Journal of Medicinal Plants Research Vol. 5(18), pp. 4511-4518, 16 September, 2011 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2011 Academic Journals Full Length Research Paper Relative antioxidant activity of Brazilian medicinal plants for gastrointestinal diseases Cibele Bonacorsi1, Luiz Marcos da Fonseca1, Maria Stella G. Raddi1*, Rodrigo R. Kitagawa2, Miriam Sannomiya3 and Wagner Vilegas4 1 Department of Clinical Analysis, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, SP, Brazil. 2 Department of Pharmaceutical Sciences, Federal University of Espírito Santo (UFES), Vitória, ES, Brazil. 3 School of Arts, Sciences and Humanities, University of São Paulo (USP), São Paulo, SP, Brazil. 4 Department of Organic Chemistry, Chemistry Institute, São Paulo State University (UNESP), Araraquara, SP, Brasil. Accepted 5 July, 2011 The free radical scavenging capacity of Brazilian medicinal plants and some of their constituents was examined in vitro using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) quantitative assay. Twelve medicinal plants, used to treat gastrointestinal disorders (Alchornea glandulosa, Alchornea triplinervia, Anacardium humile, Byrsonima crassa, Byrsonima cinera, Byrsonima intermedia, Davilla elliptica, Davilla nitida, Mouriri pusa, Qualea grandiflora, Qualea parviflora and Qualea multiflora), were selected because they showed antiulcerogenic activity in previous studies. The radical scavenging methanolic extracts activity demonstrated to be dose-dependent. The efficient concentration, which represents the amount of the antioxidant able of decrease the initial DPPH radical by 50%, vary from < 5 to 17.2 µg/ml. The lowest efficient concentration values among the analyzed plant were shown by A. humile, B. crassa and Q. parviflora. Purified phenolic compounds (amentoflavone, (+)-catechin, methyl gallate, quercetin 3-O-alpha-L-arabinopyranoside, and quercetin 3-O-beta-D-galactopyranoside) were also tested and the greatest antioxidant activities were obtained with (+)-catechin and methyl gallate, similar to quercetin, a phenolic compound used as standard. Key words: Brazilian medicinal plants, antioxidant activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH), gastric ulcers. INTRODUCTION Gastric and duodenal ulcers affect a large proportion of the world population and are induced by several factors, including stress, smoking, alcoholic beverages, nutritional deficiencies, ingestion of nonsteroidal anti-inflammatory drugs and Helicobacter pylori infection (O`Malley, 2003). Studies reveal an extensive variety of chemical compounds isolated from medicinal plants with antiulcer activity (Borrelli and Izzo, 2000). An ethnopharmacolo- gical survey carried out in the Cerrado of Central Brazil showed a high number of medicinal plants used to *Corresponding author. E-mail: raddims@fcfar.unesp.br. Tel: +55 16 33015720. treat gastric pain and gastritis (Silva et al., 2000). Among the plants used in Brazilian popular medicine for the treatment of gastrointestinal disorders, several have shown antiulcerogenic and/or anti-Helicobacter pylori activities (Bonacorsi el al., 2009; Lima et al., 2008; Luiz- Ferreira et al., 2008; Moraes et al., 2008). There are evidences in support the participation of reactive oxygen species and other free radicals in the etiology and pathophysiology of several human diseases, including gastrointestinal inflammation and gastric ulcer (Repetto and Llesuy, 2002). Compounds with