Preparation method of organic compound cystochalasin D

Abstract

The invention provides a preparation method of an organic compound cytochalasin D, and relates to the technical field of preparation of organic compounds. The preparation method of the organic compound cystochalasin D specifically comprises the following steps: S1, activation and expansion of strains; s2, strain rice amplification culture and extraction; s3, carrying out primary separation on the cytochalasin D; s4, further segmenting; and S5, separating a target product. According to the preparation method of the cytochalasin D, the purity and the yield are remarkably improved, the production time is shortened, the cost is reduced, a better solution is provided for industrial production of the cytochalasin D, and the problems that in the prior art, the purity is low, the yield is low and the cost is high are solved.

Description

Technical Field

[0001] The invention relates to the technical field of organic compound preparation, in particular to a method for preparing an organic compound cytochalasin D. Background Art

[0002] Cytochalasin D is a selective inhibitor of actin polymerization with an IC50 value of 25nM. It can be derived from fungi. Cytochalasin D has cell permeability activity. Cytochalasin D inhibits the interaction of G-actin-cofilin by binding to G-actin. Cytochalasin D can also inhibit the binding of Cofilin to F-actin, reducing the rate of actin polymerization and depolymerization in living cells. Cytochalasin D can reduce the release of exosomes, thereby reducing the amount of survivin in the tumor environment. Cytochalasin D induces phosphorylation of Yap, preventing its nuclear translocation and retention in the cytoplasm. Cytochalasin D (a natural metabolite) can regulate the expression of mesenchymal stem cell genes, thereby differentiating them into osteoblasts. By stimulating stem cells with cytochalasin D, these stem cells can differentiate into osteoblasts. After that, injection of cytochalasin D into the interstitial space of the bone marrow of mice can cause bone regeneration.

[0003] Compared with the existing preparation methods and technologies, it is very difficult to obtain cytochalasin D with high purity and high yield in a faster time. Moreover, as people's demand for cytochalasin D increases, the price of cytochalasin D is very expensive. Therefore, it is very necessary to use a suitable preparation method to obtain cytochalasin D with high purity and high yield in a faster time. The main step to obtain more cytochalasin D in a short time is to separate the target product in step S5. In this step, Fr.5.8 (22.2342g) is used to pass C18 under the isocratic condition of 50% methanol water to directly obtain the target product cytochalasin D with high purity and high yield. In this step, the target product can also be directly prepared by liquid phase, but the time consumed by using liquid phase preparation is too long, and more samples will be lost, and more analytical methanol will be consumed, and the required cost becomes high. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for preparing an organic compound cytochalasin D, which solves the problem that it is very difficult to obtain cytochalasin D with high purity and high yield using the prior art preparation method.

[0005] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for preparing an organic compound cytochalasin D, specifically comprising the following steps:

[0006] S1. Activation and expansion of bacterial strains

[0007] Endophytic fungus Xylaria officinalis (SY4) was thawed from a glycerol cryopreservation tube, transferred into PSA medium, and cultured at 28℃ for 20 days to obtain mycelium;

[0008] S2. Expansion and extraction of rice strains

[0009] The second generation strain was transferred into a solid medium containing 15 kg of rice, and fermented for 30 days, and extracted with an equal volume of ethyl acetate for 6 times to obtain 500 g of total extract, which was preliminarily segmented by macroporous resin and gradient eluted with methanol and water (0%, 30%, 60%, 90%) to obtain 120 g of extract for the next separation;

[0010] S3. Preliminary Isolation of Cytochalasin D

[0011] 120 g of the extract was segmented using a silica gel column, using dichloromethane:methanol as the mobile phase, gradient elution (200:1 to 1:1), and 15 segments (Fr.1-Fr.15) were obtained;

[0012] S4. Further segmentation

[0013] Fr.5 (33.3323 g) was segmented again on a silica gel column, using petroleum ether:acetone to chloroform:methanol as the mobile phase, gradient elution (8:1 to 4:1 to 75:1 to 1:1), to obtain 9 segments (Fr.5.1-Fr.5.9);

[0014] S5. Separation of target product

[0015] Fr.5.8 (22.2342 g) was passed through a C18 column with methanol and water as the mobile phase and gradient elution (50% to 100%) to obtain 10 segments (Fr.5.8.1-Fr.5.8.10), of which Fr.5.8.8 was the target product cytochalasin D with a mass of 4.8796 g.

