Phenolic compounds and antioxidant , antimicrobial and antimycobacterial activities of Serjania erecta Radlk . ( Sapindaceae )

Serjania erecta Radlk.(Sapindaceae) is a medicinal plant traditionally used in Brazil. We assayed the ethanolic extract of leaves and roots against seven microorganisms. The REMA (Resazurin Microtiter Assay) assay was used to measure the biological activity in vitro against Mycobacterium tuberculosis and our results showed moderate activity of the ethanolic extract. On the other hand S. aureus, P. aeruginosa, S. setubal, C. albicans, S. cerevisiae and E. coli revealed that the leaves and roots of S. erecta inhibited the growth of all microorganisms. The ethanolic extracts of leaves and roots showed low values of antioxidant activities. The ethanolic extracts of leaves and roots were analyzed by chromatographic and spectrometric methods. (-)-Epicatechin, kaempferol aglycone and five glycoside derivates were isolated: kaempferol-3-O-a-l-rhamnopyranoside, kaempferol-3-O-a-l-rhamnopyranosyl-(1→6)-b-dglucopyranoside from the roots and kaempferol, kaempferol 3,7-di-O-a-L-rhamnopyranoside, vitexin, isovitexin and (-)-epicatechin in the leaves. This is the first chemical study reported in the literature about this specie.


INTRODUCTION
The tribe Paullinieae belongs to the Sapindaceae and shows neotropical distribution and comprises a p p r o x i m a t e l y 4 5 0 s p e c i e s i n s e v e n g e n e r a : Cardiospermum, Houssayanthus, Lophostigma, Paullinia, Serjania, Urvillea and Thinouia (Ferrucci, 2000) .The genus Serjania with about 231 species is the largest one of Paullinieae and occurs in drier areas, along forest edges and in weedy and disturbed areas (Ferrucci, 2000).Serjania erecta Radlk. is known in Brazil as "cinco-folhas" or "cipó-cinco-folhas" (Guarim Neto et al., 2000;Pott et al., 2004).Its leaves are used in folk medicine against inflammation, stomach ache, ulcerative diseases, and the roots to treat hypertension (Guarim Neto et al., 2000;Pott et al., 2004).From aerial parts of the medicinal plant S. triquetra 11α-hydroperoxy-hederagenin, a sapogenin, stigmasterol, oleanolic acid, morolic acid, hederagenin were isolated (Chávez, Delgado, 1994).
Chemical investigations on S. salzmanniana have revealed the presence of antifungal and molluscicidal saponins in the roots (Ekabo et al., 1993(Ekabo et al., , 1996)).Leaf extracts of S. yucatanensis have in vitro activity against epimastigotes and trypomastigotes of Trypanosoma cruzi (Hernández et al., 2012).Some species, such as S. lethalis inhibited the production of NO by J774 macrophages showing an important anti-inflammatory activity (Mesquita et al., 2007).
In the present communication, our interest in identification of compounds from native plants in Cerrado motivated us to conduct a chemical investigation on Serjania erecta Radlk.and to evaluate antimicrobial, antimycobacterial and antioxidant activities and phenolic and flavonoids contents of polar extract of leaves and roots of this specie.

Plant material
S. erecta was collected in the municipality of Aquidauana, MS, Brazil, in November 2009, and identified by A. Pott.A voucher specimen (HMS 8355) is deposited in the Herbarium of the Embrapa Gado de Corte in Mato Grosso do Sul, Brazil.

Extraction, isolation and identification of compounds
The leaves (600 g) and roots (400 g) of S. erecta were extracted successively with hexane, ethyl acetate and ethanol at room temperature.Extracts were filtered and concentrated under vacuum.For this work we used only the ethanol extract (12.0 g and 3.4 g) of leaves and roots, respectively.Both extracts were fractionated by XAD-2 (Supelco, Bellefonte, PA, USA) resin column chromatography (30 cm × 3 cm).The leaves extract was eluted with 1.0 L of water, followed by 0.8 L of methanol and finally with 0.2 L of acetone.Roots extract was eluted with 0.5 L of water, followed by 0.5 L of methanol and finally with 0.2 L of acetone.An aliquot of 1.2 g and 0.95 g of the methanolic fraction (FRMeOH) of leaves and roots extracts were dissolved in 10 mL and 5 mL of methanol, respectively, and fractionated by Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala, Sweden), column chromatography (70 cm × 3 cm) eluted with methanol at a flow rate of 0.3 mL min -1 .We collected 40 fractions of 8 mL from ethanolic extract of leaves and 28 fractions from the root extracts.The fractions were combined according to their behavior by thin-layer chromatography (silica gel plates, ethyl acetate/n-propanol/ water, 120:8:70 by volume, upper phase).The substances obtained were further purified by repeated column chromatography either on polyvinylpolypyrrolidone (Sigma, eluted with MeOH) eluted with methanol.NMR spectra were recorded on a Bruker DPX 300 spectrometer.IR spectrum were performed in a FT-IR-Nicolet Impact IMACT-400, KBr and UV spectrum were performed in a Hitachi 110 spectrophotometer.

