Method for extracting and separating cembratriene glycol by using deep eutectic solvent

Abstract

The invention discloses a method for extracting and separating cembratriene glycol by using a deep eutectic solvent. The method comprises the following steps: synthesizing a deep eutectic solvent by selecting choline chloride as a hydrogen bond donor and propylene glycol as a hydrogen bond receptor, putting the tobacco inflorescence into the deep eutectic solvent for extraction, filtering after extraction, adding petroleum ether into a DES extracting solution, recovering a petroleum ether layer after extraction, carrying out reduced pressure distillation to dryness, adding acetonitrile for dissolving, and filtering to obtain the tobacco inflorescence. Two isomers of cembratriene glycol are separated by preparative chromatography to obtain two monomeric compounds with high purity. The technological process is simple, a large number of purification steps are not needed, the solvent is convenient to recycle, the purity is high, and the method is suitable for large-scale industrial production and has very high use value.

Description

Technical Field

[0001] The invention belongs to the field of separation and extraction of natural products, and specifically relates to a method for extracting and separating cedrintrienediol using a deep eutectic solvent. Background Art

[0002] Cephalotriene diol is a cephalosporin-type diterpene compound, which is abundant in tobacco. Cephalotriene diol in tobacco can produce flavor components such as solanone, solanofuran, and norsolani through degradation, which are important contributors to tobacco aroma components. Cephalotriene diol gives tobacco a roasted aroma, a slightly sweet herbal aroma, and a cocoa aroma. When the content is high, it produces a slight floral aroma, and when the content is low, it produces a light powdery aroma. This compound is not only an important precursor of cigarette aroma components, but also has multiple biological activities such as inhibiting tumor cell growth, inhibiting prostaglandin synthesis, inhibiting nicotine perception, and acting as a neuroprotectant.

[0003] At present, the methods for purifying and analyzing cedrinone have their own characteristics, but they still have disadvantages such as residual derivatization reagents, low solvent recovery rate, low detection sensitivity, and cumbersome pretreatment. Therefore, it is of great scientific significance and application value to study a method that can quickly and massively extract and prepare high-purity monomer cedrinone from tobacco inflorescence. Summary of the invention

[0004] In view of the above-mentioned deficiencies of the prior art, the present invention provides a method for extracting and separating cedrinone diol with a deep eutectic solvent. The method of the present invention is based on green new deep eutectic solvent extraction, can quickly prepare high-purity cedrinone diol, and provide a reference substance for the analysis and quality evaluation of the sweet and caramel aroma components of cigarettes.

[0005] The method for extracting and separating cedrintrienediol by a deep eutectic solvent of the present invention comprises the following steps: Step 1: Using tobacco inflorescence as raw material, adding a deep eutectic solvent for extraction, filtering after the extraction is completed, collecting the extract, and recording it as L1; the obtained extract is back-extracted with an organic solvent to recover the cedartriene diol component to obtain a cedartriene diol component enriched product.

[0006] Step 2: The cedartriene diol component enriched product obtained in step 1 is completely dissolved in acetonitrile, which is recorded as L2; ​​preparative chromatography liquid phase separation is performed, the eluate is collected, concentrated by rotary evaporation under reduced pressure, and freeze-dried to obtain the target product.

[0007] In step 1, the deep eutectic solvent is prepared by a method comprising the following steps: The hydrogen bond donor and the hydrogen bond acceptor are mixed in a certain molar ratio, sealed, placed in a water bath, heated and dissolved until uniform and transparent to form a DES solvent, and then allowed to stand at room temperature for use.

[0008] The hydrogen bond donor is choline chloride, the hydrogen bond acceptor is propylene glycol, the molar ratio of the two is 1:3-1:5, and the water content is 20%. The percentage content here means that the sum of the hydrogen bond donor and the hydrogen bond acceptor accounts for 80%, and the remaining 20% ​​is water. This ratio is a volume ratio.

