Potential radical-scavenging activity of Pouteria caimito leaves extracts

1 Faculdade de Ciências da Saúde, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Asa Norte, Brasília, DF, Brazil. 2 Instituto de Química. Universidade de Brasilia, Campus Universitário Darcy Ribeiro, Asa Norte, Brasília, DF, Brazil. 3 Departamento de Botânica, Instituto de Ciências Biológicas, Universidade de Brasília, Campus Universitário Darcy Ribeiro, Asa Norte, Brasília, DF, Brazil. 4 Departamento de Ensino de Graduação, Instituto Federal de Educação, Ciência e Tecnologia Goiano, Campus Rio Verde, Rod. Sul Goiana, Km 01, Setor Universitário Rio Verde, GO, Brazil.


INTRODUCTION
Oxidative stress has been considered as a major cause of aging and various chronic and degenerative diseases.These conditions include inflammation, diabetes mellitus, cancer, heart disease, and neuronal degeneration, such as Alzheimer's disease and Parkinson's disease (Ng, et al., 2007).A general recommendation to reduce systemic oxidation processes involves increasing the intake of foods considered to be rich in antioxidant compounds (e.g.polyphenols and carotenoids) owing to their well-known health-giving effects.This recommendation led to .the identification of many plants having potential antioxidant activities (Katalinic, et al., 2006).Therefore, natural antioxidants from plant extracts have attracted growing interest due to consumer concern about the safety of the synthetic antioxidants in food.Pouteria caimito (Ruiz & Pav.) Radlk.belongs to the Sapotaceae family and is commonly known as abiu, caimito, or abiurana.This species is widespread in South America (Luna, 2004), and can easily be found in Brazilian home gardens.The yellow fruits are eaten in natura or used to prepare desserts and ice cream.Additionally, several parts of P. caimito have been used as folk remedies, mainly to relieve body pain and aid in wound healing (Kramer, et al., 2002).The fruit pulp is mucilaginous and is eaten to relieve coughs, bronchitis, and other pulmonary complaints, while the latex is used as a vermifuge and a purgative.Also, it is applied to abscesses (Morton and Dowling, 1987).The Tikano people (Amazon native people) use P. caimito as a treatment to facilitate childbirth (Luz, 1996;Ruiz, et al., 2011).
The ethanol extract of P. caimito leaves showed antimicrobial activity against Pseudomonas aeruginosa, Bacillus cereus, and Candida albicans (Kramer, et al., 2002).A bark extract was evaluated against Artemia salina larvae and showed no toxicity (Quignard, et al., 2003).The methanol extract from the bark was tested against Colletotrichum lindemuthianum, a fungus causing bean anthracnose; however, it did not show any antifungal activity (Pinto, et al., 2010).Peruvian rainforest inhabitants use the leaves of P. caimito to treat malaria.However, an in vitro evaluation of a crude hydro-ethanolic extract did not show any activity against Plasmodium (a chloroquine-resistant strain) or Leishmania (Kvist et al., 2006;Ruiz et al., 2011).
Thus, P. caimito is an interesting species owingto its antioxidant and biological properties and should be thoroughly examined.The objective of this work therefore was to evaluate the hexane, ethanol, and aqueous extracts from P. caimito leaves byusing pre-screening procedures for Artemia salina toxicity and in vitro antioxidant models.

Plant material
The leaves of P. caimito were collected at Pocrane, Minas Gerais, Brazil, in December 2005 and identified by Prof. S. M. Gomes.A voucher specimen was deposited at Herbarium of Universidade de Brasília (UB) (voucher number UB 27284).

Extraction procedures
The dried and powdered plant material (506.0 g) was macerated at room temperature for seven days (repeated three times) with hexane, followed by ethanol.After filtration, the solvents were removed under reduced pressure, at a temperature below 40 o C, yielding 14.0 g of hexane and 28.4 g of crude ethanol extracts (2.8% and 5.6% yield, respectively).The aqueous extract from 300.0 g of the plant material was obtained by infusion by using 3.0 L of distilled water.After filtration, the obtained infusion was lyophilized, yielding 23.2 g of aqueous extract (7.7% yield).

Isolation procedures
Hexane extract (8.5 g) was chromatographed on silica gel 60 Merck, by using a hexane: ethyl acetate: methanol gradient.The obtained fractions were monitored by thin layer chromatography (TLC), performed on precoated ALUGRAM sil G Machery-Nagel silica gel (60/0.2mm) plates, using anisaldehyde reagent to visualize the spots (Wagner and Bladt, 1996).After analysis by TLC, the obtained fractions were collected and 15 groups were formed.After purification, groups 1, 3, 5, and 6 were characterized by 1 H and 13 C NMR spectra analysis. 1H and 13 C NMR spectra were obtained using Varian (7.05 T) MercuryPlus spectrometers, operating at 300 MHz and by using CDCl 3 .

Brine shrimp lethality test (BST)
The crude extracts were tested for brine shrimp lethality.The assay was performed according to a simplified Meyer's method (Meyer et al., 1982), with modifications.Briefly, encysted eggs of the brine shrimp Artemia salina L. (Maramar) were incubated in artificial sea water at 28 °C.Samples were dissolved in 200 µL of DMSO plus 20 mL of artificial seawater.Serial dilutions (triplicate) were prepared in the same solution.Metanauplius (10 units) was added to each set of tubes containing samples and the cultures were further incubated for 24 h.Controls containing DMSO were included in each set of experiments.Potassium dichromate was used as the reference standard.LD 50 (after 24 h) was calculated by Probit analysis (Perfeito, et al., 2005).

