Paclitaxel extraction method based on biological disruption

Abstract

The invention discloses a paclitaxel extraction method based on biological disruption, and belongs to the technical field of paclitaxel extraction. The invention provides a paclitaxel separation method combining a physical method and a biological method, the crushed tissue of taxus chinensis is subjected to natural enzymolysis by using bacillus subtilis, and ultrasonic extraction is further performed, so that the extraction rate of paclitaxel is increased. According to the method, the extraction rate of the paclitaxel is increased, the waste of raw materials is reduced, the production cost is reduced, the large-scale utilization of the paclitaxel is promoted, and the method has very important potential value.

Description

Technical Field

[0001] The invention relates to the technical field of paclitaxel extraction, and in particular to a paclitaxel extraction method based on biological crushing. Background Art

[0002] Paclitaxel (PTX) is a tricyclic diterpenoid compound and a new type of anticancer drug ingredient that can effectively promote microtubule polymerization. It is mainly used as a chemotherapy adjuvant for cancers with high incidence rates such as breast cancer and cervical cancer. Due to its unique structure and significant anticancer effect, it has become a research hotspot at home and abroad.

[0003] Natural paclitaxel is mainly extracted and separated from plants of the genus Taxus. Plants of the genus Taxus are rare species and the content of paclitaxel in Taxus is low. In addition, the content of paclitaxel in different parts of Taxus is different, and the content of bark is the highest, which leads to the restriction of the source of raw materials. Therefore, the efficient extraction of paclitaxel compounds has great economic and ecological value. At present, in the process of paclitaxel purification, since the subsequent extraction and recrystallization processes are relatively mature, the key to improving the paclitaxel acquisition rate lies in improving the yew tissue crushing and separation process, and increasing the paclitaxel acquisition rate per unit of yew raw material by improving the separation degree of paclitaxel and wood fiber substrate. However, the existing schemes generally have the disadvantages of insufficient effect of paclitaxel acquisition rate or high cost, which limits the extraction rate of paclitaxel, causes waste of raw materials, pushes up production costs, and affects the large-scale utilization of paclitaxel. Summary of the invention

[0004] In view of the above prior art, the object of the present invention is to provide a method for extracting paclitaxel based on biological disruption.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a method for extracting paclitaxel based on biological crushing, comprising the following steps:

[0007] (1) culturing fermentation bacteria to obtain a seed culture solution of the fermentation bacteria;

[0008] (2) preparing yew powder;

[0009] (3) preparing Taxus chinensis slurry;

[0010] (4) bacterial hydrolysis, inoculating the fermentation bacteria described in step (1) into the Taxus slurry described in step (3) for culturing;

[0011] (5) Taxol extraction: ultrasonically extract the Taxus slurry obtained through bacterial hydrolysis in step (4), and centrifuge to obtain a Taxol extract.

[0012] Furthermore, in step (1), the fermentation bacteria are inoculated into LB liquid culture medium, and cultured at a constant temperature of 30-40° C. and 50-300 r / min in a shaking incubator for 12-36 hours to obtain a seed culture solution of the fermentation bacteria.

[0013] Furthermore, the fermentation bacteria is Bacillus subtilis ATCC6633.

[0014] Furthermore, in step (2), the yew bark is cleaned, dried at 20-90° C., crushed, and sieved through a 10-200 mesh sieve to obtain yew powder.

[0015] Furthermore, in step (3), the yew powder obtained in step (2) is weighed, and the yew powder is fully homogenized with sterile water at a solid-liquid ratio of 1 g: (1-50) mL, and the pH value is adjusted to 6-8 to obtain a yew slurry.

[0016] Furthermore, in step (4), according to the volume of the liquid culture medium, the seed culture liquid of the fermentation bacteria is inoculated, and the culture is carried out at a constant temperature of 30-40°C and 50-300 r / min in a shaking incubator for 12-64 hours to obtain a bacterial suspension; 1 mL of the bacterial suspension is inoculated into the yew slurry, stirred sufficiently, and cultured at a constant temperature of 30-40°C and 50-300 r / min with shaking for 12-64 hours.