[0009] Further, the heating temperature is 80° C. The solid-liquid ratio of the deep eutectic solvent is 1:30-1:50 (w / v), the extraction temperature is 35-45° C., and the extraction time is 22-24 hours. Here, the liquid-to-solid ratio refers to the weight of the tobacco inflorescence and the volume ratio of the deep eutectic solvent.

[0010] In step 1, the organic solvent used for back extraction is petroleum ether, and the solid-liquid ratio is 1:2 to 1:3. Here, the solid-liquid ratio refers to the volume ratio of the extract to petroleum ether being 1:2 to 1:3.

[0011] In step 2, the parameters of the preparative chromatographic liquid phase separation are set as follows: The chromatographic column was ChromCore 120 C18 5 μm 21.2 × 250 mm; The mobile phase was acetonitrile and water in a volume ratio of 60:40 to 80:20 (v / v); The elution flow rate is 3-5 mL / min; The injection solution concentration was 3 mg / mL; The injection volume was 3 mL; The detection wavelength is 210 nm.

[0012] Further, the target components were collected according to different retention times, and the eluate corresponding to the absorption peak at 28 min was collected, which was the solution containing the compound α-cephalotriene diol; the eluate corresponding to the absorption peak at 33 min was collected, which was the solution containing the compound β-cephalotriene diol.

[0013] In step 2, the reduced pressure distillation temperature is 75°C.

[0014] The present invention uses petroleum ether to back-extract cephalotriene diol in a deep eutectic solvent, and the purity of cephalotriene diol in petroleum ether can reach 84.4%, where the purity is the purity before isomer separation. The innovation lies in that the method is simple and fast, and a purity of 84.4% can be achieved by only two steps of deep eutectic solvent extraction and petroleum ether back-extraction. The subsequent semi-preparative chromatographic separation is improved for the separation of two isomers.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The deep eutectic solvent is composed of hydrogen bond donors (such as amides, carboxylic acids and polyols) and hydrogen bond acceptors (such as quaternary ammonium salts) through hydrogen bonds, and has a significantly lower melting point than the pure substances of each component, and is green, environmentally friendly, non-toxic, low in raw material cost, and the solvent is recyclable. The present invention designs and synthesizes a deep eutectic solvent with higher extraction efficiency, combines anti-solvent extraction and preparative chromatography, and realizes the large-scale separation and preparation of cedrintrienediol in tobacco inflorescence.

[0016] The present invention uses a deep eutectic solvent combined with an anti-solvent extraction method to separate and purify cedrinone in tobacco inflorescence for the first time, and simultaneously uses preparative chromatography to separate two isomers of cedrinone. The process is simple, green and environmentally friendly, the solvent is easy to recycle, the prepared compound has high purity, is suitable for mass production, and has strong use value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the spectrum of separation by preparative HPLC in Example 1, wherein the peaks at 28 min and 33 min represent α-cephalotriene diol and β-cephalotriene diol, respectively.

[0018] Figure 2 This is the HPLC spectrum of α-cephalotriene diol prepared in Example 1, with a purity of 95.7%.

[0019] Figure 3 This is the HPLC spectrum of β-cephalotriene diol prepared in Example 1, with a purity of 95.9%.

[0020] The data here are the purity of α-cepatriene diol and β-cepatriene diol after semi-preparative HPLC separation of the two isomers of cepatriene diol. The purity in the table below is the purity of cepatriene diol after petroleum ether extraction but before semi-preparative separation.

[0021] Figure 4 This is the H-NMR characterization (600 MHz) of the α-cephalotriene diol prepared in Example 1.

[0022] Figure 5 This is the H-NMR characterization (600 MHz) of the β-cephalotriene diol prepared in Example 1. DETAILED DESCRIPTION

[0023] The following examples are provided for a better understanding of the present invention, but are not limited to the best embodiment, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention. The material-liquid ratio recorded in the present invention, unless otherwise specified, represents the ratio of the mass g of the material to the volume mL of the liquid.

