Taxol and extraction method and use

Abstract

The application provides a taxol and a preparation method and application thereof, and the extraction method of the taxol comprises the following steps: taking a yew leaf stalk, a yew flower or a yew root as an explant, inducing to form a callus, inducing differentiation of the obtained callus to obtain yew adventitious roots, and extracting taxol from the yew adventitious roots. The application successfully establishes a complete culture system from yew explants to scale-up of adventitious roots for the first time, does not rely on felling wild yew, realizes sustainable production of taxol raw materials, and solves the resource bottleneck problem of taxol production from the source.

Description

Technical Field

[0001] This invention relates to the field of plant extraction technology, specifically to a paclitaxel, its extraction method, and its applications. Background Technology

[0002] Paclitaxel is a pentacyclic triterpenoid compound derived from the yew tree and is a broad-spectrum anticancer drug widely used in clinical practice. In recent years, research has also discovered its potential applications in antibacterial and anti-inflammatory properties. However, traditional paclitaxel production methods primarily rely on direct extraction from the bark of wild yew trees. Yew trees are slow-growing and scarce resources; this method not only results in a low extraction rate, typically only 0.01-0.05% of the bark's dry weight, but also causes severe damage to wild resources and the ecological environment.

[0003] To address the issue of paclitaxel source, researchers have explored various alternative methods, such as yew cell culture and hairy root culture. For instance, patent CN103651139A discloses a method to increase paclitaxel content by inducing callus redifferentiation in *Taxus wallichiana*. While this method can increase paclitaxel content in callus to some extent, callus is prone to genetic variation during long-term subculture, leading to unstable production of secondary metabolites. Furthermore, this method remains at the callus stage and has not yet established a stable, scalable culture system.

[0004] While existing technologies attempt to obtain paclitaxel from yew cell culture or from hairy roots transformed by Agrobacterium rhizogenes, problems such as unstable culture systems, low yield of secondary metabolites, high production costs, and susceptibility to contamination exist, making it difficult to achieve large-scale production.

[0005] Furthermore, current research on paclitaxel largely focuses on its anti-tumor effects, while its potential in antibacterial and anti-inflammatory applications has not been systematically explored. There is a lack of clear activity validation data, effective concentrations, and application protocols, which fails to provide reliable support for the development of antibacterial and anti-inflammatory drugs. Therefore, the existing technology requires further development. Summary of the Invention

[0006] To address the shortcomings of existing technologies and solve the aforementioned problems, a method for preparing paclitaxel and its applications are proposed, and the following technical solution is provided: A method for extracting paclitaxel includes the following steps: using yew petioles, yew flowers, or yew roots as explants to induce callus formation; inducing the obtained callus to differentiate into adventitious roots of yew; and extracting paclitaxel from the adventitious roots of yew.

[0007] Furthermore, the preparation process of the adventitious roots of Taxus chinensis specifically involves: transferring the callus tissue to an adventitious root induction medium containing NAA and IBA to induce adventitious root differentiation; and transferring the differentiated adventitious root segments into a liquid culture medium to achieve adventitious root amplification.

[0008] Furthermore, the NAA concentration in the root-establishing induction medium is 1-2 mg / L, and the IBA concentration is 0.3-0.7 mg / L.

[0009] Furthermore, the differentiated adventitious root segments were transferred to liquid culture medium and cultured with shaking at 110-130 r / min for 20-40 days to achieve the amplification of adventitious roots.

[0010] Furthermore, the adventitious roots of the yew were pulverized, and paclitaxel was extracted using an ionic liquid-ultrasound-assisted extraction method.

[0011] Furthermore, the ionic liquid-ultrasound-assisted extraction method is as follows: pulverized adventitious roots of Taxus chinensis are added to an extractant and ultrasonically extracted to obtain paclitaxel. The extractant is an aqueous solution of 1-butyl-3-methylimidazolium trichloroferric bromide and methanol, wherein the mass concentration of 1-butyl-3-methylimidazolium trichloroferric bromide in the extractant is 1.0-1.2%, and the liquid-to-solid ratio of the extractant to the pulverized adventitious roots of Taxus chinensis is 10.5:1.

[0012] Furthermore, the extraction temperature was 40℃, the ultrasonic power was 150-250W, the ultrasonic time was 25-35 minutes, and the extraction was performed twice, with the two extracts combined.

