A rapid separation method of high purity taxadiene

Abstract

This invention relates to the field of chemical separation and purification technology, and discloses a rapid separation method for high-purity taxadiene, comprising the following steps: S1, preparing tobacco raw material into powder, adding an extraction solvent for extraction to obtain an extract, concentrating the extract to obtain a crude extract rich in taxadiene and neophytadiene; S2, transferring the crude extract to a normal-phase simple silica gel column filled with silica gel using a Buchner funnel, performing isocratic elution, collecting the fraction containing taxadiene and neophytadiene, evaporating the solvent to obtain a first concentrate; S3, dissolving the first concentrate in acetonitrile, transferring it to an activated reversed-phase solid-phase extraction column for elution, collecting the eluent in fractions, combining the fractions containing high-purity taxadiene, and evaporating to dryness to obtain the final product. This invention solves the problems of complex tobacco substrate materials, numerous interferences, high difficulty in extracting and separating taxadiene, cumbersome steps, and low separation efficiency in the existing technology for separating taxadiene from tobacco raw materials.

Description

Technical Field

[0001] This invention belongs to the field of chemical separation and purification technology, and particularly relates to a rapid separation method for high-purity taxadiene. Background Technology

[0002] Taxadiene is a key precursor compound in the biosynthetic pathway of the anticancer drug paclitaxel. Due to the extremely low content of paclitaxel in yew trees and the difficulty in extraction, the heterologous synthesis of paclitaxel and its precursors in microorganisms or plant substrates (such as tobacco) using synthetic biology methods has become a research hotspot. Tobacco, with its large biomass and rapid growth, is a promising production platform. However, the complex metabolic background of tobacco, containing numerous terpenoids with similar structures and polarities such as neophytadiene, poses a significant challenge to the efficient and high-purity separation of trace amounts of taxadiene from tobacco.

[0003] Existing methods for separating taxadiene generally suffer from low efficiency, low purity, or difficulty in scale-up. For example, patent application CN121591551A discloses a method for separating and purifying taxadiene from tobacco and quantitatively detecting it using GC-MSMS. This method includes the following steps: S1, preparing tobacco leaves into powder, adding an extractant, and performing multiple ultrasonic extractions to obtain an extract; S2, evaporating the extract to dryness, adding an organic solvent, and dissolving to form a concentrate; S3, adding the concentrate to a silica gel column for gradient elution to obtain an extract sample; S4, evaporating the extract sample to dryness, adding acetonitrile to dissolve, and recrystallizing twice to obtain purified taxadiene. While this method increases the purification efficiency from 29.51% to 69.24%, a significant improvement over traditional TLC, it still has significant drawbacks: 1. This method requires multiple gradient elution steps on a chromatography column, which is cumbersome, consumes a large amount of solvent, and has a long separation cycle. 2. The column chromatography method used has a limited throughput, and the subsequent recrystallization step increases the complexity and time cost of the process, which is not conducive to the rapid preparation of high-purity products from large quantities of raw materials in the kilogram range; 3. Although the purity of the product can reach about 69%, it has not yet reached the high-purity standard (usually referring to >90%), which may affect the subsequent quantitative analysis or its application as a standard. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a rapid separation method for high-purity taxadiene, which solves the problems of complex tobacco substrate, numerous interferences, high difficulty in extracting and separating taxadiene, and cumbersome steps and low separation efficiency in the existing technology for separating taxadiene from tobacco raw materials.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] This invention discloses a rapid separation method for high-purity taxadiene, comprising the following steps:

[0007] S1. The tobacco raw material is made into powder, and an extraction solvent is added to extract the extract. The extract is then concentrated to obtain a crude extract rich in taxadiene and neophytadiene.

[0008] S2. The crude extract is transferred to a normal-phase simple silica gel column filled with silica gel using a Buchner funnel, and isocratic elution is performed. The fraction containing taxadiene and neophytadiene is collected, and the solvent is evaporated to obtain the first concentrate.

[0009] S3. Dissolve the first enriched product in acetonitrile, transfer it to an activated reversed-phase solid-phase extraction column, elute, collect the eluent in fractions, combine the fractions containing high-purity taxadiene, and evaporate to dryness to obtain the final product.

[0010] Furthermore, the method for making tobacco raw materials into powder in step S1 is as follows: the freeze-dried tobacco leaves are ground into powder using liquid nitrogen.

