In Vitro Evaluation of the Potential Antioxidant of Bidens segetum Mart. ex Colla (Asteraceae) in Melanocyte and Melanoma Cells

Bidens segetum Martius ex Colla known as the “picão do mato”, is an herba-ceous plant that occurs in the Cerrado biome of some Brazilian states. Among the species of Bidens, we highlight B. pilosa known as “picão preto”, of which several activities are reported as antioxidant and antibacterial. Ethanolic extract from Bidens segetum (EEBs) showed antioxidant potential when analyzed by free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and antifungal activity against Cladosporium cladosporiodes and C. sphareospermum fungi. PFFR3.3 subfraction from EEBs has 81.5% of 5-O caffeoylquinic acid (5-CQA) and potential antioxidant (DPPH). However, PFFR3.3 did not de-crease superoxide anion in metastatic melanoma cells by dihydroethidium assay (DHE). PP4 subfraction is a mixture of polyacetylenes that has antifungal (Cladosporium) and antioxidant activity, since reduced superoxide anion amount in melanoma cells after 5 min of treatment. However, no dose-response and time-response curve were observed, not even with the authentic standard (5-CQA). Complementary chemical studies will be performed to confirm the polyacetylenes and 5-CQA structures present in the EEBs from B. segetum and new methodologies should be performed to confirm the antioxidant activity of these compounds and the effects on melanocytes and melanomas.


Introduction
Bidens genus pertaining to Heliantheae tribe (Asteraceae) has approximately 240 species distributed throughout the world, mainly in North and South America. In Brazil, around 13 species are found in some biomes [1] [2] [3] [4]. The main ethnopharmacological applications of the species include the treatment of diabetes, malaria, antioxidant and antibacterial activities and the most used species are Bidens pilosa and Bidens alba [5]- [10]. Among the most common chemical constituents are reported polyacetylenes (34%), chalcones (12%), flavonoids (9%) and phenylpropanoids (9%) [11]. Polyacetylenes present in Bidens are relatively unstable long chain hydrocarbons and highly sensitive to light exposure [11]. On the other hand, polyacetylenes isolated from Bidens pilosa could modulate the differentiation of human helper T cells and prevent autoimmune diabetes in mice and demonstrating antiproliferative effect on normal and rapidly growing human transformed cell lines [12] [13]. Bidens segetum is still little studied, but tests with ethanolic extract of flowers, stems and leaves showed cytotoxic effect against cancer cells and antioxidant activity from the ethyl acetate fraction [14].
Reactive oxygen species (ROS) are produced during the body's metabolic processes [14] [15] and superoxide anion ( 2 O − ) is formed by the reduction of molecular oxygen (O 2 ) [16]. The uncontrolled production of ROS overloads the antioxidant defense mechanisms, generating oxidative stress, increasing cell proliferation, apoptosis, cytotoxicity, and aging [17] [18]. In carcinogenesis, ROS are involved in all three stages (initiation-promotion-progression) and malignant transformation of melanocytes has been recognized as associated with increased ROS production [19] [20] [21].
Melanocytes are responsible for the production of melanin pigment that colors the skin and protects it from UV exposure [22] [23]. Besides increased UV exposure leads to genetic and epigenetic alterations, it also induces a chronic inflammation and ROS accumulation, which in turn, is associated with melanocytes malignant transformation and melanoma development [24]. Melanoma is the least frequent type of skin cancer (3% to 4%) of cases, but its incidence is lethal in about 75% to 80%. The prognosis of death for this type of tumor is 6 to 9 months and the chances of cure are greater the sooner the disease is diagnosed and treatment started [25] [26] [27] [28]. Therefore, antioxidant administration could be a chemoprevention strategy. This work aims to evaluate the antioxidant potential of Bidens segetum species in decreasing superoxide anion ( 2 O − ) levels in melanoma cells and melanocytes based on dihydroethidium oxidation (DHE).     Analytical HPLC-DAD and semi-preparative HPLC-DAD Analytical scale HPLC-DAD was performed on an Agilent model 1260 chromatograph consisting of a G1316A thermostatic furnace, a G1329B automatic injector, a G1330B thermostatic sample compartment, a G1311B pump equipped with a spectrum scan detector. Ultraviolet by arrangement of 60 mm flow cell photodiodes. The stationary phase for analytical scale analyzes was carried out using a reverse phase C 18 Zorbax Eclipse plus column (4.6 × 150 mm) with 3.5 µm particle diameter and a flow rate of 1.0 mL•min −1 . The elution system employed was the gradient mode initiated and consisted of a mixture of 75% acidified H 2 O with 0.1% acetic acid and 25% acetonitrile (ACN) for 2 min, from 2 to 7 min reaching 50% of ACN, from 7 to 25 min reaching 90% ACN, from 25 to 26 min 100% ACN, which was held for one more minute.

