Method for Separating 6-Formylisomaiwandiones A from High Isoflavone Mixtures

Abstract

The present invention discloses a method for separating 6-formylindomyrtone A from a high isoflavone mixture, and the steps are as follows: 1) Using the ophiopogon japonicus high isoflavone mixture as a raw material, dissolving the raw material in a mixed solution composed of ethyl acetate and a hydrophobic organic solvent to obtain a raw material solution; 2) Using a binary mixed solvent composed of an ionic liquid and water as an extractant and using the same solvent as the solvent of the raw material solution as a detergent, performing fractional extraction on the raw material solution, and collecting the raffinate; 3) Subjecting the raffinate to vacuum concentration, washing with water and drying treatment to obtain 6-formylindomyrtone A. The present invention uses an ionic liquid or a binary mixed solvent composed of an ionic liquid and a medium-polarity to polar organic solvent as an extractant, has a high selective separation ability for 6-formylindomyrtone A, and the purity and yield of the separated 6-formylindomyrtone A can both reach more than 90%.

Description

Technical Field

[0001] The invention belongs to the technical field of natural drug chemistry, and in particular relates to a method for extracting and separating 6-aldehyde isoflavone A from a high-isoflavone mixture of a traditional Chinese medicine, ophiopogon japonicus, by using ionic liquid. Background Art

[0002] Ophiopogon japonicus is a perennial evergreen herb of the genus Ophiopogonis in the family Liliaceae Ophiopogon japonicus The dried root of (Lf)ker-Gawl. is one of the traditional commonly used Chinese medicines. Ophiopogon japonicus has the effects of nourishing yin and promoting body fluid, moistening the lungs and clearing the heart; it is mainly used to treat dry cough due to lung dryness, tuberculosis cough due to yin deficiency, sore throat due to throat numbness, thirst due to body fluid injury, internal heat and thirst, restlessness and insomnia, dry intestines and constipation, etc. It has been used for the treatment of acute cough, sore throat and cardiovascular and cerebrovascular diseases for thousands of years. In my country, the main authentic production areas of Ophiopogon japonicus are located in Zhejiang Province and Sichuan Province, among which Zhejiang Ophiopogon japonicus is one of the famous Zhejiang authentic medicinal materials. Existing studies have shown that Zhejiang Ophiopogon japonicus contains high isoflavone components such as methyl Ophiopogon japonicus flavanone A, methyl Ophiopogon japonicus flavanone B, 6-aldehyde iso-Ophiopogon japonicus flavanone A and 6-aldehyde iso-Ophiopogon japonicus flavanone B. These four high isoflavones have similar structures, but their biological activities are not exactly the same.

[0003] 6-aldehyde isoophyte flavanone A (structural formula see Figure 1 ) is a relatively trace high isoflavone component in the tuberous root or fibrous root of Ophiopogon japonicus. Due to the small structural difference with other high isoflavone compounds, it is difficult to separate the monomer. The existing method for separating and purifying 6-formyl isoflavone A monomer mainly uses the tuberous root or fibrous root of Ophiopogon japonicus as raw material to obtain a high isoflavone component mixture through alcohol extraction and preliminary separation, and then uses repeated column chromatography, countercurrent extraction and other methods to separate and obtain 6-formyl isoflavone A monomer. This type of chromatographic separation method is relatively complicated and therefore not easy to apply industrially.

[0004] Extraction is a separation technology with simple equipment and convenient operation. The key lies in the selection of extractant. 6-formyl isoflavanone A has multiple hydrophobic ring structures and has low solubility in polar solvents such as water, methanol, and acetonitrile. It has good solubility only in ethyl acetate and is almost insoluble in water. Therefore, in the commonly used binary extraction system (such as ethyl acetate-water, n-butanol-water, methanol-n-hexane, acetonitrile-n-hexane, etc.), one phase is an organic solvent with high solubility for 6-formyl isoflavanone A, while the other phase is often water or weakly polar organic solvent with low solubility for 6-formyl isoflavanone A, resulting in the problem of low distribution coefficient of 6-formyl isoflavanone A and poor separation selectivity.

[0005] To improve the distribution behavior of 6-formylindimophnflavanone A, a third solvent can be added to the binary extraction system. The third solvent can be added to water or the weakly polar phase to increase the solubility of 6-formylindimophnflavanone A in this phase. Alternatively, another component can be added to the organic phase in which 6-formylindimophnflavanone A has a high solubility to reduce the solubility of 6-formylindimophnflavanone A in this phase, thereby achieving the purpose of regulating the partition coefficient of 6-formylindimophnflavanone A. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the present invention provides a method for separating 6-formylindimophnflavanone A with high purity from a mixture by using an organic solvent in which 6-formylindimophnflavanone A is highly soluble and an organic solvent in which it is slightly soluble as the raw material solvent and detergent, and a binary mixed solvent composed of an ionic liquid or an ionic liquid and a moderately polar organic solvent as the extractant, and through multi-stage fractional extraction.

