DRUG DEVELOPMENT RESEARCH 73 : 477486 (2012)

DDR

Research Overview

Biological, Chemical, and Omics Research of Taxus Medicinal Resources

Da-Cheng Hao1, Pei-Gen Xiao1,2*, Guang-Bo Ge3, and Ming Liu1 Biotechnology Institute/School of Environment, Dalian Jiaotong University, Dalian, China 2 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences/Peking Union Medical College, Beijing, China 3 Pharmaceutical resource discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
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Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I-III Regulatory, Quality, Manufacturing Postmarketing Phase IV

ABSTRACT Taxus is a botanical source for the anticancer drug taxol (paclitaxel), rst reported in 1971 as a result of bioassay guided fractionation of active extracts from stem bark samples of T. brevifolia. This led to additional discoveries of pharmacologically active taxoids in other Taxus species and to investigations on their biosynthetic precursors, analogues, and derivatives. In continuation with our studies on Taxus biochemistry, we review the genomics, transcriptomics, proteomics, metabolomics, and bioinformatics of Taxus and their endophytic fungi. The bibliometric method is used to quantify and characterize the global scientic effort in the omics technologies being applied. We conclude that systems biology and omics technologies will play an increasingly important role in future medical research involving bioactive compounds of Taxus and other natural products. Drug Dev Res 73 : 477486, 2012. 2012 Wiley
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Key words: Taxus; medicinal resource; phylogeny and evolution; biotechnology; phytochemistry; omics

INTRODUCTION

The genus Taxus, commonly known as yew, a gymnosperm in the family Taxaceae, includes 24 species [Spjut, 2007] of small trees or shrubs that are a major source of medicinal compounds, particularly the anticancer/cardiovascular drug taxol (paclitaxel). Taxol was rst isolated from T. brevifolia Nutt. [Wani et al., 1971], a species found in the Pacic Northwest of North America [Spjut, 2007]. Native species in China include the more widely distributed T. biternata Spjut, T. celebica (Warb.) H.L. Li, T. contorta Griff., T. chinensis (Pilg.) Rehder, T. kingstonii Spjut, T. mairei (Leme & Lv) S.Y. Hu ex T.S. Liu, T. sumatrana (Miq.) de Laub., T. umbraculifera (Sieb. ex Endl.) C. Lawson, T. wallichiana Zucc., T. yunnanensis W.C. Cheng & L.K. Fu, and other localized species [Spjut, 2007; http:// www.worldbotanical.com/nomenclature], all of which have been referred to as Chinese yew under a limited
2012 Wiley Periodicals, Inc.

species concept for yew in China [e.g., Rehder, 1940]. Cultivated species such as Taxus media, which include many cultivars, are largely hybrids involving wild species in Japan that include T. cuspidata Sieb. & Zucc. and European, Southwest Asian, and North African species, T. baccata L., T. fastigiata Lindley, and T. recurvata Hort. ex C. Lawson (Spjut; http://www.worldbotanical. com/introduction). These species and their cultivars provide economic and ecological benets to China. Historically, the Chinese yew has additional names [Li et al., 2011] discovered in the excavation and
*Correspondence to: Pei-Gen Xiao, Institute of Medicinal Plant Development, Chinese Academy of Medical sciences/Peking Union Medical College, Beijing 100193, China. E-mail: xiaopg@public.bta.net.cn Published online in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/ddr.21040

