Taraxacum kok-saghyz producing taxol, method for increasing the yield of taxol and its precursor substances in Taraxacum kok-saghyz, and application thereof
By applying phosphorus fertilizer during the rubber grass planting process, especially under phosphorus stress conditions, the yield of paclitaxel and its precursor substances in rubber grass is increased, and the problem of insufficient source of paclitaxel is solved and an alternative source of paclitaxel is provided.
Patent Information
- Application Number
- CN202510513679.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-23
- Publication Date
- 2025-07-25
- Estimated Expiration
- 2045-04-23
AI Technical Summary
In the prior art, the source of paclitaxel mainly depends on trees such as yew. It has low extraction efficiency and long growth cycle, resulting in serious resource damage and cannot meet market demand. Chemical synthesis depends on insufficient supply of Bacartine precursor substances.
Phosphorus fertilizer is applied when planting rubber grass, especially under phosphorus stress conditions. By increasing the phosphorus content in the planting environment, the yield of paclitaxel and its precursor substances in rubber grass, including the content of 10-deacetylbacartine III and paclitaxel.
The yield of paclitaxel and its precursor substances in rubber grass has been significantly improved, providing an alternative source of paclitaxel, meeting market demand and reducing dependence on yew.
Smart Images
Figure CN120036189B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of planting, and particularly relates to a Taraxacum kok-saghyz producing taxol, a method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, and applications thereof. Background Art
[0002] Taraxacum kok-saghyz ( Taraxacum kok-saghyz ) is a plant of the genus Taraxacum, and its roots can produce a natural rubber, so it is considered an alternative crop to rubber trees. Taraxacum kok-saghyz is also an edible plant, its leaves can be eaten or used as livestock feed. In addition, the roots of Taraxacum kok-saghyz, like most dandelion plants, have medicinal value, which can clear heat and detoxify, reduce swelling and dissipate nodules. In recent years, the anti-tumor effect of dandelion has also been widely explored, but its anti-tumor mechanism is not very clear. Whether Taraxacum kok-saghyz, as a kind of dandelion, has anti-tumor medicinal value has not been explored. Taxol, as one of the most successful anti-cancer drugs applied in the world at present, has a demand far greater than the supply.
[0003] At present, the source of taxol mainly comes from the extraction of Taxus and related plant materials. However, the extraction efficiency is relatively low. And because the growth cycle of trees such as Taxus is relatively long, the extraction of taxol causes great damage to the survival of Taxus. On the other hand, taxol can be synthesized by chemical methods, but it still largely depends on the supply of the precursor substance baccatin extracted from Taxus or related cell cultures. Therefore, it is urgent to find and develop a new alternative plant rich in taxol and increase its taxol yield to meet the growing market demand for taxol in the world. Summary of the Invention
[0004] In view of the above problems, the present invention provides a Taraxacum kok-saghyz producing taxol, a method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, and applications thereof.
[0005] To achieve the above object, the technical solutions adopted by the present invention are as follows:
[0006] A Taraxacum kok-saghyz producing taxol, wherein the Taraxacum kok-saghyz producing taxol is obtained by planting Taraxacum kok-saghyz;
[0007] The Taraxacum kok-saghyz producing taxol produces taxol.
[0008] Further, the Taraxacum kok-saghyz is planted under phosphorus stress conditions.
[0009] Further, the phosphorus content in the planting environment of the Taraxacum kok-saghyz is ≥0 mg, preferably ≥1.3 mg, and most preferably ≥8 mg.
[0010] An application of the above-mentioned Taraxacum kok-saghyz producing taxol, wherein the application is to extract 10-deacetylbaccatin III and / or taxol from the Taraxacum kok-saghyz producing taxol to obtain 10-deacetylbaccatin III and / or taxol.
[0011] A method for increasing the yield of taxol and its precursor substances in Taraxacum kok-saghyz, the method being to grow Taraxacum kok-saghyz under conditions of phosphorus stress to increase the yield of taxol and its precursor substances in Taraxacum kok-saghyz;
[0012] The precursor substance is 10-deacetylbaccatin III.
[0013] Furthermore, the method is to grow Taraxacum kok-saghyz and apply phosphate fertilizer to increase the yield of taxol and its precursor substances in Taraxacum kok-saghyz.
