Method for preparing high-purity bakuchiol by combining deep eutectic solvent with high-speed counter-current chromatography

Abstract

The invention is applicable to the technical field of bakuchiol preparation, and provides a method for preparing high-purity bakuchiol by combining a deep eutectic solvent with high-speed counter-current chromatography, which comprises the following steps: mixing choline chloride with a hydrogen bond acceptor to obtain DES; mixing the fructus psoraleae powder with DES, extracting the mixture in a microwave extraction device, and carrying out suction filtration to collect filtrate; adding an extraction solvent into the filtrate, oscillating, extracting, layering, collecting an upper-layer organic phase, and concentrating under reduced pressure until the upper-layer organic phase is dry, so as to obtain a crude extract; preparing an n-hexane-ethyl acetate-methanol-water solvent system, dissolving the fructus psoraleae crude extract by using a mixed solvent of upper and lower phases, pumping into a mobile phase by adopting a high-speed counter-current chromatograph, and collecting fractions; and concentrating under reduced pressure and drying in vacuum. The DES extraction technology and the HSCCC purification technology are combined and applied to preparation of the bakuchiol, a brand-new and green technical path is formed, the problem that the bakuchiol is difficult to separate from structural analogues is solved, extremely high purity can be achieved in one step, DES and HSCCC solvents can be recycled, and the production cost and environmental pollution are reduced.

Description

Technical Field

[0001] This invention belongs to the field of psoralen preparation technology, and particularly relates to a method for preparing high-purity psoralen using deep eutectic solvent combined with high-speed countercurrent chromatography. Background Technology

[0002] Bakuchiol is a natural coumarin derivative extracted from the psoralea plant, with the chemical structure C0.05. 20 H 22 O4, possessing significant phytoestrogen-like activity, antioxidant, anti-inflammatory, and anti-aging effects, is hailed as "phytoretinol." Due to its similar anti-wrinkle and collagen-promoting effects to retinol, but with less irritation and better stability, it has garnered significant attention in the cosmetics and pharmaceutical industries in recent years, with a promising market prospect. However, efficiently and environmentally friendly methods for obtaining high-purity psoralen from psoralen still face challenges.

[0003] Traditional methods often use highly polar organic solvents such as petroleum ether and n-hexane for Soxhlet extraction or reflux extraction, which poses a risk of solvent residue, affecting the safety of the product in cosmetics and is not in line with the trend of green chemistry.

[0004] The crude extract of Psoralea corylifolia has a complex composition, containing various coumarins, flavonoids and monoterpenoids and other structural analogs. Conventional silica gel column chromatography separation is cumbersome and takes more than 10 hours, and uses a large amount of organic solvents, making it difficult to achieve both yield and purity.

[0005] In traditional extraction and vacuum concentration processes, if the temperature is not properly controlled, psoralen is prone to isomerization or degradation, resulting in loss of activity and darkening of product color.

[0006] While supercritical CO2 extraction can avoid organic solvent residue, it requires sophisticated equipment and has limited selectivity for highly polar psoralen, necessitating the addition of entrainers, which is costly. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing high-purity psoralen using deep eutectic solvents combined with high-speed countercurrent chromatography, thereby addressing the problems mentioned in the background art.

[0008] The present invention is implemented as follows: a method for preparing high-purity psoralen using a deep eutectic solvent combined with high-speed countercurrent chromatography includes the following steps:

[0009] S1. Preparation of deep eutectic solvent and pretreatment of raw materials: Choline chloride and hydrogen bond acceptor are mixed and stirred until a homogeneous and transparent liquid is formed, which is the desired deep eutectic solvent DES; dried Psoralea seeds are crushed and sieved to obtain Psoralea powder.

[0010] S2, DES extraction: Mix Psoralea corylifolia powder with DES, place in a microwave extraction device, stir and extract, filter after extraction, and collect the filtrate;

[0011] S3. Extraction and enrichment and DES recovery: Add an equal volume of extraction solvent to the filtrate, shake to extract, let stand to separate the layers and collect the upper organic phase. Repeat the extraction several times, combine the organic phases, and concentrate the combined organic phases to dryness under reduced pressure to obtain crude extract of Psoralea corylifolia.

[0012] S4, HSCCC precise separation and purification: Prepare a solvent system of n-hexane-ethyl acetate-methanol-water, let it stand and separate into layers, the upper phase is the stationary phase and the lower phase is the mobile phase. Dissolve the crude extract of Psoralea corylifolia in the mixed solvent of the upper and lower phases, and pump the mobile phase in using a high-speed countercurrent chromatograph. Collect the target components according to the detector spectrum.

[0013] S5. Post-processing: The collected fraction containing psoralen is concentrated under reduced pressure and dried under vacuum to obtain a white crystalline powder, which is high-purity psoralen.

