CN114573474A - Method for preparing hydroxy sanshool by deep eutectic solvent extraction and dynamic axial chromatography - Google Patents

Abstract

The invention relates to a method for preparing hydroxyl sanshool by deep eutectic solvent extraction and dynamic axial chromatography, belonging to the field of natural product extraction. The method comprises the following steps: s1, supercritical CO₂Extracting the zanthoxylum oil as a raw material, adding a eutectic solvent for ultrasonic auxiliary extraction, centrifuging after extraction is finished, and collecting a lower DES layer to obtain a DES extracting solution of the hydroxysanshool compound; recovering hydroxy sanshool components from the obtained extracting solution by an organic solvent reverse extraction method to obtain a hydroxy sanshool component enrichment product; s2, dynamic axial preparative chromatography separation and purification: the hydroxy sanshool component enrichment product is completely dissolved by using a mobile phase with the mass volume of 1-3 times, and the dynamic axial compression is adopted to prepare liquid phase separation, wherein the mobile phase comprises methanol, water and the like. The process is simple, does not need a large number of purification steps, is convenient to recover the solvent, has high purity, is suitable for large-scale industrial production, and has strong use value.

Description

A kind of deep eutectic solvent extraction, dynamic axial chromatography to prepare hydroxysanshool method

technical field

The invention relates to a method for preparing hydroxysanshool by low eutectic solvent extraction and dynamic axial chromatography, and belongs to the field of natural product extraction.

Background technique

Rattan pepper (Zanthoxylum armatum DC) is a traditional Chinese medicinal and edible resource with a fragrant, soft, non-irritating and bitter taste. Studies have shown that sanshool and its amide homologues with structural changes caused by differences in alkyl unsaturation or carbon chain oxidation are regarded as the basis of the numbness of vine pepper and are also important quality assurance. -β-Sanshool, Hydroxy-γ-Sanshool, Hydroxy-ε-Sanshool mainly, may induce numbness by activating transient receptor potential (TRP) V1 and TRPA1 or blocking ion channels. Anesthesia, analgesia, anti-inflammatory, intestinal protection and other functions. However, these compounds are structurally unstable, and are prone to isomerization, hydrolysis and oxidation in the air, resulting in technical bottlenecks in the large-scale preparation of high-purity sanshools. At present, there is no standard method for the numbness evaluation of Chinese prickly ash/vinegarden pepper and the extraction of numbness substances suitable for industrial production at home and abroad, resulting in uneven quality of raw materials and products of Chinese prickly ash and rattan pepper, which seriously affects the interests of consumers and the effective supervision of the market.

Chinese Patents CN102771747B, CN102690208A, CN105237430A disclose the preparation process of sanshool compounds including, using organic solvent or supercritical CO ₂ to extract samples of Chinese prickly ash/vinegar, and then use silica gel and gel chromatography to purify, collect different elution conditions The fractions obtained from the above-mentioned fractions, and then use preparative thin-layer chromatography, preparative/semi-preparative HPLC and other instruments to obtain higher-purity monomers, the preparation process is tedious and complicated, the preparation amount is small, and the efficiency is low. , The preparation process of macro-extraction to prepare a variety of high-purity monomers has great scientific significance and engineering application value.

SUMMARY OF THE INVENTION

The purpose of the present invention is to aim at the deficiencies of the prior art, provide a kind of low eutectic solvent extraction, dynamic axial chromatography to prepare hydroxysanshool method, this method is a kind of extraction based on green new low eutectic solvent, industrial grade rapid preparation high The method for the purity of hydroxysanshool compounds provides a reference substance for the analysis and quality evaluation of the numbness components of vine pepper.

The object of the present invention is achieved through the following technical solutions:

A method for rapidly preparing high-purity sanshool compounds at industrial level, comprising the following steps:

S1, using supercritical CO ₂ extraction of vine pepper oil as a raw material, adding a deep eutectic solvent (DES), ultrasonic-assisted extraction, centrifuging after the extraction, collecting the lower DES layer, and obtaining a DES extract solution of hydroxysanshool components; the obtained extract solution Through the organic solvent back extraction method, the hydroxysanshool components are recovered to obtain the enriched products of the hydroxysanshool components.

Preferably, the deep eutectic solvent is prepared by the following method. The hydrogen bond donor and the hydrogen bond acceptor are mixed according to a certain molar ratio, and after being sealed, they are heated at 80° C. until they are clarified to form a DES solvent. set aside.

Preferably, the hydrogen bond donor is choline chloride, the hydrogen bond acceptor is one of formic acid, acetic acid, and lactic acid, and the molar ratio is 1:2.

