阅读说明：本技术 一种紫杉醇的制备方法 (Preparation method of paclitaxel ) 是由 林金新 颜武华 邓良斌 张珊珊 王一珏 杜小倩 于 2020-11-11 设计创作，主要内容包括：本发明涉及药物制备技术领域,具体涉及一种紫杉醇的制备方法；本发明中在制备紫杉醇的过程中,通过向培养基中的培养基中选用葡萄糖及乳糖作为碳源,也有效地提高了红豆杉形成层干细胞培养体系中紫杉醇的含量；再者,通过加入适量的绿原酸及水杨酸,两者之间的配合使用,能有效提高愈伤组织中紫杉醇的含量；其次,通过液体悬浮液培养基中加入的牛二醇和丙酮酸钠之间相互协同能有效地提高细胞中紫杉醇的含量；而甲基茉莉酮酸配合紫外光照的使用能显著促进刺激代谢产物的合成,有效地诱导紫杉醇含量的提高；而后依次对所得的紫杉醇浸膏进行第一洗脱、第二洗脱及多重结晶处理,最终使得所制备的紫杉醇的纯度得到极大地提高。(The invention relates to the technical field of medicine preparation, in particular to a preparation method of paclitaxel; in the process of preparing the taxol, the content of the taxol in a taxus cambium stem cell culture system is effectively improved by selecting glucose and lactose as carbon sources in a culture medium in the culture medium; furthermore, by adding a proper amount of chlorogenic acid and salicylic acid, the content of paclitaxel in the callus can be effectively increased by matching the chlorogenic acid and the salicylic acid; secondly, the content of the taxol in the cells can be effectively improved through mutual synergy between the bovine diol and the sodium pyruvate added into the liquid suspension culture medium; the methyl jasmonic acid can obviously promote the synthesis of the stimulated metabolite by matching with the use of ultraviolet light, and effectively induces the increase of the content of the taxol; and then sequentially carrying out first elution, second elution and multiple crystallization treatment on the obtained paclitaxel extract, so that the purity of the prepared paclitaxel is greatly improved.)

1. A preparation method of paclitaxel is characterized by comprising the following steps:

s1, selecting a good-growing annual taxus yunnanensis, taking mature tender leaves of the good-growing annual taxus yunnanensis as a culture substrate, cleaning the culture substrate, disinfecting, cutting off a discolored explant, carrying out induction culture on the discolored explant, selecting a fresh embryogenic callus with good growth for successive subculture for 4-7 times, inoculating the fresh embryogenic callus into a culture medium, adding 0.1-0.13 mg/L of chlorogenic acid and 0.1-0.15 mg/L of salicylic acid into the culture medium, carrying out shake culture, carrying out subculture once every 8-10 days, and establishing a stably-growing taxus yunnanensis cambium dry cell culture system by subculture for 2-3 months;

s2, adding a taxus chinensis endophytic fungi extracting solution into the culture medium, uniformly mixing, placing the mixture into a bioreactor, selecting a taxus chinensis cambium stem cell culture system with the growth rate of 8.0-10.0 mg/g.d, inoculating the taxus chinensis cambium stem cell culture system into the bioreactor according to the inoculation amount of 12-15% for suspension culture at the culture temperature of 22-25 ℃, performing aeration culture for 12 days under the condition of artificial illumination, respectively adding 0.25-0.32 mg/L of bovine diol, 0.2-0.3 mg/L of sodium pyruvate, 8-12 mg/L of methyl jasmonic acid and 0.15-0.20 mg/L of gibberellin into the liquid suspension culture medium, and assisting ultraviolet irradiation to continue culture;

