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Antioxidant activity and peroxidase inhibition of Amazonian plants extracts traditionally used as anti-inflammatory
© de Vargas et al. 2016
Received: 19 August 2015
Accepted: 19 February 2016
Published: 27 February 2016
The Erratum to this article has been published in BMC Complementary and Alternative Medicine 2016 16:119
The Amazon is the largest rainforest in the world and is home to a rich biodiversity of medicinal plants. Several of these plants are used by the local population for the treatment of diseases, many of those with probable anti-inflammatory effect. The aim of the present investigation was to evaluate the in vitro antioxidant and anti-peroxidases potential of the ethanol extracts of five plants from the Brazilian Amazon (Byrsonima japurensis, Calycophyllum spruceanum, Maytenus guyanensis, Passiflora nitida and Ptychopetalum olacoides).
DPPH, ABTS, superoxide anion radical, singlet oxygen and the β-carotene bleaching methods were employed for characterization of free radical scavenging activity. Also, total polyphenols were determined. Antioxidant activities were evaluated using murine fibroblast NIH3T3 cell. Inhibition of HRP and MPO were evaluated using amplex red® as susbtract.
The stem bark extracts of C. spruceanum and M. guyanensis provided the highest free radical scavenging activities. C. spruceanum exhibited IC50 = 7.5 ± 0.9, 5.0 ± 0.1, 18.2 ± 3.0 and 92.4 ± 24.8 μg/mL for DPPH•, ABTS+•, O2 -• and 1O2 assays, respectively. P. olacoides and C. spruceanum extracts also inhibited free radicals formation in the cell-based assay. At a concentration of 100 μg/mL, the extracts of C. spruceanum, B. japurensis inhibited horseradish peroxidase by 62 and 50 %, respectively. C. spruceanum, M. guyanensis, B. japurensis also inhibited myeloperoxidase in 72, 67 and 56 %, respectively.
This work supports the folk use these species that inhibited peroxidases and exhibited significant free radical scavenging and antioxidant activities what can be related to treatment of inflammation.
The enormous biodiversity of the Amazon jungle has potential as the source of new natural products. Many species that are still essentially unknown to science have been used for centuries by local populations for treating a variety of illnesses . The pursuit of new therapeutic alternatives and the development of new drugs starting from natural products have been the underlying motives for chemical and pharmacological studies. Regions having abundant flora, medicinal plants and a rich traditional knowledge, as is the case of the Amazon forest, are especially attractive given the relative low number of publications on plants from this part of the world .
Plants used in traditional medicine can provide diverse secondary metabolites with antioxidant potential most of which are phenolic compounds [3, 4] such as flavonoids and tannins. Flavonoids are of particular interest because of their antioxidant activity and ability to act as scavengers of oxygen radicals. The antioxidant capacity of these phenolic compounds is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers, or decomposing peroxides [3, 5].
Reactive oxygen species (ROS) such as singlet oxygen (1O2), super oxide anion (O2 -) hydroxyl radical (∙OH) and hydrogen peroxide (H2O2) are often generated as by-products of biological reactions or from exogenous factors. These reactive species cause oxidative damage in reactions with nearly every molecule found in living cells, including DNA . Thus, excess ROS must be eliminated by an antioxidant system. They play important roles in aging and in the pathogenesis of age related disorders such as cancer, hypertension, atherogenesis, Alzheimer’s disease and Parkinson’s disease .
In order to gain further knowledge of folk uses and traditional plants in the Amazon, the purpose of this research is to evaluate in vitro antioxidant and peroxidasic activity of ethanolic extract of the leaves and bark of Byrsonima japurensis A. Juss., Calycophyllum spruceanum (Benth.) Hook. f. ex K. Schum., Maytenus guyanensis Klotzch., Passiflora nitida Kunth. and Ptychopetalum olacoides Benth.
Ultrapure water was prepared using a Millipore Direct® Q3 (Millipore Corp., MA, U.S.A.) and was used throughout. All remaining reagents were of the highest purity available and obtained from the Sigma Chemical Company (St. Louis, MO, U.S.A.).
Medicinal plants used in this study
Indicated against various inflammatory disorders, especially in the uterus and prostate
For age spots, cuts, diabetes, eye infections, ovarian problems, scars, scrapes, skin fungi, skin parasites, skin problems, wrinkles, and wounds, and as an antioxidant and cosmetic
Used as a stimulant, tonic and muscle relaxant, to relieve arthritis, rheumatism, hemorrhoids, swollen kidney, skin rashes, skin cancer prevention.
Treatment of gastrointestinal disorders
Used as tonic neuromuscular, neurasthenia, impotence, menstrual disorders, dysentery. It also has stimulant properties of the central nervous system.
