N O T E
Isolation and identification of antioxidants from Pedilanthus tithymaloides
Pedro M. AbreuÆSusan MatthewÆTania Gonza´lezÆ Lucie VanickovaÆDavid CostaÆAna GomesÆ Marcela A. SegundoÆEduarda Fernandes
Received: 29 March 2007 / Accepted: 25 June 2007 / Published online: 31 July 2007 ÓThe Japanese Society of Pharmacognosy and Springer 2007
Abstract A bioassay-guided methodology utilizing 2,2- diphenyl-1-picrylhydrazyl (DPPH), the trolox equivalent antioxidant capacity (TEAC), and reducing power assays, as well as an assessment of scavenging properties against O2, H2O2, HOCl, ROO,NO, and ONOOwere used to find the main antioxidant principles ofPedilanthus tithym- aloides(Euphorbiaceae), a shrub used in traditional Cuban medicine. The principles were identified as kaempferol 3-O-b-D-glucopyranoside-600-(3-hydroxy-3-methylglutarate), quercitrin, isoquercitrin, and scopoletin. The contents of total phenolics and flavonoids were found to be 76.0 ± 4.8 mg of gallic acid equivalents/g extract and 9.8 ± 0.4 mg of rutin equivalents/g extract, respectively.
Keywords Pedilanthus tithymaloides FlavonoidCoumarinAntioxidant activity Anti-inflammatory activity
Introduction
Pedilanthus tithymaloides (L.) Poit. (Euphorbiaceae) is a low tropical American shrub that has been reported to possess a wide range of medicinal properties. So far, the search for bioactive compounds in this shrub has led to the isolation of a diterpene inhibitor of cancer cell growth, the antibacterial compounds 50-S-methylthioadenosine and 1,4- dihydroquinone, a proteolytic enzyme with oral anti- inflammatory activity, and a lectin possessing mitogenic activity with murine spleen lymphocytes and hemaggluti- nation properties. In Cuba, this species is traditionally used as a tincture in the treatment of stomatological infections.
In a previous investigation, we demonstrated the in vivo anti-inflammatory activity of the tincture by inhibiting carrageenan-induced rat paw edema, as well as its scav- enging effects against reactive oxygen species (ROS) and reactive nitrogen species (RNS) [1].
The purpose of the present study was to identify the antioxidant principles of P. tithymaloides. These com- pounds were characterized by extensive spectroscopic means as kaempferol 3-O-b-D-glucopyranoside-600-(3- hydroxy-3-methylglutarate) (1), quercitrin (2), isoquerci- trin (3), and scopoletin (4).
Materials and methods
Chemicals and plant material
Reagents and standard samples were obtained from Sigma- Aldrich (St. Louis, MO, USA), Fluka Chemie GmbH (Steinheim, Germany), and Aldrich (Milwaukee, WI, USA). Plant material was collected in February 2002, in the region of II Frente Frank Paı´s at Santiago de Cuba, and P. M. Abreu (&)S. MatthewL. Vanickova
Faculdade de Cieˆncias e Tecnologia da Universidade Nova de Lisboa,
CQFB/REQUIMTE, 2829-516 Caparica, Portugal e-mail: pma@dq.fct.unl.pt
D. CostaA. GomesM. A. SegundoE. Fernandes Departamento de Quı´mica-Fı´sica,
Faculdade de Farma´cia da Universidade do Porto, REQUIMTE, 4099-030 Porto, Portugal
T. Gonza´lez
Departamento de Farmacia, Facultad de Ciencias Naturales, Universidad de Oriente, Santiago de Cuba, Cuba
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J Nat Med (2008) 62:67–70 DOI 10.1007/s11418-007-0186-z
identified at the herbarium of Museum Toma´s Romay, Santiago de Cuba, where the voucher specimen no. 2726 has been deposited.
General experimental procedures
Optical rotations were obtained using a PerkinElmer (Waltham, MA, USA) 241-MC polarimeter. UV and FT-IR spectra were recorded on Milton Roy (Ivyland, PA, USA) Spectronic 1201 and PerkinElmer 157G spectrophotome- ters, respectively. The NMR spectra were recorded on a Bruker (Ettlingen, Germany) ARX 400 spectrometer (1H at 400 MHz;13C at 100.61 MHz). EI–MS (70 eV) and FD–
MS were carried out on a Micromass (Manchester, UK) GCTOF spectrometer, ESI–MS and HR–ESI–MS experi- ments were performed on an Agilent (Palo Alto, CA, USA) MSD1100 single-quadropole spectrometer, and on an Ag- ilent ESI–TOF instrument, respectively. Column chroma- tography was performed using Diaion HP-20 (Supelco 13605; Bellefonte, PA, USA), Sephadex LH-20 (Amer- sham 17-0090; Little Chalfont, UK), SiO2(Merck 13905;
Darmstadt, Germany), and RP-18 silica gel (Merck 13900).
