LAM. (SAPOTACEAE)
1
JOSÉ HELTON VASCONCELOS ARCOVERDE, 1RAIANA APOLINÁRIO DE PAULA, 1MYCHELY SHEILA MELO, 1BARBARA DE AZEVEDO RAMOS,
1
DANIEL RODRIGO DE ARAUJO, 2RAFAEL MATOS XIMENES, 1
ALEXANDRE GOMES DA SILVA, , 1THIAGO HENRIQUE NAPOLEÃO, 1
MÁRCIA VANUZA DA SILVA, 1MARIA TEREZA DOS SANTOS CORREIA, 1,2
MARIA DAS GRAÇAS CARNEIRO-DA-CUNHA*
1
Departamento de Bioquímica, CCB, Universidade Federal de Pernambuco, Cidade Universitária, 50670-420 Recife, Pernambuco, Brazil.
2
Laboratório de Biotecnologia, LIKA, Universidade Federal de Pernambuco, Cidade Universitária, 50670-901 Recife, Pernambuco, Brazil.
3
Departamento de Antibióticos, CCB, Universidade Federal de Pernambuco, Cidade Universitária, 50740-520 Recife, Pernambuco, Brazil.
(*) Corresponding author: Phone: +55.81.21268547; Fax: +55.81.21268576 E-mail address: [email protected] (M.G. Carneiro-da-Cunha)
Abstract - Bioactive compounds are found in many plants and are known to possess biological properties. Manilkara, which belongs to the family Sapotaceae, contains species that are popularly used to treat various diseases. The objective of this work was to evaluate the antioxidant, antibacterial, DNA- protective and anti-termite activities of organic extracts prepared from the leaves of M. rufula, which were extracted in an eluotropic series with the solvents chloroform, ethyl acetate and methanol. The phytochemical profile obtained from the extracts was traced using thin-layer chromatography and some compounds were observed, which were mostly phenolic derivatives, such as tannins and flavonoids. The extracts prepared with ethyl acetate and methanol showed significant antioxidant activity, in vitro, which was detected using DPPH and phosphomolybdenum methods. These extracts also
contained high concentrations of phenolic compounds and flavonoids, had DNA-protective activity, and were shown to be effective antimicrobial agents that acted against some strains of Gram-negative and Gram-positive bacteria. The three extracts also exhibited termiticide activity and eliminated 100% of the insects within 48 hours. This is the first study that shows M. rufula is a valuable plant species with biotechnological potential, making it a promising source of new biological compounds.
Keywords- bioactive compounds – antioxidant – DNA nicking - termiticide.
INTRODUCTION
Plants are a focus in many fields of study, mainly because they present diverse pharmacological activities and have been used for therapeutic purposes by many traditional societies for a long period of time (Sittiwet and Puangpronpitag 2009). Actually, studies have shown that pure natural compounds and standardized plant extracts offer numerous opportunities to develop new drugs with biotechnological potential (Aswathanarayan and Vittal 2013; Mendez et al. 2012).
Reactive oxygen species (ROS), a subproduct of the normal metabolism, have aroused great interest over the last few decades due to their involvement in pathologies. ROS can be produced in cells as a response to various factors, for example, chemical agents and radiation. Oxidative stress can promote DNA damage, inhibiting cellular functions and the peroxidation of lipids and proteins, contributing to the development of sicknesses, such as cancer, diabetes, arteriosclerosis, inflammation problems and premature aging (Dakah et al. 2014). Polyphenols found in many plants are reported to have good antioxidant properties and protect against cell damage caused by ROS. In recent years, there has been a growing interest in identifying scavengers of free radicals and antioxidants that specifically inhibit ROS actions in cellular genetic material (Girish et al. 2012).
The rising prevalence of multidrug resistance in pathogenic microorganisms and the undesirable side effects of certain antibiotics have
further increased an interest in searching for new antimicrobial drugs that originate from plants. This is because one of the various ways plants defend themselves against pathogenic microorganisms is the production of bioactive molecules (Hossain et al. 2011).
