Optimization of Taxol Extraction Process Using Response Surface Methodology and Investigation of Temporal and Spatial Distribution of Taxol in Taxus mairei

Taxus mairei is an important source for industrial extraction of taxol in China. However, the standard and steps of extraction are currently not uniform, which seriously affects the taxol yield. In the present study, the influence of four factors (methanol concentration, solid-liquid ratio, ultrasonic extraction temperature, and ultrasonic extraction time) on the taxol yield was successively explored in T. mairei. A response surface methodology (RSM) was used to optimize the extraction process based on the single-factor experiments above. The optimal conditions were as follows: methanol concentration was 90%, solid-liquid ratio was 1:15 (g/mL), ultrasonic extraction temperature was 40 °C and ultrasonic extraction time was 60 min. Moreover, the twigs and needles from T. mairei with different tree ages were treated by the optimum extraction process, which further revealed temporal and spatial distribution of taxol in the reproducible tissues. Interestingly, the taxol content was relatively higher in needles of T. ‘Jinxishan’ (a cultivar from T. mairei with yellow aril, FY), but was less in FY twigs. The accumulation of taxol in twigs and leaves of females (with red aril, FR) was significantly higher than that of males (M); however, the content showed a decreasing trend with the increasing tree ages. Therefore, it is suitable to increase the proportion of female trees especially the FY leaves as raw materials for the industrial production of taxol from T. mairei, and the tree ages should be better controlled at 3–7 years.

Effects of exogenous NO on antioxidant system of Taxus plants under simulated acid rain stress

Taxus is a famous medicinal and landscape tree species. The aim of this study was to explore the effect of exogenous nitric oxide (NO) on the resistance of Taxus plants to acid rain stress and to identify Taxus species with strong acid rain resistance by principal component analysis and comprehensive evaluation. In this study, sodium nitroprusside (SNP) was used as the exogenous NO donor. The effects of different SNP solution concentrations on the antioxidant systems of three Taxus species subjected to simulated acid rain stress (pH = 3.0) were compared. In order to achieve this goal, we determined the rate of O2- production, the ASA and GSH contents in leaves of three Taxus plants (Taxus mairei, Taxus chinensis, and Taxus yunnanensis). At the same time, the active leaves of some antioxidant enzymes (SOD, POD, CAT, APX and GR) were determined. For Taxus chinensis plants subjected to acid rain stress, treatment with an SNP concentration of 0.25 mmol·L-1 led to the most significant improvements in the antioxidant system. For Taxus mairei and Taxus yunnanensis, the treatment with the SNP concentration of 0.50 mmol·L-1 was best for improving their antioxidant systems under stress. Meanwhile, Taxus chinensis had the strongest resistance to simulated acid rain, followed by Taxus mairei and Taxus yunnanensis.

Metabolic Variations of Flavonoids in Leaves of T. media and T. mairei Obtained by UPLC-ESI-MS/MS

The needles of Taxus species contain a large number of bioactive compounds, such as flavonoids. In the present study, the total flavonoid content in leaves of Taxus media and Taxus mairei was 19.953 and 14.464 mg/g, respectively. A total of 197 flavonoid metabolites (70 flavones, 42 flavonols, 26 flavone C-glycosides, 20 flavanones, 15 anthocyanins, 13 isoflavones, 6 flavonolignans, and 5 proanthocyanidins) were identified for the first time by a widely targeted Ultra Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (UPLC-ESI-MS/MS) method within the two Taxus species, containing 160 common metabolites, with 37 unique metabolites merely determined in T. mairei or T. media. Moreover, 42 differential flavonoid metabolites were screened in the two Taxus species, which showed specific metabolic patterns in isoflavonoid biosynthesis, anthocyanin biosynthesis, and flavone and flavonol biosynthesis pathways. Compared to T. mairei, a more activated phenylpropanoid pathway was found in T. media, which could be responsible for the higher content of total flavonoids in T. media. Our results provide new insights into the diversity of flavonoid metabolites between T. mairei and T. media, and provide a theoretical basis for the sufficient utilization of Taxus species and the development of novel drugs.

Transcriptome Sequencing Reveals Regulatory Mechanisms of Taxol Synthesis in Taxus wallichiana var. Mairei

Taxol is one of the most potent and effective anticancer drugs and is originally isolated from Taxus species. To investigate the specific regulatory mechanisms of taxol synthesis in Taxus wallichiana var. mairei, RNA-seq was conducted to reveal the differences in transcriptional levels between wild type (WT) and “Jinxishan” (JXS), a cultivar selected from a population of Taxus mairei that shows about 3-fold higher taxol content in the needles than WT. Our results indicated that high expressions of the genes taxadienol acetyltransferase (TAT), taxadiene 5-alpha hydroxylase (T5H), 5-alpha-taxadienol-10-beta-hydroxylase (T10OH), and 2-debenzoyl-7,13-diacetylbaccatin III-2-O-benzoyl-transferase (DBBT), which catalyze a series of key acetylation and hydroxylation steps, are the main cause of high taxol content in JXS. Moreover, in the present study, the activation of jasmonic acid (JA) signal transduction and its crosstalk with gibberellin (GA), auxin, and ethylene (ET) explained the elevation of differentially expressed genes (DEGs) from the taxol biosynthesis pathway. This also indicates that taxol biosynthesis in T. mairei is associated with the balance of cell development and defense. TF-encoding (transcriptional factor) genes, represented by the ethylene-responsive transcription factor (ERF), basic/helix-loop-helix (bHLH), MYB, and WRKY families, were detected as differentially expressed between JXS and WT, further indicating that the regulation of hormone signaling on taxol biosynthesis genes was mediated by transcription factors (TFs). To our knowledge, this is the first study to illustrate the regulatory mechanisms of taxol synthesis in a new cultivar of T. mairei with a high taxol content in its needles. These transcriptome data provide reasonable explanations for the variation of taxol content between WT and JXS.