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Metabolomics analysis of major metabolites in medicinal herbs

Chin Chye Teo,*ab Swee Ngin Tan,*a Jean Wan Hong Yong,c Tenmoli Ra,a Peiling Liewab and Liya Gea
Received 8th June 2011, Accepted 22nd September 2011 DOI: 10.1039/c1ay05334e
Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs.rsc.org | doi:10.1039/C1AY05334E

A metabolomics approach with a platform consisting of GC-MS, 1H NMR and HPLC-UV was used to investigate the regulatory mechanisms of metabolic pathways in Stevia rebaudiana and Coptidis rhizoma. The data suggested that glycolysis and the oxidative pentose phosphate pathway (OPPP) remained as the main components of plant respiration in these botanicals. However, the absence of citrate, succinic acids, glutamate and fumarate in Stevia suggested that the tricarboxylic acid (TCA) cycle was not required extensively to synthesize its bioactive diterpene glycosides. The results further suggested that the carbon ow was directed between the shikimic acid and methylerythritol 4-phosphate (MEP) pathway. For the Coptidis, the use of an alternative biosynthesis route with proline, phenylalanine, catechollactate and 2-mono-isobutyrin was proposed to synthesize its major bioactive alkaloids. It was observed that different fatty acids might be needed to modulate the biosynthesis of the bioactive secondary metabolites in the medicinal herbs. Concurrently, primary and secondary metabolite proling was studied by unsupervised multivariate Principal Component Analysis (PCA) to discriminate and assign the different phenotypes under various cultivation conditions. Hence, the simultaneous proling of primary and secondary metabolites could signicantly aid the existing functional genomics approaches to study the biosynthesis function in medicinal herbs.

Introduction
Metabolomics is the study of the proles of organic compounds or metabolites present in a biological system.13 Plant metabolomics seeks to associate changes in metabolite levels with yield, disease resistance, nutritional traits and gene function.4 In brief, these metabolites could be intermediates and end-products from either primary or secondary metabolic processes.5 The primary metabolites such as amino acids, lipids and carbohydrates are involved in essential plant physiological processes like glycolysis and the tricarboxylic acid (TCA) cycle, which release energy from organic compounds by oxidative reactions.6,7 However, the secondary metabolites are structurally more diverse and they are divided into ve major groups: polyketides, isoprenoids (e.g. terpenoids and steroids), alkaloids, phenylypropanoids and avonoids.8 There are more than 100 000 bioactive secondary metabolites produced by plants via certain biosynthetic pathways such as acetate, shikimate and mevalonate.810

a Natural Sciences and Science Education Academic Group, Nanyang Technological University, 1 Nanyang Walk, Singapore, 637616, Singapore. E-mail: sweengin.tan@nie.edu.sg; chinchye_teo@yahoo.com. sg; Fax: (+65) 6896 9414 b Institute of Advanced Studies, Nanyang Technological University, Nanyang Executive Centre #02-18, 60 Nanyang View, Singapore, 639673, Singapore c Singapore University of Technology and Design, 20 Dover Road, Singapore, 138682, Singapore

In metabolomics studies, reproducible and robust spectroscopic and chromatographic analytical techniques coupled with sensitive detectors such as a mass spectrometer have been established.1123 High Performance Liquid Chromatography coupled with a Photodiode Array Detector (HPLC-PDA) is usually used for secondary metabolite proling while Gas Chromatography coupled with Mass Spectrometry (GC-MS) and Nuclear Magnetic Resonance (NMR) are popular techniques to study the primary metabolite proling in plants. However, it is currently impossible to perform the analysis of primary and secondary metabolites present in complex plant extracts using a single analytical technique. Metabolite proling of either primary or secondary metabolites present in plants by these techniques has been widely reported.20,22,2431 The obtained metabolite proles could provide snapshots to study the regulatory mechanisms of metabolic pathways in the plant system. Hence, a metabolomics approach could investigate the major glycolysis pathway that fuels respiration in plants to provide energy for biosynthesic reactions.9,10 This approach could also study pyruvate oxidation which is a linkage between glycolysis and the TCA cycle.9,10 To handle the large chemical data sets in metabolomics studies, unsupervised and supervised multivariate methods such as Principle Component Analysis (PCA), Hierarchical Cluster Analysis (HCA) and Partial Least SquareDiscriminant Analysis (PLS-DA) are popular for the global classication of samples and also provide a good summary of data concerning functional relations between metabolites.13,29,3234 These methods could provide deep insights into
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regulatory processes and were also able to directly determine the phenotype.3335 The PCA technique is usually the rst step in the evaluation and detection of patterns, trends and groups among samples in these multivariate data.5 In this study, we attempt to analyse the key metabolites in two different medicinal herbs with a platform comprising of GC-MS, 1 H NMR and HPLC-UV. Using the metabolite proling obtained with PCA, we could discriminate the herbs grown under different Good Agriculture Practice (GAP) cultivation conditions and also of different geographical origins. Concurrently, a metabolomics approach linking the primary and secondary metabolite proling was developed for the identication of intermediates in novel biosynthetic pathways for the conversion of major primary metabolites to bioactive secondary metabolites in medicinal herbs.