[0013] Furthermore, the explants are disinfected before inducing callus formation. The disinfection process is as follows: first soak in 75% ethanol for 50-60 seconds, then soak in 0.1% mercuric chloride for 7-8 minutes, and finally rinse with sterile water 4-5 times.

[0014] In addition, this application also provides paclitaxel obtained by the above extraction method.

[0015] This application also provides the use of paclitaxel in the preparation of antibacterial drugs.

[0016] Beneficial effects: 1. This invention is the first to successfully establish a complete culture system from yew explants to large-scale amplification of adventitious roots, without relying on logging of wild yew trees, thus realizing the sustainable production of paclitaxel raw materials and solving the resource bottleneck problem of paclitaxel production from the source.

[0017] 2. This invention optimizes the key conditions for adventitious root induction and amplification, with an adventitious root induction rate of up to 82.3%. After 40 days of liquid culture, the biomass can increase to more than 9 times the initial inoculum, which is significantly better than traditional callus culture or cell suspension culture.

[0018] 3. This invention applies ionic liquid-ultrasound-assisted extraction technology to adventitious root materials, obtaining high-quality paclitaxel with an extraction rate ≥0.235 mg / g dry weight and a purity ≥99.2%. This invention is the first to systematically verify the significant antibacterial and anti-inflammatory activities of paclitaxel produced by adventitious root culture technology, clarify its minimum inhibitory concentration against various Gram-positive bacteria and its inhibitory effect on arthritis in CIA mice, opening up new avenues for the pharmaceutical application of paclitaxel beyond anti-tumor therapy.

[0019] 4. The adventitious root culture system was verified by repeated experiments. The coefficients of variation for induction rate and biomass growth rate were both less than 5%; the coefficients of variation for paclitaxel extraction rate and purity were less than 3%; the antibacterial and anti-inflammatory activity experiments were repeated 3 times, and the results were consistent, indicating that the process of this invention is stable and the effect is reliable. Attached Figure Description

[0020] Figure 1 This is a comparison diagram of the inhibition zones of paclitaxel sample (100 μg / mL), blank solvent control, and chloramphenicol positive control (10 μg / mL) against Staphylococcus aureus in Example 3 of the present invention. Figure 2 This is a quantitative comparison chart of the antibacterial activity of paclitaxel in Example 3 of the present invention. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Based on the embodiments in this application, other similar embodiments obtained by those skilled in the art without creative effort should all fall within the scope of protection of this application.

[0022] According to embodiments of the present invention, paclitaxel, its preparation method, and its applications are provided. The extraction steps of paclitaxel include: using yew petioles, yew flowers, or yew roots as explants to induce callus formation; inducing the differentiation of the obtained callus to obtain adventitious roots of yew; and extracting paclitaxel from the adventitious roots of yew. This invention successfully establishes for the first time a complete culture system for large-scale expansion from yew explants to adventitious roots, without relying on the felling of wild yew trees, achieving sustainable production of paclitaxel raw materials and solving the resource bottleneck problem of paclitaxel production at its source.

[0023] Example 1 Induction and large-scale cultivation of adventitious roots of Taxus chinensis 1. Materials and Methods Materials: Explants were selected from the petioles of healthy 3-year-old Taxus chinensis plants, with a petiole length of 2-3 cm. Flowers were fresh, unopened buds. Roots were lateral roots with a diameter of 0.2-0.3 cm. MS medium (commercially available), 2,4-D, 6-BA, NAA, IBA (commercially available, purity ≥98%), 75% ethanol, 0.1% mercuric chloride (commercially available analytical grade). Instruments included a clean bench, incubator, shaker, electronic balance, and grinder.

[0024] Explant disinfection: Rinse the selected explants with tap water for 30 minutes to remove surface dust and impurities; transfer them to a clean bench and soak them in 75% ethanol for 55 seconds (petioles, flowers) or 60 seconds (roots), then rinse twice with sterile water; then soak them in 0.1% mercuric chloride for 7.5 minutes (petioles, flowers) or 8 minutes (roots), then rinse four times with sterile water, and blot dry the surface moisture with sterile filter paper for later use; divide the petioles, flowers and roots into 3 replicate groups, with 50 explants treated in each group, and calculate the disinfection effect (number of explants free from bacterial contamination after disinfection / total number of explants × 100%).

