Industrial Crops and Products 33 (2011) 488–496 Contents lists available at ScienceDirect Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop Metabolite fingerprint of “capim dourado” (Syngonanthus nitens), a basis of Brazilian handcrafts Mariana Pacifico a , Assunta Napolitano b , Milena Masullo b , Felipe Hilario a , Wagner Vilegas a , Sonia Piacente b,∗ , Lourdes Campaner dos Santos a a b UNESP, São Paulo State University, Institute of Chemistry, Organic Chemistry Department, CP 355, CEP 14800-900 Araraquara, São Paulo, Brazil Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano, SA, Italy a r t i c l e i n f o Article history: Received 30 August 2010 Received in revised form 21 October 2010 Accepted 22 October 2010 Available online 23 November 2010 Keywords: Syngonanthus nitens C-Glycoside-flavones Xanthones HPLC–ESI-MSn NMR a b s t r a c t Syngonanthus nitens is a grass-like species, whose flower stems assume a beautiful golden colour when dried, hence their common name “capim dourado”. This plant represents an important source of income for rural communities, especially in Jalapão region, being the scapes used in the craft industry to make, together with buriti palm strips, traditional handcrafts from their coils. Therefore, considering that scapes and not flowerheads are used, we carried out an analytical study to define the metabolite fingerprint of both S. nitens parts, with the aim, on one hand, to identify the molecules responsible for the characteristic golden colour of the capim dourado herb and, on the other hand, to identify the occurrence of interesting constituents in S. nitens flowerheads to increase the value of this part of the plant, considered a waste matter of golden grass handcrafts. Therefore an HPLC–ESI-MSn method, based on high-performance liquid chromatography coupled to electrospray negative ionization multistage ion trap mass spectrometry, was developed to rapidly identify and guide the isolation of the secondary metabolites occurring in flowers and scapes. On the basis of the on-line HPLC–ESI-MSn screening, 17 compounds, including 6 new molecules, were isolated, and their structures were unambiguously elucidated by NMR spectroscopic data. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Syngonanthus nitens (Bong. Ruhland) is a grass-like species of Eriocaulaceae (Schmidt et al., 2007), a pantropical, predominantly herbaceous monocotyledonous family, comprising around 1100 species in 11 genera (Giulietti et al., 2000; Sano, 2004). It is a perennial polycarpic herb with leaves arranged spirally in a basal rosette of about 4 cm (Schmidt et al., 2007). The plants are clonal, and fertile rosettes can annually produce from 1 to 10 scapes (flower stalks or stems), with each scape bearing a capitulum flower (Giulietti et al., 1996). The flower stems of S. nitens are bright and assume a beautiful golden colour when dried, acquiring a phenomenal aesthetic similarity to spun gold, hence their common name “capim dourado” (golden grass). This grass-like species occurs in all Brazilian Cerrado, in areas with intermediate humidity within the humid grasslands (Giulietti et al., 1996). In particular, in the Jalapão region, state of Tocantins, within the Cerrado area, where humid grasslands occur as a belt between the scrubland and the gallery forest, golden grass grows naturally in fields of soaked wet in the rainy season and dry during periods of drought (Ratter et al., 1997). ∗ Corresponding author. Tel.: +39 089969763; fax: +39 089969602. E-mail address: piacente@unisa.it (S. Piacente). 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.10.023 It is especially in this region of Brazilian Savannah Highland, in the cities of Tocantínia (Tocantins State) and São Domingos (Goiás State), that the scapes of S. nitens have been used in the craft industry since the late 1990s, thus representing an important economic resource for rural communities. Traditional handcrafts, made from coils of S. nitens scapes together with buriti palm (Mauritia flexuosa) strips, range from handmade objects, such as vases, placemats, baskets, to personal accessories like hats, bracelets, earrings, handbags, purses and belts, and many other decorative items (Schmidt et al., 2007). Recently, the traditional handcrafts made by women from the Mumbuca Community for more than 70 years started being commercialized in large Brazilian cities and European countries (Schmidt et al., 2007). Thereby, the handcrafts made from coils of S. nitens scapes represent an important source of income for these regions, above all for Jalapão. According to this, increasing extraction rates are observed, thus making the preservation of S. nitens habitat extremely important. There is a program of management and protection of these lands administered by the Government, allowing scape harvesting only after the second middle of September, and requiring the cutting of the flowerheads