IC4R008-Metabolomics-2014-25057267

Project Title

• Toward better annotation in plant metabolomics: isolation and structure elucidation of 36 specialized metabolites from Oryza sativa (rice) by using MS/MS and NMR analyses

The Background of This Project

• Metabolomics plays an important role in phytochemical genomics and crop breeding; however, metabolite annotation is a significant bottleneck in metabolomic studies. In particular, in liquid chromatography–mass spectrometry (MS)-based metabolomics, which has become a routine technology for the profiling of plant-specialized metabolites, a substantial number of metabolites detected as MS peaks are still not assigned properly to a single metabolite. Oryza sativa (rice) is one of the most important staple crops in the world. In the present study, the researchers isolated and elucidated the structures of specialized metabolites from rice by using MS/MS and NMR.

Plant Materials & Treatment

• Rice plants (cultivar Habataki) were grown in plastic pots containing granular soil (Bonsoru No.2; Sumitomo Chemical, Tokyo); after approximately 10 weeks of incubation, shoots were collected, lyophilized, and stored at -80 ℃ until use (Matsuda et al. 2012).

Research Findings

• 36 compounds, including five new flavonoids (6–9 and 24), were isolated and assigned from the leaves of rice using MS/MS and NMR methods (Fig. 1). To our knowledge, this is the first time that 18 of the known compounds (4, 5, 12, 13, 17–23, 29–33, 35, and 36) have been isolated from rice leaves.

Fig. 1 Structures of compounds 1–36. Glc b-Dglucopyranosyl, Rut rutinosyl, Neo neohesperidosyl, GluA glucuronopyranosyl, Ara arabinosyl, erythro and threo the forms of lignan parts of flavonolignans, asterisk new compound

• Compound 6 was obtained as a yellow amorphous powder. The molecular formula of compound 6 was established as C29H32O18 by HR-ESI-QTOF-MS. Compound 6 was assigned as tricin 7-O-(200-O-b-D-glucopyranosyl)-b-D-glucuronopyranoside.(Fig. 2). Compound 7 was obtained as a yellow amorphous powder. HR-ESI-QTOF-MS gave the molecular formula C26H26O15. Compound 7 was assigned as tricin 7-O-(600-O-malonyl)- b-D-glucopyranoside(Fig. 2). Compound 8 was obtained as a yellow amorphous powder. The molecular formula of compound 8 was determined as C34H34O16 with HR-ESI-QTOF-MS. Compound 8 was assigned as tricin 7-O-(600- (E)-sinapoyl)-b-D-glucopyranoside(Fig. 2). Compound 9 was obtained as a yellow amorphous powder. The molecular formula of compound 9 was established as C34H38O17 by HR-ESI-QTOF-MS. Compound 9 was assigned as tricin 40-O-(threo-b-syringylglyceryl) ether 700-O-b-Dglucopyranoside(Fig. 2). Compound 24 was obtained as a yellow amorphous powder. The molecular formula was found to be C26H28O15 by HR-ESI-QTOF-MS. compound 24 was assigned as luteolin 6-C-(200-O-b-D-glucopyranosyl)-a-Larabinoside(Fig. 2).

Fig. 2 Key HMBC correlations of compounds 6–9 and 24

• On comparison of the 1H- and 13C-NMR spectral data (Supplementary data file S2) with those in the literature, these compounds were assigned as tricin (1) (Jiao et al. 2007), tricin 7-O-b-Dglucopyranoside (2) (Kong et al. 2007), tricin 5-O-b-Dglucopyranoside (3) (Adjei-Afriyie et al. 2000), tricin 7-Orutinoside (4) (Hirai et al. 1986), tricin 7-O neohesperidoside (5) (Zhang et al. 2009), tricin 40-O-(erythro-b-guaiacylglyceryl) ether (10) (Bouaziz et al. 2002), tricin 40-O- (threo-b-guaiacylglyceryl) ether (11) (Bouaziz et al. 2002), tricin 40-O-(erythro-b-guaiacylglyceryl) ether 7-O-b-Dglucopyranoside (12) (Bouaziz et al. 2002), tricin 40-O- (threo-b-guaiacylglyceryl) ether 7-O-b-D-glucopyranoside (13) (Bouaziz et al. 2002), tricin 40-O-(erythro-b-guaiacylglyceryl) ether 700-O-b-D-glucopyranoside (14) (Baek et al. 2012), tricin 40-O-(threo-b-guaiacylglyceryl) ether 700-O-b-D-glucopyranoside (15) (Baek et al. 2012), tricin 40-O (erythro-b-guaiacylglyceryl) ether 900-O-b-D-glucopyranoside (16) (Baek et al. 2012), and tricin 40-O-(threob- 4-hydroxyphenylglyceryl) ether (17) (Chang et al. 2010). Comparing the 1H-NMR spectral data with those in the literature, they were assigned as syringetin 3-O-b-D-glucopyranoside (18) (Guo et al. 2010) and syringetin 3-O-rutinoside (19) (Victoire et al. 1988). Comparing the 1H-NMR spectral data with those in the literature, four flavonoid C-glycosides (compounds 20–23) were assigned as apigenin 6-C-a-L-arabinosyl-8-C-b-Larabinoside (20) (Xie et al. 2003), chrysoeriol 6-C-a-Larabinosyl- 8-C-b-L-arabinoside (21) (Shie et al. 2010), swertisin (22) (Cheng et al. 2000), and isoorientin 7,30- dimethyl ether (23) (Zhu et al. 2010). Moreover, comparing the 1H- and 13C-NMR spectral data with those in the literature, four O,C-glycosides (compounds 25–28) were assigned as isoscoparin 200-O-(6000-(E)-feruloyl)-glucopyranoside (25) (Besson et al. 1985), isoscoparin 200-O-(6000- (E)-p-coumaroyl)-glucopyranoside (26) (Besson et al. 1985), isovitexin 200-O-(6000-(E)-feruloyl)-glucopyranoside (27) (Markham et al. 1998), and isovitexin 200-O-(6000-(E)- p-coumaroyl)-glucopyranoside (28) (Markham et al. 1998).