Preparation of extracts
Experimental procedures used in preparation of extracts
B. japurensis 1
Evaporator rotative and lyophilization
Evaporator rotative and lyophilization
M. guyanensis 2
Hot air (45 °C/48 h)
Cold Maceration with ultrasound bath
Evaporator rotative and lyophilization
P. nitida 1
Evaporator rotative and lyophilization
P. olacoides 1
Hot air (45 °C/48 h)
Cold Maceration with ultrasound bath
Evaporator rotative and lyophilization
Determination of total polyphenols
The test was performed using the Folin-Ciocalteu colorimetric method as described by Singleton and Rossi . Each dried plant extract was dissolved in EtOH at a concentration of 10 mg/mL. Each test solution was transferred to a test tube. Then, distilled H2O (400 μL) and Folin-Ciocalteau reagent (160 μL) were added. After homogenization in a vortex apparatus, 10.6 % aqueous Na2CO3 (4 mL) was added. After incubation for 3 min, the absorbance was measured at 715 nm in a spectrophotometer (Ultrospec 2000 UV/Vis, Pharmacia Biotech, England). The total polyphenol content was expressed in milligrams (mg) of EGA (equivalents of gallic acid) per gram (g) of extract. All analyses were performed in triplicate.
Superoxide anion radical assay
Singlet oxygen assay
β -carotene bleaching assay
The antioxidant activity of extracts was evaluated using the β-carotene-linoleic acid method described by Miller . The samples were diluted in EtOH: H2O (1:1) to a concentration of 100 μg/mL. 10 μL of extract solutions, water (blank) or BHT (butylated hydroxytoluene, standard) at the same concentraction of the extracts were added to the wells of a 96-well plate. Next, β-carotene (2.0 mg) was dissolved in CHCl3 (1.0 mL). A portion of the resulting solution (150 μL) was added to linoleic acid (50 μL), Tween 80 emulsifier mixture (200 μL) and CHCl3 (500 μL) in an Erlenmeyer flask and homogenized. After evaporation of the CHCl3 under a flow of N2, distilled H2O (25 mL) previously saturated with air was added followed by vigorous shaking for 30 min. A portion of the resulting emulsion (240 μL) was transferred to each well and the zero time absorbance was immediately measured at 470 nm using an ELISA reader Multimode Detector DTX-800 microplate reader (Beckman Coulter, CA, USA). The emulsion system was incubated for 2 h at 50 °C and the absorbance was measured every 15 min.
Antioxidant activity in cell
This assay was performed as described by Wolfe & Liu . NIH3T3 cells (fibroblast murine) were seeded at 6 × 104/well on a 96-well plate in 100 μL of growth medium (DMEM) and incubated for 24 h at 37 °C. 24 h after seeding, the medium was removed and the cells were washed with PBS. Triplicate wells were treated for 1 h with 100 μL medium with extract at a concentration of 20 μg/mL and 25 μM DCFH-DA. After the treatment period, the cells were washed with PBS (100 μL). Then 600 μM ABAP was applied to the cells in HBSS (100 μL), and the fluorescence was measured in ELISA reader Multimode Detector DTX-800 microplate reader (Beckman Coulter, CA, USA) with emission at 538 nm and excitation at 485 nm every 5 min for 1 h. Quercetin in DMSO (20 μg/mL) and saline solution were used as positive and negative controls, respectively. Each plate included triplicate control and blank wells: control wells contained cells treated with DCFH-DA and oxidant; negative control wells contained cells treated with dye and PBS without oxidant.
Peroxidase inhibition activities
Median inhibition concentrations (IC50) were obtained by plotting the graphic regression using Microcal™ Origin® software version 6.0 (Microcal Software, Inc., Northampton, USA) and presented as graphs using Excel for Windows (Microsoft, Inc., St. Louis, USA). Milliequivalence (mEq) values were obtained by division of IC50 of plant extracts by the specific IC50 standard used in each method.
Results and discussion
Total polyphenols and free radical scavenger activity (IC50 in μg/mL) of Amazonian medicinal plants
12.3 ± 0.2
8.4 ± 0.6
44.9 ± 6.4
1408 ± 113
5.0 ± 0.1
7.5 ± 0.9
18.2 ± 3.0
92.4 ± 24.8
8.2 ± 0.3
28.4 ± 0.8
35.3 ± 3.0
517 ± 70.8
38.5 ± 1.9
49.9 ± 0.1
100 ± 9.9
8.7 ± 0.3
29.7 ± 0.3
142 ± 16.5
715 ± 195
12.5 ± 0.6
10.4 ± 0.4
180 ± 14.4
4.8 ± 0.3
2.7 ± 0.3
32.9 ± 1.6
1.0 ± 0.1
1.1 ± 0.2
7.8 ± 1.2
58.4 ± 6.0
3.9 ± 0.1
5.6 ± 0.2
The DPPH scavenging assay was chosen as a primary test to be performed in initial screening of extracts due to its relatively low cost and the high stability of this reagent. Mensor et al.  consider the DPPH method fast and easy for evaluation of the presence of antioxidant potential in biological samples with the great advantage that the test is prepared and executed at room temperature which eliminates the risk of thermal degradation of substances under study. C. spruceanum and B. japurensis extracts were the most active in the DPPH test exhibiting IC50 < 10 μg/mL (Table 3).
