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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 | 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 flow 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 profiling was studied by unsupervised multivariate Principal Component Analysis (PCA) to discriminate and assign the different phenotypes under various cultivation conditions. Hence, the simultaneous profiling of primary and secondary metabolites could significantly aid the existing functional genomics approaches to study the biosynthesis function in medicinal herbs.

Metabolomics is the study of the profiles of organic compounds or metabolites present in a biological system.1–3 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 five major groups: polyketides, isoprenoids (e.g. terpenoids and steroids), alkaloids, phenylypropanoids and flavonoids.8 There are more than 100 000 bioactive secondary metabolites produced by plants via certain biosynthetic pathways such as acetate, shikimate and mevalonate.8–10

a Natural Sciences and Science Education Academic Group, Nanyang Technological University, 1 Nanyang Walk, Singapore, 637616, Singapore. E-mail:; 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.11–23 High Performance Liquid Chromatography coupled with a Photodiode Array Detector (HPLC-PDA) is usually used for secondary metabolite profiling while Gas Chromatography coupled with Mass Spectrometry (GC-MS) and Nuclear Magnetic Resonance (NMR) are popular techniques to study the primary metabolite profiling 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 profiling of either primary or secondary metabolites present in plants by these techniques has been widely reported.20,22,24–31 The obtained metabolite profiles 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 classification of samples and also provide a good summary of data concerning functional relations between metabolites.13,29,32–34 These methods could provide deep insights into
Anal. Methods

This journal is ª The Royal Society of Chemistry 2011

The Anal. cinnamic acid. USA).06% could be obtained for retention time and ca. Italy).999.O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (purity > 99%). The second to fifth 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. Germany) for 5 min at 13 000 rpm. Germany) and ground using an IKA MF10 microfine grinder (Staufen. was This journal is ª The Royal Society of Chemistry 2011 . The herbs were ground into powders as described for Stevia.e. Tuttlingen. 40. Plant materials and reference standards preparation Stevia rebaudiana under different GAP cultivation conditions. Louis. Ultra-pure water was obtained from a Millipore Alpha-Q water system (Millipore. deuterated water (D2O) and other chemical reference standards (proline.8 mg LÀ1 respectively. we could discriminate the herbs grown under different Good Agriculture Practice (GAP) cultivation conditions and also of different geographical origins. Then 1 mL of an aqueous mixture of methanol (1 : 1) was added to the sample and vortexed for 1 min. They were freeze-dried using an Alpha 1-2 freeze dryer (Martin Christ.5 mg LÀ1 kinetin (K) (PhytoTechnology Labs. A Shimadzu GC 17A system (Kyoto.2 mg LÀ1 and from 3. leucine. The supernatant was collected and then evaporated to dryness using a Thermo Fisher Scientific model SPD 2010-230 speedvac system (MA.02 mg LÀ1 indole-3-acetic acid (IAA). Ammonium acetate and acetic acid (AA) were purchased from Merck (Darmstadt. 0. 8 h. MA. a metabolomics approach linking the primary and secondary metabolite profiling was developed for the identification of intermediates in novel biosynthetic pathways for the conversion of major primary metabolites to bioactive secondary metabolites in medicinal herbs. Analysis Gas chromatography-mass spectrometry (GC-MS).52% for peak area. i. Germany) and sieved through an insert of pore size 0. sucrose. Stock solutions of all the chemical reference standards were prepared at 200 mg LÀ1 in pure pyridine. lactose. after which 50 mL of each standard was incubated in the dark with 60 mL BSTFA for the derivatization step. To quantify the marker compounds in the botanicals.02 mg LÀ1 6-benzylaminopurine (BA) and 0. The sample was centrifuged in a Mikro 20 centrifuge (Hettich Zentrifugen. The derivatization conditions were studied with different volumes (20. maltose. MO. all plants were freeze-dried and reduced to powder form using a pestle and mortar. phenylalanine. Tuttlingen. Japan). Extraction was carried out under optimized conditions. Secondary metabolites by green-solvent microwave-assisted extraction (MAE). acetonitrile (ACN) and formic acid (FA) were purchased from Fisher Scientific (MA. 0. Concurrently. RA and BR at 1000 mg LÀ1 were prepared in methanol. It was found that a relative standard deviation (RSD) of less than 0. Methods linearity of each standard was established between 10 to 100 mg LÀ1 with correlation coefficient r2 $ 0.5 mm. Reference standards for HPLC analysis. Reference standards for GC-MS analysis.24. each of the derivatized analytes was then transferred to an amber vial for GC-MS analysis. A closed vessel system (under controlled temperature and pressure) was employed using Start E from Milestone (Sorisole. 0. The LODs and LOQs for the marker compounds were determined to be from 1. Coptidis rhizoma of different geographical origins. Berberine (BR) (purity > 98%). fructose. The reproducibility of the retention time and peak area for the marker compounds was investigated under optimum HPLC conditions.02 mg LÀ1 6(3-hydroxybenzylamino)purine meta-Toplin (mT). USA). The limits of detection (LOD) and quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise (S/N) of around 3 and 10 respectively.5 In this study.25 A sample for each extract was transferred to the 2 mL Eppendorf tube and centrifuged in a Mikro 20 centrifuge (Hettich Zentrifugen. tyrosine. a three point calibration based on the linearity established was used. Malaysia). N. trends and groups among samples in these multivariate data. Typically 50 mg of the ground herb powder (n ¼ 3) was accurately weighed in an Eppendorf tube. It was achieved by performing repeated injections (n ¼ 6) of a mixture of the standards at a concentration of 60 mg LÀ1. Germany). tryptophan.0 to 1. n ¼ 6) for each standard were found to be less than 1% on different days. lysine. The supernatant was collected for HPLC analysis. serine.33–35 The PCA technique is usually the first step in the evaluation and detection of patterns. rebaudiosides A (RA) and steviol were purchased from Chromadex Inc (St. 1 H NMR and HPLC-UV. fumaric acid and citric acid) were purchased from Sigma-Aldrich (St. we attempt to analyse the key metabolites in two different medicinal herbs with a platform comprising of | doi:10. Experimental Chemicals and standards Stevioside (SV). A 0. 60 and 80 mL) of BSTFA in pure pyridine under various incubation times (4 h. 100  C for 10 min for Stevia and 20 min for Coptidis. Bedford. Kansas). Pyridine was bought from Univar (Rotterdam. Germany) for 5 min at 12 000 rpm. Extraction methods Primary metabolites using methanol aqueous mixture. After 4 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. as described in our previous work.1 g plant sample was accurately weighed into the vessel. USA). Stock solutions of SV. Methanol (MeOH). The system precisions (RSD. The dried Coptidis rhizoma was purchased from three different Chinese medicinal halls (Kluang. mannose. USA).2 to 3. Using the metabolite profiling obtained with PCA.rsc. The Netherlands). galactose. 16 h and 24 h). Santa Ana. The sixth condition was a control. glycine. equipped with an auto-sampler model AOC-20i and a split/splitless injector. CA. glucose. The first condition (wild type) was obtained from the USA. After 12 weeks.View Online Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs. USA).1039/C1AY05334E regulatory processes and were also able to directly determine the phenotype.

