Phenolic Compounds from Limonium pruinosum

NPC 2015 Vol. 10 No. 2 319 - 321 Natural Product Communications Phenolic Compounds from Limonium pruinosum Sihem Bouderminea,b, Nicola Malafronteb, Teresa Mencherinib, Tiziana Espositob, Rita Patrizia Aquinob, Noureddine Beghidjaa, Samir Benayachea, Massimiliano D’Ambolac and Antonio Vassallod,* a Department of Chemistry, Research unit, Development of Natural Resources, Bioactive Molecules and Physiochemical and Biological Analysis, University of Constantine 1, Algeria b Dipartimento di Farmacia, Università degli Studi di Salerno, Via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy c Dipartimento di Medicina Veterinaria e Produzioni animali, Università di Napoli Federico II, Via Federico Delpino 1, 80137 Napoli, Italy d Dipartimento di Scienze, Università degli Studi della Basilicata, Viale dell’Ateneo Lucano 10, 83100 Potenza, Italy [email protected] Received: November 21st, 2014; Accepted: December 22nd, 2014 A new phenolic compound, 6’-O-sulfonyl-salidroside (1), together with fifteen known compounds, were isolated from the all parts of Limonium pruinosum. Their structures were elucidated by 1D and 2D NMR experiments including 1D TOCSY, DQF-COSY, HSQC and HMBC spectroscopy, as well as ESIMS analysis. The antioxidant activity of the extracts and all isolated compounds was evaluated by DPPH test. Keywords: Limonium pruinosum, Plumbaginaceae, Phenolic compounds, NMR. The genus Limonium (Plumbaginaceae), also known as “Sea Lavender”, “Statice”, or “Marsh-rosemary”, comprises about 300 species with a worldwide distribution. Some species are endemics in the Mediterranean region [1]. All species are herbaceous perennial plants with varying degree of colors from white to pink, produced on a branched panicle or corymb. Limonium species are traditionally used for the treatment of infections, fever, hemorrhage and other disorders [2]. Previous phytochemical studies of this genus led to the isolation and characterization of flavonoids, carbonyl compounds, hydrocarbons, fatty acids, naphthoquinone, tannins, alkaloids and amino acids [3]. Different pharmacological activities have been reported such as: antiviral, antitumor, antipyretic, hemostatic, depurative, antifungal and antimicrobial [4]. L. pruinosum Kuntze is among the plants that feed camels in the arid region of southern Tunisia. The antioxidant activity of L. pruinosum crude extract was previously reported, but phytochemical investigations have never been reported [5]. In this paper, we describe the isolation and structural identification by spectroscopic and spectrometric techniques of a new compound, together with known compounds, and the antioxidant activity of the extracts and pure compounds [6]. The ethanolic-aqueous extract of L. pruinosum whole plant was successively partitioned with n-hexane, chloroform, ethyl acetate and n-butanol, in the amounts 0.3, 0.8, 6.0 and 12.0 g, respectively. The free-radical scavenging activity of Limonium extracts was evaluated by DPPH test [6,7]. The polar ethyl acetate and n-butanol extracts showed a high potency in scavenging DPPH (EC50 5.33 and 11.64 g/mL, respectively) (Table 1), which correlated with their total phenolic content (251.7 and 158.8 g/mg, respectively, and expressed as gallic acid equivalents). The other extracts did not exhibit activity in this assay up to 400 g/mL. Fractionation and purification of ethyl acetate and n-butanol extracts by a combination of chromatographic techniques led to the isolation of one new compound 1 and fifteen known compounds (2-16). Table 1: Total phenol content, and free-radical scavenging activity (DPPH test) of L. pruinosum