First phytochemical studies of japecanga (Smilax fluminensis) leaves: flavonoids analysis

Abstract

This is the first chemical study of the antiradical potential of Smilax fluminensis Steud., Smilacaceae, leaves crude extract and fractions and the elucidation of two structurally isolated flavonoids. Quercetin-3-O-α-L-rhamnopyranoside (1-6)-O-β-D-glucopyranoside and quercetin-3-O-β-L-galactopyranoside were elucidated by spectrometric methods (¹H and ¹³C NMR and mass).

Smilax fluminensis ; Quercetin; Chromatography; NMR

Introduction

The genus Smilax is comprised of 300 to 350 species, found in the tropics and subtropics worldwide, they are bramble woody vines with a paired tendrils for climbing (Andreata, 2006). Several species of Smilax have been traditionally used as food or pharmaceutical materials in many countries, like the leaves of S. excelsa, which are widely used in some parts of Turkey in the daily diet (Ozsoy et al., 2008). Roots and young shoots of S. aspera are used as an ingredient to elaborate soft drinks and as a substitute of asparagus (Mariani et al., 2008). The roots and rhizomes of several species are used as folk medicine for their antibacterial, antifungal, anti-inflammatory and hepatoprotective activities as well as a syphilis treatment (Xu et al., 2005; Mandal et al., 2008). Phenolic compounds as gallic acid, protocatechuic acid, caffeic acid, gentisic acid, trans-o-coumaric acid and some flavonoids (Ozsoy et al., 2008; Yang et al., 2008; Zhang et al., 2009; Wungsintaweekul et al., 2011) were isolated from some species of the genus Smilax. So far, the chemical constituents of Smilax fluminensis Steud., Smilacaceae, have not been investigated. Therefore, the aim of this study was to elucidate the structure of flavonoids by ¹H and ¹³C NMR and ESI-MS.

Results and discussion

The isolated compounds were analyzed using HPLC and ¹H and ¹³C NMR. The obtained compound 1 was a dark yellow solid; the molecular formula was established as MS (ESI): m/z [M+] calculated for C₂₇H₃₀O₁₆: 610.15; found m/z 609, 12 (M-H)-. MP: 201-203ºC. The chromatogram peak for compound 1 was detected at 254 nm with 32 min RT.

The compound 2 was a dark yellow solid; the molecular formula was established as C₂₁H₂₀O₁₂, MW 464 g.mol^-1. MP: 231-233ºC. The chromatogram peak of compound 2 was detected at 254 nm with 25.8 min RT.

Compound 1: Quercetin-3-O-α-L-rhamnopyranoside(1-6)-O-β-D-glucopyranoside

¹H NMR (300 MHz CD₃OD ): 6.18/6.19 (1H, d, H-6), 6.39/6.38 (1H, d, H-8), 7.72/7.52 (1H, d/d, H-2'), 6.90/6.83 (1H, d, H-5'), 7.62/7.53 (1H, d/d, H-6'), 4.21/5.34 (1H, d/d, H-1''), 3.08 (1H, m, H-2''), 3.10 (1H, m, H-3''), 2,93 (1H, m, H-4''), 2.98 (1H, m, H-5''), 3.53 (2H, m, H-6''), 4.24/4.38 (1H, s, H-1'''), 3.07 (1H, m, H-2'''), 3.34 (1H, m, H-3'''), 2.93 (1H, m, H-4'''), 3.12 (1H, m, H-5'''), 0.94/0.98 (3H, s, H-6''').

¹³C NMR (75 MHz CD₃OD): 157.7/156.7 (C), 157.7/156.7(C), 176.6/177.4 (C), 161.3/162.3 (C), 100.2/98,2 (CH), 166.7/164.1 (C), 92.5/93.7 (CH), 155.9/156.5 (C), 104.2/104.0 (C), 122.1/121.3 (C), 116.3/116.4 (CH), 145.8/144.8 (C), 149.5/148.4 (C), 116.1/115.2 (CH), 121.7/121.7 (CH), 104.3/102.0 (CH), 74.4/74.9 (CH), 77.2/77.4 (CH), 69,7/70.5 (CH), 76.0/76.0 (CH), 67.9/69.3 (CH₂), 102.3/101.6 (CH), 71.3/70.7 (CH), 69.1/70.5 (CH), 71.7/71.9 (CH), 68,6/68,4 (CH), 17.2/17.3 (CH₃).

Compound 2: Quercetin-3-O-β-D-galactopyranoside

¹H NMR (300 MHz CD₃OD ): 6.21/6.20 (1H, d, H-6), 6.41/6.41 (1H, d, H-8), 7.54/7.54 (1H, d, H-2'), 6.82 (1H, d, H-5'), 7.67/7.66 (1H, d/d, H-6'), 5.37/5.37 (1H, d, H-1''), 3.57/3.57 (1H, m, H-2''), 3,55/3.38 (1H, d, H-3''), 3.65/3.66 (1H, m, H-4''), 3.10/3.34 (1H, m, H-5''), 3,60/3.46 (2H, m, H-6'').

¹³C NMR (75 MHz CD₃OD ): 157.0/156.9 (C), 134.0/134.0 (C), 178.0/178.0 (C), 161.0/161.7 (C), 98.9/99.3 (CH), 164.9/164.9 (C), 93.7/94.1 (CH), 157.3/156.8 (C), 104.7/104.3 (C), 121.6/121.6 (C), 116.8/116.5 (CH), 144.7/145.4 (C), 148.8/149.0 (C), 115.0/115.3 (CH), 121,9/122.5 (CH), 102.4/102.4 (CH), 71.1/71.7 (CH), 73.9/73.7 (CH), 68.5/68.5 (CH), 76.9/76.3 (CH), 60.4/60.4 (CH₂).