and the dispersal of at least part of the seeds in the grassland areas just after scape harvesting (Schmidt et al., 2007). Concerning this, a central aspect of the potential sustainability of S. nitens for handcrafts relates to the fact that scapes and not flower- M. Pacifico et al. / Industrial Crops and Products 33 (2011) 488–496 heads are used, with the exception of some regions of Bahia, Goiás and Minas Gerais states, in which scapes and flowerheads are harvested to be used as dried ornamental flowers (Giulietti et al., 1996). In this case, the economic value is determined by the colour and the structure of the flowerheads, and these characteristics change, decreasing quality and value, after seed production. To our knowledge no study has been done until now on the chemical composition of neither scapes or flowerheads of S. nitens. Only a characterization study on cellulose whiskers extracted from capim dourado fibers has been engaged, reporting their use to reinforce natural rubber and defining morphological, thermal and mechanical properties of ensuing nanocomposite films (Siqueira et al., 2010). Thereby, in the context of the previous described law provisions, and with the aim to give further scientific contribution to support and improve Brazilian handcrafts artisan economy, we decided to carry out an analytical study to define the metabolite fingerprint of S. nitens flowerheads, now considered a waste matter of golden grass handcrafts, but potentially increasing in value if containing bioactive metabolites. HPLC hyphenated techniques are playing increasingly important roles in support of phytochemical investigations, such as targeting the isolation of new active compounds, for the dereplication of known plant constituents and for metabolic profiling studies (Wolfender et al., 1998). In particular liquid chromatography coupled to mass spectrometry (HPLC–MS) is considered a powerful tool for structural elucidation and analysis of complex matrixes, providing structure specific data for the characterization of known or unknown molecular species with high selectivity and sensitivity (Mattoli et al., 2006). In fact, liquid chromatography allows complex mixtures of metabolites from crude extracts to be trapped, focused, and selectively eluted prior to introduction into the mass spectrometer. Utilization of electrospray ionization (ESI), a soft-ionization technique that generates protonated/deprotonated molecules (positive/negative ion mode, respectively), permits the analysis of non-volatile and labile components in the extract without derivatization (Mattoli et al., 2006). Tandem mass spectrometry (MS/MS) provides further structure information uniquely identifying various types of metabolites by characteristic fragmentations of their structure (Hoffmann, 1996). Thereby, HPLC–ESI-MSn technique, combining the efficient separation capability of HPLC with the great power of structural characterization and high sensitivity of mass spectrometry, results the most suitable approach to analyze complex metabolite mixtures of plant extracts. In order to this, an HPLC–ESI-MSn method, based on highperformance liquid chromatography coupled to electrospray negative ionization multistage ion trap mass spectrometry, was developed to rapidly identify and guide the isolation of flowers metabolites. The same analytical approach has been applied to S. nitens scapes, to obtain a metabolite profile giving informations on molecules responsible for characteristic golden colour of the capim dourado herb. On the basis of the on-line HPLC–ESI-MSn screening, 17 compounds, including 6 new molecules, were isolated, and their structures were unambiguously elucidated by NMR spectroscopic data. 2. Materials and methods 2.1. General experimental procedures NMR experiments were performed on a Bruker DRX-600 spectrometer at 300 K. All 2D-NMR spectra were acquired in CD3 OD (99.95%, Sigma–Aldrich) and standard pulse sequences and phase cycling were used for DQF-COSY, HSQC and HMBC. HPLC–ESI-MSn and ESI-MSn analyses were performed on a SURVEYOR MS microHPLC system coupled with an LCQ Deca XP Max ion trap mass 489 spectrometer (ThermoFinnigan, San José, CA, USA). The mass spectra were acquired in negative ion mode and processed using the Xcalibur software (version 1.3) provided by ThermoFinnigan. Column chromatography was performed over Sephadex LH-20 (Pharmacia). HSCCC separation was performed on an HSCCC apparatus (P.C. Inc.). HPLC-DAD separations were carried out on an Varian (Walnut Creek, CA, USA) ProStar 210/330 chromatographic system, Rheodyne injector (Cotati, CA, USA) 7125 sample injector with a 0.1 mL sample loop and photodiode array detector. HPLCUV separations were performed on an Agilent 1100 series liquid chromatograph, equipped with a G-1312A binary pump, a G-1328B rheodyne injector with a 0.2 mL sample loop, and a G-1365B multiple wavelength detector. HPLC-IR separations were carried out on a Knauer system equipped with a Smartline refractive index detector 2300/2400 Knauer, and a Rheodyne injector with a 0.2 mL sample loop. TLC was performed on silica gel 60 (Merck) plates. 