Microsoft Word - 27-Bio_39845 1365 Original Article Biosci. J., Uberlândia, v. 34, n. 5, p. 1365-1378, Sept./Oct. 2018 ANTIOXIDANT AND PHOTOPROTECTIVE POTENTIAL OF Moringa oleifera LAM (Moringaceae) POTENCIAL ANTIOXIDANTE E FOTOPROTETOR DE Moringa oleifera LAM (Moringaceae) Janine Mailho GIMENIS1; Amanda Costa GOMES1; Valter Henrique Marinho dos SANTOS2; Paulo Cesar FERREIRA3; Camila Areias OLIVEIRA4; André Rolim BABY5; Regildo Márcio Gonçalves da SILVA6 1. Mestre Farmacêutico, Universidade Estadual Paulista - UNESP, Instituto de Química de Araraquara, Araraquara, SP, Brasil; 2. Doutor Biólogo, Atina Ativos Naturais, Pouso Alegre, SP, Brasil; 3. Doutor Farmacêutico; 4. Professor, Doutor, Universidade de São Paulo, Departamento de Farmácia, São Paulo, SP, Brasil; 5. Mestre Farmacêutico; 6. Professor, Doutor, Universidade Estadual Paulista, Faculdade de Ciências e Letras de Assis, Assis, SP, Brasil. regildos@yahoo.com.br ABSTRACT: Moringa oleifera Lam. (Moringaceae) is a plant with several biological activities and therapeutic properties. However, the complete knowledge about its pharmacological, biological and ecological effects, and about the active components present in each vegetable part are not still completely elucidated. This study aimed to evaluate the antioxidant and photoprotective activities of different extracts from leaves and flowers of M. oleifera. These activities were assessed through in vitro tests, DPPH radical scavenging method, iron ion chelating effect (FRAP), lipid peroxidation (TBARS), nitric oxide scavenging method and assessment of the activity against the lipid peroxidation through hemolytic method. The photoprotective activity was assessed through spectrophotometric analysis and through in vitro test with Labsphere. It was also determined the extract’s phenolic content and total flavonoid through spectrophotometry and HPLC. The obtained results demonstrated that this species have components with antioxidant and photoprotective potential mainly in the extracts obtained from fresh leaves and flowers. Therefore, it was possible to verify that M. oleifera has potential to be used as source of antioxidant components with photoprotective activity mainly due to the presence of phenolic components and among these, the flavonoids. KEYWORDS: Flavonoids. Formulations. Plant-based cosmetics. Moringa oleífera. Sunscreen. Vegetables extracts. INTRODUCTION Moringa oleifera Lam., a plant species belonging to the Moringaceae family, has been used as food, in traditional medicine and in industrial applications (ABDULKARIM et al.,2005; ANWAR et al.,2007; SILVA et al.,2013; ARANTES et al.,2015). M. oleifera is native to eastern Africa and southern India, introduced to different regions around the world including Africa, Americas and Caribbean Islands for similar usage as a food component and herbal medicine (BEZERRA et al.,2004; MUHL et al.,2011). The leaves, flowers and immature pods of this plant are used as highly nutritive supplements with many pharmacological properties (BEZERRA et al.,2004; ANWAR et al.,2007; MUHL et al.,2011; SILVA et al.,2013; ARANTES et al.,2015; ARORA; ONSARE, 2015). Moreover, M. oleifera have long been recognized by the Ayurvedic and Unani systems of medicine for prevention and treatment of several diseases, e.g., gastric ulcers, skin diseases, hay fever, fatigue and bronchitis (ANWAR et al.,2007; SIXT-DANIELL et al.,2011; VONGSAK, 2013). The M. oleifera leaf extract have been reported to exhibit antioxidant activity both in vitro and in vivo, due to abundant phenolic acids and flavonoids (EILERT et al.,1981; SANCHEZ- MACHADO et al.,2006). However, the studies conducted so far did not evaluate the photoprotective potential of extracts from this species. The antioxidant components are responsible for reestablishing the equilibrium of redox metabolism and reduce the surplus levels of free radicals to prevent oxidative stress. This stress can lead to molecular and cellular damage that can promote the emergence of different diseases and premature aging (POURZAND; TYRRELL, 1999; MORITA; KRUTMANN, 2000; JAIN; JAIN, 2010). The increased production of free radicals can be of endogenous origin such as aerobic respiration, inflammation, hydrogen peroxide metabolism and cytochrome P450 enzymes, and exogenous origin such as diet, medication, smoking, pollution and ionizing radiation (mainly ultraviolet exposure) (BONDY; NADIERI, 1994). Excessive exposure to ultraviolet