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classication of the historical documents in the Si Ku Quan Shu [Guy, 1987], the largest collection of books in Chinese history from which the property and avor, the channel tropism, and the effects and functions of the Chinese yew were summarized. The use of Taxus for treating cancer has been traced back to Tang Dynasty nearly 1,200 years earlier than what has been reported for the species in Western countries [Li et al., 2011]. Nevertheless, Western science research on Taxus has led to a greater understanding of its chemistry [e.g., Wani et al., 1971; Kong et al., 2007; Wu et al., 2010; Feng et al., 2011; Watchueng et al., 2011], biotechnological applications [Yukimune et al., 1996; Gao et al., 2003; Lee et al., 2010; Hao et al., 2010a, b, c], genomics [Trapp and Croteau, 2001; Hao et al., 2009a, b; Hao et al., 2011a, b; Onrubia et al., 2011; 2011], pharmacology [Ojima et al., 1999; Zhang et al., 2008, 2009], microbial symbionts [Stierle et al., 1993; Zhan et al., 2003; Tan and Cuo, 2006; Hao et al., 2008a; Chen et al., 2009; Soca-Chafre et al., 2011], toxicology [Brown and Hull, 1951; Gausterer et al., 2012], and taxonomy [Spjut, 2007]. This review, however, does not cover the full range of such studies; instead, we focus on recent progress in molecular biology and genomics of Taxus, as well as its chemistry and metabolomics. The bibliometric method is used to quantify and characterize the global scientic production of Taxus-related research.
MOLECULAR BIOLOGY AND GENOMICS OF TAXUS

Molecular Phylogeny, Taxonomy, and Evolution Plants of the genus Taxus are sources of a number of physiologically and pharmacologically active compounds of different classes, especially the anticancer paclitaxel and many other taxane derivatives. There is a large variation in taxane content between the different species and cultivars [van Rozendaal et al., 2000; Hao et al., 2011a]. It is essential to nd suitable plants for the various production protocols of paclitaxel. T. mairei, an endemic Taxus species of southern China, is the most important source plant for 7-xylosyltaxanes that can be converted to paclitaxel and other useful taxanes via chemical transformation and biotransformation [Hao et al., 2008a]. As it is difcult to authenticate and differentiate one species from other source plants of taxanes by morphology, molecular studies are denitely required for solving the problem. The molecular studies with the limited taxon sampling have provided only few detailed insights into relationships within Taxus [Li et al., 2001; Collins et al., 2003; Shah et al., 2008]. Hao et al. [2008b] substantially increased taxonomic sampling of nuclear
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ribosomal DNA and chloroplast DNA for Taxus and provided a comprehensive picture of their phylogeny. One chloroplast (trnStrnQ spacer) and three nuclear (taxadiene synthase (TS), 10-deacetylbaccatin III-10bO-acetyltransferase (DBAT), and 18S ribosomal DNA [rDNA]) molecular markers were combined to infer the interspecic relationship. In Asia, T. chinensis, T. mairei, T. sumatrana, and T. wallichiana cluster together and are sisters to a clade containing T. baccata and T. contorta (i.e., T. fuana). T. yunnanensis is more closely related to T. wallichiana than to four other Taxus species from China; T. contorta is closer to the Euro-Mediterranean T. baccata than to the Asian species. This study provides a genetic method for authentication of economically important Taxus species and proposes a robust phylogenetic hypothesis for the genus. Using trnStrnQ spacer sequences, one is able to distinguish T. mairei from all other species of Taxus. Schirone et al. [2010] used trnStrnQ in the origin analysis of T. baccata in the Azores and found that the Azorean population represents a different evolutionary line within Taxus. In addition, other chloroplast markers, including matK, rbcL, and psbA-trnH, were combined with trnL-F and internal transcribed spacer (ITS) to infer the phylogenetic relationship between and within the families Taxaceae and Cephalotaxaceae [Hao et al., 2008c], and the results were largely congruent with those inferred by trnStrnQ, TS, DBAT, and 18S rDNA [Hao et al., 2008b]. Based on these results, Christenhusz et al. [2011] proposed that the small group Amentotaxus be assigned within the family Taxaceae. A novel alignment-free DNA barcode sequence identication algorithm, BRONX, which accounts for variability observed within taxon and hierarchic relationships among taxa, was proposed based on these DNA marker sequences [Little, 2011]. DNA barcoding is a taxonomic method that uses a short genetic marker in an organisms DNA to identify it as belonging to a particular species. It differs from molecular phylogeny in that the main goal is not to determine classication but to identify an unknown sample according to a known classication [Kress et al., 2005]. Liu et al. [2011] evaluated the utility of ve candidate plant DNA barcoding regions (rbcL, matK, trnH-psbA, trnL-F, and ITS) in Eurasian yews. As single loci, trnL-F and ITS showed the highest species discriminatory power, each resolving 11 of 11 lineages (i.e., barcode taxa). The proposed CBOL core barcode (rbcL + matK) resolved 8 of 11 lineages. Based on overall performance, trnL-F and ITS, separately or combined, are proposed as the barcode for Eurasian Taxus, which can be used to rapidly and reliably identify Taxus species in Eurasia for conservation protection and for monitoring illegal trade. In plants, establishing a

