Abstract: The study Alternanthera brasiliana (L.) Kuntze aims to prove its effectiveness in folk medicine. From the study of their chemical constitution were identified by LC-MS technique three glycosylated flavones than were called 2 ”-O-ramnosylvitexin; 4 ’,5,7-trimethoxy-2 ”-O-ramnosylvitexin and Ligustroflavone. The tests showed high antioxidant capacity of the specie and in small concentrations showed too high levels of photoprotection. In addition its high capacity for photoprotection also paves its use in cosmetology. Alternanthera brasiliana is a species extremely promising with all parameters to become a herbal medicine.
INTRODUCTION
Alternanthera brasiliana (L.) Kuntze belongs to the family Amaranthaceae and is popularly known in Brazil as "Penicillin" and "Terramycin", a herbaceus plant, is used against inflammation, cough and diarrhoea in Brazilian popular medicine (Barua et al., 2013).
Some studies were conducted with Alternanthera brasiliana (L.) Kuntze, such as its use as a food supplement (Delaporte et al., 2005). They also performed in vitro studies with a view to pigment production (Silva et al., 2005). As for the evaluation of pharmacological activities are few studies as cytotoxic activity by Nihei and Dias (2001) and Lagrota et al. (1994) to give an account of showed anti-HSV activity and one fraction showed activity on DNA synthesis of infected cells. In other studies, the plant species showed activity against HIV (Silva et al., 2005), antinociceptive (Souza et al., 1998), antimicrobial (Brochado et al., 2003) and the first phytochemical study with the objective of isolating secondary metabolites was performed in 2003, in which phytochemicals and secondary metabolites were isolated and identified six O-glycosylated flavonoids (Brochado et al., 2003).
In recent years, a substantial amount of evidence has shown the key role of free radicals and other oxidants as largely responsible for aging and degenerative diseases associated with aging such as cancer, cardiovascular disease, cataracts, immune system decline and brain dysfunction (Sousa et al., 2007).
In search of biologically active molecules and interest in the antioxidant activity of plant extracts, it was decided to conduct this study in order to evaluate the antioxidant and photoprotective Alternanthera brasiliana (L.) Kuntze.
MATERIALS AND METHODS
Plant material and preparation of extracts: The aerial parts of Alternanthera brasiliana (L.) Kuntze were collected in August 2011 in the city of Petrolina (9.37714°S The coordinates 40.528837°Altitude 391 m), state of Pernambuco, Northeastern Brazil, a region of arid climatic conditions involved for months with strong sunshine and low rainfall. A voucher specimen (19072) is deposited in the Herbarium of the São Francisco (HVASF), Federal University of São Francisco Valley.
The aerial parts of Alternanthera brasiliana (L.) Kuntze dried and pulverized (555 g) were subjected to maceration with 95% EtOH for 72 h. The solution was filtered and concentrated on rotaevaporator to 50°C, yielding 101.70 g of ethanol extract. To evaluate the activities, the ethanol extract was suspended in MeOH: H2O (3:7) and partitioned with hexane, chloroform (CHCl3) and ethyl acetate (AcOEt), in increasing order of polarity to obtain the respective phases which yielded 0.92, 0.95 and 0.35 g, respectively.
HPLC-UV/DAD/ESI+MS profile: The HPLC analyses were conducted for Analytic Central-IQUSP, using a RP-18 column (phenomenex luna, 5 μm, 250/4.6 mm), formic acid 0.1% in deuterated water, such as solvent A and methanol such as solvent B elution gradient mode: 30% of B (15 min), 30-40% of B (15 min), 40-60% of B (10 min), 60-100% of B (5 min) with flow of 1 mL min-1. The UV-DAD detector was set record between 290-320 nm. The extracts and fractions of A. brasiliana were diluted in methanol (with standard Merck). In the firgerprinting ESI-MS analysis, the general conditions were: Source temperature of 40°C, capillary voltage of 4.0 kV in the positive ion mode ESI(+)-MS. Structural analysis of single ions in the mass spectra as from A. brasiliana was performed by ESI-MS/MS. The compounds were identified by comparison of their ESI-MS/MS fragmentation spectra with the literature data.
Total phenolic determination: Total phenolic contents were assayed using the Folin-Ciocalteu reagent, it is based on the method reported by Slinkard and Singleton (1977) and only the volumes have been reduced (Almeida et al., 2011). Total phenolic contents of the extracts (three replicates per treatment) were expressed as milligram gallic acid equivalents per gram (mg GAE/g) through the calibration curve with gallic acid. The calibration curve range was 50-1000 mg L-1 (R2 = 0.9938). All samples were performed in triplicates.