radiation leads to increased free radicals and reduces endogenous antioxidants, promoting damage to Received: 14/09/17 Accepted: 15/06/18 1366 Antioxidant and photoprotective… GIMENIS, J. M. et al. Biosci. J., Uberlândia, v. 34, n. 5, p. 1365-1378, Sept./Oct. 2018 membrane lipids, proteins, carbohydrates and nucleic acids by oxidation and modification of these molecules. Such damage can lead to formation of skin cancer (photocarcinogenesis) and directly influence the process of aging (photoaging) (SEITE et al., 2000; DUALE et al.,2010; WANG et al.,2010). Recent studies have shown that active substances found in vegetables can present active components with photoprotective action. Among these are vitamins C and E, tannins, alkaloids and flavonoids (VIOLANTE et al.,2009). This effect is related to the structural similarity between synthetic chemical filters and active ingredients extracted from plants, since the ultraviolet absorption has been observed when using plant extract in pharmaceuticals and cosmetics, indicating a possible anti-sunburn action (RAMOS et al.,1996). Among the sunscreens active components rich in flavonoids, ethanolic extracts show two peaks absorption at 240-280nm and 300-550nm wavelengths, indicating a potential for absorbing UV radiation, whose wavelength is between 290 - 320 (UVB) and 320-400 (UVA) (VIOLANTE et al.,2009). Studies have shown that flavonoids inhibit enzymes that are involved in free radicals production and act as chelating agents against metal involved in the metabolism of oxygen (BROWN et al.,1998). Therefore, the current study was undertaken to evaluate the antioxidant activity of different extraction forms of M. oleifera‘s leaves and flowers correlating the way of prepare for best use in ethnopharmacology. MATERIAL AND METHODS Plants materials The leaves and flowers of M. oleifera were collected from specimens present in the Universidade Estadual Paulista (UNESP-Brazil) (22°32’20’’S and 50°22’60’’W). A voucher specimen has been deposited in the Herbarium Assisense (HASSI) under number of 760. The leaves and flowers were cleaned and a portion was dried at 40º C for 24h. The dried samples were ground and passed through a sieve (20 mesh). The powder went kept sealed and protected from luminosity in containers until use. Another portion of fresh sample was used for squeezing and obtaining extracts from the fresh plant parts. Preparation of extract Several extraction methods were performed using water, hydroethanolic solution 70% (v/v) and absolute ethanol as solvents. Each extraction was repeated several times until exhaustion. Each method was done in triplicate. Preparation of fresh and lyophilized aqueous extract Fresh leaves and flowers of M. oleifera were pounded into processor with distilled water at a ratio of 1:10 (w: v) for 5 minutes and filtered, obtaining fresh aqueous extract. A volume of aqueous extracts were frozen and freeze-dried to obtain the dry extracts. The extracts were used in the tests. Preparation of hydroethanolic and ethanolic extracts M. oleifera’s leaves and flowers were dried at 40°C and pounded. The resulting powders were used to prepare the hydroethanolic and ethanolic extracts. The hydroethanolic extracts were prepared at a ratio of 1:10 (w/v) with ethanol 70% solution in distilled water by mechanical stirring for 24 hours. The extracts were filtered and concentrated on a rotary evaporator under vacuum. The resulting extracts were frozen and lyophilized to obtain dry extracts. The ethanolic extracts were prepared at a ratio of 1:10 (w/v) ethanol PA by mechanical stirring for 24 hours. After that, they were filtered and taken to rotary evaporator and posteriorly taken to desiccator to obtain the dry extracts. (CHOI et al., 2016). Preparation of sunscreen formulations The oil-in-water emulsions were prepared using an anionic emulsifier (Ammonium Acryloyldimethyltaurate / VP Copolymer). From this emulsion, there were prepared three samples: Mo-0 emulsion (without extract and sunscreen addition), Mo-1 emulsion (without sunscreen and extract added) and Mo-2 (with sunscreen and M. oleifera hydroethanolic extract). The qualitative and quantitative composition of the formulations are described in Table1. Test of antioxidant activity DPPH radical scavenging test The antioxidant activity of crude extract and fractions were determined by the H+ donor ability to the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma, USA), according