Functionalized nano cellulose double-template imprinted aerogel microsphere for the targeted enrichment of taxanes.

Paclitaxel, a diterpenoid isolated from the bark of Taxus wallichiana var. chinensis (Pilger) Florin, is currently showing significant therapeutic effects against a variety of cancers. Baccatin III (Bac) and 10-Deacetylbaccatin III (10-DAB) are in great demand as important precursors for the synthesis of paclitaxel. This work aims to develop a simple, rapid and highly selective, safe, and non-polluting molecularly imprinted material for 10-DAB and Bac enrichment. In this study, we innovatively prepared molecularly imprinted materials with nanocellulose aerogel microspheres and 2-vinylpyridine (2-VP) as a bifunctional monomer, and 10-DAB and Bac as bis-template molecules. In particular, functionalized nanocellulose dual-template molecularly imprinted aerogel microsphere (FNCAG-DMIM) were successfully synthesized by the bifunctional introduction of functional nanocellulose aerogel microsphere (FNCAG) modified with Polyethyleneimine (PEI) as a carrier and functional monomer, which provided a large number of recognition sites for bimodal molecules. FNCAG-DMIM showed high specificity for 10-DAB and Bac specific assays. Under the optimal experimental conditions, the adsorption capacities of FNCAG-DMIM for 10-DAB and Bac reached 52.27 mg g-1 and 53.81 mg g-1, respectively. In addition, it showed good reliability and practicality in the determination of real samples. The present study extends the research on the synthesis of natural functional monomers by molecularly imprinted materials and opens up new horizons for the targeted isolation of plant compounds by dual-template molecularly imprinted materials.

An Integrated Strategy Based on 10-DAB Extraction and In Situ Whole-Cell Biotransformation of Renewable Taxus Needles to Produce Baccatin III.

Baccatin III is a crucial precursor in the biosynthesis pathway of paclitaxel. Its main sources are extraction from Taxus or chemical synthesis using 10-deacetylbaccatin III (10-DAB) as substrate. However, these preparation approaches exhibit serious limitations, including the low content of baccatin III in Taxus and the complicated steps of chemical synthesis. Heterologous expression of 10-deacetylbaccatin III-10-O-acetyltransferase (TcDBAT) in microbial strains for biotransformation of 10-DAB is a promising alternative strategy for baccatin III production. Here, the promotion effects of glycerol supply and slightly acidic conditions with a low-temperature on the catalysis of recombinant TcDBAT strain were clarified using 10-DAB as substrate. Taxus needles is renewable and the content of 10-DAB is relatively high, it can be used as an effective source of the catalytic substrate 10-DAB. Baccatin III was synthesized by integrating the extraction of 10-DAB from renewable Taxus needles and in situ whole-cell catalysis in this study. 40 g/L needles were converted into 20.66 mg/L baccatin III by optimizing and establishing a whole-cell catalytic bioprocess. The method used in this study can shorten the production process of Taxus extraction for baccatin III synthesis and provide a reliable strategy for the efficient production of baccatin III by recombinant strains and the improvement of resource utilization rate of Taxus needles.

A nanocellulose molecularly imprinted membrane: Preparation, characterization and application in targeted separation of taxane 10-deacetylbaccatin III.

Targeted separation of active phytochemicals is urgently needed in the natural medicine field. In this paper, due to the natural porosity and high biocompatibility of cellulose, a nanocellulose membrane combined with surface molecular imprinting was successfully prepared; the efficient nanocellulose-based molecular imprinted membrane (NC-MIM) provided good adsorption for the targeted separation of phytochemicals such as 10-deacetylbaccatin III (10-DAB), an essential intermediate in the synthesis of the anticancer drug paclitaxel. Through a series of characterization and adsorption experiments, the adsorption mechanism of NC-MIM was determined. At pH 8.0 and temperatures of 20 °C-40 °C, the maximum capacity of NC-MIM for adsorption of 10-DAB reached 66.90 mg g - 1, and the content of 10-DAB was dramatically increased 17.5-fold after adsorption. The specific adsorption results showed that NC-MIM had excellent capacity for targeted separation of 10-DAB from among taxane structural analogues. Even after ten cycles, NC-MIM demonstrated a remarkable adsorption capacity of 86.43%, thereby indicating exceptional selectivity and stability. The successful implementation of NC-MIM for green, safe, and efficient enrichment of phytochemicals from plants provides a promising new approach and valuable insights into its practical application.

Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin III to baccatin III.

10-Deacetylbaccatin III (10-DAB) C10 acetylation is an indispensable procedure for Taxol semi-synthesis, which often requires harsh conditions. 10-Deacetylbaccatin III-10-ß-O-acetyltransferase (DBAT) catalyzes the acetylation but acetyl-CoA supply remains a key limiting factor. Here we refactored the innate biosynthetic pathway of acetyl-CoA in Escherichia coli and obtained a chassis with acetyl-CoA productivity over three times higher than that of the host cell. Then, we constructed a microbial cell factory by introducing DBAT gene into this chassis for efficiently converting 10-DAB into baccatin III. We found that baccatin III could be efficiently deacetylated into 10-DAB by DBAT with CoASH and K+ under alkaline condition. Thus, we fed acetic acid to the engineered strain both for serving as a substrate of acetyl-CoA biosynthesis and for alleviating the deacetylation of baccatin III. The fermentation conditions were optimized and the baccatin III titers reached 2, 3 and 4.6 g/L, respectively, in a 3-L bioreactor culture when 2, 3 and 6 g/L of 10-DAB were supplied. Our study provides an environment-friendly approach for the large scale 10-DAB acetylation without addition of acetyl-CoA in the industrial Taxol semi-synthesis. The finding of DBAT deacetylase activity may broaden its application in the structural modification of pharmaceutically important lead compounds.

Synthesis and biological evaluation of novel cabazitaxel analogues.

Cabazitaxel is one of the most recently FDA-approved taxane anticancer agent. In view of the advantages in preclinical and clinical data of cabazitaxel over former toxoids, the synthesis and biological evaluation of novel cabazitaxel analogues were conducted. First, a novel semi-synthesis of cabazitaxel was described. This strategy is concise and efficient, which needs five steps from the 10-deacetylbaccatin III (10-DAB) moiety and a commercially available C13 side chain precursor with a 32% overall yield. Besides, this strategy avoids using many hazardous reagents that involved in the previously reported processes. Then, a panel of cabazitaxel analogues were prepared basing on this strategy. The cytotoxicity evaluations showed that the majority of these cabazitaxel analogues are potent against both A549 and KB cells and their corresponding drug-resistant cell lines KB/VCR, and A549/T, respectively. Further in vivo antitumor efficacies assessment of 7,10-di-O-methylthiomethyl (MTM) modified cabazitaxel (compounds 16 and 19) on SCID mice A549 xenograft model showed they both had similar antitumor activity to the cabazitaxel. Since compound 19 was observed causing more body wight loss on the mice than 16, these preliminary studies suggest 16 might be a potent drug candidate for further preclinical evaluation.

Microbial Cell Factory of Baccatin III Preparation in Escherichia coli by Increasing DBAT Thermostability and in vivo Acetyl-CoA Supply.

Given the rapid development of genome mining in this decade, the substrate channel of paclitaxel might be identified in the near future. A robust microbial cell factory with gene dbat, encoding a key rate-limiting enzyme 10-deacetylbaccatin III-10-O-transferase (DBAT) in paclitaxel biosynthesis to synthesize the precursor baccatin III, will lay out a promising foundation for paclitaxel de novo synthesis. Here, we integrated gene dbat into the wild-type Escherichia coli BW25113 to construct strain BWD01. Yet, it was relatively unstable in baccatin III synthesis. Mutant gene dbat S189V with improved thermostability was screened out from a semi-rational mutation library of DBAT. When it was over-expressed in an engineered strain N05 with improved acetyl-CoA generation, combined with carbon source optimization of fermentation engineering, the production level of baccatin III was significantly increased. Using this combination, integrated strain N05S01 with mutant dbat S189V achieved a 10.50-fold increase in baccatin III production compared with original strain BWD01. Our findings suggest that the combination of protein engineering and metabolic engineering will become a promising strategy for paclitaxel production.

Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin III to baccatin III

10-Deacetylbaccatin III (10-DAB) C10 acetylation is an indispensable procedure for Taxol semi-synthesis, which often requires harsh conditions. 10-Deacetylbaccatin III-10-

Evaluation of the anticancer activity of enzymatically synthesized Baccatin III: an intermediate precursor of Taxol®.