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standardized DNA barcoding system has been more challenging than in animals [Hao et al., 2012b]. Positive selection and repetitive sequences might inuence the discriminatory power of the commonly used DNA barcodes [Hao et al., 2010a,b,d,e]. Notwithstanding, DNA barcoding would be routinely used in the identication and authentication of the medicinal plant species during drug development. Plants synthesize an enormous number of secondary metabolites that provide an increasingly exploited reservoir for the generation of pharmaceutically active agents, and many more await discovery. Over the past 20 years, major advances have been made in the identication of genes responsible for paclitaxel biosynthesis [Croteau et al., 2006], a process requiring more than 20 enzymatic reactions involving the construction of the tetracyclic skeleton and the addition of the various oxygen and acyl functional groups. Among the intermediate steps, the cyclization of geranylgeranyl diphosphate (GGPP) to taxadiene is catalyzed by TS (Koepp et al., 1995), and the acetylation of 10-deacetylbaccatin III (10-DAB) to baccatin III is catalyzed by DBAT. Positive, diversifying selection is an important evolutionary force that accelerates divergence between homologous proteins [Swanson et al., 2001]. As paclitaxel biosynthetic enzymes catalyze the formation of important defense molecules, paclitaxel, and the related taxanes, it is intriguing to study whether the adaptive evolution affects any sites of genes coding for relevant enzymes. Evolutionary patterns of sequence divergence were analyzed in genes TS and DBAT [Hao et al., 2009b]. While both TS and DBAT are, overall, under purifying selection, a number of amino acids of TS under positive selection are identied based on the inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Interestingly, in the X-ray crystal structure of TS, the carboxyterminal catalytic domain is a class I terpenoid cyclase, which binds and activates substrate GGPP with a threemetal ion cluster [Kksal et al., 2011]. The N-terminal domain and a third insertion domain together adopt the fold of a vestigial class II terpenoid cyclase. A class II cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TS structure reveals a denitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis. Moreover, lineages, i.e., T. yunnanensis and T. mairei, with signicantly elevated rates of amino acid substitution, i.e., undergoing adaptive evolution, are identied using a genetic algorithm [Hao et al., 2009b]. Polymorphism-divergence tests of T. baccata populations pointed to positive selection for taxoid-O-

