更高。
3)实施例7-10是反应温度和时间交叉变动后的实验结果，数据表明：相较于实施例3-4(摩尔配比变动)，实施例7-10与实施例1的差距明显缩小。由此说明：在还原反应的诸多工艺参数中，起始原料与组合还原剂以及组合还原剂内部A与B两成分之间的摩尔配比参数的设置合适如否是影响反应结果的第一和第二要素。
4)表4中实施例1和实施例11的相关数据结果表明，在其他参数固定时，当以还原剂成分C替换成分B(即A+C的二元组合方式)后，相比于实施例11，实施例1的还原反应折纯收率水平液相纯度均有明显提高，说明在亚硫酸盐二元组合还原剂中，相较于A+C的二元组合，A+B的二元组合可以获得更高的还原反应收率和粗品液相纯度结果。
表5：表4中部分代表性实施例的一次重结晶实验结果
Figure PCTCN2022091499-appb-000006
表6：表5中各实施例和对比例的一次结晶物的二次结晶实验结果
Figure PCTCN2022091499-appb-000007
Figure PCTCN2022091499-appb-000008
表5-表6实验数据显示：当浓缩粗品(一次精制原料)的液相纯度较高时(如实施例1-2)时，只需实施2次简单的重结晶过程，便可以获得纯度≥99.5％的高纯二氢槲皮素(二次结晶物)。反之，当还原粗品的液相纯度低于50％(对比例1-3)时，显然缺乏经济价值。
各实施例的重结晶试验结果还表明，本公开所设置的两次醇-水混合重结晶溶剂中的醇浓度和液-固比例参数不仅合适，而且很有效。这与惯用的聚酰胺柱层析或大孔树脂吸附分离方法相比较，功效得到很大的提升。
在附图中，图1为本公开实施例2的二氢槲皮素半合成浓缩粗品的液相色谱图；图2为本公开实施例2的二氢槲皮素一次结晶物(结晶样品)的液相色谱图；图3为本公开实施例2的二氢槲皮素二次结晶物(结晶样品)的液相色谱图；图4为本公开对比例1的二氢槲皮素半合成浓缩粗品的液相色谱图；图5为本公开实施例2的二氢槲皮素二次结晶物(结晶样品)的 1H NMR谱图。
表6中实施例2的二次结晶物(以下简称供试样品)的元素分析结果如表7所示：
表7：元素分析测试结果：
元素 C H O
理论值 59.21％ 3.98％ 36.81％
分析值 59.20％ 3.98％ 36.80％
结论：表7的分析值与理论值的误差范围符合规定(＜±0.3％)。
本公开半合成二氢槲皮素供试品的 1H NMR谱数据表征
以本公开实施例2制备的二氢槲皮素二次结晶样品为例，进行 1H NMR谱数据表征。
二氢槲皮素二次结晶样品的结构式和 1H NMR谱数据如下：
Figure PCTCN2022091499-appb-000009
1H NMR谱数据： 1H NMR(400MHz,DMSO-d6):δ11.90(s,1H,OH-5),10.83(b,1H,OH-7),9.04(s,1H,OH-3'),8.99(s,1H,OH-4'),6.88(s,1H,Ar-H-2'),6.74(s,2H,Ar-H-5',6'),5.91-5.90(m,1H,Ar-H-8),5.86-5.85(m,1H,Ar-H-6),5.76(d,J＝5.8Hz,1H,OH-3),4.98(d,J＝11.3Hz,1H,H-2),4.52-4.48(m,1H,H-3).
1H NMR表征数据解析：
从 1H NMR谱看，这个化合物2，3位的氢是反式的(2R,3R和2S,3S两种异构体的1:1混合物)。根据是这两个H的偶合常数，是11.3Hz.如果这两个H处于顺式，偶合常数应该是2-3Hz.
从 1H NMR谱看，样品中顺式异构体含量极少。顺式异构体的H-2应该在δ5.34附近出现一个信号，在这个谱图里只占约1％。
结论：供试样中反式异构体占比约99％，与天然二氢槲皮素反式构型一致。
备注：以上二氢槲皮素 1H NMR谱及解析结果以及附图5源自中国科学技术大学化学系。
以上所述仅为本公开的实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开提供了一种二氢槲皮素的制备方法，由该制备方法具有反应步骤短、反应溶媒(无毒)和试剂价廉、粗品纯化过程简便、污染物少、生产成本低的显著优势；并且，还原反应后未作用完低纯度起始原料(过滤滤饼)经过本公开所述的简便、高效的纯化处理后全部回收再利用，进一步降低了半合成原料成本；尤其是本半合成方法所获得目标产品中反式异构体的占比约99％，与天然槲皮素的反式构型一致。因而具有很好的推广、应用价值，尤其适用于规模化工业生产。