2.2. Plant material Flowers and scapes of S. nitens were collected in December in Jalapào city, Tocantins State, Brazil and authenticated by Professor Dr. Paulo Takeo Sano of the São Paulo University (USP), SP. A voucher specimen (SPF 189975) was deposited at the Herbarium of the IB-USP. 2.3. Extraction Dried and powdered flowers (416 g) and scapes of S. nitens (410 g) were separately and successively extracted for a week, at room temperature, with hexane, CH2 Cl2 and MeOH. The solutions were evaporated to dryness in vacuo to give 11.8 g of MeOH crude extract for flowers (2.87%), and 16.0 g of MeOH crude extract for scapes (3.90%), respectively. 2.4. HPLC–ESI-MS analysis Methanol extracts of flowers and scapes were separately analyzed by on-line HPLC–ESI-MSn . HPLC separation was conducted on a Luna PFP(2) column (5 ␮m, 3 mm × 150 mm; 100 A; Phenomenex, Torrance, CA, USA) at a flow rate of 0.2 mL/min. A gradient elution was performed by using H2 O (A) and CH3 CN (B), both added of 0.1% acetic acid, as mobile phases. After a 3 min hold at 5% B, elution was performed according to the following conditions: from 5% B to 38% B in 11 min; hold to 38% B for 3 min, to 42% B in 8 min; hold to 42% B for 10 min; to 47% B in 5 min. The column effluent was analyzed by ESI-MS in negative ion mode. The capillary voltage was set at −32 V, the spray voltage at 5 kV and the tube lens offset at 30 V. The capillary temperature was 280 ◦ C. Data were acquired in MS1 and MSn scanning modes. 2.5. Fractionation by gel filtration 3.5 g of flowers extract and 3.0 g of scapes extract were separately dissolved in methanol (20 mL) and each mixture was centrifuged for 5 min at 3200 rpm. This step was repeated 3 times for flowers extract, and 2 times for scapes extract. After collection of relative supernatants, each solution was filtered through an Iso-Disk P-34, 3 mm diameter PTFE membrane, 0.45 ␮m pore size (Supelco, Bellefonte, PA, USA), and dried in vacuo, yielding 3.0 g from flowers filtrate and 2.6 g from scapes filtrate, respectively. 3.0 g of flowers dried filtrate and 2.6 g of scapes dried filtrate were separately fractionated on a Sephadex LH-20 column (57 cm × 3 cm), using MeOH as mobile phase, affording 190 and 260 fractions (8 mL), respectively. 490 M. Pacifico et al. / Industrial Crops and Products 33 (2011) 488–496 2.6. HSCCC analysis The sephadex fractions 69–88 (400 mg) of the scapes were fractionated by HSCCC, in ethyl acetate–n-butanol–water (3.5:1.5:5.0, v/v/v), using (H → T), triple coil, 1.6, 130 mm (large coil), flow rate 1 mL/min. The lower aqueous phase was used as stationary phase. The retention of the stationary phase for this solvent system was 87.7% at 850 rpm. 84 fractions (3 mL) were obtained. 2.7. HPLC-UV analysis HSCCC fractions 16–19 were analyzed by HPLC-UV, on a C18 reversed-phase column (␮-Bondapak C18, 300 mm × 7.6 mm i.d., 10 ␮m), using H2 O + TFA (0.1%) and CH3 CN + TFA (0.1%) as mobile phases, at a flow rate of 2 mL/min, and detecting at 280 nm. Elution was performed according to the following conditions: after a 3 min hold at 5% B, from 5% B to 42% B in 12 min; to 44% B in 2 min, hold to 44% B for 10 min; to 50% B in 6 min. The chromatographic separation yielded two compounds, that, on the basis of ESI-MSn and NMR experiments, were identified as compounds 6 (1.9 mg, tR = 15.2 min), and 7 (3.8 mg, tR = 16.1 min). HSCCC fractions 68–72 were analyzed by HPLC-UV, according to the previous conditions. The chromatographic separation yielded five compounds identified, on the basis of ESI-MSn and NMR experiments, as compounds 1 (1.8 mg, tR = 14.7 min), 2 (1.5 mg, tR = 15.3 min), 3 (2.8 mg, tR = 15.9 min), 4 (1.2 mg, tR = 16.5 min), and 5 (2.0 mg, tR = 17.2 min). 2.8. HPLC-IR analysis Scapes sephadex fractions 131–253 (240 mg) were isocratically analyzed by HPLC-IR, on a ␮-Bondapak C18 column (250 mm × 10 mm i.d., 10 ␮m), using H2 O/MeOH (58:42) as eluents, and a flow rate of 2 mL/min. The chromatographic separation yielded five compounds, that, on the basis of ESI-MSn and NMR experiments, were identified as compounds 8 (4.2 mg, tR = 16.2 min), 9 (3.8 mg, tR = 17.0 min), 10 (2.0 mg, tR = 17.7 min), 11 (11.1 mg, tR = 19.3 min), and 17 (11.2 mg, tR = 23.6 min). 2.9. HPLC-DAD analysis Flowers sephadex fractions 129–134 (129 mg) were analyzed by HPLC-DAD, on a C18 reversed-phase column (␮-Bondapak C18, 300 mm × 7.6 mm i.d., 10 ␮m), using H2 O + TFA (0.05%) and MeOH + TFA (0.1%) as mobile phases, at a flow rate of 2.5 mL/min. Elution was performed according to the following conditions: from 30% B to 70% B in 20 min; to 90% B in 20 min; and to 100% B in 5 min. The chromatographic separation yielded five compounds identified, on the basis of ESI-MSn and NMR experiments, as compounds 12 (11.2 mg, tR = 18.0 min), 13 (6.8 mg, tR = 18.5 min), 14 (5.1 mg, tR = 21.0 min), 15 (10.2 mg, tR = 21.5 min), and 16 (5.3 mg, tR = 22.0 min). 