[0014] The method for preparing high-purity bakuchiol using deep eutectic solvent combined with high-speed countercurrent chromatography provided in this invention solves the safety and environmental problems caused by the use of toxic organic solvents in traditional extraction methods. It completely replaces traditional toxic solvents such as hexane and petroleum ether with the green solvent DES, ensuring product safety and process environmental friendliness from a fundamental perspective. It overcomes the bottlenecks of existing purification technologies, such as long processes, low efficiency, and difficulty in obtaining cosmetic-grade high-purity bakuchiol. It replaces the cumbersome silica gel column chromatography with preparative-grade HSCCC technology, realizing a shift from a "adsorption-desorption" solid-liquid separation mode to a "continuous distribution" liquid-liquid separation mode, offering significant advantages such as high resolution, no irreversible adsorption, and high recovery rate. It establishes a mild, efficient, and green preparation process, maximizing the preservation of the chemical integrity and biological activity of bakuchiol. It enables integrated, scalable production of high-purity products (≥98%) from raw materials. Attached Figure Description

[0015] Figure 1 Chromatograms and results of the method for preparing high-purity psoralen using deep eutectic solvent combined with high-speed countercurrent chromatography provided in this embodiment of the invention;

[0016] Figure 2 The chromatogram and results of the hexane extraction-silica gel column chromatography method provided in the embodiments of the present invention are shown. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0018] A method for preparing high-purity psoralen using a deep eutectic solvent combined with high-speed countercurrent chromatography includes the following steps:

[0019] S1. Preparation of deep eutectic solvent (DES) and pretreatment of raw materials: Choline chloride and ethylene glycol are mixed at a molar ratio of 1:2 and stirred at 80°C until a homogeneous and transparent liquid is formed, which is the desired deep eutectic solvent; dried psoralea seeds are crushed and passed through a 40-mesh sieve to obtain psoralea powder.

[0020] S2, DES Green and Efficient Extraction: Mix Psoralea corylifolia powder and DES solvent at a ratio of 1:15 (g / mL), place in a microwave extraction device, and extract by stirring at 50℃ and 250W for 30 minutes. After extraction, filter while hot and collect the filtrate.

[0021] S3. Extraction and enrichment and DES recovery: Add an equal volume of ethyl acetate to the collected filtrate, shake to extract, allow to stand and separate into layers, collect the upper organic phase, repeat the extraction 3 times, combine the organic phases (the lower DES phase can be recycled after dehydration), concentrate the combined organic phases to dryness under reduced pressure at 40℃ to obtain crude extract of Psoralea corylifolia.

[0022] S4: Precise separation and purification by HSCCC: Prepare a solvent system of n-hexane-ethyl acetate-methanol-water (volume ratio 5:5:5:5). After standing and separating the layers, the upper phase is the stationary phase and the lower phase is the mobile phase. Dissolve the crude extract of Psoralea corylifolia in an appropriate amount of the mixed solvent of the upper and lower phases. Use a high-speed countercurrent chromatograph at 850 rpm in tail-to-head mode and pump the mobile phase at a flow rate of 2.0 mL / min. Collect the target components according to the detector chromatogram.

[0023] S5: Post-processing: The collected fraction rich in psoralen is concentrated under reduced pressure at 40°C and dried under vacuum to obtain a white crystalline powder, which is high-purity psoralen.

[0024] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0025] Example 1: A high-purity psoralen was prepared using the following method:

[0026] S1, DES preparation and raw material pretreatment: Weigh 70g of choline chloride and 124g of ethylene glycol, mix them and stir at 80℃ until clear; at the same time, 1.0 kg of dried psoralea seeds are crushed and passed through a 40-mesh sieve;

[0027] S2. Mix the psoralea powder with 15 L of DES solvent, stir and extract at 250W microwave power and 50℃ for 30 minutes, filter, and obtain the filtrate.

[0028] S3. The filtrate was extracted three times with 15 L of ethyl acetate, the organic phases were combined, and the mixture was concentrated under reduced pressure at 40 °C to obtain 85 g of crude Psoralea corylifolia extract.

[0029] S4. Prepare a 2L solvent system of n-hexane-ethyl acetate-methanol-water (5:5:5:5, v / v). Dissolve 5.0g of crude Psoralea corylifolia extract in 20mL of the mixed solvent. Start the HSCCC instrument and pump the lower phase (mobile phase) at 850 rpm and a flow rate of 2.0 mL / min. Collect the fractions with retention times in the range of 180-220 minutes according to the UV detector spectrum.

[0030] S5. The collected fraction was concentrated and vacuum dried to obtain 3.9g of white powder.

[0031] Detection results: The obtained white powder was analyzed by HPLC, and the results are as follows. Figure 1 As shown, the purity of psoralen in the above sample is as high as 98.5%. Based on the initial weight of psoralen powder, the yield of this separation is 0.39%. Based on the psoralen content in the crude extract, the recovery rate of HSCCC one-step purification reaches 91.2%.