[0017] Furthermore, the volume ratio of the seed culture solution of the fermentation bacteria to the liquid culture medium is 1 mL: (1-100) mL.

[0018] Furthermore, in step (5), the Taxus slurry after bacterial hydrolysis is subjected to ultrasonic extraction with methanol, the extraction time is 60 minutes, and the centrifugation is performed at a speed of 5000 r / min for 10 minutes, and the obtained filtrate is the paclitaxel extract.

[0019] Beneficial effects of the present invention:

[0020] The present invention proposes a "physical method + biological method" paclitaxel separation method, which uses Bacillus subtilis to naturally enzymatically hydrolyze yew pulverized tissue, and further ultrasonically extracts, thereby improving the paclitaxel extraction rate. The present invention improves the paclitaxel extraction rate, reduces the waste of raw materials, reduces production costs, and promotes the large-scale utilization of paclitaxel. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0022] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0023] The test materials used in the embodiments of the present invention that are not specifically described are all conventional test materials in the art and can be purchased through commercial channels. The Bacillus subtilis strain used in this application was purchased from the Shanghai Collection of Microorganisms Center and is numbered ATCC6633. The yew used in the present invention is Taxus chinensis var. mairei, the bark of which was legally purchased from the Taxus chinensis Trading Center, and the paclitaxel standard solution used was purchased from Shanghai McLean Biological Reagent. The LB liquid culture medium used in the present invention (referred to as liquid culture medium in subsequent embodiments) is composed of 3.0 g yeast extract, 3.0 g malt extract, 5.0 g peptone, 10.0 g glucose, and 1.0 L distilled water.

[0024] Example 1

[0025] Cultivation of Bacillus subtilis

[0026] The strain was activated and cultured using LB liquid culture medium: a single colony was picked from the glycerol tube of Bacillus subtilis using an inoculation loop and inoculated into the LB liquid culture medium in a clean bench, and cultured at 37°C and 150 rpm in a shaking incubator for 24 hours to obtain the seed culture solution of Bacillus subtilis.

[0027] Example 2

[0028] Preparation of Paclitaxel Extract

[0029] (1) Preparation of Taxus powder. The bark of Taxus was cleaned and placed in a constant temperature drying oven at 50°C for drying. After drying, it was crushed by a pulverizer and passed through a 60-mesh sieve to obtain Taxus powder.

[0030] (2) Preparation of Taxus slurry. Weigh 1.5 g of the Taxus powder obtained in step (1) into a conical flask, thoroughly homogenize the Taxus powder with sterile water at a solid-liquid ratio of 1 g:10 mL, and adjust the pH value to 7.0 with sodium hydroxide. Place the slurry in a microwave heating device, maintain the temperature at 55°C, and microwave for 2 minutes.

[0031] (3) Bacterial hydrolysis. A 250 mL conical flask was filled with 100 mL of sterilized liquid culture medium, and the seed culture solution prepared in Example 1 was added according to the volume of the liquid culture medium at a volume ratio of 1% (mL / mL). The culture was kept constant at 37°C and 150 r / min in a shaking incubator for 24 h to obtain a bacterial suspension. 1 mL of the suspension prepared by Bacillus subtilis was inoculated into the Taxus slurry, stirred thoroughly, and kept constant at 37°C and 150 r / min for 24 h.

[0032] (4) Taxol extraction. The Taxus slurry after bacterial hydrolysis was extracted with methanol by ultrasonic extraction for 60 min. The extract was centrifuged at 5000 rpm for 10 min, and the filtrate was separated from the residue. The obtained filtrate was the Taxol extract.

[0033] Example 3

[0034] Preparation of Paclitaxel Extract

[0035] (1) Preparation of Taxus powder. The bark of Taxus was cleaned and placed in a constant temperature drying oven at 50°C for drying. After drying, it was crushed by a pulverizer and passed through a 60-mesh sieve to obtain Taxus powder.