to the in vitro methodology (BLOIS, 1958). The experiment was performed in triplicate using a solution made of 1 mL of acetate buffer (pH 5.5 and 100 mM), 1.25 mL of ethanol P.A., 250µL of DPPH solution and 50 mL of samples. The extracts reacted with DPPH radical for a period of 30 min under low light and were then subjected to an UV-vis spectrophotometer (Femto-600 Plus) at an absorbance of 517 nm 1367 Antioxidant and photoprotective… GIMENIS, J. M. et al. Biosci. J., Uberlândia, v. 34, n. 5, p. 1365-1378, Sept./Oct. 2018 (BRAND-WILLIANS et al.,1995; DI MAMBRO; FONSECA, 2005). The calculation of antioxidant activity was performed according to the formula: Antioxidant Activity (%) = [(control- sample)/control] x100. The antioxidant activity of the extract or fraction can be seen by the degree of discoloration of the reagent after the 30 min required for the reaction to attain a plateau. Gallic acid (Vetec-Fine Chemicals, Brazil) was used as a standard. Table 1. Qualitative and quantitative composition of the formulations for photoprotective efficacy Compositiona Concentration % (w/w) Formulation codes Mo-0 Mo-1 Mo-2 Ammonium Acryloyldimethyltaurate / VP Copolymer 3 3 3 Propylene Glycol 5 5 5 Disodium EDTA 0.15 0.15 0.15 BHT 0.05 0.05 0.05 Phenoxyethanol (and) methylparaben (and) ethylparaben (and) butylparaben (and) propylparaben (and) isobutylparaben 0.7 0.7 0.7 Butyl Methoxydibenzoylmethane 3 - 3 Ethylhexyl PABA 8 - 8 Ethylhexyl methoxycinnamate 7.5 - 7.5 Hydroethanolic extract of M. oleifera - 2 2 Water b b b aQualitative composition was reported in accordance with INCI (International Nomenclature of Cosmetic Ingredient), bEnough to complete 100%. Ferric reducing antioxidant power (FRAP) The FRAP reagent was prepared as required by mixing 25 mL acetate buffer (300 mM, pH 3.6), 2.5 mL TPTZ solution (10 mM in HCl solution (40 mM)), and 2.5 mL FeCl3 solution (20 mM). Sample (1000, 2000, 3000, 5000 and 10000µg/mL) or Trolox standard (25-500 µ M/L) 90 µ l was mixed with 270 µL of distilled water and 2.7 mL freshly prepared FRAP reagent and incubated at 37 °C by 30 min. Maximum absorbance values were reader at 595 nm. The results were expressed as micromoles of Trolox equivalents (TE) per gram of dry extract or fraction (BENZIE; STRAIN, 1996). Nitric oxide scavenging Nitric oxide (NO) scavenging assay was carried by using sodium nitroprusside (SHAHRIAR ET AL.,2013). This can be determined by the use of the Griess reaction. 2 mL of 10 mM sodium nitroprusside in 0.5 mL phosphate buffer saline PBS (pH 7.4) was mixed with 0.5 mL of extracts at various concentrations (3000, 5000 and 10000 µ g mL-1) and incubated at 37°C for 150 minutes. After incubation the mixture was maintained 60 minutes at room temperature. The absorbance was measured at 540 nm. The percentage of inhibition was calculated according to the following equation concerning sodium nitrite calibration curve (y=0.0052x+0.0349). Lipid peroxidation assay (TBARS) A modified thiobarbituric acid reactive species (TBARS) assay using egg yolk homogenates as lipid-rich media was performed to measure the formation of lipid peroxide (AWAH et al.,2010; RUBERTO; BARATTA, 2000). Malondialdehyde (MDA), a secondary end-product of the oxidation of polyunsaturated fatty acids, reacts with two molecules of thiobarbituric acid (TBA) yielding a pinkish red chromogenic with maximum absorption at 532 nm. Egg homogenate (500 µ l) of 10%, v/v in phosphate buffered saline (pH 7.4) and 100 µl of sample (3000, 5000 and 10000 µ g mL-1) were added to 1 mL of the hydrochloride 2.2''Azobis (2- amidinopropane) dihydrochloride (AAPH) (0.12M) to induce lipid peroxidation. The mixture was incubated for 30 minutes at 37ºC. After incubating and cooling at room temperature, it was added 0.5 mL of trichloroacetic acid (ATA) (15%) and 0.5 mL of TBA (0.6%). The mixture was incubated at 97ºC for 15 minutes. After incubation, centrifugation was performed with 1 mL of n-butane (2783 rpm for 10 minutes). Then, the reading of the supernatant was performed at 532 nm. Inhibition (%) of lipid peroxidation was calculated using the equation: Inhibitory activity (%) = [(AC-AS)/AC]x100 where AC is the absorbance of the control, and AS is the absorbance of the sample. 