acetyltransferase and taxane 2a-O-benzoyltransferase and balancing selection for DBAT [Burgarella et al., 2012]. These ndings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented in Taxus, where species-specic taxane metabolism has evolved recently, under varying adaptive pressures produced by the host-enemy coevolution. The natural selection processes may have produced taxane variants still undiscovered. Information about codons that are under positive selection and purifying selection is important for studies of plant secondary metabolism and phylogenetics and could facilitate the development of more broadly applicable enzymes for biotransformation of taxanes. Biocatalytic methods offer the potential to produce metabolites that are difcult to synthesize by traditional medicinal chemistry [Hunter et al., 2011]. The substrate selectivity, regioselectivity, and other properties of the taxane biosynthetic enzymes could be altered by directed evolution to produce desired metabolites and drug candidates. Genomics During the past few years, the high demand for low-cost sequencing has driven the development of high-throughput second generation sequencing technologies that parallelize the sequencing process, producing thousands or millions of sequences at once [Hall, 2007; Schuster, 2008]. Roche 454 pyrosequencing and Illumina high-throughput sequencing are based on sequencing by synthesis, SOLiD and Polonator are based on sequencing by ligation, while Helicos and Pacic Biosciences utilize the single molecule sequencing [Egan et al., 2012]. Collins et al. [2008] demonstrated that transcriptome analysis using highthroughput short-read sequencing need not be restricted to the genomes of model organisms. The de novo transcriptome sequencing and characterization based on Illumina second generation sequencing has been performed successfully for many medicinal plants [Hao et al., 2012a]. Illumina produces orders of magnitude more sequences at a fraction of the cost of 454 pyrosequencing. The estimated genome size of Taxus is 10,000 Mb [Leitch and Hanson, 2001], and 20.8% of the fosmid end sequences of the T. mairei gonomic library are repetitive elements [Hao et al., 2011b]. Because the whole genome sequences of Taxus are not available presently, it is realistic to rst perform the transcriptome sequencing for collecting key information of the large and complex Taxus genome. The de novo assembly of T. mairei transcriptome using Illumina pairedend sequencing technology is performed [Hao et al.,
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2011a]. In a single run, 13,737,528 sequencing reads corresponding to 2.03 Gb total nucleotides are produced. These reads were assembled into 36,493 unique sequences. Based on similarity search with known proteins, 23,515 unigenes were identied to have the Blast hit with a cutoff E-value above 10-5. digital gene expression (DGE) tag sequencing is an implementation of the LongSAGE (serial analysis of gene expression) protocol on the Illumina sequencing platform that increases utility while reducing both the cost and time required to generate gene expression proles. The ultra-highthroughput sequencing capability of the Illumina platform allows the cost-effective generation of libraries containing an average of 20 million tags, a 200-fold improvement over classical LongSAGE [Morrissy et al., 2010]. The transcriptome difference of three tissues is investigated with the tag-based DGE system. A large number of genes associated with tissue specic functions and taxane biosynthetic pathway are identied. The expression of the taxane biosynthetic genes is signicantly higher in the root than in the leaf and the stem, while high activity of the taxane-producing pathway in the root is also revealed via metabolomic analyses. In addition, many antisense transcripts and novel transcripts are found; clusters with similar differential expression patterns, enriched gene ontology terms and enriched metabolic pathways with regard to the differentially expressed genes are revealed. Compared with two T. cuspidata transcriptome data sets generated on the 454 sequencing platform [Lee et al., 2010; Wu et al., 2011], this study provides the most comprehensive sequence resource available for Taxus study and will help dene mechanisms of tissue specic functions and secondary metabolism in nonmodel plant organisms. The Illumina sequencing platform was also used to identify microRNAs (miRNAs) from Taxus chinensis cells and to investigate the effect of the taxoid elicitor methyl jasmonate on miRNA expression [Qiu et al., 2009]. Metagenomics is the study of metagenomes, genetic material recovered directly from environmental samples. Basic ndings at the metagenomic levels could be applied to drug discovery and development. The regional variability of the bacterial community composition and diversity was studied by the comparative analysis of three large 16S rRNA gene clone libraries from the Taxus rhizosphere in different regions of China [Hao et al., 2008a]. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that the abundance of sequences afliated with g-proteobacteria, b-proteobacteria, and Actinobacteria was higher in the library from the Taxus media rhizosphere of the temperate region compared with the subtropical T. mairei rhizosphere. On the other hand, Acidobacteria was
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more abundant in libraries from the subtropical T. mairei rhizosphere. Richness estimates and diversity indices of three libraries revealed major differences, indicating a higher richness in the Taxus rhizosphere bacterial communities of the subtropical region and considerable variability in the bacterial community composition within this region. By enrichment culture, a novel Actinobacteria strain DICP16 was isolated from the Taxus media rhizosphere and was identied as Leifsonia shinshuensis sp. DICP16 was able to remove the xylosyl group from 7-xylosyl-10-DAB and 7-xylosyl10-deacetyltaxol (DAT), thereby making the xylosyltaxanes available as sources of 10-DAB and paclitaxel. A xylosidase isolated from DICP16 could hydrolyze 20-C, b-(1 6)-xyloside of ginsenoside Rb3 (G-Rb3) into ginsenoside Rd, but did not hydrolyze the other b-Dglucosidic bonds of G-Rb3 [Luan et al., 2008]. Moreover, two fungal strains and a bacterial strain were chosen to transform sinenxan A, an abundant taxane in Taxus [Zhan et al., 2003]. These studies illustrate the utility of metagenomics and microbial transformation in drug development. In the near future, both the culturedependent and culture-independent approaches should be integrated in the metagonomic, metatranscriptomic, and metaproteomic studies of Taxus rhizosphere and phyllosphere.
CHEMISTRY AND METABOLOMICS OF TAXUS

