New Alcamide and Anti-oxidant Activity of Pilosocereus gounellei A. Weber ex K. Schum. Bly. ex Rowl. (Cactaceae)

The Cactaceae family is composed by 124 genera and about 1438 species. Pilosocereus gounellei, popularly known in Brazil as xique-xique, is used in folk medicine to treat prostate inflammation, gastrointestinal and urinary diseases. The pioneering phytochemical study of P. gounellei was performed using column chromatography and HPLC, resulting in the isolation of 10 substances: pinostrobin (1), β-sitosterol (2), a mixture of sitosterol 3-O-β-d-glucopyranoside/stigmasterol 3-O-β-d-glucopyranoside (3a/3b), 132-hydroxyphaeophytin a (4), phaeophytin a (5), a mixture of β-sitosterol and stigmasterol (6a/6b), kaempferol (7), quercetin (8), 7′-ethoxy-trans-feruloyltyramine (mariannein, 9) and trans-feruloyl tyramine (10). Compound 9 is reported for the first time in the literature. The structural characterization of the compounds was performed by analyses of 1-D and 2-D NMR data. In addition, a phenolic and flavonol total content assay was carried out, and the anti-oxidant potential of P. gounellei was demonstrated.


Introduction
Cactaceae is a family belonging to the order Caryophyllales with 124 genera and about 1438 species [1] distributed throughout American territory in tropical and temperate dry regions