Preferably, the centrifugal speed is 4000r/mi, and the centrifugal time is 10min.

Preferably, the ultrasonic-assisted treatment conditions are that the ratio of material to liquid is 1:5-1:10 (w/v), the extraction power is 150-200w, and the extraction time is 0.5h.

Preferably, the organic solvent used in the back extraction is ethyl acetate, and the solid-liquid ratio is 1:5 to 1:10.

S2, Separation and purification by dynamic axial preparative chromatography: The sanshool concentrates are completely dissolved with 1-3 times the mass and volume of the mobile phase, and the liquid phase separation is prepared by dynamic axial compression. The mobile phase is methanol and water, and the detection wavelength is 254 nm. , the target components were collected according to different retention times, the eluate was concentrated by rotary evaporation under reduced pressure, and each monomer compound was obtained after freeze-drying.

Preferably, the dynamic axial compression column packing in the dynamic axial preparative liquid chromatography is selected from one or more of Nucifera C18, Hedera ODS-2, ACCHROM X5, and ACCHROM C18PE. Hedera ODS-2 enables efficient separation of hydroxysanshool.

Octadecyl reverse silica gel, the particle size of the silica gel is 5-10 μm, more preferably 5 μm; the size of the dynamic axial compression column is preferably 50 mm×250 mm.

Preferably, the mobile phase ratio is methanol:water=65:35～85:15(v/v).

Preferably, the flow rate of the elution is 10-40 mL/min.

Preferably, the concentration of the injection solution is 50-150 mg/mL.

Deep eutectic solvents are composed of hydrogen bond donors (such as compounds such as amides, carboxylic acids and polyols) and hydrogen bond acceptors (such as quaternary ammonium salts) through hydrogen bonding, and have significantly lower melting points than the pure substances of each component. , and green environmental protection, non-toxic, low cost of raw materials, solvent can be recycled. The patent design and synthesis of DESs with higher extraction efficiency, combined with dynamic axial preparative chromatography, realizes large-scale industrial-scale preparation of a series of high-purity sanshool compounds in vine pepper.

Compared with the prior art, the positive effects of the present invention are reflected in:

(1) In the present invention, choline chloride and acids are mixed to prepare a DES solvent, which significantly improves the extraction rate of hydroxysanshool compounds compared to known extraction solvents.

(2) For the first time, the present invention adopts a complete set of technology with deep eutectic solvent extraction/purification-dynamic axial compression column separation as the core to simultaneously separate hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ from vine pepper oil -The method of sanshool, hydroxy-ε-sanshool, establishes a process route of extracting, purifying and separating 4 monomer compounds from vine pepper, the process is simple, does not require a large number of purification steps, and the solvent is easy to recover , high purity, suitable for large-scale industrial production, and has a strong use value.

Description of drawings

Figure 1 is the HPLC chromatogram of Example 1 using DAC for separation, wherein a, b, c, d represent hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool, hydroxy-ε- Sanshool.

Fig. 2 is the HPLC spectrum of hydroxy-α-sanshool prepared in Example 1, the purity is 97.63%.

Fig. 3 is the HPLC spectrum of hydroxy-β-sanshool prepared in Example 1, the purity is 99.96%.

Fig. 4 is the HPLC spectrum of hydroxy-γ-sanshool prepared in Example 1, the purity is 98.57%.

Figure 5 is the HPLC spectrum of the hydroxy-ε-sanshool prepared in Example 1, and the purity is 100%.

Detailed ways

The following examples are provided for a better understanding of the present invention, and are not limited to the best embodiments, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by combining with the features of other prior art shall fall within the protection scope of the present invention.

The material-to-liquid ratio recorded in this application, unless otherwise specified, refers to the ratio between the mass g of the material and the liquid volume ml.

Example 1:

(1) Preparation of deep eutectic solvent (DES): Mix choline chloride and acetic acid in a molar ratio of 1:2, seal and heat at 80 °C to dissolve until clear to form DES solvent, and stand at room temperature for later use .

(2) Extraction of sanshool compounds based on deep eutectic solvent: take 50 g of vine pepper oil (preferably obtained through supercritical CO ₂ extraction, such as Yaomazi vine pepper oil) as raw material, according to the material ratio of 1:10 (w/ v) adding DES, ultrasonic-assisted extraction for 30 min, and centrifuging at 4000 rpm for 10 min to collect the DES layer to obtain a hydroxysanshool DES extract; the obtained extract was added with ethyl acetate at a material-to-liquid ratio of 1:8 to recover the sanshools, and the filtrate was collected by filtration. Steam and concentrate to obtain the enriched product of hydroxysanshools.