s3, collecting the culture suspension cultured in the step S2, adding a buffer solution into the culture suspension, washing the culture suspension for 3-4 times, and carrying out freeze drying treatment to obtain culture freeze dried powder; adding a proper amount of deionized water into the culture frozen dry powder, heating and soaking the culture frozen dry powder in a water bath environment at 40-50 ℃, performing centrifugal filtration treatment, adding water into obtained filter residues for repeatedly soaking for 3-5 times, combining filtrates, performing stirring extraction by using ethyl acetate, standing, collecting an inorganic phase after an obvious layering phenomenon occurs, and extracting the obtained inorganic phase by using ethyl acetate for 3-4 times; collecting inorganic phase, adding 60-70% ethanol solution, and concentrating under reduced pressure at low temperature to obtain extract containing paclitaxel;

s4, weighing the extract obtained in the step S3, adding chloroform into the extract to completely dissolve the extract, then adding silica gel into the extract to stir uniformly, filling the mixture into a chromatographic column after drying and screening, eluting the mixture by using first eluent, carrying out sectional combination and concentration after TLC detection to obtain a taxol crude product, then adding acetone into the taxol crude product to completely dissolve the taxol crude product, adding silica gel and stirring uniformly; drying, sieving, loading into chromatographic column, gradient eluting with second eluent, detecting by TLC, and concentrating by stage;

s5, crystallizing the obtained concentrate for 3-4 times by using a mixed solvent, performing vacuum reduced pressure drying at 45-55 ℃ after suction filtration, performing chromatography separation on the dried product under the pressure of 14-18 MPa, performing TLC detection, performing segmentation, merging and concentration, performing recrystallization on the obtained concentrate by using the mixed solvent, and finally performing suction filtration and drying treatment to obtain the taxol finished product.

2. The method of claim 1, wherein the paclitaxel is prepared by the following steps: and in the step S1, ethanol solution with the concentration of 65-75% is selected for disinfection for 60-80S.

3. The method of claim 1, wherein the paclitaxel is prepared by the following steps: selecting ripe potatoes with smooth surfaces, cleaning and peeling, then weighing 180-220 g of potatoes, slicing the potatoes into slices, putting the slices into a pot, adding water, boiling until the slices are broken, filtering the slices with 4 layers of gauze, adding 13-18 g of agar powder, 40-50 g of glucose and 35-50 g of lactose into the filtrate, boiling to fully dissolve the agar powder, and then adding water to a constant volume of 1000 mL; and then carrying out high-pressure sterilization for 30-35 min at the temperature of 115-125 ℃, and obtaining a finished culture medium product after the sterilization is finished.

4. The method of claim 1, wherein the paclitaxel is prepared by the following steps: the preparation method of the endophytic fungi extract in the step S2 comprises the following steps: inoculating taxus chinensis endophytic fungi into the culture medium in the step S1, performing shake culture for 5-7 days in a shaking table with the temperature of 25-30 ℃ and the rotating speed of 60-80 r/min, performing ultrasonic crushing for 5-8 min, adding ethyl acetate with the same volume as the culture medium, removing a water layer after the solution generates obvious layering phenomenon, performing reduced pressure drying treatment on the obtained extract liquor, and adding deionized water with the volume of 0.25-0.32 time of the extract liquor into the product obtained after the reduced pressure drying to obtain the endophytic fungi extract liquor.

5. The method of claim 1, wherein the paclitaxel is prepared by the following steps: in the step S3, the illumination time is 4-6 h during the ultraviolet light irradiation, and the illumination intensity is 2000-2200 lux.

6. The method of claim 1, wherein the paclitaxel is prepared by the following steps: the buffer solution in the step S3 is phosphate buffer solution, and the concentration of the phosphate buffer solution is 100-120 mM.

7. The method of claim 1, wherein the paclitaxel is prepared by the following steps: the first eluent used in the step S4 is methanol containing 10-20% of chloroform.

8. The method of claim 1, wherein the paclitaxel is prepared by the following steps: the second eluent used in the step S4 is petroleum ether containing 5-8% of acetone.

9. The method of claim 1, wherein the paclitaxel is prepared by the following steps: the mixed solvent used in the step S5 is prepared by mixing acetone and petroleum ether according to a volume ratio of 1: 1.6-2.0.

Technical Field

The invention relates to the technical field of medicine preparation, in particular to a preparation method of paclitaxel.