• On comparing the 1H- and 13C-NMR spectral data with those in the literature, these compounds were assigned as 1,3-Odiferuloylglycerol (29) (Luo et al. 2012), 1-O-feruloyl-b-Dglucose (30) (Miyake et al. 2007), 1-O-sinapoyl-b-D-glucose (31) (Miyake et al. 2007), 3-O-p-coumaroylquinic acid (32) (Ma et al. 2007), and 3-O-feruloylquinic acid (33) (Ida et al. 1994). The MS spectra of compound 34 in the negative ionization mode showed a deprotonated molecular ion at m/z 299. The MS/MS spectra of the precursor ion at m/z 299 gave a major fragment ion at m/z 137 [(M - H)- 162]-, suggesting the presence of a hexose group. Compound 34 was assigned as salicylic acid 2-O-b-D-glucopyranoside (Grynkiewicz et al. 1993) by comparing the 1Hand 13C-NMR spectral data with those in the literature.

• This compound was assigned as kynurenic acid (35) (Beretta et al. 2007) by comparing the MS/MS and 1H-, 13C-NMR spectral data with those in the literature. On comparing the 1H-NMR spectral data with those in the literature, compound 36 was assigned as lycoperodine-1 (Yahara et al. 2004).

• MS/MS data of tricin 40-O-(erythro-b-guaiacylglyceryl) ether 7-Ob-D-glucopyranoside (12) and tricin 40-O-(threo-b guaiacylglyceryl) ether 7-O-b-D-glucopyranoside (13) showed major product ions at m/z 527 [(M + H)-162]+, corresponding

to the loss of glucose and aglycone fragment ions, and at m/z 331 [(M + H)-162-196]+, indicating the loss of glucose and guaiacylglyceryl groups (Fig. 3).

Fig. 3 Mass spectra of tricin 40-O-(erythro-b-guaiacylglyceryl) ether 7-O-b-D-glucopyranoside ( 12) ( m/z 688) and tricin 40-O-(threo-bguaiacylglyceryl) ether 7-O-b-D-glucopyranoside (13) (m/z 688) at ramped collision energy from 10 to 50 eV in positive ionization mode

• The MS/MS spectra of the flavonoid O,C-glycosides isoscoparin 200-O-(6000-(E)-feruloyl)-glucopyranoside (25), isoscoparin 200-O-(6000-(E)-p-coumaroyl)-glucopyranoside (26), isovitexin 200-O-(6000-(E)-feruloyl)-glucopyranoside (27), and isovitexin 200-O-(6000-(E)-p-coumaroyl)-glucopyranoside (28) in the positive ionization mode showed fragment ions of the C-glycoside at m/z 463 and 433, which were formed by the neutral loss of glucose and acyl substituents (feruloyl or coumaroyl moiety). Fragment ions of the feruloyl moiety at m/z 177 and coumaroyl moiety at m/z 147 were also observed (Fig. 4).

Fig. 4 Mass spectra of isoscoparin 200-O-(6000-(E)-feruloyl)-glucopyranoside (25) (m/z 800) at ramped collision energy from 10 to 50 eV in positive ionization mode. The upper figure shows the display range at m/z 50–850, the lower figure shows the expanding range at m/z 300–480

Labs working on this Project

• RIKEN Center for Sustainable Resource Science, 1-7-22
• School of Pharmacy, Nihon University, 7-7-1 Narashinodai
• School of Pharmacy, Showa University, 1-5-8 Hatanodai
• Graduate School of Pharmaceutical Sciences, Chiba University

Corresponding Author

• K. Saito: kazuki.saito@riken.jp