Chemotaxonomy, also called chemosystematics, is the attempt to classify and identify organisms (originally plants) according to conrmable differences and similarities in their biochemical compositions. Chemotaxonomy-based plant selection is a prerequisite for the successful natural products research. Because of difculty in polymerase chain reaction amplication, molecular markers are very often inapplicable for yew extracts. More importantly, the gene variations cannot represent the variations at the metabolite level that are closely related to the manufacturing process of taxanes. Novel classications based on metabolic analysis are thus highly desirable. During the past 20 years, some substantial quantitative and qualitative variations among different Taxus species have been found using modern analytical techniques [van Rozendaal et al., 2000; Wang et al., 2011]. However, there was no practical chemical classication that can be applied to yew species identication. The systemic analysis of yew constituents is a big challenge, due to the numerous constituents from different classes and varying metabolite levels caused by many nongenetic factors such as development stage, climate, elevation, slope exposure, and others. As a rapid, cost-efcient, and popular analysis method, high performance liquid chromatography (LC)

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ngerprinting has been regarded as the rst choice for medicinal plant identication and quality control [Lu et al., 2005; Xie et al., 2006]. A holistic approach of ngerprint analysis, prole similarity-based clustering, and choice of taxonomic markers capable of capturing the greatest chemical variations is proposed for Taxus classication [Ge et al., 2008b]. Thirty samples representing eight Taxus species are collected and analyzed, and the ngerprint-based data are extracted and processed by hierarchical clustering analysis and principal component analysis (PCA). Based on the PCA loadings, 12 chemical constituents, identied by LC/mass spectrometry (MS), are selected as the chemotaxonomic markers that can be used to establish a more sensible classication. Eight studied species are divided into six well-supported groups, and most samples can be assigned to the correct species. Traditional chemotaxonomic and chemosystematic studies are frequently used to infer relationships among plant taxa, by using the average concentration of several preselected compounds [van Rozendaal et al., 1999]. However, they could not be used to examine the variations within species and may result in wrong conclusions in cases where the intraspecic variation is large [Becerra, 2003; Wink, 2003]. In contrast, prole-based classication can investigate variations within and among species by comparison of ngerprints [Vieira et al., 2003]. Moreover, the ngerprint similarity-based taxonomy, which relies on the ratio of selected constituents, can improve the misclassications caused by large quantitative differences. Metabolomics is the systematic study of the unique chemical ngerprints that specic cellular processes leave behind [Daviss, 2005], i.e., the study of their small-molecule metabolite proles. Gas chromatography coupled to time-of-ight MS was used to prole metabolite changes of T. cuspidata cells under laminar shear stress [Han and Yuan, 2009a]. Yet, LC/MS has been recognized as the most appropriate analytical technique of metabolomics for structural assignment of taxanes in complex samples due to its specicity and structure-characterization ability [Tanaka et al., 2011; Wang et al., 2011]. A lipidomic approach using LC/electrospray ionization (ESI)/MS was employed to prole phospholipid species of T. cuspidata cells under laminar shear stress [Han and Yuan, 2009b]. However, rapid metabolic proling of yew materials is still a challenge, because there are numerous trace ingredients from different classes and many analogues with similar chromatographic behavior, as well as many regio- and stereoisomers [Vivekanandan et al., 2006]. In recent years, ultra-performance LC (UPLC), which employs sub-2 mm stationary phase particles to achieve superior theoretical plates and