multiple mechanism of protective action may be highly effective in minimizing tissue injury in human diseases (Umamaheswari et al., 2007). A number of methods and variations in methods to measure antioxidants capacity in botanicals have been 4512 J. Med. Plant. Res. proposed. The radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) is still highly utilized in antioxidant research due to its simple reaction system, which involves only the direct reaction between the radical and the antioxidant, without other interference, such as enzyme inhibition or the presence of multiple radicals (Cheng et al., 2006). The aim of this work was to evaluate the antioxidant capacity of methanolic (MeOH) extracts and isolated phenolic compounds from Brazilian medicinal plants for the treatment of gastroduodenal diseases using the DPPH free radical quantitative assay. MATERIALS AND METHODS Extract preparation The MeOH extracts used in this study were the same employed in others studies and the preparation have already been described by Calvo et al. (2007) for Alchornea glandulosa, Lima et al. (2008) for Alchornea triplinervia, Luiz-Ferreira et al. (2008) for Anacardium humile, Cardoso et al. (2006) for Byrsonima crassa, Moleiro (2007) for Byrsonima basiloba, Sannomiya et al. (2007) for Byrsonima intermedia, Biso et al. (2010) for Davilla species, Santos et al. (2008) for Mouriri pusa, and Nasser (2008) for Qualea species. Briefly, the air-dried and powdered leaves were extracted with methanol at room temperature (48 h). Solvent was evaporated at temperature of 60°C under reduced pressure to yield the methanol solid extract. The reference material, extract and phytochemical screening of all plants are synthesized on Table 1. The plants employed in this study are part of the BIOTA/FAPESP Program. Purified phenolic compounds The air-dried and powdered leaves (2.0 kg) of B. crassa were extracted with methanol at room temperature (48 h). The solvent was evaporated at 60°C under reduced pressure to produce the MEOH extract. The yield (w/w) of the extract from the dried powdered B. crassa leaves was 7.91% (158.3 g). An aliquot of the extract (4.0 g) was permeated on a Sephadex LH-20 column (100 x 5 cm) and then eluted with MeOH. Fractions (8 ml) were collected and analyzed by thin-layer chromatography on silica gel eluted with CHCl3/MeOH (80:20) and revealed by spraying with either NP/PEG (diphenylaminoborate/polyethyleneglycol) or an anisaldehyde/sulfuric acid solution. Fractions 129 to 141 (95.0 mg) were purified by repeated column chromatography (CC) on microcrystalline cellulose using with CHCl3/MeOH (80:20) as the eluent, yielding the biflavonoid amentoflavone (6.0 mg). Fractions 88 to 95 (69.0 mg) were further purified by high-performance liquid cromatograph (HPLC), with MeOH/H2O (1:1) as the eluent, to yield quercetin-3-O-beta-D-galactopyranoside (15.0 mg). Fractions 82 to 87 (122.0 mg) were purified by silica CC using EtOAc/n-PrOH/H2O 140:8:80 (upper phase) as the eluent, yielding quercetin-3-O-alpha- L-arabinopyranoside (14.0 mg) and a mixture of (-)-epicatechin and (+)-catechin (30.0 mg). Epicatechin and (+)-catechin were separated by HPLC with MeOH/H2O (20:80) as the eluent to yield 10 mg of each purified compound. Fractions 55 to 60 (112.0 mg) were purified by silica gel CC with CHCl3/MeOH (75:25) as the eluent, yielding methyl gallate (8.0 mg), which was confirmed by NMR and TLC (Sannomiya et al., 2004). The extract and the purified major