The ¹H NMR spectrum of compounds 1 and 2 elicited a signal typical of an A ring between δH 6.18-6.21 ppm (1H, H-6) and δH 6.39-6.41 ppm (1H, H-8). Furthermore, two typical B ring signals, were observed at δH 7.72-7.54 ppm (1H, H-2'), δH 6.82-6.90 ppm (1H, H-5') and at δH 7.62-7.67 ppm (1H, H-6'). These groups suggest two polyphenolic compounds from quercetin derivatives. The sugar moieties of compound 1 identified as α-L-rhamnopyranoside(1-6)-O-β-D-glucopyranoside with chemical shifts for (H-1'') at δH 4.21/5.34 ppm (1H, d/d, J = 1.0/6.0 Hz, H-1''), at δH 4.24/3.38 (1H, s, H-1'''), at δH 0.94/0.98 ppm (3H, s,H-6''') and (H-sugar) at δH 2.93-3.53 ppm (m). The sugar moieties of compound 2 identified β-D-galactopyranosyl with chemical shifts for (H-1'') at δH 5.37/5.37 ppm (1H, d, J = 9.0 Hz, H-1'') and H-sugar at δH 3.55-3.66 ppm (m).

The ¹³C NMR spectrum (75 MHz in CD₃OD) for compound 1signaled 27 carbons, with a methyl group at carbon at 67.9 ppm confirming the presence of a glycoside chain (Niassy et al., 2004; Moura et al., 2011). A carbonyl signal was detected at 176.6 ppm, and signals at 102.3 and 104.3 ppm were determined to be the anomeric carbons of rhamnose (C1''') and glucose (C1''), respectively. A spectrum band at 67.9 ppm was suggested to be a methylene carbon of glucose (C6''). The binding region rhamnosyl unit was assigned to (C6''), due to the signal at 6.2 ppm relative to the unsubstituted monomer. The methyl group of the rhamnose signals at 17.2 ppm (C6'''). So the heteroside chain was assigned the structure α-L-rhamnopyranoside-(1'''→6'')-O-β-D-glucopyranoside. The aromatic carbons signals assigned were (C8) 92.5 ppm, (C6) 100.2 ppm, (C5') 116.1ppm, (C2') 116.3 ppm and (C6') 121.7 ppm. The signal on the carbonyl was assigned to (C4) and non-hydrogenated carbons were assigned based on data retrieved from literature (Pizzolatti et al., 2003; Braca et al., 2004; Niassy et al., 2004; Silva et al., 2005; Cha and Lee, 2007; Maisuthisakul et al., 2007; Peres et al., 2009; Moura et al., 2011) (C3) 132.4 ppm, (C5) 161.3 ppm, (C7) 166.7 ppm, (C9) 155.9 ppm and (C10) 104.2 ppm, (C1') 122.1 ppm, (C3') 145.8 ppm and (C4') 149.5 ppm (Pizzolatti et al., 2003; Yang et al., 2008). Compound 1 was identified by HPLC and confirmed by ¹H and ¹³C NMR, and mass spectrometry, which showed m/z 609.12 [M-H]-. Fragmentation produced a fragment representing m/z 301.21 [M-147-163-H]-, indicating the loss of rhamnose (m/z 147) and glucose (m/z 163), respectively, (calculated for C₂₇H₃₀O₁₆).

The ¹³C NMR spectrum (75 MHz in CD₃OD) of compound 2 displayed 21 carbon signals, and a methyl carbon at 60.4 ppm confirming the presence of a glycoside chain (Pizzolatti et al., 2003; Braca et al., 2004; Silva et al., 2005; Moura et al., 2011). A carbonyl was identified at 178.0 ppm, and signal at 102.4 ppm were assigned to the anomeric carbons of rhamnose (C1''). The methylenic carbon of glucose (C6'') signal was determined at 60.4 ppm. Thus the glycoside chain was assigned the structure β-D-galactopyranosyl. The aromatic carbons were determined at (C8) 93.7 ppm, (C6) 98.9 ppm, (C5') 115.0 ppm (C2') 116.8 ppm and (C6') 121.9 ppm. The signal of the carbonyl, assigned to C4, and non-hydrogenated carbons were assigned by comparison with data from literature (Pizzolatti et al., 2003; Braca et al., 2004; Mariani et al., 2008; Moura et al., 2011) (C3) 134.0 ppm, (C5) 161.0 ppm, (C7) 164.9 ppm, (C9) 157.3 ppm and (C10) 104.7 ppm, (C1') 121.6 ppm, (C3') 144.7 ppm and (C4') 148.8 ppm (Niassy et al., 2004, Moura et al., 2011). Compound 2 was identified by HPLC and confirmed by ¹H NMR and ¹³C NMR.

Conclusion

This is the first phytochemical study of S. fluminensis leaves and the flavonoids described in the literature. Considering the results obtained from the fractions of the studied extract, we isolated and elucidated the structure of quercetin-3-O-α-Lramnopyranoside (1-6)-O-β-D-glucopyranoside and quercetin-3-O-β-D-galactopyranoside.

Acknowledgment

The authors would like to thank Professor Virgínia Claudia da Silva (UFMT-ICET) for her important contributions to this work; we also thank Professor Lourdes Campaner dos Santos for equipment accessibility (Mass Spectra acquisition, Department of Chemistry, UNESP-Araraquara) and for CNPq for financial support (Process Nº 558225/2009-8).

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Publication Dates

• Publication in this collection
  Jul-Aug 2014

History

• Received
  05 June 2014
• Accepted
  21 June 2014