Baccatin III is an important precursor for the synthesis of clinically important anticancer drug Taxol. Previously, we have characterized a key enzyme of 10-deacetylbaccatin III-10-ß-O-acetyltransferase (DBAT) which catalyses the 10-deacetylbaccatin III into baccatin III in taxol biosynthesis. Here, in the present study, we have evaluated and compared the cytotoxic properties of the enzymatically synthesized baccatin III (ESB III) with standard baccatin III in different human cancer cell lines, namely human cervical cancer (HeLa), human lung cancer (A549), human skin cancer (A431) and human liver cancer cells (HepG2). Among the various cancer lines tested, HeLa was more susceptible to ESB III with IC50 of 4.30 µM while IC50 values for A549, A431 and HepG2 ranged from 4 to 7.81 µM. Further, it showed G2/M phase cell cycle arrest, production of reactive oxygen species and depolarised mitochondrial membrane potential. In addition, annexin V-FITC staining was performed which showed the apoptotic cell death of HeLa cells, when treated with ESB III. Hence, ESB III was capable to show anticancer activities by inducing apoptotic cell death which could further be used for the semisynthesis of taxol in future.

High-effective biosynthesis of baccatin â ¢ by using the alternative acetyl substrate, N-acetyl-d-glucosamine.

AIMS: Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17 709 677·4 USD mol-1 ), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin Ⅲ, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production. METHODS AND RESULTS: In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin Ⅲ-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30·95 USD mol-1 , about 572 202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2·13-fold of that when using acetyl-CoA. In the in vivo system, the yield increased 24·17%, which may indicate its cooperation with acetyl-CoA. CONCLUSION: The success of open-whole-cell synthesis and in vivo synthesis of baccatin Ⅲ by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin Ⅲ. SIGNIFICANCE AND IMPACT OF THE STUDY: All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin Ⅲ semi-synthesis.

Study on methods for screening anti-tumor active components of Taxus chinensis based on integrity of traditional Chinese medicine / 中草药

Objective: Two screening methods for drug active constituents were established based on the integrity of traditional Chinese medicine to screen the anti-breast cancer active components, which were hollow fiber cell fishing-high performance liquid chromatography (HFCF-HPLC) and cell fishing-dispersive liquid-liquid microextraction-high performance liquid chromatography (CF-DLLME-HPLC). Methods: According to HFCF-HPLC, MCF-7 cells were planted into the cavity of polypropylene hollow fiber, and then the hollow fiber was put into the extract of Taxus chinensis to screen and capture the bioactive component groups combined with cells in a simulated human body environment. According to CF-DLLME-HPLC, on the basis of normal cell metabolism, MCF-7 cells were co-incubated with T. chinensis extract, supernatant of different incubation time was taken for liquid phase microextraction, and then chromatographic analysis was performed to find potential active component groups according to chromatographic peak changes. Results: Anti-breast cancer active components of T. chinensis were obtained by these two methods. 10-Deacetylbaccatin III, baccatin III, taxinine M, 10-deacetyltaxol, cephalomannine, taxol, taxuyunnanine C were obtained by HFCF-HPLC, and 10-deacetyltaxol, baccatin III, taxinine M, 10-deacetyltaxol, sciadopitysin were obtained by CF-DLLME-HPLC. Conclusion: The two methods can screen the effective antitumor active ingredients, and provide a convenient, universal and efficient new method for the screening of active ingredients and the comprehensive evaluation of the quality of traditional Chinese medicine.

Hyper-production of taxol from Aspergillus fumigatus, an endophytic fungus isolated from Taxus sp. of the Northern Himalayan region.