Background

The taxol is a natural secondary metabolite separated and purified from the bark of a gymnosperm yew, and has good anti-tumor effect through clinical verification, and particularly has special effects on ovarian cancer, uterine cancer, breast cancer and the like with high incidence rate of cancer. Paclitaxel is taken as diterpene alkaloid compound with anticancer activity, has novel and complex chemical structure, wide and remarkable biological activity, completely new and unique action mechanism and scarce natural resources, is greatly favored by phytologists, chemists, pharmacologists and molecular biologists, and becomes an anticancer star which draws attention in the next half of the 20 th century and research focus.

However, in the prior art, the paclitaxel source is obtained from the bark of yew, the content is very low, the extraction is difficult, the extraction purity is relatively low, and the ever-increasing demand of people for high-purity paclitaxel cannot be met. Therefore, it is of great significance to improve the purity of paclitaxel.

Disclosure of Invention

Aiming at the technical problems in the background technology, the invention provides a preparation method of paclitaxel, and the paclitaxel prepared by the invention has high recovery rate and high purity; the paclitaxel preparation process provided by the invention has wider market prospect and is more suitable for popularization.

Technical scheme

In order to achieve the purpose, the invention is realized by the following technical scheme:

a preparation method of paclitaxel comprises the following steps:

s1, selecting a good-growing annual taxus yunnanensis, taking mature tender leaves of the good-growing annual taxus yunnanensis as a culture substrate, cleaning the culture substrate, disinfecting, cutting off a discolored explant, carrying out induction culture on the discolored explant, selecting a fresh embryogenic callus with good growth for successive subculture for 4-7 times, inoculating the fresh embryogenic callus into a culture medium, adding 0.1-0.13 mg/L of chlorogenic acid and 0.1-0.15 mg/L of salicylic acid into the culture medium, carrying out shake culture, carrying out subculture once every 8-10 days, and establishing a stably-growing taxus yunnanensis cambium dry cell culture system by subculture for 2-3 months;

s2, adding a taxus chinensis endophytic fungi extracting solution into the culture medium, uniformly mixing, placing the mixture into a bioreactor, selecting a taxus chinensis cambium stem cell culture system with the growth rate of 8.0-10.0 mg/g.d, inoculating the taxus chinensis cambium stem cell culture system into the bioreactor according to the inoculation amount of 12-15% for suspension culture at the culture temperature of 22-25 ℃, performing aeration culture for 12 days under the condition of artificial illumination, respectively adding 0.25-0.32 mg/L of bovine diol, 0.2-0.3 mg/L of sodium pyruvate, 8-12 mg/L of methyl jasmonic acid and 0.15-0.20 mg/L of gibberellin into the liquid suspension culture medium, and assisting ultraviolet irradiation to continue culture;

s3, collecting the culture suspension cultured in the step S2, adding a buffer solution into the culture suspension, washing the culture suspension for 3-4 times, and carrying out freeze drying treatment to obtain culture freeze dried powder; adding a proper amount of deionized water into the culture frozen dry powder, heating and soaking the culture frozen dry powder in a water bath environment at 40-50 ℃, performing centrifugal filtration treatment, adding water into obtained filter residues for repeatedly soaking for 3-5 times, combining filtrates, performing stirring extraction by using ethyl acetate, standing, collecting an inorganic phase after an obvious layering phenomenon occurs, and extracting the obtained inorganic phase by using ethyl acetate for 3-4 times; collecting inorganic phase, adding 60-70% ethanol solution, and concentrating under reduced pressure at low temperature to obtain extract containing paclitaxel;

s4, weighing the extract obtained in the step S3, adding chloroform into the extract to completely dissolve the extract, then adding silica gel into the extract to stir uniformly, filling the mixture into a chromatographic column after drying and screening, eluting the mixture by using first eluent, carrying out sectional combination and concentration after TLC detection to obtain a taxol crude product, then adding acetone into the taxol crude product to completely dissolve the taxol crude product, adding silica gel and stirring uniformly; drying, sieving, loading into chromatographic column, gradient eluting with second eluent, detecting by TLC, and concentrating by stage;

s5, crystallizing the obtained concentrate for 3-4 times by using a mixed solvent, performing vacuum reduced pressure drying at 45-55 ℃ after suction filtration, performing chromatography separation on the dried product under the pressure of 14-18 MPa, performing TLC detection, performing segmentation, merging and concentration, performing recrystallization on the obtained concentrate by using the mixed solvent, and finally performing suction filtration and drying treatment to obtain the taxol finished product.