[0014] Furthermore, the amount of phosphate fertilizer applied per plant per day is ≥0 mg, preferably ≥1.3 mg, and most preferably ≥8 mg;
[0015] The application of phosphate fertilizer starts after Taraxacum kok-saghyz germinates and grows for 0.5 to 2 months;
[0016] The types of phosphate fertilizer include but are not limited to: at least one of phosphorus-containing culture solution, potassium dihydrogen phosphate, and phosphate fertilizer.
[0017] Furthermore, the method is to grow Taraxacum kok-saghyz in soil applied with phosphate fertilizer to increase the yield of taxol and its precursor substances in Taraxacum kok-saghyz.
[0018] Furthermore, the soil applied with phosphate fertilizer is to apply ≥0 mg of phosphate fertilizer in the soil, preferably ≥1.3 mg, and most preferably ≥8 mg.
[0019] The types of phosphate fertilizer include but are not limited to: at least one of phosphorus-containing culture solution, potassium dihydrogen phosphate, and phosphate fertilizer.
[0020] An application of Taraxacum kok-saghyz, wherein the application is to extract 10-deacetylbaccatin III and / or taxol from the Taraxacum kok-saghyz obtained by the above method to obtain 10-deacetylbaccatin III and / or taxol.
[0021] The beneficial effects of a Taraxacum kok-saghyz producing taxol, a method for increasing the yield of taxol and its precursor substances in Taraxacum kok-saghyz, and the application of the present invention are:
[0022] The present invention detects paclitaxel in rubber grass and finds that the rubber grass contains a certain amount of paclitaxel and other related components. Further, by exploring the influence of phosphorus content on paclitaxel production by rubber grass, it is found that when the phosphorus content in the planting environment (such as culture medium) is low, the paclitaxel content in the rubber grass is also low, while when the phosphorus content in the planting environment (such as culture medium) increases, the paclitaxel content in the rubber grass significantly increases; finally, by applying phosphorus-containing culture solution to rubber grass cultivated in soil, the content of paclitaxel and its precursor baccatin in the rubber grass is greatly increased, laying a solid theoretical foundation and technical support for rubber grass to become a substitute for existing paclitaxel extraction source plants. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 In Example 1 of the present invention, rubber grass grown in a phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate and a phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate for 16 days respectively; the left side shows rubber grass grown in a phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate, and the right side shows rubber grass grown in a phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate; the rubber grass on the left side is slightly smaller in size, has a shorter root, and has a purple-red color at the stem and leaf part and the root, which may contain anthocyanins; the rubber grass on the right side is larger in size, has a longer root, and has a greener color at the stem and leaf part and the root;
[0024] Figure 2 The content of inorganic phosphorus in the rubber grass grown in the phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate and the phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate in Example 1 of the present invention; wherein the content of inorganic phosphorus in the rubber grass cultured in the phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly higher than that in the rubber grass cultured in the phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate;
[0025] Figure 3 The content of paclitaxel in the rubber grass grown in the phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate and the phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate in Example 1 of the present invention; wherein the content of paclitaxel in the rubber grass cultured in the phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly higher than that in the rubber grass cultured in the phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate;
[0026] Figure 4is the content of anthocyanin in Taraxacum kok-saghyz grown in the phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate and the phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate in Example 1 of the present invention; wherein, the content of anthocyanin in Taraxacum kok-saghyz cultured in the phosphorus-free 1 / 2MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly lower than that in Taraxacum kok-saghyz cultured in the phosphorus-free 1 / 2MS medium supplemented with 10 μM potassium dihydrogen phosphate;
[0027] Figure 5 is the phenotype of 2-month-old Taraxacum kok-saghyz seedlings grown in the soil irrigated with the phosphorus-free 1 / 2MS culture solution supplemented with 10 μM potassium dihydrogen phosphate and the phosphorus-free 1 / 2MS culture solution supplemented with 600 μM potassium dihydrogen phosphate in Example 2 of the present invention; wherein, the leaves of Taraxacum kok-saghyz irrigated with the phosphorus-free 1 / 2MS culture solution supplemented with 600 μM potassium dihydrogen phosphate for 1 month are significantly larger than those of Taraxacum kok-saghyz treated with the phosphorus-free 1 / 2MS culture solution supplemented with 10 μM potassium dihydrogen phosphate for 1 month;
[0028] Figure 6 is the content of 10-deacetylbaccatin III and paclitaxel in Taraxacum kok-saghyz grown in the soil irrigated with the phosphorus-free 1 / 2MS culture solution supplemented with 10 μM potassium dihydrogen phosphate and the phosphorus-free 1 / 2MS culture solution supplemented with 600 μM potassium dihydrogen phosphate in Example 2 of the present invention; wherein, the contents of 10-deacetylbaccatin III and paclitaxel in Taraxacum kok-saghyz irrigated with the phosphorus-free 1 / 2MS culture solution supplemented with 600 μM potassium dihydrogen phosphate for 1 month are significantly higher than those in Taraxacum kok-saghyz irrigated with the phosphorus-free 1 / 2MS culture solution supplemented with 10 μM potassium dihydrogen phosphate for 1 month. Detailed implementation manners
[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.