[0007] Specifically, it is realized through the following technical solutions:

[0008] A method for separating 6-formylindimophnflavanone A from a high isoflavone mixture comprises the following steps:

[0009] 1) Using a high isoflavone mixture containing methylophiopogonone A, methylophiopogonone B, 6-formylindimophnflavanone A and 6-formylindimophnflavanone B as the raw material, dissolving the raw material in a mixed solution of ethyl acetate and a hydrophobic organic solvent to prepare a raw material solution;

[0010] 2) Using a binary mixed solvent composed of an ionic liquid and water as the extractant and a solvent the same as that of the raw material solution as the detergent, performing fractional extraction on the raw material solution, and collecting the raffinate;

[0011] 3) Sequentially subjecting the raffinate collected in step 2) to vacuum concentration, water washing and drying treatments to obtain 6-formylindimophnflavanone A.

[0012] Furthermore, the mass fraction of 6-formylindimophnflavanone A in the raw material is greater than 25%.

[0013] Furthermore, the concentration of 6-formylindimophnflavanone A in the raw material solution is 1-100 g / L. If the concentration of 6-formylindimophnflavanone A in the raw material solution is too high, it is not conducive to the effective separation of the two substances; if the concentration is too low, the raw material throughput is small and the solvent loss is large, which is not conducive to the economy of the process.

[0014] Further, the solvent in the raw material liquid and the detergent is a mixed solvent of n - hexane and ethyl acetate, and the volume ratio of n - hexane to ethyl acetate is 5 - 10:1 - 5. Different volume ratios of n - hexane and ethyl acetate form a mixed solvent, which not only has a high solubility for the high - isoflavone mixture but also can form a two - phase system with a small mutual solubility with the ionic liquid - water mixed solvent.

[0015] Further, during the fractional extraction process, the flow ratio of the extractant, the detergent, and the raw material liquid is 1 - 60∶1 - 20∶1, and the temperature of the fractional extraction is 20 - 45°C. If the temperature is too low, the viscosity of the extractant is large, the mass transfer rate decreases, and the throughput is small, which is not conducive to production operations; if the temperature is too high, the solvent volatilizes severely, which will reduce the distribution ratio and selectivity of the fractional extraction.

[0016] Further, the molar ratio of the ionic liquid to water in the extractant is 5:95 - 15:85.

[0017] Further, the selectivity of the ionic liquid for 6 - formylisomaiwandanone A is mainly determined by the alkyl chain length of its cation and the anion N -. When the cation is an imidazolium group composed of a carbon chain of hexyl - octyl, and the polarity and hydrogen - bond basicity of the anion N - are within an appropriate range and are easy to form a π - π interaction with 6 - formylisomaiwandanone A, it has a good separation effect on 6 - formylisomaiwandanone A. After optimization, it is determined that the cation M+ is one of a series of cations with substituents, including but not limited to imidazolium - type cations with substituents, pyridinium - type cations with substituents, pyrrolidinium - type cations with substituents, piperidinium - type cations with substituents, or quaternary ammonium - type cations with substituents, etc., and the anion N - is chloride ion, bromide ion, tetrafluoroborate, acetate, or hydrosulfide ion.

[0018] Further, the substituents in the cation M+ can be selected from one or two of C1 - C8 alkyl groups and hydroxyethyl groups, and the number of substituents is single - substitution or multi - substitution. When it is multi - substitution, the substituents at different sites can be the same or different.

[0019] Further, the extraction device used for fractional extraction includes common extraction devices such as packed towers, sieve - plate towers, rotating - disk towers, and centrifugal extractors, including an extraction section and a washing section. The schematic diagram of the device is as Figure 2As shown in the figure, where 1 is the first stage of the extraction section, 2 is the second stage of the extraction section, 1' is the first stage of the washing section, 2' is the second stage of the washing section, N is the number of stages of the extraction section, and N' is the number of stages of the washing section. The extractant enters the fractional extraction system from the first stage of the extraction section, the raw material liquid enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, and the raw material liquid is combined at the last stage of the extraction section and then enters the extraction section. The extract phase and the washing phase undergo multi-stage countercurrent contact. An extraction liquid rich in methylophiopogonone A, methylophiopogonone B, and 6-formylindimophiopogonone B flows out from the first stage of the washing section, and a raffinate liquid enriched in 6-formylindimophiopogonone A flows out from the first stage of the extraction section. The raffinate liquid is collected; the raffinate liquid is concentrated under vacuum, washed with water, and dried to obtain 6-formylindimophiopogonone A.

