Identification of Minor Secondary Metabolites from the Latex of Croton lechleri (Muell-Arg) and Evaluation of Their Antioxidant Activity

Dragon’s blood (Sangre de drago), a viscous red sap derived from Croton lechleri Muell-Arg (Euphorbiaceae), is extensively used by indigenous cultures of the Amazonian basin for its wound healing properties. The aim of this study was to identify the minor secondary metabolites and test the antioxidant activity of this sustance. A bio-guided fractionation of the n-hexane, chloroform, n-butanol, and aqueous extracts led to the isolation of 15 compounds: three megastigmanes, four flavan-3-ols, three phenylpropanoids, three lignans, a clerodane, and the alkaloid taspine. In addition to these known molecules, six compounds were isolated and identified for the first time in the latex: blumenol B, blumenol C, 4,5-dihydroblumenol A, erythro-guaiacyl-glyceryl-β-O-4’-dihydroconiferyl ether, 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol and floribundic acid glucoside. Combinations of spectroscopic methods (1H-, 13C- NMR and 2D-NMR experiments), ESI-MS, and literature comparisons were used for compound identification. In vitro antioxidant activities were assessed by DPPH, total antioxidant capacity and lipid peroxidation assays. Flavan-3-ols derivatives (as major phenolic compounds in the latex) exhibited the highest antioxidant activity.

The distribution of phenolic flavan-3-ols between the n-butanol and the CHCl 3 extracts revealed some interesting features. The principal component in the n-butanol extract was gallocatechin (10; 11.8 mg), followed by epigallocatechin (9; 5.2 mg) and epicatechin (7; 3.0 mg). Epicatechin has also been isolated, in lower concentrations, as a component of the CHCl 3 extract, along with catechin (8; 4.0 mg); reflecting these compositions, the highest antioxidant activity was detected in the n-butanol extract, compared to the CHCl 3 extract. In the chloroformic extract, flavan-3-ol components were accompanied by several other phenolic constituents.
Compound 5, also termed erythro-guaiacyl-glycerol-β-O-4'-dihydroconiferyl ether, was identified on the basis of its spectroscopic data. The EI-MS spectrum showed a molecular ion peak at m/z 378 [M] + which is consistent with a molecular formula of C 20 H 26 O 7.
Several clerodane diterpenoids have been identified in previous phytochemical studies [18], but until now, floribundic acid glucoside (14) had never been detected in any species of the genus Croton.

Antioxidant activity.
All crude extracts (n-hexane, CHCl 3 , n-BuOH and aqueous residue) obtained from the C. lechleri latex were analyzed for their phenolic content. The highest concentration of phenols (306 mg/g) was found in the n-BuOH extract and the lowest one in the n-hexane extract (4.84 mg/g). Consequently, the latter was not further investigated. The CHCl 3 , n-BuOH and aqueous residues were tested to evaluate their antioxidant potential ( Table 2). As expected, the n-BuOH extract showed the highest activity; in particular it exhibited a scavenging action toward the stable radical DPPH which was stronger than that of all three reference compounds, i.e. quercetin, Trolox ® , and ascorbic acid. Only the Sephadex ® fractions (1, 2, 3 and 4) obtained from this extract were examined for their antioxidant activity ( Table 2). The scavenging abilities of the four fractions were comparable with those of the standards; the overall antioxidant capacity yielded similar results, but only at 10 μM concentrations. In both tests, fractions 2 and 3 showed the strongest activity. From these fractions, epigallocatechin and gallocatechin were isolated and then investigated. Two pure compounds exhibited remarkable antioxidant activities ( Table 2) both in terms of DPPH removal (with values lower than those of quercetin and Trolox ® ) and of antioxidant capacity (with results similar to those of the quercetin and Trolox ® ). Epicatechin was isolated from fraction 4; the activity of this molecule (IC 50 : 19.3 μM; Table 2) was in line with that reported in the literature [3]. The presence of epicatechin and catechin in only trace amounts in the CHCl 3 extracts explains their lack of antioxidant activity.

