Parameter Optimization of Ultrasonic-Microwave Synergistic Extraction of Taxanes from Taxus cuspidata Needles.

Taxanes are the best-known compounds in Taxus cuspidata owing to their strong anticancer effects. However, the traditional taxanes extraction method is the solid-liquid extraction method, which is limited by a large energy consumption and low yield. Therefore, it is urgent to find an efficient method for taxanes extraction. The ultrasonic microwave synergistic extraction (UME) method integrates the cavitation effect of ultrasound and the intensifying heat transfer (ionic conduction and dipole rotation of molecules) effect of microwave to accelerate the release of intracellular compounds and is used in active ingredient extractions. This study aimed to evaluate the performance of UME in extracting taxanes from T. cuspidata needles (dichloromethane-ethanol as extractant). A single-factor experiment, Plackett-Burman design, and the response surface method showed that the optimal UME parameters for taxanes extraction were an ultrasonic power of 300 W, a microwave power of 215 W, and 130 sieve meshes. Under these conditions, the taxanes yield was 570.32 µg/g, which increased by 13.41% and 41.63% compared with the ultrasound (US) and microwave (MW) treatments, respectively. The reasons for the differences in the taxanes yield were revealed by comparing the physicochemical properties of T. cuspidata residues after the UME, US, and MW treatments. The cell structures were significantly damaged after the UME treatment, and numerous tiny holes were observed on the surface. The absorption peaks of cellulose, hemicellulose, and lignin increased significantly in intensity, and the lowest peak temperature (307.40 °C), with a melting enthalpy of -5.19 J/g, was found after the UME treatment compared with the US and MW treatments. These results demonstrate that UME is an effective method (570.32 µg/g) to extract taxanes from T. cuspidata needles by destroying cellular structures.

Deciphering the Antimicrobial Potential of Taxus wallichiana Zucc: Identification and Characterization Using Bioassay-Guided Fractionation.

Taxus wallichiana Zucc. is a high valued medicinal plant and has been mainly studied for its anti-cancer properties. However, research on its other important biological activities, such as its antimicrobial potential, still needs attention. The focus of the present study is to investigate the antimicrobial activity of secondary metabolites of T. wallichiana needles against 3 different groups of microorganisms, i. e., bacteria, actinobacteria, and fungi. Bioactive compounds from T. wallichiana needles were separated through column chromatography, and, TLC-bioautography. Mobile phases were optimized using Snyder's selectivity triangle. Antimicrobial spots were fractionated and compounds were identified by gas chromatography-mass spectroscopy (GC/MS) and liquid chromatography-mass spectrometry (LC/MS). Functional groups were characterized using Fourier transform infrared spectrometry (FTIR) and nuclear magnetic resonance (NMR) was used to identify the molecular structures. GC/MS and LC/MS data analysis confirm the presence of fatty acids (arachidic acid, behenic acid, palmitic acid, and stearic acid), vitamins (nicotinamide), and alkaloids (cinchonine, timolol), aminobenzamides (procainamide), carbocyclic sugar (myoinositol), and alkane hydrocarbon (hexadecane), having antimicrobial activity in the needles of T. wallichiana. To the best of our knowledge, this is the first report on the isolation and characterization of antimicrobial compounds from the needles of Taxus wallichiana (Himalayan yew). The data obtained from the present study will be supportive to the new drug discoveries in modern medicine with various combinations of medicinal plant's active constituents that can be used for curing many diseases.

Analytical toxicology of yew constituents in human blood and urine by liquid chromatography-high-resolution tandem mass spectrometry.

The active, poisonous constituents in Taxus baccata, the yew plants, are taxine alkaloids whose main action is suggested to be a block of calcium and sodium channels. The main alkaloids are taxine B (30%) and taxine A (1.3%). Symptoms can include bradycardia, bradypnea, diastolic, and cardiac standstill. The current investigation reports the analytical toxicology of human blood and urine to confirm a suspected ingestion of yew needles. This includes the qualitative detection of several yew ingredients, including the main alkaloids, the validated quantification of 3,5-dimethoxyphenol, and the discussion of suitable analytical targets. After analyzing human specimens and yew needle extracts using the developed procedures, the five alkaloids 1-deotaxine B, taxicatin, taxine A, taxine B, and taxine I could be detected and tentatively identified. Finally, taxine A and B can be recommended as analytical targets besides 3,5-dimethoxyphenol.