extracts and compounds. Extract or Compound ext- Ethyl acetate ext- n-BuOH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 -tocopherolc L-ascorbic Acidc a Phenolic content (g/mg extract) a 251.7 ± 3.7 158.8 ± 2.6 DPPH test [IC50 (g/mL)]b 5.3 ± 0.2 11.6 ± 0.6 10.6 ± 1.5 10.8 ± 1.7 13.3 ± 2.0 7.2 ± 0.5 1.5± 0.2 17.0 ± 1.5 156.2 ± 5.4 1.2 ± 0.09 1.9 ± 0.1 7.4± 0.6 35.7 ± 2.9 227.8 ± 6.7 7.0 ± 0.4 17.0 ± 1.8 16.0 ± 1.0 103.3 ± 4.4 10.1± 1.3 5.8± 0.9 gallic acid equivalents. bMean ± S.D. of three determinations. cPositive control. OSO3H HO HO O O OH OH Figure 1: Compound 1. The NMR and MS data of compound 1 demonstrated the molecular formula C14H20O10S (HRESIMS at m/z 379.3597 [M-H]-), which suggested the presence of a sulfate group in the molecule. Acid hydrolysis of 1 followed by treatment with BaCl2, confirmed the 320 Natural Product Communications Vol. 10 (2) 2015 Table 2: NMR data of compound 1 (CD3OD, 600 MHz)a. position 1 2 3 4 5 6 7 8 1’ 2’ 3’ 4’ 5’ 6’ 1 H 6.72 d (8.0) 7.29 d (8.0) 7.29 d (8.0) 6.72 d (8.0) 2.86 t (6.7) 4.02, m 3.73, m 4.32 3.23 dd (8.5, 9.0) 3.38 t (9.5) 3.36 t (9.5) 3.50 m 4.35 dd (12.0, 2.5) 4.16 dd (12.0, 5.0) C 155.2 115.0 130.1 129.6 130.1 115.0 35.9 70.0 102.9 74.1 76.7 70.4 74.9 67.0 a J values are in parentheses and reported in Hz; chemical shifts are given in ppm; assignments were confirmed by COSY, HSQC and HMBC experiments. presence of a sulfate group. The ESI-MS in negative ion mode showed the [M-H]- ion peak at m/z 379, and fragments in the MS/MS analysis at m/z 241 [M-H-C8H10O2]-), and at m/z 97 corresponding to the sulfate group for the loss of a sugar moiety. The 1H NMR spectrum (Table 2) showed signals of four aromatic protons [H 7.29 (2H, d, J = 8.0 Hz, H-2/6), 6.72 (2H, d, J = 8.0, H-3/5)], and of four methylene protons at [H 2.86 2H, br t, J = 6.7 Hz), 4.02 (1H, m), 3.73 (1H, m)]. The 1H NMR spectrum also showed the presence of a -glucopyranosyl unit (H 4.32, 1H, d, J = 7.8 Hz). A combination of 1D-TOCSY and COSY experiments provided evidence for the presence in the molecule of the segments H-1’H2-6’, H-7H-8, and H-2/5H-3/6. The elucidation of the whole basic carbon skeleton from the above subunits was achieved on the basis of a series of 1J (HSQC) and 3J (HMBC) correlations. The HMBC spectrum exhibited correlations between the proton signal at H 2.86 and the carbons at C 129.6 (C-1) and 130.2 (C-2 and C-5) confirming the position of the 4-hydroxyphenylethanol structure. The location of glucopyranose was obtained on the basis of the HMBC correlations between the proton signal at H 4.32 (H-1’) and the carbon signal at 70.0 (C-1’) ppm, and between the proton signals at  4.02 and 3.73 (H2-8) and the carbon signal at 102.9 ppm [8]. The position of the sulfate group was established at C-6 of the glucose unit on the basis of the downfield shifts of H2-6’ (H 4.35, dd, J = 12.0, 2.5 Hz; and 4.16, dd, J = 12.0, 5.0 Hz) and of C-6’ c 67.0, consistent with the presence of an ester moiety [9]. Thus compound 1 was established as the new 6’-O-sulfonylsalidroside. The fifteen known compounds were identified as 1,6-O-digalloyl-β(2) [10], myricetin-3-O-galactopyranoside (3), myricetin-3-O-(6”-galloylglucoside) (4) [11], trans-N-caffeoyltyramine (5) [1], myricetin 3-O-glucopyranoside (6) [11], kaempferol 3-O-rhamnopyranoside (7), gallic acid (8), methyl gallate (9), limoniastramide (10) [1], avicularoside (11) [2], kaempferol (12) [12], myricetine-3-O-arabinofuranoside (13) [4], quercitin-3-O-galactopyranoside (14), quercitin-3-O-rhamnopyranoside (15) [11], and astragalin 6”-O-gallate (16) [12] by detailed NMR and MS analyses and comparison with literature data. D-glucopyranose In order