and held for 2 min with a total run time of 22 min. A total of 200 peaks for each botanical were computed for their peak areas. The migration time of the peaks was found to be stable both intraday and interday with variation less than 0. The metabolites detected using GC-MS on different plant extracts were summarized in Table 1 (RSD varies from 0. thereby partially accounting for concentration due to the different sample sizes used. (n ¼ 6). 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. and Coptidis at 230. The assignment of metabolites in the 1D 1H NMR spectra was performed by 2D NMR using protocols published elsewhere. This journal is ª The Royal Society of Chemistry 2011 a multi-dimensional vector was constructed manually to characterize the biochemical patterns. was detected in the extracts of the two plants using GC-MS (Tables 1 and 3) and also showed a signal at d ¼ 5. 1D 1H NMR spectra were obtained using a Bruker 400 MHz US2 spectrometer operating at 400.1% FA) as mobile phase A (MPA) and acidified MeOH (0.5% (relative standard deviation (RSD). gradient elution was carried out with acidified water (0. PCA translates the peak areas obtained from n-dimensional variables into principal components (PC) where there is a score describing different chromatograms obtained. pH 6) was prepared with D2O as the solvent. USA). Helium was the carrier gas (>99. Results and discussion Metabolite profiling with PCA The primary metabolite profiling obtained by the GC-MS and 1H NMR techniques for the aqueous methanol extracts of Stevia and Coptidis are shown in Fig.40 in the 1H NMR spectra (Fig.24 to 10. gradient elution was carried out with acidified 50 mM ammonium acetate (0. Among the detected peaks. 0. 10 mL of standards and sample extracts were injected. fructose. Phosphate buffer (0. 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. All these results were input into a Simca-P+ Software Package (Umetrics.. The identification of peaks was based on the use of reference standards and NIST 98 library. the peak areas of the chromatographic fingerprints at different wavelengths were obtained as follows: Stevia at 210. 240. 5 mm) maintained at 40  C. 260.. Umea. other sugar molecules such as glucose. 250. with reference to internal TMS (d 0. Following the injection of 1 mL of sample. and were used as input data. The water resonance was eliminated by applying a secondary radio frequency irradiation during the relaxation delay of 1.18. galactose. interfaced to a Shimadzu QP5000 MS singlequadrupole system (Kyoto. The MS was performed in positive electron ionization (EI) mode with an ionization energy of 70 eV. 260.9 mm.99%).45% respectively.rsc.37 High performance liquid chromatography-photodiode array detector (HPLC-PDA).25 mm film thickness (Santa Clara. Germany). A Waters 2790 Alliance HPLC (Milford.76 to 9.4 s. n ¼ 6). Massachusetts.5 version.d. n ¼ 3) and in Table 2 (RSD varies from 1.19. The NMR spectra were manually processed using XWIN-NMR software (3. 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.12 MHz observation frequency.36. Anal.09%. The inlet was operated in splitless mode.1039/C1AY05334E used with a HP-5MS capillary column (5-phenyl)-methylpolysiloxane. which were products of the glycolysis process. The distribution pattern generated from the data in these plots can be correlated with the general characteristics of the samples analyzed. For Stevia. 230. Japan). The precision and accuracy of the GC-MS method were investigated by analyzing six injections of different plant extracts. 254. The mass spectra obtained was inspected manually and only those compounds with probability matching higher than 90% were considered.11. Aqueous extracts were analyzed using a pre-saturation pulse sequence (relaxation decay-901 acquisition).org | doi:10. 7 inch length.1% AA) as MPB. the oven temperature programme was as follows: an initial temperature of 100  C was raised to 300  C at 10  C minÀ1. 0. The analysis of the extracts was performed with a Waters XTerra C18 (150 Â 3. The detector signal was recorded from 3 min after injection until 40 min. Data analysis with PCA For GC-MS. The GC inlet temperature was set at 280  C and MS interface 300  C. Sweden) for subsequent evaluation of the similarities of different chromatograms based on PCA.1% AA) as MPA and acidified ACN (0. 270 and 280 nm. Nuclear magnetic resonance (1H NMR).D.12. in the absence of internal standard. Using the two complementary techniques.06 to 7. 254. with 0.1% FA) as mobile phase B (MPB).0 mL minÀ1 was used with a detection wavelength of 254 nm. 250.0 s. Each vector was normalized to the total sum of vector intensity. the metabolites detected in these plant extracts could be classified into amino acids. A flow rate of 0.05% trimethyl silane propionic acid sodium salt (TSP) as an internal standard (IS) and 200 mL buffer. For Coptidis. From Tables 1 and 3. SigmaAldrich).View Online Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs. Sucrose. 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. 2).7 mL minÀ1 was used with a detection wavelength at 210 nm. and ions were scanned across the range of 50–720 atomic mass units (amu). each sample was represented by a Total Ion Chromatogram (TIC). Bruker. CA.2 M. a major metabolite involved in glycolysis pathways. PCA score plots have been used for the classification of samples from their measured properties. For all experiments. A flow rate of 1.22. 30 m. showed high reproducibility both on the same day and on different days. were also detected by the GC-MS method.23. Each dried plant extract was reconstituted with 400 mL D2O containing 0. sugars and others (inclusive of organic acids) (Tables 1 to 4).04%. For HPLC-PDA. The peak area for each chromatogram was normalized to a constant sum. with a constant flow of 1 mL minÀ1. Methods .25 mm i. USA) was employed to separate and quantify the amount of extracted marker compounds. The RSD values for normalized peak areas for different compounds for intraday (n ¼ 6) and interday (n ¼ 6) analyses were found to vary from 1. This solution was then transferred into an NMR tube (5 mm O. 1 and 2. 270 and 280 nm. The peaks due to column bleed and derivatization reagent were removed.7 min purge-on time.38–42 These data showed that the current method.0) and auto phase correction was used.