Identification and in-silico characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) gene in Corylus avellana L.

Paclitaxel® (PC) is one of the most effective and profitable anti-cancer drugs. The most promising sources of this compound are natural materials such as tissue cultures of Taxus species and, more recently, hazelnut (Corylus avellana L.). A large part of the PC biosynthetic pathway in the yew tree and a few steps in the hazelnut have been identified. Since understanding the biosynthetic pathway of plant-based medicinal metabolites is an effective step toward their development and engineering, this paper aimed to identify taxadiene-5α-ol-O-acetyltransferase (TDAT) in hazelnut. TDAT is one of the key genes involved in the third step of the PC biosynthetic pathway. In this study, the TDAT gene was isolated using the nested-PCR method and then characterized. The cotyledon-derived cell mass induced with 150 µM of methyl jasmonate (MeJA) was utilized to isolate RNA and synthesize the first-strand cDNA. The full-length cDNA of TDAT is 1423 bp long and contains a 1302 bp ORF encoding 433 amino acids. The phylogenetic analysis of this gene revealed high homology with its ortholog genes in Quercus suber and Juglans regia. Bioinformatics analyses were used to predict the secondary and tertiary structures of the protein. Due to the lack of signal peptide, protein structure prediction suggested that this protein may operate at the cytoplasm. The homologous superfamily of the T5AT protein, encoded by TDAT, has two domains. The highest and lowest hydrophobicity of amino acids were found in proline 142 and lysine 56, respectively. T5AT protein fragment had 24 hydrophobic regions. The tertiary structure of this protein was designed using Modeler software (V.9.20), and its structure was verified based on the results of the Verify3D (89.46%) and ERRAT (90.3061) programs. The T5AT enzyme belongs to the superfamily of the transferase, and the amino acids histidine 164, cysteine 165, leucine 166, histidine 167, and Aspartic acid 168 resided at its active site. More characteristics of TDAT, which would aid PC engineering programs and maximize its production in hazelnut, were discussed.

Bioactive Components, Volatile Profile and In Vitro Antioxidative Properties of Taxus baccata L. Red Arils.

This study aimed at assessing the composition of bioactive compounds, including ascorbic acid, carotenoids and polyphenols, the volatile compound profile and the antioxidant activity of red arils (RAs) of Taxus baccata L. grown in diverse locations in Poland. Among the carotenoids assayed in high quantities (3.3-5.42 µg/g), the lycopene content (2.55-4.1 µg/g) was remarkably higher than that in many cultivated fruits. Samples collected from three sites were distinguished by higher amounts of ascorbic acid (125 mg/100 g, on average) than those found in many cultivated berries. Phenylpropanoids quantitatively dominated among the four groups of phenolic compounds. Chromatographic separation enabled the detection of two phenylpropanoid acids: ferulic and p-coumaric. Irrespectively of the growth site, RAs contained substantial amounts of (-)-epicatechin (1080 µg/100 g, on average). A higher ability to scavenge DPPH● and ABTS●+ radicals was found in the hydrophilic fraction of RAs from two sites (Warsaw and Koszalin) compared with the other two sites. The volatile compound profile of RAs was dominated by alcohols, followed by ketones, esters and aldehydes. The presence of some volatiles was exclusively related to the specific growth site, which may be regarded as a valuable indicator. The combination of bioactive and volatile compounds and the fairly good antioxidant potential of RAs render them an attractive source for preparing functional foods.

Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases.

Taxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10ß-hydroxylase (T10ßOH) and a newly putative ß-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.

Red Arils of Taxus baccata L.-A New Source of Valuable Fatty Acids and Nutrients.