[0025] Callus induction: Sterilized explants were inoculated into callus induction medium (MS + 2.0 mg / L 2,4-D + 1.0 mg / L 6-BA, pH 5.8, with 0.8% agar added, autoclaved at 121℃ for 20 min), with 3 explants per bottle; incubated in a constant temperature incubator at 25±2℃ for 28 days in the dark, with 3 replicates, 30 bottles per group; after culture, the callus induction rate was calculated as (number of explants forming callus / number of inoculated explants × 100%), and the callus morphology was observed.

[0026] Adventitious root differentiation: Loose-textured, vigorously growing callus tissue (0.8-1.2 cm in diameter) was selected and transferred to adventitious root induction medium (MS medium, supplemented with different concentrations of NAA and IBA, pH 5.8, with 0.8% agar added, and sterilized). Five treatment groups were set up for adventitious root induction medium, with three replicates per group and 30 bottles per group. One piece of callus tissue was inoculated in each bottle. The culture conditions were 25±2℃, diffused light culture (light intensity 1500 lx, light duration 12 h / d), and cultured for 30 days. The adventitious root induction rate (number of callus tissues differentiating adventitious roots / number of inoculated callus tissues × 100%), the number of adventitious roots per callus tissue, and the length of adventitious roots were counted for each group to screen the optimal combination of NAA and IBA.

[0027] The specific treatment groups for the adventitious root induction medium are set up as follows: Group 1: MS + 1.0mg / L NAA + 0.3mg / L IBA Group 2: MS + 1.0mg / L NAA + 0.5mg / L IBA Group 3: MS + 1.5mg / L NAA + 0.5mg / L IBA Group 4: MS + 2.0mg / L NAA + 0.5mg / L IBA Group 5: MS + 1.5mg / L NAA + 0.7mg / L IBA Adventitious root liquid amplification culture: Adventitious roots cultured from the optimal treatment group were selected, cut into 2cm long segments, and inoculated into liquid amplification medium (with the same composition as the adventitious root induction medium of group 3, but without agar). 10g of adventitious root segments were inoculated into each 500mL Erlenmeyer flask. The flasks were placed in a shaker at 120r / min, 25±2℃, and cultured under diffused light (1500lx light intensity, 12h / d light duration) for 40 days. Three replicate groups were set up, with 10 flasks in each group. At 10, 20, 30, and 40 days of culture, samples were taken and the fresh weight of the adventitious roots was weighed. The biomass growth rate was calculated as (current fresh weight / initial fresh weight × 100% - 100%). After the culture was completed, the adventitious roots were harvested, washed, dried at 60℃ to constant weight, pulverized, and passed through an 80-mesh sieve for later use.

[0028] 2. Experimental Results The experimental results of this embodiment are shown in Tables 1-3 below.

[0029] Table 1. Disinfection and callus induction of Taxus chinensis As shown in Table 1, the disinfection effect of the three types of explants was over 98.7%, with the petiole explant showing the best disinfection effect (99.3±0.5%). The callus induction rate was over 83% for all three types of explants, with the root explant showing the highest induction rate (86.0±1.0%), followed by the petiole explant at 85.7±1.2% and the flower explant at 83.3±1.5%. The callus induced by the petiole and root explants showed better morphology and no browning, making them suitable for subsequent adventitious root differentiation. Therefore, the petiole or root was preferred as the explant.

[0030] Table 2. Effects of different concentrations of auxin on the growth of adventitious roots of Taxus chinensis. As shown in Table 2, different combinations of NAA and IBA concentrations had a significant effect on the adventitious root induction rate, number of adventitious roots, and length (P<0.05). Group 3 (NAA 1.5 mg / L + IBA 0.5 mg / L) had the highest adventitious root induction rate (78.0±1.5%), which was significantly higher than other groups. Each callus tissue had the most adventitious roots (9.8±0.7 roots) and the longest adventitious root length (2.5±0.2 cm), exhibiting the best morphology. Therefore, this combination was determined to be the optimal hormone combination for adventitious root induction.

[0031] Table 3. Effects of different cultivation times on the morphology and growth of adventitious roots of Taxus chinensis. As shown in Table 3, the biomass of adventitious roots gradually increased with the culture time during liquid amplification culture, and the growth trend was significant (P<0.01). After 40 days of culture, the biomass increased to 9.2 times the initial inoculum, and the biomass growth rate reached 820.0±15.0%. The adventitious roots grew vigorously, had well-developed root systems, and were free from contamination by other microorganisms, which met the requirements for large-scale production. The excellent morphology of the adventitious roots after liquid amplification could be clearly observed, further verifying the effectiveness of the culture method of this invention.

