Ocimum gratissimum L. leaf flavonoid-rich fraction suppress LPS-induced inflammatory response in RAW 264.7 macrophages and peritonitis in mice

Author’s Accepted Manuscript Ocimum gratissimum L. leaf flavonoid-rich fraction suppress LPS-induced inflammatory response in RAW 264.7 macrophages and peritonitis in mice Abayomi Mayowa Ajayi, Domingos Tabajara de Oliveira Martins, Sikiru Olaitan Balogun, Ruberlei Godinho de Oliveira, Sérgio Donizeti Ascêncio, Olusegun George Ademowo

PII: DOI: Reference:

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S0378-8741(17)30526-3 http://dx.doi.org/10.1016/j.jep.2017.04.005 JEP10815

To appear in: Journal of Ethnopharmacology Received date: 8 February 2017 Revised date: 7 April 2017 Accepted date: 7 April 2017 Cite this article as: Abayomi Mayowa Ajayi, Domingos Tabajara de Oliveira Martins, Sikiru Olaitan Balogun, Ruberlei Godinho de Oliveira, Sérgio Donizeti Ascêncio and Olusegun George Ademowo, Ocimum gratissimum L. leaf flavonoid-rich fraction suppress LPS-induced inflammatory response in RAW 264.7 macrophages and peritonitis in mice, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ocimum gratissimum L. leaf flavonoid-rich fraction suppress LPS-induced inflammatory response in RAW 264.7 macrophages and peritonitis in mice. Abayomi Mayowa Ajayia,c, Domingos Tabajara de Oliveira Martinsa, Sikiru Olaitan Baloguna,b, Ruberlei Godinho de Oliveiraa, Sérgio Donizeti Ascêncioc, Olusegun George Ademowod* a

Department of Basic Health Sciences, Faculty of Medicine, Federal University of Mato Grosso (UFMT), Av. Fernando Correa da Costa, no. 2367, Coxipó, Boa Esperança, Cuiabá 78060-900, Mato Grosso, Brazil b

Curso da Farmácia, AJES- Faculdades de Vale do Juruena. Avenida Gabriel Müller, s/n AJES Módulo I, 78320-000, Juína Mato Grosso, Brazil c

Natural Products Research Laboratory, Faculty of Medicine, Federal University of Tocantins (UFT), Av. NS15, Palmas 77020-210, Tocantins, Brazil d

Department of Pharmacology & Therapeutics, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Oyo – State, Nigeria. *

Corresponding author. Professor O.G. Ademowo, dDepartment of Pharmacology & Therapeutics, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Oyo – State, Nigeria. Mobile No: +2348023342856. [email protected] Abstract Ethnopharmacological relevance: Ocimum gratissimum L. is a herbaceous plant that has been reported in several ethnopharmacological surveys as a plant readily accessible to the communities and widely used for the treatment of inflammatory diseases. The main goal of this study was to investigate the in vitro and in vivo anti-inflammatory activity and mechanism of action of the ethylacetate fraction of O. gratissimum leaf (EAFOg) and to chemically characterize this fraction. Materials and Methods: EAFOg was obtained from a sequential methanol extract. The safety profile was evaluated on RAW 264.7 cells, using the alamarBlue® assay. Phenolic contents were determined by spectrophotometry, and metabolites quantified by high performance liquid chromatography. The anti-inflammatory activity of EAFOg and its ability to acts on leucocytes infiltration, inflammatory mediators as NO, IL-1β, TNF-α, and IL-10 in lipopolysaccharide-induced

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peritonitis in mice and LPS-stimulated RAW 264.7 macrophage were evaluated. In addition, the anti-inflammatory activity of EAFOg was also investigated in arachidonic acid-related enzymes. Results: Total phenolic and flavonoid contents of EAFOg were 139.76 ± 1.07 mg GAE/g and 109.95 ± 0.05 mg RE/g respectively. HPLC analysis revealed the presence of rutin, ellagic acid, myricetin and morin. The fraction exhibited no cytotoxic effects on the RAW 264.7 cells. The EAFOg (10, 50 and 200 mg/kg) significantly reduced (p < 0.05) neutrophils (38.8, 58.9, and 66.5%) and monocytes (38.9, 58.0 and 72.8%) in LPS-induced peritonitis. Also, EAFOg (5, 20 and 100 µg/mL) produced significant reduction in NO, IL-1β, and TNF-α in RAW 264.7 cells. However, IL-10 level was not affected by the EAFOg, and it preferentially inhibits COX-2 (IC50 = 48.86 ± 0.02 µg/mL) than COX-1 and 15-LO (IC50 > 100 µg/mL). Conclusion:

The flavonoid-rich fraction of O. gratissimum leaves demonstrated anti-inflammatory activity via mechanisms that involves inhibition of leucocytes influx, NO, IL-1β, and TNF-α in vivo and in vitro, thus supporting its therapeutic potential in slowing down inflammatory processes in chronic diseases.

Keyword: Flavonoid; phenolic compounds; inflammation; in vivo; in vitro.

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1. Introduction Inflammation is fundamentally a protective response, a non-specific, innate and complex response to vascularized tissue with the major goal to bring immune cells and molecules of host defense from the circulation and get rid of the infectious or noxious stimuli. However, inappropriately triggered or uncontrolled inflammation may cause tissue damage and thus needs pharmacological treatment to control its symptoms (Sousa et al., 2013). The inflammation comprises two phases: acute and chronic. Acute inflammation is the initial response and begins with vascular events that involve arterial vasodilatation, contraction of endothelial cells and increase in the microvasculature permeability. The acute inflammatory response is characterized by recruitment of inflammatory cells (i.e neutrophils, eosinophils, and basophils) to the inflammatory site (Maskrey et al., 2011). This acute pro-inflammatory phase induces pain, swelling, redness and heat, which are indicators that cellular destruction is taking place. The acute inflammatory response is self-limiting and normally will result in resolution and return of tissue homeostasis (Nathan and Ding, 2010). If the pro-inflammatory phase continues at a low, but chronic level that is below the perception of pain, its presence can become a driver for many chronic diseases (Sears and Ricordi, 2011). Non-resolving inflammation contributes significantly to the pathogenesis of many chronic diseases including asthma, atherosclerosis, rheumatoid arthritis, multiple sclerosis, Alzheimer’s disease and cancer (Nathan and Ding, 2010). Inflammatory diseases affect many people in the world and represent the greatest collective burden of suffering and economic cost (Jacobs et al., 2011). Although existing antiinflammatory drugs are effective for suppressing inflammation but they also have intrinsic potential for harm and are not definitive for chronic inflammation (Suleyman et al., 2007; Whitehouse, 2011). In addition, many of these drugs are single target, whereas, it has become more evident that, for complex diseases like inflammation, an interference with multiple targets is superior to targeting a single key factor regarding drug efficiency, side-effects and adverse compensatory mechanisms (Koeberle and Werz, 2014). Plants extracts and their derivatives have been shown to be multi targets in nature, due to the pleiotropic effects of natural products compounds and their synergistic effects, in many cases (Koeberle and Werz, 2014).