(3) Dynamic axial compression separation: The chromatographic column is equilibrated with Nucifera C18U (5μm, 50×250mm) with the mobile phase until the chromatographic baseline is stable, and the enriched products of hydroxysanshool are prepared with a mobile phase of 80 mg/mL, and filtered to obtain The sample solution was separated by industrial preparative chromatography. The mobile phase was methanol with a volume fraction of 65%, the flow rate was 40 mL/min, and the detection wavelength was 254 nm. The target components were collected according to different retention times. Freeze drying under vacuum gave each monomer compound.

(4) The monomer compounds were detected by HPLC, the purity of hydroxy-α-sanshool was 97.63%, the purity of hydroxy-β-sanshool was 99.96%, the purity of hydroxy-γ-sanshool was 98.57%, the purity of hydroxy-ε- The purity of sanshool is 100.00%.

Example 2:

(1) Preparation of deep eutectic solvent (DES): Mix choline chloride and formic acid in a molar ratio of 1:2, seal and heat at 80 °C to dissolve until clarification to form DES solvent, and stand at room temperature for later use .

(2) Extraction of sanshool compounds based on deep eutectic solvent: take 100 g of supercritical CO ₂ extracted vine pepper oil as a raw material, add DES according to the material ratio of 1:10, ultrasonic-assisted extraction for 30 min, and centrifuge at 4000 rpm for 10 min to collect the DES layer to obtain Sanshool DES extract; the obtained extract is added with ethyl acetate according to a material-to-liquid ratio of 1:8 to recover sanshools, the filtrate is collected by filtration, and concentrated by rotary evaporation to obtain an enriched product of hydroxysanshools.

(3) Dynamic axial compression separation: The chromatographic column adopts Nucifera C18U (5μm, 50×250mm), and the mobile phase is used to balance the chromatographic baseline until the chromatographic baseline is stable. The sample solution was obtained, and the sample solution was separated by industrial preparative chromatography, using gradient elution, 0min-60min, methanol/water=65:35 (v/v), 60min-80min, methanol/water=85:15, flow rate The concentration was 40 mL/min, and the detection wavelength was 254 nm. The target components were collected according to different retention times. The collected solution was concentrated under reduced pressure at 35°C to 45°C, and freeze-dried under vacuum to obtain each monomer compound.

(4) The monomer compounds were detected by HPLC. The purity of hydroxy-α-sanshool was 97.57%, the purity of hydroxy-β-sanshool was 96.38%, the purity of γ-sanshool was 98.30%, and the purity of hydroxy-ε-sanshool was 97.70%. %.

Example 3

(1) Preparation of deep eutectic solvent (DES): Mix choline chloride and acetic acid in a molar ratio of 1:2, seal and heat at 80 °C to dissolve until clear to form DES solvent, and stand at room temperature for use .

(2) Extraction of sanshool compounds based on deep eutectic solvent: take 100 g of supercritical CO ₂ extracted vine pepper oil as a raw material, add DES according to the material ratio of 1:10, ultrasonic-assisted extraction for 30 min, and centrifuge at 4000 rpm for 10 min to collect the DES layer to obtain Sanshool DES extract; the obtained extract is added with ethyl acetate according to a material-to-liquid ratio of 1:8 to recover sanshools, the filtrate is collected by filtration, and concentrated by rotary evaporation to obtain an enriched product of hydroxysanshools.

(3) Dynamic axial compression separation: Hedera ODS-2 (5μm, 50×250mm) was used for the chromatographic column, and the mobile phase was used to equilibrate to a stable chromatographic baseline. The sample solution was obtained by filtration, and the sample solution was separated by industrial preparative chromatography. The mobile phase was methanol with a volume fraction of 65%, the flow rate was 45 mL/min, and the detection wavelength was 254 nm. The target components were collected according to different retention times. Concentrate under reduced pressure at 35°C to 45°C and freeze-dry under vacuum to obtain each monomer compound.

(4) The monomer compounds were detected by HPLC. The purity of hydroxy-α-sanshool was 97.24%, the purity of hydroxy-β-sanshool was 98.23%, the purity of hydroxy-γ-sanshool was 96.54%, and the purity of hydroxy-ε-sanshool was 96.54%. is 99.39%.

Comparative Example 1

Compared to Example 1, the DES solvent was not used, but methanol was used as the extraction solvent.

Comparative Example 2

Compared with Example 1, urea and acetamide (amide group) were used as hydrogen bond donors.