DPPH assay
The free radical-scavenging activity of the plant extracts and standards was assessed based on Blois' method with slight modification (Yildirim, et al., 2001), using the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical.Butylated hydroxytoluene (BHT), a well-known and widely used synthetic antioxidant, was used as a positive control.DPPH (2.0 mg) was dissolved in ethanol (10 mL) to obtain a final concentration of 500 M.This solution was preserved in a dark flask at room temperature.
DPPH solution (300 L), prepared daily, was added to 300 L of each extract or BHT solution in various concentrations and placed in the dark at 25 o C.After incubation for 30 min, the absorbance of each solution was measured at 517 nm.
A negative control was prepared using 300 L of the solution, previously obtained from each extract and 300L of ethanol, to account for the pigments of the crude extracts.Absorbance values of the solutions were measured and subtracted from those obtained with DPPH.
Data were used to calculate the concentration necessary to consume one-half of the initial amounts of DPPH (EC 50 ).Each of the measurements described above was performed in at least three replicated experiments, and the results were reported as mean and standard deviation values.The results were expressed ing/mL.

Proanthocyanidin content
Proanthocyanidin quantification was performed using an adapted vanillin/sulfuric acid method (Morais, et al., 1999).Briefly, extracts were dissolved in a 10% hydroethanolic solution at 1000 g/mL concentration.An aliquot (1 mL) of these solutions was mixed with 2 mL of freshly made vanillin/sulfuric acid solution (0.2 g of vanillin in 100 mL of 30% sulfuric acid) and incubated for 15 minutes at 25 o C. The absorption was measured at 500 nm.A calibration curve was constructed with catechin as a reference standard in the 1000-62.5 g/mL concentration range.Results were expressed in mg equivalents of catechin per g of extract.

Total phenolic content
The total phenolic content (TPC) was determined by the Folin-Ciocalteu method as previously described (Morais, et al., 1999;Souza, et al., 2007), with slight adaptation to the semi-micro scale.A 5% aqueous solution of Folin-Ciocalteu reagent was prepared and 300 L was transferred to disposable semi-micro cuvettes, which contained aliquots of the extract solutions (100 to 200 L).After 5 minutes, 300 L of an aqueous solution of sodium carbonate (7.5%) was added and the final volume was adjusted to 900 L with water.
This mixture was incubated for 2 hours at 25 o C. Absorbance was measured at 760 nm.A calibration curve was constructed with gallic acid solutions in the 10-40 g/mL concentration range.
The TPC was determined by interpolation of absorbance values with the calibration curve obtained (TPC = Abs*0.0072+ 0.0029), with a correlation coefficient of 0.9956, and detection and quantification limits of 0.5 and 1.5g/mL gallic acid, respectively.Results were expressed as mg equivalents of gallic acid (GAE) per g of extract.

RESULTS AND DISCUSSION
The NMR spectra of compound 1 showed a typical steroidal profile.In 1 H NMR spectrum, in addition to other signals, a multiplet at 3.57, from hydrogen linked to C3 of the steroidal skeleton and two double doublets at 5.02 and  5.16, attributed to olefinic hydrogens,were observed.The 13 C NMR spectrum showed 29 signals (Table 1).A comparison of NMR spectra data with those reported in the literature (Villasenor et al., 1996) allowed identification of compound 1 as spinasterol.
The Artemia salina lethality test presents a correlation with an in vivo oral acute toxicity model (Parra, et al., 2001).Mathews (1995) proposed the use of the Artemia salina test as a model to evaluate compounds for their ability to protect against superoxide-mediated toxicity (Matthews, 1995).For both evaluations, an LD 50 value lower than 1 mg/mL was considered active (Meyer, et al., 1982).Based on this classification, P. caimito leaf extracts can be regarded as non-toxic, as well as presenting no protective activity against superoxide-mediated toxicity.
On the other hand, by using a DPPH radical-scavenging test, one of the shortest tests available to investigate the overall hydrogen/electron-donating activity of single antioxidants, the scavenging ability of the aqueous extract, despite being lower than that of the controls (ascorbic acid and BHT), was found to be good.Moreover, it seemed that the inhibition percentage (antioxidant activity) and total phenolics could be correlated.By comparison of the obtained values for antioxidant activity and phenolic content, it is possible to observe that as phenol content rises, radical-scavenging activity increases, as shown by other authors (Chaouche, et al., 2014;Haddouchi, et al., 2014;Karabegović, et al., 2014)

CONCLUSION
While studies of the antioxidant activity of P. caimito fruit can be found (Canuto, et al., 2010;Nascimento, et al., 2008), there are few studies involving P. caimito leaves.With regard to chemical composition, although spinasterol has been described as a common phytosterol of the Sapotaceae family (Khallouki, et al., 2003), as far we know, this is the first time this compound has been found in P. caimito.The presence of this compound can justify, at least in part, the biological activity shown by this plant species.

Table 2 :
Total phenol and proanthocyanidin content and radical scavenge potential of Pouteria caimito leaves extracts.