13. 10. inositol. 9. 11. 8. 6. tyrosine. arabinoic acid. lactose. glucofuranoside. 19. 8. 7. butanedioic acid. 2. fructose. cinnamic acid. glucose. 7. glucose. citric acid. 14. D-ribofuranose. fructose. proline. 12. 11. 2. linoleic acid. 10. maltose. | doi:10. sucrose and 12. oleic acid. 2. Methods This journal is ª The Royal Society of Chemistry 2011 . sucrose and 13. lactose. (C) Coptidis rhizoma: 1.1039/C1AY05334E Fig. tyrosine. octadecanoic acid. sucrose and 20. ribitol. 3. 6. glucose. phenylalanine. cinnamic acid. palmitic acid. phenylalanine. 4. 18. 6. 2-monoisobutyrin. 16. b-DL-arabinopyranose. lactose. phenylalanine. 5. leucine. 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. 3. 10. arabinose. serine. Anal. 15. 12. 9.View Online Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs. 5. 4. catechollactate. proline. 11. leucine.rsc. 9. 17. citric acid. serine. octadecanoic acid. 7. 5. fructose. 3. 8. proline. 1 (A) A mixture of pure standards: 1. lysine.

mT and K conditions suggested that this key molecule might be used extensively to promote the glycolysis process to synthesize the other | doi:10. These molecules are important building blocks for the C6–C3 class of secondary metabolites such as the flavonoids and terpenoid quinones. pyruvate. 1. The findings were consistent with the GC-MS data shown in Table 1. the method used for the profiling of secondary metabolites in the current work was validated in our earlier work. Hence. 1C).24 Hence. 2. their profiles obtained with PCA could be used to classify plants of different origins for quality control purposes. Stevia contains mainly secondary metabolites such as monoterpene. proline. 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. the lower concentration of sucrose in IAA. Compared to the control group. 5. 1. were also detected by GC-MS and 1H NMR analysis (Tables 1 to 4). Other important metabolites such as phenylalanine and tyrosine. 8. In general. the results revealed that the primary metabolites were mainly carbohydrates and lipids. It was noted that the concentration of metabolites extracted for each growth condition was rather different for each botanical (Tables 1. phenylalanine. 6. phenylalanine. BA. 4A and 5A).44. which has antifungal and antibacterial activity.24 To assign metabolic phenotype of the medicinal herbs analyzed. Thus. 3.78. 2. acetic acid. the extraction method could have an important effect on the chromatographic profiles obtained for the determination and profiling of secondary metabolites in medicinal plants. 2).51 in the 1H NMR spectra obtained for Coptidis extracts (Fig. 5 and 6). citrate. 2). due to the diversity in the biosynthesis of these metabolites. However. 6.View Online Fig. was absent from the Stevia extracts but it showed a signal at d ¼ 2. the physical environmental conditions such as nature of soil. 4. and thus they were not detected (Fig. fructose. 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. 3. diterpene and sesquiterpene. 3. Due to the complexity of the botanical extracts. The various marker compounds (SV.23) signals which are usually present in NMR spectra were eliminated by the water suppression programme. the b-glucose (d. tyrosine and 7. the Coptidis was found to possess smaller amounts of amino acids but greater amounts of sugars and fatty acids compared to Stevia (Table 3).70. climate changes and light intensity could influence the biosynthesis of the metabolites in plants. 4B and 5B). d ¼ 4. 3.46 Metabolomics approach and biosynthesis of major plant metabolites Terpenes in Stevia rebaudiana. The observation indicated that the biosynthesis pathways for certain metabolites in the two medicinal herbs might be different. RA and BR) present in these plant extracts were quantified by the same technique (Tables 5 and 6). which is consistent with another report43 (Table 3). the profiling of secondary metabolites alone in Stevia could not distinguish botanicals from different sources (Fig.64) and a-glucose (d. For Coptidis. However. while significant amounts of phenolic Anal. a combination of chromatographic fingerprints based on the primary and secondary metabolite profiling 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). which was a typical product of the TCA cycle. In contrast.rsc. The fructose molecule from the breakdown of starch showed signals at d ¼ 3. d ¼ 5. Note: Absence of citrate peak at d ¼ 2. leucine. alanine. sucrose.88 and 4. Methods Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs. which were synthesized from phosphoenolpyruvate (PEP) in glycolysis via the shikimate pathway. sucrose and 9. the primary and secondary metabolite profilings could clearly distinguish their different origins (Fig. was consistent with various reports. However. lactate. 7.10 in the extracts of the two plants (Tables 2 and 4).54ppm and (B) Coptidis rhizoma: IS: internal standard. The tissue-cultured controlled conditions with auxin (IAA) and cytokinins (6BA. Hence. citrate. succinic acid. The detection of butanedoic acid (Peak 2 in Fig. the concentrations of key metabolites were rather different for the Stevia cultivated under different nutrient-controlled conditions compared to the wild type (Table 1). fructose.1039/C1AY05334E . 5. 3. 4 and 5). proline. It was noted that the types of metabolites detected for Stevia grown under nutrient-controlled conditions with different growth hormones were similar (Table 1). 3.45 The secondary metabolite profiling obtained by HPLC-UV for Stevia and Coptidis were shown in Fig. mT and K) hormones might have sufficient nutrients to promote the This journal is ª The Royal Society of Chemistry 2011 glycolysis process to generate higher concentrations of metabolites compared to the wild type. Compared to primary metabolites. The PCA score plots showed the similarities and differences in the primary and secondary metabolite profiling obtained from medicinal herbs of different sources and cultivation under GAP conditions (Fig. 2 A representative 1H NMR spectrum of the aqueous mixture fraction obtained from plant extracts of (A) Stevia rebaudiana: IS: internal standard. pattern recognition tools such as PCA were used. 4.