Furthermore, the ethanol solution with the concentration of 65-75% is selected for sterilization treatment in the step S1 for 60-80S.

Further, the preparation method of the culture medium used in the step S1 comprises the steps of selecting ripe potatoes with smooth surfaces, cleaning and peeling, weighing 180-220 g of the potatoes, slicing the potatoes, putting the potatoes into a pot, adding water, boiling the potatoes until the potatoes are broken, filtering the potatoes by using 4 layers of gauze, adding 13-18 g of agar powder, 40-50 g of glucose and 35-50 g of lactose into the filtrate, boiling the mixture to fully dissolve the agar powder, the glucose and the lactose, and adding water to the mixture until the volume is 1000 mL; and then carrying out high-pressure sterilization for 30-35 min at the temperature of 115-125 ℃, and obtaining a finished culture medium product after the sterilization is finished.

Further, the method for preparing the endophytic fungi extract in the step S2 comprises the following steps: inoculating taxus chinensis endophytic fungi into the culture medium in the step S1, performing shake culture for 5-7 days in a shaking table with the temperature of 25-30 ℃ and the rotating speed of 60-80 r/min, performing ultrasonic crushing for 5-8 min, adding ethyl acetate with the same volume as the culture medium, removing a water layer after the solution generates obvious layering phenomenon, performing reduced pressure drying treatment on the obtained extract liquor, and adding deionized water with the volume of 0.25-0.32 time of the extract liquor into the product obtained after the reduced pressure drying to obtain the endophytic fungi extract liquor.

Furthermore, the illumination time in the step S3 is 4-6 h and the illumination intensity is 2000-2200 lux.

Furthermore, the buffer solution in step S3 is phosphate buffer solution, and the concentration of the phosphate buffer solution is 100 to 120 mM.

Further, the first eluent used in the step S4 is methanol containing 10 to 20% of chloroform.

Further, the second eluent used in the step S4 is petroleum ether containing 5-8% of acetone.

Further, the mixed solvent used in the step S5 is prepared by mixing acetone and petroleum ether in a volume ratio of 1: 1.6-2.0.

Advantageous effects

Compared with the known public technology, the technical scheme provided by the invention has the following beneficial effects:

in the process of preparing the taxol, the glucose and the lactose are selected as carbon sources in the culture medium, and the obtained fermentation product hardly secretes any pigment, so that the quality of the obtained callus is ensured. Meanwhile, the content of paclitaxel in the taxus cambium stem cell culture system is effectively improved. In addition, by adding a proper amount of chlorogenic acid and salicylic acid, the content of paclitaxel in the callus can be effectively increased by matching the chlorogenic acid and the salicylic acid. Secondly, the content of the taxol in the cells can be effectively improved through mutual synergy between the bovine diol and the sodium pyruvate added into the liquid suspension culture medium. The methyl jasmonic acid can be used with ultraviolet light to remarkably promote the synthesis of the stimulated metabolite and effectively induce the increase of the content of the paclitaxel. And then sequentially carrying out first elution, second elution and multiple crystallization treatment on the obtained paclitaxel extract, so that the purity of the prepared paclitaxel is greatly improved.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The present invention will be further described with reference to the following examples.