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Although there is a growing interest in anti-inflammatory activity of plant extracts by academic researchers as well as phytopharmaceutical industry, a high proportion of the plant species have not been studied in detail (Iwalewa et al., 2007). Thus, the search for new anti – inflammatory agents from nature continues. Methodical identification of plant constituents that can promote the resolution of inflammation in a way that is homeostatic, modulatory and well tolerated by the body need such an important consideration. The characterization and development of standardized herbal medicines with proven efficacy and safety is of great importance, not only for increasing accessibility but also for offering new and affordable therapeutic options (Recio et al., 2012; Fürst and Zündorf, 2014). Ocimum gratissimum L. (Lamiaceae), is a shrub popularly known as “Efinrin nla” in Yoruba, “Ebavbokho” in Bini, “Dadoya” in Hausa, and “Nchanwu” by Igbo speaking people of Nigeria. Its leaves are prepared in form of maceration, decoction or infusion in water or alcohol. A cupful of the macerated leaf is drunk 2-3 times daily for five days in the treatment of inflammatory diseases (Olorunisola, 2015); hemorrhoids (Ajibesin et al., 2008; Erinoso and Aworinde, 2012), malaria (Iyamah and Idu, 2015), diabetes (Gbolade, 2009; Ezuruike and Prieto, 2014), and liver diseases (Mukazayire et al., 2011). Pharmacological studies have reported anti-inflammatory activity of its aqueous or alcohol extracts (Onasanwo et al., 2005; Tanko et al., 2008 and Ajayi et al., 2014); as well as antinociceptive activities of the essential oil (Rabelo et al., 2003; Paula-Freire et al., 2012). Also, results of the preliminary studies on sequential n-hexane, chloroform and methanol extracts of O. gratissimum leaves showed the methanol extract as the most active anti-inflammatory extract (unpublished data). Several phytochemical studies showed presence of phenolic acids and flavonoids in O. gratissimum. Among the phenolic compounds identified includes: xanthomicrol, rutin, kaempferol 4 O-rutinoside, rosmarinic acid, vicenin-2, caffeic acid, cichoric acid, cirsimaritin, nevadensin, transferulic acid, and quercetin (Grayer et al., 2000; Ola et al., 2009: Ouyang et al., 2013). One important characteristics of natural product extract is the complex mixtures of active, partially active and inactive components (Heinrich, 2010). Sequential extraction of O. gratissimum leaves with hexane and chloroform provides suitable less polar solvents that helps to remove non-polar extraneous compounds (waxes, oils, sterols, and chlorophyll) from the plant matrix. Waxes and chlorophylls are ubiquitous compounds that could interfere with activity of 4

the extract (Porterat and Harmburger, 2006; Park et al., 2007). Bulk polyphenols like tannins on the other hand may also form tight complexes with metal ions, proteins and polysaccharides (Wall et al., 1996). According to Picker et al., (2014), clearance of bulk compounds represents a valuable strategy for cell-based anti-inflammatory evaluation of plant extracts. They also identified liquid-liquid partitioning as the optimal method for the elimination of both chlorophyll and polyphenols (tannins). Thus, subjecting sequential methanol extract of O. gratissimum leaf to liquid-liquid partitioning with ethylacetate was a strategy to optimize the enrichment of flavonoids into the fraction. The main objective of our study was to investigate the effect of the flavonoid-rich ethylacetate fraction (EAFOg) on LPS-induced inflammation in mice and RAW 264.7 cells. In addition, its inhibitory effects on arachidonic acid-related enzymes and its chemical characterization was investigated. 2.

MATERIAL & METHODS

2.1

Plant Material Leaves were harvested from the fully matured plants cultivated in a garden in Ibadan,

Nigeria. The plant was authenticated by botanist from the herbarium of Forestry Research Institute of Nigeria (FRIN), Ibadan, Nigeria, and the plant name was checked using the Plant List (http://www.theplantlist.org/). A voucher specimen with flowers was deposited at herbarium with specimen number F.H.I. 110191.

2.1.1 Preparation of extracts and flavonoid-rich fraction Dried leaves were ground into powder using a mechanical grinder. Two hundred and fifty grams of powdered leaves was sequentially extracted by maceration in n-hexane (1:5, w/v), Chloroform (1:5, w/v) for 48 h each. The residue was dried in a laboratory oven at 30oC to remove all the traces of chloroform. The residue (210 g) was extracted by maceration in 80% methanol (1:5, w/v) for 48 hours to obtain the methanol extract. The methanol was evaporated under reduced pressure (600 mmHg) at 40

o

C in a rotary evaporator (model 801, Fisatom;

Brazil). The methanol extract concentrate (130 mL) was partitioned repeatedly with ethylacetate (1:1 v/v) to obtain the ethylacetate and residual aqueous fractions. The solvent was evaporated under reduced pressure; residual solvent was eliminated in a hot air circulating oven (model TE5

394/4 Tecnal, Brazil) at 40 ± 4 o C. Both ethylacetate and residual aqueous fractions were then lyophilized (Lyophilizer model LL 1500, Heto, Italy) to obtain light dark green and brown fractions, stored in an amber bottle and kept in a freezer at – 30 ºC, respectively. The percentage yield of the EAFOg was 1.0% from the dry plant, and 36.55% from the methanol extract. At the time of use, it was dissolved in Tween 80 (1%) for in vivo and DMSO for in vitro studies. 2.2

Phytochemical Standardization

2.2.1 Preliminary phytochemical analysis. Preliminary phytochemical analysis of the EAFOg was performed as described by Matos (2009), which rely on chemical reactions of coloration, precipitation and foam formation. The secondary metabolites qualified were quantified in spectrophotometer and confirmed by highperformance liquid chromatography HPLC.