The ABTS radical scavenging test has been described exhaustively by many different authors and in general is useful for the evaluation of antioxidant activity of substances having lipophilic or hydrophilic properties, including flavonoids e carotenoids [14, 19, 20]. In this test, the most active extracts were those of C. spruceanum, M. guyanensis and P. olacoides which exhibited IC50 < 10 μg/mL (Table 3). IC50 values were lower than values for plant extracts considered antioxidant in the literature, such as Calpurnia aurea, an effective scavenger of the ABTS radical, with percentage inhibition 100 % . Only samples exhibiting IC50 < 10 μg/mL are considered very active antioxidants as they have activity comparable to the antioxidant standards quercertin, β-carotene, ascorbic acid, gallic acid and Trolox® .
The ability of some extracts to scavenge free radicals in tests of antioxidant capacity, such as those based on DPPH and ABTS, does not mean that these extracts will perform readily where complex mechanisms are operating such as those in physiological substrates. For this reason, there is a need to verify the antioxidant effect in scavenging specific species such as superoxide anion radical (O2 •-). O2 •- is produced constantly in organisms by diverse cellular processes, such as the electron transport chain in mitochondria, in microsomes and through enzymes like xanthine oxidase and NADPH oxidase and can be increased as part of certain pathologies . Extracts of C. spruceanum, M. guyanensis and B. japurensis exhibited the highest superoxide anion radical scavenging activity as evidenced by IC50 of 18–45 μg/mL. It is noteworthy that these extracts are as active on a weight basis as several of the standards used, such as ascorbic acid .
Singlet oxygen (1ΔgO2) is the excited electronic state of molecular oxygen and is produced in general by photochemical reactions. It is reactive with a large number of biologically important molecules, including lipid membranes, through which peroxidation processes are initiated. Rubrene is a polycyclic hydrocarbon which is highly soluble in organic solvents and which auto-oxidizes in the presence of ambient light, generating 1ΔO2 from triplet oxygen (3ΔgO2), the ground state of molecular oxygen, present in air. For this reason, the assay involving rubrene is useful for the characterization of 1ΔgO2 scavenging action in the extracts tested . The test has been used for the study of photoprotective and 1ΔO2 scavenging actions of substances isolated from fruit and other parts of plants, as well as fruit and vegetable juices [14, 22, 24]. The rubrene method did not provide good results in the present study. This may be due to the interference of the highly and diversely colored extracts at the relatively low visible light wave length used (440 nm). The lowest IC50 value was for C. spruceanum extract, which was comparable to that of the DABCO standard and lower than that for quercetin (Table 3).
In general, total polyphenols levels were related to antioxidant potential revealed in the other assays for the plant extracts under study. The plant extracts exhibiting the greatest antioxidant potential were those with the highest levels of total polyphenols, namely B. japurensis, C. spruceanum and M. guyanensis. This result is similar to that found by Cai et al.  who used the ABTS method to evaluate the antioxidant capacity of more than 112 medicinal plant species used in the treatment and prevention of cancer in chinese traditional medicine. Those results indicated a strong correlation between antioxidant activity and high levels of phenolic compounds.
MPO is a member of the heme peroxidase-cyclooxygenase superfamily and is abundantly expressed in neutrophils and to a lesser extent in monocytes and certain type of macrophages. MPO participates in an innate immune defense mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity of MPO is its ability to use chloride as a co-substrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, evidence has emerged that MPO-derived oxidants contribute to tissue damage and the initiation and propagation of acute and chronic vascular inflammatory disease. The fact that circulating levels of MPO have been shown to predict risks for major adverse cardiac events and that levels of MPO-derived chlorinated compounds are specific biomarkers for disease progression, has attracted considerable interest in the development of therapeutically useful MPO inhibitors . Thus, the anti-inflammatory activities of the medicinal plants evaluated in this study may be related to inhibition of the enzyme MPO demonstrated herein. This inhibition can occur in several ways: (i) due to presence of oxidizable constituents capable of acting on the prosthetic group causing enzyme inhibition, (ii) not directly by enzyme inhibition, but by inhibition of oxidable species generated and (iii) by chelation of metals such iron or copper which are necessary for enzyme activity .
In conclusion, low IC50 values demonstrated the excellent free radical scavenging and antioxidant potential of B. japurensis, C. spruceanum and M. guyanensis. It is important to emphasize that the data presented in this study are not yet available for any of these species and reveal the great potential of the Amazon medicinal flora as a source of new bioactive, antioxidant extracts with potential therapeutic uses.
The authors recognize support provided by the Brazilian Science and Technology Ministry (MCT) through grants (CT-AMAZONIA) from the National Council for Scientific and Technological Development (CNPq) and Financier of Studies and Projects (FINEP) and a scholarship for F.V.S. (CNPq). Access to the genetic patrimony was authorized by CGEN (Authorization number 0032/2008). E.S.L is a member of National Institute of Science and Technology- INCT Redoxoma of MCT/CNPq. A.P.A.B received a grant from DCR/CNPq/FAPEAM.
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