Total flavonoid determination: Total flavonoid contents were determined by using a colorimetric method described previously (Dewanto et al., 2002). The results were expressed as milligram of catechin equivalents per gram of extracts (mg CE/g) through the calibration curve with catechin (R2 = 0.9948). The calibration curve range was 50-1000 mg L-1.
DPPH free-radical scavenging activity: The free radical scavenging activity was measured using the 2,2-diphenyl-1-picrylhydrazil (DPPH) assay (De Paola, 2001; Falcon et al., 2006). The absorbance values were measured at 518 nm and converted into the percentage Antioxidant Activity (AA) using the following equation:
Ethanol (1.0 mL) plus plant extracts solutions (2.5 mL) were used as a blank. DPPH solution (1.0 mL) plus ethanol (2.5 mL) was used as a negative control. The positive controls (ascorbic acid, BHA and BHT) were those using the standard solutions. Assays were carried out in triplicate. The IC50 values were calculated by linear regression using by GraphPad Prism 5.0 program.
β-carotene bleaching test: The β-carotene bleaching method is based on the loss of the yellow colour of β-carotene due to its reaction with radicals formed by linoleic acid oxidation in an emulsion (Wannes et al., 2010). Ascorbic acid, BHA and BHT were used as positive control. In the negative control, the extracts were substituted with an equal volume of ethanol. The antioxidant activity (%) was evaluated in terms of the bleaching of the β-carotene using the following equation:
where, A0 is the initial absorbance and At is the final absorbance measured for the test sample, A00 is the initial absorbance and At0 is the final absorbance measured for the negative control (blank). The results are expressed as percentage of antioxidant activity (AA %). Tests were carried out in triplicate.
Determination of the maximum absorption wavelength and Sun Protection Factor (SPF) in vitro: For determining of the maximum absorption wavelength (λmax), the dried extracts were diluted in absolute ethanol, obtaining concentrations of 5, 25, 50 and 100 mg L-1. Subsequently, was performed spectrophotometric scanning at wavelengths between 260-400 nm, with intervals of 5 nm. The readings were performed using 1 cm quartz cell and ethanol used as blank (Violante et al., 2009). Calculation of SPF was obtained according to the equation developed by Mansur et al. (1986).
Statistical analysis: The data obtained in the experiments was statistically analyzed using the GraphPad Prism® version 5.0 and expressed as Mean±SD. The differences at p<0.05 were considered significative.
RESULTS AND DISCUSSION
This study, identified the presence of flavonoid compounds in the extract and phases of A. brasiliana. The ESI-MS technique with direct infusion was used to characterize the presence of compounds with potent free-radical scavenging and photoprotective activities presented in this study.
The analysis by ESI-MS showed that of some constituents which absorb in the UV range in the samples Ab-ETOH and Ab-AcOEt were coincided with the mass of the flavones: (1) 2’=-O-ramnosylvitexin, (2) 4’,5,7-trimethoxy-2’=-O-ramnosylvitexin and (3) Ligustroflavone (Table 1, Fig. 1). Structural analysis of single ions in the mass spectra was made by comparison with the literature data (Zhang et al., 2010; Pieroni et al., 2000).
| Fig. 1: | Glycosilated flavones identified in the extract and phase ethyl acetate of Althernathera brasiliana |
| Table 1: | Compouds identified in the ethanolic extract (Ab-EtOH) and phase ethyl acetate (Ab-AcOEt) from Althernathera brasiliana |
| Table 2: | Total Phenolics (TP), Total Flavonoids (TF) and antioxidant activity of the dried extracts of Alternanthera brasiliana |
| EC50 values were obtained by interpolation from linear regression analysis with 95% of confidence level, EC50 is defined as the concentration sufficient to obtain 50% of a maximum effect estimate in 100%, Values are given as Mean±SD (n = 3) | |
The peaks were justified according the fragmentations the following and retention factor (Rf): (1) C27H30O14, Rf = 20.0, 601 [M+Na]+, 579 [M+H]+, 433 [M+H+-rhamnose], 271 [M+H+-rhamnose-glucose]; (2) C30H36O14, Rf = 36.0, 643 [M+Na]+, 621 [M+H]+, 475 [M+H+-rhamnose], 313 [M+H+-rhamnose-glucose]; (3) C33H40O18, Rf = 38.0, 747 [M+Na]+, 725 [M+H]+, 578 [M+H+-rhamnose], 271 [M+H+-rhamnose-glucose-rhamnose] (Fig. 1). Factors such as byosynthesis, analysis of MS/MS, range characteristic UV absorption and comparison with literature data were considered to define the structures. The similarity between the aglycone and the agreement of the data with the classes of secondary metabolites isolated from Alternanthera brasiliana and published in the literature (Facundo et al., 2012) assisted in the structural proposition.