2.10. ESI-MSn analysis Negative ESI-MSn analyses of each isolated compound were performed using the same conditions as those for HPLC–ESI-MSn analysis. Each compound was dissolved in CH3 OH and infused in the ESI source by using a syringe pump (flow rate 5 ␮L/min). 3. Results 3.1. S. nitens flowerheads In order to obtain a preliminary metabolite fingerprint of S. nitens flowerheads, the methanol extract was analyzed by HPLC–ESI-MSn in negative ion mode (ESI), with a mobile phase consisting of an aqueous acetonitrile gradient acidified with acetic acid (Fig. 1). Under these conditions, a good chromatographic profile was achieved, showing a clean separation between molecules mainly belonging to two different classes, but all producing the [M−H]− ion as the main peak. A more careful analysis of ESIMSn spectra recorded for each chromatographic peak allowed us to ascribe the earlier eluting molecules (1–11) to the flavonoid class and the later eluting compounds (12–16) to the xanthone class (Fig. 2). In particular, according to literature data, the study of ESI-MSn fragmentation pattern of each flavonoid metabolite permitted to distinguish between the major structural types of this class, ascertaining the presence of 8 flavone (1–4, 6–8, 11) and 3 flavanone (5, 9–10) derivatives (McNab et al., 2009; Ferreres et al., 2003). On the basis of the online screening by HPLC–ESI-MSn , to confirm mass spectral structural assignment, each compound was isolated and its structure was unambiguously elucidated by NMR experiments. 3.1.1. Structure elucidation of compound 2 The negative ESI-MS spectrum of 2 showed the [M−H]− ion at m/z 477; the ESI-MS/MS fragmentation pattern of this ion was consistent with a C-glycoside flavone (Table 1). The fragmentation pathway of C-glycoside flavonoids is rather different from that of O-glycoside flavonoids (Kazuno et al., 2005). In fact, while the O-glycoside flavonoids produce only [M−H-162]− ions due to the cleavage of the O-glycosidic linkage, C-glycoside flavonoids undergo to cross-ring cleavages in the sugar moiety, originating characteristic fragment ions giving diagnostic informations about the aglycone, the type of sugar moiety, and the glycosidation site in mono-C-glycoside isomers (Ferreres et al., 2007), that is generally limited to the C-6 or C-8 position of the aglycone (Becchi and Fraisse, 1989). In the negative ion mode, a diagnostic [M−H120]− product ion is generated only by hexose units, thereby readily indicating the type of sugar moiety (Wu et al., 2004). Furthermore, being the sugar residue of 8-C-glycosides more stable than that of 6-C-glycosides, preferential fragmentations occur in case of C-6 linked sugars, yielding more abundant fragment ions and allowing to ascertain the glycosidation site (Ferreres et al., 2003). In particular, the [M−H-90]− ion can be also observed, being more abundant in the 6-C-glycoside than in the 8-C-glycoside derivatives where it may show very low abundance. Moreover, the [M−H-18]− ion is detected with greater frequency in the former isomers than in the latter ones. Finally, the presence in the ESI-MS/MS spectra of the [M−H-120]− and [M−H-90]− diagnostic ions gives immediately account of the type of aglycone, deriving the above cited ions from the aglycone plus the residue of the sugar that remains linked to it, thereby indicating the substitution pattern (hydroxyl/methoxy groups) of the aglycone itself (Ferreres et al., 2003). According to this C-glycoside flavone behaviour, the presence in the ESI-MS/MS spectrum of the [M−H]− ion at m/z 477 of two abundant ion peaks at m/z 387 and 357, corresponding to the [M−H-90]− and the [M−H120]− product ions, respectively, together with the [M−H-18]− ion were indicative of a hexose sugar linked to C-6 of the aglycone (Table 1). The ions at m/z 387 and 357 afforded useful informations about the nature of the aglycone, corresponding to the A + 41 and A + 71 ions (A = aglycone), generated by neutral losses of part of the hexose sugar, and suggesting an aglycone molecular weight of 316 a.m.u. Further indications about the substitution of the aglycone could be deduced observing the presence of the [M−H-15]•− ion at m/z 462, due to the neutral loss of a methyl group. Moreover, the well-known retro-Diels-Alder (RDA) fragmentation mechanism, common to flavone skeleton, provided information about the position of this group, as well as about the number of hydroxyl groups, on A or B ring (McNab et al., 2009). In fact, the presence in the ESI-MS/MS spectrum of the characteristic product ion at m/z M. Pacifico et al. / Industrial Crops and Products 33 (2011) 488–496 491 Fig. 1. HPLC–ESI-MS profile of the methanol extract of S. nitens flowerheads. 