[0032] Example 2: Extraction and purification of paclitaxel 1. Materials and Methods Materials: Adventitious root powder of Taxus chinensis harvested after 40 days of liquid amplification culture in Example 1 (passed through an 80-mesh sieve), 1-butyl-3-methylimidazolium trichloroferric bromide (ionic liquid, commercially available, purity ≥99%), methanol, chloroform, acetonitrile (commercially available analytical grade), paclitaxel standard (Sigma-Aldrich, purity ≥99.5%); Instruments included ultrasonic extractor, subcritical fluid extractor, silica gel column (100~200 mesh), preparative HPLC, vacuum distillation unit, oven, electronic balance, high performance liquid chromatograph, etc.

[0033] Comparative experiment of two extraction methods: Two extraction methods were set up: ionic liquid-ultrasound-assisted extraction and subcritical fluid extraction. Three replicates were set up for each method. 10g of adventitious root powder was taken from each group to extract paclitaxel. The extraction rates of the two methods were compared. The specific operation is as follows: Method 1: Ionic liquid-ultrasound-assisted extraction: Take 10g of adventitious root powder, add 1.17% of 1-butyl-3-methylimidazolium trichloroferric bromide and 60% methanol aqueous solution (liquid-to-solid ratio 10.5:1, v / w), place in an ultrasonic extractor, and extract at 40℃ and 200W for 30 minutes. Extract twice, and combine the two extracts; concentrate under reduced pressure (50℃, 0.08MPa) to remove the solvent and obtain crude paclitaxel extract for later use.

[0034] Method 2: Subcritical fluid extraction: Take 10g of adventitious root powder and put it into a subcritical fluid extraction vessel. Use carbon dioxide as the extractant and 85% ethanol as the entrainer (the amount of entrainer is 20% of the raw material mass). Set the extraction pressure to 30MPa, the extraction temperature to 47℃, the CO2 flow rate to 25L / h, and the extraction time to 3.5h. Use a combination of static extraction for 1h and dynamic extraction for 2.5h. After extraction, separate the vessel I and II at 30℃ and 6.5MPa. Collect the extract, concentrate under reduced pressure to remove the entrainer, and obtain crude paclitaxel extract for later use.

[0035] Purification: The crude paclitaxel extracts obtained by the two methods were purified separately, as follows: ① Silica gel column chromatography: The crude extract was dissolved in a small amount of chloroform and loaded onto a silica gel column (50 cm long, 2 cm diameter). Chloroform-methanol (9:1 v / v) was used as the eluent at a rate of 1 mL / min. The eluent was collected and detected by thin-layer chromatography (TLC) (developing solvent: chloroform-methanol = 95:5, UV light at 254 nm). The eluents containing paclitaxel were combined and concentrated to dryness under reduced pressure. ② Preparative HPLC purification: The sample purified by silica gel column chromatography was dissolved in methanol to prepare a 1 mg / mL solution and loaded onto a preparative HPLC system (reversed-phase C18 column, 250 mm × 10 mm, 5 μm). The mobile phase was acetonitrile-water (containing 0.1% phosphoric acid) gradient elution (0-5 min 40% acetonitrile, 5-25 min). The column was prepared using 40-80% acetonitrile, with a flow rate of 3.0 mL / min, a column temperature of 30℃, and a detection wavelength of 227 nm. The eluent corresponding to the paclitaxel peak was collected, concentrated under reduced pressure, and freeze-dried to obtain high-purity paclitaxel.

[0036] Extraction rate and purity detection: The extraction rate and purity of paclitaxel were detected using high-performance liquid chromatography (HPLC). The chromatographic conditions were consistent with those of preparative HPLC (flow rate adjusted to 1.0 mL / min, injection volume 20 μL, other conditions unchanged). First, a paclitaxel standard curve was plotted: 10 mg of paclitaxel standard was accurately weighed, dissolved in methanol, and diluted to 100 mL to prepare a 0.1 mg / mL standard stock solution. 0.5, 1.0, 2.0, 4.0, and 8.0 mL of the standard stock solution were respectively pipetted and diluted to 10 mL with methanol to obtain a series of standard solutions with concentrations of 5, 10, 20, 40, and 80 μg / mL. These solutions were injected sequentially for detection. A standard curve was plotted with paclitaxel concentration as the abscissa (X) and peak area as the ordinate (Y). The regression equation (R² ≥ 0.999) was calculated for quantitative calculation of paclitaxel content in the samples.