[0036] (2) Preparation of Taxus slurry. Weigh 1.5 g of the Taxus powder obtained in step (1) into a conical flask, thoroughly homogenize the Taxus powder with sterile water at a solid-liquid ratio of 1 g:10 mL, and adjust the pH value to 7.0 with sodium hydroxide. Place the slurry in a microwave heating device, maintain the temperature at 55°C, and microwave for 2 minutes.

[0037] (3) Bacterial hydrolysis. A 250 mL conical flask was filled with 100 mL of sterilized liquid culture medium, and the seed culture solution prepared in Example 1 was added according to the volume of the liquid culture medium at a volume ratio of 1% (mL / mL). The culture was kept constant at 37°C and 150 r / min in a shaking incubator for 24 h to obtain a bacterial suspension. 1 mL of the suspension prepared by Bacillus subtilis was inoculated into the Taxus slurry, stirred thoroughly, and kept constant at 37°C and 150 r / min for 36 h.

[0038] (4) Taxol extraction. The Taxus slurry after bacterial hydrolysis was extracted with methanol by ultrasonic extraction for 60 min. The extract was centrifuged at 5000 r / min for 10 min, and the filtrate was separated from the residue. The obtained filtrate was the Taxol extract.

[0039] Example 4

[0040] Preparation of Paclitaxel Extract

[0041] (1) Preparation of Taxus powder. The bark of Taxus was cleaned and placed in a constant temperature drying oven at 50°C for drying. After drying, it was crushed by a pulverizer and passed through a 60-mesh sieve to obtain Taxus powder.

[0042] (2) Preparation of Taxus slurry. Weigh 1.5 g of the Taxus powder obtained in step (1) into a conical flask, thoroughly homogenize the Taxus powder with sterile water at a solid-liquid ratio of 1 g:10 mL, and adjust the pH value to 7.0 with sodium hydroxide. Place the slurry in a microwave heating device, maintain the temperature at 55°C, and microwave for 2 minutes.

[0043] (3) Bacterial hydrolysis. A 250 mL conical flask was filled with 100 mL of sterilized liquid culture medium, and the seed culture solution prepared in Example 1 was added according to the volume of the liquid culture medium at a volume ratio of 1% (mL / mL). The culture was kept constant at 37°C and 150 r / min in a shaking incubator for 24 h to obtain a bacterial suspension. 1 mL of the suspension prepared by Bacillus subtilis was inoculated into the Taxus slurry, stirred thoroughly, and kept constant at 37°C and 150 r / min for 48 h.

[0044] (4) Taxol extraction. The Taxus slurry after bacterial hydrolysis was extracted with methanol by ultrasonic extraction for 60 min. The extract was centrifuged at 5000 r / min for 10 min, and the filtrate was separated from the residue. The obtained filtrate was the Taxol extract.

[0045] Example 5

[0046] Preparation of Paclitaxel Extract

[0047] (1) Preparation of Taxus powder. The bark of Taxus was cleaned and placed in a constant temperature drying oven at 50°C for drying. After drying, it was crushed by a pulverizer and passed through a 60-mesh sieve to obtain Taxus powder.

[0048] (2) Preparation of Taxus slurry. Weigh 1.5 g of the Taxus powder obtained in step (1) into a conical flask, thoroughly homogenize the Taxus powder with sterile water at a solid-liquid ratio of 1 g:10 mL, and adjust the pH value to 7.0 with sodium hydroxide. Place the slurry in a microwave heating device, maintain the temperature at 55°C, and microwave for 2 minutes.

[0049] (3) Bacterial hydrolysis. A 250 mL conical flask was filled with 100 mL of sterilized liquid culture medium, and the seed culture solution prepared in Example 1 was added according to the volume of the liquid culture medium at a volume ratio of 1% (mL / mL). The culture was kept constant at 37°C and 150 r / min in a shaking incubator for 24 h to obtain a bacterial suspension. 1 mL of the suspension prepared by Bacillus subtilis was inoculated into the Taxus slurry, stirred thoroughly, and kept constant at 37°C and 150 r / min for 60 h.