The aim of this study, focused on the nutritional value of wild berries, was to determine the contents of macronutrients, profiles of fatty (FAs) and amino acids (AAs), and the contents of selected elements in red arils (RA) of Taxus baccata L., grown in diverse locations in Poland. Protein (1.79-3.80 g/100 g) and carbohydrate (18.43-19.30 g/100 g) contents of RAs were higher than in many cultivated berries. RAs proved to be a source of lipids (1.39-3.55 g/100 g). Ten out of 18 AAs detected in RAs, mostly branched-chain AAs, were essential AAs (EAAs). The EAAs/total AAs ratio approximating were found in animal foods. Lipids of RA contained seven PUFAs, including those from n-3 family (19.20-28.20 g/100 g FA). Polymethylene-interrupted FAs (PMI-FAs), pinolenic 18:3Δ5,9,12; sciadonic 20:3Δ5,11,14, and juniperonic 20:4Δ5,11,14,17, known as unique for seeds of gymnosperms, were found in RAs. RAs may represent a novel dietary source of valuable n-3 PUFAs and the unique PMI-FAs. The established composition of RAs suggests it to become a new source of functional foods, dietary supplements, and valuable ingredients. Because of the tendency to accumulate toxic metals, RAs may be regarded as a valuable indicator of environmental contamination. Thus, the levels of toxic trace elements (Al, Ni, Cd) have to be determined before collecting fruits from natural habitats.

Effect of Taxus baccata L. Extract on Hydatid Cyst Protoscolices In vitro.

Hydatidosis is the most important global parasitic infectious disease both in humans and animals, which can lodge at different organs of the host, such as liver, lung (even heart), and brain which may lead to death. Surgery is the main method for the treatment of hydatidosis. In surgical therapy of hydatidosis, the use of sporicidal agents is very important since these agents inactivate live protoscolices and prevent recurrence of infection. Presently, numerous scolicidal chemical agents have been administrated to inactivate the hydatid cyst contents. Recently, there has been a high tendency among researchers to evaluate and present herbal plants as alternative option due to inexpensiveness, availability, low side effects, and toxicity. This study aimed to evaluate the scolicidal effect of hydro alcoholic Taxus baccata L. extract in vitro for the first time. The scolicidal activities of the extract were tested in concentrations of 50, 100, and 150 mg/ml following 10, 30, and 60 min of incubation, and the experiments were performed in triplicate. Viability of protoscolices was confirmed by 0.1% eosin vital staining. The data were analyzed in SAS software (version 9.4). The results showed that the hydroalcoholic extract of Taxus baccata L. at the concentration of 150 mg/ml led to killing 66.6% of protoscolices at 60 min. according to the results of this investigation, it is recommended to use this plant as a scolicidal plant. The findings of the present study showed that Taxus baccata L. had potent scolicidal effects. However, further studies are required to evaluate the efficacy of Taxus baccata L. in vivo.

A Potent and Safer Anticancer and Antibacterial Taxus-Based Green Synthesized Silver Nanoparticle.

PURPOSE: Paclitaxel is a generic drug produced based on Taxol which is an extract of Taxus tree, well known for its anticancer and antibacterial effects. This study was aimed at building up an agent with the antibacterial and anticancer benefits of both the silver ions and Taxol, together with less cytotoxic effects. MATERIALS AND METHODS: Colloidal silver nanoparticles (AgNPs) were synthesized by reducing aqueous AgNO3 with aqueous Taxus leaf extract at nonphotomediated conditions, without any catalyst, template or surfactant. The AgNP production was confirmed by ultraviolet-visible (UV-VIS) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier-transform infrared (FTI) spectroscopy. The MTT assay for human breast cancer cells as well as the DAPI fluorescent staining microscopy tested the biocompatibility and anticancer effects of AgNPs, silver nitrate, and Taxol. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques were performed to determine the shape and size of the nanoparticles. MTT assay showed the best inhibitory concentration of AgNPs on cancer cells. The antibacterial activity of the three case study materials was tested for gram-positive (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) using well diffusion test. RESULTS: This work proposes more anticancer effects for AgNP made by Taxus brevifolia extract, comparing Taxol solution. IC50 was observed as 3.1 mM for Taxol while 1.5 mM for new AgNP. Moreover, Taxus showed no antibacterial effects while the new AgNP showed a dose-dependent biocompatibility along with slightly more antibacterial effects (MIC: 1.6 and 6.6mM for gram-positive and -negative bacteria, respectively) comparing with silver nitrate solution (MIC: 1.5 and 6.2 mM for gram-positive and -negative bacteria, respectively). CONCLUSION: The production of herbal-mediated silver nanoparticles may be an efficient substitution for the silver nitrate-based medicines with less side effects.

