Special metabolites isolated from Urochloa humidicola (Poaceae)

This study aims to identify special metabolites in polar extracts from Urochloa humidicola (synonym Brachiaria humidicola ) that have allelopathic effects and induce secondary photosensitization in ruminants. The compounds were isolated and identified via chromatographic and spectroscopic techniques. The compounds 4-hydroxy-3-methoxy-benzoic acid, trans -4-hydroxycinnamic acid, and p -hydroxy-benzoic acid; the flavonols isorhamnetin-3-O -β-d-glucopyranoside and methyl quercetin-3-O -β -d- glucuronate; and kaempferitrin, quercetin-3- O-α-l-rhamnopyranoside, and tricin were identified in the extract from the leaves of Urochloa humidicola . Two furostanic saponins, namely, dioscin and 3-O -α-l- rhamnopyranosyl-(1-4)-[α-l- rhamnopyranosyl-(1-2)] -β -d- glucopyranosyl–penogenin, as well as catechin-7-O-β -d- glucopyranoside were identified in the methanolic extract obtained from the roots of this plant. This species features a range of metabolites that may be toxic for animals if used in food and may interfere with the growth medium, thereby inhibiting the development of other species


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The Urochloa genus belongs to the Poaceae family, Paniceae tribe, also known as Brachiaria (Morrone andZuloaga 1992, Veldkamp 1996).The species of this genus have adapted to different soil types (Lapointe 1993) and are used as dead matter for protecting the agricultural soil system.Among Urochloa species, U. decumbens, U. brizantha, and U. humidicola are the most frequently used as animal feed in Brazil.
The frequent use of these species in animal production need a detailed understanding of their potential dangers.In particular, their secondary metabolites include compounds that adversely affect animal health, such as photosensitization in ruminants and horses (Tokarnia et al. 2012).These problems are attributed to the steroidal saponins synthesized by fodder, especially protodioscin (25-R and 25-S) (Brum et al. 2009).An antinutritional effect has also been reported, which is attributed to the reduction in food intake and digestibility of animals due to the presence of metabolites such as terpenoids and flavonoids (Silva et al. 2012).
In terms of allelopathic effects, these species also have adverse effects on cropping systems; for example, the cinnamic acid derivative metabolized by Brachiaria species inhibits seed germination in Euphorbia heterophylla and Bidens pilosa (Oliveira et al. 2014).However, there may be both negative as well as positive allelopathic effects of different species depending upon crops consortiums.In this context, Rodrigues et al. (2012) cited negative allelopathic effects of the extracts from U. brizantha and U. decumbens on the seed germination of Stylosanthes guianensis and of U. decumbens on the germination of Stylosanthes capitata; they also reported a positive allelopathic effect of U. brizantha on the seeds of Stylosanthes macrocephala.The exudates of U. humidicola roots inhibit nitrification by Nitrosomonas europaea bacteria because of the action of two compounds identified in a phytochemical study of this exudate: p-coumaric and ferulic acids.These acids can permeate the cell membrane of these bacteria and inhibit the action of enzymes responsible for their nitrification (Gopalakrishnan et al. 2007, Subbarao et al. 2006).To the best of our knowledge, no study has investigated the isolation and identification of metabolites in the extracts from U. humidicola.
Terpenoids and flavonoids in fodder are known to have qualitative effects, such as antinutritional effects resulting from a reduction in consumption and digestibility (Silva et al. 2012).However, flavonoids such as tricin, quercetin-3-O-α-l-rhamnoside, and isorhamnetin 3-O-β-dglucoside are cited as beneficial, as they have antiinflammatory and antioxidant activities (Luyenn et al. 2014, 2015, Riethmüller et al. 2015, Kim et al. 2009) as well as antifungal activity, which are associated with increased feed efficiency (Aderogba et al. 2013).The saponin dioscin has also been reported to promote beneficial effects, particularly against liver fibrosis (Zhang et al. 2015), beyond its anti-inflammatory (Wu et al. 2015), antitumoral (Kaskiw et al. 2009), and antioxidant (Gao et al. 2012) activities.Additionally, Jayanegara et al. (2014) reported beneficial effects of secondary plant metabolites, particularly in the case of saponins