The family Sapotaceae has a tropical and subtropical distribution and includes approximately 50 genera and 1000 species of trees and shrubs. In Brazil there are 200 species within 14 genera of Sapotaceae, such as Chrysophyllum, Manilkara, Micropholis, Pouteria, Pradosiaos and Sideroxylon (Almeida-Junior 2010). Chemically, species of this family are characterized by having various classes of substances, for example, saponins, flavonoids, polyphenols, triterpenes, carotenoids, steroids, fatty acids and tannins; scientifically, these compounds have already been shown to have diverse biological activities (Barbosa-Filho et al. 2008; Kushima 2005; Montenegro et al. 2006). Some species of Manilkara, have been popularly used as medicinal plants to heal wounds and to treat various illnesses, such as inflammation, fever, postpartum hemorrhages and, upset stomachs. In vitro, species of this genus have shown antimicrobial, anti-parasitic, anti-tumor and antioxidant activity (Fernandes et al. 2011). Manilkara rufula, also known as maçaranduba, is used by some traditional communities to treat stomach problems, as an antipyretic, to heal wounds and to control worm infestations.
There is a continuous necessity to search for new species of plants that have biotechnological potential. The objective of this work was to evaluate the antioxidant, antimicrobial, DNA-protective and anti-termite activities of organic extracts prepared from the leaves of Manilkara rufula, which were extracted in an eluotropic series with the solvents chloroform, ethyl acetate and methanol.
METHODS AND MATERIALS
Collection of Plant Material. Leaves of M. rufula, free of diseases, were collected in an area of Parque Nacional do Catimbau (PARNA do Catimbau), northeastern Brazil, in January (dry season) and June (rainy season) of 2012. Voucher specimens were identified and deposited in the herbarium at the Instituto Agronômico de Pernambuco (IPA) under the numbers 86769 and
86770. The collected leaves were dried in a dryer at 37ºC for 72h. The dried material was ground and stored at 4°C until use.
Organic Extracts. The extracts, from the powder of the leaves of M. rufula, were made in a Soxhlet apparatus using organic solvents, chloroform (CHCl3), ethyl acetate (AcOEt) and methanol (MeOH), in an eluotropic series. One hundred grams of the sample was packed in muslin cloth and used for the extraction at a temperature below the boiling point of each solvent. All of the samples were submitted to reflux until saturation (24 h). After this period, the extracts were filtered through Whatman filter paper (nº 1). In all cases, the solvents were removed until the organic extracts were dry, separately concentrated using a rotary evaporator at 45°C under reduced pressure, and stored at -10 ºC. The concentrated residue was dissolved in sterilized dimethyl sulfoxide (DMSO- 1:9), methanol (PA) or NaCl (0.15 M) when necessary. The extracts were filtered with a 0.22µm filter (type GV Millipore) and used for the study
Phytochemical Screening. To determine the phytochemical compounds present in the extracts, standard reference assays were developed. The identification of flavonoid aglycone, flavonoid heterosides, cinnamic derivatives and alkaloids was made according to Wagner and Bladt (1996); mono and sesquiterpenes, triterpenes and steroids according to Harborne (1998); and condensed proanthocyanidins and leucoanthocyanidins according to Roberts et al. (1957). Assays of Antioxidant Activity
Scavenging activity of the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Antioxidant activity was determined from the reactions of scavenging the stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl) by the molecular components present in the extracts of M. rufula; the methodology was based on Blois (1958). A methanol solution of DPPH (20 mg/ml) was prepared and its concentration adjusted for an absorbance between 0.6 and 0.7 at 517nm. A 250μl aliquot of this solution was mixed with 40μl of different concentrations of the extracts (10, 25, 50, 100, 200, 300 and 400 µg /ml dissolved in methanol) and the absorbance was read after thirty minutes. Quercetin and ascorbic acid were used as reference compounds for the assay. The tests were performed in triplicate.