Thus, we determined the total phenolic and flavonoid contents of the crude ethanol extract (Ab-EtOH) and phases hexane (Ab-Hex), chloroform (Ab-CHCl3), ethyl acetate (Ab-AcOEt) of Alternanthera brasiliana. As show in the Table 2, of the four samples Ab-AcOEt (ethyl acetate phase) showed the highest total phenolic content (167.43 mg g-1) and the highest total flavonoid content (87.06 mg g-1) about a half of the total phenolic content.
DPPH is one of a few stable available organic nitrogen radicals and has a UV-Vis absorption maximum at 515-518 nm. The data showed that the Ab-AcOEt sample exhibited excellent free radical scavenging activity, with a value of IC50 of 28.90±1.03 μg mL-1 (Table 2). The antioxidant activity of extracts was also evaluated too by the β-carotene-linoleic acid bleaching method. The rate of the β-carotene bleaching can be slowed down in the presence of antioxidants (Kulisic et al., 2004). In this model, the samples showed high antioxidant activity (41-81%) and the most active extract was the Ab-Hex with percentage of antioxidant activity of 81.82±15.91 (Table 2).
This specie to possess, in the chemical constituents, phenolics and flavonoids compounds that are important antioxidant substances that are obtained from most natural plants. According to these results, it was concluded that dried samples from Alternanthera brasiliana have potent antioxidant activity, mainly by scavenging abilities observed against DPPH radical. Previous studies have identified flavonoids and other phenolic compounds isolated from A. brasiliana (Facundo et al., 2012). The existing data give new information for the antioxidant potential and polyphenolic content of plant species that have been traditionally used as medicinal plant.
Table 3 shows the spectrophotometric absorption profile of the dried extracts of A. brasiliana. Analyzing the data can be observed that phase Ab-AcOEt showed characteristic absorption bands in regions UVC, UVB and UVA in a concentration dependent manner, suggesting a possible photoprotective potential. The SPF in vitro was determined by the spectrophotometric method developed by Mansur et al. (1986) using the UVB region, considered to be the region of greatest incidence during the day in which people are exposed for longer period (Dutra et al., 2004). In Table 4, it can be observed that the phase Ab-AcOEt showed higher SPF at concentration 100 mg L-1 (11.320±3.47).
The results obtained from photoprotective potential can be justified by the high content of flavonoids present mainly in Ab-AcOEt. The maximum absorption wavelength (λmax = 400 nm), with absorption bands in regions UVA, was observed for all other samples Ab-EEB, Ab-Hex and Ab-CHCl3 (Table 3). According to the literature, the content of flavonoids produced by a plant is considered an important factor for protecting plants against ultraviolet radiation (Souza et al., 2005). The ultraviolet absorption spectrum of the flavonoids shows in general two peaks of maximum absorption (240-280 and 300-550 nm) (Bobin et al., 1995).
| Table 3: | Maximum absorption wavelength (λmax) and absorption type of dried extracts of Alternanthera brasiliana |
| UVC: 200-290 nm, UVB: 290-320 nm, UVA: 320-400 nm | |
| Table 4: | Sun Protection Factor (SPF) in vitro of the dried extracts of Alternanthera brasiliana |
| Values are given as Mean±SD (n = 3) | |
The results about SPF also showed standard concentration-dependent (Table 4). Although, the test has been carried out in vitro, it was demonstrated that this method correlates well with in vivo tests, because relates the absorbance of the substance in question with the erythematogenic effect of radiation and intensity of light at specific wavelengths between 290 and 320 nm (UVB region) (Violante et al., 2009). As the population already uses the species as a healing skin protection factor adds value to herbal treatments.
CONCLUSION
The excellent results of photoprotective and antioxidant activity obtained for Alternanthera brasiliana suggest that this species, which is already widely used in folk medicine, may become an herbal.