[0030] The phosphorus-free 1 / 2MS medium was purchased from Beijing Coolaber Technology Co., Ltd., and potassium dihydrogen phosphate was purchased from Sinopharm Chemical Reagent Co., Ltd.
[0031] Example 1 Effect of planting environment on the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz
[0032] In this example, Taraxacum kok-saghyz seeds were planted in media with different phosphorus concentrations to investigate the effect of phosphorus content on the yield of paclitaxel and its precursor substances in Taraxacum kok-saghyz, as follows:
[0033] Take the phosphorus-free 1 / 2MS medium, and add 10 μM and 600 μM potassium dihydrogen phosphate respectively for standby;
[0034] Take Taraxacum kok-saghyz seeds and disinfect them with 20% 84 disinfectant for 15 minutes, then rinse them 6 times with distilled water. Sow the disinfected seeds in a phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate and a phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate respectively. Place them in an incubator at 26 °C (16 h light / 8 h dark) and take pictures after culturing for 16 days. As Figure 1 shown, then take samples as test tissues respectively and detect the contents of inorganic phosphorus, anthocyanin and paclitaxel in them. The inorganic phosphorus contents in Taraxacum kok-saghyz grown in media with different phosphorus concentrations are shown in Figure 2 ; the paclitaxel contents are shown in Figure 3 ; and the anthocyanin contents are shown in Figure 4 . As Figure 1 can be seen, the overall plants of Taraxacum kok-saghyz cultured in the phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate are slightly smaller, with shorter roots. At the same time, the colors of the stems, leaves and roots show purplish red, which may contain anthocyanin. While the overall plants of Taraxacum kok-saghyz cultured in the phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate are larger, with longer roots, and the colors of the stems, leaves and roots are greener. As Figure 2 can be seen, the inorganic phosphorus content in Taraxacum kok-saghyz cultured in the phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly higher than that in Taraxacum kok-saghyz cultured in the phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate. As Figure 3 can be seen, Taraxacum kok-saghyz contains paclitaxel components, and the paclitaxel content in Taraxacum kok-saghyz cultured in the phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly higher than that in Taraxacum kok-saghyz cultured in the phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate, indicating that increasing the phosphorus addition amount in the medium can significantly increase the paclitaxel content in Taraxacum kok-saghyz. At the same time, as Figure 4 can be seen, the anthocyanin content in Taraxacum kok-saghyz cultured in the phosphorus-free 1 / 2 MS medium supplemented with 600 μM potassium dihydrogen phosphate is significantly lower than that in Taraxacum kok-saghyz cultured in the phosphorus-free 1 / 2 MS medium supplemented with 10 μM potassium dihydrogen phosphate.
[0035] In summary, it can be seen that by increasing the phosphorus content in the planting environment (such as applying phosphate fertilizer), the inorganic phosphorus content and paclitaxel content in the planted Taraxacum kok-saghyz can be effectively increased, while the anthocyanin content can be reduced.
[0036] Among them, the detection method of inorganic phosphorus content is as follows:
[0037] Take potassium dihydrogen phosphate and add purified water to make an inorganic phosphorus standard solution with a concentration of 1 mmol / L.
[0038] Take 0.1 g of the tissue to be tested, add 1 mL of distilled water, homogenize thoroughly on ice, then centrifuge at 4°C and 10,000 rpm for 10 min, and take the supernatant as the test solution.