[0020] The present invention uses a mixed solvent of ethyl acetate in a certain proportion to dissolve the high isoflavone mixture, and uses a long-chain cationic imidazolium ionic liquid-water as the extractant for the selective separation of 6-formylindimophiopogonone A. For example, in a two-phase system single-stage extraction and separation of the high isoflavone mixture at 30 °C with n-hexane:ethyl acetate (8:2) as the solvent and 1-hexyl-3-methylimidazolium bromide as the extractant, the selectivity coefficient of 6-formylindimophiopogonone A for the other three high isoflavones can reach 19.36. It can be seen that using a suitable ternary two-phase extraction system for the separation of 6-formylindimophiopogonone A and other high isoflavones can obtain a distribution coefficient more favorable for industrial production and a better selectivity coefficient.

[0021] The purity of 6-formylindimophiopogonone A obtained by the separation method of the present invention is: absolute purity > 95%.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1) The present invention uses an ionic liquid or a binary mixed solvent composed of an ionic liquid and a medium-polarity to polar organic solvent as the extractant, which has a high selective separation ability for 6-formylindimophiopogonone A. At the same time, the extractant used is not only easy to recycle and reuse, but also relatively green and environmentally friendly, with less pollution to the environment, and has broad application prospects;

[0024] 2) The present invention uses the fractional extraction technology, with less consumption of traditional Chinese medicine raw materials, high production capacity, and low cost;

[0025] 3) The method of the present invention adopts optimized conditions, and the purity and yield of 6-formylindimophiopogonone A can both reach more than 90%. Description of the Drawings

[0026] Figure 1 is the structural formula of 6-formylindimophiopogonone A;

[0027] Figure 2 It is a schematic structural diagram of an existing multi-stage fractional extraction device;

[0028] Figure 3 Effect of ionic liquids with different structures on the extraction and separation of 6-formylindomauvignone A

[0029] Figure 4 Effect of ionic liquid concentration on the extraction and separation of 6-formylindomauvignone A;

[0030] Figure 5 Effect of the ratio of n-hexane to ethyl acetate on the extraction and separation of 6-formylindomauvignone A. Specific implementation mode

[0031] The following further describes the present invention in detail with specific examples to better understand the technical solution.

[0032] In the following examples, the concentrations of methyl domauvignone A and methyl domauvignone B were analyzed by high performance liquid chromatography. The specific analysis conditions of the liquid chromatography were as follows: C18 silica gel column (4.6×250mm, particle size 5μm), column temperature 35°C, mobile phase acetonitrile∶0.1% formic acid = 65∶35 (V / V), flow rate 1 mL / min, detector was an ultraviolet detector, and the wavelength was 285 nm.

[0033] The calculation methods of the yield and purity in the present invention are as follows:

[0034] Yield = mass of 6-formylindomauvignone A in the product / mass of 6-formylindomauvignone A in the raw material × 100%.

[0035] Purity = mass of 6-formylindomauvignone A in the product / total mass of the product × 100%.

[0036] Example 1: Selection and verification of the method conditions for separating 6-formylindomauvignone A from the mixture of high isoflavones in Ophiopogon japonicus

[0037] Main instruments and reagents:

[0038] Take 15 kg of the dried fibrous roots of Ophiopogon japonicus, pulverize them, and extract them ultrasonically twice with 200 L of 80% ethanol for 1 h each time. After filtration and combination of the filtrates, recover the extraction solvent using a rotary evaporator. Dilute the obtained extract with water and extract it with ethyl acetate. After concentration and drying under reduced pressure of the ethyl acetate extract, obtain an extract (156.2 g). Pass it through a silica gel column chromatography, and elute it with petroleum ether:ethyl acetate (100:0, 50:1, 20:1, 10:1, 5:1) respectively. Combine 5000 mL of eluates according to HPLC identification. Finally, obtain 14.7 g of a high isoflavone mixture containing 6-formylisomaiwandflavone A in the elution part of petroleum ether:ethyl acetate (20:1). Among them, the weight percentages of methyl maiwandflavone A, methyl maiwandflavone B, 6-formylisomaiwandflavone A, and 6-formylisomaiwandflavone B are 25.5%, 38.3%, 29.2%, and 7.0% respectively).