in livestock systems due to the reduction of ruminal methane, which increases animal productivity and provides environmental benefits.These benefits have been yet not explored in animal production, nor has the possibility that metabolites other than cinnamic acid derivatives or a combination of other compounds are responsible for the allelopathic activities.To address this gap, this manuscript presents the first phytochemical study of the extracts from this plant, entailing the isolation of the metabolites in the leaves and roots of U. humidicola, to clarify the possible positive and negative effects of its use in animal feed and crop production.Ac) extract from the leaves and the methanol (UHRM-Ac) extracts from the roots were selected for chromatographic fractionation.First, 6.079 g of UHFMH 2 O-Ac was subjected to chromatographic fractionation on 70-230 mesh silica gel (224.00g) using dichloromethane, ethyl acetate, and methanol as the mobile phase, in a gradient of increasing polarity.In total, 320 fraction of 100 ml were collected.The fractions were pooled according to their thinlayer chromatography (TLC) profiles.The group containing fractions 51-56 (0.1342 g), which were obtained with dichloromethane:ethyl acetate (8:2) as the mobile phase, was analyzed by 13 C and 1 H NMR and GC-MS.These analyses allowed the identification of 4-hydroxy-3-methoxy-benzoic acid (1), trans-4-hydroxycinnamic acid (2), and 4-hydroxy-benzoic acid (3).The group containing fractions 80-86 (0.1024 g), which were obtained with dichloromethane: ethyl acetate (6: 4), was purified on a silica gel column.The purified fractions were then analyzed by 13 C and 1 H NMR to identify isorhamnetin-3-O-β-d-glucopyranoside (4) and methyl-quercetin-3-O-β-d-glucuronate (5).The group containing fractions 133-136 (0.4668 g), which were obtained with ethyl acetate: methanol (8:2), were combined and chromatographed on a Sephadex LH-20 column with methanol elution.This process produced 34 fractions, and kaempferitrin (6) was obtained.
The ethyl acetate fraction UHFM-Ac (1.59 g) was chromatographed on 70-230 mesh silica gel column (54.50 g) with dichloromethane, ethyl acetate, and methanol elution in a gradient of increasing polarity.In total, 152 fractions of 25 ml were collected.Fractions were combined on the basis of TLC analysis.The group of fractions 22-26 (0.0821 g), obtained with ethyl acetate as eluent, was eluted on Sephadex LH-20 column using methanol as eluent.The analysis DÉBORA R. DE OLIVEIRA et al. of the fractions led to the identification of tricin (8).Fractions 97-99 (0.1240 g), obtained with ethyl acetate and methanol (4:6), were subjected to additional chromatographic fractionation on a silica gel column, and the analysis of the fraction led to the identification of quercetin-3-O-α-lrhamnopyranoside (7).
The fraction UHRM-Ac (5.74 g) was subjected to fractionation on 70-230 mesh silica gel column (190.00 g) by elution with ethyl acetate and methanol in a gradient of increasing polarity.In total, 227 fractions of 100 ml were collected.The fractions were pooled according to their chromatographic profiles observed by TLC analysis.The group of fractions 120-124 (0.3082 g), obtained with ethyl acetate: methanol (6:4), was subjected to fractionation by HPLC.This chromatographic fractionations were composed of water as solvent (A) and acetonitrile as solvent (B) in a 4:6 ratio.The mobile phase was filtered before use and delivered isocratically at a flow rate of 4 ml min -1 .The analysis was conducted at room temperature using a Phenomenex C18 semipreparative column (250 mm × 10 mm i.d., 5 µm), and the analytes were monitored at 205 nm.In addition to some unidentified components, saponins represented by peaks at 3.084 and 5.801 min were isolated, analyzed by 1 H and 13 C NMR and HRMS, and identified as the steroidal saponins dioscin (9) and 3-O-α-l-rhamnopyranosyl (1-4)-[α-lrhamnopyranosyl-(1-2)]-β-d-glucopyranosides penogenin (10), respectively.The group of fractions 129-133 (0.5340 g), eluted with ethyl acetate and methanol (6:4), was subjected to fractionation on a Sephadex LH-20 column using methanol as the eluent.This fractionation produced 47 fractions, among which fractions 36-40 (0.0197 g) were recombined and filtered on (230-400 mesh) flash silica column using ethyl acetate:methanol (8:2) as the eluent.The NMR analysis of fractions 1-3 (0.025 g) then enabled the identification of catechin-7-O-β-d-glucopyranoside (11).