Taxol® (generic name Paclitaxel) is a chemotherapeutic drug, effective against head, neck, breast, lung, bladder, ovary, and cervix cancers. Rising demands in chemotherapy and limited supply of natural taxol have ultimately increased the cost of the drug. Semi synthesis using taxol precursors is not able to meet the global supply and has intensified the need to find alternative ways of taxol production. In the present study, 34 different endophytes were isolated from Taxus sp. collected from Shimla, Himachal Pradesh (India). Primary screening of taxol-producing fungi was carried out based on the presence of dbat gene, essential for the taxol biosynthetic pathway. A fungal isolate TPF-06 was screened to be a taxol-producing strain based on the PCR amplification results. It was characterized and identified as Aspergillus fumigatus by 18S rRNA (Accession No. KU-837249). Multiple sequence alignment (MSA) of nuclear ribosomal internal transcribed spacer (ITS) region and phylogenetic analysis confirmed that strain belonged to A. fumigatus clade (Accession No. MF-374798) and is endophytic in nature. Presence of taxol was detected and quantified by High-Performance Liquid Chromatography (HPLC) and characterized by using Thin Layer Chromatography (TLC), Ultraviolet (UV) spectroscopy, Mass spectrometry (MS), Fourier-Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopy. Microbial fermentation in the S7 medium yielded 1.60 g/L of taxol, which to the best of our knowledge is the highest taxol production from an endophytic fungus. Findings of the present study suggest that the A. fumigatus is an excellent alternate source for taxol supply, and it may become a highly potent strain on a commercial scale. The involvement of dbat gene in A. fumigatus KU-837249 strain further suggested a way of increasing taxol yield in fungi by medium engineering and recombinant DNA technology in the future.

Characterizing an engineered carotenoid-producing yeast as an anti-stress chassis for building cell factories.

BACKGROUND: A microorganism engineered for non-native tasks may suffer stresses it never met before. Therefore, we examined whether a Kluyveromyces marxianus strain engineered with a carotenoid biosynthesis pathway can serve as an anti-stress chassis for building cell factories. RESULTS: Carotenoids, a family of antioxidants, are valuable natural products with high commercial potential. We showed that the free radical removal ability of carotenoids can confer the engineered host with a higher tolerance to ethanol, so that it can produce more bio-ethanol than the wild type. Moreover, we found that this engineered strain has improved tolerance to other toxic effects including furfurals, heavy metals such as arsenate (biomass contaminant) and isobutanol (end product). Furthermore, the enhanced ethanol tolerance of the host can be applied to bioconversion of a natural medicine that needs to use ethanol as the delivery solvent of hydrophobic precursors. The result suggested that the engineered yeast showed enhanced tolerance to ethanol-dissolved hydrophobic 10-deacetylbaccatin III, which is considered a sustainable precursor for paclitaxel (taxol) bioconversion. CONCLUSIONS: The stress tolerances of the engineered yeast strain showed tolerance to several toxins, so it may serve as a chassis for cell factories to produce target products, and the co-production of carotenoids may make the biorefinary more cost-effective.

A Chemical Screen Identifies Compounds Capable of Selecting for Haploidy in Mammalian Cells.

The recent availability of somatic haploid cell lines has provided a unique tool for genetic studies in mammals. However, the percentage of haploid cells rapidly decreases in these cell lines, which we recently showed is due to their overgrowth by diploid cells present in the cultures. Based on this property, we have now performed a phenotypic chemical screen in human haploid HAP1 cells aiming to identify compounds that facilitate the maintenance of haploid cells. Our top hit was 10-Deacetyl-baccatin-III (DAB), a chemical precursor in the synthesis of Taxol, which selects for haploid cells in HAP1 and mouse haploid embryonic stem cultures. Interestingly, DAB also enriches for diploid cells in mixed cultures of diploid and tetraploid cells, including in the colon cancer cell line DLD-1, revealing a general strategy for selecting cells with lower ploidy in mixed populations of mammalian cells.

Taxus ingredients in the red arils of Taxus baccata L. determined by HPLC-MS/MS.

INTRODUCTION: Taxus baccata L. is an evergreen conifer whose plant parts are cardiotoxic. Only the red arils of the berries are described as non-toxic and taxane-free. OBJECTIVE: Extraction and HPLC-MS/MS methods were developed for the investigation of the Taxus compounds 3,5-dimethoxyphenol, 10-deacetylbaccatin III, baccatin III, cephalomannine, taxol A and taxinine M in the red arils of the yew berries. METHODOLOGY: A liquid-liquid extraction method for the red arils of the fruits from three yews were developed. An accurate (ESI+) HPLC-MS/MS method was performed for the simultaneous detection and determination of the target compounds in multiple reaction monitoring (MRM) mode. RESULTS: All Taxus agents obtained were detected in the red arils. Highest concentrations were determined for baccatin III and 10-deacetylbaccatin III. CONCLUSION: The developed quantitative method is reliable and selective and was successfully applied for quantification of selected Taxus ingredients in red arils of Taxus baccata. It was disproved that the red arils of the berries do not contain the selected Taxus compounds.