[0037] Crude paclitaxel extracts and purified concentrates obtained from two extraction methods were dissolved in methanol and diluted to appropriate concentrations. After passing through a 0.22 μm filter membrane, the samples were injected and analyzed under the chromatographic conditions described above. The peak areas were recorded and substituted into the standard curve regression equation to calculate the paclitaxel content in the samples. The extraction rate was calculated as follows: Extraction rate (mg / g dry weight) = Mass of purified paclitaxel concentrate (mg) / Mass of adventitious root powder (g); the purity was calculated as follows: Purity (%) = Paclitaxel peak area / Total peak area of ​​the sample × 100%. All assays were performed in triplicate. Data are expressed as mean ± standard deviation, and significance analysis was performed using SPSS software.

[0038] 2. Experimental Results The comparison results of paclitaxel extraction rate, purity, and crude extract yield of the two extraction methods in this embodiment are shown in Table 4 below: Table 4 Effect of different extraction methods on the extraction rate of Taxus chinensis Table 4 shows that both extraction methods can effectively extract paclitaxel from the adventitious roots of Taxus chinensis, and the purity after purification is ≥99.2%. Among them, the preferred ionic liquid-ultrasound-assisted extraction method of this invention achieves an extraction rate of 0.235±0.006 mg / g dry weight, which is significantly higher than that of the subcritical fluid extraction method (0.229±0.007 mg / g dry weight, P<0.05); and the extraction time is only 1.5 h, much shorter than that of the subcritical fluid extraction method (3.5 h). At the same time, the crude extract yield and paclitaxel content are slightly higher than those of the subcritical fluid extraction method, demonstrating the advantages of high efficiency and time saving.

[0039] Furthermore, the 1-butyl-3-methylimidazolium trichloroferric bromide ionic liquid used in the ionic liquid-ultrasound-assisted extraction method can be recycled and reused (with a recycling rate of over 85%), reducing the consumption of organic solvents and lowering environmental pollution. This aligns with the beneficial effects of the present invention, namely "environmentally friendly process and low cost," making it more suitable for large-scale industrial production.

[0040] Example 3: Verification of the antibacterial and anti-inflammatory activity of paclitaxel 1. Materials and Methods Materials: High-purity paclitaxel (purity 99.3±0.1%) obtained by ionic liquid-ultrasound-assisted extraction in Example 2; Staphylococcus aureus (ATCC 6538), Streptococcus pneumoniae (ATCC 49619), and Bacillus subtilis (ATCC 6633) (all commercially available standard strains); chloramphenicol (positive control, antibacterial); ibuprofen (positive control, anti-inflammatory); LB medium and MH medium; SPF-grade Kunming mice (weight 20±2g, half male and half female); type II collagen (Sigma-Aldrich); Freund's complete adjuvant and Freund's incomplete adjuvant; TNF-α, IL-6, and IL-1β ELISA kits; instruments included a biosafety cabinet, incubator, ELISA reader, electronic balance, and mouse paw edema meter.

[0041] Antibacterial activity verification (in vitro antibacterial experiment): The inhibitory activity of paclitaxel against three Gram-positive bacteria was verified using the agar diffusion method (inhibition zone determination) and the microdilution method (minimum inhibitory concentration MIC determination). Three replicate groups were set up, and the specific operation is as follows: Agar diffusion method: Three standard strains were inoculated into LB liquid medium and cultured at 37℃ with shaking at 180 rpm for 12 h. The bacterial concentration was adjusted to 1×10⁶ CFU / mL. 100 μL of bacterial solution was evenly spread onto MH agar plates. After absorption, sterile Oxford cups (6 mm in diameter) were used to make wells. Different concentrations of paclitaxel solution (25, 50, 100 μg / mL) were added to each well. A positive control group (chloramphenicol, 10 μg / mL) and a blank control group (methanol solvent) were also set up. After incubation at 37℃ for 24 h, the diameter of the inhibition zone (mm) was measured to determine the antibacterial activity (inhibition zone diameter ≥15 mm indicates high sensitivity, 10-14 mm indicates moderate sensitivity, and <10 mm indicates no sensitivity). The comparison of inhibition zones of paclitaxel sample (100 μg / mL), blank solvent control, and chloramphenicol positive control (10 μg / mL) against Staphylococcus aureus is shown in the figure. Figure 1 As shown.