[0039] Respectively take the inorganic phosphorus standard solution and the test solution, detect the absorbance of inorganic phosphorus in them by the molybdenum blue method, calculate the content of inorganic phosphorus in the test solution by the external standard single-point method, and then calculate the content of inorganic phosphorus in the tissue to be tested.
[0040] The detection method for the content of paclitaxel is as follows:
[0041] Take paclitaxel and add methanol to prepare standard curve solutions with final concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, and 10,000 ng / mL.
[0042] Take each standard solution and perform liquid chromatography-tandem mass spectrometry detection respectively, obtain the chromatogram corresponding to each standard solution and the peak area of paclitaxel in the chromatogram, and draw the standard curve of paclitaxel using the concentration of paclitaxel in each standard solution and its corresponding peak area of paclitaxel.
[0043] Place the tissue to be tested in liquid nitrogen for grinding and pulverization, accurately weigh 1 g of the obtained sample, add 10 mL of methanol, ultrasonicate in an ice-water bath for 30 min, centrifuge at 11,000 rpm for 30 min to obtain the supernatant and precipitate; add 5 mL of methanol to the precipitate for extraction again, combine the supernatants, add 70 mg of C18 (octadecylsilane-bonded silica gel) and 40 mg of GCB (graphitized carbon black), centrifuge at 4°C and 5,000 rpm for 10 min, take the supernatant, dry it with nitrogen, then add 400 μL of methanol for reconstitution, and filter through a 0.22 μm organic phase filter membrane to obtain the test solution.
[0044] Take the test solution for liquid chromatography-tandem mass spectrometry detection to obtain the chromatogram of the test solution and the peak area of paclitaxel contained therein, substitute the peak area of paclitaxel into the standard curve of paclitaxel, calculate the content of paclitaxel in the test solution, and then calculate the content of paclitaxel in the tissue to be tested.
[0045] Among them, the chromatographic conditions for liquid chromatography-tandem mass spectrometry detection are:
[0046] The chromatographic column is waters ACQUITY UPLC BEH HSS T3 1.8um 2.1╳100mm;
[0047] The column temperature is 30°C;
[0048] Mobile phase A is an aqueous solution of 0.1% formic acid, and mobile phase B is acetonitrile;
[0049] The flow rate is 0.3 mL / min;
[0050] The injection volume is 1 μL;
[0051] The elution mode of liquid chromatography is gradient elution, and the specific elution program is as follows:
[0052] From 0 to 2 min, 99% mobile phase A and 1% mobile phase B;
[0053] From 2 to 6 min, 99% → 0% mobile phase A and 1% → 100% mobile phase B;
[0054] From 6 to 8 min, 0% mobile phase A and 100% mobile phase B;
[0055] From 8 to 8.1 min, 0% → 99% mobile phase A and 100% → 1% mobile phase B;
[0056] From 8.1 to 10 min, 99% mobile phase A and 1% mobile phase B.
[0057] The mass spectrometry conditions are as follows:
[0058] The capillary voltage is 3 kV, the source compensation electrophoresis is 50 V, the ion source temperature is 150 °C, the desolvation temperature is 500 °C, the cone hole gas flow is 150 L / Hr, and the desolvation gas flow is 1000 L / Hr.
[0059] The detected polarity of paclitaxel is +, the parent ion (m / z) is 854.37, and the daughter ions (m / z) are 286.2 / 509.1, the declustering voltage is 10 V, and the collision energy is 18 / 17 V. Among them, represents the quantitative ion.
[0060] The detection method for the total anthocyanin content is as follows:
[0061] Determination principle: Anthocyanins are red in acidic solutions, and the depth of their color is proportional to the concentration of anthocyanins. The absorption peak wavelength of the anthocyanin acidic solution is 530 nm, and the molar extinction coefficient is 4.62×10 4 , so the spectrophotometric method can be used to determine its content. However, chlorophyll often exists in some extracts, interfering with the determination. Therefore, it is necessary to simultaneously measure the optical density values of the extract at wavelengths of 620 nm (soluble sugar) and 650 nm (absorption value of chlorophyll), and accurately calculate the optical density value of anthocyanins using the Greey formula before calculating the content of anthocyanins.