Transcriptome profiling reveals specific patterns of paclitaxel synthesis in a new Taxus yunnanensis cultivar.

The difference in contents of paclitaxel and 10-deacetylbaccatin III (10-DABIII) in needles between wildtype (WT) and a new cultivar (Zhongdayihao, ZD1) of Taxus yunnanensis was examined. Transcriptome profiling was conducted for different tissues of the ZD1 and WT to illustrate the regulation mechanism of paclitaxel biosynthesis. It was observed that average contents of paclitaxel and 10-DABIII in ZD1 were 4 folds and 32 folds higher than those in WT, respectively. More significant elevations of differential expressed genes (DEGs) from paclitaxel biosynthesis pathway were revealed in ZD1 rather than WT, which should be responsible for the higher contents of paclitaxel and 10-DABIII in the ZD1. Special tissues-dependent expression patterns of paclitaxel biosynthesis DEGs in ZD1 compared to WT were unraveled. The relative higher expressions of paclitaxel biosynthesis genes in needles than other tissues supported the higher content of paclitaxel and 10-DABIII content in needles of ZD1. Attenuation of plant hormone signal transduction pathway led to the lower expression of TFs in ZD1 rather than WT. Besides, the significant negative correlations between differential expressed TFs and DEGs from paclitaxel biosynthesis pathway displayed a possibly negative regulation pattern of these TFs on paclitaxel biosynthesis pathway genes. These results provided new insights into the molecular process of paclitaxel synthesis in Taxus.

The Combination Process for Preparative Separation and Purification of Paclitaxel and 10-Deacetylbaccatin III Using Diaion® Hp-20 Followed by Hydrophilic Interaction Based Solid Phase Extraction.

There is no other naturally occurring defense agent against cancer that has a stronger effect than paclitaxel, commonly known under the brand name of Taxol®. The major drawback for the more widespread use of paclitaxel and its precious precursor, 10-deacetylbaccatin III (10-DAB III), is that they require large-scale extraction from different parts of yew trees (Taxus species), cell cultures, taxane-producing endophytic fungi, and Corylus species. In our previous work, a novel online two-dimensional heart-cut liquid chromatography process using hydrophilic interaction/ reversed-phase chromatography was used to introduce a semi-preparative treatment for the separation of polar (10-deacetylbaccatin III) and non-polar (paclitaxel) taxanes from Taxus baccata L. In this work, a combination of the absorbent (Diaion® HP-20) and a silica based solid phase extraction is utilized as a new, efficient, and cost effective method for large-scale production of taxanes. This process avoids the technical problem of two-dimensional preparative liquid chromatography. The first stage of the process involves discarding co-extractive polar compounds including chlorophylls and pigments using a non-polar synthetic hydrophobic absorbent, Diaion® HP-20. Extract was then loaded on to a silica based hydrophilic interaction solid phase extraction (silica 40-60 micron). Taxanes was eluted using a mixture of water and methanol at the optimized ratio of 70:30. Finally, the fraction containing taxanes was applied to semi-preparative reversed phase HPLC. The results revealed that using this procedure, paclitaxel and 10-DAB III could be obtained at 8 and 3 times more, respectively than by the traditional method of extraction.

Infrared and Raman spectroscopic methods for characterization of Taxus baccata L.--Improved taxane isolation by accelerated quality control and process surveillance.

Different yew species contain poisonous taxane alkaloids which serve as resources for semi-synthesis of anticancer drugs. The highly variable amounts of taxanes demand new methods for fast characterization of the raw plant material and the isolation of the target structures during phyto extraction. For that purpose, applicability of different vibrational spectroscopy methods in goods receipt of raw plant material and in process control was investigated and demonstrated in online tracking isolation and purification of the target taxane 10-deacetylbaccatin III (10-DAB) during solvent extraction. Applying near (NIRS) and mid infrared spectroscopy (IRS) the amount of botanical impurities in mixed samples of two different yew species (R(2)=0.993), the leave-to-wood ratio for Taxus baccata material (R(2)=0.94) and moisture in dried yew needles (R(2)=0.997) can be quantified. By partial least square analysis (PCA) needles of different Coniferales species were successfully discriminated by Attenuated Total Reflectance-Fourier-Transform Infrared Spectroscopy (ATR-FT-IR). The analytical potential of ATR-FT-IR and Fourier Transform-Raman Spectroscopy (FT-RS) in process control of extraction and purification of taxanes is demonstrated for determination of the water content in methanolic yew extracts (R(2)=0.999) and for quantification of 10-DAB (R(2)=0.98) on a highly sophisticated level. The decrease of 10-DAB in the plant tissue during extraction was successfully visualized by FT-IR imaging of thin cross sections providing new perspectives for process control and design.