01 6 7 8 a d m d Sucrose Phenylalanine Tyrosine Values are represented as mean Æ SD.84%) 6.78.01 (RSD: 5.04 (RSD: 3.05 (RSD: 2.65 Cinnamic acidb Octadecanoic 20.22%) 0.23 Æ (RSD: 1.52%) 2.77%) 9.401 7.53%) 0.55%) 2.06 (RSD: 5.1039/C1AY05334E L-Lysine b Leucineb Phenylalanine 10.01 (RSD: 0.009 0.06 0.28 Æ 0.55%) 1.84%) 0.71.01 (RSD: 0.001 0.05 16.78%) 6. Table 2 1 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.87%) 6.05 (RSD: 1.01 (RSD: 3.07 (RSD: 2.11 Æ 0. 18.09 Æ 0.70 Æ 0.67 Æ 0.26 Æ 0.79%) 0.04 0.01 (RSD: 2.88%) 0.85 Æ 0.24 Æ 0.14 Æ 0.67 Æ 0.00%) 6.13 (RSD: 1.11 (RSD: 2.13 Æ 0.61 Æ 0.25%) 3.52%) 3.16%) 3.23 (RSD: 5.97 Æ 0.65%) 6.15 (RSD: 2.60 Æ 0.06 (RSD: 5.35%) — 0.99 Æ 0.07 (RSD: 3. Methods pathway.82%) — 0.81 Æ 0.59%) 4.40%) 2.78.35 Æ | doi:10.03 (RSD: 3.360 1.50 4.28%) 0.35%) 2.01 (RSD: 0.02 mg LÀ1 mT (n ¼ 3) 0.65 31.46 Æ 0.13 Æ 0.81 Æ 0.01 (RSD: 2.11 Æ 0.59%) 0.58 Sugars b D-Fructose b D-Glucose b D-Galactose 16.09%) 1.91 Æ 0.42%) 3.52%) 1.35 b b L-Tyrosine Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs.01 (RSD: 2.36 (RSD: 5.03 (RSD: 5.21 Æ 0.01 (RSD: 9.17 Æ 0.32%) 3.38%) 9.69%) 0.71 Æ 0.01 (RSD: 2.02 (RSD: 2.04 (RSD: 5.18 (RSD: 6.11 (RSD: 5.08 (RSD: 2.53 (RSD: 7.709 3.32 Æ 0.16%) 1.37 (RSD: 7.89 Æ 0.63%) 4.55 Æ 0.02 mg LÀ1 BA (n ¼ 3) (n ¼ 3) 2.01 (RSD: 0.53%) 2. 30.20 Æ 0.52%) 3.08 (RSD: 1.01 (RSD: 0.17%) 0.11 (RSD: 0.16 (RSD: 4.11 Æ 0.99 32.0%) 0.77%) 0.54%) 1. 19.04 (RSD: 2.14 Æ 0.88%) 0.01 (RSD: 3.78%) 0.02 (RSD: 0.77%) 1.02 (RSD: 2.08 (RSD: 5.05 (RSD: 1. 32. Ctr: Control.82.09 (RSD: 2. compounds and phenylpropanoids are not found.83%) 2.68 17.32 17.67%) 3.065 Æ 0.74 Æ 0.02 (RSD: 4.56%) 9.109 5.06 (RSD: 1.51 (RSD: 5.786 3.01 (RSD: 4.32%) 0.12 (RSD: 1.71 Æ 0.05 0.39 Æ 0. 18.76 Æ 0.64%) 0.53%) 1.39 Æ 0.01 (RSD: 1.05 0.123 Æ 0.35 Æ 0.10 (RSD: 1.53 Æ 0.87%) 3.21 Æ 0.17 Æ 0.03 (RSD: 1.009 0.63 Æ 0.02 (RSD: 1.99 Æ 0.341 3.22 Æ 0.82%) Wild type (n ¼ 3) 0.76 Æ 0.03 0.06%) 0.85 Æ 0.62%) 1.39%) 0.25 Æ 0.78 (RSD: 8.89 Æ 0.43 Æ 0.63.20 Æ 0.86%) 0. 32.94 Æ 0.15%) 1.12 (RSD: 1.41.00%) 1.34 Æ 0.56 Æ 0.16%) 2.02 Æ 0.03 Æ 0.37%) 9.01 0.14 (RSD: 3.13 5.885 4.16 Æ 0.40 Æ 0.38 Æ 0.76%) 7.00%) 1.02 (RSD: 2.16%) 4.10 (RSD: 2.340 3.04 (RSD: 0.961 2.5 mg LÀ1 K (n ¼ 3) 0.56 Æ 0. BA: cultured in different growth media Normalized peak intensitya Compounds Amino acids b L-Proline Serine b Retention time (min) 0.00%) 4.02 (RSD: 0.96.06 (RSD: 5.76 Æ 0.01 (RSD: 1.75 Æ 0.31%) 0.37 Æ 0.16 Æ 0.25%) 3. K.61%) 5.23 Æ 0.401 6.69%) 11.83 Æ 0.65 Sucroseb Lactose Inositol Fatty acids 19.56 Æ 0.9.03 (RSD: 2. 33.43 Æ 0.74%) 3.07%) 1.37%) 0.48 Æ 0.70%) 2.02 (RSD: 3.52. 32.01 0.73 Æ 0.01 (RSD: 2.22 (RSD: 3. 28.12 Æ 0.47 The biosynthesis of steviol glycosides was proposed by the plastid localized methylerythritol 4-phosphate (MEP) Anal.970 Multiplicity t m m s d m Normalized peak intensitya(n ¼ 3) 0.54 Æ 0.View Online Table 1 GC-MS analysis of primary metabolites in Stevia rebaudiana under different cultivation conditions.21%) Control (n ¼ 3) 1.24%) 7.33 Æ 0.62 Æ 0.69%) 0.01 (RSD: 1.94 1.72 Æ 0.25%) Æ 0. b denotes those metabolites identified with standards and searches from NIST Mass Spectral Library 2002.65%) 0.09 (RSD: 4.18 Æ 0.88 Æ 0.01 (RSD: 0.30 17.57. 19.03 (RSD: 1.79 Æ 0.rsc.36%) 1.45 Æ 0.02 mg LÀ1 IAA 0.01 (RSD: 2.03 (RSD: 4.01 (RSD: 5.16 Æ 0.56 Æ 0.97%) 2.08 (RSD: 4.48 Steviol glycosides are diterpenoids whose biosynthesis pathway shares four common steps with gibberellic acid formation.43 Æ 0.24.29%) 0.79 Æ 0.50%) 6. W: Wild. 35.04 (RSD: 10. 11. 1 2 3 4 5 Chemical shift (ppm) 0.02%) 4.83 Æ 0.06 (RSD: 2.06 (RSD: 1.31 Æ 0.47.95 18.86%) 0.46 Æ 0.43 Æ 0.60 Æ 0. IAA.42 Æ 0.48 The plant gene for the first step in the MEP pathway was deoxyxyulose-5-phosphate synthase. mT.80 acid a Values are represented as mean Æ SD.029 Æ 0.11 Æ 0.85%) 0.45 (RSD: 6.02 (RSD: 9.07 (RSD: 3.01 (RSD: 7.01 (RSD: 6. 18.47%) 1. 20. Steviol was proposed as a key intermediate for the biosynthesis of SV and RA but it was not This journal is ª The Royal Society of Chemistry 2011 . which led to the synthesis of deoxyxyulose-5-phosphate from pyruvate and glyceraldehyde 3-phosphate. 17.39.19 Æ 0.