Identification of Isolated Compounds
Chromatographic procedures led to the isolation of 10 compounds from P. gounellei (Figures 1 and 2). The isolated compounds were identified by analysis of their 1-D and 2-D NMR data and comparisons with the literature. Compound 1 was isolated as colourless crystals soluble in chloroform and it was identified as the flavonoid pinostrobin [22]. Compounds 2, 3a/3b and 6a/6b were identified as steroids: β-sitosterol, the mixture of sitosterol 3-O-β-D-glucopyranoside/stigmasterol, 3-O-β-D-glucopyranoside [23] and sitosterol/stigmasterol [24], respectively. These phytosteroids are widespread in plants, being important components of vegetable cell walls and membranes. In addition, they are known as anti-inflammatory agents and precursors of vitamin D [25].
Compounds 4 and 5 were isolated as dark green amorphous solids. The 1 H-and 13 C-NMR indicated that the compounds are chlorophyll derivatives. When compared with the literature data, they were identified as 13 2 -hydroxyphaeophytin a (4) [26] and phaeophytin a (5) [27]. These porphyrinic compounds are derived from chlorophyll a and are widely present in the vegetable kingdom [28]. The substances 7 and 8 were isolated as a yellow amorphous powder and were identified as the flavonoids kaempferol (7) [29] and quercetin (8) [30]. Flavonoids have great relevance in the pharmaceutical field, displaying anti-oxidant, anti-inflammatory and antimicrobial activities [31], and have been previously reported from several species of the genera Opuntia and Pilosocereus [8]. The 1 H-NMR spectrum of compound 9 ( Figure 2) showed two doublets (δ H 7.11 and δ H 6.75) integrating for two protons each, suggesting the presence of a para-substituted ring. Two doublets in δ H 6.79 (H-5), coupling ortho, and δ H 7.11 (H-2), coupling meta with H-6 and a double doublet at δ H 6.98 (H-6) coupling ortho and meta with H-5 and H-2, respectively, indicated the presence of an AMX ring system. The presence of a methoxy group in one aromatic ring was shown by a singlet at δ H 3.79 (3H). The presence of the coumaroyl and tyramine units was suggested by the following signals: a pair of doublets at δ H 6.52 (H-8) and δ H 7.31 (H-7), both with J = 16 Hz, characteristic of trans olefin protons; an interesting triplet at δ H 8.00, referring to a proton bonded to N [32]; two double doublets at δ H 3.29 (H-8a I ) and 3.25 (H-8b I ) referring to the methylene carbon adjacent to N and one triplet on δ H 4.27 (H-7 I ), attributed to a proton on the oximethinic carbon [33]. Other relevant signals in the 1 H-NMR spectra of 9 were two signals at δ H 3.27 (q) and δ H 1.07 (t) attributed to an ethoxy group attached to an oximethinic carbon whose proton was found at δ H 4.27 (t) [33]. The 13 C-NMR spectrum showed signals for 20 carbons, supporting the information provided by the 1 H-NMR spectrum about the presence of the coumaroyl and tyramine portions of compound 9. The coumaroyl portion was defined by the signals at δ C 165.8 (amide α,β-unsaturated carbonyl) and two signals at δ C 139.46 and δ C 119.01 (the α,β-unsaturated carbons C-7 and C-8). The tyramine portion was confirmed by the signals at δ C 45.61 (CH 2 , C-8 I ) and an oximethinic carbon δ C 79.6 (CH, C-7 I ), indicating the position the ethoxy group is attached to [32]. The presence of one ethoxy group in compound 9 was reinforced by the signals at δ C 63.51 (CH 2 , C-1 II ) and δ C 15.42 (CH 3 , C-2 II ). The 13 C-NMR spectrum confirmed the presence of two aromatic rings, with δ C 128.07 (C-2 I /6 I ), δ C 115.34 (C-3 I /5 I ) of one AA I BB I system and δ C 110.9 (C-2), δ C 115.83 (C-5) and δ C 121.82 (C-6) of the AMX system, showing one methoxy group at δ C 55.75 (H 3 CO-C-3) [32,34].
The absence of a correlation for proton δ H 8.00 (t, N-H) with any carbon in the HMQC spectrum, and the correlation shown by the proton at δ H 8.00 (t, 1H) and the carbon at δ C 165.8 ( 2 J) in the HMBC spectrum demonstrated that the compound possesses an amide carbonyl [32][33][34], being a trans-feruloyl derivative [32][33][34]. The HMBC spectrum showed correlations ( 3 J) that confirmed the presence of the coumaroyl moiety: H-7 with C-2, C-6 and C-9; H-8 with C-1; H-5 with C-3 and C-1. Other correlations in the HMBC spectrum suggest the presence of the ethoxy group and also the trans-feruloyl tyramine portion: H-1 II with C-7 I and H-8 I with C-9 and C-1 I . These data allowed identification of compound 9 as 7 I -ethoxy-trans-feruloyl-tyramine, reported herein for the first time. The COSY spectrum supported the proposed structure by showing correlations between hydrogen (N-H) δ H 8.00 (t) with H-8 I ; H-8 I and H-7 I ; H-1 II and H-2 II ( Table 1). The optical rotation of 9 was found to be ¡10 ¥ (0.01; MeOH) establishing the S(¡) absolute configuration at the C-7 chiral center [33].  17, confirming the proposed structure of compound 9. The hypotheses that compound 9 is an artefact was eliminated based on previous studies that described the isolation of trans-feruloyltyramine and its derivatives by several different extraction methods [33,35]. Liang et al. performed the extraction of eight nitrogenated substances from Portulaca oleracea using microwave irradiation and different solvents such as dichloromethane, ethyl acetate, methanol, ethanol, ethanol 70%, ethanol 30% and water. The eight isolated substances, including N-feruloylnormetanephrine and the N-trans-feruloyltyramine, were extracted with all the tested solvents, showing that those solvents did not promote the formation of different radicals or artefacts [35].
Compound 10 was obtained as a pale amorphous solid and its spectra showed a structure similar to compound 9, differing in the absence of signals related to the ethoxy group. Comparisons of the spectral data of compound 10 with compound 9 and literature data allowed identification of compound 10 as trans-feruloyltyramine, previously isolated from other plant species [32][33][34]. Compound 10 was shown to possess action against weeds, improvement of seed germination [34] and anti-inflammatory activity by inhibiting COX enzymes [36].