[0011] Furthermore, the extraction solvent in step S1 is n-hexane, and the extraction is performed using an ice bath with ultrasonication for 15-25 minutes, with the number of extractions being ≥2.

[0012] Furthermore, the filling method of the normal phase simple silica gel column includes: laying filter paper at the bottom of the Buchner funnel, mixing 300-400 mesh silica gel with n-hexane to form a slurry, and then pouring it into the Buchner funnel, allowing it to settle naturally to form a silica gel column layer with a height of 5-8 cm.

[0013] Furthermore, in step S2, isocratic elution is performed using n-hexane.

[0014] Furthermore, the packing material of the reversed-phase solid-phase extraction column is selected from one of C18, C8, and C18E.

[0015] Furthermore, in step S3, the packing material of the reversed-phase solid-phase extraction column is C18 or C18E. Before loading the sample, the reversed-phase solid-phase extraction column needs to be fully wetted and activated with 30-50 mL of acetonitrile.

[0016] Furthermore, in step S3, the packing material of the reversed-phase solid-phase extraction column is C8. Before loading the sample, the reversed-phase solid-phase extraction column needs to be fully wetted and activated with 30-50 mL of acetonitrile, and then equilibrated with 30-50 mL of acetonitrile / water mixture with a volume ratio of 90:10.

[0017] Furthermore, in step S3, a mixed solution of acetonitrile and water is used for elution, with a volume ratio of acetonitrile to water of 70:30 or 90:10.

[0018] Furthermore, in step S3, gas chromatography-mass spectrometry is used to perform real-time or offline detection on the fractionally collected eluent. By comparing the characteristic peaks of taxadiene and neophytadiene, the fractions containing high-purity taxadiene are identified and merged.

[0019] In summary, this application has the following beneficial effects:

[0020] 1. This invention creatively employs a two-step method: preliminary enrichment using a normal-phase simple silica gel column followed by high-purity separation using a reverse-phase SPE column. The first step utilizes a Buchner funnel silica gel column and solvent to rapidly remove most impurities and enrich the target analyte. The second step utilizes reverse-phase chromatography to efficiently separate taxadiene and neophytadiene. This completely eliminates the complex multi-step gradient elution and subsequent recrystallization steps of existing technologies, simplifying the process from at least "multi-step gradient elution + recrystallization" to "isocratic elution + isocratic / simple gradient elution," making the operation simple and rapid.

[0021] 2. The first step of this invention, the Buchner funnel silica gel column treatment, has a high throughput, capable of processing kilogram-level crude tobacco extracts and rapidly completing preliminary purification. The second step, reversed-phase SPE separation, is highly targeted and can efficiently separate the difficult-to-separate substances taxadiene and neophytadiene. Example data shows that the taxadiene prepared by this method has extremely high purity, reaching over 90%, significantly higher than the 69.24% purity level of existing technologies.

[0022] 3. The core separation device of this invention is a common Buchner funnel and a commercially available SPE column, avoiding the use of expensive, low-throughput semi-preparative high-performance liquid chromatography equipment. This method consumes less solvent and is easy to scale up in parallel, facilitating the transition from laboratory to large-scale preparation and laying a technological foundation for potential future industrial applications.

[0023] 4. This invention effectively solves the problems of complex tobacco substrate, numerous interferences, difficulty in extracting and separating taxadiene, and cumbersome steps with low separation efficiency; it is particularly suitable for rapidly obtaining high-purity taxadiene from tobacco matrices with complex composition. Attached Figure Description

[0024] Figure 1 The GC-MS chromatograms of the hexane extract (YY) and the separation solution (CF) obtained by Buchner funnel simple silica gel column are shown.

[0025] Figure 2 The GC-MS chromatogram of the separated products in segments 18-26 of a C18 column;

[0026] Figure 3 The mass spectrum of taxadiene-3 (product 1) is shown.

[0027] Figure 4The GC-MS chromatogram of the separated products in segments 27-36 of the C18 column;

[0028] Figure 5 The mass spectrum of neophydiene (product 2);

[0029] Figure 6 The GC-MS chromatograms of the separated products in segments 13-24 of the C8 column are shown.