Example 1

A preparation method of paclitaxel comprises the following steps:

s1, selecting annual taxus yunnanensis with good growth vigor, taking mature tender leaves of the annual taxus yunnanensis as a culture substrate, cleaning the culture substrate, then sterilizing, cutting off the discolored explants, then carrying out induction culture on the explants, selecting fresh embryogenic callus with good growth vigor and continuous subculture for 4 times, inoculating the fresh embryogenic callus into a culture medium, adding 0.1mg/L of chlorogenic acid and 0.1mg/L of salicylic acid into the culture medium, carrying out shake culture, subculturing once every 8 days, and establishing a stably-growing taxus yunnanensis cambium dry cell culture system through subculture for 2 months;

s2, adding a taxus chinensis endophytic fungi extracting solution into the culture medium, uniformly mixing, placing the mixture into a bioreactor, selecting a taxus chinensis cambium stem cell culture system with the growth rate of 8.0 mg/g.d, inoculating the taxus chinensis cambium stem cell culture system into the bioreactor according to the inoculation amount of 12% for suspension culture at the culture temperature of 22 ℃, respectively adding 0.25mg/L of bovine diol, 0.2mg/L of sodium pyruvate, 8mg/L of methyl jasmonic acid and 0.15mg/L of gibberellin into the liquid suspension culture medium after carrying out aeration culture for 12 days under the condition of artificial illumination, and continuing the culture under the auxiliary irradiation of ultraviolet light;

s3, collecting the culture suspension cultured in the step S2, adding a buffer solution into the culture suspension, washing the culture suspension for 3 times, and then carrying out freeze drying treatment to obtain culture freeze dried powder; adding a proper amount of deionized water into the culture frozen dry powder, heating and soaking the culture frozen dry powder in a water bath environment at 40 ℃, adding water into obtained filter residues for repeatedly soaking for 3 times after centrifugal filtration treatment, then mixing filtrates, stirring and extracting with ethyl acetate, standing, collecting inorganic phases after obvious layering occurs, and extracting the obtained inorganic phases with ethyl acetate for 3 times; collecting inorganic phase, adding 60% ethanol solution, and concentrating at low temperature under reduced pressure to obtain extract containing paclitaxel;

s4, weighing 283.5mg of extract 400g obtained in the step S3, adding trichloromethane into the extract to completely dissolve the trichloromethane, adding silica gel into the extract, uniformly stirring, drying in the air, sieving, filling into a chromatographic column, and eluting with a first eluent; obtain column chromatography product 362.4mg containing paclitaxel, contain paclitaxel 268.9mg after TLC detection, the recovery rate is 94.85%; combining and concentrating by sections to obtain a crude taxol product, adding acetone into the crude taxol product to completely dissolve the acetone, adding silica gel, and stirring uniformly; drying, sieving, filling into chromatographic column, gradient eluting with second eluent to obtain 267.5mg of column chromatography product containing paclitaxel, detecting by TLC to obtain 259.3mg of paclitaxel with recovery rate of 96.43% and purity of 96.93%; carrying out segmentation, merging and concentration;

s5, crystallizing the obtained concentrate for 3 times by using a mixed solvent, performing vacuum reduced pressure drying at 45 ℃ after suction filtration, performing chromatography separation on the dried product under the pressure of 14MPa, performing TLC detection, combining and concentrating in sections, performing recrystallization on the obtained concentrate by using the mixed solvent, and finally performing suction filtration and drying treatment to obtain a taxol finished product, wherein the purity of the taxol is 99.67% by using TLC detection.

In the step S1, an ethanol solution with a concentration of 65% is selected for disinfection for 60S.

The preparation method of the culture medium used in the step S1 comprises the steps of selecting mature potatoes with smooth surfaces, cleaning and peeling the potatoes, weighing 180g of the potatoes, slicing the potatoes into slices, putting the slices into a pot, adding water, boiling the slices until the slices are broken, filtering the slices by using 4 layers of gauze, adding 13g of agar powder, 40g of glucose and 35g of lactose into the filtrate, boiling the filtrate to fully dissolve the agar powder, the glucose and the lactose, and adding water to fix the volume to 1000 mL; then carrying out high-pressure sterilization for 30min at the temperature of 115 ℃, and obtaining a finished culture medium product after the sterilization is finished.