to identify the compounds responsible for the observed activity of Limonium crude extracts, the free radical scavenging activity of the new compound 1 and the known compounds (2-16) was evaluated. The new sulfate phenylpropanoid glycoside (1) showed a DPPH scavenging activity superimposable with that of the positive control -tocopherol (EC50 10.6 and 10.1 g/mL, respectively,) and about two fold lower than that of L-ascorbic acid (EC50 5.85 g/mL). Gallic acid (8), methyl gallate Bouderim et al. (9), and trans-N-caffeoyltyramine (5) were the most active compounds (EC50 1.20, 1.92, and 1.50 g/mL, respectively). Also the dimer of the phenolic acid amide, limoniastramide (10) displayed a strong capacity to scavenge the free radical DPPH (EC50 7.4 g/mL). Moreover, myricetin and quercetin derivatives (3, 4, 6, 11, 13-15) exhibited considerable antioxidant activities with EC50 values ranging from 7.0 to 35.7 g/mL. On the other hand, kaempferol (12) and its derivatives (7, 16) had a weak free radical scavenging activity. Experimental General experimental procedures: Optical rotations were measured on a Perkin-Elmer 241 polarimeter equipped with a sodium lamp (589 nm) and a 1 dm microcell. UV spectra were recorded on a Perkin-Elmer-Lambda spectrophotometer. NMR experiments were performed on a Bruker DRX-600 spectrometer at 300 K. All the 2D NMR spectra were acquired in CD3OD. Standard pulse sequences and phase cycling were used for DQF-COSY, TOCSY, HSQC, and HMBC experiments. HRESIMS were acquired in positive ion mode on a Q-TOF premier spectrometer equipped with a nanoelectrospray ion source (Waters-Milford, MA, USA). Column chromatography was performed over Sephadex LH-20. HPLC separations were conducted on a Shimadzu LC-8A series pumping system equipped with a Shimadzu RID-10A refractive index detector and Shimadzu injector on a C18-Bondapak column (30 cm x 7.8 mm, 10 µm Waters, flow rate 2.0 mL min-1) [13]. Plant material: The whole plant of L. pruinosum was collected during the flowering period, in April 2012, in the region of Bechar, southwest Algeria. The plant was identified by Prof. Mouhammed Kaabache of the University of Setif, Algeria. A voucher specimen, number 116LP/VAREBIOL/412, was deposited in the Herbarium of the Chemistry Department at the University of Constantine 1, Algeria. Extraction and isolation: The whole plant of L. pruinosum (1.8 Kg) was powdered and successively extracted by exhaustive maceration in ethanol (80%), filtered and the solvent removed under vacuum at 40°C in a rotary evaporator obtaining 27.3 g of dried extract. Part of this (25.0 g) was partitioned with n-hexane, chloroform, ethyl acetate and n-butanol to yield 0.3, 0.8, 6.0 and 12 g of the respective residues. The BuOH extract was successively partitioned between n-BuOH and H2O. A portion (2.5 g) of the n-BuOH-soluble fraction was separated by Sephadex LH-20 with methanol as eluent (flow rate 1.1 mL/min). Fractions of 10 mL were collected, analyzed by TLC on silica 60 F254 gel-coated glass sheets with CHCl3:MeOH:H2O (40:9:1 v/v/v) and n-BuOH–AcOH–H2O (60:15:25 v/v/v) and grouped to obtain 8 fractions (A–H). Fraction D (432 mg) was purified by RP-HPLC using MeOH–H2O (15:85) to give compound 1 (3 mg, tR 10 min). Fraction E (226 mg) was purified by RP-HPLC using MeOH–H2O (1:4) to give 1,6-Odigalloyl-β-D-glucopyranose (2) (1.4 mg, tR 22 min) and myricetin3-O-galactopyranoside (3) (1.4 mg, tR 37 min). Fraction G (70 mg) was a pure compound myricetin 3-O-(6”-galloylglucoside) (4). All the ethyl acetate extract (6.0 g) was subjected to CC using silica gel and eluting with CH2Cl2, followed by increasing concentrations of MeOH (between 