constituents were solubilized in dimethyl sulfoxide (DMSO). DPPH photometric assay The free radical scavenging activity of MeOH extracts and the phenolic compounds were measured in vitro by DPPH assay. Briefly, ethanol solutions of the methanol solid extracts and phenolic compounds (50 µl), at different concentrations (5 to 100 µg/ml), were mixed with 100 µl of DPPH (2,2-diphenyl-1- picrylhydrazyl, Sigma-Aldrich, USA) solution (0.3 mM, in methanol). The absorbance was measured at 517 nm (ABS sample) after 30 min of reaction at 25°C. Quercetin (Sigma-Aldrich, USA) was used as a standard antioxidant and DPPH solution plus ethanol was used as negative control (ABS control). Measurements were performed at least in triplicate. The capability to scavenge the DPPH radical was calculated as DPPH scavenging effect (%) = [(ABScontrol - ABSsample/ABScontrol) x 100]. The efficient concentration (EC50), concentration of the antioxidant required to decrease the initial DPPH radical concentration by 50%, was also determined. A lower value of EC50 indicates a higher antioxidant activity (Siddhuraju and Manian, 2007). RESULTS The percentages of antioxidant activity for the MeOH extracts are shown on Table 2. The DPPH radical scavenging activity was dose-dependent. In terms of efficient concentration, the radical scavenging activity varied from < 5 to 17.2 µg/ml. The lowest efficient concentrations among the analyzed plants were shown by A. humile, B. crassa and Q. parviflora. The most effective purified phenolic compounds studied for antioxidant activities were (+)-catechin and methyl gallate, similar to quercetin, the flavonol used as standard (Table 3). DISCUSSION Gastritis and peptic ulcers are important gastrointestinal disorders that affect a considerable number of people. Products on the market for the treatment these diseases, including antacids, proton pump inhibitors, anticholinergic and histamine H2-antagonists, can produce several adverse reactions (Yacyshyn and Thomson, 2002). Medicinal plants are among the most attractive sources of new drugs and have been shown to give promising results in treatment of gastric ulcers (Borrelli and Izzo, 2000). There is considerable interest in the antioxidant activity of plants, due to the fact that free radicals have been related to some diseases, as well as to the aging process (Nijveldt et al., 2001; Willcox et al., 2004). In this study, differences on the free radical scavenging activity of the MeOH extract from plants used in Brazilian Bonacorsi et al. 4513 Table 1. Plants employed in this study, reference material, obtained extract, phytochemical screening, and phenolic compounds. Plant Herbarium voucher number Plant part Methanolic extract residue (%) Phytochemical screening Phenolic compound Alchornea glandulosa Campinas University Nº 132828 Leaf 11.8 (Calvo et at., 2007) Phenolic acids, alkaloids, steroids, flavonoids, isocumarins, saponins, tannins, terpenes (Calvo, 2007; Calvo et al., 2007; Lopes et al., 2005) Amentoflavone, methyl gallate, quercetin- 3-O-alpha-L-arabinopyranoside, quercetin-3-O-beta-D-galactopyranoside (Calvo et al.., 2007) Alchornea triplinervia São Paulo State University Nº 14873 Leaf 15 (Lima et al., 2008) Phenolic acids, ellagic acids, alkaloids, flavonoids, isocumarins, saponins, tannins (Calvo 2007; Lima et al., 2008) Amentoflavone, methyl gallat, quercetin- 3-O-alpha-L-arabinopyranosid, quercetin- 3-O-beta-D-galactopyranoside (Calvo, 2007) Anacardium humile