serine.08 (RSD: 2.002 0.0221 Æ 0.94 Æ 0.31 Æ 0.70 Æ 0.13 12.10 (RSD: 2.03 (RSD: 3.16%) 1.004 0.514 2.18 Æ 0.01 (RSD: 2.56 Æ 0.32 Æ 0.42 Æ 0.01 0.27 Æ 0.001 0. 18.017 Æ 0.59.98 Æ 0. 33.04 0.88 17.25%) 0.03 1.01 (RSD: 9. Tables 1 and 2.30 Æ 0.287 Æ 0.02 (RSD: 5.79 16.023 Æ 0. 33.042 Æ 0.78%) 0.59 Æ 0.09 (RSD: 1.009 0.41 Æ 0.38%) Æ 0.63 Æ 0.70%) Amino acids 3.03 (RSD: 5.24%) 0.29%) Æ 0.01 (RSD: 3.42 (RSD: 7.55 Æ 0.01 0.0002 0.06 Æ 0.39 Æ 0. 33.033 Æ 0.170 Æ 0.64 Æ 0.39%) 0.88%) 0.65 Æ 0.0004 0.64 0.46 2.01 (RSD: 2.406 2.007 14 15 a d m Sucrose Phenylalanine Values are represented as mean Æ SD.18 (RSD: 6.28 Æ 0. 18.33%) Values are represented as mean Æ SD.02 0.105 Æ 0.02 (RSD: 3.84%) 0.40%) 0.007 Source 3 (n ¼ 6) 0.66 Æ 0.191 Æ 0. 11.13 (RSD: 4.076 Æ 0.36 Æ 0.002 0. were also Table 4 1 H NMR chemical shift assignments of the metabolites of the aqueous methanol extracts of Coptidis rhizoma Normalized peak intensitya No.0145 Æ 0.06 (RSD: 3.97%) 1.401 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 ¼ 6) 0.73%) 0.009 0.02 (RSD: 1.04 (RSD: 4.81 Æ 0. 32.91 0.064 Æ 0.81%) 0.99 32.46 3.04%) 0.61%) 4.91%) Æ 0.28 Æ 0.22%) 2.0803 Æ 0.02 (RSD: 1.39.568 3. 9.29 0.14 (RSD: 7.78.0%) 0.01 0.03 0. galactose and inositol to provide the energy for synthesis of amino acids such as proline.65%) 17.02 0.66 Æ 0.06 Æ 0.18.01 0.26 Æ 0.41%) 10.72%) 0. 13.0003 0.07 (RSD: 1.071 Æ 0.02 0.32 17. Methods .206 Æ 0.32 (RSD: 1. 6A.009 0.67 16.74%) 2.01 (RSD: Æ 0.08 2.1202 Æ 0. 19.155 Æ 0. sucrose was the major transport form of fixed carbon where carbohydrate was translocated from source to sink tissues converting into glucose.27 Æ 0.70%) 2. 17.07 5.09 Æ 0. 32.78 Æ 0.01 (RSD: 3.002 Source 2 (n ¼ 6) 0.33%) 0.25 Æ 0. lysine and phenylalanine.rsc.00%) 0.88 3.82.66 Æ 0.706 3.43 Æ 0.11 Æ 0.01 (RSD: 10.63%) 1.01 (RSD: 3.107 Æ 0.07 (RSD: 1.01 0.27 Æ 0.90 21.01 (RSD: 1.42%) Æ 0.1039/C1AY05334E 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 20.61 Æ 0.01 (RSD: 1.233 Æ 0.09%) 4.44%) Æ 0.58 17. 15.009 0.002 0. 18.01 (RSD: 0.41.23 | doi:10.06 (RSD: 2.64 Æ 0.01 (RSD: 8.40 Æ 0.01 (RSD: 2. From Fig.348 3.23 1.14 Æ 0.089 Æ 0.28 Æ 0.367 2.30 (RSD: 1.01 0.03 (RSD: 10.14.03 0.52%) 0. 17.01 (RSD: 2.16 Æ 0.68%) Æ 0.32 Æ 0.219 Æ 0. 35.78.33%) 19.12 Æ 0.66 Æ 0.018 Æ 0.70 0.27 Æ 0.11 Æ 0.12%) Source 3 (n ¼ 6) 0.48 1.04 (RSD: 1.12 0.98.01 (RSD: 7.054 Æ 0.04 Æ 0.78%) 3.09 Æ 0.20 Æ 0.004 1.38%) Source 2 (n ¼ 6) 0.005 0.004 0.06 0.01 (RSD: 6.08 0.35.001 0.58 Æ 5.001 0.886 4.02 Æ 0.63%) 0.005 0.34%) Æ 0.06 (RSD: 3.96.119 Æ 0.052 Æ 0.01 0.24%) Æ 0.01 (RSD: 3.054 Æ 0.51%) Æ 0.0197 Æ 0.44 20.108 5.35 Æ 0. b denotes those metabolites identified with standards and searches from NIST Mass Spectral Library 2002.35 Æ 0.02 (RSD: 1. which form the basic aromatic ring structures in compounds.01 (RSD: 1.69.42 1.09%) 0.157 Æ 0.003 0.00 Æ 0.04 0.43%) 2.005 0.02 (RSD: 2.57%) 3.71 Æ 0.89 Æ 0.007 0.58.30 31.003 0.09 0.01 (RSD: 1.02 0.07 (RSD: 3.10 Æ 0.52.01 (RSD: 9.0166 Æ 0. The key amino acids such as phenylalanine and tyrosine.003 0.14%) 1.407 7.82%) 0.13 (RSD: 5.01 0.89%) 0.73%) 2.57%) 0.04%) 2.45%) 2.17 Æ 0.43%) 0.31 (RSD: 8.06%) 1.98 1.006 0.43%) 0. tyrosine.12%) Æ 0.78 0.05 16.67%) 0.001 0.03 (RSD: 1.47 Æ 0.12%) 2.74 23.20 3. 28. 15.79.97 7.65 16.73 Æ 0.01 (RSD: 3.0005 0.82 2.71 (RSD: 7.51.51 3. lactose.121 Æ 0.67 Æ 0.09 0.34 Æ 0.29 15.58.97%) Æ 0.441 2.01 (RSD: 3.View Online Table 3 GC-MS analysis of primary metabolites in Coptidis rhizoma obtained from different sources (S1–S3) Normalised peak intensitya Compounds b L-Proline Retention time (min) Source 1 (n ¼ 6) 0. 