Experimental
Chemicals and standards Stevioside (SV), rebaudiosides A (RA) and steviol were purchased from Chromadex Inc (St. Santa Ana, CA, USA). Berberine (BR) (purity > 98%), N,O-bis(trimethylsilyl)triuoroacetamide (BSTFA) (purity > 99%), deuterated water (D2O) and other chemical reference standards (proline, serine, tyrosine, lysine, phenylalanine, glycine, leucine, tryptophan, fructose, glucose, galactose, sucrose, lactose, maltose, mannose, cinnamic acid, fumaric acid and citric acid) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol (MeOH), acetonitrile (ACN) and formic acid (FA) were purchased from Fisher Scientic (MA, USA). Ammonium acetate and acetic acid (AA) were purchased from Merck (Darmstadt, Germany). Pyridine was bought from Univar (Rotterdam, The Netherlands). Ultra-pure water was obtained from a Millipore Alpha-Q water system (Millipore, Bedford, MA, USA). Plant materials and reference standards preparation Stevia rebaudiana under different GAP cultivation conditions. The rst condition (wild type) was obtained from the USA. They were freeze-dried using an Alpha 1-2 freeze dryer (Martin Christ, Germany) and ground using an IKA MF10 microne grinder (Staufen, Germany) and sieved through an insert of pore size 0.5 mm. The second to fth conditions were grown in vitro in Murashige & Skoog Medium (MS basal salt medium) spiked with 2% (w/v) sucrose with 5 different growth hormones: 0.02 mg L1 indole-3-acetic acid (IAA), 0.02 mg L1 6(3-hydroxybenzylamino)purine meta-Toplin (mT), 0.02 mg L1 6-benzylaminopurine (BA) and 0.5 mg L1 kinetin (K) (PhytoTechnology Labs, Kansas). The sixth condition was a control. After 12 weeks, all plants were freeze-dried and reduced to powder form using a pestle and mortar. Coptidis rhizoma of different geographical origins. The dried Coptidis rhizoma was purchased from three different Chinese medicinal halls (Kluang, Malaysia). The herbs were ground into powders as described for Stevia. Reference standards for HPLC analysis. Stock solutions of SV, RA and BR at 1000 mg L1 were prepared in methanol. The
Anal. Methods

linearity of each standard was established between 10 to 100 mg L1 with correlation coefcient r2 $ 0.999. To quantify the marker compounds in the botanicals, a three point calibration based on the linearity established was used. The system precisions (RSD, n 6) for each standard were found to be less than 1% on different days. The reproducibility of the retention time and peak area for the marker compounds was investigated under optimum HPLC conditions. It was achieved by performing repeated injections (n 6) of a mixture of the standards at a concentration of 60 mg L1. It was found that a relative standard deviation (RSD) of less than 0.06% could be obtained for retention time and ca. 0.52% for peak area. The limits of detection (LOD) and quantication (LOQ) under the present chromatographic conditions were determined at a signal-to-noise (S/N) of around 3 and 10 respectively. The LODs and LOQs for the marker compounds were determined to be from 1.0 to 1.2 mg L1 and from 3.2 to 3.8 mg L1 respectively. Reference standards for GC-MS analysis. Stock solutions of all the chemical reference standards were prepared at 200 mg L1 in pure pyridine, after which 50 mL of each standard was incubated in the dark with 60 mL BSTFA for the derivatization step. After 4 h, each of the derivatized analytes was then transferred to an amber vial for GC-MS analysis. Extraction methods Primary metabolites using methanol aqueous mixture. Typically 50 mg of the ground herb powder (n 3) was accurately weighed in an Eppendorf tube. Then 1 mL of an aqueous mixture of methanol (1 : 1) was added to the sample and vortexed for 1 min. The sample was centrifuged in a Mikro 20 centrifuge (Hettich Zentrifugen, Tuttlingen, Germany) for 5 min at 13 000 rpm. The supernatant was collected and then evaporated to dryness using a Thermo Fisher Scientic model SPD 2010-230 speedvac system (MA, USA). The derivatization conditions were studied with different volumes (20, 40, 60 and 80 mL) of BSTFA in pure pyridine under various incubation times (4 h, 8 h, 16 h and 24 h). The optimised conditions were then determined to be an addition of 100 mL pure pyridine to reconstitute the dried sample followed by 60 mL BSTFA before incubation in the dark for 24 h prior to GC-MS analysis. Secondary metabolites by green-solvent microwave-assisted extraction (MAE). A closed vessel system (under controlled temperature and pressure) was employed using Start E from Milestone (Sorisole, Italy). A 0.1 g plant sample was accurately weighed into the vessel. Extraction was carried out under optimized conditions, i.e. 100  C for 10 min for Stevia and 20 min for Coptidis, as described in our previous work.24,25 A sample for each extract was transferred to the 2 mL Eppendorf tube and centrifuged in a Mikro 20 centrifuge (Hettich Zentrifugen, Tuttlingen, Germany) for 5 min at 12 000 rpm. The supernatant was collected for HPLC analysis. Analysis Gas chromatography-mass spectrometry (GC-MS). A Shimadzu GC 17A system (Kyoto, Japan), equipped with an auto-sampler model AOC-20i and a split/splitless injector, was
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used with a HP-5MS capillary column (5-phenyl)-methylpolysiloxane; 30 m, 0.25 mm i.d., 0.25 mm lm thickness (Santa Clara, CA, USA), interfaced to a Shimadzu QP5000 MS singlequadrupole system (Kyoto, Japan). The GC inlet temperature was set at 280  C and MS interface 300  C. Following the injection of 1 mL of sample, the oven temperature programme was as follows: an initial temperature of 100  C was raised to 300  C at 10  C min1, and held for 2 min with a total run time of 22 min. The inlet was operated in splitless mode, with 0.7 min purge-on time. Helium was the carrier gas (>99.99%), with a constant ow of 1 mL min1. The MS was performed in positive electron ionization (EI) mode with an ionization energy of 70 eV. The detector signal was recorded from 3 min after injection until 40 min, and ions were scanned across the range of 50720 atomic mass units (amu). Nuclear magnetic resonance (1H NMR). Phosphate buffer (0.2 M, pH 6) was prepared with D2O as the solvent. Each dried plant extract was reconstituted with 400 mL D2O containing 0.05% trimethyl silane propionic acid sodium salt (TSP) as an internal standard (IS) and 200 mL buffer. This solution was then transferred into an NMR tube (5 mm O.D., 7 inch length, SigmaAldrich). 1D 1H NMR spectra were obtained using a Bruker 400 MHz US2 spectrometer operating at 400.12 MHz observation frequency. Aqueous extracts were analyzed using a pre-saturation pulse sequence (relaxation decay-901 acquisition). The water resonance was eliminated by applying a secondary radio frequency irradiation during the relaxation delay of 1.0 s. A total of 64 scans was accumulated over a spectral width of 4844 Hz into 64 K data points with an acquisition time of 3.4 s. The NMR spectra were manually processed using XWIN-NMR software (3.5 version, Bruker, Germany), with reference to internal TMS (d 0.0) and auto phase correction was used. The assignment of metabolites in the 1D 1H NMR spectra was performed by 2D NMR using protocols published elsewhere.18,19,22,23,36,37 High performance liquid chromatography-photodiode array detector (HPLC-PDA). A Waters 2790 Alliance HPLC (Milford, Massachusetts, USA) was employed to separate and quantify the amount of extracted marker compounds. The analysis of the extracts was performed with a Waters XTerra C18 (150 3.9 mm, 5 mm) maintained at 40  C. For all experiments, 10 mL of standards and sample extracts were injected. For Stevia, gradient elution was carried out with acidied water (0.1% FA) as mobile phase A (MPA) and acidied MeOH (0.1% FA) as mobile phase B (MPB). The initial condition was set at 10% MPB and ramped to 100% MPB over 25 min before returning to the initial condition for the next 10 min. A ow rate of 0.7 mL min1 was used with a detection wavelength at 210 nm. For Coptidis, gradient elution was carried out with acidied 50 mM ammonium acetate (0.1% AA) as MPA and acidied ACN (0.1% AA) as MPB. The initial condition was set at 30% MPB with a gradient up to 100% MPB over 15 min before returning to the initial condition for the next 10 min. A ow rate of 1.0 mL min1 was used with a detection wavelength of 254 nm. Data analysis with PCA For GC-MS, each sample was represented by a Total Ion Chromatogram (TIC). Among the detected peaks,
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a multi-dimensional vector was constructed manually to characterize the biochemical patterns. Each vector was normalized to the total sum of vector intensity, thereby partially accounting for concentration due to the different sample sizes used. The peaks due to column bleed and derivatization reagent were removed. The identication of peaks was based on the use of reference standards and NIST 98 library. The mass spectra obtained was inspected manually and only those compounds with probability matching higher than 90% were considered. For HPLC-PDA, the peak areas of the chromatographic ngerprints at different wavelengths were obtained as follows: Stevia at 210, 230, 250, 254, 260, 270 and 280 nm, and Coptidis at 230, 240, 250, 254, 260, 270 and 280 nm, and were used as input data. A total of 200 peaks for each botanical were computed for their peak areas. The peak area for each chromatogram was normalized to a constant sum. All these results were input into a Simca-P+ Software Package (Umetrics, Umea, Sweden) for subsequent evaluation of the similarities of different chromatograms based on PCA. PCA translates the peak areas obtained from n-dimensional variables into principal components (PC) where there is a score describing different chromatograms obtained. PCA score plots have been used for the classication of samples from their measured properties. The distribution pattern generated from the data in these plots can be correlated with the general characteristics of the samples analyzed.