[0030] Figure 7 The GC-MS chromatograms of the separated products in segments 22-34 of the C18 column are shown. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1

[0033] This embodiment presents a rapid separation method for high-purity taxadiene, using a C18 reverse-phase SPE column to separate taxadiene. The steps are as follows:

[0034] S1. Raw material extraction: Take 1 kg of transgenic tobacco leaves (which can be heterologously synthesized into taxadiene) ground into powder using liquid nitrogen, add 500 mL of n-hexane, and extract by ultrasonic extraction in an ice bath for 15 minutes. Repeat the extraction three times, combine all extracts, and evaporate the solvent to dryness using a rotary evaporator. Add 100 mL of n-hexane to the evaporated product, dissolve by ultrasonication, and obtain a dark green concentrated solution.

[0035] S2. First Step Separation and Enrichment (Buchner Funnel and Silica Gel Column): Line a Buchner funnel with filter paper, weigh an appropriate amount of 300-400 mesh silica gel, add n-hexane and stir to form a homogenate. Pour the homogenate into the Buchner funnel and allow it to settle naturally to form a silica gel column layer approximately 5 cm high. Slowly add the concentrated solution obtained in S1 to the top of the silica gel column. Elute directly with n-hexane, observing the color of the eluent. Begin collecting the eluent when a pale yellow target stream appears, and stop collecting when the eluent is essentially colorless. Evaporate the collected stream to dryness using a rotary evaporator to obtain a pale yellow oily substance (first enrichment). Dissolve a small amount of this enrichment in acetonitrile and perform GC-MS analysis. The results are as follows: Figure 1As shown in the figure, after this step, a large number of complex impurity peaks with retention times of 30-45 min in the original extract (YY) basically disappeared, and the enriched product (CF) mainly contained neophytadiene with a retention time of 16.89 min and taxadiene with a retention time of 22.62 min. This indicates that this step effectively removed most of the highly polar impurities such as pigments and achieved the initial enrichment of the target analyte.

[0036] S3. Second-step high-purity separation (C18 reversed-phase SPE column): Dissolve the first enrichment obtained in S2 in 30 mL of acetonitrile. Take one C18 solid-phase extraction column (e.g., 500 mg / 6 mL) and activate and equilibrate it sequentially with 30 mL of acetonitrile and then 30 mL of acetonitrile:water (90:10, v / v) mixture. Load the acetonitrile-dissolved sample. After loading, wash with 50 mL of acetonitrile:water (70:30, v / v) mixture, then elute with 200 mL of pure acetonitrile, collecting the eluent in 5 mL fractions per tube. Analyze each eluent fraction using GC-MS.

[0037] Test results as follows Figures 2-4 As shown. Figure 2 (Segments 18-26) show that taxadiene (22.62 min) mainly elutes in segments 19-25, with sharp and single peaks. Figure 3 (Sections 27-36) show that neophytadiene (16.89 min) eluted in segments 27-33. The elution times of the two fractions were clearly separated with no overlap. The taxadiene-rich fractions from segments 19-25 were combined and evaporated by rotary evaporation to obtain a high-purity taxadiene white solid. GC-MS area normalization analysis showed a purity exceeding 97.3%.

[0038] A distinct product peak 2 appeared at a retention time of 16.89 min in the 27-36 segment of the C18 column, with its peak height becoming increasingly pronounced. In the 30-31 segment, the peak of product 2 remained relatively stable. From the 33-36 segment, the peak of product 2 began to decline continuously, exhibiting an overall trend of rapid rise-stable-rapid decline. Mass spectrometry analysis of product 2 is shown below. Figure 5 As shown, it was identified as neophytadiene;

[0039] The above results indicate that the C18 chromatography column can effectively separate the two products mentioned above in tobacco leaves.

[0040] Example 2

[0041] This embodiment presents a second rapid separation method for high-purity taxadiene, using a C8 reversed-phase SPE column to separate taxadiene. The steps are as follows:

[0042] S1. Raw material extraction: Take 1 kg of transgenic tobacco leaves (which can be heterologously synthesized into taxadiene) ground into powder using liquid nitrogen, add 500 mL of n-hexane, and extract by ultrasonic extraction in an ice bath for 17 minutes. Repeat the extraction three times, combine all extracts, and evaporate the solvent to dryness using a rotary evaporator. Add 100 mL of n-hexane to the evaporated product, dissolve by ultrasonication, and obtain a dark green concentrated liquid.