[0016] The present invention provides a method for preparing an organic compound cytochalasin D. The method has the following beneficial effects:

[0017] The invention provides a method for preparing an organic compound cytochalasin D. Compared with the prior art, the method for preparing cytochalasin D of the invention can obtain cytochalasin D with high purity and high yield in a shorter time. Specifically, through separation of a C18 column of Fr.5.8 section (22.2342 g), a target product cytochalasin D with high purity and high yield can be directly obtained under an isocratic condition of 50% methanol and water, and the mass reaches 4.8796 g. The optimization of this step significantly improves the purity and yield of the product, and meets the market demand for high-quality cytochalasin D. It is difficult to obtain a large amount of high-purity cytochalasin D in a short time by a traditional method, while the method of the invention can quickly separate the target product under optimized conditions, and a C18 column with an isocratic condition of 50% methanol and water is used. The separation method saves time and improves efficiency. Compared with the liquid phase preparation method, although the target product can also be obtained in the liquid phase, the time is long and the sample loss and cost are high. The present method effectively avoids these problems. In the preparation process, a C18 column and 50% methanol-water isocratic conditions are used for separation, which greatly reduces the consumption of methanol and avoids excessive sample loss, thereby reducing the overall cost. Compared with the liquid phase preparation method, the required time and resource investment are significantly reduced, making the production more economical and efficient. In the C18 column separation process of the Fr.5.8 segment (22.2342 g), the present invention uses 50% methanol-water isocratic conditions to directly obtain high-purity and high-yield cytochalasin D. This optimization step effectively simplifies the separation process, improves efficiency and yield, and saves time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the method flow chart of the present invention;

[0019] Figure 2 is a schematic diagram of the hydrogen spectrum of cytochalasin D of the present invention;

[0020] Figure 3 is a DEPT schematic diagram of cytochalasin of the present invention;

[0021] Figure 4 This is a schematic diagram of the preliminary experimental results of Fr.5 through positive silica gel of the present invention;

[0022] Figure 5 This is a schematic diagram of the Fr.5 gel pre-experimental results of the present invention;

[0023] Figure 6 It is a schematic diagram of the experimental results of the formal experiment of Fr.5 (33.3323g) through positive silica gel of the present invention;

[0024] Figure 7 It is a schematic diagram of the experimental results of the liquid phase pre-experiment of Fr.5.8 (33.3323g) of the present invention;

[0025] Figure 8 This is a schematic diagram of the Fr.5.8 (22.2342g) passing through the C18 point plate of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] like Figure 1-7 As shown, the embodiment of the present invention provides a method for preparing an organic compound cytochalasin D, which specifically comprises the following steps:

[0028] S1. Activation and expansion of bacterial strains

[0029] Endophytic fungus Xylaria officinalis (SY4) was thawed from a glycerol cryopreservation tube and transferred to PSA medium for culture to obtain mycelium;

[0030] S2. Expansion and extraction of rice strains

[0031] The second generation strain was transferred into a solid medium containing 15 kg of rice and fermented for 30 days. The extract was extracted with an equal volume of ethyl acetate for 6 times to obtain 500 g of total extract. The total extract was preliminarily segmented by macroporous resin and gradient eluted with methanol and water to obtain 120 g of extract for the next separation.