[0039] Experimental method

[0040] Pre-equilibration experiment: Mix ionic liquid and water in a certain proportion to prepare a composite extractant A, and mix n-hexane and water in a certain proportion to prepare a raw material liquid dissolving agent B (washing liquid). Subsequently, add a certain amount of solution A and solution B into a 50 mL stoppered conical flask, then place it in a constant temperature oscillator, oscillate it at a speed of 200 rpm for 2 h at a certain temperature, and then let it stand until the two phases are completely separated; Take out the completely separated upper and lower phases and place them in a conical flask for use.

[0041] Extraction experiment: Use the equilibrated n-hexane:ethyl acetate to prepare a raw material liquid of 3 mg / mL of the powdered high isoflavone mixture. Take the same volume of the raw material liquid and the equilibrated composite extractant and add them into a stoppered conical flask. Place the conical flask in a constant temperature oscillator, oscillate it according to the method in the pre-equilibration experiment and then let it stand for phase separation; Sample the upper and lower phases respectively with a syringe, and then perform high performance liquid chromatography (HPLC) analysis. -1 Take the same volume of the raw material liquid and the equilibrated composite extractant and add them into a stoppered conical flask. Place the conical flask in a constant temperature oscillator, oscillate it according to the method in the pre-equilibration experiment and then let it stand for phase separation; Sample the upper and lower phases respectively with a syringe, and then perform high performance liquid chromatography (HPLC) analysis.

[0042] Calculation of distribution coefficient and separation selectivity:

[0043] The distribution coefficient and separation selectivity are important parameters to describe the extraction equilibrium.

[0044] The calculation formula for the distribution coefficient of solute A is as follows:

[0045] D A = C 萃取相 / C 萃余相

[0046] The separation selectivity S of solute A for solute B A / B The calculation formula is as follows:

[0047] SA / B =D A / D B

[0048] Experimental results

[0049] Selection of extractant: Influence of ionic liquids with different structures on the extraction and separation of 6-formylisomaiwandflavanone A

[0050] Select different anion structures, such as Cl - , Br - , OAc - , HSO4 - , BF4 - , and different cations such as [N 444 (C7H7)] + , [N 111 (C7H7)] + , [N 2222 + , [N 4444 + , [Hmin] + , [Emin] + , [Epy] + , [Bpy] + , [Pyr12] + , [BMMIm] + , [Dmin] + and [Omin] + . Investigate the influence of different anion and cation structures on the extraction and separation effect. The distribution coefficient and selectivity coefficient of 6-formylisomaiwandflavanone A (H3) are as Figure 3 shown.

[0051] Selection of dilution concentration of ionic liquid: Using the mixture of [Hmim]Br and water as the extractant, under the same extraction experimental conditions, change the content of [Hmim]Br in the extractant, and investigate the influence of the change in the concentration of ionic liquid on the extraction and separation effect. The results are as Figure 4 shown.

[0052] Selection of raw material liquid solvent and detergent: Using the mixture of [Hmim]Br / water as the extractant, under the same extraction experimental conditions, change the ratio of n-hexane:ethyl acetate in the raw material liquid solvent B, and investigate the influence of the change in the ratio of n-hexane:ethyl acetate on the extraction and separation effect. The results are as Figure 5 shown.

[0053] Conclusion

[0054] ​​The present invention provides a method for separating 6-formylindimophllin A from four ophiopogonoid mixtures using n-hexane-ethyl acetate / ionic liquid-water as the medium. When the molar concentration of ionic liquid [Hmim]Br in the composite extractant is 10%, the D H3 and S H3 / H124 reach ideal values, which are 5.26 and 12.46 respectively. When the molar concentration of ionic liquid [Hmim]Br in the composite extractant is 10%, the D H3 and S H3 / H124 reach ideal values, which are 3.92 and 17.08 respectively.

[0055] Example 2

[0056] The ophiopogonoid mixture prepared in Example 1 was formulated into a raw material solution of 10 g / L with n-hexane-ethyl acetate (9:1). 1-Hexyl-3-methylimidazolium bromide ([Hmin]Br)-water mixed solvent was used as the extractant (mole fraction of [Hmin]Cl was 10%), and n-hexane:ethyl acetate (9:1) was used as the detergent. The flow ratio of the extractant, detergent, and raw material solution was 3∶2.7∶1. Fractional extraction was carried out in a fractional extraction device at 40°C. The fractional extraction was divided into an extraction section and a washing section. Among them, the extraction section had 10 stages, and the washing section had 8 stages. The extractant entered the fractional extraction system from the first stage of the extraction section, the raw material solution entered the fractional extraction system from the last stage of the extraction section, the detergent entered the fractional extraction system from the first stage of the washing section, and the raw material solution was merged at the last stage of the washing section and then entered the extraction section together. The extraction phase and the washing phase were subjected to multi-stage countercurrent extraction. The raffinate enriched with 6-formylindimophllin A flowed out from the first stage of the extraction section, and the raffinate was collected; the raffinate was concentrated under vacuum to remove ethyl acetate, then washed repeatedly with water to remove the ionic liquid, and finally dried to remove water to obtain the final product. By HPLC analysis, the absolute purity of 6-formylindimophllin A in the product was 95.2%, and the yield was 92.1%.

