HPLC-DAD Analysis and Antioxidant Activity of Hymenaea Martiana Hayne (Fabaceae)
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Available online www.jocpr.com Journal of Chemical and Pharmaceutical Research, 2012, 4(2):1160-1166 ISSN : 0975-7384 Research Article CODEN(USA) : JCPRC5 HPLC-DAD analysis and antioxidant activity of Hymenaea martiana Hayne (Fabaceae) Maria Eduarda Gomes da Cruz Silva 1, Amanda Leite Guimarães 1, Ana Paula de Oliveira 1, Camila de Souza Araújo 1, José Alves de Siqueira Filho 2, André Paviotti Fontana 2, Patrícia Kauanna Fonseca Damasceno 3, Carla Rodrigues Cardoso Branco 3, Alexsandro Branco 3, Jackson Roberto Guedes da Silva Almeida 1* 1Núcleo de Estudos e Pesquisas de Plantas Medicinais, Universidade Federal do Vale do São Francisco, 56.304-205, Petrolina, Pernambuco, Brazil 2Centro de Referência para Recuperação de Áreas Degradadas da Caatinga (CRAD), 56.300- 000, Petrolina, Pernambuco, Brazil 3Laboratório de Fitoquímica, Departamento de Saúde, Universidade Estadual de Feira de Santana, 44.036-900, Feira de Santana, Bahia, Brazil ______________________________________________________________________________ ABSTRACT A qualitative characterization of the main phenolic compounds from ethanol extract of Hymenaea martiana was carried out by high performance liquid chromatography with diode array detection (HPLC-DAD). The total phenolics content of the plant extracts was determined by the Folin-Ciocalteu method. Total flavonoid content also was measured. Antioxidant activities of the extracts were evaluated by using DPPH radical scavenging and β- carotene-linoleic acid bleaching and compared with ascorbic acid, BHA and BHT used as reference compounds. The total phenolic content was of 428.50 ± 5.91 and 705.50 ± 7.22 mg of gallic acid equivalent/g for EtOH and AcOEt extracts, respectively. The total flavonoids content was of 394.90 ± 8.43 and 479.60 ± 10.38 for two extracts, respectively. All extracts exhibited good antioxidant activities. The EtOH extract showed better antioxidant activity than ascorbic acid and BHA using by DPPH method, with a value of IC50 of 0.84 ± 0.26 µg/ml. BHT was the most effective antioxidant. The results obtained show that phenolic compounds contribute to the antioxidant activity of the extract. Further studies will be conducted to isolate the chemical constituents responsible for antioxidant activity. Keywords: Hymenaea martiana , Fabaceae, antioxidant activity. ______________________________________________________________________________ INTRODUCTION Hymenaea (Fabaceae) is a genus widely distributed from Central to South America, mainly in the Amazon basin [1]. Approximately 25 species from the American continent have been described [2]. Plants from the genus Hymenaea are commonly used in Brazilian traditional medicine to treat inflammatory process, bacterial infections, rheumatism and anaemia [3, 4]. The trunk exudes a resin, which is used locally in folk medicine for treatment of wounds, bronchitis and stomach disorder [5]. Hymenaea species are known to mainly contain the diterpenoids compounds in the trunk resin and bark extract [6]. Diterpenes of the enantio -labdanoic type in the trunk resin and bark extract and ent -halimane in the seed pod resin as well as clerodane-type diterpenes were isolated from this genus [7, 8 & 9]. Hymenaea martiana Hayne is a native tree known in the Northeastern region of Brazil as “jatobá”. A few examples of biological activities for extracts of this specie have been reported. The previous studies have showed antinociceptive and anti-inflammatory activities related with hydroalcoholic extract obtained from the bark of H. 1160 J R G da Silva Almeida et al J. Chem. Pharm. Res., 2012, 4(2):1160-1166 ______________________________________________________________________________ martiana . The exact mechanism that underlies its analgesic and anti-inflammatory profiles remains unclear, but may result from its ability to inhibit the generation of lipoxygenase and/or cyclooxygenase products of the arachidonic acid pathway [10]. Carneiro et al. [11] demonstrated in the bark of H. martiana , the presence of glycoside compounds such as astilbin, eucryphin and engelitin, which were capable of antagonising bradykinin responses. This effect may explain the analgesic and anti-inflammatory actions of this plant. Astilbin is a flavonoid initially identified as an active principle present in a crude extract from the bark of this specie which shows antioxidant activity [12]. Astilbin and some related compounds were evaluated for antinociceptive and anti-oedematogenic activities. The results indicated that taxifolin and its tetramethylated derivative exhibited potent and dose-dependent antinociceptive action. Both compounds showed significant anti-oedematogenic effect being more effective than astilbin [13]. In our continuing search of the Brazilian Caatinga medicinal plants to combine biodiversity conservation with drug discovery we demonstrated the antinociceptive effect of the ethanolic extract of Amburana cearensis in mice [14] and anti-ulcer activity of ethanolic extract of Encholirium spectabile in rodents [15]. Selective