The preparation method of the endophytic fungi extract in the step S2 comprises the following steps: inoculating taxus endophytic fungi into the culture medium in the step S1, carrying out shake culture for 5d in a shaking table with the temperature of 25 ℃ and the rotating speed of 60r/min, carrying out ultrasonic crushing for 5min, adding ethyl acetate with the same volume as the culture medium, removing a water layer after the solution generates obvious layering phenomenon, carrying out reduced pressure drying treatment on the obtained extract liquor, and adding deionized water with 0.25 time of the volume of the extract liquor into the product obtained after the reduced pressure drying, thus obtaining the endophytic fungi extract liquor.

In the step S3, the illumination time is 4h and the illumination intensity is 2000lux during the ultraviolet light illumination.

In step S3, the buffer solution is phosphate buffer solution with concentration of 100 mM.

The first eluent used in step S4 was methanol containing 10% chloroform.

The second eluent used in step S4 was petroleum ether containing 5% acetone.

The mixed solvent used in the step S5 is prepared from acetone and petroleum ether in a volume ratio of 1: 1.6 mixing.

Example 2

A preparation method of paclitaxel comprises the following steps:

s1, selecting annual taxus yunnanensis with good growth vigor, taking mature tender leaves of the annual taxus yunnanensis as a culture substrate, cleaning the culture substrate, then sterilizing, cutting off the discolored explants, then carrying out induction culture on the explants, selecting fresh embryogenic callus with good growth vigor for 5 times of continuous subculture, inoculating the fresh embryogenic callus into a culture medium, adding 0.12mg/L of chlorogenic acid and 0.13mg/L of salicylic acid into the culture medium, carrying out shake culture, subculturing once every 9 days, and establishing a stably-growing taxus yunnanensis cambium dry cell culture system through subculture for 2 months;

s2, adding a taxus chinensis endophytic fungi extracting solution into the culture medium, uniformly mixing, placing the mixture into a bioreactor, selecting a taxus chinensis cambium stem cell culture system with the growth rate of 9.0 mg/g.d, inoculating the taxus chinensis cambium stem cell culture system into the bioreactor according to 13% of inoculation amount for suspension culture, carrying out aeration culture at the culture temperature of 23 ℃ under the condition of artificial illumination for 12 days, respectively adding 0.30mg/L of bovine diol, 0.25mg/L of sodium pyruvate, 10mg/L of methyl jasmonic acid and 0.18mg/L of gibberellin into the liquid suspension culture medium, and assisting ultraviolet irradiation to continue the culture;

s3, collecting the culture suspension cultured in the step S2, adding a buffer solution into the culture suspension, washing the culture suspension for 3 times, and then carrying out freeze drying treatment to obtain culture freeze dried powder; adding a proper amount of deionized water into the culture frozen dry powder, heating and soaking the culture frozen dry powder in a water bath environment at 45 ℃, adding water into obtained filter residues for repeatedly soaking for 4 times after centrifugal filtration treatment, then mixing filtrates, stirring and extracting with ethyl acetate, standing, collecting inorganic phases after obvious layering occurs, and extracting the obtained inorganic phases with ethyl acetate for 3 times; collecting inorganic phase, adding 65% ethanol solution, and concentrating at low temperature under reduced pressure to obtain extract containing paclitaxel;

s4, weighing 400g of extract containing 281.7mg obtained in the step S3, adding trichloromethane into the extract to completely dissolve the extract, then adding silica gel into the extract, uniformly stirring the mixture, carrying out air drying and sieving treatment, filling the mixture into a chromatographic column, and eluting the chromatographic column with first eluent to obtain 372.3mg of column chromatography product containing paclitaxel, wherein the recovery rate is 97.44% after TLC detection, and the paclitaxel is 274.5 mg; combining and concentrating by sections to obtain a crude taxol product, adding acetone into the crude taxol product to completely dissolve the acetone, adding silica gel, and stirring uniformly; drying, sieving, filling into chromatographic column, gradient eluting with second eluent to obtain column chromatography product 268.2mg containing paclitaxel, detecting by TLC to obtain paclitaxel 262.5mg, recovering 95.63%, and purity of paclitaxel 97.87%; carrying out segmentation, merging and concentration;