[0024] The present invention will be described in detail below with reference to specific embodiments. Embodiment 1:

[0025] The method for extracting and separating cedrintrienediol by deep eutectic solvent in this embodiment comprises the following steps: (1) The preparation method of the deep eutectic solvent is to mix the hydrogen bond donor choline chloride and the hydrogen bond acceptor propylene glycol in a molar ratio of 1:4 with a water content of 20%, seal the mixture, place it in an 80°C water bath, heat and dissolve it until it is uniform and transparent to form a DES solvent, and then let it stand at room temperature for use.

[0026] (2) Using discarded tobacco inflorescence as raw material, a deep eutectic solvent was added at a liquid-to-solid ratio of 1:40 for extraction, and the extraction was carried out at 40°C for 24 hours. After the extraction was completed, gauze was used to filter and DES was recovered to obtain a DES extract, which was recorded as L1. 100 mL of petroleum ether was added to the obtained extract and back-extracted at 40°C. After 6 hours, the petroleum ether layer was recovered and distilled to dryness under reduced pressure at 45°C to obtain a product enriched in cedrinone.

[0027] (3) Preparative chromatography separation and purification: The enriched product of cedrinone component was completely dissolved in acetonitrile so that the solution after dissolution was 3 mg / mL, recorded as L2; ​​preparative liquid phase separation was adopted, the preparative chromatographic column was ChromCore 120 C18 5μm 21.2×250 mm, the mobile phase was acetonitrile and water, the mobile phase ratio was acetonitrile: water = 70:30 (v / v), the flow rate was 4 mL / min, and the detection wavelength was 210 nm.

[0028] (4) Collect the target components according to different retention times. Collect the eluate corresponding to the absorption peak at 28 minutes, which is the solution containing the compound α-cepatriene diol; collect the eluate corresponding to the absorption peak at 33 minutes, which is the solution containing the compound β-cepatriene diol. The eluate is distilled under reduced pressure at 75°C, concentrated to dryness, and freeze-dried to obtain the monomer compounds.

[0029] (5) The extraction rate of L1 was calculated by HPLC, the purity of L2 was calculated by HPLC, and the purity of the monomer compound was tested by HPLC. The high performance liquid chromatograph was equipped with a C18 reverse phase column (C18 4.6×250 mm, 5μm), the detection wavelength was 210nm, the mobile phase was V(acetonitrile):V(water)=60:40, the flow rate was 1.00 mL / min; the column temperature was 30℃, and the injection volume was 20μL. Embodiment 2:

[0030] The method for extracting and separating cedrintrienediol by deep eutectic solvent in this embodiment comprises the following steps: (1) The preparation method of the deep eutectic solvent is to mix the hydrogen bond donor choline chloride and the hydrogen bond acceptor propylene glycol in a molar ratio of 1:3 with a water content of 20%, seal the mixture, place it in an 80°C water bath and heat to dissolve until it becomes uniform and transparent to form a DES solvent, and then let it stand at room temperature for use.

[0031] (2) Using discarded tobacco inflorescence as raw material, deep eutectic solvent was added at a liquid-to-solid ratio of 1:30 for extraction, and the extraction was carried out at 40°C for 24 hours. After the extraction was completed, gauze was used to filter and DES was recovered to obtain a DES extract, which was recorded as L1. 80 mL of petroleum ether was added to the obtained extract and back-extracted at 40°C. After 6 hours, the petroleum ether layer was recovered and distilled to dryness under reduced pressure at 45°C to obtain a product enriched in cedrinone.

[0032] (3) Preparative chromatographic separation and purification: The enriched product of cedrinone component was completely dissolved in acetonitrile so that the solution after dissolution was 3 mg / mL, recorded as L2; ​​preparative liquid phase separation was adopted, the preparative chromatographic column was ChromCore 120 C18 5μm 21.2×250 mm, the mobile phase was acetonitrile and water, the mobile phase ratio was acetonitrile: water = 60:40 (v / v), the flow rate was 3 mL / min, and the detection wavelength was 210 nm.