Anticancer activity and molecular mechanisms of α-conidendrin, a polyphenolic compound present in Taxus yunnanensis, on human breast cancer cell lines.

α-Conidendrin is a polyphenolic compound found mainly in Taxus yunnanensis, as the source of chemotherapy drug paclitaxel, which has been used in traditional medicine for treatment of cancer. This study aimed to investigate the anticancer activity and molecular mechanisms of α-conidendrin on breast cancer cell lines. The results of the present study show that α-conidendrin possesses potent antiproliferative effects on breast cancer cell lines MCF-7 and MDA-MB-231. α-Conidendrin significantly induced apoptosis in breast cancer cells via reactive oxygen species generation, upregulation of p53 and Bax, downregulation of Bcl-2, depolarization of mitochondrial membrane potential (MMP), release of cytochrome c from mitochondria, and activation of caspases-3 and -9. α-Conidendrin remarkably inhibited the proliferation of breast cancer cells through induction of cell cycle arrest by upregulating p53 and p21 and downregulating cyclin D1 and CDK4. Unlike breast cancer cells, the antiproliferative effect of α-conidendrin on human foreskin fibroblast cells (normal cells) was very small. In normal cells, reactive oxygen species levels, loss of MMP, release of cytochrome c, mRNA expression of p53, p21, cyclin D1, CDK4, Bax, and Bcl-2 as well as mRNA expression and activity of caspases-3 and -9 were significantly less affected by α-conidendrin compared with cancer cells. These results suggest that α-conidendrin can be a promising agent for treatment of breast cancer with little or no toxicity against normal cells.

Brevifoliol ester induces apoptosis in prostate cancer cells by activation of caspase pathway.

Prostate cancer is fourth most abundant cancer type around the globe. Brevifoliol, a rearranged taxoid from Taxus walllichiana needles has been derivatized as C5 esters using Steglich esterification reaction. Seventeen diverse analogues were evaluated against a panel of human cancer cell lines by MTT assay. Among these, two of the semi-synthetic analogues, that is, 13 and 16 exhibited potent cytotoxicity, selectively against PC-3, prostate cancer cell lines. In cell cycle analysis, analogue 13 induced S and G2/M phase arrest and induced apoptosis by activating caspase-3. Compound 13 showed moderate efficacy in in-vivo Ehrlich ascites carcinoma in Swiss albino mice. Further, compound 13 was found to be safe in Swiss albino mice up to 1,000 mg/kg dose in acute oral toxicity. Brevifoliol ester 13 may further be optimized for better efficacy.

Extraction, Purification, and Biological Activities of Polysaccharides from Branches and Leaves of Taxus cuspidata S. et Z.