Results and discussion

Metabolite proling with PCA The primary metabolite proling obtained by the GC-MS and 1H NMR techniques for the aqueous methanol extracts of Stevia and Coptidis are shown in Fig. 1 and 2. Using the two complementary techniques, the metabolites detected in these plant extracts could be classied into amino acids, sugars and others (inclusive of organic acids) (Tables 1 to 4). It was worthy of note that common polar and slightly non-polar metabolites in these plants could be determined using a simple single step extraction and the number of metabolites detected was consistent with other reports.11,12,3842 These data showed that the current method, in the absence of internal standard, showed high reproducibility both on the same day and on different days. The precision and accuracy of the GC-MS method were investigated by analyzing six injections of different plant extracts. The migration time of the peaks was found to be stable both intraday and interday with variation less than 0.5% (relative standard deviation (RSD), n 6). The RSD values for normalized peak areas for different compounds for intraday (n 6) and interday (n 6) analyses were found to vary from 1.06 to 7.45% respectively. The metabolites detected using GC-MS on different plant extracts were summarized in Table 1 (RSD varies from 0.76 to 9.09%, n 3) and in Table 2 (RSD varies from 1.24 to 10.04%, (n 6). Sucrose, a major metabolite involved in glycolysis pathways, was detected in the extracts of the two plants using GC-MS (Tables 1 and 3) and also showed a signal at d 5.40 in the 1H NMR spectra (Fig. 2). From Tables 1 and 3, other sugar molecules such as glucose, fructose, galactose, which were products of the glycolysis process, were also detected by the GC-MS method.
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Fig. 1 (A) A mixture of pure standards: 1, proline; 2, serine; 3, leucine; 4, phenylalanine; 5, tyrosine; 6, glucose; 7, citric acid; 8, cinnamic acid; 9, octadecanoic acid; 10, fructose; 11, sucrose and 12, lactose. A representative Total Ion Chromatogram (TIC) from the GC-MS analysis of derivatized components in the methanol aqueous mixture fraction of (B) Stevia rebaudiana: 1, proline; 2, serine; 3, leucine; 4, phenylalanine; 5, inositol; 6, tyrosine; 7, glucose; 8, lysine; 9, cinnamic acid; 10, octadecanoic acid; 11, fructose; 12, sucrose and 13, lactose. (C) Coptidis rhizoma: 1, proline; 2, butanedioic acid; 3, phenylalanine; 4, arabinose; 5, 2-monoisobutyrin; 6, b-DL-arabinopyranose; 7, D-ribofuranose; 8, glucofuranoside; 9, arabinoic acid; 10, citric acid; 11, glucose; 12, fructose; 13, ribitol; 14, palmitic acid; 15, catechollactate; 16, linoleic acid; 17, oleic acid; 18, lactose; 19, sucrose and 20, maltose.