2.2.2 Determination of total phenolics content. The quantification of the total phenolic content was performed by the Folin–Ciocalteu method, as described by Amorim et al., (2008) using galic acid as a standard. Methanolic solutions (0.2 mL) of the extract (1 mg/mL, w/v) or standard (0.1-1.0 µg/mL w/v) aqueous solution were mixed with the Folin–Ciocalteu reagent (0.5mL of 10%, v/v), sodium carbonate (1 mL of 7.5%, w/v) and 8.3 mL of Milli-Q water, gently agitated and kept for 30 min in the dark. The absorbance was measured at 760 nm in a UV–vis Spectrophotometer (Biospectro® SP-220) equipped with quartz cells of 1cm path length, calibrated with Milli-Q water. Total phenolic content was determined by interpolation of the absorbance of the samples against a calibration curve constructed with different concentrations of gallic acid standard, expressed as mg gallic acid equivalents (GAE) per gram of EAFOg (mg GAE/g). All experiments were performed in triplicate. 2.2.3 Determination of total flavonoid contents The quantification of total flavonoids was performed per the description of Soares et al. (2014) with modifications, using rutin as standard. The reactions were performed in triplicate by mixing 0.5 mL of methanolic solutions of the extract (1 mg/mL; w/v) or standard (1-10 mg/mL w/v) with an aqueous solution of 0.5 mL of 60% acetic acid, 2 mL methanolic solution of 20% 6

pyridine (v/v), 1 mL of 5% aluminum chloride (w/v) and 6 mL of water milli-Q. A white was constructed by joining all of the reaction components and the extract or standard, replacing the aluminum chloride with methanol. This reaction mixture was gently stirred and kept for 30 min in the dark and its absorbance was measured at 420 nm in a spectrophotometer. The total flavonoid contents were determined by interpolating the absorbance of the samples (subtracting the absorbance of blank) against a calibration curve constructed with different concentrations of rutin standard and expressed as milligrams of rutin equivalents (RE) per gram of dry EAFOg (mg RE/g).

2.2.4 High performance liquid chromatography (HPLC) analysis HPLC of the EAFOg was developed by Shimadzu® chromatograph (LC-10 Avp series, Japan) equipped with a (LC-10 AD) pump, (DGU-14A) degasser, UV–vis (SPD-0A) detector, column oven (CTO-10A), manual injector Rheodyne (loop 20 μL) and CLASS (LC-10A) integrator. The separation was carried out by a gradient system, using a reverse-phase Phenomenex Luna 5mm C18 (2) (250 x 4.6 mm2) column with direct-connect C18 Phenomenex Security Guard Cartridges (4 x 3.0 mm2) filled with similar material as the main column. Phases were mobile phase A = 0.1% phosphoric acid in Milli-Qwater and mobile phase, 0.1% phosphoric acid in Milli-Q water/acetonitrile/ methanol (54:35:11, v/v). Program gradient: 0-5 min, 0% B; 5 - 10 min, 50% B, 10 - 20 min, 70% B, 20 - 30 min 80% B, 30-50 min, 100% B, 5080 min 100% B. Flow rate: 1 mL/min, temperature: 22 °C. UV detection was done at 340 nm. The compounds were identified by comparing the retention times of samples and authentic standards such as rutin, ellagic acid, myricetin and morin (Sigma®). The content of the compounds was expressed as micrograms per milligram of EAFOg, which was calculated after correlating the area of the analyte with the calibration curves of the standards constructed at concentrations of 4.5 – 18 μg/mL. The EAFOg solutions and standards were prepared with “B phase” and filtered through a Millipore ® (0.22 mM pore size) membrane.

2.3

Animals

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Female Swiss-Webster mice (23 – 30 g), 2 - 2.5 age in months, obtained from the ‘Biotério Central’ of Universidade Federal de Mato Grosso (UFMT) were used for the studies. Animals were maintained in propylene cages at 26 ± 1 o C in a 12 h dark/12 h light cycle, with free access to standard laboratory chow (Purina, Labina®, Brazil) and water ad libitum. The protocol for the study followed the ethical principles for animal experimentation and was approved by the Institutional Committee for Ethics in the Use of Animals (CEUA/UFMT) with protocol number 23108.139319/2016-05. 2.4

Cell Culture Murine macrophage cell line RAW 264.7 (ATCC TIB-71) was obtained from the Rio de

Janeiro Cell Bank cell Collection. Cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL), and maintained at 37 oC in a humidified incubator of 5% CO2. 2.5

Drugs and Reagents Lipopolysaccharides (LPS) from Escherichia coli (serotype 055:B5), dexamethasone

acetate, N-ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), sodium nitrite Griess reagent were obtained from Sigma (USA). Doxorubicin was obtained from Fluka (USA). AlamarBlue® was obtained from Invitrogen (Life Technologies, USA). IL-1β, TNF-α and IL-10 kits (eBiosciences®, USA). COX and LOX colorimetric assay kit (Cayman Chemical Company, USA). 2.6 Peritonitis induced by lipopolysaccharide Acute inflammation in mouse was produced by injection of bacterial LPS (250 ng/cavity/0.2 mL) into the peritoneal cavity as described by Souza et al., (2003). Mice were distributed into six groups (n=8 /group) and orally pretreated (0.1 ml/10g) with vehicle (1% Tween 80), EAFOg (10, 50 and 200 mg/kg) or dexamethasone (0.5 mg/kg). After 1 h, LPS dissolved in pyrogen free sterile saline (0.9%) was administered (250 ng/cavity/0.2 mL) intraperitoneally to control and treatment groups, while the normal control group received sterile saline. Six hours after LPS, mice were anaesthetized with ketamine (180 mg/kg) and xylazine (30 mg/kg) by intraperitoneal (i.p.) route, and the peritoneal cavity was washed with 3 mL of 8