Claims (12)

  1. 一种二氢槲皮素的制备方法，其特征在于，包括以下步骤：
    将反应溶剂水用碱化试剂调节至碱性后，得碱性水溶液；
    将二水槲皮素溶解于碱性水溶液中，并加入亚硫酸盐二元组合还原剂进行还原反应，得到终点还原反应液；
    将所述终点还原反应液用水稀释后经酸化、陈化、过滤，得到滤液和滤饼；
    将所述滤液作萃取、洗涤、干燥和真空浓缩后得到浓缩粗品；
    将浓缩粗品经重结晶后，得到二氢槲皮素。
  2. 根据权利要求1所述二氢槲皮素的制备方法，其特征在于，所述二水槲皮素与反应溶剂水的质量比为1:80-200；
    可选地，所述二水槲皮素与反应溶剂水的质量比为1:100-180；
    更可选地，所述二水槲皮素与反应溶剂水的质量比为1:120-160。
  3. 根据权利要求1或2所述二氢槲皮素的制备方法，其特征在于，所述二水槲皮素与所述碱化试剂的摩尔比例为1：3.5-5.5；
    可选地，所述二水槲皮素与所述碱化试剂的摩尔比例为1：4.0-5.0。
  4. 根据权利要求1-3中任一项所述二氢槲皮素的制备方法，其特征在于，所述二水槲皮素与所述亚硫酸盐二元组合还原剂的摩尔比为1:3-8；
    可选地，所述二水槲皮素与所述亚硫酸盐二元组合还原剂的摩尔比为1:4.5-7.5；
    更可选地，所述二水槲皮素与所述亚硫酸盐二元组合还原剂的摩尔比为1:5.0-6.0。
  5. 根据权利要求1-4中任一项所述二氢槲皮素的制备方法，其特征在于，所述亚硫酸盐二元组合还原剂包括连二亚硫酸钠与焦亚硫酸钠的二元组合，或连二亚硫酸钠与亚硫酸氢钠的二元组合；
    可选地，所述亚硫酸盐二元组合还原剂包括连二亚硫酸钠与焦亚硫酸钠的二元组合；
    可选地，所述连二亚硫酸钠与焦亚硫酸钠的摩尔比为1:0.15-0.45，优选为1:0.2-0.40，更优选为1:0.25-0.35。
  6. 根据权利要求1-5中任一项所述二氢槲皮素的制备方法，其特征在于，所述还原反应的反应温度为60-120℃，反应时间为55-115min；
    可选地，所述还原反应的反应温度为80-100℃，反应时间为70-100min。
  7. 根据权利要求1-6中任一项所述二氢槲皮素的制备方法，其特征在于，所述反应溶剂水与还原反应后的稀释用水的质量比为1：0.1-0.6；
    可选地，所述反应溶剂水与还原反应后的稀释水的质量比为1:0.2-0.5。
  8. 根据权利要求1-7中任一项所述二氢槲皮素的制备方法，其特征在于，所述酸化是指将所述终点还原反应液用水稀释后用稀酸酸化；
    可选地，所述酸化终点pH值为0.5-4.0，优选为1.5-3.0；
    可选地，所述酸化过程中的溶液温度为0-35℃，优选为15-25℃；
    可选地，所述稀酸包括稀盐酸、稀硫酸或稀醋酸，优选为稀硫酸；
    可选地，所述稀硫酸的质量浓度为5％-15％，优选为8％-12％。
  9. 根据权利要求1-8中任一项所述二氢槲皮素的制备方法，其特征在于，将所述滤饼经用热水洗涤纯化后回收再利用；
    可选地，所述热水为50℃-100℃的水，优选为65℃-85℃的水；
    更可选地，所述洗涤时间为10-30min，优选为15-25min。
  10. 根据权利要求1-8中任一项所述二氢槲皮素的制备方法，其特征在于，所述萃取时所用的萃取溶剂为憎水性有机溶剂；
    可选地，所述憎水性有机溶剂包括脂肪族醚、脂环族醚、脂肪族酯和憎水性脂族酮中的至少一种；
    更可选地，所述憎水性有机溶剂包括异丙基醚、叔丁基醚、乙酸乙酯、乙酸异丙酯、甲基异丁基酮中的至少一种。
  11. 根据权利要求1-9中任一项所述二氢槲皮素的制备方法，其特征在于，所述重结晶过程包括：将浓缩粗品经一次结晶后得到一次结晶物；将一次结晶物进行二次结晶后得到二次结晶物；
    可选地，所述重结晶所用溶剂包括低级脂肪醇和水的混合溶剂；优选地，所述低级脂肪醇包括甲醇、乙醇、异丙醇中的至少一种；更优地，所述低级脂肪醇包括甲醇和乙醇；
    可选地，所述重结晶过程所用溶剂中低级脂肪醇的浓度为20％-70％，优选为30％-60％；
    可选地，所述浓缩粗品与所述重结晶所用溶剂的料液比为1:3-10，优选为1:5-8；
    可选地，所述一次结晶品与所述重结晶所用溶剂的料液比为1:5-10，优选为1:6-9。
  12. 由权利要求1-11中任一所述的制备方法制备获得的二氢槲皮素。
PCT/CN2022/091499 2021-05-10 2022-05-07 一种二氢槲皮素的制备方法 WO2022237689A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/008,044 US11753389B2 (en) 2021-05-10 2022-05-07 Method for preparing dihydroquercetin