Tocantins University Nº 1922 Leaf 29.7 (Luiz-Ferreira et al., 2008) Phenolic acids, catechins, flavonoids, saponins, tannins (Luiz-Ferreira et al., 2008) Amentoflavone, (+)-catechin, methyl gallate, quercetin-3-O-beta-D- galactopyranoside (Luiz-Ferreira et al., 2008) Byrsonima basiloba São Paulo State University Nº 24163 Leaf 9.4 Moleiro, 2007 Phenolic acids, catechins, flavonoids, steroids, tannins (Lira et al., 2008; Moleiro, 2007) Amentoflavone, (+)-catechin, methyl gallate, quercetin-3-O-alpha-L- arabinopyranoside, quercetin-3-O-beta-D- galactopyranoside (Lira et al., 2008) Byrsonima crassa Tocantins University Nº 3377 Leaf 7.91 (Cardoso et al., 2006) Phenolic acids, catechins, steroids, flavonoids, tannins, terpenes (Cardoso, 2006; Cardoso et al., 2006; Sannomiya et al., 2005) Amentoflavone, (+)-catechin, methyl gallate, quercetin-3-O-alpha-L- arabinopyranoside, quercetin-3-O-beta-D- galactopyranoside (Cardoso et al., 2006; Sannomiya et al., 2005) Byrsonima intermedia São Paulo State University Nº 24164 Leaf 4.3 (Cardoso, 2006) Phenolic acids, catechins, steroids, flavonoids, tannins (Cardoso, 2006; Sannomiya et al., 2007) Amentoflavone, (+)-catechin, methyl gallate, quercetin-3-O-alpha-L- arabinopyranoside, quercetin-3-O-beta-D- galactopyranoside (Sannomiya et al., 2007) 4514 J. Med. Plant. Res. Table 1. Contd. Davilla elliptica Tocantins University Nº 4593 Leaf 7.1 (Rodrigues, 2007) Phenolic acids, catechins, steroids, flavonoids, tannins, terpenes (Biso et al., 2010; Carlos et al., 2005; Michelin et al., 2005) Quercetin-3-O-alpha-L- arabinopyranoside, quercetin-3-O-beta-D- galactopyranoside (Rodrigues, 2007) Davilla nitida Tocantins University Nº 3849 Leaf 11.7 (Rodrigues, 2007) Phenolic acids, catechins, flavonoids (Biso et al., 2010; Rodrigues, 2007) Mouriri pusa Tocantins University Nº 3341 Leaf 10 Santos et al. (2008) Catechins, flavonoids, tannins (Santos et al., 2008; Vasconcelos et al., 2008) (+)-catechin, quercetin-3-O-alpha-L- arabinopyranoside, quercetin-3-O-beta-D- galactopyranoside (Andreo et al., 2007; Santos et al., 2008) Qualea grandiflora Tocantins University Nº 4158 Bark 10.9 Nasser (2007) Ellagic acids, catechins, steroids, flavonoids, saponins, tannins, terpenes (Nasser, 2007; Nasser et al., 2008) Qualea multiflora Tocantins University Nº 9226 Bark 10.2 Nasser (2007) Phenolic acids, ellagic acids, flavonoids, saponins, tannins, terpenes (Nasser, 2007; Nasser et al., 2008) Qualea parviflora Tocantins University Nº 3379 Bark 9.2 Nasser (2007) Ellagic acids, steroids, flavonoids, saponins, tannins, terpenes (Nasser et al., 2006; Nasser, 2007; Nasser et al., 2008) popular medicine to treat gastritis and ulcers were demonstrated. It is apparent from this data that levels of potential antioxidant activity have a wide variation in lower concentrations. Previously published investigations have demonstrated the effects of MeOH extract of A. glandulosa (Calvo et al., 2007) and B. crassa (Sannomiya et al., 2005) on different models of gastric lesion induced in mice. The pathogenesis of ethanol-induced gastric mucosal damage is still unknown but the formation of oxygen-derived free radicals and neutrophil infiltration into gastric mucosa is considering the main sources of mucosal damage (Chow et al., 1998). Plants containing antioxidant properties are effective in preventing this kind of lesion and a presence the phenolic compounds may probably explain the antiulcerogenic effect of these plants (Calvo et al., 