313 allowed to locate one methoxyl and two hydroxyl substituents at the B-ring. This result was supported by the [M−H-138]− ion, originated by the neutral loss of a methoxy-cyclohexadiendione unit. The 1 H NMR spectrum of compound 2 showed two singlets at ı 6.61 and 6.42, assigned to H-3 and H-8 of a flavone skeleton, respectively, and a signal at ı 7.12, assigned to H-2′ and H-6′ , which suggested the presence of a 3′ ,4′ ,5′ -trisubstituted ring B of a flavone (Table 2). Moreover, a signal at ı 4.00 (s) corresponding to a methoxyl group was evident. The 1 H NMR spectrum displayed also a signal corresponding to an anomeric proton at ı 4.82 (d, J = 8.8 Hz). The 13 C NMR spectrum of 2 displayed 22 carbon signals, including 16 carbons attributable to a methoxyflavone aglycone and 6 carbons corresponding to a hexose unit. The chemical shifts of all the individual protons of the sugar unit were ascertained from a combination of 1D-TOCSY and DQF-COSY spectral analysis, and the 13 C NMR chemical shifts of their attached carbons could be assigned unambiguously from the HSQC spectrum (Table 2). These data showed the presence of a ␤-glucopyranosyl unit. According to ESI-MS/MS results, the chemical shift of C-1glc (ı 74.2) confirmed the attachment of the glucose unit via a C-glycosidic linkage (Krafczyk and Glomb, 2008). The HMBC correlation between the proton signal at ı 4.82 (H-1glc ) and the carbon resonance at ı 109.9 (C-6) allowed to confirm the position of the glucose unit at C-6 (Fig. 3). The methoxyl group was located at C-3′ on the basis of the HMBC correlation between the proton signal at ı 4.00 and the carbon resonance at ı 149.4. Consequently, 2 was determined to be the new 5,7,4′ ,5′ -tetrahydroxy-3′ -methoxy-6-C-␤-dglucopyranosylflavone. 3.1.2. Structure elucidation of compound 4 The full negative ESI-MS spectrum of compound 4 showed an [M−H]− ion at m/z 491 (Table 1). Analogously to 2, the ESIMS/MS fragmentation pattern of 4 allowed to define the nature of this compound as a 6-C-glycoside-flavone, showing a diagnostic base peak at m/z 371, together with a minor peak at m/z 401, originating from neutral losses of 120 and 90 a.m.u., and corresponding to A + 41 and A + 71, respectively. This information, together with the observation of the [M−H-164]− ion originated by the RDA fragmentation mechanism, and the [M−H15]·− fragment ion, due to a loss of a methyl group, allowed the characterization of the aglycone as a derivative of 2, having an additional methyl group on A ring. Accordingly, NMR data for aglycone moiety of 4, in comparison to those of 2, confirmed that 4 differed from 2 only by the presence of a methoxyl group instead of a hydroxyl group at C-7 (Table 2). Furthermore, NMR data of the sugar unit of 4 showed the presence of a ␤glucopyranosyl unit (ı 4.86, 1Hglc ), which gave rise to splitted signals. Signal duplication in the NMR spectra is due to the presence of rotameric conformers, created by rotational hindrance at the C(sp3 )–C(sp2 ) glucosyl-flavone linkage in C-glucosyl-substituted flavones. These splits are due to the presence of rotameric conformers of flavone 6-C-glycosides (Davoust et al., 1980; Rayyan et al., 2005). Davoust et al. (1980) reported that only 6-C-␤glucosylflavones substituted by a methoxyl or an O-␤-glucose unit at C-7 position show splitted signals. This was explained by steric hindrance of the 7-substituents in the ortho-position to 6-C-glucose unit (Davoust et al., 1980), in accordance with the reports concerning rotamers of 6-C-glucosylflavones. Therefore, 4 was deter- M. Pacifico et al. / Industrial Crops and Products 33 (2011) 488–496 492 Fig. 2. Compounds isolated from S. nitens. mined to be the new 5,4′ ,5′ -trihydroxy-7,3′ -dimethoxy-6-C-␤-dglucopyranosylflavone. 3.1.3. Structure elucidation of compound 5 The negative ESI-MS/MS spectrum of compound 5 showed the [M−H]− ion at m/z 463 (Table 1). The 6-C-glycosylation could be inferred by the occurrence of [M−H-120]− and [M−H-90]− ions, the presence of a methyl group was suggested by the [M−H-15]·− ion, and the RDA pathway gave information about the presence of two hydroxyl groups on B-ring, showing a product ion at m/z 327, and consequently allowing the assignment of the methyl group at the A-ring. This latter assignment was further supported by the presence of the [M−H-108]− ion, produced by neutral loss of B- ring as cyclohexadiendione (Table 1). The 1 H NMR spectrum of compound 5 revealed the presence of non-equivalent methylene protons at ı 2.99 (dd, J = 16.5, 13.5 Hz, H-3ax) and 2.60 (dd, J = 16.5, 2.5 Hz, H-3eq), and a double doublet signal at ı 5.23 (dd, J = 13.5, 2.5 Hz, H-2), commonly found in a flavanone nucleus (Magela et al., 2008). Additionally, the 1 H NMR spectrum showed signals for the aromatic protons at ı 5.98 (s, H-8), 6.78 (dd, J = 8.5, 1.9 Hz, H-6′ ), 6.83 (d, J = 1.9 Hz, H-2′ ), 6.89 (d, J = 8.5 Hz, H-5′ ), which revealed the substitution patterns in the A and B