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110519620.6 2021-05-10
CN202110519620.6A CN113214210B (zh) 2021-05-10 2021-05-10 一种二氢槲皮素的制备方法

Publications (1)

Publication Number Publication Date
WO2022237689A1 true WO2022237689A1 (zh) 2022-11-17

Family

ID=77095417

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/091499 WO2022237689A1 (zh) 2021-05-10 2022-05-07 一种二氢槲皮素的制备方法

Country Status (3)

Country Link
US (1) US11753389B2 (zh)
CN (1) CN113214210B (zh)
WO (1) WO2022237689A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113214210B (zh) 2021-05-10 2022-03-04 合肥立方制药股份有限公司 一种二氢槲皮素的制备方法
CN113185485B (zh) 2021-05-10 2022-03-04 合肥立方制药股份有限公司 一种二氢槲皮素的半合成方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101863869A (zh) * 2010-06-03 2010-10-20 东北林业大学 一种落叶松木片提取二氢槲皮素的方法
CN103044379A (zh) * 2012-12-31 2013-04-17 李玉山 一种二氢槲皮素的绿色制备技术
CN113214210A (zh) * 2021-05-10 2021-08-06 合肥立方制药股份有限公司 一种二氢槲皮素的制备方法

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2744920A (en) * 1952-09-09 1956-05-08 Ervin F Kurth Converting dihydroquercetin to quercetin
RU2359666C2 (ru) * 2007-03-29 2009-06-27 Николай Эдуардович Нифантьев Способ выделения секоизоларицирезинола и дигидрокверцетина из древесины (варианты)
CN101993429A (zh) 2010-12-08 2011-03-30 天津市尖峰天然产物研究开发有限公司 从落叶松木屑中提取二氢槲皮素的方法
WO2013025972A1 (en) 2011-08-17 2013-02-21 Globeimmune, Inc. Yeast-muc1 immunotherapeutic compositions and uses thereof
HUE031673T2 (en) * 2011-09-14 2017-07-28 Lek Pharmaceuticals Synthesis of triazolopyrimidine compounds
CN104387357A (zh) 2011-12-09 2015-03-04 成都欧康医药有限公司 槲皮素的生产工艺
LT6013B (lt) * 2012-05-18 2014-03-25 Uab "Rokiå Kio Pragiedruliai" Ypatingai gryno dihidrokvercetino gamybos būdas
PL2981520T3 (pl) * 2013-04-04 2019-09-30 Lek Pharmaceuticals D.D. Nowy sposób syntezy 1-(2-((2,4-dimetylofenylo)tio)fenylo)piperazyny
CN103570663B (zh) 2013-11-25 2015-08-26 广东众生药业股份有限公司 一种高纯度槲皮素的制备方法
EP2894154A1 (en) * 2014-01-14 2015-07-15 LEK Pharmaceuticals d.d. Synthesis of vortioxetine via (2-(piperazine-1-yl)phenyl)aniline intermediates
