Free radical scavenging activity and chemical constituents of Urvillea ulmaceae

Abstract

The methanol extract of Urvillea ulmaceae Kunth (Sapindaceae) aerial parts and the hexane, ethyl acetate, and hydromethanol fractions were evaluated for their free radical scavenging activity with the DPPH assay. Among all the tested fractions, the ethyl acetate fraction was the most active, exhibiting an IC₅₀ of 16.33 μg/mL, comparable to that of the commercial antioxidant BHT. Fractionation of the ethyl acetate fraction through chromatographic methods afforded trans-N-methyl-5-hydroxypipecolic acid, epicatechin, and proanthocyanidin A2 as the main constituents. Epicatechin and proanthocyanidin A2 showed potent DPPH radical scavenging activity, with IC₅₀ values of 18.34 and 11.45 μg/mL, respectively. A new compound, trans-N-methyl-5-hydroxypipecolic acid, did not show any antioxidant effect (IC₅₀ > 500 μg/mL).

Keywords::

• Urvillea ulmaceae
• Sapindaceae
• trans-N-methyl-5-hydroxypipecolic acid
• epicatechin
• proanthocyanidin A2
• DPPH free radical scavenging activity

Introduction

Reactive oxygen species (ROS), including free radicals, are reported to cause tissue damage of biological systems, and to be involved in aging and in the pathogenesis of some diseases such as arthritis, atherosclerosis, diabetes, and cancer (Ames, 1983; Feher et al., 1987; Halliwel & Gutteridge, 1989; Aruoma, 1998). Antioxidants are compounds that protect cells against free mediated processes. Several studies have demonstrated that plants are natural antioxidant sources due, mainly, to the presence of flavonoids, which act in reducing free radical formation and also in scavenging free radicals (Miller, 1996; Pietta, 2000; Knekt et al., 2002). In this context, our research group has continued studying plants that occur naturally in the Brazilian stretch of the Upper Paraná River, Porto Rico (PR), to investigate the chemical composition and antioxidant properties of the species present in that area (Nazari et al., 2006).

The genus Urvillea is a group of perennial climbers with 13 species distributed in tropical and subtropical areas of the American continent (Nogueira et al., 1995). Urvillea ulmaceae Kunth (Sapindaceae) is a naturally occurring species in the Brazilian stretch of the Upper Paraná River, PR, Brazil. A decoction from the crushed stems of this plant is used by the French Guianan Wayapi as an antidiarrheic (Grenand et al., 1987). Reported phytochemical studies on the Urvillea genus are restricted to the U. uniloba Radlk., from which fatty acids and cyanolipids were isolated (Mikolajczak et al., 1970; Spitzer, 1996). There are no chemical or biological studies reported for Urvillea ulmaceae.

In the present study, we evaluated the free radical scavenging activity towards 1,1-diphenyl-1-picrylhydrazyl (DPPH) of the methanol extract and its fractions and of an isolated compound from Urvillea ulmaceae aerial parts. Also, the chemical composition of the active fraction was investigated through chromatographic methods. The isolated compounds were characterized by 1D and 2D nuclear magnetic resonance (NMR) spectral data and comparison with those reported in literature.

Materials and methods

Plant material

Aerial parts of Urvillea ulmaceae were collected in May 2004 in a Brazilian stretch of the Upper Paraná River, area of Porto Rico, state of Paraná, Brazil, and identified by Dr. Maria Conceição de Souza. A voucher specimen has been deposited at the Herbarium of the Departamento de Biologia, Universidade Estadual de Maringá, Paraná, Brazil.

Extraction and isolation of the chemical constituents

Air-dried aerial parts of Urvillea ulmaceae (950 g) were exhaustively extracted by maceration with methanol at room temperature. Evaporation of the solvent afforded the methanol extract (27.5 g). Part of this extract (24.5 g) was dissolved in MeOH–H₂O 1:1 and partitioned with n-hexane and ethyl acetate. The solvents were evaporated to give the hexane (HF, 5.3 g), ethyl acetate (EAF, 12.5 g), and hydromethanol (HMF, 1.60 g) fractions.