30.0008 0.12 Æ 0.03 0.85%) Æ 0.56%) 2.51 16.01 (RSD: 5.34 Æ 0.0006 0.18 (RSD: 8.43 Æ 0.29 (RSD: 6.03 (RSD: 4.583 2.001 0.13.01 (RSD: 4.096 Æ 0.12 Æ 0.56%) 0. 17. 20. 32.01 (RSD: 3. This journal is ª The Royal Society of Chemistry 2011 Anal. 1 2 3 4 5 6 7 8 9 10 11 12 13 Chemical shift (ppm) 1.783 3.77 Æ 0.003 0.005 0.33.79 0.78 1.84 18.88%) 0.094 Æ 0.008 0. 17.94 2.001 0.55 Æ 0.154 Æ 0.58 15.58 0.014 Æ 0.448 Æ 0.160 Æ 0.68%) Æ 0.009 0.02 0.081 Æ 0.55 Æ 0.04 0.02 (RSD: 3.21 (RSD: 10.36 Æ 0.50 10.01 (RSD: 5.47%) 0.07 (RSD: 3.80 Æ 1.01 (RSD: 1.41 Æ 0.02 Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs.04 (RSD: 6.18 Æ 0. detected in the extracts48 (Table 5).01 (RSD: 5.96 Æ 0.002 0.186 Æ 0.72%) Æ 0.35 Æ 0.27 Æ 0.79 23.71%) 0.01 (RSD: 2.17 Æ 0.

rebaudioside A and steviol extracted from Stevia rebaudiana cultivated under different conditions.12%.5 473.0 (RSD: 1.6 (RSD: 0.03%. n ¼ (RSD: 1. K. n ¼ 3) Extraction solvent: water.5 (RSD: 1.7 (RSD: 4.64%.05%.8 501.1 348.3 Æ 4. Investigation of the MEP/terpenoids and shikimate/ phenylpropanoids pathways using a proteomics-based approach and GC-MS profiles of non polar extracts from basil Fig. 6A and 6B).2 Æ 1. the absence of the phenolic compounds in Stevia indicated that cinnamic acid might be the end product of this pathway.2 Æ 8. n ¼ 3) 101. n ¼ 3) 138. n ¼ (RSD: 1.45%.1 Æ 5.0 (RSD: 1. Ctr: Control. n ¼ 3) 156. IAA. Thus. 4 PCA score plots for methanol aqueous mixture extracts of (A) Stevia rebaudiana under different cultivation conditions. extraction time: 20 min.52%.rsc. n ¼ 3) 4578 Æ 23 (RSD: 0.0 Æ 2.04%. Most plant phenolic compounds were derived from phenylalanine produced via the shikimic acid pathway. However. n ¼ 3) Amount of Steviol (mg/100 g) Æ SD ND ND ND ND ND ND Anal.7 (RSD: 1. Methods This journal is ª The Royal Society of Chemistry 2011 .9 (RSD: 0. n ¼ 3) 5071 Æ 39 (RSD: 0.48%. using a MAE method with water as the extraction fluid. n ¼ (RSD: 0.4 Æ 1.8 Æ 0.15%. BA: cultured in different growth media and (B) Coptidis rhizoma of different sources (S1–S3) generated using a combination of PC1 and PC2.37%.6 Æ 2.4 Æ 1.30%. n ¼ 3) 115.View Online Table 6 Amount of berberine extracted from Coptidis rhizoma obtained from different sources using MAE method with water as extraction fluid 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) Æ SD 5237 Æ 2 (RSD: 0. detected by 1H NMR analysis (Fig.2 338.3 Æ 1. Stevia also used the MEP pathway for the biosynthesis of SV and | doi:10. n ¼ 3) 96. 2A).6 521. n ¼ (RSD: 0. W: Wild.71%.1039/C1AY05334E Fig. it was proposed that steviol was used up extensively in the biosynthesis of SV and RA in the MEP pathway. Compared to other intermediates such as cinnamic acid in the shikimic acid pathway (Fig. Table 5 Amount of stevioside.71%.83%. Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs. the concentration of steviol was at trace levels in the extracts and not detected by the current method (Table 5). mT.1 Æ 3.77%. n ¼ 3) 3) 3) 3) 3) 3) Amount of Rebaudioside A (mg/100 g) Æ SD 166. 3 A representative HPLC chromatogram with bioactive marker compounds identified with standards in MAE extracts for (A) Stevia rebaudiana and (B) Coptidis rhizoma. n ¼ (RSD: 0. ‘‘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) Æ SD 944.0 Æ 5.