[0062] Experimental steps:
[0063] a. Sample extraction
[0064] Weigh 0.05 - 1 g of the tissue to be tested and place it in a centrifuge tube. Add 10 mL of 0.1 mol / L hydrochloric acid ethanol solution, tighten the tube mouth, extract it in a water bath at 60 °C for 30 min, pour the extract into a 25 mL volumetric flask, add another 5 mL of 0.1 mol / L hydrochloric acid ethanol solution to extract for 15 min, pour the extract into the 25 mL volumetric flask, add another 5 mL of 0.1 mol / L hydrochloric acid ethanol solution to extract for 15 min, and pour the extract into the 25 mL volumetric flask. The total extraction time is 1 h. Rinse the residue and make up the volume to 25 mL to obtain the test solution.
[0065] b. Content determination
[0066] Using 0.1 mol / L hydrochloric acid ethanol solution as the reference solution, take the test solution and measure the optical density values of the extract at wavelengths of 530 nm, 620 nm, and 650 nm with a spectrophotometer, calculate the optical density value of anthocyanin, and then calculate the content of anthocyanin.
[0067] Example 2 Influence of planting environment on the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz
[0068] In this example, different amounts of phosphate fertilizer were added during the process of planting Taraxacum kok-saghyz seeds to investigate the influence of phosphate fertilizer on the yields of paclitaxel and its precursor substances, as follows:
[0069] Take phosphorus-free 1 / 2 MS culture solution and add 10 μM and 600 μM potassium dihydrogen phosphate respectively for standby;
[0070] Directly sow Taraxacum kok-saghyz seeds in moist soil. After germination and growth for 1 month, continuously irrigate and culture with phosphorus-free 1 / 2 MS culture solution containing 10 μM potassium dihydrogen phosphate and phosphorus-free 1 / 2 MS culture solution containing 600 μM potassium dihydrogen phosphate for 1 month. Irrigate 100 mL per plant per day, and the daily irrigation amount per plant is 60 μmol (equivalent to 8 mg of potassium dihydrogen phosphate). Take pictures as Figure 5 shown, and then take the roots and above-ground parts respectively to detect the contents of 10-deacetylbaccatin III and paclitaxel. The results are as Figure 6 shown.
[0071] From Figure 5 it can be seen that the leaves of Taraxacum kok-saghyz irrigated with phosphorus-free 1 / 2 MS culture solution containing 600 μM potassium dihydrogen phosphate for 1 month are significantly larger than those of Taraxacum kok-saghyz treated with phosphorus-free 1 / 2 MS culture solution containing 10 μM potassium dihydrogen phosphate for one month.
[0072] From Figure 6It can be seen that the contents of 10-deacetylbaccatin III and paclitaxel in Taraxacum kok-saghyz plants irrigated with a phosphorus-free 1 / 2MS culture solution containing 600 μM potassium dihydrogen phosphate for 1 month are significantly higher than those in Taraxacum kok-saghyz plants irrigated with a phosphorus-free 1 / 2MS culture solution containing 10 μM potassium dihydrogen phosphate for 1 month, indicating that the application of phosphate fertilizer can increase the contents of 10-deacetylbaccatin III and paclitaxel in Taraxacum kok-saghyz plants.
[0073] In summary, it can be seen that during the cultivation of Taraxacum kok-saghyz, increasing the application rate of phosphate fertilizer can promote the growth of Taraxacum kok-saghyz and increase the contents of 10-deacetylbaccatin III and paclitaxel in Taraxacum kok-saghyz plants.
[0074] Among them, the detection method for the content of paclitaxel is the same as that in Example 1;
[0075] The detection method for the content of 10-deacetylbaccatin III is as follows:
[0076] Take 10-deacetylbaccatin III and add methanol to prepare standard curve solutions with final concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, and 10000 ng / mL.
[0077] Take each standard solution for liquid chromatography-tandem mass spectrometry detection, obtain the chromatogram corresponding to each standard solution and the peak area of 10-deacetylbaccatin III in the chromatogram, and use the concentration of 10-deacetylbaccatin III in each standard solution and its corresponding peak area of 10-deacetylbaccatin III to plot the standard curve of 10-deacetylbaccatin III.