Taxus cuspidata S. et Z. is an excellent natural source of bioactive polysaccharides and has various biological activities. The objective of this study was to evaluate the effect of antidiabetic and antitumor activities of polysaccharides from Taxus cuspidata branches and leaves (TCBL) and to determine the optimum extraction technology of TCBL using a low-temperature and high-efficiency enzyme and ultrasound-assisted coupled extraction (EUCE) method. Optimal technology parameters were determined as follows: an extraction temperature of 51 °C, an extraction time of 33 min, a ratio of material to liquid of 1:19 (g:mL), and an enzyme concentration of 0.10 mg·mL-1. Under the optimized conditions, the polysaccharide yield from TCBL obtained by EUCE was 4.78% ± 0.18%. The four purified polysaccharides (Pe1, Pe2, Pe3, Pe4) from TCBL are mainly composed of arabinose, galactose, glucose, a small amount of xylose, and mannose. This composition was assessed by HPIC analysis. The antidiabetic activity and antitumor activity of polysaccharides from TCBL were assayed in vitro. Among the four purified polysaccharides from TCBL, purified Pe4 had the highest inhibitory capacity against α-glucosidase, and its IC50 value was 123.0 µg·mL-1. Pe1 had the highest antitumor capacity against MCF7 cells and HepG2 cells, with IC50 values of 169.0 and 132.0 µg·mL-1. Pe4 had the highest antitumor effect on human cervical cancer cells (Hela), and its IC50 value was 89.9 µg·mL-1. Pe4 polysaccharide demonstrated a good α-glucosidase inhibitory activity and antitumor capacity against Hela cells. Therefore, Pe4 polysaccharide from TCBL is a beneficial source of potential inhibitors of type II diabetes and human cervical cancer activity.

Identification of the antiglycative components of Hong Dou Shan (Taxus chinensis) leaf tea.

The formation of advanced glycation end products (AGEs) may affect life quality and cut lifespan. With the increasingly utilization of new foods and booming development of international trade, large amounts of newly emerging teas have come into our sight. In this study, we evaluated the antiglycative capabilities of five popular herbal teas. Results showed that Chinese Hong Dou Shan (Taxus chinensis; HDS) leaf tea had particularly strong antiglycative activity via scavenging methylglyoxal (MGO) in both chemical (glucose-BSA, fructose-BSA, and MGO-BSA) and cell models (HUVECs). Furthermore, the intracellular AGEs level was alleviated to normal state by the two HDS leaf tea fractions, H-47 and H-57 which were almost as effective as EGCG when added at the same level (50 µg/mL). The effective components against glycation were further separated and tentatively identified as catechin, epicatechin, gallocatechin, epigallocatechin, procyanidin B2, a dihydromyricetin dimer, and a quercetin glucoside by HPLC-DAD and LC-Q-TOF/MS analysis.

Phenolic Compounds of Catalpa speciosa, Taxus cuspidate, and Magnolia acuminata have Antioxidant and Anticancer Activity.

Tree bark represents an important source of medicinal compounds that may be useful for cancer therapy. In the current study, high-performance liquid chromatography with diode-array detection (HPLC-DAD) was used to determine the profile of the phenolic compounds of Catalpa speciosa, Taxus cuspidata, and Magnolia acuminata bark extracts. The antioxidant and anticancer bioactivities against different cancer cell lines were investigated. M. acuminata exerted significantly higher antioxidant activities in the diphenyl picrylhydrazine and ß-carotene-linoleic acid assays than the other species. In C. speciosa, novel profiles of phenolic acids (ferulic acid was the predominant compound) and catechin were detected. In T. cuspidata, six phenolic acids were detected; the predominant compounds were hydroxycaffeic acid and protocatechuic acid. In M. acuminata, two phenolic acids and three catechins were detected; catechin was the predominant compound. The three species exerted clear anticancer activity against MCF-7, HeLa, Jurkat, T24, and HT-29 cells, with the strongest activity found in the extracts from M. acuminata. No antiproliferative activity against normal cells was found. Flow cytometry revealed greater accumulation of necrotic and early/late apoptotic cells in various treated cancer cells than in untreated control cells, and protocatechuic acid induced a similar accumulation of necrotic cells to that of the bark extracts. Caspase-3 and -7 activity was increased in cancer cells treated with different bark extracts; the highest activity was found in the M. acuminata treatment. Our results suggested that the treatment of cancer cells with bark extracts of M. acuminata, C. speciosa, and T. cuspidata, and protocatechuic acid induced apoptosis, suggesting an association between anticancer activities and individual phenolic compounds.

Taxus chinensis ameliorates diabetic nephropathy through down-regulating TGF-ß1/Smad pathway.