BA: cultured in different growth media and (B) Coptidis rhizoma of different sources (S1–S3) generated using a combination of PC1 and PC2. However. mT.56 The octadecanoic pathway was proposed to modulate the biosynthesis of antibiotic compounds which were integrated into plant defense.9. K. oxidative pentose phosphate pathway (OPPP) and the TCA cycle were the main components of plant respiration. 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. The addition of phenylalanine was shown to double the berberine secretion and also the content of phenolic compounds in the cell culture.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.49 Hence. the results suggested that the carbon flow was directed between the shikimic acid and MEP pathways in Stevia. maltose and glucose were probably the exclusive products of starch breakdown. linoleic acid and hexadecanoic acid. the biosynthesis function of Coptidis generated more types of sugars (Table 3).1039/C1AY05334E Fig. proline.9. Glycolysis.41 However. 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. At the same time.39.43. the absence of citrate. 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). 6B) in Coptidis.57 For Coptidis. From Table 5.54 However. Compared to Stevia. a principal carbohydrate storage in plants. 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. W: Wild. 6A).9. it was proposed that starch. lysine and octadecanoic acid were depleted significantly (more than 2 times) for the biosynthesis of a higher level of bioactive secondary metabolites in these medicinal plants (Tables 1 and 5).10 For the biosynthesis of bioactive secondary metabolites such as berberine.10 It was observed that 2-mono-isobutyrin and catechollactate which were not present in Stevia (Tables 1 and 3). it was proposed that the biosynthesis occurred in the plastids through the sequential addition of two carbon units. 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).53 To the best of our knowledge. sucrose. Ctr: Control. 4A and 5A indicated that the types and concentrations of the primary and secondary metabolites were rather different for the wild type. For fatty acids. The PCA score plots in Fig. 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. galactose. respiration and biosynthesis of alkaloids.41 The detection of disaccharides such as maltose in Coptidis suggested that these sugars were exported from the plastid to support sucrose synthesis.10. Alkaloids are a diverse group of low molecular weight compounds mostly synthesized from amino acids.41.52.View Online metabolism in cultured potato plant cells50 and altered plant mitochondrial proton conductance.38 effects of polyunsaturated fatty acids and N-acetylglucosamine on free-radical This journal is ª The Royal Society of Chemistry 2011 . Alkaloids in Coptidis rhizoma. might be intermediates for the synthesis of alkaloids (Fig.rsc. Anal. leaves had shown multiple levels of metabolic control.9. IAA. | doi:10. 6B).52. the key elements in modulating biosynthesis of secondary metabolites for antibacterial activities might involve a significant amount of fatty acids such as oleic acid. The biosynthesis of benzylisoquinoline alkaloids from tyrosine. palmatine and epiberberine in Coptidis.10. The roles of fatty acids in plant physiological processes include oxidation at the b-carbon to provide biosynthetic precursors. 1C and 2B. 2A) proposed that the TCA cycle was not used extensively for biosynthesis in Stevia (Fig. the breakdown of sucrose to glucose and fructose was essential to mobilize the carbon resources.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. Tables 3 and 4). could be broken down to glucose units as a source of energy and carbon skeletons. glutamate and fumarate in the chemical data obtained (Fig. 1A and Fig. dopamine and others were reported earlier.43. the wild type contains a higher concentration of SV and RA than the other nutrient-controlled conditions. Methods Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs. 5 PCA score plots for the MAE extracts obtained for (A) Stevia rebaudiana under different cultivation conditions. succinic acids.51 Hence.55 Plant defense against microbial pathogens relies on the induction of defense proteins and low molecular weight antibiotics such as alkaloids. for the biosynthesis of alkaloids.

org | doi:10. Lastly. The thin line indicates the biosynthetic pathway for conversion of metabolites. 6 Metabolite profiling 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.rsc. The variation of metabolite profiles in different medicinal plants had biological causes reflecting the flexibility of the metabolic network under different external environmental conditions. 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 identified primary metabolites found in these herbs are shown with bold black lines. Methods botanicals. deeper insights into the qualitative and quantitative This journal is ª The Royal Society of Chemistry 2011 . Conclusions The simultaneous profiling of primary and secondary metabolites could significantly extend and enhance the power of existing functional genomics approaches for the study of the biosynthesis function in medicinal herbs. The arrow indicates the product of a certain biosynthetic pathway.View Online Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs.1039/C1AY05334E Fig. The current method used a simple extraction step with various analytical tools to provide a snapshot of a significant number metabolites present in the Anal.