[0078] Place the tissue to be tested in liquid nitrogen for grinding and pulverization, accurately weigh 1 g of the obtained sample, add 10 mL of methanol, ultrasonicate in an ice-water bath for 30 min, centrifuge at 11000 rpm for 30 min to obtain the supernatant and precipitate; add 5 mL of methanol to the precipitate for extraction again, combine the supernatants, add 70 mg of C18 (octadecylsilyl-bonded silica gel) and 40 mg of GCB (graphitized carbon black), centrifuge at 4 °C and 5000 rpm for 10 min, take the supernatant, dry it under nitrogen, and then redissolve it in 400 μL of methanol, and filter it through a 0.22 μm organic phase filter membrane to obtain the test solution.
[0079] Take the test sample solution for liquid chromatography - tandem mass spectrometry detection to obtain the chromatogram of the test sample solution and the peak area of 10 - deacetylbaccatin III contained therein. Substitute the peak area of 10 - deacetylbaccatin III into the standard curve of 10 - deacetylbaccatin III to calculate the content of 10 - deacetylbaccatin III in the test sample solution, and then calculate the content of 10 - deacetylbaccatin III in the tissue to be tested.
[0080] Among them, the chromatographic conditions for liquid chromatography - tandem mass spectrometry detection are as follows:
[0081] The chromatographic column is waters ACQUITY UPLC BEH HSS T3 1.8um 2.1╳100mm;
[0082] The column temperature is 30 °C;
[0083] Mobile phase A is an aqueous solution of 0.1% formic acid, and mobile phase B is acetonitrile;
[0084] The flow rate is 0.3 mL / min;
[0085] The injection volume is 1 μL;
[0086] The elution mode of liquid chromatography is gradient elution, and the specific elution procedure is as follows:
[0087] 0 - 2 min, 99% mobile phase A, 1% mobile phase B;
[0088] 2 - 6 min, 99% → 0% mobile phase A, 1% → 100% mobile phase B;
[0089] 6 - 8 min, 0% mobile phase A, 100% mobile phase B;
[0090] 8 - 8.1 min, 0% → 99% mobile phase A, 100% → 1% mobile phase B;
[0091] 8.1 - 10 min, 99% mobile phase A, 1% mobile phase B.
[0092] The mass spectrometry conditions are as follows:
[0093] The capillary voltage is 3 kV, the source compensation electrophoresis is 50 V, the ion source temperature is 150 °C, the desolvation temperature is 500 °C, the cone hole gas flow is 150 L / Hr, and the desolvation gas flow is 1000 L / Hr.
[0094] The polarity of the detected 10 - deacetylbaccatin III is +, the parent ion (m / z) is 545.2, and the daughter ions (m / z) are 121.2 / 527.1, the declustering voltage is 40 V, and the collision energy is 20 / 10 V. Among them, Represent quantitative ions.
[0095] Example 3 A method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0096] This example provides a method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, which are specifically as follows:
[0097] I. Method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0098] Sow Taraxacum kok-saghyz seeds directly in moist soil. After germination and growth for 1 month, apply 8 mg of potassium dihydrogen phosphate to each plant daily to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and taxol.
[0099] II. Application
[0100] Extract 10-deacetylbaccatin III and taxol from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and taxol.
[0101] Among them, the method for extracting taxol can be to place Taraxacum kok-saghyz in a flask, add an appropriate amount of methanol, cover the flask with a stopper, place it on a constant temperature water bath, soak at 70 ± 5 °C for 12 - 20 hours, filter the soaking solution into an Erlenmeyer flask, wash the residue with methanol more than three times until the washing solution is colorless, combine the extraction solutions, and blow the extraction solutions to near dryness under nitrogen blowing conditions to obtain taxol.
[0102] The method for extracting 10-deacetylbaccatin III and taxol can also be to take Taraxacum kok-saghyz, add an appropriate amount of methanol, perform ultrasonic treatment in an ice-water bath, centrifuge to obtain supernatant and precipitate; repeat the extraction once for the precipitate by adding an appropriate amount of methanol, combine the supernatants, add C18 (octadecylsilyl-bonded silica gel) and GCB (graphitized carbon black), centrifuge, take the supernatant, and dry it under nitrogen to obtain 10-deacetylbaccatin III and taxol.
[0103] Similarly, 10-deacetylbaccatin III and / or taxol can also be obtained by other extraction methods.
[0104] Example 4 A method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0105] This example provides a method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, which are specifically as follows:
[0106] I. Method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0107] Sow the seeds of Taraxacum kok-saghyz directly in moist soil. After germination and growth for 0.5 months, apply 0.1 mg of potassium dihydrogen phosphate per plant per day to obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.