[0043] S2. First Step Separation and Enrichment (Buchner Funnel and Silica Gel Column): Line a Buchner funnel with filter paper, weigh an appropriate amount of 300-400 mesh silica gel, add n-hexane and stir to form a homogenate. Pour the homogenate into the Buchner funnel and allow it to settle naturally to form a silica gel column layer approximately 6 cm high. Slowly add the concentrated solution obtained in S1 to the top of the silica gel column. Elute directly with n-hexane, observing the color of the eluent. Begin collecting the eluent when a pale yellow target stream appears, and stop collecting when the eluent is essentially colorless. Evaporate the collected stream to dryness using a rotary evaporator to obtain a pale yellow oily substance (first enrichment). Dissolve a small amount of this enrichment in acetonitrile and perform GC-MS analysis. The results are as follows: Figure 1 As shown in the figure, after this step, a large number of complex impurity peaks with retention times of 30-45 min in the original extract (YY) basically disappeared, and the enriched product (CF) mainly contained neophytadiene with a retention time of 16.89 min and taxadiene with a retention time of 22.62 min. This indicates that this step effectively removed most of the highly polar impurities such as pigments and achieved the initial enrichment of the target analyte.

[0044] S3. Second step of high-purity separation (C8 reversed-phase SPE column): Dissolve the first concentrate obtained in S2 in 50 mL of acetonitrile. Take one C8 solid-phase extraction column and activate and equilibrate it sequentially with 30 mL of acetonitrile and 30 mL of acetonitrile:water (90:10, v / v) mixture. Load the acetonitrile-dissolved sample. After loading, wash with 150 mL of acetonitrile:water (90:10, v / v) mixture, then elute with 100 mL of pure acetonitrile, collecting the eluent in 5 mL fractions per tube. Analyze each eluent fraction using GC-MS.

[0045] Test results as follows Figure 6 As shown in segments 13-24. Taxadiene (22.62 min) showed significant elution in segments 14-20, reaching a peak in segments 19-20, followed by a rapid decline. Neophytadiene was eluted or retained earlier under these elution conditions, achieving effective separation from taxadiene. The fractions from segments 14-20 (or the purest fraction selected based on peak shape) were combined and evaporated to dryness to obtain high-purity taxadiene. Calculated by GC-MS area normalization, its purity was higher than 97.9%.

[0046] Example 3

[0047] This embodiment describes a rapid separation method for high-purity taxadiene, using a C18E reversed-phase SPE column. The steps are as follows:

[0048] S1. Raw material extraction: Take 1 kg of transgenic tobacco leaves (which can be heterologously synthesized into taxadiene) ground into powder using liquid nitrogen, add 500 mL of n-hexane, and extract by ultrasonic extraction in an ice bath for 20 minutes. Repeat the extraction twice, combine all extracts, and evaporate the solvent to dryness using a rotary evaporator. Add 100 mL of n-hexane to the evaporated product, dissolve by ultrasonication, and obtain a dark green concentrated solution.

[0049] S2. First Step Separation and Enrichment (Buchner Funnel and Silica Gel Column): Line a Buchner funnel with filter paper, weigh an appropriate amount of 300-400 mesh silica gel, add n-hexane and stir to form a homogenate. Pour the homogenate into the Buchner funnel and allow it to settle naturally to form a silica gel column layer approximately 8 cm high. Slowly add the concentrated solution obtained in S1 to the top of the silica gel column. Elute directly with n-hexane, observing the color of the eluent. Begin collecting the eluent when a pale yellow target stream appears, and stop collecting when the eluent is essentially colorless. Evaporate the collected stream to dryness using a rotary evaporator to obtain a pale yellow oily substance (first enrichment). Dissolve a small amount of this enrichment in acetonitrile and perform GC-MS analysis. The results are as follows: Figure 1 As shown in the figure, after this step, a large number of complex impurity peaks with retention times of 30-45 min in the original extract (YY) basically disappeared, and the enriched product (CF) mainly contained neophytadiene with a retention time of 16.89 min and taxadiene with a retention time of 22.62 min. This indicates that this step effectively removed most of the highly polar impurities such as pigments and achieved the initial enrichment of the target analyte.