Diabetic nephropathy (DN) is one of the common microvascular complications of diabetes mellitus. Renal fibrosis is closely related to the deterioration of renal function. The present study aimed to investigate protective effect of Taxus chinensis on high-fat diet/streptozotocin-induced DN in rats and explore the underlying mechanism of action. The rat DN model was established via feeding high fat diet for 4 weeks and subsequently injecting streptozotocin (30 mg·kg-1 body weight) intraperitoneally. The rats with blood glucose levels higher than 16.8 mmol·L-1 were selected for experiments. The DN rats were treated with Taxus chinensis orally (0.32, 0.64, and 1.28 g·kg-1) once a day for 8 weeks. Taxus chinensis significantly improved the renal damage, which was indicated by the decreases in 24-h urinary albumin excretion rate, blood serum creatinine, and blood urea nitrogen. Histopathological examination confirmed the protective effect of Taxus chinensis. The thickness of glomerular basement membrane was reduced, and proliferation of mesangial cells and podocytes cells and increase in mesangial matrix were attenuated. Further experiments showed that Taxus chinensis treatment down-regulated the expression of TGF-ß1 and α-SMA, inhibited phosphorylation of Smad2 and Smad3. These results demonstrated that Taxus chinensis alleviated renal injuries in DN rats, which may be associated with suppressing TGF-ß1/Smad signaling pathway.

Ultrasonic Assisted Extraction of Paclitaxel from Taxus x media Using Ionic Liquids as Adjuvants: Optimization of the Process by Response Surface Methodology.

(1) Background: Ionic liquids (ILs) are considered "green" solvents and have been widely used in the extraction and separation field in recent years; (2) Methods: In this study, some common ILs and functionalized magnetic ionic liquids (MILs) were used as adjuvants for the solvent extraction of paclitaxel from Taxus x media (T. x media) using methanol solution. The extraction conditions of methanol concentration, IL type and amount, solid-liquid ratio, extraction temperature, and ultrasonic irradiation time were investigated in single factor experiments. Then, three factors of IL amount, solid-liquid ratio, and ultrasonic irradiation time were optimized by response surface methodology (RSM); (3) Results: The MIL [C4MIM]FeCl3Br was screened as the optimal adjuvant. Under the optimization conditions of 1.2% IL amount, 1:10.5 solid-liquid ratio, and 30 min ultrasonic irradiation time, the extraction yield reached 0.224 mg/g; and (4) Conclusions: Compared with the conventional solvent extraction, this ultrasonic assisted extraction (UAE) using methanol and MIL as adjuvants can significantly improve the extraction yield, reduce the use of methanol, and shorten the extraction time, which has the potentiality of being used in the extraction of some other important bioactive compounds from natural plant resources.

Chemical Components, Biological Activities, and Toxicological Evaluation of the Fruit (Aril) of Two Precious Plant Species from Genus Taxus.

The fruit (aril) of the endangered genus Taxus plants is an abandoned herbal resource. Traditionally, people enthusiastically focus on its bark, its renewable, tremendous arils fall into the soil with seeds after they are mature. The present research investigated the fruit of two species from the genus Taxus, Taxus chinensis var. mairei, and Taxus media, with regards to their antioxidant and antihyperglycaemic activities, safety, and bioactive constituents. Results showed that T. chinensis var. mairei and T. media both had certain biological activities with T. chinensis var. mairei better in antioxidant activity and T. media better in antihyperglycaemic activity. Correlation analysis revealed that the differences in bioactivities depended on content of their mainly chemical components. The mice acute oral toxicity test indicated that the methanol extracts of the two biotypes of Taxus were safe. And nineteen compounds were tentatively assigned from the two varieties, via tandem mass spectrometry using a LC-ESI-Q-TOF-MS instrument, which included phenols, flavonoids, and terpenes. These results indicate a possible application of Taxus fruit extracts in various fields like in food industry, however, this still needs further investigations.

Improving 10-deacetylbaccatin III-10-ß-O-acetyltransferase catalytic fitness for Taxol production.