[0032] Comparative Analysis: The commonly used n-hexane extraction-silica gel column chromatography method was selected as the control example for the experiment. The same batch of psoralen powder was used, and hexane hot reflux extraction was employed. The extract was then subjected to gradient elution on a silica gel column (200-300 mesh) (petroleum ether:ethyl acetate from 20:1 to 5:1). The fraction containing psoralen was collected and concentrated. HPLC analysis showed the following results: Figure 2 As shown, the purity of psoralen was 95.2%, and the recovery rate was only 70%.

[0033] In summary, the method provided by the embodiments of the present invention has the following advantages:

[0034] High purity and high recovery rate: The purity of the final product is ≥98.5%, meeting the standards for high-end cosmetic raw materials; the single-step recovery rate of bakuchiol by HSCCC exceeds 90%;

[0035] Green and environmentally friendly: Using low-toxicity, biodegradable DES as an extraction solvent replaces traditional toxic solvents, achieving green manufacturing from the source;

[0036] High efficiency and time-saving: DES combined with microwave-enhanced extraction is highly efficient; the HSCCC separation process does not require a solid support, avoiding irreversible adsorption loss, and the entire purification process can be completed in 3-4 hours, which is much faster than traditional column chromatography.

[0037] Activity preservation: The entire process is carried out at medium and low temperatures, which effectively prevents the thermal degradation of psoralen and ensures the bioactivity of the product.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing high-purity psoralen using deep eutectic solvent combined with high-speed countercurrent chromatography, characterized in that, Includes the following steps: S1. Preparation of deep eutectic solvent and pretreatment of raw materials: Choline chloride and hydrogen bond acceptor are mixed and stirred until a homogeneous and transparent liquid is formed, which is the desired deep eutectic solvent DES; dried Psoralea seeds are crushed and sieved to obtain Psoralea powder. S2, DES extraction: Mix Psoralea corylifolia powder with DES, place in a microwave extraction device, stir and extract, filter after extraction, and collect the filtrate; S3. Extraction and enrichment and DES recovery: Add an equal volume of extraction solvent to the filtrate, shake to extract, let stand to separate the layers and collect the upper organic phase. Repeat the extraction several times, combine the organic phases, and concentrate the combined organic phases to dryness under reduced pressure to obtain crude extract of Psoralea corylifolia. S4, HSCCC precise separation and purification: Prepare a solvent system of n-hexane-ethyl acetate-methanol-water, let it stand and separate into layers, the upper phase is the stationary phase and the lower phase is the mobile phase. Dissolve the crude extract of Psoralea corylifolia in the mixed solvent of the upper and lower phases, and pump the mobile phase in using a high-speed countercurrent chromatograph. Collect the target components according to the detector spectrum. S5. Post-processing: The collected fraction containing psoralen is concentrated under reduced pressure and dried under vacuum to obtain a white crystalline powder, which is high-purity psoralen.

2. The method for preparing high-purity psoralen using a deep eutectic solvent combined with high-speed countercurrent chromatography according to claim 1, characterized in that, In step S1, the hydrogen bond acceptor is one of ethylene glycol, glycerol, or lactic acid.

3. The method for preparing high-purity psoralen using a deep eutectic solvent combined with high-speed countercurrent chromatography according to claim 2, characterized in that, In step S1, the molar ratio of choline chloride to hydrogen bond acceptor is 1:1-3.

4. The method for preparing high-purity psoralen using a deep eutectic solvent combined with high-speed countercurrent chromatography according to claim 1, characterized in that, In step S2, the ratio of psoralen powder to DES in liquid form (g:mL) is 1:10-20.

5. The method for preparing high-purity psoralen using a deep eutectic solvent combined with high-speed countercurrent chromatography according to claim 1, characterized in that, In step S2, the temperature of the stirring extraction is 40-60℃, the microwave power is 200-300W, and the extraction time is 20-40 minutes.

6. The method for preparing high-purity psoralen using a deep eutectic solvent combined with high-speed countercurrent chromatography according to claim 1, characterized in that, In step S3, the extraction solvent is one of ethyl acetate, methyl tert-butyl ether, and butyl acetate.

7. The method for preparing high-purity psoralen using a deep eutectic solvent combined with high-speed countercurrent chromatography according to claim 1, characterized in that, In step S4, the volume ratio of the solvent system n-hexane-ethyl acetate-methanol-water is 4-6:4-6:4-6:4-6.

8. The method for preparing high-purity psoralen using a deep eutectic solvent combined with high-speed countercurrent chromatography according to claim 1, characterized in that, In step S4, the rotation speed of the mobile phase pumped in using the high-speed countercurrent chromatograph is 800-900 rpm, and the flow rate of the mobile phase is 1.5-2.5 mL / min.

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