[0050] (4) Taxol extraction. The Taxus slurry after bacterial hydrolysis was extracted with methanol by ultrasonic extraction for 60 min. The extract was centrifuged at 5000 r / min for 10 min, and the filtrate was separated from the residue. The obtained filtrate was the Taxol extract.

[0051] Example 6

[0052] Preparation of Paclitaxel Extract

[0053] (1) Preparation of Taxus powder. The bark of Taxus was cleaned and placed in a constant temperature drying oven at 50°C for drying. After drying, it was crushed by a pulverizer and passed through a 60-mesh sieve to obtain Taxus powder.

[0054] (2) Preparation of Taxus slurry. Weigh 1.5 g of the Taxus powder obtained in step (1) into a conical flask, thoroughly homogenize the Taxus powder with sterile water at a solid-liquid ratio of 1 g:10 mL, and adjust the pH value to 7.0 with sodium hydroxide. Place the slurry in a microwave heating device, maintain the temperature at 55°C, and microwave for 2 minutes.

[0055] (3) Bacterial hydrolysis. A 250 mL conical flask was filled with 100 mL of sterilized liquid culture medium, and the seed culture solution prepared in Example 1 was added according to the volume of the liquid culture medium at a volume ratio of 2% (mL / mL). The culture was kept constant at 37°C and 150 r / min in a shaking incubator for 24 h to obtain a bacterial suspension. 1 mL of the suspension prepared by Bacillus subtilis was inoculated into the Taxus slurry, stirred thoroughly, and kept constant at 37°C and 150 r / min for 48 h.

[0056] (4) Taxol extraction. The Taxus slurry after bacterial hydrolysis was extracted with methanol by ultrasonic extraction for 60 min. The extract was centrifuged at 5000 r / min for 10 min, and the filtrate was separated from the residue. The obtained filtrate was the Taxol extract.

[0057] Example 7

[0058] Preparation of Paclitaxel Extract

[0059] (1) Preparation of Taxus powder. The bark of Taxus was cleaned and placed in a constant temperature drying oven at 50°C for drying. After drying, it was crushed by a pulverizer and passed through a 60-mesh sieve to obtain Taxus powder.

[0060] (2) Preparation of Taxus slurry. Weigh 1.5 g of the Taxus powder obtained in step (1) into a conical flask, thoroughly homogenize the Taxus powder with sterile water at a solid-liquid ratio of 1 g:10 mL, and adjust the pH value to 7.0 with sodium hydroxide. Place the slurry in a microwave heating device, maintain the temperature at 55°C, and microwave for 2 minutes.

[0061] (3) Bacterial hydrolysis. A 250 mL conical flask was filled with 100 mL of sterilized liquid culture medium, and the seed culture solution prepared in Example 1 was added according to the volume of the liquid culture medium at a volume ratio of 4% (mL / mL). The culture was kept constant at 37°C and 150 r / min in a shaking incubator for 24 h to obtain a bacterial suspension. 1 mL of the suspension prepared by Bacillus subtilis was inoculated into the Taxus slurry, stirred thoroughly, and kept constant at 37°C and 150 r / min for 48 h.

[0062] (4) Taxol extraction. The Taxus slurry after bacterial hydrolysis was extracted with methanol by ultrasonic extraction for 60 min. The extract was centrifuged at 5000 r / min for 10 min, and the filtrate was separated from the residue. The obtained filtrate was the Taxol extract.

[0063] Example 8

[0064] Preparation of Paclitaxel Extract

[0065] (1) Preparation of Taxus powder. The bark of Taxus was cleaned and placed in a constant temperature drying oven at 50°C for drying. After drying, it was crushed by a pulverizer and passed through a 60-mesh sieve to obtain Taxus powder.

[0066] (2) Preparation of Taxus slurry. Weigh 1.5 g of the Taxus powder obtained in step (1) into a conical flask, thoroughly homogenize the Taxus powder with sterile water at a solid-liquid ratio of 1 g:10 mL, and adjust the pH value to 7.0 with sodium hydroxide. Place the slurry in a microwave heating device, maintain the temperature at 55°C, and microwave for 2 minutes.