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Fig. 2 A representative 1H NMR spectrum of the aqueous mixture fraction obtained from plant extracts of (A) Stevia rebaudiana: IS: internal standard; 1, leucine; 2, lactate; 3, proline; 4, fructose; 5, sucrose; 6, tyrosine and 7, phenylalanine. Note: Absence of citrate peak at d 2.54ppm and (B) Coptidis rhizoma: IS: internal standard; 1, alanine; 2, acetic acid; 3, proline; 4, pyruvate; 5, succinic acid; 6, citrate; 7, fructose; 8, sucrose and 9, phenylalanine.

However, the b-glucose (d, d 4.64) and a-glucose (d, d 5.23) signals which are usually present in NMR spectra were eliminated by the water suppression programme, and thus they were not detected (Fig. 2). The fructose molecule from the breakdown of starch showed signals at d 3.70, 3.78, 3.88 and 4.10 in the extracts of the two plants (Tables 2 and 4). Other important metabolites such as phenylalanine and tyrosine, which were synthesized from phosphoenolpyruvate (PEP) in glycolysis via the shikimate pathway, were also detected by GC-MS and 1H NMR analysis (Tables 1 to 4). These molecules are important building blocks for the C6C3 class of secondary metabolites such as the avonoids and terpenoid quinones. However, citrate, which was a typical product of the TCA cycle, was absent from the Stevia extracts but it showed a signal at d 2.51 in the 1H NMR spectra obtained for Coptidis extracts (Fig. 2). The ndings were consistent with the GC-MS data shown in Table 1. The observation indicated that the biosynthesis pathways for certain metabolites in the two medicinal herbs might be different. It was noted that the types of metabolites detected for Stevia grown under nutrient-controlled conditions with different growth hormones were similar (Table 1). However, the concentrations of key metabolites were rather different for the Stevia cultivated under different nutrient-controlled conditions compared to the wild type (Table 1). The tissue-cultured controlled conditions with auxin (IAA) and cytokinins (6BA, mT and K) hormones might have sufcient nutrients to promote the
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glycolysis process to generate higher concentrations of metabolites compared to the wild type. Compared to the control group, the lower concentration of sucrose in IAA, BA, mT and K conditions suggested that this key molecule might be used extensively to promote the glycolysis process to synthesize the other metabolites. Thus, these auxins and cytokinins were deemed suitable to promote the biosynthesis of key primary metabolites for subsequent conversion into bioactive secondary metabolites in these plants. In contrast, the Coptidis was found to possess smaller amounts of amino acids but greater amounts of sugars and fatty acids compared to Stevia (Table 3). The detection of citrate indicated that the TCA cycle was involved in the biosynthesis of other metabolites such as proline and butanedioic acid (Tables 3 and 4). Since the Coptidis was dried rhizomes, the results revealed that the primary metabolites were mainly carbohydrates and lipids, which is consistent with another report43 (Table 3). The detection of butanedoic acid (Peak 2 in Fig. 1C), which has antifungal and antibacterial activity, was consistent with various reports.44,45 The secondary metabolite proling obtained by HPLC-UV for Stevia and Coptidis were shown in Fig. 3. The various marker compounds (SV, RA and BR) present in these plant extracts were quantied by the same technique (Tables 5 and 6). Due to the complexity of the botanical extracts, the extraction method could have an important effect on the chromatographic proles obtained for the determination and proling of secondary metabolites in medicinal plants.24 Hence, the method used for the proling of secondary metabolites in the current work was validated in our earlier work.24 To assign metabolic phenotype of the medicinal herbs analyzed, pattern recognition tools such as PCA were used. The PCA score plots showed the similarities and differences in the primary and secondary metabolite proling obtained from medicinal herbs of different sources and cultivation under GAP conditions (Fig. 4 and 5). It was noted that the concentration of metabolites extracted for each growth condition was rather different for each botanical (Tables 1, 3, 5 and 6). Hence, due to the diversity in the biosynthesis of these metabolites, their proles obtained with PCA could be used to classify plants of different origins for quality control purposes. Compared to primary metabolites, the proling of secondary metabolites alone in Stevia could not distinguish botanicals from different sources (Fig. 4A and 5A). Hence, a combination of chromatographic ngerprints based on the primary and secondary metabolite proling with PCA could provide more comprehensive and insightful information to evaluate the quality of medicinal plants from different GAP cultivation conditions (Fig. 4A and 5A). For Coptidis, the primary and secondary metabolite prolings could clearly distinguish their different origins (Fig. 4B and 5B). In general, the physical environmental conditions such as nature of soil, climate changes and light intensity could inuence the biosynthesis of the metabolites in plants.46 Metabolomics approach and biosynthesis of major plant metabolites Terpenes in Stevia rebaudiana. Stevia contains mainly secondary metabolites such as monoterpene, diterpene and sesquiterpene, while signicant amounts of phenolic
Anal. Methods

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Table 1 GC-MS analysis of primary metabolites in Stevia rebaudiana under different cultivation conditions. W: Wild, Ctr: Control, IAA, mT, K, BA: cultured in different growth media Normalized peak intensitya Compounds Amino acids b L-Proline Serine
b

Retention time (min)