[0033] (4) Collect the target components according to different retention times. Collect the eluent corresponding to the absorption peak at 28 minutes, record it as eluent L1, which is the solution containing the compound α-cepatriene diol; collect the eluent corresponding to the absorption peak at 33 minutes, record it as eluent L2, which is the solution containing the compound β-cepatriene diol. The eluent is distilled under reduced pressure at 75°C, concentrated to dryness, and freeze-dried to obtain each monomer compound.

[0034] (5) The extraction rate of L1 was calculated by HPLC, the purity of L2 was calculated by HPLC, and the purity of the monomer compound was tested by HPLC. The high performance liquid chromatograph was equipped with a C18 reverse phase column (C18 4.6×250 mm, 5μm), the detection wavelength was 210nm, the mobile phase was V(acetonitrile):V(water)=60:40, the flow rate was 1.00 mL / min; the column temperature was 30℃, and the injection volume was 20μL. Embodiment 3:

[0035] The method for extracting and separating cedrintrienediol by deep eutectic solvent in this embodiment comprises the following steps: (1) The preparation method of the deep eutectic solvent is to mix the hydrogen bond donor choline chloride and the hydrogen bond acceptor propylene glycol in a molar ratio of 1:5 with a water content of 20%, seal the mixture, heat and dissolve in a water bath at 80°C until the mixture becomes uniform and transparent to form a DES solvent, and then leave the mixture to stand at room temperature for use.

[0036] (2) Using discarded tobacco inflorescence as raw material, deep eutectic solvent was added at a liquid-to-solid ratio of 1:50 for extraction, and the extraction was carried out at 40°C for 24 hours. After the extraction was completed, gauze was used to filter and DES was recovered to obtain a DES extract, which was recorded as L1. 120 mL of petroleum ether was added to the obtained extract and back-extracted at 40°C. After 6 hours, the petroleum ether layer was recovered and distilled to dryness under reduced pressure at 45°C to obtain a product enriched in cedrinone.

[0037] (3) Preparative chromatography separation and purification: The enriched product of cedrinone component was completely dissolved in acetonitrile so that the dissolved solution was 3 mg / mL, recorded as L2; ​​preparative liquid phase separation was adopted, the preparative chromatographic column was ChromCore 120 C18 5μm 21.2×250 mm, the mobile phase was acetonitrile and water, the mobile phase ratio was acetonitrile: water = 80:20 (v / v), the flow rate was 5 mL / min, and the detection wavelength was 210 nm.

[0038] (4) Collect the target components according to different retention times. Collect the eluent corresponding to the absorption peak at 28 minutes, record it as eluent L1, which is the solution containing the compound α-cepatriene diol; collect the eluent corresponding to the absorption peak at 33 minutes, record it as eluent L2, which is the solution containing the compound β-cepatriene diol. The eluent is distilled under reduced pressure at 75°C, concentrated to dryness, and freeze-dried to obtain each monomer compound.

[0039] (5) The extraction rate of L1 was calculated by HPLC, the purity of L2 was calculated by HPLC, and the purity of the monomer compound was tested by HPLC. The high performance liquid chromatograph was equipped with a C18 reverse phase column (C18 4.6×250 mm, 5μm), the detection wavelength was 210nm, the mobile phase was V(acetonitrile):V(water)=60:40, the flow rate was 1.00 mL / min; the column temperature was 30℃, and the injection volume was 20μL. Comparative Example 1:

[0040] Compared to Example 1, glycerol was used as the hydrogen bond donor. Comparative Example 2:

[0041] Compared to Example 1, butanediol was used as the hydrogen bond donor. Comparative Example 3:

[0042] Compared with Example 1, ethyl acetate was used as the stripping solvent. Comparative Example 4:

[0043] Compared with Example 1, dichloromethane was used as the stripping solvent. Technical effect evaluation:

[0044] The examples of the present invention and the comparative examples were compared, and the results are shown in Table 1. The data are the extraction rate and purity of cedrintrienediol after petroleum ether extraction and before isomer separation.