Phenolic and flavonoid tests and antioxidant activity
The phenolic and flavonoid contents and antioxidant activity, in two different models (Coutinho et al., 2008;Lin, Tang, 2007;Blois, 1958), were measured in the leaves and roots.Total soluble phenolics in the leaves and root extract of S. erecta were determined with Folin-Ciocalteau reagent according to the method using gallic acid as a standard phenolic compound.Aliquots of 0.1 mL samples were mixed with 0.5 mL (1/10 dilution) of the Folin-Cicauteau reagent and 1 mL of ultrapurified water.The solutions were mixed and incubated at room temperature for 1 min.After 1 min, 1.5 mL of 20% sodium carbonate solution was added and incubated at room temperature for 40 min.The reaction mixture absorbance was measured at 760 nm on a spectrophotometer.The blanks were prepared with all the reagents except the sample.Gallic acid was chosen as a standard using 6 point standard curve (10, 50, 100, 150, 300 and 400 μg mL -1 ).All the tests were performed in triplicate.The data were expressed as milligram gallic acid g extract -1 .
Flavonoids were determined in the leaf and root extracts of S. erecta, with 1.50 mL of ethanol 95%, 0.10 mL of AlCl 3 .6H 2 O 10%, 0.10 mL NaC 2 H 3 O 2 .3H 2 O 1 mol/L and 2.80 mL of ultrapurified water.The solutions were mixed and incubated at room temperature for 40 min.The reaction mixture absorbance was measured at 415 nm on a spectrophotometer.The blanks were prepared with all the reagents except the sample.Quercetin was chosen as a standard using 6 point standard curve (10, 50, 100, 150, 300 and 400 μg mL -1 ).All the tests were conducted in triplicate.Data were expressed as milligram quercetin g extract -1 .
The antioxidant activity of ethanolic extract, based on free radical scavenging and b-carotene/linoleic acid assays, was determined by the method described by Blois, 1958 andCoutinho et al., 2008, respectively.For these assays the extracts were evaluated at concentrations at 40, 80, 160, 320 and 640 μg mL -1 .The radical scavenging effect of each sample was calculated and compared with that of quercetin (Sigma) (2.5,5,10,20, and 40 µg mL -1 ), and the experimental values in b-carotene/linoleic acid assay were compared with those of tert-butylated hydroxytoluene.All the tests were conducted in triplicate.