Fig. 1. Sturcture of taxine B and taxinine M, two prominent side-route metabolites of Taxus. Highly abundant taxine B was found in the needles of T. baccata and T. cuspidata, while abundant taxinine M was detected in the needles of T. mairei, T. yunnanensis, and T. chinensis.

extremely high resolution in very short analytical times, has attracted the wide attention of pharmaceutical and biochemical analysts [OConnor et al., 2006]. UPLC and its hyphenated system also showed excellent sensitivity and improved peak capability [Novakova et al., 2006]. In 2008, an efcient and sensitive proling approach was developed for the complex yew samples, using UPLC/ESI/MS [Ge et al., 2008a]. The appropriate in-source collision-induced dissociation energy was employed to produce informative characteristic ions that could be used for stereochemical and substructural assignment of yew constituents. The method was successfully applied in the rapid screening of yew hair roots from various species, and 53 constituents including 47 taxoids were detected from the partially puried root extract. C-7 hydroxytaxane stereoisomers could be identied based on their different fragment ions under the optimal proling conditions. Hair roots from different Taxus species exhibited nearly identical chemical distribution, indicating that they had similar metabolic framework [Hao et al., 2011a]. Taxus root resources display benign medicinal prospect because they have relatively simple chemical proles and possess high yields of valuable taxanes such as paclitaxel, cephalomannine, 10-DAT, and 7-xylosyltaxanes. Furthermore, these pharmaceutically important taxanes comprise over 55% of the peak areas in the entire chromatogram (019 min), which is quite different from the needles that usually have major divergent pathways apart from the paclitaxel biosynthesis such as the formation of abundant taxine B and taxinine M (Fig. 1) [Ketchum et al., 2003]. These detected taxanes and their connectivity facilitate further studies on the taxane drug discovery and development from the Taxus root. To date, more than 400 natural taxanes and hundreds of synthetic analogues have been discovered [Wu et al., 2010; Wang et al., 2011]. The core structure of paclitaxel and its analogues consists of four rings with substituents at different carbon sites. Most taxanes have a b-hydroxyl at the C-7 site, and 7a stereoisomers
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Fig. 2. Semisynthesis of docetaxel, the analogue of the anticancer drug paclitaxel.