Total Phenolic, Total Flavone and Flavonol Contents and Anti-Oxidant Activity of Extracts and Methanolic Fraction from P. gounellei
The values of total phenolic, flavone and flavonol contents and anti-oxidant activity (DPPH and ABTS) of ethanol extracts of stems, roots, flowers, fruits and methanol fraction are shown in Table 2.
The fruit extract showed the best antiradical activity in the ABTS test (IC 50 = 10.4¨0.24) and the flower extract showed the lowest anti-oxidant activity (IC 50 = 76.9¨0.61); the cladode extract showed a slightly higher activity (IC 50 = 62.4¨0.44) than the flower extract. The scavenging activity of free radicals of the roots extract showed an activity (IC 50 = 41.6¨1.06) similar to the methanol fraction (IC 50 = 40.9¨0.69). Thus, we can classify the activity of extracts and methanol fraction of P. gounellei (Pg) as follows: Pg fruits > MeOH fraction = Pg roots > Pg cladodes > Pg flowers. In the DPPH test, the fruit extract showed the best anti-oxidant activity (IC 50 = 11.3¨0.12), followed by the root extract (IC 50 = 102.1¨1.49). The methanol fraction (IC 50 = 130.1¨3.02) showed similar activity to the cladode extract (IC 50 = 136.0¨3.48) and the least potent was the flower extract (IC 50 = 194.3¨2.33). A correlation has been shown between anti-oxidant activity and total phenolic content in natural products, especially between extracts with the two highest values. The fruit extract showed a greater amount of phenolics (127.9¨1.67 mg GAE/g) and also showed the greater anti-oxidant activity in DPPH (IC 50 = 11.3¨0.12) and ABTS (IC 50 = 10.4¨0.24) test. The flower extract presented the lowest anti-oxidant activity in DPPH (IC 50 = 194.3¨2.33) and ABTS (IC 50 = 76.9¨0.61) tests as well as the lowest phenolic content (43.5¨2. 16). When analysing the other extracts and methanol phase of the plant, a sequence of linear correlations between total phenolic content and radical scavenging activity was not observed.
Among all tested samples, only the fruit extract has sufficient anti-oxidant activity to be considered for nutraceutical use. According to the anti-oxidant activity index (AAI), all extracts and phases of P. gounellei showed an index lower than 0.5, thus presenting poor activity. Surprisingly, the fruit extract presented an AAI value of 2.01, which is considered as very strong.
Many studies have reported the relationship between total phenolics assay results and anti-oxidant activity [37]; our study confirms these findings. The relationships between the total phenolic content and the antiradical activity of DPPH (1/EC 50 ), and antiradical activity of ABTS ¤+ (1/EC 50 ) are shown in Figure 3. The Pearson correlation coefficients (r) of these plots were approximately 0.943 for the DPPH assay and 0.944 for the ABTS assay. This result suggests that 94% of the anti-oxidant capacity of extracts and methanol fraction from P. gounellei is due to the contribution of phenolic compounds. It is interesting to mention that there was no inverse correlation between the total phenolic content and the anti-oxidant activity by the DPPH and ABTS methods and flavones/flavonols content, at least when comparing the extract with the highest anti-oxidant activity, the extract from the fruits. Therefore, it is possible that the anti-oxidant activity may be attributed to other phenolic compounds than flavonols and/or flavones.  The solvents used in the chromatographic procedures were p.a. grade: n-hexane, dichloromethane, chloroform and ethyl acetate. Methanol HPLC grade (Tedia , Rio de Janeiro, Brazil). Water was obtained from a Millipore MilliQ system (Millipore, São Paulo, Brazil).
Isolated compounds were identified using 1-D and 2-D NMR analysis acquired on the following spectrometers: Varian Oxford (200 MHz), Varian (500 MHz) (Varian, Palo Alto, CA, USA) and Avance III (Bruker, Coventry, UK) using deuterated solvents. The high-resolution mass spectra were obtained using LC-HRMS analysis performed on an Accela 600 HPLC system combined with an Exactive (Orbitrap) mass spectrometer from Thermo Fisher Scientific (Bremen, Germany). EIMS was obtained with a Shimadzu QP-2000 spectrometer (Kyoto, Japan). The rαs 25 D 25 ¥ C was determined using a MCP 200 polarimeter (Anton Paar, Saint Laurent, QC, Canada).