[0067] (3) Bacterial hydrolysis. A 250 mL conical flask was filled with 100 mL of sterilized liquid culture medium, and the seed culture solution prepared in Example 1 was added according to the volume of the liquid culture medium at a volume ratio of 6% (mL / mL). The culture was kept constant at 37°C and 150 r / min in a shaking incubator for 24 h to obtain a bacterial suspension. 1 mL of the suspension prepared by Bacillus subtilis was inoculated into the Taxus slurry, stirred thoroughly, and kept constant at 37°C and 150 r / min for 48 h.

[0068] (4) Taxol extraction. The Taxus slurry after bacterial hydrolysis was extracted with methanol by ultrasonic extraction for 60 min. The extract was centrifuged at 5000 r / min for 10 min, and the filtrate was separated from the residue. The obtained filtrate was the Taxol extract.

[0069] Example 9

[0070] Preparation of Paclitaxel Extract

[0071] (1) Preparation of Taxus powder. The bark of Taxus was cleaned and placed in a constant temperature drying oven at 50°C for drying. After drying, it was crushed by a pulverizer and passed through a 60-mesh sieve to obtain Taxus powder.

[0072] (2) Preparation of Taxus slurry. Weigh 1.5 g of the Taxus powder obtained in step (1) into a conical flask, thoroughly homogenize the Taxus powder with sterile water at a solid-liquid ratio of 1 g:10 mL, and adjust the pH value to 7.0 with sodium hydroxide. Place the slurry in a microwave heating device, maintain the temperature at 55°C, and microwave for 2 minutes.

[0073] (3) Bacterial hydrolysis. A 250 mL conical flask was filled with 100 mL of sterilized liquid culture medium, and the bacterial suspension prepared in Example 1 was inoculated at a volume ratio of 8% (mL / mL) according to the volume of the liquid culture medium, and cultured in a shaker at 37°C and 150 r / min for 24 h. 1 mL of the suspension prepared by Bacillus subtilis was inoculated in the Taxus slurry, stirred thoroughly, and cultured at 37°C and 150 r / min for 48 h.

[0074] (4) Taxol extraction. The Taxus slurry after bacterial hydrolysis was extracted with methanol by ultrasonic extraction for 60 min. The extract was centrifuged at 5000 r / min for 10 min, and the filtrate was separated from the residue. The obtained filtrate was the Taxol extract.

[0075] Comparative Example 1

[0076] Preparation of Paclitaxel Extract

[0077] (1) Preparation of Taxus powder. The bark of Taxus was cleaned and placed in a constant temperature drying oven at 50°C for drying. After drying, it was crushed by a pulverizer and passed through a 60-mesh sieve to obtain Taxus powder.

[0078] (2) Preparation of Taxus slurry. Weigh 1.5 g of Taxus powder prepared in step (1) and extract it with methanol ultrasonically for 60 min. Centrifuge the extract at 5000 r / min for 10 min, separate the filtrate from the residue, and the obtained filtrate is the paclitaxel extract.

[0079] Test Example 1:

[0080] The paclitaxel extracts obtained in Examples 2 to 9 and Comparative Example 1 were analyzed by high performance liquid chromatography. The chromatographic conditions were as follows: the column was a C18 column, the mobile phase was methanol-water (volume ratio of 58 mL:42 mL), the flow rate was 1.0 mL / min, the injection volume was 20 μL, the detection wavelength was 227 nm, and the column temperature was 30°C.

[0081] Take 4 mg of paclitaxel standard, prepare a paclitaxel standard solution with a concentration of 2 mg / mL with 2 mL of methanol, take 6 tubes of 1 mL of standard solution, dilute to 2, 1, 0.5, 0.25, 0.125, 0.0625 mg / mL concentrations, perform high performance liquid chromatography analysis and draw a standard curve. The content of paclitaxel in the paclitaxel extracts in Examples 2 to 9 and Comparative Example 1 is obtained through the standard curve.