The radical scavenging of DPPH was calculated by the following formula: Scavenged [DPPH](%) = (Abs sample Abs control)Abs control x 100
Total Antioxidant Activity. The total antioxidant activity (TAA) was evaluated based on the method used by Pietro et al. (1999). An aliquot (100 µL) of the sample in methanol (1mg/mL) was added to 1000 µL of phosphomolybdenum (600 mM of sulfuric acid, 28 mM of sodium phosphate and 4 mM of ammonium molybdate) and incubated in a water bath (bain-marie) at 95°C for 90 minutes. The same procedure was used to prepare the control, which was a methanol and phosphomolybdenum solution. After cooling to 25°C, the absorbance of the samples was measured at 695 nm. The test was performed in triplicate and the total antioxidant activity was expressed in relation to the ascorbic acid and calculated by the following formula:
TAA%=(Aa – Ac)(Aaa – Ac) x 100
Where: Ac=Absorbance of the control, Aa=Absorbance of the sample, Aaa=Absorbance of the ascorbic acid.
Dosage of Phenolic Compounds. The total phenolic content was determined by the Folin-Ciocalteu method according to McDonald et al. (2001). The extracts were adjusted to a concentration of 1 mg/ml in methanol, and 900 µL of distilled water, 100 µL of the sample and 500 µL of the Folin-Ciocalteu reagent (1:10) were added to each sample tube. After 10 minutes in the dark, 2.5 ml of 7.5% sodium carbonate was added and left for 20 more minutes in the dark at room temperature (25 ± 2°C). After this period the absorbance of the samples, at 765nm, was read against white. A curve of the calibration pattern was prepared using gallic acid dissolved in methanol and the following linear equation was found: y=0.023x + 0.62, r²= 0.995. The concentration of total phenol in the sample was determined from this curve. The total phenol in the extract was expressed in terms of the equivalent of gallic acid (GAE mg/g of extract).
Dosage of Flavonoids. Determining the flavonoids was based on the methodology described by Woisky and Salatino (1998), with modifications. The results were expressed as quercetin equivalents (EQ) per milligram of sample. The extracts were prepared at 1mg/mL.
DNA Nicking Assay. The DNA nicking assay was performed using pBR 322 plasmid DNA. The reaction mixture contained 0.3 µL of plasmid DNA, 10 µL of Fenton’s reagent (30 mM H2O2, 50 mM ascorbic acid, and 80 mM FeCl3) followed by the addition of extracts. The final volume of the mixture was brought up to 25 µL using distilled water. The mixture was then incubated for 30 min at 37 °C and the DNA was analyzed on 1% agarose gel (prepared by dissolving 1 g of agarose in 100 mL of 0.5 x TBE Buffer) (Kaur et al. 2008). The measurement of DNA damage protection was analyzed by Total Lab Quant.
Antibacterial activity
Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). The bacteria was provided in nutrient agar (DifcoTM) by the Departamento de Antibióticos (DA) at the Universidade Federal de Pernambuco (UFPE), Brazil, and stored at 4°C. The Gram-positive strains were Staphylococcus aureus (UFPEDA-02), Micrococcus luteus (UFPEDA-100) and Bacillus subtilis (UFPEDA-86), and the Gram-negative strains were Pseudomonas aeruginosa (UFPEDA-416), Escherichia coli (UFPEDA-224) and Klebsiella pneumoniae (UFPEDA-396). The bacteria were cultivated in nutrient broth (DifcoTM) in 250 ml, shaken bottles, and incubated overnight in an orbital agitator at 100 rpm and 37°C. The cellular concentration was determined by measuring the turbidity of the suspension using calibration curves (turbidity equivalent to 0.5 on the MacFarland scale).
The susceptibility tests for determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were performed using CLSI (2011). The bacteria were cultivated for 24 hours at 37oC. The extracts were dissolved in 10% DMSO and the dilutions were prepared in microtiter plates with 96 wells containing culture medium; final concentrations varied from 0.190 to 50 mg/ml. Posteriorly, 20 µl of the bacterial suspension was inoculated in the microplate and the tests were carried out using a final volume of 200 µl. The plates were incubated at 37°C for 24 hours. Chloramphenicol was used as a positive control. Cell viability was observed using Resazurin solution (0.01%) and was indicated by a change in color. Changes from pink to purple were considered to have bacterial growth. For each extract corresponding to the lowest concentrations tested, which did not
change color after macroscopic evaluation, the MIC was determined and expressed in mg/mL. Using the results of the MIC test, the concentrations that were shown to be completely absent of bacterial growth were identified and 10 µl of each culture was transferred to Petri plates containing Mueller-Hinton agar and incubated for 24 h at 37°C to determine the MBC. The lowest sample concentration, where there was no growth on the agar surface, was defined as the MBC.