1368 Antioxidant and photoprotective… GIMENIS, J. M. et al. Biosci. J., Uberlândia, v. 34, n. 5, p. 1365-1378, Sept./Oct. 2018 Inhibition oxidative hemolysis human erythrocyte Ethics Committee of the Universidade Estadual Paulista - UNESP approved all the procedures in this study protocol and all subjects involved gave a written informed consent (no 833.386, approved in 16/09/2014). For this study, blood was obtained from healthy donors who were non-smokers and not receiving any pharmacological treatment and collected in tubes containing EDTA anticoagulant. Every experiment was performed daily with the erythrocytes that came from the same donors. Anticoagulated blood was centrifuged during 5 min at 2500 rpm, plasma and the white cell layer (buffy coat) were discarded after and the remaining erythrocytes were washed three times with phosphate buffered saline (PBS). An erythrocyte suspension was prepared at 10% concentration in PBS. AAPH (2,2′-azobis (2- amidinopropane) hydrochloride solution was prepared using the same buffer at the moment of its use, and also kept away from the light. The extract was dissolved in 50% stock-solution of propylene glycol, and later diluted in PBS at the time of use. The incubation of erythrocyte suspension with AAPH in the presence or absence of extracts at different concentrations was performed in a 37ºC water-bath with a gentle shaking motion and in the absence of light for 6h. Positive (100% hemolysis) and negative controls were incubated with and without AAPH, respectively, included in the assay to examine a protective effect of extracts. Aliquots were removed every one hour, transferred to eppendorf tubes and centrifuged at 2500 rpm for 10 minutes and then was performed spectrophotometric reading of supernatant. Hemolysis was determined by reading the absorbance of hemoglobin at 540 nm. The test samples were done in triplicate for statistical analyzes and percentage hemolysis was calculated by the following equation: % Hemolysis=100.[(AS- ANC)/(APC-ANC)] where AS is the absorbance of the sample, ANC is the absorbance of the control negative and APC is the absorbance of the control positive. In vitro photoprotective efficacy Photoprotective efficacy was performed in vitro by reflectance spectrophotometry with an integrated sphere (RSIS) (UV-2000S Ultraviolet Transmittance Analyzer, Labsphere, USA). Sun Protection Factor (SPF) and critical wavelength were calculated. Samples were weighed and uniformly applied with a glove-coated finger on the rough side of PMMA (polymethyl methacrylate) plates, at a ratio of 1.3 mg/cm2. The samples were then allowed to dry at room temperature for 20 min, protected from light. The analyses were carried out in replicates of three, and nine different points per plate were measured for each sample (VELASCO et al.,2008; COLIPA GUIDELINES, 2011; WANG AND LIM, 2011). In vitro photostability assay After the in vitro photoprotection assay, sample plates with the sunscreens were irradiated for one hour using a solar simulator. Solar simulator emission was maintained at 580.08 W/m2, corresponding to a UV irradiance of 55 W/m2 (irradiation dose, 198 kJ/m2). SPF and critical wavelength parameters were analyzed post- irradiation and compared with the pre-irradiation results. The analyses were prepared in replicates of three, and nine different points per plate were measured for each sample (VELASCO et al.,2008; SCALIA; MEZZENA, 2010; COLIPA GUIDELINES, 2011; WANG; LIM, 2011). Phytochemical screening Total phenols and flavonoids quantification The quantification of total phenols and flavonoids was performed with the extract diluted in ethanol at concentrations of 100, 250, 500, 1000, 1500 and 2000µg.mL-1. The Folin-Ciocalteu method was performed to determine total phenols (SINGLETON; ROSSI, 1965). For each 0.5 mL of extract at different concentrations it was added 5mL of distilled water and 0.25 mL of Folin-Ciocalteu reagent. After 3 minutes, 1mL of saturated Na2CO3 solution at 10% was added and the mixture was stored for 1 hour. The absorbance was measured at 725nm using a UV-Vis spectrophotometer (model: SP220, Biospectro, Brazil). All the tests were performed in triplicate and the results were expressed in mg of gallic acid per gram of extract. For the extract’s total flavonoids quantification the determination by UV-Vis spectrophotometer was performed and the samples were prepared based on flavonoids complexation with AlCl3 (ZHISHEN et al.,1999). An aliquot of 250 µ L of each different concentration of extract was mixed with 1.25 mL of distilled water and 75 µ L of NaNO2 solution at 5%. After 6 minutes, 150 µ L of AlCl3/H2O solution at 10% was