ABSTRACT
Erythrina senegalensis DC (Fabaceae) is a plant used in traditional medicine in Burkina Faso (West Africa) to cure several diseases such as malaria, rheumatism, abdominal pain, fibroma and infections which are always accompanied by oxidative stress. This study aimed to highlight the antioxidant activities in dichloromethane (DCM) and ethyl acetate (EtOAc) extracts of E. senegalensis stem bark and roots. We used the 1, 1 diphenyl-2-picryl hydrazyl (DPPH) radical scavenging test and the 12-lipoxygenase I-B inhibitory method. We previously characterized the phytochemical groups by thin layer chromatography and colored reactions in tubes. The extracts in DCM (IC50-root: 5.18 ± 0.06 and IC50-bark: 5.76 ± 0.68) showed strong scavenging activity. However, as a 12-lipoxygenase inhibition, the DCM extracts were almost inactive. The EtOAc extracts from root (IC50-LOX: 7.21 ± 2.31; IC50-DPPH: 7.27 ± 0.13) and from stem bark (IC50-LOX : 4.95 ± 1.12; IC50-DPPH : 11.4 ± 1.3) presented both the radical scavenging and the 12-lipoxygenase inhibitory effects. Polyphenols (flavonoids, tannins), steroids and terpenoids characterized in all extracts may be involved in the observed 12-lipoxygenase inhibition and radical scavenging.
Key words: E. senegalensis, antioxidant, 12-lipoxygenase I-B inhibitory.
Erythrina senegalensis DC (Fabaceae) is a plant used in traditional medicine in Burkina Faso (West Africa) to cure several diseases such as malaria, rheumatism, abdominal pain, fibroma (Nacoulma et al., 1999), skin diseases and amenorrhea in Mali (Togola et al., 2008). Aqueous stem bark extracts are effective against venereal and pulmonary infectious diseases (Iwu, 1993). The antioxidant properties of extracts from E. senegalensis are rarely assessed while phytochemicals are good antioxidants because they are able to scavenge free radicals or inhibit oxidative enzymes such as cycloxygenase and lipoxygenase. And then, phytochemicals are also less mutagenic and teratogen than the synthetic antioxydants.
Lipoxygenases (LOXs EC1.13.11.12) are nonheme iron-containing dioxygenases that catalyze the formation of corresponding hydroperoxides from polyunsaturated fatty acids such as linoleic and arachidonic acids. They are mainly called 5-, 12-, and 15-LOX based on their ability to insert molecular oxygen at the 5-, 12-, or 15-carbon atom of arachidonic acid (Wisastra and Dekker, 2014). LOX enzymes expressed in immune, epithelial, and tumor cells are an important source of reactive oxygen species (ROS) that display a variety of functions, including inflammation, skin disorder, and tumorigenesis (Mashima et al., 2015). So, implicated in the pathogenesis of inflammatory and hyperproliferative diseases, the LOXs represent potential targets for pharmacological intervention.
Free radicals increase in the body during inflammation, exercise or after exposure to exogenous sources such as pollution, smoking, certain medications and radiations (Lobo et al., 2010). An excess of oxidative stress can lead to the oxidation of lipids and proteins, which is associated with changes in their structure and functions (Lobo et al., 2010). Free radicals may oxidize and modify the DNA’s genes or the cellular regulatory proteins and lipids leading to many metabolic and cellular disturbances such as cancer, asthma, atherosclerosis, cataract and inflammatory diseases (Lobo et al., 2010).
The antioxidant system of the body, essentially enzymatic (superoxide dismutase, catalase, glutathione peroxidase) is often swamped and free radicals become dangerous for the health without an appropriate treatment. For all these reasons, it is very important to find vegetal antioxidant compounds from E. senegalensis that are able to reduce oxidative damage. The aim of this study is to characterize main phytochemical groups in the extracts of E. senegalensis and to show their free radical scavenging and 12-lipoxygenase inhibitory effects.
Plant materials
Fresh roots and stem bark of Erythrina senegalensis (Fabaceae) DC, after locating the plant (30P0641573 UTM132 7922), were collected in June 2009 from their natural habitat in Saponé, at 50 km from Ouagadougou, Burkina Faso (West Africa). The specimen was certified by Dr. Souleymane GANABA, Department of Forestry of the National Centre for Scientific Research and Technology of Ouagadougou. A voucher specimen was deposited at Burkina National Herbarium (HNBU) and attributed No. 8709.
Collected plant materials were dried at room temperature under shade to prevent the direct effect of sun. The resultant dried plant parts were individually reduced to powder with mortar and pestle, sieved and kept in a clean dried cupboard before use.
Extraction of plant materials
The extraction of phytochemicals is made by successive exhaustion with increasing polarity solvents. Three hundred grams of powder (300 g) were initially defatted with ether, dried and successively exhausted with dichloromethane and ethyl acetate. The exhaustion of the drugs by a solvent is continued until the percolating liquid becomes limpid. Each liquid obtained by filtration was freeze-dried in a rotavapor to yield a solid residue. Appropriate concentrations of the extract were made and used in experiments. Dried plant drugs powder was weighed (M). After a complete exhaustion, the dry extract obtained from the rotavapor was also weighed (m).