1% and 100%). Fractions of 50 mL were collected, analyzed by TLC (silica gel plates, in CH2Cl2 and mixtures CH2Cl2−MeOH ) and grouped into 7 fractions (A−G). Fraction B (82 mg) was purified by RP-HPLC using MeOH–H2O (3:7) to give trans-N-caffeoyltyramine (5) (1.4 mg, tR 90 min). Fraction C (92 mg) was purified by RP-HPLC using MeOH–H2O (45:55) to give myricetin 3-O-glucopyranoside (6) (1.1 mg, tR 12 min) and kaempferol 3-O-rhamnopyranoside (7) (2.1 mg, tR 60 min). Fraction Phenolic compounds from Limonium pruinosum D (217 mg) was purified by RP-HPLC using MeOH–H2O (2:3) to give gallic acid (8) (2.7 mg, tR 4 min), methyl gallate (9) (1.7 mg, tR 6 min), limoniastramide (10) (1.6 mg, tR 30 min), avicularoside (11) (1.8 mg, tR 55 min) and kaempferol (12) (22.0 mg, tR 55 min). Fraction E (422 mg) and F (430 mg) were separately purified by RP-HPLC using MeOH–H2O (35:65) to give methyl gallate (9) (2.2 mg, tR 13 min), myricetin-3-O-arabinofuranoside (13) (8.2 mg, tR 47 min), quercetin-3-O-galactopyranoside (14) (2.2 mg, tR 52 min) and quercetin-3-O-rhamnopyranoside (15) (2.8 mg, tR 13 min) from fraction E, and myricetin-3-O-galactopyranoside (3) (1.5 mg, tR 40 min), quercetin-3-O-galactopyranoside (14) (3.4 mg, tR 52 min), and astragalin 6”-O-gallate (16) (1.3 mg, tR 64 min) from fraction F. The purity of each compound was determined by NMR and HPLC. Compound 1 [α]D:-20.2.4 (c 0.11, MeOH). 1 H and 13C NMR: Table 2. ESIMS m/z: 379 [M + H]-, 241 [(M – H) –C8H10O2]-, HRESIMS m/z: 379,3597 [M-H]- (calcd for C14H20O10S, 379.3577). Quantitative determination of total phenols: Ethyl acetate and nbutanol extracts of L. pruinosum, dissolved in MeOH, were analyzed for their total phenolic content according to the FolinCiocalteau colorimetric method. Total phenols were expressed as gallic acid equivalents (μg/mg extract) [14]. Natural Product Communications Vol. 10 (2) 2015 321 Bleaching of the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH• test): The antiradical activities of L. pruinosum extracts, pure compounds (1-16), -tocopherol, and L-ascorbic acid were determined using the stable1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) and the procedures described by Mencherini et al. [15]. In its radical form, DPPH• has an absorption band at 517 nm, which disappears upon reduction by an antiradical compound. An aliquot (37.5 μL) of the MeOH solution containing different amounts of the extract or compounds from L. pruinosum was added to 1.5 mL of daily prepared DPPH• solution (0.025 g/L in MeOH); the maximum concentration employed was 400 μg/mL. An equal volume (37.5 μL) of the vehicle alone was added to the control tubes. Absorbance at 517 nm was measured on a Shimadzu UV-1601UV–visible spectrophotometer 10 min after starting the reaction. The DPPH• concentration in the reaction medium was calculated from a calibration curve analyzed by linear regression. The percentage of remaining DPPH• (%DPPH• REM) was calculated as follows: % DPPH• REM) [DPPH•]T/[ DPPH•]0 × 100, where T is the experimental duration time (10 min).  -Tocopherol and L-ascorbic acid were used as positive controls in the test. All experiments were carried out in triplicate, and the mean effective scavenging concentrations (EC50) were calculated by using the Litchfield and Wilcoxon test. Results are reported in Table 1. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] Trabelsi N, Oueslati S, Ksouri R, Nassra M, Marchal A, Krisa S, Abdelly C, Mérillon JM, Waffo-Téguo P. (2014) The antioxidant properties of new dimer and two monomers of phenolic acid amides isolated from Limoniastrum guyonianum. 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