rings, and a signal at ı 3.98 (s), corresponding to a methoxyl group (Table 2). The 1 H NMR spectrum displayed also a signal corresponding to an anomeric proton at ı 4.75 (d, J = 9.0 Hz). The 13 C NMR spectra displayed 22 carbon signals, including 16 of a methoxyflavanone aglycone, Table 1 ESI-MS and ESI-MSn product ions of new flavones and xanthones occurring in the methanol extracts of S. nitens flowers and scapes. [M−H]− Product ions 2 477 ESI-MS/MS: 4 5 491 463 ESI-MS/MS: ESI-MS/MS: 6 9 491 463 ESI-MS/MS: ESI-MS/MS: 12 273 ESI-MS/MS: ESI-MS3 : 462 [M−H-15]• − , 459 [M−H-18]− , 387 [M−H-90]− , 357 [M−H-120]− , 339 [M−H-138]− , 313 [M−H-164]− , 269 [M−H-164-44]− 476 [M−H-15]• − , 473 [M−H-18]− , 401 [M−H-90]− , 371 [M−H-120]− , 353 [M−H-138]− , 327 [M−H-164]− 448 [M−H-15]• − , 445 [M−H-18]− , 435 [M−H-28]− , 419 [M−H-44]− , 373 [M−H-90]− , 343 [M−H-120]− , 355 [M−H-108]− , 327 [M−H-136]− , 283 [M−H-136-44]− 476 [M−H-15]• − , 473 [M−H-18]− , 401 [M−H-90]− , 371 [M−H-120]− , 353 [M−H-138]− , 327 [M−H-164]− 448 [M−H-15]• − , 445 [M−H-18]− , 435 [M−H-28]− , 419 [M−H-44]− , 373 [M−H-90]− , 343 [M−H-120]− , 355 [M−H-108]− , 327 [M−H-136]− , 283 [M−H-136-44]− 258 [M−H-15]• − 230 [M−H-15-28]• − , 229 [M−H-15-29]• − , 202 [M−H-15-56]• − , 174 [M−H-15-84]• − , 135 [M−H-15-123]• − M. Pacifico et al. / Industrial Crops and Products 33 (2011) 488–496 493 Table 2 NMR data (600 MHz, CD3 OD) for compounds 2, 4, 5, 6 and 9.a 2 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ OCH3 (C7) OCH3 (C3′ ) 4 ıH (J in Hz) ıC 6.61, s 166.4 104.0 7.12, s 182.3 161.2 109.9 164.2 94.1 157.1 104.6 122.1 103.1 149.4 139.0 147.2 108.8 4.00, s 57.1 6.42, s 7.12, s C-Glucose at C-6 5 6 ıH (J in Hz) ıC ıH (J in Hz) 6.65, s 166.4 104.1 5.23 (dd, 2.5, 13.5) 2.99 (dd, 13.5, 16.5) 2.60 (dd, 2.5, 16.5) 6.76, s 7.13, (d, 1.9) 7.11, (d, 1.9) 3.98, s 4.00, s 182.3 161.8 110.2 165.7 91.3 159.1 106.2 122.1 103.2 149.6 139.1 147.0 108.7 56.4 57.1 ıC 198.0 163.0 106.1 167.1 95.2 162.3 101.9 131.0 115.9 145.8 147.2 115.2 118.8 56.4 5.98, s 6.83 (d, 1.9) 6.89 (d, 8.5) 6.78 (dd, 1.9, 8.5) 3.98, s C-Glucose at C-6 79.7 43.6 C-Glucose at C-6 9 ıH (J in Hz) ıC ıH (J in Hz) 6.62, s 166.5 104.0 5.23 (dd, 2.5, 13.5) 2.99 (dd, 13.5, 16.5) 2.60 (dd, 2.5, 16.5) 6.51, s 7.13, (d, 1.9) 7.09, (d, 1.9) 3.95, s 4.00, s 182.5 157.2 95.6 165.3 106.5 163.0 106.0 122.1 103.2 149.6 139.0 147.1 108.8 56.5 57.1 5.90, s 6.80 (d, 1.9) 6.89 (d, 8.5) 6.83 (dd, 1.9, 8.5) 3.95, s C-Glucose at C-8 ıC 79.7 43.6 198.0 166.8 96.2 167.2 106.9 162.9 102.3 131.0 116.0 145.7 147.2 115.2 119.2 56.6 56.6 C-Glucose at C-8 ıH (J in Hz) ıC ıH (J in Hz) ıC ıH (J in Hz) ıC ıH (J in Hz) ıC ıH (J in Hz) ıC 1 4.82, d (8.8) 74.2 2 4.44, dd (8.8, 9.0) 71.3 3 4 5 6 3.45, dd (9.0, 9.0) 3.40, dd (9.0, 9.0) 3.40, m 3.90, dd (2.5, 12.0) 3.70, dd (4.5, 12.0) 80.3 71.8 82.6 62.6 4.86, d (8.8) 4.92, d (8.8) 4.26, dd (8.8, 9.0) 4.47, dd (8.8, 9.0) 3.46, dd (9.0, 9.0) 3.41, dd (9.0, 9.0) 3.41, m 3.90, dd (2.5, 12.0) 3.70, dd (4.5, 12.0) 74.2 74.2 72.2 71.3 80.3 71.8 82.6 62.6 4.75, d (9.0) 4.87, d (9.0) 4.21, dd (9.0, 9.0) 4.26, dd (9.0, 9.0) 3.45, dd (9.0, 9.0) 3.40, dd (9.0, 9.0) 3.42, m 3.90, dd (2.5, 12.0) 3.70, dd (4.5, 12.0) 74.2 74.2 71.3 71.1 80.3 71.8 82.6 62.6 4.99, d (7.8) 5.02, d (7.8) 4.12, dd (7.8, 9.0) 4.34, dd (7.8, 9.0) 3.53, dd (9.0, 9.0) 3.71, dd (9.0, 9.0) 3.48, m 3.99, dd (2.5, 12.0) 3.87, dd (4.5, 12.0) 75.1 75.1 72.4 72.1 80.1 72.4 82.7 63.2 4.89, d (7.8) 4.97, d (7.8) 4.12, dd (7.8, 9.0) 4.26, dd (7.8, 9.0) 3.47, dd (9.0, 9.0) 3.63, dd (9.0, 9.0) 3.42, m 3.95, dd (2.5, 12.0) 3.65, dd (4.5, 12.0) 75.1 75.1 72.5 72.3 80.1 72.4 82.7 63.2 a Assignments were confirmed by DQF-COSY, HSQC and HMBC experiments. Two chemical shift values are given for rotameric conformers. and 6 of a sugar unit (Table 2). These data, in combination with 1D-TOCSY, HSQC, HMBC, DQF-COSY correlations, and with those obtained from mass spectrometric analyses, suggested that 5 was a derivative of the 7-methoxyeriodictyol, with an additional substitution at C-6 (ı 106.1), as shown by the absence of the proton at position 6. The chemical shift of C-1glc (ı 74.2) showed the presence of a C-glycoside flavonoid. The NMR data allowed to identify the presence of rotameric conformers of ␤-glucopyranosyl unit. A cross-peak in the HMBC spectrum between the proton signal at ı 4.75 (H-1glc ) and the carbon resonance at ı 106.1 (C-6) allowed us to locate the sugar unit at C-6. The methoxyl group was located at C-7 on the basis of the HMBC correlation between the proton signal at ı 3.98 and the carbon value at ı 167.1. On the basis of these observations, 5 was identified as the new 3′ ,4′ ,5-trihydroxy7-methoxy-6-C-glucopyranosylflavanone. 