added. After 5 minutes, 0.5 mL of NaOH 1M solution was added and then the total volume was completed by adding 2.5 mL of distilled water. The samples were shaken in a vortex mixer and the absorbance was measured at 510 nm. All the tests were performed in triplicate 1369 Antioxidant and photoprotective… GIMENIS, J. M. et al. Biosci. J., Uberlândia, v. 34, n. 5, p. 1365-1378, Sept./Oct. 2018 and the results were expressed in mg of rutin per gram of extract. The total phenolic and flavonoid analyzes are generic quantitative tests for the determination of these compounds, which represent the presence or absence of these compounds in the evaluated extracts. This analysis is not directly related to the antioxidant activity of the extracts, therefore, the determination of total phenolics and flavonoids is correlated with the compounds present in the extracts, forming a preliminary phytochemical profile. Analysis by high performance liquid chromatography (HPLC-PDA) With aid of PDA detector performing scan in the spectral range of 200-600nm, spectra in UV region for eluted peaks were obtained. Peaks with absorption bands typical of flavonoids (Figure 1) which are recognized for presenting the Band II, with maximum wavelength in spectral range of 250- 280nm, assigned to A-ring and Band I, with maximum length in spectral range of 300-320nm, assigned to B-ring, presenting higher incidence of molecules from group of flavones and flavonoids. Polyphenol analysis by HPLC / PDA The stock solutions of leaves and flowers were diluted in the proportions of 1:50 (v / v) to 1:20 (v / v) methanol, respectively. The system used was the DionexUltimate 3000 HPLC (Dionex, Idstein, Germany) coupled with a photodiode detector 3000 and the Ultimate Chormeleon software to quantify and qualify the phenolic components. A column (Acclaim® 120) was used, C18 5µm 120A (4.6 mm x 250 mm) for separation of components. The column was maintained at 40°C throughout the analysis and detection was performed at three wavelengths (225, 280, 300 and 340 nm). The sample injection volume was 10 uL. The mobile phase (A) was composed of acidified water with 1% phosphoric acid and the step (B) of methanol. The identification and quantification of components were performed by comparison of retention time and spectrum of purified standards (Sigma-Aldrich, Brazil) (ESCARPA; GONZÁLEZ, 2001). Statistical analysis The data are expressed as the mean±SD by measuring three independent replicates. Analysis of variance using one-way ANOVA followed by Tukey’s test was performed to test the significance of differences between means obtained among the treatments at the α≤0.05 level of significance using BioEstat software version 5.0. RESULTS Antioxidant activity Table 2 shows the results obtained with the in vitro tests for antioxidant activity and determination of phenols and flavonoids of the hydroethanolic and ethanolic extracts prepared from dried leaves and flowers of M. oleifera and fresh dry and aqueous extracts of M. oleifera’s leaves and flowers. The hydroethanolic and ethanolic extracts demonstrated dose-dependent activity at the highest concentration (10 mg/mL) to the DPPH test. The hydroethanolic extracts of leaves and flowers in the concentration of 5 to 10 mg / mL did not differ significantly from the standard Gallic acid. Similarly, in the FRAP test was observed that the higher values were obtained for the concentration of 10 mg/mL of hydroethanolic and ethanolic extracts of leaves and flowers. In determining the content of phenolics and total flavonoids for all extracts evaluated, there was no significant difference in the concentrations tested. However the ethanol extracts of leaf and flower at a concentration of 3 mg/mL showed the highest concentration of phenols and flavonoids. For fresh dry and aqueous extracts of M. oleifera’s leaves and flowers, it was found that the antioxidant activity behaved in dose dependence, and the highest observed value was 100.0% for the concentration, added to the fact that they did not differ statistically from control Gallic acid for the DPPH test. In determining the content of phenolics and total flavonoids all evaluated extracts also showed higher levels in the concentration of 100.0%. 