s5, crystallizing the obtained concentrate for 3 times by using a mixed solvent, performing vacuum reduced pressure drying at 50 ℃ after suction filtration, performing chromatography separation on the dried product under the pressure of 16Mpa, performing TLC detection, combining and concentrating in sections, performing recrystallization on the obtained concentrate by using the mixed solvent, performing suction filtration and drying to obtain a finished product of paclitaxel, and detecting the purity of the paclitaxel to be 99.78% by using TLC.

In the step S1, an ethanol solution with a concentration of 70% is selected for disinfection for 70S.

The preparation method of the culture medium used in the step S1 comprises the steps of selecting ripe potatoes with smooth surfaces, cleaning and peeling, weighing 200g of the potatoes, slicing the potatoes into slices, putting the slices into a pot, adding water, boiling the slices until the slices are broken, filtering the slices by using 4 layers of gauze, adding 15g of agar powder, 45g of glucose and 40g of lactose into the filtrate, boiling the filtrate to fully dissolve the agar powder, 45g of glucose and 40g of lactose, and adding water to fix the volume to 1000 mL; then carrying out high-pressure sterilization for 30min at the temperature of 120 ℃, and obtaining a finished culture medium product after the sterilization is finished.

The preparation method of the endophytic fungi extract in the step S2 comprises the following steps: inoculating the taxus endophytic fungi into the culture medium in the step S1, carrying out shake culture for 6d in a shaking table with the temperature of 28 ℃ and the rotating speed of 70r/min, carrying out ultrasonic crushing for 6min, adding ethyl acetate with the same volume as the culture medium, removing a water layer after the solution generates obvious layering phenomenon, carrying out reduced pressure drying treatment on the obtained extract liquor, and adding deionized water with 0.3 time of the volume of the extract liquor into the product obtained after the reduced pressure drying, thus obtaining the endophytic fungi extract liquor.

In the step S3, the illumination time is 5h and the illumination intensity is 2100lux during the ultraviolet light illumination.

In step S3, the buffer solution is phosphate buffer solution with a concentration of 110 mM.

The first eluent used in step S4 was methanol containing 15% chloroform.

The second eluent used in step S4 was petroleum ether containing 6% acetone.

The mixed solvent used in the step S5 is prepared from acetone and petroleum ether in a volume ratio of 1: 1.8 are mixed.

Example 3

A preparation method of paclitaxel comprises the following steps:

s1, selecting a good-growth annual Yunnan taxus chinensis, taking mature tender leaves of the yew as a culture substrate, cleaning the culture substrate, then sterilizing, cutting off a discolored explant, carrying out induction culture on the explant, selecting a fresh embryogenic callus with good growth for 7 times of continuous subculture, inoculating the fresh embryogenic callus into a culture medium, adding 0.13mg/L of chlorogenic acid and 0.15mg/L of salicylic acid into the culture medium, carrying out shake culture, subculturing once every 10 days, and establishing a stably-growing taxus chinensis cambium dry cell culture system through 3-month subculture;

s2, adding a taxus chinensis endophytic fungi extracting solution into the culture medium, uniformly mixing, placing the mixture into a bioreactor, selecting a taxus chinensis cambium stem cell culture system with the growth rate of 10.0 mg/g.d, inoculating the taxus chinensis cambium stem cell culture system into the bioreactor according to the inoculation amount of 15% for suspension culture, carrying out aeration culture at the culture temperature of 25 ℃ under the condition of artificial illumination for 12 days, respectively adding 0.32mg/L of bovine diol, 0.3mg/L of sodium pyruvate, 12mg/L of methyl jasmonic acid and 0.20mg/L of gibberellin into the liquid suspension culture medium, and assisting ultraviolet irradiation to continue the culture;