[0042] Microdilution method: Paclitaxel solution was serially diluted with MH medium to 12.5, 25, 50, 100, and 200 μg / mL, and 100 μL was added to each well of a 96-well plate; then 100 μL of bacterial suspension (1×10⁶ CFU / mL) was added. Positive control group (chloramphenicol), blank control group (culture medium), and solvent control group (methanol) were set up. After incubation at 37℃ for 24 h, the OD value was measured using a microplate reader. The lowest paclitaxel concentration (MIC) with an OD value ≤ 50% of the blank control group was defined as the minimum inhibitory concentration. A quantitative comparison of the antibacterial activity of the paclitaxel group (100 μg / mL), paclitaxel group (50 μg / mL), paclitaxel group (25 μg / mL), positive control group, and blank control group is shown in the figure. Figure 2 As shown.

[0043] Anti-inflammatory activity verification (in vivo experiment): Using a collagen-induced arthritis (CIA) mouse model, the in vivo anti-inflammatory activity of paclitaxel was verified. Four groups were set up, with 10 mice in each group. The specific operation is as follows: Model establishment: Type II collagen was dissolved in 0.1 mol / L acetic acid solution (concentration 2 mg / mL), mixed with an equal volume of Freund's complete adjuvant, and fully emulsified to prepare an immunogen; 100 μL of immunogen was subcutaneously injected into the back of each mouse, and a booster immunization was administered intraperitoneally on day 21 (50 μL of immunogen mixed with an equal volume of Freund's incomplete adjuvant per mouse); 7 days after model establishment, the swelling of the paws of the mice was observed to confirm successful model establishment (the degree of paw swelling increased by ≥30% compared with normal mice).

[0044] Grouping and administration: ① Normal control group (no modeling): 10 mL / kg / day of normal saline by gavage; ② Model group (no administration after modeling): 10 mL / kg / day of normal saline by gavage; ③ Paclitaxel treatment group: 5 mg / kg / day of paclitaxel dissolved in normal saline by gavage; ④ Ibuprofen positive control group: 10 mg / kg / day of ibuprofen dissolved in normal saline by gavage. All groups were administered the medication continuously for 14 days at a fixed time each day.

[0045] Index detection: On days 0, 2, 4, 6, 8, 10, 12, and 14 after drug administration, the paw swelling degree (mm³) of mice was measured using a mouse paw swelling meter, and the swelling inhibition rate was calculated (swelling inhibition rate % = (swelling degree of model group - swelling degree of treatment group) / swelling degree of model group × 100%). After 14 days of drug administration, blood was collected from the eyeballs of mice, and serum was collected by centrifugation (3000 r / min, 10 min). The levels of TNF-α, IL-6, and IL-1β in the serum were detected using an ELISA kit, strictly following the instructions of the kit.

[0046] 2. Experimental Results The results of the antibacterial and anti-inflammatory activity verification in this embodiment are shown in Tables 5 and 6 below, which clearly demonstrate the antibacterial and anti-inflammatory effects of paclitaxel: Table 5. Antibacterial activity of different concentrations of paclitaxel against three Gram-positive bacteria. As shown in Table 5, the paclitaxel extracted in this invention has significant inhibitory effects on three Gram-positive bacteria: Staphylococcus aureus, Streptococcus pneumoniae, and Bacillus subtilis, exhibiting a clear concentration-dependent effect (the higher the concentration, the larger the diameter of the inhibition zone). Specifically, at a concentration of 100 μg / mL, the inhibition zone diameter against Staphylococcus aureus reached 15.2 ± 0.3 mm, against Streptococcus pneumoniae 14.8 ± 0.2 mm, and against Bacillus subtilis 15.0 ± 0.3 mm, all ≥ 14.8 mm. The minimum inhibitory concentration (MIC) was 25–50 μg / mL, indicating that paclitaxel possesses good in vitro antibacterial activity.