2007; Luiz-Ferreira et al., 2008; Sannomiya et al., 2005). Although, the DPPH assay is usually classified as single electron transfer reaction, this radical may be Bonacorsi et al. 4515 Table 2. Percentage of DPPH radical scavenging activity and antioxidant efficient concentration for methanolic extract of Brazilian plants used to treat gastritis. Scavenging (%) a Botanical name (popular name) Methanolic extract concentration (µg/ml) EC50 b 5 10 20 40 50 80 100 Alchornea glandulosa (tapiá) 28.5 ± 0.8 50.3 ± 0.4 72.4 ± 0.9 78.3 ± 1.1 80.1 ± 0.9 80.6 ± 0.9 83.8 ± 1.2 10 Alchornea triplinervia (tanheiro) 46.0 ± 0.5 67.2 ± 0.5 77.4 ± 0.9 79.8 ± 1.0 76.9 ± 0.9 79.6 ± 0.7 78.5 ± 1.0 5.9 Anacardium humile (cajuzinho-do-cerrado) 59.4 ± 0.7 73.2 ± 1.2 74.3 ± 0.5 74.1 ± 0.9 73.7 ± 0.8 73.4 ± 1.0 73.7 ± 1.3 < 5 Byrsonima basiloba (murici-de-ema) 22.6 ± 0.6 42.1 ± 1.1 64.8 ± 0.6 73.4 ± 0.8 73.2 ± 0.7 74.2 ± 1.0 74.6 ± 0.9 14.2 Byrsonima crassa (murici) 61.9 ± 1.2 81.9 ± 1.5 85.2 ± 1.1 86.4 ± 0.9 85.9 ± 0.7 86.3 ± 1.2 86.2 ± 0.6 < 5 Byrsonima intermedia (murici-do-campo) 27.0 ± 1.3 47.1 ± 1.1 73.1 ± 1.0 76.3 ± 0.9 77.7 ± 1.2 77.8 ± 1.3 78.0 ± 0.6 12.0 Davilla elliptica (cipó-de-carijó) 36.0 ± 0.7 59.7 ± 0.8 76.3 ± 1.1 76.9 ± 1.1 77.0 ± 1.1 77.1 ± 0.9 77.8 ± 0.7 8.8 Davilla nitida (cipó-de-carijó) 16.8 ± 0.7 31.2 ± 0.8 57.6 ± 0.7 77.0 ± 0.7 78.4 ± 0.8 78.7 ± 0.9 78.4 ± 0.9 17.2 Qualea grandiflora (pau-da-terra) 34.5 ± 0.8 62.5 ± 0.8 77.5 ± 0.5 80.1 ± 1.2 80.0 ± 0.9 80.5 ± 0.4 81.5 ± 0.9 8.6 Qualea parviflora (ipê-cascudo) 53.0 ± 0.7 71.7 ± 1.2 74.1 ± 1.0 77.0 ± 1.0 78.8 ± 1.0 81.3 ± 0.9 81.2 ± 1.0 < 5 Qualea multiflora (Cerra-do-campo) 50.9 ± 1.1 72.7 ± 1.1 74.6 ± 0.7 77.4 ± 1.3 78.6 ± 0.9 81.3 ± 0.7 80.7 ± 0.7 5 Mouriri pusa (puçá) 39.9 ± 0.7 57.4 ± 0.8 59.6 ± 0.8 63.0 ± 0.6 64.2 ± 0.5 67.5 ± 0.5 67.1 ± 0.8 7.9 a means of triplicate readings; b antioxidant efficient concentration. neutralized either by direct reduction via electron transfers or by radical quenching via H atom transfer (Jiménez et al., 2004). Based on the mechanism of reduction of the DPPH molecule and on previous knowledge of the chemistry of some plants, it is possible to infer that the strong antioxidant activity of some polar extracts is due, at least in part, to the presence of substances with an available hydroxyl group. Compounds able to donate hydrogen are derived from the shikimate pathway, as for example, flavonoids (Huang et al., 2002). Flavonoids and gallic acid derivatives (methyl gallate, ethyl gallate, propyl gallate and others gallate esters) are major classes of phenolic compounds that are widely spread throughout the plant kingdom, whose structure- antioxidant activity relationships have been reported (Nenadis et al., 2004). In general, the free-radical scavenging and antioxidant activities of phenolics depend primarily on the number and positions of the hydrogen-donating hydroxyl groups on the aromatic ring of these molecules, but is also affected by other factors, such as glycosylation of aglycones and other H-donating groups (Cai et al., 2004). The results of this study showed that the flavonoid quercetin (a standard antioxidant) had a better antioxidant activity than its 3-O-glycoside derivatives (quercetin-3-O- alpha-L-arabinopyranoside and quercetin-3-O- beta-D-galactopyranoside). This corroborates reports that flavonoid aglycones are more potent antioxidants than their corresponding glycosides (Heim et al., 2002). Catechin and quercetin belong to the