cold sterile phosphate buffered saline (10 mM, pH 7.4) containing EDTA (3 mM). The abdomen was gently massaged, and blood free cell suspension was carefully aspirated with a syringe. Aliquots of peritoneal fluid were placed in eppendorff tubes and stained with Turk solution, total cell count was performed in a Neubauer chamber, while an aliquot of this lavage was used to make smear for differential cell counting. Aliquots of the peritoneal fluids were stored in a freezer at -80 o C for determination of cytokines. 2.6.1 In vivo quantification of NO and cytokines in peritoneal lavage. The nitrite levels in peritoneal lavage were determined per the Griess reaction by adding 100 µL of Griess reagent to 100 µL of peritoneal fluid for 10 min at room temperature. Absorbance was measured at 540 nm in a microplate reader and nitrite concentration was determined using a sodium nitrite standard curve. Concentrations of IL-β, TNF-α, and IL-10 were determined with ELISA kit (eBioscience, USA), in accordance with manufacturer’s instruction using Multiskan® EX ELISA plate reader (Thermo Scientific, USA). 2.7

In vitro evaluation of COX-1 and COX-2 inhibitory activity Direct enzyme inhibitory activity of EAFOg towards COX-1 and COX-2 activity was

determined using the COX colorimetric inhibitor screening assay kit (Cayman, No 701050). The assay measures the peroxidase activity of the COX enzyme by colorimetrically monitoring the appearance of oxidized N, N, N1, N1-tetramethyl-p-phenylenediamine (TMPD). Inhibitory effects of EAFOg was compared to indomethacin (COX-1/2 non-selective inhibitor), SC560 (COX-1 selective inhibitor) and NS398 (COX-2 selective inhibitor). 2.8

Lipoxygenase inhibitory assay The lipoxygenase enzyme inhibitory effect was measured using the Cayman 15-

Lipoxygenase inhibitor screening assay kit (Cayman Chemical Company, Ann Aref? or, USA). The assay detects and measures the hydroperoxides produced in the lipoxygenation reaction using a purified lipoxygenase (LO). The inhibitory activity of EAFOg was tested for 15-LO using the LOX inhibitor screening kit according to the manufacturer’s instruction. 2.9

Cell viability assay in RAW 264.7 cells 9

Briefly, RAW 264.7 cells (2 x 104 cells/well in 96-well culture plate) were incubated at 37o C (5% CO2) overnight. The cells were treated with/without EAFOg (200 - 3.125 µg/mL) and doxorubicin (58 - 0.0058 µg/mL, serial dilution) was used as a positive control. After incubation for 24 or 72 h, the treatments were removed and 200 µL of 10% alamarBlue© solution was added. Six hours after, the absorbance was read at 540 nm (oxidized state) and 620 nm (reduced state) with Multiskan® EX ELISA plate reader (Thermo Scientific, USA). Cell viability was expressed as percentage of control (Riss et al., 2004). 2.10

Determination of nitrite production in RAW 264.7 macrophages RAW 264.7 cells (2.5 x 105/well) were plated in a 24-well plate overnight. Cells were

pre-treated with EAFOg (5, 20 and 100 µg/mL) or L-NAME (2.69 µg/mL), for 1 h before stimulation with LPS (1 µg/mL), and incubated at 37 oC and 5% CO2 for 24 h. The nitrite production in the medium was measured according to the Griess reaction by adding 100 µL of Griess reagent to 100 µL of medium and incubated for 10 min at room temperature. Absorbance was measured at 540 nm in a microplate reader and nitrite concentration was determined using a sodium nitrite standard curve prepared in medium devoid of phenol red. Nitrite level was expressed as percentage of control. 2.11

Determination of cytokine production in RAW 264.7 macrophages RAW 264.7 cells (2.5 x 105/well) were plated in a 24-well plate overnight. Cells were

pre-treated with EAFOg at concentrations of 5; 20 and 100 µg/mL or dexamethasone (10 µg/mL) for 1 h before stimulation with LPS (1 µg/mL), and incubated at 37 oC and 5% CO2 for 24 h. The supernatants were collected and stored at - 80 o C before analysis. The concentrations (pg/mL) of IL-1β, TNF-α, and IL-10 in the cell supernatants were measured using an ELISA kit (eBiosciences, USA) according to the manufacturer’s instruction. 2.12

Data analysis The results were expressed as mean ± standard error of mean (X ± SEM). Comparisons

between means were analyzed by one-way analysis of variance (ANOVA). When significant, it was followed by Student-Newman–Keuls test for multiple comparisons. p values < 0.05 were 10

considered significant. The IC50 was determined from a linear regression relating the percentage of inhibition versus the logarithm of the concentrations tested and assuming a confidence level of 99% (p < 0.01) for the curve obtained. For in vitro assays that do not involve statistical analysis, we used the mean ± SEM three independent experiments performed in triplicate. All values were analyzed with GraphPad Prism® software version 5 (GraphPad Software, San Diego, CA, USA). .

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3.

RESULTS

3.1

Phytochemical analysis The preliminary phytochemical analysis of ethylacetate fraction of the leaves of O.

gratissimum (EAFOg) revealed the presence of phenolic compounds, flavonoids and tannins. The contents of total phenolics and total flavonoids were: 139.764 ± 1.072 mg GAE/g; 109.954 ± 0.046 mg RE/g respectively. Analysis by HPLC confirmed the presence of phenolic compounds

detected

in

the

preliminary

phytochemical

analysis

and

quantified

spectrophotometrically. The data show the presence of rutin (time: 22.4 min), ellagic acid (time: 23.5 min), myricetin (time: 28.7 min) and morin (time: 31.1 min). The concentrations are as follows: rutin (1.131 μg/mg) ellagic acid (11.655 μg/mg), myricetin (6.535 μg/mg) and morin (0.073 μg/mg), in the fraction (Fig. 1).

Fig. 1. High performance liquid chromatography (HPLC) fingerprint of the ethylacetate fraction of Ocimum gratissimum leaf (EAFOg) detected at 280 nm. Peak 1: rutin, 2: ellagic acid, 3: myricetin and 4: morin. Insert HPLC of authentic standards phenolic compounds.