[0050] S3. Second step of high-purity separation (C18E reversed-phase SPE column): Dissolve the first concentrate obtained in S2 in 50 mL of acetonitrile. Take a C18E (terminated C18) solid-phase extraction column and activate it with 30 mL of acetonitrile. Load the acetonitrile-dissolved sample onto the column. After loading, wash with 50 mL of acetonitrile:water (70:30, v / v) mixture, then elute with 200 mL of pure acetonitrile, collecting the eluent in 5 mL fractions per tube. Analyze each eluent fraction using GC-MS.

[0051] Test results as follows Figure 7 As shown in segments 22-34. Taxadiene (22.62 min) showed significant elution in segments 23-31 with good peak shape. The target fractions were combined and evaporated to dryness to obtain high-purity taxadiene. Calculated by GC-MS area normalization, its purity was higher than 99.1%.

[0052] The above embodiments demonstrate that the two-step separation method provided by this invention, through rapid enrichment using a Buchner funnel silica gel column in the first step and efficient separation of taxadiene and neophytadiene using different types of reversed-phase SPE columns (C18, C8, C18E) in the second step, yields high-purity (>90%) taxadiene. This method has the advantages of simple operation, rapid separation, low cost, and high purity, and is particularly suitable for the large-scale preparation of high-purity taxadiene standards or intermediates from complex tobacco matrices.

[0053] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A rapid separation method for high-purity taxadiene, characterized in that, Includes the following steps: S1. The tobacco raw material is made into powder, and an extraction solvent is added to extract the extract. The extract is then concentrated to obtain a crude extract rich in taxadiene and neophytadiene. S2. The crude extract is transferred to a normal-phase simple silica gel column filled with silica gel using a Buchner funnel, and isocratic elution is performed. The fraction containing taxadiene and neophytadiene is collected, and the solvent is evaporated to obtain the first concentrate. S3. Dissolve the first enriched product in acetonitrile, transfer it to an activated reversed-phase solid-phase extraction column, elute, collect the eluent in fractions, combine the fractions containing high-purity taxadiene, and evaporate to dryness to obtain the final product.

2. The rapid separation method for high-purity taxadiene according to claim 1, characterized in that, The method for making tobacco raw materials into powder in step S1 is as follows: freeze-dried tobacco leaves are ground into powder using liquid nitrogen.

3. The rapid separation method for high-purity taxadiene according to claim 1, characterized in that, The extraction solvent in step S1 is n-hexane, and the extraction is performed by ultrasonication in an ice bath for 15-25 minutes, with the number of extractions being ≥2.

4. A rapid separation method for high-purity taxadiene according to any one of claims 1-3, characterized in that, The filling method of the normal phase simple silica gel column includes: laying filter paper at the bottom of the Buchner funnel, mixing 300-400 mesh silica gel with n-hexane to form a slurry, and then pouring it into the Buchner funnel, allowing it to settle naturally to form a silica gel column layer with a height of 5-8 cm.

5. The rapid separation method for high-purity taxadiene according to claim 1, characterized in that, In step S2, isocratic elution is performed using n-hexane.

6. The rapid separation method for high-purity taxadiene according to claim 4, characterized in that, The packing material of the reversed-phase solid-phase extraction column is selected from one of C18, C8, and C18E.

7. The rapid separation method for high-purity taxadiene according to claim 6, characterized in that, In step S3, the packing material of the reversed-phase solid-phase extraction column is C18 or C18E. Before loading the sample, the reversed-phase solid-phase extraction column needs to be fully wetted and activated with 30-50 mL of acetonitrile.

8. The rapid separation method for high-purity taxadiene according to claim 6, characterized in that, In step S3, the packing material of the reversed-phase solid-phase extraction column is C8. Before loading the sample, the reversed-phase solid-phase extraction column needs to be fully wetted and activated with 30-50 mL of acetonitrile, and then equilibrated with 30-50 mL of acetonitrile / water mixture with a volume ratio of 90:

10.

9. A rapid separation method for high-purity taxadiene according to claim 7 or 8, characterized in that, In step S3, a mixed solution of acetonitrile and water is used for elution, with a volume ratio of acetonitrile to water of 70:30 or 90:

10.

10. A rapid separation method for high-purity taxadiene according to claim 7 or 8, characterized in that, In step S3, gas chromatography-mass spectrometry is used to detect the collected eluent in real time or offline. By comparing the characteristic peaks of taxadiene and neophytadiene, the fractions containing high-purity taxadiene are identified and merged.

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