The natural concentration of the anticancer drug Taxol is about 0.02% in yew trees, whereas that of its analogue 7-ß-xylosyl-10-deacetyltaxol is up to 0.5%. While this compound is not an intermediate in Taxol biosynthetic route, it can be converted into Taxol by de-glycosylation and acetylation. Here, we improve the catalytic efficiency of 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) of Taxus towards 10-deacetyltaxol, a de-glycosylated derivative of 7-ß-xylosyl-10-deacetyltaxol to generate Taxol using mutagenesis. We generate a three-dimensional structure of DBAT and identify its active site using alanine scanning and design a double DBAT mutant (DBATG38R/F301V) with a catalytic efficiency approximately six times higher than that of the wild-type. We combine this mutant with a ß-xylosidase to obtain an in vitro one-pot conversion of 7-ß-xylosyl-10-deacetyltaxol to Taxol yielding 0.64 mg ml-1 Taxol in 50 ml at 15 h. This approach represents a promising environmentally friendly alternative for Taxol production from an abundant analogue.

Laser Desorption/Ionisation Mass Spectrometry Imaging of European Yew (Taxus baccata) on Gold Nanoparticle-enhanced Target.

INTRODUCTION: European yew (Taxus baccata) is a plant known to man for centuries as it produces many interesting and important metabolites. These chemical compounds were repeatedly analysed by various analytical techniques, but none of the methods used so far allowed the localisation of the chemical compounds within the tissue and also correlation between plant morphology and its biochemistry. OBJECTIVE: Visualisation of the spatial distribution of yew metabolites with nanoparticle-based mass spectrometry imaging. METHODOLOGY: Compounds occurring on cross-section of a one-year yew sprig has been transferred to gold nanoparticle-enhanced target (AuNPET) by imprinting. The imprint was then subjected to mass spectrometry imaging analysis. RESULTS: Nanoparticle-enhanced mass spectrometry imaging made it possible to study the distribution of selected compounds in the European yew tissue, including taxanes - terpene alkaloids characteristic for the Taxus genus. Results prove that aspartate, taxinine M, baccatin IV and taxine B are located mainly in the cortex. Taxuspine W was located in the vascular tissue. Maleate was found to be located mainly in the phloem tissue. In contrast, the proton adduct of chlorophyll b was found in the external layer of twigs. CONCLUSION: The results presented a high correlation between the location of compounds and the morphology of the plant, thus giving the opportunity to see the selected details of chemical structure of the analysed tissue for the first time. Copyright © 2017 John Wiley & Sons, Ltd.

Separation and purification of two taxanes and one xylosyl-containing taxane from Taxus wallichiana Zucc.: A comparison between high-speed countercurrent chromatography and reversed-phase flash chromatography.

10-Deacetylbaccatin III, an important semisynthetic precursor of paclitaxel and docetaxel, can be extracted from Taxus wallichiana Zucc. A process for the isolation and purification of 10-deacetylbaccatin III (1), baccatin III (2), and 7ß-xylosyl-10-deacetyltaxol (3) from the leaves and branches of Taxus wallichiana Zucc. via macroporous resin column chromatography combined with high-speed countercurrent chromatography or reversed-phase flash chromatography was developed in this study. After fractionation by macroporous resin column chromatography, 80% methanol fraction was selected based on high-performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis. A solvent system composed of n-hexane, ethyl acetate, methanol, and water (1.6:2.5:1.6:2.5, v/v/v/v) was used for the high-speed countercurrent chromatography separation at a flow rate of 2.5 mL/min. The reversed-phase flash chromatography separation was performed using methanol/water as the mobile phase at a flow rate of 3 mL/min. The high-speed countercurrent chromatography separation produced compounds 1 (10.2 mg, 94.4%), 2 (2.1 mg, 98.0%), and 3 (4.6 mg, 98.8%) from 100 mg of sample within 110 min, while the reversed-phase flash chromatography separation purified compounds 1 (9.8 mg, 95.6%) and 3 (4.9 mg, 97.9%) from 100 mg of sample within 120 min.