Antimycobacterial and antimicrobial activities
The anti-M.tuberculosis activity of the ethanolic extract of leaves and roots was determined using the REMA (Resazurin Microtiter Assay) assay as analytical method (Collins, Franzblau, 1997).The minimal inhibitory concentration (MIC) values of these compounds necessary to inhibit 90% of growth of M. tuberculosis H 37 Rv ATCC 27294 were determined in triplicate in sterile 96-well plates (Falcon 3072; Becton Dickinson, Franklin Lakes, NJ, USA), in a SPECTRAfluor Plus (Tecan, Männedorf, Switzerland) microfluorimeter.For the standard test, the MIC value of isoniazid was determined each time.The acceptable MIC of isoniazid ranges from 0.015 to 0.03 µg mL -1 .
The antimicrobial activity of the ethanolic extracts was assayed using the broth microdilution method (Silva et al., 2009) ).Standard strains of microorganisms were obtained from the American Type Culture Collection (Manassas, VA, USA), and the standard antibiotics chloramphenicol and nystatin were used in order to control the sensitivity of the microbial test.The minimal inhibitory concentration (MIC) was determined in 96-well culture plates by a microdilution method using a microorganism suspension at a density of 10 5 colonyforming units/mL with soy casein broth incubated for 24 hours at 37 °C for bacteria and Sabouraud broth incubated for 72 hours at 25 °C for yeasts.Proper blanks were assayed simultaneously, and samples were tested in triplicate.
The identification of all compounds (Figure 1) was achieved by the experimental data (IV, NMR, and ultraviolet).We also confirmed their structures by comparing them with the previously reported respective literature data (Agrawal, 1989;Harborne, 1996).
The UV spectrum showed typical results for flavonoids for compounds 1-7, with bands between 250-280 nm relative to ring A and 310-365 nm relative to ring B (Mabry, Markham, Thomas, 1970).The IV spectrum revealed absorption bands that range between 3242 and 3417 cm -1 that typically result from the OH link axial deformation.Bands close to 1650 cm -1 suggest the presence of carbonyl group.Furthermore, the presence of an aromatic ring may be observed, evidenced by a set of bands that range between 1600 and 1450 cm -1 , which denotes the axial deformation of the aromatic C=C.(Silverstein, Bassler, Morril, 1994).
The 1 H and 13 C NMR signals for compounds 1, 2, 6 and 7 were in good agreement with those of kaempferol, the difference between them was located at the glycoside linked to the C-3 and C-7 of the aglycone.These glycosides were identified by the signals at sugar region δ (16-18 and 60-102) in the spectrum by comparing against those described in the literature.For compound 1, no signals were identified at this region.The signals for compound 2 indicated two rhamnopyranoside moieties, for compound 6 one rhamnopyranoside moiety and for the compound 7 one rhamnopyranoside and one glucoside moieties (Agrawal, 1989;Harborne, 1996).The 1 H NMR signals for these compounds were found in the aromatic region δ (6-8) and sugar region δ 3-4.For compound 2, the sugars showed the anomeric hydrogen at δ 5.54 (ls, H1'') and δ 5.29 (ls, H1''') and signals at δ 1.26 and δ 0.92 of H6'' and H6'''indicating the presence of one unit of rhamnose at C-7 and C-3, respectively (Harborne, 1996).Compound 6 showed anomeric proton at δ 5.36 with J = 1.8 Hz, showing a a configuration for rhamnopyranoside and value for H6'' at δ 0.90.For compound 7, the presence of two anomeric signals at δ 5.30 (d, J = 7.6 Hz) and δ 4.40 (brs) ) in the 1 H NMR spectrum and two anomeric carbons at δ 101.5 and δ 100.7 in the 13 C NMR spectrum suggested that compound to be a disaccharide.Furthermore, the presence of one doublet with J = 6.5 Hz at δ 1.14 in the 1 H-NMR spectrum and at δ 17.5 in the 13 C NMR spectrum indicated the presence of one rhamnose moiety.The connecting position of the sugars was established using HMBC experiments.Correlations were observed between the anomeric signal of glucose (δ 5.30) and the C-3 of the kaempferol (δ 133.4) and the anomeric hydrogen signal of the rhamnose (δ 4.40, d, J = 1.8 Hz) and the C-6 of glucose (δ 66.4) (Agrawal, 1989;Harborne, 1996).
For compounds 4 and 5, the 1 H and 13 C spectra presented characteristic resonance of a glycoside flavone.The H-3 exhibited as a singlet at d 6.72 and 6.75, respectively.The two anomeric protons of glucose units showed a duplet at d 4.58 (J = 10 Hz) for 4 and d 4.53 (J = 10 Hz) for 5. Characteristic 1 H and 13 C chemical shift values and coupling constant data indicated that the structure of 4 was based on that of apigenin-6-C-b-Dglucopyranoside (isovitexin) and 5 was based on that of FIGURE 1 -Substances isolated from leaves and roots of S. erecta.
1 H NMR spectrum of 3 presented chemical shifts of characteristic patterns of catechins, with two double doublets at d 2.86 (H-4α) and d 2.74 (H-4β) bonded to C-4, a multiplet at d 4.21 assigned to H-3 and a broad singlet at d 4.88 referring to H-2.This set of signals is typical of the C ring of epicatechin.The 13 C spectrum showed 15 signals which could be assigned to the epicatechin (Harborne, 1996;Agrawal, 1989).Signals assigned to C-2 (δ 79.5) and C-3 (δ 67.04) characterizing the relative stereochemistry of epicatechin 3-OHα that differentiates of its catechin epimer.