[0032] S3. Preliminary Isolation of Cytochalasin D

[0033] 120 g of the extract was segmented using a silica gel column, using dichloromethane:methanol as the mobile phase, gradient elution (200:1 to 1:1), and 15 segments (Fr.1-Fr.15) were obtained;

[0034] S4. Further segmentation

[0035] Fr.5 (33.3323 g) was segmented again on a silica gel column, using petroleum ether:acetone to chloroform:methanol as the mobile phase, gradient elution (8:1 to 1:1), to obtain 9 segments (Fr.5.1-Fr.5.9);

[0036] S5. Separation of target product

[0037] Fr.5.8 (22.2342 g) was passed through a C18 column with methanol and water as the mobile phase and gradient elution (50% to 100%) to obtain 10 segments (Fr.5.8.1-Fr.5.8.10), of which Fr.5.8.8 was the target product cytochalasin D with a mass of 4.8796 g.

[0038] In step S1, the activation and expansion of the strain requires constant temperature cultivation at 28° C. for 20 days to obtain mycelium.

[0039] Step S2: The rice culture and extraction of the bacterial strain are carried out by gradient elution with methanol and water. Specifically, the following steps are performed:

[0040] First, use pure water for elution to remove the highly polar components that are insoluble in methanol. Then use a mixed solution of 30% methanol and 70% water for elution. This step can elute some medium polar components. Then use a mixed solution of 60% methanol and 40% water for further elution to separate more medium to low polar components. Finally, use a mixed solution of 90% methanol and 10% water for elution to remove the remaining low polar components and target compounds.

[0041] Step S3: Gradient elution of cytochalasin D for preliminary separation, obtaining 15 segments:

[0042] A mixed solvent of dichloromethane: methanol = 200:1 is used, i.e. 200 parts of dichloromethane and 1 part of methanol, and the ratio is changed gradually: during the elution process, the ratio of dichloromethane and methanol is gradually changed, as 200:1, 150:1, 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, and finally to 1:1. At each ratio, the eluate flowing out of the silica gel column is collected, and the entire elution process is divided into 15 sections, i.e., a part of each eluate collected at a different gradient ratio. These 15 eluates are respectively marked as Fr.1 to Fr.15. By analyzing the components of each eluate, such as using TLC thin layer chromatography method, the substances contained in each section are determined, and the sample is separated into 15 parts. Each part may contain different components, and these parts can be further used for analysis and research.

[0043] Step S4 further divides the gradient elution into segments to obtain 9 segments:

[0044] Fr.5 (33.3323 g) was again segmented using a silica gel column, using a mixed solvent of petroleum ether: acetone = 8:1, and then gradually changing the ratio to petroleum ether: acetone = 6:1, 4:1, and then switching to a mixed solvent of chloroform and methanol, with the ratios of chloroform: methanol = 75:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1. At each ratio stage, the eluate flowing out of the silica gel column was collected, and after spot plate merging, a total of 9 segments were collected, which were marked as Fr.5.1 to Fr.5.9. Each segment (Fr.5.1 to Fr.5.9) was further analyzed, such as TLC thin layer chromatography, to determine the specific components contained in each separated segment. Through this gradient elution method, the Fr.5 sample was successfully further purified and separated into 9 segments. These segments can be used for more detailed chemical analysis, purity inspection or subsequent experimental research.

[0045] Step S5: Gradient elution in the separation of target product, 10 sections are obtained:

[0046] A mixed solvent of 50% methanol and 50% water was used. During the elution process, the proportion of methanol was gradually increased and finally reached 100% methanol. The specific proportion was gradually increased from 50% methanol to 60%, 70%, 80%, 90%, until 100% methanol. At each proportion stage, the eluate flowing out of the C18 column was collected and divided into 10 sections, marked as Fr.5.8.1 to Fr.5.8.10, and each section (Fr.5.8.1 to Fr.5.8.10) was further analyzed, such as TLC thin layer chromatography, to determine the specific components contained in each separation section. Through this gradient elution method, the Fr.5.8 sample was successfully further purified and separated into 10 sections. These sections can be used for more detailed chemical analysis, purity inspection or subsequent experimental research.