TLC was performed on normal- and reversed-phase pre- coated Si gel F254 (MN 818133 and Merck 5559, respectively), HPTLC-Si gel 60 F254 (Merck 105628), and HPTLC-cellulose (Merck 105887) plates. Cartridges of polymer sorbent (Strata X33, Phenomenex, Torrance, CA, USA) were used for solid-phase extraction (SPE). LPLC was performed on Michel–Miller columns filled with RP- 18 silica gel (Merck 13900).
Extraction and isolation
Powdered stems and leaves of P. tithymaloides (1.5 kg) were percolated with 70% EtOH, yielding a residue of 294 g. A portion of the extract (50 g) was fractionated by RP-18 CC using a gradient elution of H2O, H2O/MeOH (1:1), MeOH, and CH2Cl2. The active fractions were assembled and submitted to Diaion HP-20 CC (H2O, MeOH, and CH2Cl2 gradient elution). Fractions eluted with H2O/MeOH 70:30–0:100 were grouped and submit- ted to RP-18 CC (H2O/MeOH 100:0–0:100) to yield a total of 60 fractions. Fractions eluted with H2O/MeOH 80:20–60:40 were then submitted to successive RP-18 and SiO2 CC to afford compound 4 (4 mg). Fractions eluted with H2O/MeOH 50:50 yielded3(4 mg), after purification on a Sephadex LH-20 column and SPE cartridge. Com- pounds 1 (6 mg) and 2 (11 mg) were obtained from fractions eluted with H2O/MeOH 60:60–20:80, and further chromatographed by RP-18 flash CC and RP-18 LPLC.
Compound 5 (15 mg) was isolated from the CH2Cl2
fraction by flash SiO2 CC. Physical and spectral data of 1–3 and 5 were in agreement with those reported in the literature for kaempferol-3-O-b-D-glucopyranoside-600- (3-hydroxy-3-methylglutarate) [2], quercitrin [3, 4], iso- quercitrin [5], and 3b-friedelinol [6, 7], respectively.
Compound4was identified by comparison with a standard sample of scopoletin.
Kaempferol-3-O-b-D-glucopyranoside-600-(3-hydroxy-3 -methylglutarate) (1) pale yellow amorphous powder.
½ a25D61°(c0.11, EtOH). UVkmax(MeOH) nm (loge) 209 (3.43), 267 (3.21), 350 (3.13). IR (KBr) cm13409, 1718, 1654, 1608, 1507, 1360, 1181, 1081. HR–ESI–MS m/z:
615.1330 [M + Na]+ (calculated for C27H28O15Na:
615.1326). FD–MS m/z: 593 [M + H]+ (100), 593 [M C6H9O4+ 2H]+ (10), 286 [M C12H19O9+ H ]+ (38), 145 [C6H9O4]+(5).1H- and13C-NMR, see [2].
Scavenging assays
DPPH, TEAC, and reducing power scavenging assays were performed in triplicate according to a previously described procedure [8]. ROS and RNS scavenging assays on superoxide radical (O2), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), peroxyl radical (ROO), peroxynitrite (ONOO), and nitric oxide (NO) were performed in a microplate reader (Synergy HT, Bio-Tek, Winooski, VT, USA), using spectrophotometric, fluori- metric or chemiluminescence detection [1]. Each study corresponds to four experiments performed in triplicate.
Determination of total phenolics and flavonoids
The total soluble phenolic content of the ethanolic extract was determined by the Folin–Ciocalteu colorimetric method [9], and expressed as gallic acid equivalents (GAE) in milligrams of GAE per gram of dried extract. The content of flavonoids was determined by a previously described method using rutin as a reference compound [10], and expressed in rutin equivalents (RE).
Results and discussion
In a previous investigation on the anti-inflammatory and antioxidant activity of a medicinal tincture from P. tithymaloides, we suggested that the scavenging activity could result from the presence of phenolic compounds [1].