often coexist because the conversion of 7-hydroxyl from b-equatorial into a-axial orientation is very facile [Tian and Stella, 2008]. 7-epi-paclitaxel and other 7a-hydroxyltaxanes have been detected from natural sources, in the manufacturing process for paclitaxelactive pharmaceutical ingredients, as well as from taxane-related metabolic samples [Ge et al., 2009]. As a common impurity and metabolite of taxane drugs, the monitoring of 7-epi-paclitaxel and other 7a-hydroxyltaxanes is important for quality control and pharmacological research of taxane drugs. In 2009, different ESI-MS methods were utilized to analyze several pairs of taxane stereoisomers including paclitaxel and 7-epi-paclitaxel [Ge et al., 2009]. Both ESI-MS and tandem MS provided stereochemically dependent mass spectra in negative-ion mode, and all studied stereoisomers could be easily discriminated based on their feature ions or distinct fragmentation patterns. MS/MS experiments for several taxane analogues at various collision energies were performed to elucidate potential dissociation pathways. The gas-phase deprotonation potentials were calculated to estimate the most thermodynamically favorable deprotonation site. The results of the theoretical studies agreed well with the fragmentation patterns of paclitaxel and 7-epi-paclitaxel observed from MS/MS experiments. LC/ESI-MS was a useful and sensitive technique for assignment of C-7 taxane stereoisomers from realistic samples. Several solutions have been utilized to overcome the supply crisis and to produce taxane drugs, including plant cell culture [Zhao and Yu, 2005] and semisynthesis from common natural precursors that can be isolated from renewable yew resources such as needles and roots (Fig. 2) [Ge et al., 2008a,b; Hao et al., 2011a]. Several natural taxanes including 10-DAT, 10-DAB, baccatin III, 9-dihydro-13-acetylbaccatin III, cephalomannine, and 10-deacetyl-7-xylosylpaclitaxel can be converted to paclitaxel or docetaxel via a few steps, and they are recognized as the paclitaxel-equivalents or valuable taxanes [Ge et al., 2008a,b; Hao et al., 2011a]. Paclitaxel and these precursors are found in most Taxus species, but their distribution and level are highly variable with species and tissues. Therefore, screening of the valuable yew constituents from different species
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and tissues are crucial to the cultivation of yew trees and cost-effective manufacturing of taxane drugs. However, the rapid, sensitive, and simultaneous determination of target taxanes from yew materials was difcult due to the chemical complexity of the crude yew extract that contains trace amounts of taxanes. UPLC and ultra-fast LC (UFLC), which employ ne stationary phase particles to achieve extreme high resolution with short analytical time as well as good sensitivity, have attracted wide attention of pharmaceutical analysts for its rapid determination of trace constituents from complex samples [Dong et al., 2009]. A rapid and valid method incorporating UFLC with MS and UV detection has been developed for simultaneous determination of paclitaxel and its six semisynthesis precursors in needles and hair roots from various Taxus species [Ge et al., 2010]. All target analytes could be identied by comparing their retention times as well as UV and MS spectra with authentic standards, while seven valuable taxanes in botanical samples can be rapidly determined by UFLC-diode array detector with excellent sensitivity. Analysis of more than 100 yew samples from nine species showed signicant variations in distribution and content of seven evaluated taxanes [Hao et al., 2011a]. Thus, different development strategies should be used for the sustainable utilization of various yew resources.
BIBLIOMETRIC ANALYSIS OF TAXUS RESEARCH

A bibliometric analysis is performed to evaluate the global scientic production of Taxus research, to characterize Taxus research activities, and to identify patterns, tendencies, and regularities of Taxus-related articles. Data are based on the Science Citation Index Expanded from the Web of Science database. Articles referring to Taxus are assessed by the trend of publication output during 19912010. The records are downloaded into Microsoft Excel 2007 (Redmond, WA), and additional coding is manually performed for all data analysis. Globally, 2,916 papers were published during the 20-year study period. The mainstream research on Taxus was in the plant sciences, biochemistry and molecular biology, cardiac and cardiovascular systems, biotechnology, and applied microbiology. The G7