[0047] Experimental Example 1

[0048] The 90% part of the macroporous resin (120g of extract) was segmented using a silica gel column, with dichloromethane:methanol as the mobile phase, and the elution gradient was dichloromethane:methanol = 200:1, 150:1, 100:1, 75:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, and the spot plates were merged to form 15 segments (Fr.1-Fr.15). 800mg of Fr.5 (33.3323g) was taken out and passed through the gel. The experiment found that the gelation effect of Fr.5 (33.3323g) was not good, and the target product and fatty acids could not be completely separated.

[0049] Experimental Example 2

[0050] 90% of the macroporous resin (120g of extract) was segmented using a silica gel column, with dichloromethane:methanol as the mobile phase, and the elution gradient was dichloromethane:methanol = 200:1, 150:1, 100:1, 75:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, and the spot plate was merged to form 15 segments (Fr.1-Fr.15). 800mg of Fr.5 (33.3323g) was taken out and passed through normal silica gel, with a system of chloroform:methanol. The experiment found that the use of this system was not as good as the system of petroleum ether:acetone to chloroform:methanol.

[0051] Experimental Example 3

[0052] The 90% portion of the macroporous resin (120 g of extract) was segmented using a silica gel column with dichloromethane:methanol as the mobile phase. The elution gradient was dichloromethane:methanol = 200:1, 150:1, 100:1, 75:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1. After the spot plates were combined, they were segmented into 15 segments (Fr.1-Fr.15). Fr.5 (33.3323 g) was further segmented through the previous step S4 to obtain 10 components (Fr.5.1 to Fr.5.9), among which Fr.5.8 could also be prepared as the target product using the liquid phase.

[0053] In combination with the above experimental series, the method of the present invention is to segment Fr.5 (33.3323 g) again using a silica gel column, using petroleum ether: acetone to chloroform: methanol as the mobile phase, and the elution gradient is petroleum ether: acetone = 8:1, 6:1, 4:1 to chloroform: methanol 75:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, the method is to pass Fr.5 (33.3323 g) through a gel and find a more suitable system to pass Fr.5 (33.3323 g) through a silica gel column.

[0054] like Figure 4 and Figure 5 As shown, Fr.5 (33.3323 g) had a poor gelling effect and could not completely separate the target product from the fatty acid. However, using petroleum ether: acetone to chloroform: methanol could completely separate the fatty acid from the target product.

[0055] like Figure 7 As shown, the present invention uses C18 to prepare the target product. Preliminary experiments have shown that the target product can also be prepared by liquid phase, but the liquid phase preparation takes too long and will lose more samples and consume more analytical methanol, which increases the cost. However, using C18 can directly purify a large amount of target product, with low cost and good effect. Figure 8 shown.

[0056]

[0057]

[0058]

[0059] By comparing the above tables, we can clearly see the economic benefits and output effects of different methods. Finally, the use of petroleum ether: acetone to chloroform: methanol system for further segmentation, and then methanol-water gradient elution on a C18 column can not only successfully separate the target product cytochalasin D, but also maintain low cost and high efficiency.

[0060] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an organic compound cytochalasin D, characterized in that: The specific steps include: S1. Activation and expansion of bacterial strains Endophytic fungus Xylaria officinalis (SY4) was thawed from a glycerol cryopreservation tube and transferred to PSA medium for culture to obtain mycelium; S2. Expansion and extraction of rice strains The second generation strain was transferred into a solid medium containing 15 kg of rice and fermented for 30 days. The extract was extracted with an equal volume of ethyl acetate for 6 times to obtain 500 g of total extract. The total extract was preliminarily segmented by macroporous resin and gradient eluted with methanol and water to obtain 120 g of extract for the next separation. S3. Preliminary Isolation of Cytochalasin D 120 g of the extract was segmented using a silica gel column, using dichloromethane:methanol as the mobile phase, and gradient elution to obtain 15 segments; S4. Further segmentation Fr.5 (33.3323 g) was segmented again on a silica gel column, using petroleum ether:acetone to chloroform:methanol as the mobile phase, and gradient elution to obtain 9 segments; S5. Separation of target product Fr.5.8 (22.2342 g) was passed through a C18 column with methanol and water as the mobile phase and gradient elution to obtain 10 segments, of which Fr.5.8.8 was the target product cytochalasin D with a mass of 4.8796 g.