[0082] According to the mass of the yew bark and the content of paclitaxel in the extract, the extraction rate of paclitaxel in Examples 2 to 9 and Comparative Example 1 was calculated. The calculation formula of the extraction rate is:

[0083] Extraction rate (mg / g) = the content of paclitaxel in the extract / the mass of yew bark

[0084] 1. Effect of bacterial hydrolysis time on paclitaxel extraction

[0085] The extraction rates of paclitaxel in Examples 2 to 5 and Comparative Example 1 were statistically analyzed, and the results are shown in Table 1.

[0086] Table 1 Extraction rate of paclitaxel at different bacterial hydrolysis times

[0087]

[0088] As shown in Table 1, the extraction rate of paclitaxel extracted by the method of the present invention is improved, and the optimal bacterial hydrolysis time is 48 hours.

[0089] 2. Effect of inoculum dose on paclitaxel extraction

[0090] The extraction rates of paclitaxel in Example 4, Example 6-Example 9 and Comparative Example 1 were statistically analyzed, and the results are shown in Table 2.

[0091] Table 2 Extraction rate of paclitaxel under different inoculation conditions

[0092]

[0093] It can be seen from Table 2 that the extraction rate of paclitaxel extracted by the method of the present invention is improved, and the optimal inoculation amount is 6%.

[0094] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for extracting paclitaxel based on biological crushing, characterized in that: Here are the steps: (1) culturing fermentation bacteria to obtain a seed culture solution of the fermentation bacteria; (2) preparing yew powder; (3) preparing Taxus chinensis slurry; (4) bacterial hydrolysis, inoculating the fermentation bacteria described in step (1) into the Taxus slurry described in step (3) for culturing; (5) Taxol extraction: ultrasonically extract the Taxus slurry obtained through bacterial hydrolysis in step (4), and centrifuge to obtain a Taxol extract.

2. The method for extracting paclitaxel based on biological crushing according to claim 1, characterized in that: In step (1), the fermentation bacteria are inoculated into LB liquid culture medium, and cultured at a constant temperature of 30-40° C. and 50-300 r / min in a shaking incubator for 12-36 hours to obtain a seed culture solution of the fermentation bacteria.

3. The method for extracting paclitaxel based on biological crushing according to claim 2, characterized in that: The fermentation bacteria is Bacillus subtilis ATCC6633.

4. The method for extracting paclitaxel based on biological crushing according to claim 1, characterized in that: In step (2), the yew bark is cleaned, dried at 20-90° C., crushed, and sieved through a 10-200 mesh sieve to obtain yew powder.

5. The method for extracting paclitaxel based on biological crushing according to claim 4, characterized in that: In step (3), the yew powder obtained in step (2) is weighed, and the yew powder is fully homogenized with sterile water at a solid-liquid ratio of 1 g: (1-50) mL, and the pH value is adjusted to 6-8 to obtain a yew slurry.

6. The method for extracting paclitaxel based on biological disruption according to claim 2, characterized in that: In step (4), according to the volume of the liquid culture medium, the seed culture liquid of the fermentation bacteria is inoculated, and the culture is carried out at a constant temperature of 30-40°C and 50-300 r / min in a shaking incubator for 12-64 hours to obtain a bacterial suspension; 1 mL of the bacterial suspension is inoculated into the yew slurry, stirred sufficiently, and cultured at a constant temperature of 30-40°C and 50-300 r / min with shaking for 12-64 hours.

7. The method for extracting paclitaxel based on biological disruption according to claim 6, characterized in that: The volume ratio of the seed culture solution of the fermentation bacteria to the liquid culture medium is 1 mL: (1-100) mL.

8. The method for extracting paclitaxel based on biological crushing according to claim 1, characterized in that: In step (5), the Taxus slurry after bacterial hydrolysis is extracted with methanol by ultrasonic extraction, the extraction time is 60 minutes, and the centrifugation is carried out at a speed of 5000 r / min for 10 minutes. The obtained filtrate is the paclitaxel extract.

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