0.02 mg L1 IAA 0.02 mg L1 BA (n 3) (n 3) 2.63 0.02 (RSD: 0.76%) 7.17 0.18 (RSD: 6.69%) 0.20 0.01 (RSD: 5.00%) 1.81 0.07 (RSD: 3.86%) 0.46 0.01 (RSD: 2.17%) 0.13 0.01 (RSD: 7.69%) 11.38 0.11 (RSD: 0.97%) 2.19 0.03 (RSD: 1.37%) 9.91 0.04 (RSD: 0.40%) 2.70 0.01 (RSD: 0.37%) 0.74 0.01 (RSD: 1.35%) 2.76 0.07 (RSD: 2.53%) 2.71 0.01 (RSD: 0.36%) 1.21 0.01 (RSD: 0.83%) 2.12 0.09 (RSD: 4.24%) 7.34 0.13 (RSD: 1.77%) 0.79 0.02 (RSD: 2.53%) 0.79 0.04 (RSD: 5.06%) 0.40 0.04 (RSD: 10.0%) 0.85 0.01 (RSD: 2.50%) 6.97 0.22 (RSD: 3.15%) 1.14 0.01 (RSD: 0.87%) 3.64 0.10 (RSD: 2.74%) 3.62 0.06 (RSD: 1.65%) 0.75 0.03 (RSD: 4.00%) 4.76 0.37 (RSD: 7.77%) 9.37 0.12 (RSD: 1.28%) 0.56 0.01 (RSD: 1.78%)

0.02 mg L1 mT (n 3) 0.61 0.01 (RSD: 1.63%) 4.53 0.11 (RSD: 2.42%) 3.13 0.05 (RSD: 1.59%) 4.16 0.09 (RSD: 2.16%) 1.16 0.01 (RSD: 0.86%) 0.39 0.01 (RSD: 2.56%) 9.67 0.78 (RSD: 8.07%) 1.73 0.02 (RSD: 1.16%) 2.32 0.05 (RSD: 2.16%) 3.88 0.05 (RSD: 1.29%) 0.89 0.02 (RSD: 2.25%) 3.23 0.08 (RSD: 2.47%) 1.89 0.01 (RSD: 0.53%) 1.18 0.04 (RSD: 3.39%)

0.5 mg L1 K (n 3) 0.54 0.02 (RSD: 3.70%) 2.11 0.07 (RSD: 3.31%) 0.26 0.01 (RSD: 3.84%) 0.55 0.01 (RSD: 1.82%) 0.16 0.01 (RSD: 6.25%) 3.35 0.16 (RSD: 4.77%) 1.20 0.06 (RSD: 5.00%) 6.56 0.10 (RSD: 1.52%) 3.35 0.06 (RSD: 1.79%) 0.83 0.03 (RSD: 3.61%) 5.60 0.15 (RSD: 2.67%) 3.94 0.14 (RSD: 3.55%) 2.48 0.03 (RSD: 1.21%)

Control (n 3) 1.85 0.04 (RSD: 2.16%) 4.71 0.08 (RSD: 1.69%) 0.43 0.01 (RSD: 2.32%) 0.56 0.03 (RSD: 5.35%) 0.42 0.01 (RSD: 2.38%) 9.23 0.51 (RSD: 5.52%) 2.11 0.06 (RSD: 2.84%) 6.39 0.12 (RSD: 1.87%) 6.56 0.45 (RSD: 6.85%) 0.99 0.02 (RSD: 2.02%) 4.09 0.23 (RSD: 5.62%) 1.76 0.08 (RSD: 4.55%) 1.03 0.06 (RSD: 5.82%)

Wild type (n 3) 0.11 0.01 (RSD: 9.09%) 1.99 0.11 (RSD: 5.52%) 3.14 0.02 (RSD: 0.64%) 0.31 0.01 (RSD: 3.22%) 0.17 0.01 (RSD: 5.88%) 0.22 0.01 (RSD: 4.54%) 1.60 0.08 (RSD: 5.00%) 1.33 0.03 (RSD: 2.25%) 0.21 0.02 (RSD: 9.52%) 1.02 0.06 (RSD: 5.88%) 0.43 0.02 (RSD: 4.65%) 6.81 0.53 (RSD: 7.78%) 6.43 0.36 (RSD: 5.59%) 0.43 0.01 (RSD: 2.32%)

3.52, 11.50 4.68 17.63, 18.95 18.71, 19.13 5.35

b

b L-Tyrosine

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L-Lysine

Leucineb

Phenylalanine 10.58 Sugars b D-Fructose

b D-Glucose b D-Galactose

16.78, 28.18, 30.13, 32.30 17.24, 17.78, 18.39, 20.32 17.74, 18.65 31.96, 32.41, 32.82, 33.14, 35.99 32.05 16.57, 19.65

Sucroseb Lactose Inositol

Fatty acids 19.65 Cinnamic acidb Octadecanoic 20.80 acid

a

Values are represented as mean SD. b denotes those metabolites identied with standards and searches from NIST Mass Spectral Library 2002.

Table 2

H NMR chemical shift assignments of the metabolites of the aqueous methanol extracts of Stevia rebaudiana (wild type) Aqueous components assignment Leucine Proline Acetic acid Lactate Fructose

No. 1 2 3 4 5

Chemical shift (ppm) 0.961 2.341 3.360 1.94 1.340 3.709 3.786 3.885 4.109 5.401 7.401 6.970

Multiplicity t m m s d m

Normalized peak intensitya(n 3) 0.123 0.009 0.065 0.009 0.72 0.05 0.83 0.05 0.46 0.03 0.45 0.04 0.67 0.06 0.24 0.01 0.11 0.01 0.029 0.001 0.25 0.01 0.28 0.01

6 7 8
a

d m d

Sucrose Phenylalanine Tyrosine

Values are represented as mean SD.