Extraction r (%) performance is given by the formula:
r = [m / M] × 100
Phytochemical screening
Standard screening colorimetric tests of the extract were carried out for various constituents according to Ciulei et al. (1982) and completed by thin layer chromatography. The extracts were screened for the presence of alkaloids, flavonoids, saponins, coumarins, tannins, steroids and triterpens.
DPPH (1, 1-diphenyl-2-picryl hydrazyl) free radical scavenging activity
The test was carried out according to Kim et al. (2003) with a slight modification. The reaction mixture contained test sample (extract or quercetin) in dimethyl sulfoxid and DPPH in methanol (101 µM, Sigma, Germany).The reaction mixture was incubated at 37°C for 30 min. The absorbance was measured at 520 nm. The percentage of radical scavenging activity was determined by comparison with a DMSO-containing control. Inhibitory concentration 50 (IC50) values represented the concentration of compounds to scavenge 50% of DPPH radicals. Quercetin was used as a positive control. All the chemicals used were of analytical grade (Sigma, Germany).
In vitro lipoxygenase inhibition assay
Lipoxygenase inhibiting activity was measured by the spectrometric method developed by Lyckander and Malterud (1992) and adapted according to our working conditions. Lipoxygenase (1.13.11.12) type I-B and linoleic acid were purchased from Sigma (Germany). All other chemicals were of analytical grade. 400 µL of sodium borate buffer (0.2M, pH 9.0) containing lipoxygenase (167 U/mL) and 100 µL of test compound solution were mixed and incubated for 10 min at 25°C. The reaction was then initiated by the addition of 500 µL linoleic acid (substrate) solution. This reaction led to the formation of (9Z, 11E)-(13S)-13-hydroperoxyoctadeca- 9,11-dienoate, and the change of absorbance at 234 nm for 10 min. Test compounds and the control were dissolved in methanol. Measurements of increase in absorbance at 234 nm for 30–90 s give extract inhibitory activities. All the reactions were performed in quadriplicate.
Treatment of data
All experiments were performed in quadruplicate (n = 4). The data were given as mean ± SEM. Values of 50% inhibitory concentration (IC50) were determined from the dose curves / effects obtained using the PRISM version 5.0 software.
Yields of extractions
Successive exhaustion by permeation of plant to obtain extracts masses whose catches in tests are presented in Table 1. It showed that by percolation of the plant, the masses of extracts vary according to the solvents used and according to the parts of the plant. E. senegalensis stem bark was the richer source of lipophilic extracts, more than roots. Dichloromethane gave higher yields of extraction than ethyl acetate extracts.
Colored reactions in tubes
Steroids, triterpens and coumarins were detected in all the parts of the plant. Flavonoids and tannins were found more in stem bark than in roots (Table 2).
Thin layer chromatography
TLC (Figure 1) has indicated a predominance of catechic tannins over gallic tannins in all the parts of the plant. Flavonoids were the most abundant in the stem bark extracts. Root extracts contained more phenolic acids than flavonoids. Triterpens and steroids have been characterized in all the parts of plant.
Radical scavenging activity in extracts of E. senegalensis
Extracts have shown dose-dependent effects, as in Figure 2. DCM extracts of root and stem bark have globally shown the best dose-dependent radical scavenging effect (IC50-root: 5.1 µg/mL and IC50-bark: 5.7 µg/mL).
Lipoxygenase (LOX) inhibitory activity in extracts of E. senegalensis
Extracts of E.senegalensis DC showed a dose-dependent inhibition of 12-LOX (Figure 3). EtOAc extracts were the most active (IC50: 4.95 ± 1.12). These presented inhibitory potentials far exceeding those of DCM extracts (Table 3).
Inhibitory effects of DPPH and 12 - LOX in extracts of E. senegalensis DC.
We have observed (Table 4) that stem bark extract in EtOAc presented the best LOX inhibitory effect (IC50-LOX: 4.95 ± 1.12) and a moderate radical-scavenging effect (IC50-DPPH: 11.4 ± 1.3). However, root extract in EtOAc was as well radical-scavenging (IC50-LOX: 7.21 ± 2.31) as inhibitory of the 12-LOX of soybean (IC50-DPPH: 7.27 ± 0.13).
Our study focused on Erythrina senegalensis DC (Fabaceae), a medicinal plant of Burkina Faso. Roots and stem bark of the plant concentrate flavonoids, tannins, coumarins, and sterols and triterpens.
DCM extracts showed powerful anti-radical (DPPH) effects without 12-LOX inhibitory effect. However, EtOAc extracts possess both types of effects with a predominance of the inhibitory effect of 12-LOX of soybean.
We show in this study that extracts of E. senegalensis DC (Fabaceae) have an antioxidant potential and provide interesting data for the research of purified phytochemicals or for the development of phytomedicines for the prevention of cancer diseases, cardiovascular diseases or chronic inflammatory diseases.
The authors have not declared any conflict of interest
This study was supported by Conférence Episcopale Italienne (CEI), Union Economique Monétaire Ouest Africaine (UEMOA) for FS, CG, JBN, JS and Health’s Ministry of Burkina Faso for FS and IPG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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