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3.2 In vivo anti-inflammatory activity 3.2.1 EAFOg reduces leucocytes migration into the peritoneum. The total number of leucocytes in the peritoneal fluid in the sham group treated with vehicle (1% Tween 80) was 9.37 ± 0.69 x 106 cells. Leucocytes migration into the peritoneal cavity was increased (46.8%; p < 0.001) after six hours of i.p. injection of LPS (250 ng /cavity). Pretreatment with 10; 50 and 200 mg/kg of EAFOg before LPS injection resulted in a reduction in the number of leucocytes, with significant effect at 50 mg/kg (38.9%, p < 0.05) and 200 mg/kg (48.9%, p < 0.01), while, dexamethasone (0.5 mg/kg) inhibited leucocytes migration by 38.9% (p < 0.05) when compared to the vehicle group (Fig 2A). In the sham group, the number of neutrophils present in the peritoneal cavity was 2.02 ± 0.59 x 106 cells. In the vehicle group, LPS injection caused significant increase (78.4%, p < 0.001) in the number of neutrophils that migrated to the peritoneal cavity compared to sham group. EAFOg reduced at all doses tested (p < 0.001), the number of neutrophils by 38.8, 58.9, and 66.5% respectively. Dexamethasone (0.5 mg/kg) also attenuated neutrophils increase by 68.5% (p < 0.001) when compared to the vehicle group (Fig 2B). Similarly, monocytes influx into the peritoneum after LPS challenge was significantly increased (p < 0.001). In comparison to vehicle, EAFOg (10, 50 and 200 mg/kg) reduced monocytes count by 38.9% (p < 0.05), 58.0% (p < 0.01) and 72.8% (p < 0.01) respectively, while dexamethasone (0.5 mg/kg) caused reduction in the monocytes count by 70.4% (p < 0.001) as shown in Fig 2C. The lymphocytes counts in all groups were not statistically different (Fig 2D).

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Fig 2: Effect of vehicle (1% Tween 80), ethylacetate fraction of Ocimum gratissimum leaf (EAFOg, 10; 50 and 200 mg/kg) and dexamethasone (Dexa 0.5 mg/kg) administered orally on peritoneal fluid of female mice with lipopolysaccharide (LPS) peritonitis. The sham group received only vehicle and intraperitoneal injection of 0.9% saline (0.1 mL/10 g). Mean ± SEM for 6-7 animals. One-way analysis of variance, followed by Student-Newman-Keuls test. ††† p < 0.001 vs. sham; * p < 0.05, **p < 0.01, and *** p < 0.001 vs. vehicle (LPS). (A) Total leukocytes, (B) Neutrophils, (C), Monocytes, and (D) Lymphocytes. 15

3.2.2 EAFOg attenuated LPS-induced nitrite production in the peritoneal fluid Nitrite level in peritoneal lavage of saline group treated with vehicle (1% Tween 80) was 0.91 ± 0.10 µM. Intraperitoneal challenge with LPS induced significant increase (69.2%, p < 0.001) in nitrite production compared to saline group. Pretreatment with 10, 50 and 200 mg/kg of EAFOg significantly attenuated LPS-induced nitrite production in a dose-dependent manner by 30.8% (p < 0.01), 50.8% (p < 0.001), and 57.3% (p < 0.001), respectively. Dexamethasone (0.5 mg/kg) inhibited nitrite production by 56.6% (p < 0.001) when compared to the vehicle group (Fig 3).

Peritoneal cavity Nitrite (M)

3.5

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Fig 3: Effect of vehicle (1% Tween 80), ethylacetate fraction of Ocimum gratissimum leaf (EAFOg, 10; 50 and 200 mg/kg) and dexamethasone (Dexa, 0.5 mg/kg) administered orally on nitrites production in the peritoneal fluid of female mice with lipopolysaccharide (LPS) peritonitis. The sham group received only vehicle and intraperitoneal injection of 0.9% saline (0.1 mL/10 g). Mean ± SEM for 6-7 animals. One-way analysis of variance, followed by Student-Newman-Keuls test. ††† p < 0.001 vs. sham; ** p< 0.01 and *** p < 0.001 vs. vehicle (LPS).

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3.2.3 EAFOg attenuated LPS-induced IL-1β, TNF-α but not 1L-10 generation in the peritoneal fluid. Intraperitoneal injection of LPS into mice markedly increased the levels of IL-1β, TNF-α and IL-10 in the peritoneal fluids when compared with the saline group (Fig 4 A-B). The EAFOg (10; 50 and 200 mg/kg) significantly reduced the levels of IL-1β by 17% (p < 0.05), 40.8% and 60.65% (p < 0.001); and TNF-α by 25.8% (p < 0.05) and 33.9% (p <0.01) at the 50 and 200 mg/kg dose, respectively (Fig 4B). However, the EAFOg did not alter the levels of IL-10 in the peritoneal fluid when compared with the vehicle group (data not shown). Dexamethasone (0.5 mg/kg) reduced the levels of IL-1β (55.7%; p < 0.001), TNF-α (38.9%; p < 0.01) but did not alter IL-10 (Fig 4 A-B). A. 1000

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Dexa

LPS

Fig 4: Effect of vehicle (1% Tween 80), ethylacetate fraction of Ocimum gratissimum leaf (EAFOg, 10, 50 and 200 mg/kg) and dexamethasone (Dexa, 0.5 mg/kg) administered orally on cytokines production in the peritoneal fluid of female mice with lipopolysaccharide (LPS) peritonitis. The sham group received only vehicle and intraperitoneal injection of 0.9% saline (0.1 mL/10 g). Mean ± SEM for 6 animals. One-way analysis of variance, followed by StudentNewman-Keuls test. ††† p < 0.001 vs. sham; * p< 0.05 and *** p < 0.001 vs. vehicle. (A) IL-1β and (B) TNF-α. ND – Not detectable

3.3

EAFOg potently inhibits COX-2 but marginally inhibits COX-1 in vitro EAFOg (6.25 – 100 µg/mL) in all tested concentrations showed moderate inhibition of

COX-1 (29.6 – 46.8 %, p < 0.001), as seen in Fig.5A. However, EAFOg (50 – 100 µg/mL) caused strongly inhibition (54.0 – 91.9%, p < 0.001) of COX-2 activity (Fig 5B). The COX-1 selective inhibitor SC-560 inhibits COX-1 activity ~ 37.4% at 1 µM. Similarly, the COX-2 selective inhibitor NS398 (3.8 µM) inhibited COX-2 activity ~ 56.9%. NSAIDs used in this study (indomethacin non-selectively inhibited COX-1 (32.9%) and COX-2 (94.8%) at 100 µM. The EAFOg median inhibitory concentration (IC50) value to COX-1 was > 100 µg/mL and to COX-2 was 48.86 µg/mL. The selectivity index ratio (COX-1/COX-2) of EAFOg was greater than 2.