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The identification of compounds 1, 2, and 3 (Figure 1) in mixture was accomplished by interpretation of the mass spectra and the 1 H and 13 C NMR spectra, including such two-dimensional experiments as heteronuclear single-quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), and 1 H-1 H correlation spectroscopy (COSy).Analysis of the 1 H NMR and 1 H-1 H COSy spectra allowed the identification of signals compatible with trans-carboxylic acid (2), para-substituted aromatic rings in 2 and 3, and an ABC system in 1.The doublets at δ H 7.48, and 6.31 (J = 16 Hz), represent hydrogens 7 and 8, respectively, of compound 2. The singlet at δ H 7.52, and the doublets at δ H 6.80 (J = 8.5 Hz) of the hydrogens assigned to the 2, 6 and 3, 5 positions in compound 2; the signals at δ H 6.84 and 7.43 assigned to the hydrogens of the p-substituted aromatic ring of 3; and the remaining signals at δ H 7.78 (d, J = 8.5Hz), 7.43 (s), and 6.82 (d, J = 8.5 Hz) represent the hydrogens of the aromatic ring of compound 1.Analysis of the 13 C-NMR and two-dimensional (HSQC and HMBC) spectra and comparison with the literature data confirmed the proposed structures of 4-hydroxy-3-methoxy-benzoic acid for 1 (Pouchert and Behnke 1993a), trans-4hydroxycinnamic acid for 2 (Wang et al. 2011), and p-hydroxybenzoic acid for 3 (Pouchert and Behnke 1993b).GC-MS analysis of the fraction containing these compounds confirmed the proposed structures and even the presence of a methoxyl group in 1.
Compounds 4-8 (Figure 1) were identified as flavonoids on the basis of their 1 H and 13 C NMR spectra, which are consistent with the molecular skeleton of flavonols.Compounds 4 and 5 were identified in mixture from the 1 H NMR spectrum, which presented signals at δ H 6.21 (d, J = 2.2 Hz), corresponding to H-6 (both 4 and 5), and δ H 6.42 (d, J = 2.2 Hz) and 6.45 (d, J = 2.2 Hz), corresponding to H-8 of 4 and 5, respectively.Additional signals The location of two methoxyl groups and the sugar moieties were determined from the HMBC and nuclear Overhauser effect (NOE) spectra.These analyses allowed the location of the sugar unit at C-3 of each flavonoid, a methoxyl group at C-3′ of 4, and a methyl ester in the sugar moiety of 5.These assignments were confirmed by the absence of CH 2 -6″ and the connection of this group to a carboxyl group.The 1 H and 13 C chemical shifts were in agreement with the literature data reported for isorhamnetin-3-O-β-d-glucopyranoside (4) (Yuan et al. 2013) and methyl-quercetin-3-O-β-dglucuronate (5) (Hilbert et al. 2015).
The  .These data allowed the identification of compound 6 as kaempferitrin (Pizzolatti et al. 2003).
The 1 H NMR spectrum showed signals corresponding to five hydrogens in aromatic systems, which were attributed to the A and B rings of quercetin as the basic skeleton of compound 7.The chemical shifts observed in the 13 C NMR spectrum confirmed this assignment.The signal at δ H 5.37 (s), characteristic of a proton at an anomeric carbon, along with the observed signal at δ H 0.97 (d, J = 7.0 Hz) in the 1 H NMR spectrum led us to propose that a rhamnose unit was part of the structure.The position of the sugar was determined from the HMBC spectrum, which revealed a coupling between the hydrogen represented by the singlet at δ H 5.37 (H-1″) and C-3 (δ C 134.82).Further analysis of the 1 H and 13 C NMR spectra and comparison with the literature led to the identification of compound 7 as quercetin 3-O-β-d-rhamnoside (Ozgem et al. 2010).Compound 8 was obtained as a yellow crystalline solid, soluble in methanol.Its 1 H NMR spectrum showed only five signals: one singlet integrating to two hydrogens at δ H 7.40, which was attributed to the H-2′,6′; two doublets at δ H 6.27 (d, J = 2.2 Hz) and 6.57 (d, J = 2.2 Hz) for the H-6 and H-8 of ring A of a flavonoid; one singlet at δ H 3.98 integrating to six hydrogens related to two methoxyls; and a singlet at δ H 6.75, which was assigned to the H-3 of a flavonoid.The 2D NMR spectra allowed the location of the methoxyl at C-3′ and C-5′.These analyses and comparison with the literature led to the identification of compound 8 as tricin (Zielinska et al. 2008).
The 13 C NMR spectra of 9 and 10 both with signals of 45 carbons, including 4 and 5 quaternary carbons in compounds 9 and 10, respectively; 24 and 23 methyne carbons in compounds 9 and 10, respectively; and 11 methylene and 6 methyl carbons in both compounds.The chemical shift of the quaternary carbon at δ C 110 of a spiro system is characteristic of the tetrahydrofuran and pyran rings in the structure.The relative stereochemistry of C-25 was identified as (R) by comparison of the chemical shifts of methyl-27 and CH 2 -23 (Pires et al. 2002, Espejo et al. 1982).The presence of three methyne carbons with chemical shifts near 100 ppm was compatible with the presence of pyranosyl units, on what differences in the mass spectra confirmed the existence of two rhamnose and one glucose units in each compound.