Botanical Material
Pilosocereus gounellei was collected in Boa Vista City-PB (Brazil) in November 2010. The plant was identified by Prof. Dr. Leonardo Person Felix (CCA/UFPB) and a voucher specimen (15437) was deposited in the Herbarium Prof. Jaime Coelho Morais of the Agricultural Sciences Center (CCA/UFPB).

Extraction and Isolation
The botanical material (cladodes) was dried in an oven with circulating air at 40 ¥ C and ground using a mechanical mill, yielding 5.18 kg of powder, which was macerated with 10 L of EtOH at room temperature, for 72 h. The extraction solution was concentrated in a rotary evaporator at 40 ¥ C, yielding 237.13 g of crude ethanolic extract (CCEE). CCEE (10 g) was submitted to vacuum liquid chromatography (VLC) using silica gel and eluted with hexane (Hex), chloroform (CHCl 3 ) and methanol (MeOH) to obtain the corresponding fractions. The chloroform fraction (8.0 g) was chromatographed in a silica column using solvents of increasing polarity: Hex, dichloromethane (CH 2 Cl 2 ) and MeOH. The fractions Hex-CH 2 Cl 2 (3:7), CH 2 Cl 2 and CH 2 Cl 2 -MeOH (9:1) were combined and chromatographed using the same method yielding 188 fractions that were analysed using TLC. The fractions 35/38 (8.00 mg), after recrystallization, gave pure colourless crystals of compound 1. The fractions 54/59 (3.51 g) contained compound 2 and the fractions 146/150 (2.72 g) afforded a white powdery precipitate of compound 3. CCEE (5.00 g) was dissolved in CHCl 3 :H 2 O (1:1) and separated using a separating funnel, yielding an aqueous fraction and a chloroform fraction. A portion of the chloroform fraction (2.0 g) was subjected to successive column chromatography on silica gel, following the methodology previously described, resulting in the isolation of compound 4 (20.50 mg). CCEE (131.84 g) was submitted to column chromatography using Amberlite XAD as the stationary phase and eluted with H 2 O, MeOH, Hex, ethyl acetate (EtOAc) and acetone. The hexane fraction from XAD was chromatographed using VLC on silica gel 60 with hexane, CH 2 Cl 2 and MeOH. The hexane fraction was chromatographed on flash silica column yielding the subfractions 28/33 (92.30 mg) that were purified using preparative TLC, eluted with Hex-EtOAc (80:20), to give compound 5 (10.20 mg).
The roots were dried in an oven at 40 ¥ C and ground in a mechanical mill, yielding 1.37 kg of powder that was macerated with 10 L of EtOH at room temperature, for 72 h. The obtained solution was concentrated in a rotary evaporator at 40 ¥ C, resulting in 27.00 g of root crude ethanol extract (RCEE).
To isolate nitrogen-containing compounds, the method described by Souza and Silva [38] was used. The acidified chloroform fraction (ACF, 3.38 g) was chromatographed on a silica column eluted with hexane, EtOAc and MeOH, yielding 159 fractions. The combined fractions 88/114 (580.80 mg) were analysed by HPLC-DAD using a semipreparative column at room temperature. As the mobile phase, Milli-Q water and MeOH were used gradient-wise, and the concentration of the MeOH was increased from 50 to 100% in a 20-min run. The chromatogram showed three peaks, and the one at higher retention time was found to be the major component; thus, it was isolated and identified as compound 9 (8.00 mg). RCEE (10.00 g) was solubilized in H 2 O and yielded 5.00 g of precipitate. A sample (2.50 g) of it was dissolved in MeOH-CHCl 3 (1:1) and submitted to filtration on Sephadex LH-20 with MeOH-CHCl 3 (1:1), resulting in 10 fractions. The subfraction 9/10 (10.00 mg) was proved to be pure by TLC and identified as 10. The remaining 2.50 g of the precipitate was chromatographed on silica gel 60 column and eluted with hexane, EtOAc and MeOH, yielding the pure fractions 5/7 which corresponded to compound 3.