Bioproduction of baccatin III, an advanced precursor of paclitaxol, with transgenic Flammulina velutipes expressing the 10-deacetylbaccatin III-10-O-acetyl transferase gene.

BACKGROUND: 10-Deacetylbaccatin III (10-DAB) and baccatin III are intermediates in the biosynthesis of Taxol (an anti-cancer drug) and useful precursors for semi-synthesis of the drug. In this study, a bioconversion system was established for the production of baccatin III, an advanced precursor of paclitaxel, in the transgenic mushroom Flammulina velutipes expressing the 10-deacetylbaccatin III-10ß-O-acetyltransferase gene. The expression vector pgFvs-TcDBAT containing the 10-deacetylbaccatin III-10ß-O-acetyltransferase (DBAT) gene was constructed and transformed into the cells of F. velutipes by polyethylene glycol-mediated protoplast transformation. RESULTS: Polymerase chain reaction and Southern blotting analysis verified the successful integration of the exogenous DBAT gene into the genome of F. velutipes. Reverse transcription polymerase chain reaction and enzyme activity analyses confirmed that the DBAT gene was expressed in F. velutipes, and DBAT is able to convert substrate into baccatin III. CONCLUSION: The DBAT gene from the plant Taxus chinensis can be functionally expressed in F. velutipes. Transgenic F. velutipes expressing the DBAT gene is able to produce the target product, baccatin III. This is the first report about the transformation and expression of paclitaxel biosynthetic gene in the edible mushroom F. velutipes. This represents a significant step towards bio-production of paclitaxel and its advanced precursor baccatin III in an edible fungus.

Smashing Tissue Extraction and HPLC Determination of Paclitaxel and 10-Deacetylbaccatin from Taxus x media / 中草药·英文版

Objective To optimize the extraction technology of Taxus x media by using the contents of Paclitaxel and 10-deacetylbaccatin(10-DAB),two representative active diterpene alkaloids of taxane type from T.x media,as evaluation standard.Methods The smashing tissue extraction(STE)of Paclitaxel and 10-DAB from T.x media,was investigated by comparing with ultrasonic extraction(UE)which was one of the modern technologies of extraction.Results STE was more efficient than UE,and the contents of 10-DAI3 and Paclitaxel in the extracts obtained by STE were higher than those by UE.Conclusion STE is a fast,high-performance,and energy-saving technology for the extraction of diterpene alkaloids of taxane type.STE also provides a simple,component-safe,workable,and highly efficient method for the extraction of active natural product.

Effects of different elicitors on 10-deacetylbaccatin III-10-O-acetyltransferase activity and cytochrome P450 monooxygenase content in suspension cultures of Taxus cuspidata cells.

The effects of four elicitors, including 100 µmol/l MeJA (methyl jasmonate), 40 µl/l hydrogen peroxide (30%, w/w), 80 mg/l SA (salicylic acid) and 0.4 g/l F3 (fungal elicitor), on suspension cultures of Taxus cuspidata were studied. After addition of the above four elicitors, the enzyme activity of 10-DBAT (10-deacetylbaccatin III-10-O-acetyltransferase) was induced and reached its maximum of 5.47, 0.97, 3.30 and 6.82 U, respectively. After elicitation, the concentrations of cytochrome P450 monooxygenase were also induced to its maximum values of 0.069, 0.336, 0.321 and 0.193 nmol/ml, respectively. In addition, under the elicitation, the change in 10-DBAT activity was similar to that of cytochrome P450 monooxygenase concentration. The products of these two enzymes changed after the variety of the enzymes, and the taxol content increased through the cultivation.