1370 Antioxidant and photoprotective… GIMENIS, J. M. et al. Biosci. J., Uberlândia, v. 34, n. 5, p. 1365-1378, Sept./Oct. 2018 Table 2. Antioxidant activity through DPPH radical scavenging method (%) and iron chelate (FRAP in mM) and determination of phenols (FT) and total flavonoids (FlT) in ethanolic (Eth) and hydroethanolic (HEth) extracts of dried leaves and flowers and aqueous extract of fresh leaves and flowers of Moringa oleifera. Extracts Concentration (µ g/mL) Leaves (dried) of M. oleifera Flowers (dried) of M. oleifera DPPH FRAPa FTb FlTc DPPH FRAPa FTb FlTc Alcoolic HEth 3000 23.66±0.65a 26.14±0.49a 16.49±1.53a 171.54±4.72a 09.06±6.01a 25.09±0.65a 18.06±1.60a 32.84±7.22a 5000 39.43±2.61b 24.57±0.29a 18.61±0.16a 191.88±8.22a 20.01±1.09b 23.80±0.12a 13.11±0.00a 103.55±6.31 b 10000 74.33±0.58c 16.12±0.21b 16.16±1.33a 180.11±6.31a 46.67±2.58c 15.56±0.00a 11.61±0.80a 89.31±4.20c Eth 3000 18.25±5.11a 98.63±2.49c 25.47±0.56a 670.72±33.13b 41.24±0.88c 16.26±1.13a 32.14±1.21b 575.36±4.25 d 5000 42.63±2.54b 28.13±0.58a 19.00±0.35a 509.11±4.20c 61.40±2.66d 19.55±0.24a 28.72±1.13b 536.88±2.54 d 10000 56.47±5.98b 15.99±0.06b 16.52±0.39a 360.66±2.20d 63.31±2.07d 14.34±0.34a 21.05±0.36b 373.44±4.82e Aqueous Dried 10,0 7.76±1.65a 16.22±10.04a 23.64±1.73a 32.05±7.56a 13.3±2.7a - 1.69±0.48a 15.11±0.48a 50,0 32.03±1.17b 143.25±3.34b 90.86±1.27b 81.49±8.78b 43.55±5.31b 53.81±2.24a 38.64±3.75b 17.89±1.27a 100,0 82.04±1.69c 155.29±1.60b 157.8±4.74b 152.33±7.26c 80.91±1.99c 160.6±1.92b 83.64±1.92c 24.00±1.66a Fresh 10,0 29.22±3.59b 66.22±2.0c 16.97±0.96a - 13.24±2.63a - 16.97±0.96d - 50,0 82.35±1.27c 148.81±2.74b 64.47±2.09c 129.00±0.83c 62.63±3.43b 117.33±4.0c 64.47±2.09c 45.11±3.94b 100,0 74.62±10.48c 153.81±0.32b 107.8±6.73b 241.22±3.15d 93.23±1.14c 146.9±1.69b 107.8±6.73e 113.72±2.68c Gallic Acid 80(µ g/mL) 74.33±0.52c 74.33±0.52c Values are expressed as mean ±SE. Same letters within the same column indicate no significant differences among samples by Tukey test (α≤0.05).aPercentage values of DPPH radical scavenging activity; bValues of total phenol levels (mg of gallic acid equivalent/g of extract); cTotal flavonoid levels in quercetin equivalent per mg/g of extract 1371 Antioxidant and photoprotective… GIMENIS, J. M. et al. Biosci. J., Uberlândia, v. 34, n. 5, p. 1365-1378, Sept./Oct. 2018 Table 3 shows the results of antioxidant activity by the method of TBARS and the kidnapping of NO radical to hydroethanolic extract of dry leaf and aqueous extract of fresh leaves and flowers. The highest activities were observed at the concentration of 10 mg/mL, to hydroethanolic extract, but there was no significant difference between the analyzed concentrations. The results of these tests for the aqueous extract of fresh leaves and flowers were observed at the concentration of 100%. As for the aqueous extract of fresh flowers the highest percentage of anti-lipid peroxidation activity was observed in the concentration of 50% and also, the concentration of 100% showed greater sequestration of NO ion. The TBARS and NO tests were performed for the hydroethanolic extract of the leaves because it was the extract that presented better results in the previous tests. Table 3. Inhibition of lipid peroxidation (TBARS) and NO radical scavenging at different concentration of hydroethanolic extract and fresh leaves and flowers aqueous extract of Moringa oleifera. Extract Concentrations (µ g mL-1) Leaves TBARS- Inhibitory activity (%) % NO radical scavenging Alcoolic HEth 3000 64.36±02.55a 75.59±0.51a 5000 54.40±10.57a 76.55±1.67a 10000 56.13±21.60a 77.58±0.22a Aqueous Fresh Concentration (%) Leaves Flowers TBARS- Inhibitory activity (%) % NO radical scavenging TBARS- Inhibitory activity (%) % NO radical scavenging 10% 12.59±0.35a 74.76±3.31a 15.25±0.76a 74.18±2.80a 50% 28.12±0.64b 74.44±2.08a 27.70±0.54a 73.03±3.86a 100% 52.57±1.05c 75.01±1.99a 22.68±1.37a 74.35±3.11a Values are expressed as mean ±SE. Same letters within the same column indicate no significant differences among samples by Tukey test (α≤0.05) The analysis of the antioxidant activity by hemolytic method is shown in Figure 1. It shows that the hydroethanolic leaf extract has the potential to inhibit the hemolytic action AAPH for all concentrations tested, describing an action dependent concentration profile. The concentration of 1mg/mL showed the highest percentage inhibition of hemolysis hour for the third and fourth hour. Figure 1. Antioxidant activity through hemolysis of Moringa oleifera’s dried leaves hydroethanolic extract during 6 hours of incubation and measured in spectrophotometer at 540nm. 1372 Antioxidant and photoprotective… GIMENIS, J. M. et