Total Phenol Content Assay
The total phenolics content was evaluated by the method of Gulcin et al., [39] with some modifications, using the Folin-Ciocalteu reagent and gallic acid as a positive control. Samples of cladodes, roots, flowers and fruit and the methanolic fraction from P. gounellei, from a stock solution of 5 mg/mL, solubilized in EtOH, were transferred to a 1.0 mL Eppendorf tube by adding 20.0 µL of Folin-Ciocalteu reagent, stirring for 1 min. Then Na 2 CO 3 (60.0 µL, 15%) was added to the mixture and stirred for 30 s. Finally, distilled water (900 µL) was added to give a final concentration of 100 µg/mL. After 2 h, the absorbance of the samples was measured at 760 nm. The concentration of the phenolic compounds was determined as equivalent milligram of gallic acid per gram of sample (mg GAE/g), from the calibration curve constructed with gallic acid standard (2.5 to 15.0 µg/mL), considering the average standard error (SEM).

Total Flavones and Flavonols Content Assay
The flavones and total flavonols content were determined adapting the methodology described by Mihai et al. [40]. Stock solutions (1.0 mg/mL) of extracts from cladodes, roots, flowers and fruits and methanolic fraction from P. gounellei were prepared. Each sample solution (400 µL) and methanolic solution of aluminium chloride (200 µL, 2%) were added in a volumetric flask. The final volume was adjusted with the same solvent to 10 mL. Reaction occurred for 30 min in the dark. The reading was performed at a wavelength of 425 nm. The analysis was evaluated in triplicate and the total flavones and flavonols content was determined from the calibration curve constructed with straight line equation of quercetin solutions (1.0 to 40.0 µg/mL) and expressed in equivalent milligrams of quercetin by gram of extract (mg QE/g), considering the average standard error (SEM).
where Abs control is the absorbance of the control containing only the ethanol solution of ABTS + and Abs sample is the absorbance of the radical in the presence of the sample or standard ascorbic acid.
The antiradical efficiency was established using linear regression analysis and the 95% confidence interval (p < 0.05) obtained using the statistical program GraphPad Prism 5.0. The results were expressed as EC 50¨S EM (sample concentration required to eliminate 50% of the DPPH radicals available, plus or minus the SEM).

Statistical Analyses
The results are expressed as the mean¨standard error of the mean (SEM). Analysis of variance (ANOVA one-way and Tukey's post hoc test) were used to evaluate the differences of the means between groups. The antiradical efficiency was established using linear regression analysis. Pearson correlation coefficients (r) were used to express correlations and confidence interval of 95% (p < 0.05) obtained using the statistical program GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). The results were expressed by sample concentration required to eliminate 50% of the DPPH radicals available, plus or minus the SEM (EC 50¨S EM).

Conclusions
The phytochemical study of Pilosocereus gounellei led to the isolation and identification of 10 compounds: pinostrobin, β-sitosterol, a mixture of β-sitosterol/stigmasterol, 13 2 -hydroxyphaeophytin a, phaeophytin a, sitosterol 3-O-β-D-glucopyranoside/stigmasterol 3-O-β-D-glucopyranoside, kaempferol, quercetin, the new substance 7 I -ethoxy-trans-feruloyltyramine and trans-feruloyltyramine. The evaluation of anti-oxidant activity from P. gounellei demonstrated that the fruit ethanol extract possesses excellent anti-oxidant activity, mainly because of the presence of phenolic compounds reported in the genus and the Cactaceae family.