The ethyl acetate fraction (3.5 g) was fractionated on Sephadex LH-20 using H₂O, H₂O:MeOH 75:25, 50:50, 25:75, and MeOH as solvent, to give five sub-fractions (UM-1 to UM-5). Sub-fraction UM-1 (924. 0 mg) was purified on a chromatographic column of silica gel and eluted with a mixture of CHCl₃:MeOH, in increasing polarity, to afford trans-N-methyl-5-hydroxypipecolic acid (27. 3 mg). Purification of sub-fraction UM-3 (606. 5 mg) on Sephadex LH-20 using H₂O, H₂O:MeOH 75:25, 50:50, 25:75, and MeOH as solvent furnished epicatechin (69. 7 mg). The same procedure was used for purification of the sub-fraction UM-4 (439. 4 mg), which resulted in isolation of proanthocyanidin A2 (60. 0 mg).

Identification of the isolated compounds

The isolated compounds were identified by analysis of their NMR and electron ionization-mass spectroscopy (EI-MS) data. NMR measurements were carried out on a Varian Mercury Plus BB spectrometer operating at 300 MHz for ¹H and 75.5 for ¹³C, using D₂O or CD₃OD as solvent and tetramethylsilane (TMS) as internal standard. EI-MS spectra were recorded on a Thermoelectron Corporation Focus-DSQ II spectrometer.

The structures of epicatechin and proanthocyanidin A2 were elucidated by comparison of their NMR data with those reported in the literature (Foo et al., 1997). Compound trans-N-methyl-5-hydroxypipecolic acid was identified on the basis of MS and NMR spectroscopic data. EI-MS at 70eV, m/z (relative abundance): 159 (M⁺, 2), 130 (5), 114 (100), 96 (35), 94 (13), 81 (7), 70 (10). ¹H NMR (300 MHz, CD₃OD), δ ppm, (mult., J in Hz): 3.50 (m, H-2); 1.75 (q, J = 13 Hz, H-3ax); 2.32 (dd, J = 13 and 1.4, H-3eq); 1.56 (q, J = 11 Hz, H-4ax); 2.15 (m, H-4eq); 3.95 (m, H-5); 2.87 (m, H-6ax); 3.55 (m, H-6eq); 2.93 (N-CH₃). ¹³C NMR (75.5 MHz; CD₃OD, δ ppm): 70.8 (C-2), 28.7 (C-3), 32.6 (C-4), 66.6 (C-5), 60.2 (C-6), 45.6 (N-CH₃), 176.4 (COOH).

DPPH free radical scavenging assay

Free radical scavenging activities of the test samples and of the positive control butylhydroxytoluene (BHT) were determined using the DPPH free radical method. Various concentrations of the samples were added to 3 mL of daily-prepared methanol DPPH solution (0.1 mM). The mixture was shaken and left to stand at room temperature in the dark. After 30 min, absorbance was measured at 517 nm against a blank (containing all reagents except the test samples). Assays were carried out in triplicate. The concentrations of the samples for 50% inhibition of DPPH (IC₅₀) were obtained from the graph of I% (inhibition percentage) versus concentration of the sample in μg/mL. The percentage inhibition of DPPH (I%) was calculated using the equation: where A_blank is the absorbance of the blank solution and A_sample is the absorbance of the test sample.

Results and discussion

The free radical scavenging activity was initially evaluated for the crude methanol extract of Urvillea ulmaceae. The results (Table 1) showed that the crude methanol extract presented significant free radical scavenging activity, with an IC₅₀ of 26.98 μg/mL. Fractionation of this extract by solvent partition furnished the hexane (HF), ethyl acetate (EAF), and hydromethanol (HMF) fractions, which were tested for their antioxidant capacity toward the radical DPPH. Comparison of the obtained IC₅₀ data (Table 1) indicated a potent activity for the EAF fraction (IC₅₀ = 16.33 μg/mL), which was comparable to that of the commercial antioxidant BHT, and a moderate free radical scavenging effect for HF (IC₅₀ = 111.96 μg/mL) and HMF (IC₅₀ = 74.16 μg/mL) fractions. The most active fraction (EAF) was purified by chromatography column on Sephadex LH-20 or silica gel to afford the compound trans-N-methyl-5-hydroxypipecolic acid (Figure 1), epicatechin, and proanthocyanidin A2.