Conclusions
In summary, we have revealed for the first time the presence in C. lechleri latex, of minor secondary metabolites as megastigmane, lignan, and clerodane derivatives. The results obtained by testing the n-BuOH extract may be ascribable to flavan-3-ols, that are the strongest antioxidants among latex phenols. The complexity of the chemical profile suggested that the role of each individual compound in the latex is important in the interpretation of the pharmacological effects exhibited by sangre de drago, which deserve further investigations.

Experimental
General 1 H-and 13 C-NMR spectra were determined at 500.13 and 125.77 MHz, respectively, on a Varian Unity INOVA spectrometer equipped with an indirect detection probe. Chemical shifts were referenced to the solvent signals of deuterated methanol (CD 3 OD), residual CHD 2 OD: δ H 3.31, δ C 49.0. Electron ionization mass spectrometry (EI-MS) and ESI-MS were recorded on a Fisons VG Prospec instrument. Droplet counter-current chromatography (DCCC) was performed on a DCC-A apparatus (Tokyo Rikakikai Co., Tokyo-Japan) equipped with 250 glass-columns. HPLC was performed using a Waters 510 pump equipped with a Waters U6K injector and a Waters 401 differential refractometer as detector, using a 30 cm x 3.9 mm; i.d., C 18 μ−Bondapak (Waters, Milford, MA, USA) columns; flow rate was 1 mLmin -1 . The secondary metabolites were identified by a combination of spectroscopic methods ( 1 H, 13 C NMR and 2D-NMR experiments), ESI-MS and comparison with the literature data.

Plant Material
The sap of C. lechleri (Euphorbiaceae) was collected in 2006 from the tropical region of Upper Huallaga Valley (Tingo Maria, Peru) and kept at 4 °C in the dark. A 100 mL sample of the reddish latex is deposited (N° SD-518) in the herbarium of the University of Molise (Pesche, Isernia, Italy).

Extraction and Isolation
A small portion (150 mL) of sap was dissolved in MeOH-H 2 O (9:1, v/v, 200 mL) and extracted with n-hexane (3 x 150 mL), following Kupckan's partitioning method [27]. The water content (% v/v) of the MeOH was adjusted to 40% and the resulting solution was partitioned against CHCl 3 . The MeOH was removed from the aqueous phase, which was then extracted with n-BuOH. This gave three extracts: n-hexane (80.3 mg), CHCl 3 (1.2 g), n-BuOH (3.6 g) and an aqueous residue (7. Isolation and identification of alkaloids C. lechleri latex (50 mL) was lyophilized to yield 7.6 g of reddish-powdered latex. A portion (3.0 g) of the powdered latex was combined with distilled H 2 O (150 mL) and acidified with concentrated HCl. The aqueous layer was continuously extracted with CHCl 3 for 12 h and its pH was then adjusted to 8 with concentrated NH 4 OH. This fraction was then continuously extracted with CHCl 3 over 12 h to yield the first basic fraction. Following adjustment of pH to 10 with NH 4 OH, the aqueous layer was extracted for an additional 12 h with CHCl 3 to yield the second basic fraction. TLC examination of the two basic fractions with Dragendorff's reagent showed reagent-positive component in the second basic fraction. This fraction was dissolved in MeOH with warming, and 15 (11.3 mg) was obtained as a white precipitate upon refrigeration.  Table 1.  Table 1.

Determination of phenolic content
Total phenols of the extracts and fractions were quantified by the Folin-Ciocalteau spectrophotometric assay, using gallic acid as the reference standard [28]. Therefore, molarity refers is expressed as gallic acid equivalents.

Total antioxidant capacity
The total antioxidant capacity of the samples at concentrations of 10 and 1 μM was determined by a validated method based upon the copper reduction (Cu 2+ to Cu + ). (BIOXYTECH ® AOP-490™, Oxis Research™, Portland, OR) [29]. The reference compound was uric acid and then results are expressed as mEq uric acid.

DPPH scavenging test
Suitable aliquots of a methanolic solution containing each sample, at increasing concentrations from 1 μM to 100 μM, were added to 15 μM ethanol solution of 2,2-diphenyl-2-picrylhydrazyl radical (DPPH). Absorbance was read at 517 nm after 15 min of incubation in the dark [30]. The IC 50 was calculated by employing Prism ® 4 (GraphPad Software Inc.).