18

Absorbance 590 nm (COX-1 activity)

A. 0.12 0.10 29.6%

0.08

34.6%

32.9%

36.5%

37.5%

*** *** *** ***

0.06

46.8%

***

37.4%

***

***

0.04 0.02 0.00 0.5

6.25 12.5

DMSO (%)

25

50

EAFOg

100

100

(g/mL)

Indo

1

(M)

SC-560

Absorbance 590 nm (COX-2 activity)

B. 0.4 1.7% 8.1%

12.7%

0.3

54.0%

0.2

56.9%

***

***

0.1 91.9%

*** 0.0

0.5 DMSO (%)

6.25 12.5

25 EAFOg

50

94.8%

***

100

100

3.8

(g/mL)

Indo

NS-398

(M)

Fig 5: In vitro inhibitory screening assay of (A) COX-1, (B) COX-2 by ethylacetate fraction of Ocimum gratissimum leaf (EAFOg, 100 - 6.25 µg/mL). Absorbance are Mean ± SEM (n=3), *** p < 0.001 vs control. Values in parenthesis are the percent inhibition of enzyme activity. DMSO

19

- Dimethyl sulfoxide; Indo – Indomethacin; SC-560 – COX-1 selective inhibitor; NS-398 COX-2 selective inhibitor.

3.4

EAFOg marginally inhibits 15-LOX enzymes in vitro.

EAFOg (6.25 – 100 µg/mL) displayed significant (p < 0.01) but weak 15-LO inhibitory activity, with an IC50 greater than 100 µg/mL. The 15-LO selective inhibitor NDGA inhibits lipoxygenase activity ~ 67.4% at 30 µg/mL (Fig. 6).

Absorbance 492 nm (15-LO activity)

0.4

0.3

11.4%

12.5%

**

**

16.4%

17.3%

**

**

14.3%

**

0.2 67.4%

***

0.1

0.0 0.5 Methanol (%)

6.25 12.5

25 EAFOg

50

100

30

(g/mL)

NDGA

Fig 6: In vitro inhibitory screening assay of 15-LOX by ethylacetate fraction of Ocimum gratissimum leaf (EAFOg, 6.25 – 100 µg/mL) and Lipooxygenase inhibitor (NDGA 30 µg/mL). Absorbance are mean ± SEM (n=3). ** p < 0.01 and *** p < 0.001 vs control (Methanol 0.5%). Values in parenthesis are percent inhibition of enzyme activity.

20

3.5 Effects of Flavonoid-rich ethylacetate fraction of O. gratissimum leaf (EAFOg) on RAW 264.7 cells viability The cell viability results (Fig. 7A and B) demonstrated that for the concentrations 3.123, 6.25, 12.5, 25, and 50 µg/mL of EAFOg were higher than 85% at 24 and 72 h of treatment, that is EAFOg treated cells had a viability similar to that of the control (0.5% DMSO). The EAFOg showed IC50 values > 200 µg/mL at 24 h and at 72 h, IC50 value = 170.00 ± 2.83 µg/mL. The results also showed that the standard cytotoxic agent (doxorubicin) gave an IC50 value > 58 µg/mL at 24h, but was highly cytotoxic at 72 h with IC50 = 0.16 ± 0.02 µg/mL.

A. RAW 264.7 cells at 24 h B. RAW 264.7 cells at 72 h

120

Cell viability (% of control)

Cell viability (% of control)

100

100 80 60 40 20 0

0

3.125

6.25

12.5

25

EAFOg

50

100

200

0.0058 0.058

0.58

5.8

Doxorubucin

58

g/mL

80 60 40 20 0

3.125 6.25 12.5

25

EAFOg

50

100

200

0.00580.058 0.58

5.8

58

g/mL

Doxorubucin

Fig 7: Effect of ethylacetate fraction of Ocimum gratissimum leaf (EAFOg 3.125- 200 µg/mL) and doxorubicin (0.0058 – 58 µg/mL) on RAW 264.7 cells viability at (A) 24 h and (B) 72 h exposure.

3.6

EAFOg attenuated LPS-induced nitrite production in RAW 264.7 cells.

LPS stimulation resulted in significant (p < 0.001) increase by 84.4% in NO2- production as compared to that in control (basal unstimulated cells). However, EAFOg at 5, 20 and 100 µg/mL significantly (p < 0.001) inhibited NO2- production by 22.9, 44.7 and 75.5%, respectively, as compared to the LPS reaching the peak with 100 µg/mL. L-NAME (2.69 µg/mL) used as a positive control caused significant (70.3%, p < 0.001) reduction in NO2- level in the cell supernatant (Fig. 8).