flavanol and flavonol classes, respectively and they have been studied intensely because of their high ROS scavenging activities. These polyphenol molecules have the same number of hydroxyl groups on the A and B rings. In the chemical structure of quercetin there is a double bond at position 2,3 and an oxo group at position 4 in the C ring which seem to provide better antioxidant activity then that of catechin (Mamede and Pastore, 2004). Our data demonstrated that (+)- catechin and methyl gallate had a similar 4516 J. Med. Plant. Res. Table 3. Percentage of DPPH radical scavenging activity and antioxidant efficient concentration for the phenolic compounds. Scavenging (%) a Phenolic compound Concentration (µg/ml) EC50 b 5 10 20 40 50 80 100 Amentoflavone 36.4 ± 0.6 50.3 ± 0.7 67.2 ± 1.0 90.6 ± 1.1 95.7 ± 1.0 95.8 ± 1.3 95.6 ± 1.5 10 (+)-Catechin 93.6 ± 1.1 95.3 ± 1.0 96.3 ± 1.0 94.9 ± 0.9 94.7 ± 0.9 94.3 ± 1.1 94.0 ± 1.2 < 5 Methyl gallate 95.1 ± 0.5 97.2 ± 0.5 96.6 ± 0.5 96.8 ± 0.5 97.1 ± 0.5 97.3 ± 0.5 96.9 ± 0.6 < 5 Quercetin-3-O-alpha-L-arabinopyranoside 40.8 ± 0.6 56.6 ± 0.7 83.7 ± 0.9 94.1 ± 0.8 96.1 ± 1.1 95.6 ± 1.0 96.0 ± 1.1 8 Quercetin-3-O-beta-D-galactopyranoside 38.3 ± 0.3 53.9 ± 0.7 80.3 ± 0.7 92.2 ± 0.9 94.0 ± 1.0 93.4 ± 1.1 93.4 ± 0.9 9.9 Quercetin (standard) 94.6 ± 1.1 97.2 ± 0.9 96.9 ± 1.8 97.2 ± 1.8 96.9 ± 1.5 97.4 ± 1.5 97.0 ± 1.7 < 5 a means of triplicate readings; b antioxidant efficient concentration. radical-scavenging activity even at a lower concentration. Methyl gallate has been shown to be an effective antioxidant in a variety of acellular experiments (Hsieha et al., 2004). The lower antioxidant activity of amentoflavone was consistent with other studies (Baggett et al., 2005; Hyun et al., 2006). This work has no intending to prove the total antioxidant capacity in the studied plants because multiple reaction characteristics and mechanisms are involved and no single assay will accurately reflects all of the radical sources or all antioxidants in a mixed or complex system. The antioxidant capacity demonstrated by a single method (DPPH) using different concentrations, may be useful to demonstrate the variable levels of compounds with free radical (DPPH) scavenging activity present in plants tissue but not necessarily correspondent to total of phenolic constituents and derivatives. However, it is apparent from this data that levels of potential antioxidant activity have a wide variation at low concentration. These results suggested that the optimal effective dose for evaluating the potential of medicinal plants for its antiulcer activity on experimental models may be related, at least in part, to the concentration of antioxidants required to decrease the DPPH radical, since the antiulcerogenic potential of A. glandulosa, A. humile and B. crassa has also been attributed to the presence of oxygen radical scavengers (Calvo et al., 2007; Luiz-Ferreira et al., 2008; Sannomiya et al., 2005). The high free radical scavenging activity of some tested extracts in the DPPH test suggest that the antioxidant potential may be one of the mechanisms that explains the popular use of plants for the treatment of gastrointestinal diseases, as ulcer, because both ulcerous and inflammatory process are related to an increase of free radicals. The most effective species confirmed for their lowest antioxidant efficient concentration were A. humile, B. crassa and Q. parviflora. 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