compounds and phenylpropanoids are not found.9,24,47,48 Steviol glycosides are diterpenoids whose biosynthesis pathway shares four common steps with gibberellic acid formation.47 The biosynthesis of steviol glycosides was proposed by the plastid localized methylerythritol 4-phosphate (MEP)
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pathway.9,48 The plant gene for the rst step in the MEP pathway was deoxyxyulose-5-phosphate synthase, which led to the synthesis of deoxyxyulose-5-phosphate from pyruvate and glyceraldehyde 3-phosphate. Steviol was proposed as a key intermediate for the biosynthesis of SV and RA but it was not
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Table 3 GC-MS analysis of primary metabolites in Coptidis rhizoma obtained from different sources (S1S3) Normalised peak intensitya Compounds
b L-Proline

Retention time (min)

Source 1 (n 6) 0.18 0.01 (RSD: 5.56%) 0.18 0.01 (RSD: 5.56%) 2.98 1.79 0.70 0.91 0.58 0.23 0.29 0.42 1.77 0.08 (RSD: 2.68%) 0.07 (RSD: 3.91%) 0.01 (RSD: 1.42%) 0.03 (RSD: 3.29%) 0.01 (RSD: 1.72%) 0.01 (RSD: 4.34%) 0.01 (RSD: 3.44%) 0.01 (RSD: 2.38%) 0.06 (RSD: 3.38%)

Source 2 (n 6) 0.30 0.01 (RSD: 3.33%) 0.28 0.01 (RSD: 3.57%) 3.59 0.31 (RSD: 8.63%) 1.35 0.01 (RSD: 0.74%) 2.94 0.18 (RSD: 6.12%) 2.09 0.21 (RSD: 10.04%) 2.27 0.13 (RSD: 5.72%) 0.34 0.02 (RSD: 5.88%) 0.26 0.01 (RSD: 3.84%) 0.55 0.01 (RSD: 1.82%) 0.27 0.01 (RSD: 3.70%) 2.70 0.13 (RSD: 4.81%) 0.16 0.01 (RSD: 6.25%) 0.28 0.01 (RSD: 3.57%) 0.14 0.01 (RSD: 7.14%) 1.89 0.14 (RSD: 7.40%) 0.41 0.01 (RSD: 2.43%) 2.09 0.18 (RSD: 8.61%) 4.66 0.29 (RSD: 6.22%) 2.06 0.07 (RSD: 3.39%) 0.28 0.03 (RSD: 10.71%) 0.47 0.01 (RSD: 2.12%)

Source 3 (n 6) 0.10 0.01 (RSD: 10.0%) 0.11 0.01 (RSD: 9.09%) 4.04 0.58 5.80 1.20 0.67 0.36 0.55 0.98 0.27 0.07 (RSD: 1.73%) 0.04 (RSD: 6.89%) 0.42 (RSD: 7.24%) 0.02 (RSD: 1.67%) 0.03 (RSD: 4.47%) 0.01 (RSD: 2.78%) 0.02 (RSD: 3.63%) 0.02 (RSD: 2.04%) 0.01 (RSD: 3.70%)

Amino acids 3.52, 11.50 10.58 17.24, 17.78, 18.39, 20.32 17.74, 18.65 16.78, 28.18, 30.13, 32.30 31.96, 32.41, 32.82, 33.14, 35.99 32.05 16.69, 33.58, 33.88 17.51, 18.59, 19.84 18.13 12.33, 13.07 5.52, 9.44 20.74 23.79 23.90 21.58 15.67 16.58, 17.51 16.79 16.34, 17.29 15.35, 15.79, 15.98, 17.05 17.02

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Phenylalanineb Sugars b D-Glucose b D-Galactose b D-Fructose Sucroseb Lactose Maltoseb Mannoseb Ribitolb D-Arabinose Fatty acids Butanedioic acid Palmitic acid Linoleic acid Oleic acid Others Catechollactate 2-Mono-isobutyrin Beta-DL-Arabinopyranose D-Ribofuranose Glucofuranoside Arabinoic acid Citric acidb
a

20.96 0.30 (RSD: 1.43%) 0.11 0.01 (RSD: 9.09%) 0.66 0.01 (RSD: 1.52%) 0.56 0.01 (RSD: 1.78%) 3.78 1.51 3.97 7.23 1.78 0.64 0.12 0.07 (RSD: 1.85%) 0.06 (RSD: 3.97%) 0.10 (RSD: 2.51%) 0.09 (RSD: 1.24%) 0.03 (RSD: 1.68%) 0.02 (RSD: 3.12%) 0.01 (RSD: 8.33%)

19.36 0.32 (RSD: 1.65%) 0.17 0.01 (RSD: 5.88%) 0.41 0.01 (RSD: 2.43%) 0.55 0.03 (RSD: 5.45%) 2.31 0.04 (RSD: 1.73%) 2.02 0.06 (RSD: 2.97%) 1.42 0.02 (RSD: 1.41%) 10.06 0.71 (RSD: 7.06%) 1.71 0.02 (RSD: 1.16%) 1.00 0.04 (RSD: 4.00%) 0.12 0.01 (RSD: 8.33%)

Values are represented as mean SD. b denotes those metabolites identied with standards and searches from NIST Mass Spectral Library 2002.

detected in the extracts48 (Table 5). From Fig. 6A, Tables 1 and 2, sucrose was the major transport form of xed carbon where carbohydrate was translocated from source to sink tissues converting into glucose, lactose, galactose and inositol

to provide the energy for synthesis of amino acids such as proline, serine, tyrosine, lysine and phenylalanine. The key amino acids such as phenylalanine and tyrosine, which form the basic aromatic ring structures in compounds, were also

Table 4

H NMR chemical shift assignments of the metabolites of the aqueous methanol extracts of Coptidis rhizoma Normalized peak intensitya