Cho. J. Proc. J. 2008. Chem. 1998. 155. Goeminne and B. Dauwe. 54.. H. RP4/06TSN) of this work by the Nanyang Technological University (NTU). 2007.. Science. Chem. Zeeman and S. A. W. The authors are also grateful to Dr E. 873. Biol. Markovic. Zhang. Plant Biol. Y. Ali. Beijing. R. Vertes. Abingdon. H. M. K. X. J. S. Whang. 2007... Browne. J. C. Gaevskaya. Bairamashvili. 865. L.. 49(2). A. 56. K. 2008. P. L. J. 387. L. Teo. Willmitzer. Chromatogr. Methods . Inze. H. Bio. Zhang. 2008.. J. Chichester. F. 44 W. H. Agric. J. 170. J. 33 V. Carrari and L. 41. Shu and K. China. Maucourt. Food Chem. Y. B. Han. A. 2006. Biotechnol. 24. Food Chem. Garland Science. W. Y. S. Z. J. M. K. W. Abdel-Farid. Biais. Downloaded by Massachusetts Institute of Technology on 27 October 2011 Published on 26 October 2011 on http://pubs. 26 X. Willmitzer and A. Planta. C. Peng. Anal. H. Smith. Genet. Swarup. 18. 68. 2000. K. O. Biomed. Plant Physiol. 5. J. 19. 2008. Masako and E. 267. Gullberg. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 W. 29 K. 5. F. Plant Biol. Zachowski and F. Choi. Anal. 2008. Xia and L. 73. Rev. Nat. 2009.. 302. Fernie. Migaud and R. C. Hourton-Cabassa. Anal. Kim. J. Plant Physiol.. C. Linthorst and R. Bernillon. R. Biotechnol. 2007. Trends Plant Sci. Roussaux. S. 2007. M. 1157. Ko. 42 J. 2008. 11612. D. Phytochemistry. Wang. L.View Online variation of plant metabolites could provide information to improve the nutritional status of food crops. Lu. 52 A. B. R. 105. Plant Cell Rep. 1047. Miroux. Li. Biochem. Metabolomics. Trends Plant Sci. G. T. A. Biol. Weckwerth and O. 73. Moritz. J. Abramovic. Physiol. Choi. T. G. 1187. 2005. Lee and Y. Guy. Rex. R. 2008. Zheng and D. Moing. 80. 2002. Biosci. 2008. W. Greenberg. Anal. 34. M. M. 27 W. A. D. Chem. G. Gomez-Cadenas. J. 10(3). M. 54(2). J. Jumtee. Graham. 45 X.. G.. 13. M. Daruvar. 2009. Hayes and M. Pharm. Ong. Z. 2006. 562. Yong. M. Smolke. J. 28(6). 50 P. CRC Press. Wiley. Nat. Biotechnol. Nat.. W.. Thomas. Cho. H.1039/C1AY05334E This journal is ª The Royal Society of Chemistry 2011 Anal.. 69 (1). 1186. Biomed. B. P. Lam. H.. 433.. Jacob. H.. Kashulin. Gukasova. Fiehn. S.S. Choi. Shinano. Ammer and D. A. M. J. Merzlyak. Acad. A. Alonso. F. Anal. Ganf. F. 130. Shrestha and A. 331. Kim. Liu. Biochem. H. 9599. 2002. Kim and H. B. The technical assistance rendered by A. 36. 43 J. Xu. N. Davey. 263. Deborde. T. Kammerer. P. B. 43. Bamba. Biochem. Q. Kurt. Oka. N. 283. M.. 67. A. Okazaki. Abingdon. O. M. H. Blechert. Lieven. G. 138(4). M. Ricquier. Q. J. Narasimham and S. P. 37. Jung.. 2005. K. 7338. H. Y. 31 H. Ye. 407. J. Brodschelm. L. Plant J. Altman. Int. R. Exp. 35 L. 35. F. Shu. Trans. Kim. Talon. Baldet. A. 24. 74. K. Singapore. Deng. Woodward and W. 40 J. Tschaplinski. Y.. Michael. C. Trethewey and L. H. Ritte. Y. Kobayashi and E. 2009. Moreau. Hwang. M. Frank. Zhirov. 56. T. Paul. Z. Xie. Raamsdonk. Soc. S. 13. Guo. C. Fiehn. 39 K. Arbona. Steer. 349. Chem. Ren. 2009. S. Biomed. J. Tobin. Biotechnol. 54 K. B. 2008. 2009. C.. 2007.. 36 Y. 1.. Nordstrom. Sjostrom and T.. H. Choi. Zou. Verpoorte and P. Kopka. 38 C. 59. A. D.. Sep. M. M.. Food Res. Kim. M. Ellis. Ong. UK. Y. Wang. Li. 2009. 2009. 48 I. Fernie. A.rsc. Cai and D. He. 57. E.. Food Chem. Vigeolas. 564.. 7. G. Tarachiwin. 156. Klinkhamer. Brandle and P. A. 56 H. 53 S. Pharm. Anal. Weckwerth. 2009. H. 32.. D. H. A. Takebe. H. 51 C. L.. Jonsson. 2009. Biomed. L. Xia and M. D. J. Ong for his help with the SIMCA-P+ software program. C. B. Park. B. J. I. H. 1995. Bamba. K. Metabolomics. Food Chem. 2008. F. Meng and X. M. Sci. Agric. Verpoorte. 47 J. 669. Kim. E. K. Lloyd. Chen. Choi. M. Mesneau. Li. H. Djarmati and B. S. Phytochem. 5827. 621. A. 2008. B: Anal. G. J. K. C. 871. Holder. D. Kossmann and G. 2002. Kim. Kobayashi. Chromatogr. Kopa and M. Jan. Tenmoli Ra is grateful for the study leave granted by Ministry of Education. Opin. Oksman-Caldentey and D.. Sci. D. D.. 2008. T. H. 25 C. Engel. 285. England. R. I. A. A. 2001. 42. K. L. I. Smith. W. 254. Angela Lim. Talon and A. G. Zhiboedov and V. P. 2005. Son. H. O. J. Y. Fiehn. Y. Singapore. D. 13. S. Hew and E. Y. 2006. 41 R. J. Karyagina. C. Smith and A. J. Tan. Schauer. 55 T. J. Glazebrook and J. Wang. L. M. Chem. Wu. Okazawa. 2008. R. Zhang and Y. Yu. F. 401. W. 277(44). 38. T. J. 2009. Fukusaki and A. 45. 2503. Chromatogr.. Takachiwin.. S.. Lin. Hawkins and C. D. Ruben.. M. T. P. Nat. C. H. E. N. 613. K. Chinese Materia Medica. Chemistry Press. U. Serb. 2220. Lisec.. 2008. Rolin and M. O. Life Sci. E.. Phytochem. S. 567.. Maltese. | doi:10. 138. Wang. W. S. J. Natl. Plant Mol. Verpoorte. F. 52. 5. J. J. S. 2986. Zenk. J. 92(10). J.. 2009. Yang. Y. S. Amigues. H.. 2008. Qiu is acknowledged. Zeeman. TrAC. 655. Anna Lee and H. H. Y. 4(9). Soc. R. L. 1182. Iglesias. 56. 49 Z. Smith. J. 46 W. M. Agric. K. W. 49. Teo.. Biomed. P. Opin. M.. K. Zang. 41533. S. Genomics. Zhu. Agric. 54(3). H. J. 2008. Trends Anal. Tan. Protoc. Bowsher.. Lee and H. Ahn. D. Hew and E. 412. Kapteyn and D. Agric. B. S. W. Yong. R. Acknowledgements The authors are grateful for the financial support (Grant No. W. 1855. Bot. H. 48. Walsh. Food Chem. 132. J. Rev. Saeed.. A. First ed.. Curr. S. T. Quick. Timchenko and D. J. Plant Biochemistry. 2008.. M. T. Chem. 112. F. S. K. Fukusaki. S. Kutchan. N. S. J. Broadhurst. 2004.. V. Qu. J. Annu. P. 56. Dormann. 2006. Schliemann. K. Lee and W. N. Peter. 45. 1483. Liang. 30 O. L. J. 283. S. 2005. S. Y. Introduction to Multivariate Statistical Analysis in Chemometrics. Ferry-Dumazet. Zyprian. Hong. Dunn and D. S. 28 K. W. N. A. Annu. Tianniam. 89. S. Sweetlove. H. Xu and B. 2003. 2004. Noubhani. M. B. Riu. Dai. Ferrand. Planta Med. D. 9. Morreel. 2004. L. A. 2005. Curr. K. B. Q. 4099. 324. M. Gielen. Metabolomics. Biol. C. N. Telmer. 183. A. Raymond. J. Qiao. 2008. 219. E. Strack. 2008. Fukusaki. M. J. Choi and R. Kopka. Ma. Bioeng. Plant Cell Physiol. 37 S. J. Bhalla. Molecules. Teusink. M. Pharm. I. S.. 57 S. 900. S. Technol. M. Mueller. 113. 2007. Leiss. Pharm. 2009. S. X. Rolin and A. V. T. Medicinal Natural Products A Biosynthetic Approach 2nd Edition. Plant J. 57. 34 M. UK. Russ. 11132. 32 V. J. L. I. Z. Guo. Osaki and M. Dieter and V. C.