Sapindaceae is a rich source of secondary metabolites and their studies have previously been identified in phytochemical analyses: isoprenoids and polyphenols, saponins, triterpenes, diterpenes, flavonols, flavones, tannins and catechins (Gomig et al., 2008;Albiero et al., 2002).Our phytochemical study led to the isolation of seven flavonoids (Figure 1).The isolation of C-glycoside flavonoids in S. erecta is reported for the first here for this species and also for the genus.
Flavonoids are widely present in the plant kingdom, being found in almost all fruits and plants as colouring pigments.Phenolic compounds, such as flavonoids, could also protect membrane lipids from oxidation acting as antioxidants due to their redox properties, allowing them to act as reducing agents, hydrogen donors, free radical quenchers and metal chelators (Shukla et al., 2009).
The results showed that the antioxidant activity measured for the free radical scavenging assay in the same concentrations was greater than that of the b-carotene/ linoleic acid assay.This might be attributed to the structures present in this extract as flavonoids, which exhibit their antioxidant activity by donating hydrogen (Rice-Evans et al., 1996).
Radical-scavenging activity of phenolics depends, among other factors, on the number and position of hydroxyl (-OH) group substituents in the molecules.The presence of the carboxyl, alkyl or other groups affects the antioxidant activity of phenol compounds.The maximum effectiveness for radical scavenging apparently requires the two hydroxyl groups in the ortho-diphenolic arrangement in the B ring and 3-OH group attached to the 2,3-double bond and adjacent to the 4-carbonyl in the C ring.The glycosylation of flavonoids reduces their activity when compared to the corresponding aglycones (Rice-Evans et al., 1996).
The substances 2, 4-7 isolated from S. erecta have one hydroxyl group in the B ring and glycosylation in the rings A and C. The substances 1 and 3 are aglycones and they present one and two hydroxyl groups in the B ring, respectively.
Because S. erecta is a plant largely used as medicinal, we also decided to investigate the effect of ethanol extract against seven microorganisms: Mycobacterium tuberculosis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella setubal, Candida albicans, Saccharomyces cerevisiae and Escherichia coli.
The ethanolic leaf extract showed an MIC value of 128.0 μg mL -1 and root extract MIC value of 256.0 μg mL -1 for the anti-M.tuberculosis activity.According to Gu et al., 2003, a sample with an MIC value of ≤128.0 μg mL -1 is defined as active.According to Pauli et al. (2005) the MIC of a crude natural extract may or may not be a reliable indicator of the chances for success in isolating a potent antimycobacterial agent from that extract.
The literature reports that flavonoids and saponins are inactive or weakly to moderately active against M. tuberculosis.And because of presence of polar substances in ethanolic extracts, the results led to a dramatic decrease or complete loss of activity, since the high lipophilicity is probably the main factor that allows penetration of the compounds through the mycobacterial cell wall (Lin et al., 2002), besides the synergistic effect among the compounds present in the extract could affect the final results of analysis (Inui et al., 2007).
On the other hand, the antimicrobial activity of the ethanolic extracts examined against the other six microorganisms: S. aureus, P. aeruginosa, S. setubal, C. albicans, S. cerevisiae e E. coli, revealed that the leaves and roots of S. erecta inhibited the growth of all microorganisms and that P. aeruginosa with MICs of 5.0 mg mL -1 and 10.0 mg mL -1 , respectively, was the most sensitive.C. albicans was the most resistant microorganisms with an MIC of 25.0 mg mL -1 for ethanolic extracts leaves (Table I).Our results showed interesting antimicrobial activity of the ethanol extract from S. erecta.This is probably due to substances present in the extract, as flavonoids.
Studies of antimicrobial activity indicate that crude extracts containing high content of flavonoids have showed significant activity against various strains of Staphylococcus aureus, Streptococcus faecalis and Escherichia coli (Chattopadhay et al., 2001).
The crude extract and eight isolated compounds from Castanea sativa was evaluated against twenty strains of Gram-positive and Gram-negative bacteria.The bioactive compounds were mainly flavonoids (luteolin, kaempferol and apigenin) and triterpenoids (Schinor et al., 2007).
Serjania lethalis is yet another example.Stems and leaves of S. lethalis showed particularly strong inhibition of growth of strains of Staphylococcus aureus with minimum inhibitory concentration (MIC) values ≤100 µg mL -1 .S. lethalis showed good results for this activity, although the screened plant tissue can differ from that indicated by the traditional use of the plant (Lima et al., 2006).In northeastern Brazilian region, the leaves of this species are used after cooked against kidney problems (Guarim Neto et al., 2000).The same occurs with S. erecta that although people use the leaves and roots against a number of diseases (Guarim Neto et al., 2000;Pott et al., 2004), there is no knowledge about the use of this species as antimicrobial.

TABLE I -
Antimicrobial and antimicobacterial activities of the ethanolic extracts of leaves and roots of S. erecta Data are mean values of three replicates in µg mL -1 ± standard desviation.MIC, minimal inhibitory concentration.a Obtained from the American Type Culture Collection.