Figure 1.  NOESY correlations for trans N-methyl-hydroxypipecolic acid.

Table 1.  DPPH free radical scavenging activity (IC₅₀) for methanol extract, HF, EAF, and HMF fractions, and isolated compounds from Urvillea ulmaceae.

Sample	IC50 (μg/mL) (95% confidence limit)
Methanol extract	26.98 (24.95–29.35)
Hexane fraction (HF)	111.96 (102.31–106.88)
Ethyl acetate fraction (EAF)	16.33 (15.44–17.22)
Hydromethanol fraction (HMF)	74.16 (77.64–81.93)
Proanthocyanidin A2	11.45 (10.03–13.13)
Epicatechin	18.34 (15.91–21.01)
trans-N-methyl-5-hydroxypipecolic acid	> 500
BHT^a	16.9 (14.3–20.1)

^aBHT (butylhydroxytoluene) was used as positive control.

The compound trans-N-methyl-5-hydroxypipecolic acid (Figure 1) is being reported for the first time, according to our knowledge. Its structure determination was based on 1D and 2D NMR data (see “Materials and methods” section) and comparison with those of trans-5-hydroxypipecolic acid (Mester et al., 1979). Its ¹H NMR spectrum was characteristic of a pipecolic acid derivative, showing signals for an oxymethine hydrogen at δ 3.95 (m, H-5), a carboxymethine hydrogen at δ 3.50 (m, H-2), and for three groups of methylene hydrogens at δ 1.75 (H-3ax) and 2.32 (H-3eq); δ 1.56 (H-4ax) and 2.15 (H-4eq); δ 2.87 (H-6ax) and 3.55 (H-6eq). The singlet at δ 2.93 (3H) was attributed to the methyl group on the nitrogen atom. The ¹³C NMR spectrum confirmed the proposed structure by the signals for a carboxyl group at δ 176.4, an N-CH₃ at 45.6, two methine carbons at δ 70.8 (C-2) and 66.6 (C-5), and three methylene carbons at δ 28.7 (C-3), 32.6 (C-4), and 60.2 (C-6). Localization of the hydroxyl group at C-5 was confirmed from the J² or J³ HMBCs (heteronuclear multiple bond correlations) between the signal at δ 66.6 (C-5) and those of H-3, H-4, and H-6. The NOESY (nuclear Overhauser enhancement spectroscopy) spectra permitted establishment of the relative stereochemistry of the isolated compound. The correlation between the signals for H-2 (δ 3.50) and H-3eq (δ 2.32) indicated that the group -COOH occupied an equatorial position. The equatorial position for the hydroxyl group was confirmed by the interactions observed between H-5 and H-3ax, H-4eq and H-6eq. EI-MS spectra showed the molecular peak at m/z 159, consistent with the proposed structure. From these data the isolated compound was identified as trans-N-methyl-5-hydroxylpipecolic acid. The new compound showed no free scavenging activity in the DPPH test (IC₅₀ > 500 μg/mL; Table 1).

Epicatechin and proanthocyanidin A2 are widespread in nature, occurring in various plants (Foo et al., 1997; Okawa et al., 2001; Ploss et al., 2001; Lin et al., 2002; Verstraeten et al., 2005; Shahat, 2006; Liu et al., 2007), including species belonging to the Sapindaceae family (Sarni-Manchado et al., 2000; Sakane et al., 2005). The isolated epicatechin and proanthocyanidin A2 demonstrated potent free radical scavenging activity in the DPPH assay, with IC₅₀ values of 11.45 and 18.34 μg/mL, respectively. The antioxidant activities of these compounds are well described in the literature (Okawa et al., 2001; Verstraeten et al., 2005; Liu et al., 2007).

In conclusion, our study demonstrated the potential antioxidant properties of the plant Urvillea ulmaceae as a scavenger of free radicals and showed that this effect can be attributed to the compounds epicatechin and proanthocyanidin A2.

Declaration of interest: We are grateful to CAPES and CNPq for fellowships (S.A.D., F.P.C.), and to Fundação Araucária (Paraná State, Brazil) for financial support.