Anti-termite Activity
Insects. A colony of Nasutitermes corniger was collected from an area of Atlantic Forest located on the campus of the Universidade Federal Rural de Pernambuco, Brazil. The termite colony was selected according to overall integrity criteria. The nest was carefully removed from the trunk of a tree using a machete and then transferred to the laboratory. The colony was maintained at 28 ± 2°C (70 ± 5% relative humidity) in the dark for 6 h and subsequently used in the bioassays.
Termiticidal Assay. Termiticidal activity was evaluated by a no-choice bioassay based on the method described by Kang et al. (1990). Each experimental unit consisted of a Petri dish (90 x 15 mm, TPP-Techno Plastic Products, Trasadingen, Switzerland) with the bottom covered with filter paper. A filter paper disk (4 cm in diameter) impregnated with 200ml of sample in 0.15 M NaCl was put in each dish. Termiticidal activity was evaluated for the organic extracts made from the leaves M. rufula with chloroform, ethyl acetate and methanol (2.5, 5 and 10 mg/ml of extract). For the negative controls, the filter paper was impregnated with 0.15 M NaCl. A total of 20 active termites (at a worker-to- soldier ratio of 4:1) were transferred to each dish; these were maintained at 28°C in the dark. Insect survival was evaluated daily until all insects were dead. Bioassays were carried out in four replicates for each concentration and survival rates (as percentages) were obtained for each treatment.
Statistical Analysis. Each experiment was performed at least three times and results are presented as the mean ± SD. The statistical analysis was performed using the Student’s t-test. Differences were considered significant at p< 0.05. The concentration needed for 50% inhibition (IC50) was estimated graphically by a linear regression analysis.
RESULTS
Extracts and Phytochemical Screening. The extracts obtained with the chloroform, ethyl acetate and methanol solvents yielded 3.71%, 2.35% and15.71% (p/p respectively) of dry weight in relation to the powder of the leaves (100g) used to prepare the extracts.
The phytochemical analyses using M. rufula revealed the presence of cyanogenic heterosides, phenolic compounds (flavonoids and tannins), triterpenes and steroids; alkaloids, cinnamic derivatives and saponins were not observed (Table 1).
Table 1: Phytochemical profile obtained by thin layer chromatography (TLC) of chloroform, ethyl acetate and methanol extracts prepared from the leaves of M. rufula.
Presence (+) or absence (-) of compound.
Visualization of a band with low color intensity (+), intermediate intensity (++) and high intensity (+++) using TLC.
Scavenging of the DPPH Radical. The molecule DPPH (2,2-diphenyl- picrilidrazil) is characterized as a free radical that possesses the ability to donate a free electron to an antioxidant molecule, making it stable. This transfer causes the reagent to lose its purple color (Md, et al., 2013) and the degree of discoloration indicates the antioxidant potential of the sample. In this study, the extracts of Manilkara rufula exhibited a high degree of scavenging of the DPPH radical compared to the controls used (quercetin and ascorbic acid).
Class of secondary metabolites Manilkara rufula Leaves Cyanogenic heterosides + CHCl3 AcOEt MeOH Flavonoids (aglycones) - + + Flavonoids ( heterosides) - ++ ++ Cinnamic derivatives - - - Triterpenes +++ ++ - Steroids +++ ++ -
Monoterpenes and Sesquiterpenes - - -
Alkaloids - - -
Condensed proanthocyanidins and
Of the three organic extracts tested, two (AcOEt and MeOH) decolorized the DPPH reagent at concentrations of 84.15 µg/ml and 42.93 µg/ml, respectively, and the potential to eliminate free radicals was in the following order: extract MeOH > AcOEt > CHCl3 (Table 2). These results show that Manilkara rufula is an important source of bioactive compounds with antioxidant potential.
Table 2: Concentrations (µg/ml) of the MeOH, AcOEt and CHCl3 extracts, from the leaves of M.
rufula, able to scavenge 50% of the free radicals of DPPH.
IC50 Values Confidence interval
CONTROLS