The 1 H and 13 C NMR spectra of compound 11 showed characteristic signals of a flavanol, with saturation in the C ring.Analysis of the 1D and 2D 1 H and 13 C NMR spectra and comparison with the values for catechin led to identifying it as flavane.The additional signals were compatible with a structure containing glucose, and HMBC analysis indicated that it is located at C-7.These analyses led to the identification of compound 11 (Figure 1) as catechin-7-O-β-d-glucopyranoside (Benavides et al. 2006, Agrawal 1989).coNcluSioNS Twelve substances, including flavonoids, have been reported in this species for the first time in this work.The saponin diosgenin, which was identified in the extracts from the roots of U. humidicola, has been isolated from the leaves U. decumbens, synonym Brachiaria decumbens (Pires et al. 2002).This is the first identification of the steroidal saponinin penogenina the Urochloa genus.
The identification of p-coumaric acid and other benzoil acid derivatives is consistent with the literature witch reports that phenolic compounds, such as those present in U. humidicola, promote allelopathic action, and prevent pasture invasion plants in monoculture systems, independent of organic acids (Souza Filho et al. 2005, Kobayashi andNoguchi 2015).However, for pasture system consortiums, the presence of these substances has disadvantages, as the resulting inefficiency of the systems hinders the establishment of different plant species (Rodrigues et al. 2012).The identified constituents improve our understanding of the diversity of metabolites produced by these species.In the literature, metabolites responsible only for the photosensitization and/or allelopathic effects of Urochloa genus have been reported.In contrast, the saponins and flavonoids identified in this study have not been associated with these effects, which merit careful consideration.The identified flavonoids have biological activities and can therefore endow ruminants with beneficial functions, increasing their nutritional value.

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The authors thank to Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and to Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) for scholarship and financial support, Dr. Norberto Peporini Lopes (NPPNS-Núcleo de Pesquisa em Produtos Naturais e Sintéticos) for the HRMS.
1 H NMR spectrum of flavonoid 7 presented signals at δ H 7.85 (d, J = 8.5 Hz) corresponding to H-2′,6′ of ring B, and δ H 6.95 (d, J = 8.5 Hz), at δ H 7.51 (d, J = 2.2 Hz, H-2′), 7.57 (dd, J = 8.5 Hz and 2.2 Hz, H-6′), and 6.85 (d, J = 8.5 Hz, H-5′) were attributed to the protons of ring B of compound 4. The signals at δ H 7.52 (d, J = 2.0 Hz), 6.93 (d, J = 8.5 Hz), and 7.59 (dd, J = 8.0 and 2.0 Hz) were proposed to correspond to the protons H-2′, H-5′, and H-6′ of ring B in 5, respectively.Additional analysis of the 13 C and 2D NMR spectra confirmed the proposed flavonoid skeleton.These analyses allowed the identification of the signals of two sugar moieties that were linked in each flavonol.Two doublets at δ H 5.59 (J = 7.25 Hz) and 5.49 (J = 7.25 Hz) were assigned to the hydrogens at the anomeric carbons of the β-d-glycopyranosyl unit.These proposed assignments were confirmed by additional signals observed in the HSQC spectrum.

eXPerimeNtal PLANT MATERIAL The roots and leaves of U. humidicola were collected in April 2013 in an area already established at the Goat Sector of the Animal Science Institute of the Universidade Federal Rural do Rio de
analyses were performed using an instrument equipped with pump LC-10AS, SPD-10A detector, CBM-20A-Comunications Module (Shimadzu), and Rheodyne injector with loop of 500 µL.All other equipment commonly used for the preparation and fractionation of extracts belongs to the Laboratory of Natural Products Chemistry at UFRRJ.ExTRACTION AND ISOLATIONThe botanical material was dried at room temperature without exposure to sunlight.The 2.1 kg of leaves and 1.81 kg of roots obtained were then ground separately in a hammer mill at the Laboratory of Animal Nutrition Science, Animal Science Institute, UFRRJ.The milled material was subjected to extraction by maceration using hexane, methanol, and methanol/water (8:2) as solvents.The extractions were performed thoroughly at 7-day intervals, and each solution was concentrated on a rotary evaporator under vacuum.This process yielded six extracts: hexane leaves (UHFH), methanol leaves (UHFM), methanol: water leaves (UHFMH 2 O), hexane roots (UHRH), methanol roots (UHRM), and methanol: water roots (UHRMH 2 O).The hydromethanolic and methanolic extracts were solubilized in methanol: water (8:2) subjected to liquid-liquid partitioning by organic solvents of increasing polarity: hexane, dichloromethane, ethyl acetate, and butanol.The ethyl acetate fractions obtained from the methanol (UHFM-Ac) and hydromethanolic (UHFMH 2 O-