s3, collecting the culture suspension cultured in the step S2, adding a buffer solution into the culture suspension, washing the culture suspension for 4 times, and then carrying out freeze drying treatment to obtain culture freeze dried powder; adding a proper amount of deionized water into the culture frozen dry powder, heating and soaking the culture frozen dry powder in a water bath environment at 50 ℃, adding water into obtained filter residues for repeatedly soaking for 5 times after centrifugal filtration treatment, then mixing filtrates, stirring and extracting with ethyl acetate, standing, collecting inorganic phases after obvious layering occurs, and extracting the obtained inorganic phases with ethyl acetate for 4 times; collecting inorganic phase, adding 70% ethanol solution, and concentrating at low temperature under reduced pressure to obtain extract containing paclitaxel;

s4, weighing 400g of extract containing 285.6mg obtained in the step S3, adding trichloromethane into the extract to completely dissolve the extract, then adding silica gel into the extract, uniformly stirring, drying in the air, sieving, filling into a chromatographic column, and eluting with a first eluent to obtain 375.7mg of column chromatography product containing paclitaxel, wherein the recovery rate is 95.38% after TLC detection, and 272.4mg of paclitaxel is contained; combining and concentrating by sections to obtain a crude taxol product, adding acetone into the crude taxol product to completely dissolve the acetone, adding silica gel, and stirring uniformly; drying, sieving, filling into chromatographic column, gradient eluting with second eluent to obtain column chromatography product containing paclitaxel 266.8mg, detecting by TLC to obtain paclitaxel 262.3mg, with recovery rate of 96.29% and purity of paclitaxel 98.31%; carrying out segmentation, merging and concentration;

s5, crystallizing the obtained concentrate for 4 times by using a mixed solvent, performing vacuum reduced pressure drying at 55 ℃ after suction filtration, performing chromatography separation on the dried product under the pressure of 18Mpa, performing TLC detection, combining and concentrating in sections, performing recrystallization on the obtained concentrate by using the mixed solvent, performing suction filtration and drying to obtain a finished product of paclitaxel, and detecting the purity of the paclitaxel to be 99.82% by using TLC.

In the step S1, an ethanol solution with a concentration of 75% is selected for disinfection for 80S.

The preparation method of the culture medium used in the step S1 comprises the steps of selecting mature potatoes with smooth surfaces, cleaning and peeling, weighing 220g of the potatoes, slicing the potatoes into slices, putting the slices into a pot, adding water, boiling the slices until the slices are broken, filtering the slices by using 4 layers of gauze, adding 18g of agar powder, 50g of glucose and 50g of lactose into the filtrate, boiling the filtrate to fully dissolve the agar powder, 50g of glucose and 50g of lactose, and adding water to fix the volume to 1000 mL; then, carrying out autoclaving for 35min at the temperature of 125 ℃, and obtaining a finished culture medium product after the sterilization is finished.

The preparation method of the endophytic fungi extract in the step S2 comprises the following steps: inoculating the taxus endophytic fungi into the culture medium in the step S1, carrying out shake culture for 7d in a shaking table with the temperature of 30 ℃ and the rotating speed of 80r/min, carrying out ultrasonic crushing for 8min, adding ethyl acetate with the same volume as the culture medium, removing a water layer after the solution generates obvious layering phenomenon, carrying out reduced pressure drying treatment on the obtained extract liquor, and adding deionized water with 0.32 time of the volume of the extract liquor into the product obtained after the reduced pressure drying, thus obtaining the endophytic fungi extract liquor.

In the step S3, the illumination time is 6h and the illumination intensity is 2200lux during the ultraviolet light illumination.

In step S3, the buffer solution is phosphate buffer solution with a concentration of 120 mM.

The first eluent used in step S4 was methanol containing 20% chloroform.

The second eluent used in step S4 was petroleum ether containing 8% acetone.

The mixed solvent used in the step S5 is prepared from acetone and petroleum ether in a volume ratio of 1: 2.0, and mixing.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.