al. Biosci. J., Uberlândia, v. 34, n. 5, p. 1365-1378, Sept./Oct. 2018 Evaluation of photoprotective potential Determination of the maximum wavelength and the maximum absorbance of the hydroethanolic extract of M. oleifera’s leaves. A test was performed for the scanning spectrum of the hydroethanolic extract of the leaves. The estimated extract had a maximum absorbance between 1.6 and 2.0 in lengths from 290 to 400nm, being greater than the spectrophotometric profile described by the positive control (OMC - octyl methoxycinnamate) that presented maximum absorbance between 1.2 and 1.6. In vitro determination of hydroethanolic extract of M. oleifera’s leaves photoprotective efficacy Figure 2-A illustrates the spectrophotometric profiles of the formulations with hydroethanolic extract of M. oleifera leaves without addition of UV filters (Mo-1) and containing the UV filters (Mo-2) compared to control (Mo-0 - no addition of extract and adding sunscreen). The Mo-1 sample showed no photoprotective effect, as expected since it is a UV filter-free formulation. It was also observed a synergistic effect of the combination of M. oleifera extract with the associated sun filter (Mo-2) compared preparation with only the chemical sunscreen (Mo-0). Figure 2-B illustrates the absorption spectrum profiles of formulations with hydroethanolic extract of M. oleifera leaves (Mo-2 with sunscreen after 30 and 60 minutes) and control (Mo-0 without extract and with added sunscreen after 30 and 60 minutes) to determine the photostability. It was observed that the formulation with M. oleifera and adding UV filters, contributed to the stability of the formulation after 30 and 60 minutes of irradiation compared to the same formulation without the M. oleifera. Determination of phenolic components by HPLC-PDA In quantification and identification of polyphenolic components present in the extracts of the leaves and flowers of M. oleifera using HPLC / PDA it was possible to observe the presence of four components to the leaves: Chlorogenic Acid (238.95 µ g/g), Gallic Acid (9.974 µ g/g), Isoorientin (3,52 µ g/g) and Rutin (1358,04 µ g/g), and six for the flowers - Trans-ferulic Acid (44.42 µ g/g), p- coumaric Acid (164.44 µ g/g), Isoorientin (1,13 µ g/g), Vitexin (1,88 µ g/g), Orientin (0,66 µ g/g) and Rutin (901,78 µ g/g). In the leaves was observed highest concentration of rutin and chlorogenic acid components and for the most abundant flowers were rutin and p-coumaric acid. A B Figure 2. A - Absorbance spectra profiles of formulations containing M. oleifera‘s leaves hydroethanolic extract (Mo-1 without sunscreen and Mo-2 with sunscreen) and control (Mo-0 without extract and with sunscreen) for analysis of photoprotective activity. B - Absorbance spectra profiles of formulations containing M. oleifera‘s leaves hydroethanolic extract (Mo-2 with sunscreen after 30 and 60 minutes) and control (Mo-0 without extract and with sunscreen after 30 and 60 minutes) for photostability analysis. 1373 Antioxidant and photoprotective… GIMENIS, J. M. et al. Biosci. J., Uberlândia, v. 34, n. 5, p. 1365-1378, Sept./Oct. 2018 DISCUSSION Currently, different studies about the use of Moringa oleifera species, both as food and as preventive and treatment against different diseases, have shown that this species presents several phenolic components with nutritional and medicinal properties with significant impact in both human and animal health (GHASI et al.,2000; WATERMAN et al.,2014). In this study, we demonstrated that M. oleifera represents an important source of antioxidant components present within its leaves and flowers, regardless of how the extract is obtained, when evaluated through DPPH radical scavenging method and the iron chelate effect (FRAP) (SINGH et al.,2009; VERMA et al.,2009; MOYO et al.,2012; ALHAKMANI et al.,2013; RODRÍGUEZ-PÉREZ et al.,2015). However, these studies evaluated the extracts and antioxidant components from vegetable parts (leaves, flowers, fruits and seeds) separately and with unique acquisition methodology. Thus, the present study differs, since it is based on the evaluation of different vegetable parts (leaves and flowers) and acquisition methodologies of the extracts and consequently antioxidant components, mainly on the highest evaluated concentration (10 mg/mL). Given the proven antioxidant potential of M. oleifera’s extracts, this study determined the phenolic and total flavonoid contents. The results were according