[0108] II. Application
[0109] Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz separately to obtain 10-deacetylbaccatin III and paclitaxel.
[0110] Example 5 A method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application
[0111] This example is a method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is as follows:
[0112] I. Method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz
[0113] Sow the seeds of Taraxacum kok-saghyz directly in moist soil. After germination and growth for 2 months, apply 4 mg of potassium dihydrogen phosphate per plant per day to obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.
[0114] II. Application
[0115] Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz separately to obtain 10-deacetylbaccatin III and paclitaxel.
[0116] Example 6 A method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application
[0117] This example is a method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application, which is as follows:
[0118] I. Method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz
[0119] Sow the seeds of Taraxacum kok-saghyz directly in moist soil. After germination and growth for 1 month, apply 136 mg of potassium dihydrogen phosphate per plant per day to obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.
[0120] II. Application
[0121] Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz separately to obtain 10-deacetylbaccatin III and paclitaxel.
[0122] Example 7 A method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz and its application
[0123] This example is a method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, which are as follows:
[0124] I. Method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0125] Sow Taraxacum kok-saghyz seeds directly in moist soil. After germination and growth for 1 month, apply 1.3 mg of potassium dihydrogen phosphate per plant per day to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and taxol.
[0126] II. Application
[0127] Extract 10-deacetylbaccatin III and taxol in Taraxacum kok-saghyz separately to obtain 10-deacetylbaccatin III and taxol.
[0128] Example 8 A method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0129] This example is a method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, which are as follows:
[0130] I. Method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0131] Sow Taraxacum kok-saghyz seeds directly in moist soil. After germination and growth for 1 month, apply 50 mg of phosphate fertilizer per plant per day to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and taxol.
[0132] II. Application
[0133] Extract 10-deacetylbaccatin III and taxol in Taraxacum kok-saghyz separately to obtain 10-deacetylbaccatin III and taxol.
[0134] Example 9 A method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0135] This example is a method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, which are as follows:
[0136] I. Method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0137] Sow Taraxacum kok-saghyz seeds directly in moist soil. After germination and growth for 1 month, apply 100 mg of phosphorus-containing culture solution per plant per day to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and taxol.
[0138] II. Application
[0139] Extract 10 - deacetylbaccatin III and paclitaxel from Taraxacum kok - saghyz respectively to obtain 10 - deacetylbaccatin III and paclitaxel.
[0140] Example 10 A Method and Application for Increasing the Yield of Paclitaxel and Its Precursors in Taraxacum kok - saghyz
[0141] This example is a method and application for increasing the yield of paclitaxel and its precursors in Taraxacum kok - saghyz, which are as follows:
[0142] I. Method for Increasing the Yield of Paclitaxel and Its Precursors in Taraxacum kok - saghyz
[0143] Sow Taraxacum kok - saghyz seeds directly in moist soil. After germination and growth for 1 month, apply 16 mg of phosphorus - containing culture solution per plant per day to obtain Taraxacum kok - saghyz with higher contents of 10 - deacetylbaccatin III and paclitaxel.
[0144] II. Application
[0145] Extract 10 - deacetylbaccatin III and paclitaxel from Taraxacum kok - saghyz respectively to obtain 10 - deacetylbaccatin III and paclitaxel.
[0146] Example 11 A Method and Application for Increasing the Yield of Paclitaxel and Its Precursors in Taraxacum kok - saghyz
[0147] This example is a method and application for increasing the yield of paclitaxel and its precursors in Taraxacum kok - saghyz, which are as follows:
[0148] I. Method for Increasing the Yield of Paclitaxel and Its Precursors in Taraxacum kok - saghyz
[0149] Apply 0.1 mg of potassium dihydrogen phosphate per plant equivalent in moist soil, and then sow Taraxacum kok - saghyz seeds directly to obtain Taraxacum kok - saghyz with higher contents of 10 - deacetylbaccatin III and paclitaxel.
[0150] II. Application
[0151] Extract 10 - deacetylbaccatin III and paclitaxel from Taraxacum kok - saghyz respectively to obtain 10 - deacetylbaccatin III and paclitaxel.