No. 1 2 3 4 5 6 7 8 9 10 11 12 13

Chemical shift (ppm) 1.48 1.94 2.08 2.406 2.348 3.367 2.441 2.46 2.514 2.583 2.82 2.88 3.20 3.46 3.568 3.706 3.783 3.886 4.108 5.407 7.401

Multiplicity d s m s m m s m dd m m s t m

Aqueous components assignment Alanine Acetic acid Glutamate Pyruvate Proline Succinic acid Glutamine Citrate Aspartate Asparagine Choline Trehalose Fructose

Source 1 (n 6) 0.155 0.009 0.076 0.009 0.170 0.009 0.014 0.001 0.206 0.002 0.81 0.02 0.160 0.003 0.096 0.002 0.0145 0.0002 0.094 0.004 0.12 0.01 0.287 0.007 0.121 0.03 0.32 0.02 0.65 0.04 0.63 0.03 0.78 0.04 0.43 0.03 1.17 0.12 0.054 0.001 0.219 0.002

Source 2 (n 6) 0.017 0.003 0.107 0.008 0.27 0.01 0.191 0.005 0.1202 0.0006 0.157 0.009 0.140 0.002 0.018 0.001 0.0197 0.0003 0.0803 0.0008 0.071 0.005 0.25 0.02 0.39 0.04 0.32 0.01 0.61 0.09 0.64 0.08 0.40 0.01 0.66 0.06 0.35 0.01 0.042 0.001 0.052 0.007

Source 3 (n 6) 0.064 0.003 0.119 0.005 0.089 0.001 0.233 0.009 0.0166 0.0004 0.154 0.005 0.054 0.004 0.023 0.003 0.0221 0.0005 0.081 0.001 0.105 0.002 0.35 0.03 0.43 0.01 0.448 0.004 1.27 0.09 0.66 0.01 0.73 0.01 0.64 0.02 0.34 0.02 0.033 0.006 0.186 0.007

14 15
a

d m

Sucrose Phenylalanine

Values are represented as mean SD.

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Table 6 Amount of berberine extracted from Coptidis rhizoma obtained from different sources using MAE method with water as extraction uid Extraction conditions MAEa at 100  C (Source 1) MAEa at 100  C (Source 2) MAEa at 100  C (Source 3)
a

Amount of Berberine (mg/100 g) SD 5237 2 (RSD: 0.05%, n 3) 5071 39 (RSD: 0.77%, n 3) 4578 23 (RSD: 0.52%, n 3)

Extraction solvent: water, extraction time: 20 min.

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Fig. 3 A representative HPLC chromatogram with bioactive marker compounds identied with standards in MAE extracts for (A) Stevia rebaudiana and (B) Coptidis rhizoma.

detected by 1H NMR analysis (Fig. 2A). Most plant phenolic compounds were derived from phenylalanine produced via the shikimic acid pathway. However, the absence of the phenolic compounds in Stevia indicated that cinnamic acid might be the end product of this pathway. Stevia also used the MEP pathway for the biosynthesis of SV and RA. Compared to other intermediates such as cinnamic acid in the shikimic acid pathway (Fig. 6A and 6B), it was proposed that steviol was used up extensively in the biosynthesis of SV and RA in the MEP pathway. Thus, the concentration of steviol was at trace levels in the extracts and not detected by the current method (Table 5). Investigation of the MEP/terpenoids and shikimate/ phenylpropanoids pathways using a proteomics-based approach and GC-MS proles of non polar extracts from basil

Fig. 4 PCA score plots for methanol aqueous mixture extracts of (A) Stevia rebaudiana under different cultivation conditions. W: Wild, Ctr: Control, IAA, mT, K, BA: cultured in different growth media and (B) Coptidis rhizoma of different sources (S1S3) generated using a combination of PC1 and PC2.

Table 5 Amount of stevioside, rebaudioside A and steviol extracted from Stevia rebaudiana cultivated under different conditions, using a MAE method with water as the extraction uid. ND indicates Not Detected MAE at 100  C for 10 min Growth Conditions Wild Tissue-cultured with IAA Tissue-cultured with BA Tissue-cultured with mT Tissue-cultured with K Control Amount of Stevioside (mg/100 g) SD 944.3 4.2 338.0 2.1 348.4 1.6 521.0 5.8 501.2 8.5 473.1 3.9 (RSD: 0.45%, n (RSD: 0.64%, n (RSD: 0.48%, n (RSD: 1.12%, n (RSD: 1.71%, n (RSD: 0.83%, n 3) 3) 3) 3) 3) 3) Amount of Rebaudioside A (mg/100 g) SD 166.3 1.7 (RSD: 1.04%, n 3) 96.8 0.6 (RSD: 0.71%, n 3) 115.2 1.5 (RSD: 1.37%, n 3) 156.6 2.0 (RSD: 1.30%, n 3) 138.1 5.7 (RSD: 4.15%, n 3) 101.4 1.0 (RSD: 1.03%, n 3) Amount of Steviol (mg/100 g) SD ND ND ND ND ND ND