spasmolytic effect of a new furanoflavoquinone derivative from diplotropin, a furanoflavonoid isolated from Lonchocarpus araripensis also have been demonstrated [16]. There is no previous report on the analysis of the antioxidant activity of Hymenaea martiana . The aim of this work was to characterize qualitatively by HPLC-DAD the main phenolic compounds present in the ethanolic extract and to evaluate the antioxidant activity in vitro of this plant. EXPERIMENTAL SECTION Plant material The trunk barks of Hymenaea martiana Hayne were collected in Petrolina, State of Pernambuco, Brazil, in November 2009. A voucher specimen (6444) is deposited at the Herbarium Vale do São Francisco (HVASF) of the Universidade Federal do Vale do São Francisco. Extraction The dried and powdered trunk barks (3000 g) were repeatedly extracted three times during 72 h with 95% EtOH at room temperature. The extractive solution was concentrated under vacuum yielding after distillation of solvent, 90 g of crude ethanol extract (Hm-EtOH). The Hm-EtOH was suspended in a mixture of H 2O:MeOH (7:3) and extracted successively with hexane, CHCl 3 and AcOEt in crescent order of polarity to obtain the respective extracts. Preliminary phytochemical screening Preliminary phytochemical analysis of the ethanol extract was carried. The presence of alkaloids was tested with Dragendorff’s and Mayer’s reagents, flavonoids with HCl and Mg powder, phenols with ferric chloride and steroids and terpenoids by Liebermann-Burchard reaction [17]. HPLC-DAD analysis of phenolic compounds The solvents used in high performance liquid chromatography are of analytic grade from Merck ®. A Milli-Q System® (Bedford, MA, USA) was used to purify the water. Analyses of high performance liquid chromatography was performed on a Merck-Hitachi liquid chromatograph LaChrom Elite® equipped with a VRW HITACHI L- 2130 pump, a VRW HITACHI L-2300 Diode-Array Detector (DAD), and an auto sampler with a 100 µL loop. The data were acquired and processed using Ezchrom Elite software. The extract was analyzed using a reverse-phase HPLC column: Purospher ® STAR RP-18e (250 mm X 4.6 mm i.d., 5 µm) column (Merck). The mobile phase was composed of solvent (A) H 2O/H 3PO 4 0.1% and solvent (B) MeOH. The solvent gradient was composed of A (75- 0%) and B (25-100%) for 20 min, then 100% B for 4 min, then again at the initial conditions (75% A and 25% B) for 10 min. A flow rate of 1.0 ml/min was used in a 30 ºC oven, and 20 µL of each sample was injected. The procedure was repeated three times for each sample. Samples and mobile phases were filtered through a 0.22 µm Millipore filter prior to HPLC injection. Spectra data were recorded from to 200 to 400 nm during the entire run. Total phenolic content Total phenolic contents were assayed using the Folin-Ciocalteu reagent, it is based on the method reported by Slinkard and Singleton [18], only the volumes have been reduced [19, 20]. An aliquot (40 µl) of a suitable diluted ethanolic extract was added to 3.16 ml of distilled water and 200 µl of the Folin–Ciocalteu reagent, and mix well. The mixture was shaken and allowed to stand for 6 min, before adding 600 µl of sodium carbonate solution, and shake to mix. The solutions were left at 20 °C for 2 hours and the absorbance of each solution was determined at 765 nm against the blank and plot absorbance vs. concentration. Total phenolic contents of the extracts (three replicates per treatment) were expressed as mg gallic acid equivalents per gram (mg GAE/g) through the calibration curve with gallic acid. The calibration curve range was 50–1000 mg/l (R 2 = 0.9993). All samples were performed in triplicates. 1161 J R G da Silva Almeida et al J. Chem. Pharm. Res., 2012, 4(2):1160-1166 ______________________________________________________________________________ Determination of Total Flavonoid Content Total flavonoid content was determined by using a colorimetric method described previously [21]. Briefly, 0.30 ml of the EtOH and AcOEt extracts or (+)-catechin standard solution were mixed with 1.50 ml of distilled water in a test tube followed by addition of 90 µl of a 5% NaNO 2 solution. After 6 min, 180 µl of a 10% AlCl 3.6H 2O solution was added and allowed to stand for another 5 min before 0.6 ml of 1 M NaOH was added. The mixture was brought to 330 µl with distilled water and mixed well. The absorbance was measured immediately against the blank at 510 nm using a spectrophotometer (QUIMIS, Brazil) in comparison with the standards prepared similarly with known (+)-catechin concentrations. The results were expressed as mg of catechin equivalents per gram of extracts (mg CE/g) through the calibration curve with catechin. The calibration curve range was 50-1000 mg/l. DPPH Free Radical Scavenging Assay The free radical scavenging activity was measured using the 2,2-diphenyl-1-picrylhydrazil (DPPH) assay [22, 23]. Sample stock solution (1.0 mg/ml) of Hm-EtOH was diluted to final concentrations of 243, 81, 27, 9, 3 and 1 µg/ml, in ethanol. One ml of a 50 µg/ml DPPH ethanol solution was added to 2.5 mL of sample solutions of different concentrations, and allowed to react at room temperature.