3.1.4. Structure elucidation of compound 6 The 1 H and 13 C NMR spectra of 6 were almost superimposable to those of 4, except for the signal corresponding to A ring (Table 2). The 1 H NMR showed two singlet protons at ı 6.62 and 6.51, assigned to H-3 and H-6, respectively, and two meta-coupled protons at ı 7.13 (d, J = 1.9 Hz), and 7.09 (d, J = 1.9 Hz), assigned to H2′ and H-6′ , which suggested the presence of a 3′ ,4′ ,5′ -trisubstituted ring B. Moreover, signals at ı 4.00 (s) and ı 3.95 (s), corresponding to two methoxy groups, were evident. The 1 H NMR spectrum displayed also a signal corresponding to an anomeric proton at ␦ 4.99 (d, J = 7.8 Hz). The NMR data revealed the presence of rotameric conformers of a ␤-glucopyranosyl unit. The detailed comparison of NMR data of 4 and 6 suggested that the two compounds differed only by the position of the glucose unit, located at C-8 in 6, instead of at C-6 in 4 (Fig. 3). The presence of rotamers leading to double signals in the NMR spectra has been reported for flavones contain- ing an 8-C-hexosyl (sugar hexose) substituent, independently from the occurrence of a methoxyl group or a sugar residue at C-7. As an explanation of this, interactions between the flavone B-ring and the 8-C-hexosyl substituent leading to restricted rotation of the Bring and/or the hexose, has been suggested (Rayyan et al., 2005). This result was supported by negative ESI-MS and ESI-MS/MS analyses of 6, indicating for this latter the same molecular weight of 4 and a very similar fragmentation pattern, showing for [M−H120]− and [M−H-90]− ions lower intensities than those observed for 4 (Table 1). According to literature data (Ferreres et al., 2003), in these cases the comparison of both the retention times and the m/z of ions obtained by ESI-MS/MS of the [M−H]− ion of each isomer pair are required to establish the C-glycosylation position. In fact, under the same HPLC–ESI-MS conditions, 8-C-glycoside derivatives are reported to elute at retention times close each others, but well spaced out if compared with 6-C-glycoside derivatives (Ferreres et al., 2003). Thereby, being the HPLC–ESI-MS behaviour displayed by 6 in agreement with this report, and supported by the comparison of NMR data with those reported in literature for C-6 and C-8 flavonoid glycosides (Krafczyk and Glomb, 2008), compound 6 was identified as the new 5,4′ ,5′ -trihydroxy-7,3′ -dimethoxy-8-C-␤-dglucopyranosylflavone. 3.1.5. Structure elucidation of compound 9 Analogously to compound 6, also the 1 H and 13 C NMR spectra of 9 were almost superimposable to that of 5, except for the signal corresponding to A ring (Table 2). Once again, negative ESI-MS and ESI-MS/MS spectra, together with HPLC–ESIMS profile and NMR data, were in agreement to indicate compound 9 as the C-8-isomer of 5 (Krafczyk and Glomb, 2008), corresponding to the new 3′ ,4′ ,5-trihydroxy-7-methoxy-8C-glucopyranosylflavanone molecule (Table 1). M. Pacifico et al. / Industrial Crops and Products 33 (2011) 488–496 494 OH OH HO O OH OCH3 O HO HO OH OH O 2 HO OH OH OH HO OH O H3CO O OCH3 OH O 6 Fig. 3. HMBC key correlations of compounds 2 and 6. 3.1.6. Structure elucidation of compound 12 Finally, according to HPLC–ESI-MS results, the analysis of full negative ESI-MS spectrum of compound 12 showed a [M−H]− ion at m/z 273. In this case, the study of the fragmentation pattern in the ESI-MS/MS experiment allowed to observe a different behaviour respect to previously described molecules, being the [M−H-15]·− ion as main peak. Moreover, the ESI-MS3 spectrum of this latter product ion was characterized by the presence of fragment ions at m/z 230, 202 and 174, originating from consecutive neutral losses of CO molecules. A similar fragmentation pattern was consistent with a xanthone molecule, as supported by the check in ESI-MS3 spectrum of the RDA product ion at m/z 135. Accordingly, the 1 H NMR spectrum of 12 showed aromatic protons at ı 6.41 (s), 6.77 (d, J = 1.8 Hz), 6.87 (dd, J = 8.7, 1.8 Hz) and 8.02 (d, J = 8.7 Hz) and a singlet at ı 3.89 corresponding to a methoxy group. The 13 C NMR spectrum (Table 3) displayed 14 carbon signals. One methoxy, four methines and nine quaternary carbons (one for a carbonyl carbon) were observed. These data suggested that 12 was a xanthone with a disubstituted A ring and a trisubstituted B ring. The substitution pattern was further established on the basis of the HMBC spectrum. The methoxy group was located at C-2 on the basis of the correlation between the proton signal at ı 3.89 and the carbon signal at ı 131.6. These observations allowed us to determine the structure of 12 as the new 1,3,6-trihydroxy-2-methoxyxanthone. 