Extraction and Bio-Guided Fractionation
Semi-preparative scale HPLC-DAD was performed on Agilent model 1200 chromatograph and a C 18 reverse phase Zorbax eclipse plus LC-18 column (25 cm × 10 mm) with 5 µm diameter particles and a flow rate of 4.0 mL•min −1 . The elution system employed was the same as that used in the analytical scale HPLC. All solvents used were HPLC grade (T. J. Baker).

Bioautographic Assay
Strains used in the antifungal assays belonged to C. sphaerospermum Penzig and C. cladosporioides (Fresen) de Vries, from the mycology collection of Instituto de Botânica, São Paulo, Brazil (CCIBt 491; CCIBt140). To obtain spore solutions for the bioautographic assay, C. sphaerospermum and C. cladosporioides were maintained on plates with potato-dextrose-agar (PDA) medium, incubated at 27˚C in the dark for 14 days. Conidial suspensions of fungi were obtained in salt and glucose solution prepared as follows: a stock solution contains 8.4 g KH 2 PO 4 ; 2.4 g Na 2 HPO 4 + 2H 2 O; 4.8 g KNO 3 ; 1.2 g MgSO 4 , 7H 2 O; 1.2 g NaCl per 1200 mL of distilled water. The solution is homogenized at 120˚C for 20 min. Just before making the conidial suspension 10 mL of a 30% aqueous solution of glucose is added per 60 ml of this solution. The conidial suspension is stored in a freezer.

Conclusion
Ethanolic extract (EEBs) from Bidens segetum leaves present in their composition glycoside polyacetylenes and phenolic compounds such as 5-O-caffeoylquinic acid (5-CQA), like other Bidens species. The 5-CQA was identified in the subfraction PFFR3.3 (81.5% purity) and showed potential antioxidant when evaluated in the DPPH assay. Treatment of melanocytes for 30 min with PFFR3.3 decreased superoxide anion amount; however, in metastatic melanoma cells superoxide anion levels increased after 15 min. PP4 subfraction is a mixture, among polyacetylenes, has antifungal (C. cladosporioides and C. shaerospermum) and antioxidant activity. PP4 reduced superoxide anion amount in metastatic melanoma cells Lu1205 after 5 min at concentrations of 1.0 μg•mL −1 . However, superoxide levels increased after 15 min. Therefore, we cannot say that PFFR3.3 subfractions have activity in inhibiting superoxide anion amount because the results of inhibition of 2 O − were not conclusive; in most of the analyzed concentrations, there is no concentration-response relationship and time of action. In vitro assays to determine inhibition of superoxide anion production in melanoma and melanocytes using caffeoylquinic acids and polyacetylenes have not been reported in the literature. Complementary chemical studies will be performed to confirm the polyacetylenes and 5-CQA structures present in B.segetum leaves and new methodologies should be performed to confirm the antioxidant activity of these compounds and the effects on melanocytes and melanomas.
S. D. Franco et al.