21

NO2- (% of LPS control)

120

††† 100

*** 80 60 40

***

20 0

LPS (1g/mL) EAFOg (g/mL) L-NAME (g/mL)

-

+

+

+

+

-

-

5 -

20 -

100 -

+ 2,69

Fig 8: Effect of ethylacetate fraction of Ocimum gratissimum leaf (EAFOg 5, 20 and 100 µg/mL) and N-Nitro-L-arginine methyl ester (L-NAME 2.69 µg/mL) on nitrite production in LPS-stimulated RAW 264.7 cells. The cells were treated with EAFOg for 1 h before stimulation with LPS (1 µg/mL) and incubation for 24 h. The values are means ± SEM of two independent experiments. One-way analysis of variance, followed by Student-Newman-Keuls test. ††† p < 0.001 vs. basal; *** p < 0.001 vs. LPS. 3.7 EAFOg attenuated LPS-induced IL-1β, TNF-α and IL-10 production in LPSstimulated RAW 264.7 cells Treatment of RAW 264.7 cells with LPS resulted in significant (p < 0.001) increases in cytokines (IL-1β, TNF-α, and IL-10) as compared to basal wells (Fig 9A-C). As shown in 9A, EAFOg (20 and 100 µg/mL) significantly reduced IL-1β production (inhibition of 27.8% and 77.7 %) after stimulation with LPS for 24 h (Fig. 9A). Our results also show that EAFOg (20 and 100 µg/mL) produced significant (p < 0.01 and 0.001) reduction in TNF- α level (inhibition of 12.3% and 10.1%) following stimulation of RAW 264.7 cells with LPS (Fig 9B). Furthermore, EAFOg (5, 20 and 100 µg/mL) exhibited dose-dependent reduction on IL-10 production (Fig 9C); the inhibitory effect of EAFOg reached significant level (p < 0.001) at 100 µg/mL (inhibition of 33.3%). Dexamethasone (10 µg/mL), the positive control significantly (p < 0.001) reduced the levels of IL-1β (66.2%), TNF-α (10.4%), and IL-10 (36.7%) when compared to LPS

22

A. 120

IL-1 (% of LPS control)

100

*

80

60

*** 40

*** 20

0 LPS (1g/mL) EAFOg (g/mL) Dexa (g/mL)

-

+

+

+

+

+

-

-

5 -

20 -

100 -

10

B. 120 †††

TNF- (% of LPS control)

100

***

**

**

80

60

40

20

0 LPS (1g/mL) EAFOg (g/mL) Dexa (g/mL)

-

+

+

+

+

-

-

5 -

20 -

100 -

+ 10

23

120

IL-10 (% of LPS Control)

†††

100 80

***

***

+ 10

60 40 20 0

LPS (1g/mL) EAFOg (g/mL) Dexa (g/mL)

-

+

+

+

+

-

-

5 -

20 -

100 -

Fig 9: Effect of ethylacetate fraction of Ocimum gratissimum leaf EAFOg (5, 20 and 100 µg/mL) and Dexamethasone (Dexa 10 µg/mL) on cytokine production in LPS-stimulated RAW 264.7 cells. The cells were treated with EAFOg for 1 h before stimulation with LPS (1 µg/mL) and incubation for 24 h. The values are means ± SEM of two independent experiments. Oneway analysis of variance (Anova), followed by Student-Newman-Keuls test. ††† p < 0.001 vs. basal; *** p < 0.001 vs. LPS. (A) IL-1β, (B) TNF-α, and (C) IL-10.

24

4.

DISCUSSION In this study, we optimized the extraction of flavonoids from bioactive sequential

methanol extract of O. gratissimum leaf and quantified the major peaks in the HPLC. Phytochemical standardization of purified flavonoid-rich ethylacetate fraction of O. gratissimum leaves revealed the presence of high total flavonoid content. The optimized flavonoid-rich fraction showed flavonoid to phenolic acid ratio of about 0.78. Rutin, myricetin, morin and ellagic acid were identified and quantified by HPLC. Interestingly, no literature report on the identification of ellagic acid, morin and myricetin was found on O. grattissimum leaf. These compounds have individually been shown to have anti-inflammatory activities in vitro and in vivo (Kassim et al., 2010; Semwal et al., 2016).

In vivo anti-inflammatory activity of EAFOg was evaluated in the model of acute inflammation induced by intraperitoneal LPS injection in mice. Lipopolysaccharides as a component of gram negative bacterial cell wall promotes the release of cytokines by recruiting inflammatory cells from the blood to the affected tissues by the activation of Toll-like receptors4 (TLR-4) (Noreen et al., 2012). We observed that treatment of mice with EAFOg inhibited LPS-induced recruitment of leucocytes into the peritoneum. Polymorphonuclear leucocytes (PMNs; over 90% of which are neutrophils) are rapidly recruited into sites of acute infection and dominate the initial influx of leucocytes by responding to a chemoattractant gradient generated by resident macrophages (Henderson et al., 2003, Laurin et al., 2012). It is therefore important for anti-inflammatory agent to inhibit the process of recruitment of leucocyte cell types. In our study, we observed that treatment with EAFOg significantly reduced the number of leucocytes into the peritoneum without any specificity for neutrophils or monocytes, suggesting that inhibition of cell migration may be a key event for the anti-inflammatory mechanism. Furthermore, during LPS-induced peritonitis, inflammatory macrophages synthesize and release numerous mediators, including nitric oxide (NO), pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) amongst others (Laurin et al., 2012; Borges et al., 2014). In acute peritonitis, increased NO release mediated by the inducible nitric oxide synthase (iNOS or NOS2) has been implicated. NO couples with O2 to form peroxynitrite (ONOO−) that is believed to cause cellular 25

damage, while NO has also been shown to be chemically reactive to cause DNA damage by nitration, nitrosation and oxidation (Le Grande et al., 2001). Thus, compounds able to reduce NO production by inhibiting iNOS pathway represents an important strategy to mitigate inflammatory diseases. We observed that pretreatment of mice with EAFOg significantly reduced peritoneal NO levels. Thus, possible inhibition of iNOS may be a contributing mechanism of anti-inflammatory activity of EAFOg.