Using a different extraction procedure (percolation with 70% EtOH), the content of total phenolics was improved (76.0 ± 4.8 mg of GAE/g extract), whereas the content of flavonoids was determined as 9.8 ± 0.4 mg of RE/g
68 J Nat Med (2008) 62:67–70
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Table1ScavengingeffectsofP.tithymaloidesextract,chromatographicfractions,andcompounds14onROS,RNS,DPPH,ABTS,andreducingpower SampleO2H2O2HOClROONOONOODPPHABTSReducing power Absence ofNaHCO3
Presence of25mmol NaHCO3 IC50 (lg/ml)IC50 (mg/ml)IC50 (lg/ml)ORAC (lmoltrolox equivalent/g sample)
IC50 (lg/ml)IC50(lg/ml)IC50(lg/ml)IC50 (lg/ml)TEAC (mmol)cIC50 (lg/ml) Extract–a 1.28±0.1510.60±0.70359±4551±1.710.7±0.68.6±0.9310.0±9.021.60±0.10950.0±5.1 H2Ofr.–a3.80±0.3619.56±1.7222±1–a307.0±34.6140.3±18.7218.3±7.800.88±0.01600±4.0 MeOH/H2O (1:1)fr.100±5–a9.20±1.261157±5311.9±2.22.5±0.21.9±0.3110.0±3.102.01±0.0631.0±0.2 MeOHfr.–a0.84±0.0536.9±9.20422±3921.1±8.416.3±1.15.6±1.1160±5.801.70±0.0340.06±0.3 CH2Cl2fr.–a0.90±0.03130.4±13.467±18–a–a–a>1,000d>1,000 Positive control618±20 (trolox)0.182±0.001 (ascorbicacid)0.36±0.01 (lipoicacid)3995 (trolox)1337±58 (glutathione) 1631±55 (ascorbicacid)
0.317±0.012 (rutin)0.606±0.100 (cysteine)0.962±0.135 (cysteine) 1–b –b –b –b –b –b –b 101.5±2.71.85±0.0350.3±0.2 2–b –b –b –b –b –b –b 34.5±1.73.20±0.0728.1±0.1 3–b –b –b –b –b –b –b 11.7±0.72.90±0.0635.3±0.2 4–b –b –b –b –b –b –b 93.0±2.01.50±0.0260.8±0.3 Quercetin–b –b –b –b –b –b –b 8.01±0.604.00±0.1221.9±0.3 a-Tocopherol–b–b–b–b–b–b–b143.0±7.901.00±0.05136.83±0.2 aActivity<50%intherangeoftestedconcentrations bNottested cTEACvalueswerecalculatedat15min d Noactivityobserved
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extract. The extract presented a remarkable capacity to scavenge all ROS and RNS tested except for O2, moderate scavenging activity against DPPH and ABTS, and lower reducing power Fe3+/Fe2+ (Table1). Following RP-18 fractionation of the extract, it was observed that the frac- tion eluted with MeOH/H2O (50:50) presented the highest scavenging activity against ROS and RNS (Table1), though it was not effective against H2O2. All fractions except that eluted with CH2Cl2 showed activity against ONOO, which was enhanced in the presence of NaHCO3. A high scavenging activity in DPPH, TEAC, and reducing power assays was also observed for this fraction (Fig.1).
A bioguided procedure using a TLC autographic DPPH assay [8] led to the isolation of compounds 1–4, whereas the inactive CH2Cl2 fraction afforded compound 5. In DPPH, ABTS, and reduction power assays, the acylated flavonoid 1 showed weaker activities when compared to those of2and3(Table1), which is due to the lack of the 30-OH in the B-ring [11]. The antioxidant and anti- inflammatory properties of compounds 2–4 have been evidenced in a wide range of scavenging assays [5,12–16]
and animal experimental models [17–20].
The results presented here appear to validate our previous assumption that the phenolic constituents of P. tithymaloidesare responsible by the therapeutic efficacy of this plant in traditional Cuban medicine [1]. Moreover, this is the first reported isolation of flavonoids and cou- marins fromPedilanthusspp.
Acknowledgments REQUIMTE for financial support, Fundac¸a˜o para a Cieˆncia e Tecnologia and Fundo Social Europeu for post-doc (Matthew S) and PhD (Costa D) grants, and Programme Alban for scholarship E0D033475CU.
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O
O OH HO
OH O OH
O OH
O OH
O
HO
O OH
1 Fig. 1 Structure of compound1
70 J Nat Med (2008) 62:67–70