[0045]

[0046] The extraction rates of Examples 1, 2, and 3 were 4.49 mg / g, 4.33 mg / g, and 4.41 mg / g, respectively, and the purities were 80.7%, 84.4%, and 82.2%. The extraction rates and purities of Comparative Examples 1 and 2 using glycerol and butanediol as hydrogen bond donors were greatly reduced compared to the examples, indicating that DES solvent plays a vital role in the extraction of the target compound. By observing the purities of Comparative Examples 3 and 4, it was found that the purity of the target compound using ethyl acetate and dichloromethane as the stripping solvent was far lower than that of petroleum ether as the stripping solvent.

[0047] In summary, the DES solvent prepared by mixing choline chloride and propylene glycol in the present invention can achieve a high extraction rate and purity in combination with petroleum ether back extraction, and then separate high-purity α-cephalotriene diol and β-cephalotriene diol from the cephalotriene diol component enriched product in combination with preparative high-performance liquid chromatography. A process route for extracting, purifying and separating two isomers of cephalotriene diol from tobacco inflorescences has been established. The process is simple, does not require a large number of purification steps, and the solvent is easy to recover with high purity, suitable for large-scale industrial production, and has strong use value.

[0048] 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 in the protection scope of the present invention.

Claims

1. A method for extracting and separating cedrin by deep eutectic solvent, characterized in that The steps include: Step 1: using tobacco inflorescence as raw material, adding a deep eutectic solvent for extraction, filtering after the extraction is completed, collecting the extract, and recording it as L1; the obtained extract is back-extracted with an organic solvent to recover the cedartrienediol component, thereby obtaining a cedartrienediol component-enriched product; Step 2: The cedartriene diol component enriched product obtained in step 1 is completely dissolved in acetonitrile, which is recorded as L2; ​​preparative chromatography liquid phase separation is performed, the eluate is collected, concentrated by rotary evaporation under reduced pressure, and freeze-dried to obtain the target product.

2. The method according to claim 1, characterized in that The deep eutectic solvent is prepared by a method comprising the following steps: The hydrogen bond donor and the hydrogen bond acceptor are mixed, sealed, placed in a water bath, heated and dissolved until uniform and transparent to form a DES solvent, and then left to stand at room temperature for use; the hydrogen bond donor is choline chloride, and the hydrogen bond acceptor is propylene glycol.

3. The method according to claim 2, characterized in that: The molar ratio of the hydrogen bond donor and the hydrogen bond acceptor is 1:3-1:5, and the water content of the deep eutectic solvent is 20%.

4. The method according to claim 1, characterized in that: The solid-liquid ratio of the deep eutectic solvent is 1:30-1:50, the extraction temperature is 35-45° C., and the extraction time is 22-24 h.

5. The method according to claim 1, characterized in that: In step 1, the organic solvent used for back extraction is petroleum ether, and the solid-liquid ratio is 1:2 to 1:

3.

6. The method according to claim 1, characterized in that: In step 2, the parameters of the preparative chromatographic liquid phase separation are set as follows: The chromatographic column was ChromCore 120 C18 5 μm 21.2 × 250 mm; The mobile phase was acetonitrile and water in a volume ratio of 60:40 to 80:20; The elution flow rate is 3-5 mL / min; The injection solution concentration was 3 mg / mL; The injection volume was 3 mL; The detection wavelength is 210 nm.

7. The method according to claim 6, characterized in that: The target components were collected according to different retention times. The eluate corresponding to the absorption peak at 28 min was collected, which was the solution containing the compound α-cephalotriene diol; the eluate corresponding to the absorption peak at 33 min was collected, which was the solution containing the compound β-cephalotriene diol.

8. The method according to claim 1, characterized in that: In step 2, the reduced pressure distillation temperature is 75°C.

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