We also investigated the involvement of key inflammatory cytokines in the antiinflammatory activity of EAFOg. Our results showed that pre-treatment of mice with EAFOg attenuated the production of IL-1β and TNF-α in the LPS-induced peritonitis in mice. Cytokines such as TNF-α and IL-1β exerts strong pro-inflammatory activities and they are released predominantly by activated macrophages and to a lesser extent by neutrophils (Gabay et al., 2010). They induce expression of adhesion molecules on endothelial cells, together with the induction of chemokines, they stimulate the infiltration of inflammatory and immunocompetent cells. In addition, IL-1β and TNF-α causes fever, vasodilatation, hypotension and enhances pain sensitivity (Dinarello 2009; Feldmeyer, 2010). Thus, controlling these cytokines in inflammatory disease models is an important strategy that has contributed significantly to the successes recorded in anti-inflammatory drug development (Moller and Villiger, 2006). IL-10 is another important cytokine whose biological function is key to the regulation of the degree and duration of inflammatory response. IL-10 is known to act as a potent antiinflammatory cytokine in conditioning the activation and function of innate and Ag-specific immune cells, it selectively blocks the expression of pro-inflammatory genes encoding cytokines (TNF-α, IL-1β and IL-6) and chemokines (MCP-1, IL-8, MIP-10, and MIP-2) in myeloid cells (Moore et al., 2001; Bazzoni et al., 2010). However, pre-treatment of mice with EAFOg in our study did not alter the level of IL-10 in the peritoneal fluid. EAFOg might be acting independent of 1L-10 modulation in the LPS-induced peritonitis in mice. Taken together, it is possible to postulate that the anti-inflammatory activity of EAFOg may be due to the suppression of Th1 (type 1) immunity. Arachidonic acid is metabolized by the cyclooxygenase (COX) and lipoxygenase (LOX) enzymes in inflammatory cells to produce prostaglandins and leukotrienes. COX exists in at least two isoforms COX–1 and COX–2. Whilst COX-1 is constitutively expressed, and has a 26

“housekeeping” functions (e.g gastric cytoprotection), COX-2 is induced during inflammation and tends to facilitate the inflammatory response (Smith, 2011). Lipoxygenase converts arachidonic acid into various cytokines that also play important role in inflammation. Most cell types that participate in inflammatory reactions generate leukotrienes, including mast cells, basophils, eosinophils, neutrophils and monocytes. Investigation of the inhibitory effects of EAFOg on both COX-1/-2 and 15-LOX enzymes in vitro revealed interesting findings. EAFOg inhibited COX-2 enzyme activity, the IC50 being 48.86 µM, but showed weak activity against COX-1 and LOX with IC50 values greater than 100 µM. EAFOg inhibited COX-2 to the same degree as indomethacin (100 µM) and showed selectivity index approximately greater than 1. These findings demonstrated a direct inhibitory effect of EAFOg on COX-2 enzymes, but we are yet to ascertain if EAFOg could also inhibit COX-2 induction and prostaglandin biosynthesis in activated macrophages. Previous studies on flavonoids or flavonoid-rich extracts have demonstrated that flavonoids are selective inhibitor of COX-2 either by direct inhibition or modulation of COX-2 gene transcription and translation (Larry et al. 2004, Garcia-Lafuente et al. 2009). Some flavonoids such as luteolin, galangin or morin were among the first flavonoids to be described as inhibitors of COX (Bauman et al., 1980). Owing to the multiple constituents of flavonoids in EAFOg, inhibition of prostaglandin synthesis by interfering with COX-mediated conversions of free arachidonic acid to prostaglandin may be the major target by which it mediates its anti-inflammatory activity. Recognition of external stimuli and initiation of inflammatory process strongly depends on the resident cells (Ajuebor et al. 1999). We further extended our investigation of the antiinflammatory activity of EAFOg in vitro cell-based assays. Firstly, we investigated the effects of the EAFOg on viability of RAW 264.7 cells. Monitoring the effects of compounds on cell proliferation or direct cytotoxicity is usually done in cell based systems. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. Thus, we investigated the effects of EAFOg on cell viability using the alamarBlue® assay which involves the addition of a fluorogenic redox indicator to growing cells in culture (Nakayama et al. 1997). In RAW 264.7 cells, we demonstrated that EAFOg did not reduce cell viability at concentrations below 100 µg/mL when incubated with cells at 24 and 72 h. However, EAFOg showed reduced cell viability in RAW 264.7 cells at 72 h exposures, showing IC50 values of 170.00 ± 2.83 µg/mL. The inclusion of an in vitro cytotoxicity assay 27

provides an important advantage in identifying potentially cytotoxic compounds. Cytotoxicity assays can provide a useful way to compare and ranks extracts or compounds (Hamid et al., 2004). Suffness and Pezzuto (1990) postulates that extract/fraction and pure substance showing IC50 values greater than 30 and 4 µg/mL respectively, were considered non-toxic to cells. Thus, EAFOg could be considered non-cytotoxic to both RAW 264.7 since its IC50 is greater than 30 µg/mL. Furthermore, we investigated the anti-inflammatory effects of EAFOg on LPS-induced RAW 264.7 macrophages. Our data revealed that EAFOg inhibited LPS-induced NO, IL-1β and TNF-α production in RAW 264.7 cells. NO produced by activated macrophages play critical roles in inflammatory diseases. The present results showed that EAFOg strongly inhibits the production of NO in LPS-stimulated RAW 264.7 cells in vitro, confirming the results presented in the in vivo assay. Thus, it is possible that EAFOg might be acting directly on iNOS or another form of intracellular control, however this claims needs further investigation in future studies. Similarly, our data on the effects of EAFOg on cytokine production in LPS-induced RAW 264.7 cells agrees with LPS-induced peritonitis in mice, wherein EAFOg significantly reduced the production of IL-1β and TNF- α but did not increase the anti-inflammatory cytokine (IL-10). Considering these facts, natural compounds able to inhibit the early stage production of pro-inflammatory cytokines may be attractive as anti-inflammatory agents (Joung et al., 2012). In conclusion, the present findings demonstrate that flavonoid-rich ethylacetate fraction of Ocimum gratissimum leaf (EAFOg) possesses anti-inflammatory activity. We suggest that the activity may be related to inhibition of leucocytes recruitments, and suppression of key proinflammatory mediators NO and cytokines (IL-1β and TNF- α). Furthermore, our findings suggest that EAFOg has selective inhibition of COX-2 over COX-1. Further studies aimed at elucidating the effect on key downstream inflammatory signaling molecules as well as the identification of main active principles are clearly needed.

28

Acknowledgements This study was supported by academic staff training and development (AST&D) fellowship of the Tertiary Education Tax Fund (TETFUND) of the Federal Republic of Nigeria awarded to Abayomi M. Ajayi, Instituto Nacional de Ciência e Tecnologia em Áreas Úmidas (INAU)/CNPq/MCTI (Process No. 704792/2009) for the financial support for the present study and for the award of research fellowship (DTI 1A) to Dr. Sikiru Olaitan Balogun (Process No. 380909/2015-4) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for schorlaships to masters students that participated in the studies.

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