[0057] Example 3

[0058] The prepared mixture of ophiopogonoid in Example 1 was formulated into a raw material solution with a concentration of 30 g / L using n-hexane-ethyl acetate (8:2). 1-Hexyl-3-methylimidazolium bromochloride ([Hmin]Cl)-water mixed solvent was used as the extractant (mole fraction of [Hmin]Cl was 10%), and n-hexane:ethyl acetate (8:2) was used as the detergent. The flow ratio of the extractant, detergent, and raw material solution was 4:5:1. Fractional extraction was carried out in a fractional extraction device at 40 °C. The fractional extraction was divided into an extraction section and a washing section. There were 12 stages in the extraction section and 10 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the raw material solution entered the fractional extraction system from the last stage of the extraction section, and the detergent entered the fractional extraction system from the first stage of the washing section. The raw material solution was combined at the last stage of the washing section and then entered the extraction section together. The extraction phase and the washing phase were subjected to multi-stage countercurrent extraction. The raffinate enriched with 6-formylophiopogonanone A flowed out from the first stage of the extraction section, and the raffinate was collected; the raffinate was concentrated under vacuum to remove ethyl acetate, then washed repeatedly with water to remove the ionic liquid, and finally dried to remove water to obtain the final product. Analyzed by HPLC, the absolute purity of 6-formylophiopogonanone A in the product was 96.2%, and the yield was 94.1%.

Claims

1. A method for separating 6 - aldehyde isomaiwandflavone A from a high - isoflavone mixture, characterized in that The following steps are adopted: 1) Using a high isoflavone mixture containing methylophiopogonone A, methylophiopogonone B, 6-formylisoochnaenin A and 6-formylisoochnaenin B as raw materials, dissolving the raw materials in a mixed solution composed of ethyl acetate and n-hexane to obtain a raw material solution; the volume ratio of n-hexane to ethyl acetate is 5-10:1-5; 2) Using a binary mixed solvent composed of ionic liquid-water as the extractant and the same solvent as the raw material solution solvent as the detergent, subjecting the raw material solution to fractional extraction, and collecting the raffinate; The molar ratio of ionic liquid to water in the extractant is 5:95-15:85; Ionic liquids are composed of two parts: cations M+ and anions N-, wherein the cations M+ are selected from [N 444 (C7H7)] + , [N 4444 ] + , [Hmim] + , [Dmim] + and [Omim] + , anion N- is chloride ion and bromide ion; 3) Subjecting the raffinate collected in step 2) to vacuum concentration, water washing and drying treatments in sequence to obtain 6-formylisoochnaenin A.

2. The method for separating 6 - aldehyde isomaiwandflavone A from a high - isoflavone mixture according to claim 1, characterized in that The mass fraction of 6-formylisoochnaenin A in the raw materials is greater than 25%.

3. The method for separating 6 - aldehyde isomaiwandflavone A from a high - isoflavone mixture according to claim 1, characterized in that The concentration of 6-formylisoochnaenin A in the raw material solution is 1-100 g / L.

4. The method for separating 6 - aldehyde isomaiwandflavone A from a high - isoflavone mixture according to claim 1, characterized in that During the fractional extraction process, the flow ratio among the extractant, the detergent and the raw material solution is 1-60:1-20:1, and the temperature of the fractional extraction is 20-45 °C.

5. The method for separating 6 - aldehyde isomaiwandflavone A from a high - isoflavone mixture according to claim 1, characterized in that The fractional extraction includes an extraction section and a washing section. Both the extraction section and the washing section include multiple stages. The extractant enters the fractional extraction system from the first stage of the extraction section, the raw material solution enters the fractional extraction system from the last stage of the extraction section, the detergent enters the fractional extraction system from the first stage of the washing section, and the raw material solution is combined and enters the extraction section at the last stage of the extraction section. The extraction phase and the washing phase carry out multi-stage countercurrent contact. An extraction solution rich in methylophiopogonone A, methylophiopogonone B and 6-formylisoochnaenin B flows out from the first stage of the washing section, and a raffinate enriched in 6-formylisoochnaenin A flows out from the first stage of the extraction section. The raffinate is collected.

Images