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metabolism in cultured potato plant cells50 and altered plant mitochondrial proton conductance.51 Hence, the depletion of octadecanoic acid for the wild type plants with higher levels of SV and RA indicated that this fatty acid might be essential in modulating the biosynthesis of secondary metabolites (Tables 1 and 5). Alkaloids in Coptidis rhizoma. Alkaloids are a diverse group of low molecular weight compounds mostly synthesized from amino acids.9,10 The biosynthetic pathways for alkaloids are complex including a number of branch points as well as a high degree of tissue and sub-cellular compartmentation.9,10 For the biosynthesis of bioactive secondary metabolites such as berberine, palmatine and epiberberine in Coptidis, the breakdown of sucrose to glucose and fructose was essential to mobilize the carbon resources. It was proposed that sucrose was needed to synthesize phenylalanine which was a key precursor for the secondary metabolites in Coptidis (Table 3 and Fig. 6B). However, for the biosynthesis of alkaloids, it was proposed that starch, a principal carbohydrate storage in plants, could be broken down to glucose units as a source of energy and carbon skeletons.10,41,43,52,53 To the best of our knowledge, maltose and glucose were probably the exclusive products of starch breakdown.43,52,53 The utilization of sucrose and starch for monolignol biosynthesis had been reported and the release of glucose from starch breakdown in tobacco might be destined directly for conjugation with accumulating phenylpropanoids.41 The detection of disaccharides such as maltose in Coptidis suggested that these sugars were exported from the plastid to support sucrose synthesis, respiration and biosynthesis of alkaloids. Compared to Stevia, the biosynthesis function of Coptidis generated more types of sugars (Table 3). Amino acids such as tyrosine had been proposed as the key metabolites for the biosynthesis of berberine and production of benzylisoquinoline alkaloids in medicinal plants and Saccharomyces cerevisiae.9,10,54 However, the absence of tyrosine in the GC-MS and NMR analysis showed that phenylalanine was important in the biosynthesis of bioactive secondary metabolites in Coptidis (Fig. 1C and 2B, Tables 3 and 4). The addition of phenylalanine was shown to double the berberine secretion and also the content of phenolic compounds in the cell culture.55 Plant defense against microbial pathogens relies on the induction of defense proteins and low molecular weight antibiotics such as alkaloids. Plants develop systemic defense responses when locally infected by pathogens and the systemic acquired resistance appears to require secondary metabolites such as azelaic acid and others.56 The octadecanoic pathway was proposed to modulate the biosynthesis of antibiotic compounds which were integrated into plant defense.57 For Coptidis, the key elements in modulating biosynthesis of secondary metabolites for antibacterial activities might involve a signicant amount of fatty acids such as oleic acid, linoleic acid and hexadecanoic acid. The biosynthesis of benzylisoquinoline alkaloids from tyrosine, L-dopa, dopamine and others were reported earlier.9,10 It was observed that 2-mono-isobutyrin and catechollactate which were not present in Stevia (Tables 1 and 3), might be intermediates for the synthesis of alkaloids (Fig. 6B) in Coptidis.
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Fig. 5 PCA score plots for the MAE extracts obtained for (A) Stevia rebaudiana under different cultivation conditions. W: Wild, Ctr: Control, IAA, mT, K, BA: cultured in different growth media and (B) Coptidis rhizoma of different sources (S1S3) generated using a combination of PC1 and PC2.

leaves had shown multiple levels of metabolic control.49 Hence, the results suggested that the carbon ow was directed between the shikimic acid and MEP pathways in Stevia. Glycolysis, oxidative pentose phosphate pathway (OPPP) and the TCA cycle were the main components of plant respiration.10,39,41 However, the absence of citrate, succinic acids, glutamate and fumarate in the chemical data obtained (Fig. 1A and Fig. 2A) proposed that the TCA cycle was not used extensively for biosynthesis in Stevia (Fig. 6A). From Table 5, the wild type contains a higher concentration of SV and RA than the other nutrient-controlled conditions. The PCA score plots in Fig. 4A and 5A indicated that the types and concentrations of the primary and secondary metabolites were rather different for the wild type. A higher concentration of secondary metabolites was found to be consistent with a higher level of cinnamic acid and tyrosine from medicinal plants obtained under different growth conditions (Tables 1 and 5). At the same time, sucrose, galactose, proline, lysine and octadecanoic acid were depleted signicantly (more than 2 times) for the biosynthesis of a higher level of bioactive secondary metabolites in these medicinal plants (Tables 1 and 5). For fatty acids, it was proposed that the biosynthesis occurred in the plastids through the sequential addition of two carbon units. The roles of fatty acids in plant physiological processes include oxidation at the b-carbon to provide biosynthetic precursors,38 effects of polyunsaturated fatty acids and N-acetylglucosamine on free-radical
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Fig. 6 Metabolite proling to study the biosynthetic pathways in the production of major classes of secondary metabolites present in the two different plants: (A) Diterpene glycosides such as stevioside and rebaudioside A produced by Stevia rebaudiana and (B) Alkaloids such as berberine produced by Coptidis rhizoma. The identied primary metabolites found in these herbs are shown with bold black lines. The thin line indicates the biosynthetic pathway for conversion of metabolites. The arrow indicates the product of a certain biosynthetic pathway.

Conclusions
The simultaneous proling of primary and secondary metabolites could signicantly extend and enhance the power of existing functional genomics approaches for the study of the biosynthesis function in medicinal herbs. The current method used a simple extraction step with various analytical tools to provide a snapshot of a signicant number metabolites present in the
Anal. Methods

botanicals. It was thus a simple, low cost and rapid approach for the study of the primary and secondary metabolites in the biochemical pathways responsible for the biosynthesis of nutritional compounds such as terpenes and alkaloids in botanicals. The variation of metabolite proles in different medicinal plants had biological causes reecting the exibility of the metabolic network under different external environmental conditions. Lastly, deeper insights into the qualitative and quantitative
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variation of plant metabolites could provide information to improve the nutritional status of food crops.

Acknowledgements
The authors are grateful for the nancial support (Grant No. RP4/06TSN) of this work by the Nanyang Technological University (NTU), Singapore. The technical assistance rendered by A. B. Angela Lim, S. G. Anna Lee and H. L. Qiu is acknowledged. Tenmoli Ra is grateful for the study leave granted by Ministry of Education, Singapore. The authors are also grateful to Dr E.S. Ong for his help with the SIMCA-P+ software program.

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