[0047] Table 6. Anti-inflammatory effects of paclitaxel on mice in different groups As shown in Table 6, the paw swelling of mice in the model group was significantly increased (172±6 mm). 3The levels of TNF-α, IL-6, and IL-1β in the serum were significantly higher than those in the normal control group (P<0.01), indicating that the CIA mouse model was successfully established. After 14 days of continuous administration of paclitaxel, the paw swelling of mice decreased to 105±4 mm3, with a swelling inhibition rate of 38.7±1.5%, ≥35%; the serum levels of TNF-α, IL-6, and IL-1β decreased to 298±10 pg / mL, 217±8 pg / mL, and 199±7 pg / mL, respectively, representing decreases of 38.7%, 38.3%, and 38.8% compared to the model group, all ≥38%.

[0048] In mice treated with paclitaxel, paw swelling gradually decreased with increasing administration time, reaching an inhibition rate of 38.7% on day 14, an effect close to that of the ibuprofen positive control group. Serum inflammatory factor levels also decreased significantly, further confirming the good in vivo anti-inflammatory activity of paclitaxel. In summary, the paclitaxel extracted in this invention exhibits both significant antibacterial and anti-inflammatory activities.

[0049] Through experiments in Examples 1-3, the adventitious root culture method of Taxus chinensis established in this invention can achieve efficient induction of adventitious roots (induction rate ≥78%) and large-scale expansion (biomass increase of 8.2-9.2 times). Using ionic liquid-ultrasound-assisted extraction, paclitaxel with high extraction rate (0.235±0.006 mg / g dry weight) and high purity (≥99.2%) can be obtained. The extracted paclitaxel has significant antibacterial activity against a variety of Gram-positive bacteria and has a good alleviating effect on inflammation in vivo.

[0050] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for extracting paclitaxel, characterized in that the steps include... include: Using yew petioles, yew flowers, or yew roots as explants, callus tissue was induced to form. The obtained callus tissue was then induced to differentiate into adventitious roots of yew, from which paclitaxel was extracted.

2. The method for extracting paclitaxel according to claim 1, characterized in that, The specific preparation process of the adventitious roots of Taxus chinensis is as follows: the callus tissue is transferred to an adventitious root induction medium containing NAA and IBA to induce adventitious root differentiation; the differentiated adventitious root segments are transferred to a liquid culture medium to achieve the amplification of adventitious roots.

3. The method for extracting paclitaxel according to claim 2, characterized in that, The root-establishing induction medium contains NAA concentrations of 1-2 mg / L and IBA concentrations of 0.3-0.7 mg / L.

4. The method for extracting paclitaxel according to claim 2, characterized in that, The differentiated adventitious root segments were transferred into liquid culture medium and cultured with shaking at 110-130 r / min for 20-40 days to achieve the amplification of adventitious roots.

5. The method for extracting paclitaxel according to claim 1, characterized in that, The adventitious roots of Taxus chinensis were crushed, and paclitaxel was extracted using an ionic liquid-ultrasound-assisted extraction method.

6. The method for extracting paclitaxel according to claim 5, characterized in that, The ionic liquid-ultrasound-assisted extraction method is as follows: pulverized adventitious roots of Taxus chinensis are added to an extractant and ultrasonically extracted to obtain paclitaxel. The extractant is an aqueous solution of 1-butyl-3-methylimidazolium trichloroferric bromide and methanol, wherein the mass concentration of 1-butyl-3-methylimidazolium trichloroferric bromide in the extractant is 1.0-1.2%, and the liquid-to-solid ratio of the extractant to the pulverized adventitious roots of Taxus chinensis is 10.5:

1.

7. The method for extracting paclitaxel according to claim 6, characterized in that, The extraction temperature was 40℃, the ultrasonic power was 150-250W, the ultrasonic time was 25-35 minutes, and the extraction was performed twice. The two extracts were then combined.

8. The method for extracting paclitaxel according to claim 1, characterized in that, Before inducing callus formation, the explants are disinfected. The disinfection process is as follows: first soak in 75% ethanol for 50-60 seconds, then soak in 0.1% mercuric chloride for 7-8 minutes, and finally rinse with sterile water 4-5 times.

9. Paclitaxel obtained by the extraction method according to any one of claims 1-8.

10. The application of paclitaxel according to claim 9, characterized in that, Applications in the preparation of antibacterial drugs.

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