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industrial countries, as well as China and India, held the majority of total world production. Research on the various economically important Taxus species remained the hotspot during the 20-year study period, whereas that on the related topic paclitaxel eluting stents increased dramatically since 2002 [Chen and Zhan, 2011]. The analysis of single words in abstracts is performed to make specic inferences about the scientic literature and identify the subjective focus and emphasis specied by authors. Similar to the distribution of paper titles, taxol, cell(s), and baccata were among the most frequently used single words during 19912010. Interestingly, isolated, analysis, and species were also emphasized in abstracts. Isolation, purication, and phytochemical analyses of paclitaxel and taxoids from the respective Taxus species and endophytic fungi are the hot issues [Guenard et al., 1993; Baloglu and Kingston, 1999]. The analysis of the distribution of the author keywords suggests that, among various Taxus species, T. cuspidata was most commonly studied [Ge et al., 2008b], followed by T. baccata [Parmar et al., 1999], T. chinensis [Eisenreich et al., 1996], T. mairei [Hao et al., 2011a,b], T. yunnanensis [Parmar et al., 1999; Ge et al., 2008a,b], and T. canadensis [Ketchum et al., 1999], while T. wallichiana (Himalayan yew) [Parmar et al., 1999; Ge et al., 2008b], T. brevifolia [Trapp and Croteau, 2001], and T. media [Hao et al., 2008a,b,c] were less commonly studied. Among various Taxus tissues, the stem bark was most frequently studied [Wani et al., 1971; Eldridge et al., 2002], followed by needles [Ge et al., 2008b; Hao et al., 2011a,b], pollen [OLeary et al., 2007; Zimmermann, 2010], seeds [Li et al., 2005; Huo et al., 2007], and roots [Onrubia et al., 2011; Hao et al., 2011a]. With synthetic analysis of the word in article title, author keyword, abstract, and KeyWords Plus (table available upon request), some Taxus research hotspots have been noticed. Taxus cell culture systems, which are commercially successful, allow for sustainable production of Taxus secondary metabolites that is not limited by the low yields associated with natural harvest or the high cost associated with complex chemical synthesis [Wilson and Roberts, 2012]. Semisynthesis from the precursor baccatin III or 10-DAB, which can be extracted from Taxus leaves and roots, proved to be another valid method for commercial production of paclitaxel [Fu et al., 2009]. Encouraging ndings with endophytic fungi of Taxus resulted in much interest in the prospect of using endophytes as the producer of paclitaxel and other taxanes [Miller et al., 2008; Soca-Chafre et al., 2011]. The genome size of fungi is much smaller than that of Taxus, making it easier to perform the whole genome sequencing, assembly, and bioinformatic prediction for the endophytes, which would lay a solid foundation for the biosynthesis studies,

metabolic engineering and high-yield strain breeding. The application of compounds derived from Taxus in clinical cardiology, pharmacology, and oncology, and research related to Taxus chemistry, metabolism, cytology, and microbiology are the ongoing Taxus-related research in the 21st century. Gaps are still present in knowledge about the genomics, epigenomics, transcriptomics, proteomics, metabolomics, and bioinformatics of Taxus and their endophytic fungi. For example, only the conventional two-dimensional gel electrophoresis was used to study the responses of T. cuspidata cells to local microenvironments in different zones of immobilized support matrices [Cheng and Yuan, 2006]. The proteomic study of Taxus is still in its infancy. Epigenomics is the study of the complete set of epigenetic modications on the genetic material of a cell, known as the epigenome. It is unknown how the epigenomic mechanisms regulate the biosynthesis of secondary metabolites in Taxus.
CONCLUSION AND PROSPECTS

Plant-derived natural products hold great promise for discovery and development of new pharmaceuticals. Careful consideration of the entire process of discovery and development, a systems approach, will be required to realize this promise effectively. The biological and chemical studies of Taxus with respect to the emerging use of omics technologies provide a paradigm for the active integration of various state-of-the-art methodologies into early stage drug research and development. Both aspects are complementary and indispensable in taxonomy and authentication of Taxus, as well as in the elucidation of the correlation between the genotype and the metabolic phenotype. Although semisynthesis and subsequent plant cell culture-based production efforts have decreased the need for harvesting the endangered yew tree, production still depends on plant-based processes. Recent developments in metabolic engineering and synthetic biology offer new pathways for the overproduction of complex natural products by optimizing more technically amenable microbial hosts (e.g., Escherichia coli) [Ajikumar et al., 2010; Morrone et al., 2010]. Confronting the environmental challenge, the regulation of Taxus biological processes has to be of multiple levels, i.e., genomic level, epigenomic level, transcriptional and posttranscriptional levels, and translational and posttranslational levels. Knowledge about the regulation of almost all these levels is lacking, although it is essential for the sustainable development and utilization of the Taxus medicinal resources. Systems biology and various omics technologies will play an increasingly important role in the coming decades.
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ACKNOWLEDGMENTS

The study is supported by the National Project Seed Fund (20112012) of Dalian Jiaotong University and the Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, China. The authors thank Richard W. Spjut (World Botanical Associates, Bakerseld, CA, USA) for his reviewing and polishing the original manuscript.

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