CN105348063B (zh) 2014-08-18 2017-09-12 合肥立方制药股份有限公司 一种药用丹皮酚合成和精制新方法
US9611255B2 (en) 2015-06-10 2017-04-04 Council Of Scientific And Industrial Research Process for total synthesis of flavonoid compounds and isomers thereof
CN107880081B (zh) 2017-12-22 2021-07-06 陕西嘉禾生物科技股份有限公司 一种异槲皮素的合成方法
CN110467592B (zh) 2019-09-12 2021-07-02 广东药科大学 一种木犀草素的半合成方法
CN112375043B (zh) * 2020-11-26 2022-03-08 江苏食品药品职业技术学院 一种制备达克替尼的方法
CN113185485B (zh) 2021-05-10 2022-03-04 合肥立方制药股份有限公司 一种二氢槲皮素的半合成方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101863869A (zh) * 2010-06-03 2010-10-20 东北林业大学 一种落叶松木片提取二氢槲皮素的方法
CN103044379A (zh) * 2012-12-31 2013-04-17 李玉山 一种二氢槲皮素的绿色制备技术
CN113214210A (zh) * 2021-05-10 2021-08-06 合肥立方制药股份有限公司 一种二氢槲皮素的制备方法

Also Published As

Publication number Publication date
US11753389B2 (en) 2023-09-12
CN113214210B (zh) 2022-03-04
CN113214210A (zh) 2021-08-06
US20230192639A1 (en) 2023-06-22

Similar Documents

Publication Publication Date Title
WO2022237689A1 (zh) 一种二氢槲皮素的制备方法
CN101812009B (zh) 一种从发酵液中分离提取l-色氨酸的工艺
CN110845328B (zh) 一种以迷迭香油膏副产品制备高纯度鼠尾草酸的方法
WO2022237690A1 (zh) 一种二氢槲皮素的半合成方法
CN109761867B (zh) 一种以羊毛脂为原料生产维生素d3的工业化新方法
CN108047029A (zh) 一种从藤黄果中提取纯化羟基柠檬酸的制备方法
CN101489970A (zh) 琥珀酸的制备方法
CN104311620A (zh) 一种纯化鹅去氧胆酸的方法
CN111138443A (zh) 一种4’-去甲基表鬼臼毒素全合成的制备方法
CN104693081B (zh) 用混合溶剂精制双酚s的方法
CN109422642B (zh) 苯甲酸的精制方法、药用级苯甲酸钠的生产方法和设备
CN104592004B (zh) 一种精制长链有机酸的方法
CN111018819B (zh) 一种木犀草素的制备方法
CN104356105A (zh) 一种高含量egcg的制备方法
CN104591999A (zh) 一种长链有机酸的提纯方法
CN103275049A (zh) 用藤茶制备杨梅素的方法和焦亚硫酸盐的应用
CN103980481B (zh) 水溶性维生素e的制备方法
CN104418825B (zh) 利普司他汀的提纯方法
CN105481809B (zh) 一种丹酚酸b的分离纯化方法及丹酚酸b镁盐的制备方法
CN107033114B (zh) 一种二氢杨梅素的分离纯化方法
CN113045583B (zh) 唑啉草酯代谢物的制备方法
CN113880697A (zh) 一种大麻二酚的提取方法
CN103467428A (zh) 一种柚皮素的制备方法
CN104513151A (zh) 一种从丁二酸盐发酵液中提取高纯度丁二酸的方法
CN114478195B (zh) 一种从微生物发酵产物中提取大麻萜酚的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22806655

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22806655

Country of ref document: EP

Kind code of ref document: A1