2. The method for preparing cytochalasin D according to claim 1, characterized in that: In the activation and expansion of the strain in step S1, the strain needs to be cultured at a constant temperature of 28° C. for 20 days to obtain mycelium.

3. The method for preparing cytochalasin D according to claim 1, characterized in that: The step S2 of expanding the culture and extracting the rice strain using methanol-water gradient elution is as follows: First, use pure water for elution to remove the highly polar components that are insoluble in methanol. Then use a mixed solution of 30% methanol and 70% water for elution. This step can elute some medium polar components. Then use a mixed solution of 60% methanol and 40% water for further elution to separate more medium to low polar components. Finally, use a mixed solution of 90% methanol and 10% water for elution to remove the remaining low polar components and target compounds.

4. The method for preparing cytochalasin D according to claim 1, characterized in that: In step S3, the gradient elution of the initial separation of cytochalasin D is performed to obtain 15 segments, specifically: A mixed solvent of dichloromethane: methanol = 200:1 is used, i.e. 200 parts of dichloromethane and 1 part of methanol, and the ratio is changed gradually: during the elution process, the ratio of dichloromethane and methanol is gradually changed, as 200:1, 150:1, 100:1, 50:1, 30:1, 20:1, 10:1, 5:1, and finally to 1:

1. At each ratio, the eluate flowing out of the silica gel column is collected, and the entire elution process is divided into 15 sections, i.e., a part of each eluate collected at a different gradient ratio. These 15 eluates are respectively marked as Fr.1 to Fr.

15. By analyzing the components of each eluate, such as using TLC thin layer chromatography method, the substances contained in each section are determined, and the sample is separated into 15 parts. Each part may contain different components, and these parts can be further used for analysis and research.

5. The method for preparing cytochalasin D according to claim 1, characterized in that: The step S4 further divides the gradient elution into segments to obtain 9 segments: Fr.5 (33.3323 g) was again segmented using a silica gel column, using a mixed solvent of petroleum ether: acetone = 8:1, and then gradually changing the ratio to petroleum ether: acetone = 6:1, 4:1, and then switching to a mixed solvent of chloroform and methanol, with the ratios of chloroform: methanol = 75:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 1:

1. At each ratio stage, the eluate flowing out of the silica gel column was collected, and after spot plate merging, a total of 9 segments were collected, which were marked as Fr.5.1 to Fr.5.

9. Each segment (Fr.5.1 to Fr.5.9) was further analyzed, such as TLC thin layer chromatography, to determine the specific components contained in each separated segment. Through this gradient elution method, the Fr.5 sample was successfully further purified and separated into 9 segments. These segments can be used for more detailed chemical analysis, purity inspection or subsequent experimental research.

6. The method for preparing cytochalasin D according to claim 1, characterized in that: In step S5, the target product is separated by gradient elution, and 10 sections are obtained, specifically: A mixed solvent of 50% methanol and 50% water was used. During the elution process, the proportion of methanol was gradually increased and finally reached 100% methanol. The specific proportion was gradually increased from 50% methanol to 60%, 70%, 80%, 90%, until 100% methanol. At each proportion stage, the eluate flowing out of the C18 column was collected and divided into 10 sections, marked as Fr.5.8.1 to Fr.5.8.10, and each section (Fr.5.8.1 to Fr.5.8.10) was further analyzed, such as TLC thin layer chromatography, to determine the specific components contained in each separation section. Through this gradient elution method, the Fr.5.8 sample was successfully further purified and separated into 10 sections. These sections can be used for more detailed chemical analysis, purity inspection or subsequent experimental research.

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