3.1.7. Structure elucidation of compounds 1, 3, 7–8, 10–11, 13–16 Analogously, by the same NMR and ESI-MSn experiments the structures of compounds 1, 3, 7–8, 10–11, 13–16 were established. By comparison of their spectroscopic data with those reported in literature, they were identified as known compounds. In detail five known flavone derivatives namely 3′ ,4′ ,5,7-tetrahydroxy6-C-glucopyranosylflavone (1) (Slikel and Bushnell, 1959), 3′ ,4′ ,5-trihydroxy-7-methoxy-6-C-glucopyranosylflavone (3) (Komatsu and Tomimori, 1966), 3′ ,4′ ,5-trihydroxy-7-methoxy8-C-glucopyranosylflavone (7) (Komatsu and Tomimori, 1966), 4′ ,5-dihydroxy-3′ ,7-dimethoxy-6-C-glucopyranosylflavone (8) (Valant et al., 1980), and 3′ ,4′ ,5,6,7-pentahydroxyflavone (11) (Santos et al., 2004), along with one known flavanone derivative Table 3 NMR Data (600 MHz, CD3 OD) of compound 12.a 12 ıH (J in Hz) 1 2 3 4 4◦ 4b 5 6 7 8 8◦ 8b 9 OCH3 a 6.41, s 6.77, (d, 1.8) 6.87, (dd, 1.8, 8.7) 8.02, (d, 8.7) 3.89, s Assignments were confirmed by HSQC and HMBC experiments. ıC 150.2 131.6 154.5 94.2 159.4 159.5 103.0 165.7 115.2 127.4 103.3 103.0 181.4 60.5 M. Pacifico et al. / Industrial Crops and Products 33 (2011) 488–496 495 Fig. 4. HPLC–ESI-MS profile of the methanol extract of S. nitens scapes. namely 4′ ,5,7-trihydroxy-8-C-glucopyranosylflavanone (10) (Ito et al., 2000), and four known xanthone derivatives namely 1,3,6trihydroxy-2,5-dimethoxyxanthone (13) (Aziz-ur-Rehman et al., 2004), 3,4,8-trihydroxy-2,6-dimethoxyxanthone (14) (Ghosal et al., 1977), 1,3,6,8-tetrahydroxy-2,5-dimethoxyxanthone (15) (Meyer et al., 2008) and 1,3,6,8-tetrahydroxy-2-methoxyxanthone (16) (Iinuma et al., 1997), were identified. 3.2. S. nitens scapes The same HPLC–ESI-MS conditions used for flowerheads were applied to S. nitens scapes methanol extract, obtaining the metabolite profile shown in Fig. 4. A quick look of this profile allowed to immediately ascertain the clear prevalence of flavonoids to xanthones. In order to confirm the structure assignment inferable by the study of HPLC–ESI-MSn spectra and the rating of retention times of each compound, the same analytical approach followed for the metabolite characterization of S. nitens flowerheads was applied. Thus the identification of seven compounds (1, 3, 5, 7–10), occurring also in the flowers, and one constituent (17) (Markham, 1989), only present in scapes and identified as 3′ ,4′ ,5,7-tetrahydroxyflavone, was obtained. which cause lipid peroxidation in the cell membrane, protein modification, and DNA damage (Pietta, 2000). Thereby, they may contribute to the protection against various diseases characterized by the involvement of oxidative species (Halliwell, 2000; Scabra et al., 2006). In particular, C-glycosylflavones, common constituents of flowering plants, are reported to show wide biological activities, such as antioxidant, anticancer, antiinflammatory, anti-HIV (Wu et al., 2004), while xanthone derivatives, display many pharmacological effects, such as MAO inhibition, antitumor activity, cytotoxicity, antibacterial and antifungal activities, antiinflammatory properties, antioxidant and tuberculotic activity (Bo and Liu, 2004). Moreover, considering the type of metabolites occurring in S. nitens, it can be reasonably considered that flavonoid derivatives are responsible for the characteristic golden colour of the “capim dourado” herb. Flavonoid derivatives are in fact the most common yellow natural colourants (McNab et al., 2009). In particular, flavones per se exhibit pale yellow colour (Asenstorfer et al., 2006; Surowiec et al., 2008); however, co-pigmentation, in addition to the metal complex-pigment, is well known to cause chromatic shift by an intermolecular association that coloured pigments and colourless/transparent co-pigments (such as certain flavone glycosides) stack hydrophobically in an aqueous solution (Ono et al., 2010). 4. Discussion 4.1. Conclusions In the study reported here, S. nitens flowers and scapes showed to be differently characterized by the presence of flavonoid and xanthone compounds. In particular, noteworthy is the great abundance of these metabolites in flowerheads. These metabolites have attracted a great deal of attention for their antioxidant activity due to their ability to scavenge reactive oxygen species (ROS), In conclusion, this study allowed to obtain a fingerprint of the metabolites characterizing the flowerheads and scapes of S. nitens. Interestingly, the flower profile was more complex than that of scapes, showing a good presence of flavonoids and xanthones, the first being likely responsible for the characteristic golden colour of 496 M. Pacifico et al. / Industrial Crops and Products 33 (2011) 488–496 the “capim dourado” herb. The finding of these metabolites in flowerheads of S. nitens could increase the value of a part of the plant commonly considered as waste matter of golden grass handcraft, or, at best, as an ornamental article. Acknowledgments The authors gratefully acknowledge the financial support of the PROPG-UNESP, FAPESP Program which provided the fellowship to the project to L.C.S and a fellowship to F.H. We also thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for grants to W.V. and L.C.S. and the Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for a fellowship to M.P. References Asenstorfer, R.E., Wang, Y., Mares, D.J., 2006. 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