[0152] Example 12 A Method and Application for Increasing the Yield of Paclitaxel and Its Precursors in Taraxacum kok - saghyz
[0153] This example is a method and application for increasing the yield of paclitaxel and its precursors in Taraxacum kok - saghyz, which are as follows:
[0154] I. Method for Increasing the Yield of Paclitaxel and Its Precursors in Taraxacum kok - saghyz
[0155] Apply 8 mg of potassium dihydrogen phosphate per plant in moist soil, then directly sow the seeds of Taraxacum kok-saghyz, and obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.
[0156] II. Application
[0157] Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.
[0158] Example 13 A method and application for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz
[0159] This example is a method and application for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz, which are specifically as follows:
[0160] I. Method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz
[0161] Apply 136 mg of potassium dihydrogen phosphate per plant in moist soil, then directly sow the seeds of Taraxacum kok-saghyz, and obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.
[0162] II. Application
[0163] Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.
[0164] Example 14 A method and application for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz
[0165] This example is a method and application for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz, which are specifically as follows:
[0166] I. Method for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz
[0167] Apply 50 mg of phosphorus-containing culture solution per plant in moist soil, then directly sow the seeds of Taraxacum kok-saghyz, and obtain Taraxacum kok-saghyz with higher contents of 10-deacetylbaccatin III and paclitaxel.
[0168] II. Application
[0169] Extract 10-deacetylbaccatin III and paclitaxel from Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and paclitaxel.
[0170] Example 15 A method and application for increasing the yields of paclitaxel and its precursor substances in Taraxacum kok-saghyz
[0171] This example is a method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, which are as follows:
[0172] I. Method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0173] Apply nutrient solution containing 100 mg of phosphorus per plant to moist soil, and then directly sow the seeds of Taraxacum kok-saghyz to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and taxol.
[0174] II. Application
[0175] Extract 10-deacetylbaccatin III and taxol in Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and taxol.
[0176] Example 16 A method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0177] This example is a method and application for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz, which are as follows:
[0178] I. Method for increasing the yields of taxol and its precursor substances in Taraxacum kok-saghyz
[0179] Apply potassium dihydrogen phosphate equivalent to 4 mg per plant to moist soil, and then directly sow the seeds of Taraxacum kok-saghyz to obtain Taraxacum kok-saghyz with relatively high contents of 10-deacetylbaccatin III and taxol.
[0180] II. Application
[0181] Extract 10-deacetylbaccatin III and taxol in Taraxacum kok-saghyz respectively to obtain 10-deacetylbaccatin III and taxol.
[0182] Obviously, the described examples are only a part of the examples of the present invention, rather than all examples. All other examples obtained by those of ordinary skill in the art based on the examples in the present invention without creative efforts shall fall within the protection scope of the present invention.
Claims
1. Application of Taraxacum kok-saghyz producing paclitaxel, characterized in that, The application is to obtain Taraxacum kok-saghyz producing taxol by planting Taraxacum kok-saghyz under phosphorus stress conditions, and extracting 10-deacetylbaccatin III and / or taxol from the Taraxacum kok-saghyz producing taxol to obtain 10-deacetylbaccatin III and / or taxol; The phosphorus stress condition means that the amount of phosphate fertilizer applied per plant per day is 1.3 - 136 mg.
2. A method for increasing the yield of taxol and its precursor substances in Taraxacum kok-saghyz, characterized in that, The method is to plant Taraxacum kok-saghyz under phosphorus stress conditions to increase the yields of taxol and its precursor substances in Taraxacum kok-saghyz; The phosphorus stress condition means that the amount of phosphate fertilizer applied per plant per day is 1.3 - 136 mg.
3. The method for increasing the yield of taxol and its precursor substances in Taraxacum kok-saghyz Rodin according to claim 2, characterized in that, The method is to plant Taraxacum kok-saghyz and apply phosphate fertilizer to increase the yields of taxol and its precursor substances in Taraxacum kok-saghyz.
4. The method for increasing the yield of taxol and its precursor substances in Taraxacum kok-saghyz Rodin according to claim 2, characterized in that, The method is to plant Taraxacum kok-saghyz in soil applied with phosphate fertilizer to increase the yields of taxol and its precursor substances in Taraxacum kok-saghyz.
5. An application of Taraxacum kok-saghyz, characterized in that, The application is to extract 10-deacetylbaccatin III and / or taxol from the Taraxacum kok-saghyz obtained by the method according to any one of claims 2 - 4 to obtain 10-deacetylbaccatin III and / or taxol.