Comparative Example 3

Compared with Example 1, the chromatographic column adopts ACCHROM C18PE (7um, 50*250mm). The obtained monomer compound was detected by HPLC, and the purity of hydroxy-α-sanshool was 90.64%, the purity of hydroxy-β-sanshool was 64.68%, the purity of hydroxy-γ-sanshool was 62.45%, and the purity of hydroxy-ε-sanshool was 85.82%. . Compared with Examples 1, 2, and 3, the impurity content is more, and the purity decreases.

Test Example 1

Spectral detection was performed on the substances prepared above, and high performance liquid chromatography (HPLC) was used for detection. The chromatographic conditions were: mobile phase A: water, mobile phase B: methanol; 0～50min, A:B=50:50～25 : 75; flow rate: 1mL/min, column temperature: 30℃; UV detection wavelength: 254nm, injection volume: 10uL. The extraction rate of each extraction method was calculated, and the extraction rate was equal to the percentage of the content of sanshool compounds in the extraction solvent to the total content in the two phases.

Among them, the extraction rates of Examples 1, 2, and 3 are 24%, 23%, and 23%, respectively; Comparative Example 1, in which methanol is used instead of DES solvent, belongs to the traditional extraction of amides in Zanthoxylum bungeanum/vine pepper, compared with the examples. Substance method, the extraction rate was 20%, indicating that DES solvent has a crucial role in the extraction of target compounds. By observing the extraction rate of Comparative Example 2, when urea is used as the hydrogen bond donor, the extraction rate is only 15%, so the extraction efficiency of acid hydrogen bond donors is higher, which may be due to the hydrogen bond between amide compounds and acids Donor binding occurs, resulting in mass transfer of amides to the solution, thereby promoting adequate extraction of amides.

To sum up, in the present invention, the DES solvent prepared by mixing choline chloride and acids is extracted in combination with ultrasonic-assisted extraction, and the extraction effect is significantly improved under the synergistic effect. Then combined with the method of dynamic axial compression column separation and simultaneous separation of hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool and hydroxy-ε-sanshool from vine pepper oil, a method was established from The process route of extracting, purifying and separating 4 monomeric compounds from vine pepper, the process is simple, does not require a large number of purification steps, the solvent is easy to recover, the purity is high, it is suitable for large-scale industrial production, and has strong use value.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (10)

1. a method for the rapid preparation of high-purity hydroxysanshool compounds of industrial grade, is characterized in that comprising the following steps: S1, using supercritical CO ₂ extraction of vine pepper oil as a raw material, adding a deep eutectic solvent for ultrasonic-assisted extraction, centrifuging after the extraction, collecting the lower DES layer, and obtaining a sanshool compound DES extract; The obtained extract is subjected to organic solvent reaction Extraction method, recovering hydroxy sanshool components, and obtaining hydroxy sanshool components enrichment products; S2, separation and purification by dynamic axial preparative chromatography: the enriched products of hydroxysanshool are completely dissolved with 1-3 times the mass and volume of the mobile phase, and the liquid phase separation is prepared by dynamic axial compression. The mobile phase is methanol and water, and the detection wavelength The target component was collected according to different retention times, the eluate was concentrated by rotary evaporation under reduced pressure, and each monomer compound was obtained after freeze-drying. 2. The method according to claim 1, characterized in that, the preparation method of the deep eutectic solvent is that the hydrogen bond donor and the hydrogen bond acceptor are mixed in a certain molar ratio, and then placed at 60-80° C. after sealing. Heating and co-dissolving until clear, to form DES solvent, stand at room temperature for later use. 3. The method of claim 2, wherein the hydrogen bond donor is choline chloride; the hydrogen bond acceptor is any one of formic acid, acetic acid, and lactic acid. 4. The method of claim 2, wherein the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:1-2. 5. The method of claim 1, wherein in S1, the rotational speed used in the centrifugation is 4000 r/mi, and the centrifugation time is 10 min. 6. The method of claim 1, wherein in S1, the ultrasonic-assisted treatment conditions are that the ratio of solid to liquid is 1:5 to 1:10 (w/v), and the extraction power is 150 to 200w , the extraction time is 0.5h. 7. The method of claim 1, wherein, in S1, the organic solvent used in the back-extraction is ethyl acetate, and the solid-liquid ratio is 1:5 to 1:10. 8. The method of claim 1, wherein in S2, the dynamic axial compression column packing in the dynamic axial preparative liquid chromatography is octadecyl reverse silica gel, and the particle size of the silica gel is 10-20 μm ; The size of the dynamic axial compression column is 50mm×250mm. 9 . The method of claim 1 , wherein, in S2 , the mobile phase is methanol:water=65:35～85:15 (v/v). 10 . 10. The method of claim 1, wherein in S2, the flow rate of the elution is 10-40 mL/min; the concentration of the injection solution is 50-150 mg/mL.

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