with previous experiments carried, in which they have highlighted the presence of different phenolic and flavonoid components as constituents of the analyzed extracts (ATAWODI et al.,2010; SREELATHA, S. AND PADMA, 2011; COPPIN et al.,2013; JAISWAL et al.,2013). In addition to the antioxidant evaluations carried out in this study, we did the analysis of the extracts with highest antioxidant activity, about lipid peroxidation, through TBARS method and Nitric Oxide radical scavenging assay. So it was possible to observe a higher activity in the extracts from the leaves of M. oleifera, and these results are according with the findings in the literature (MOYO et al.,2012). As regards the assessment of the antioxidant activity through the hemolithic method, the hydroethanolic extract from M. oleifera‘s leaves has presented a protective effect against AAPH inducted hemolysis during the whole incubation period. These results are according with the studies in the literature. The concentration of 500µ inhibited less than the concentration of 250µ. In the incubation and centrifugation processes there may be variations between the concentration of hemoglobin present in the aliquot. In this way we will have differences in the percentage of hemolysis (YANG et al.,2006; PHRUEKSAN et al.,2014). Given the different evidence of antioxidant activity found on M. oleifera’s extracts though this study, we carried out for the first time assessment of M. oleífera‘s photoprotective potential, since the chronic or repeated exposure to ultraviolet radiation promotes photoaging and photocarcinogenesis (GUINEA et al.,2012). We observed that M. oleifera’s extracts has photoprotective potential, once they have presented spectral absortion in the range of UV (290-400nm). Besides that, a synergistic effect was observed in the hydroethanolic extract from the leaves in association with chemical UV filters, also increasing photostability of the formulations evaluated in this study. The correlation between antioxidant activity and photoprotective effect must be considered, since studies demonstrated that UVB radiation generates reactive oxygen species (ROS), reacting with photosensitive molecules resulting in an imbalance between the ROS and promotes damage to molecular structures such as DNA, proteins and lipids (OBERLEY 2002; MUKHTAR 2003; PETROVA et al.,2011). In addition to these results, the extracts from M. oleifera‘s leaves and flowers were also assessed through chromatography (HPLC-PAD) and the calibration curves were set with different patterns of phenolic and flavonoid components. Analyses showed different phenolic classes present in the leaves, which were not found in the flowers, as well these presented unique phenolic classes in their composition. However, the rutin quantification, as major component in both extracts, was observed. This finding may be directly correlated with the antioxidant and photoprotective activities highlighted in this study, because experiments carried has demonstrated that the rutin flavonoid presents such potentials and activities (OLIVEIRA et al.,2015; OLIVEIRA et al.,2016). CONCLUSION M. oleifera has a potential to be used as source of components with antioxidant and photoprotective activities, as well as, a photostabilizing adjuvant for sunscreen systems, mainly due to the presence of phenolic components, and among them, the flavonoids. 1374 Antioxidant and photoprotective… GIMENIS, J. M. et al. Biosci. J., Uberlândia, v. 34, n. 5, p. 1365-1378, Sept./Oct. 2018 ACKNOWLEDGMENTS The authors thank UNESP and CAPES (National Council for the Improvement of Higher Education) for financial support. RESUMO: Moringa oleifera Lam. (Moringaceae) é uma planta com várias atividades biológicas e propriedades terapêuticas. No entanto, o conhecimento completo sobre seus efeitos farmacológicos, biológicos e ecológicos, e sobre os componentes ativos presentes em cada parte vegetal não são ainda completamente elucidados. Este estudo teve como objetivo avaliar as atividades antioxidantes e fotoprotetoras de diferentes extratos de folhas e flores de M. oleifera. Estas atividades foram avaliadas através de testes in vitro, método de eliminação de radicais DPPH, efeito de quelação de íons de ferro (FRAP), peroxidação lipídica (TBARS), método de eliminação de óxido nítrico e avaliação da atividade contra a peroxidação lipídica através do método hemolítico. 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