General experimental procedures
All the chemicals used in this study were of analytical grade and including methanol xylene, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and carrageenan was purchased from a local representative of Sigma-Aldrich (Sigma-Aldrich, Germany). Levofloxacin, indomethacin, and Diclofenac sodium were purchased from Pharmacy (Hovid Pharmaceuticals).
Harvesting of the plant leaves
Fresh leaves of T. superba were collected from a forest in Enugu state, Nigeria, and authenticated by a botanist at Bioresources Development and Conservation Program (BDCP) Center, Nsukka, Enugu State, Nigeria, where a voucher specimen was deposited (INTERCEDD/203). The name was also confirmed with the plant list website. The leaves were hand-picked, free from debris, and other soil remains into a clean container. The fresh leaves were dried under the shade at a temperature of less than 40 °C to minimize the loss of volatile compounds. The dried leaves were pulverized to a coarse powder using a milling machine (Laboratory Mill, Serial No. 4745, Christy and Norris Limited, England). The coarse powder was stored in an air-tight container ready for extraction.
Extraction of plant material
The pulverized leaves of T. superba (1100 g) were extracted by cold maceration in 5 L of methanol for 72 h with intermittent vigorous shaking every 2–4 h. The extract was strained with a muslin cloth and filtered with Whatman No. 1 filter paper. The filtrate was concentrated using a rotary evaporator set at 40 °C to reduce the volume to 1/10 of its original volume and dried in a water bath set at 35 °C to obtain the methanol extract (METS). The extract was stored in an airtight amber-colored bottle and stored at 4 °C in a refrigerator until use [49].
Chemicals
All the chemicals used in this study were of analytical grade and including methanol xylene, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and carrageenan was purchased from a local representative of Sigma-Aldrich (Sigma-Aldrich, Germany). Levofloxacin, indomethacin, and Diclofenac sodium were purchased from Pharmacy (Hovid Pharmaceuticals).
Preliminary phytochemical analysis
The extract was subjected to phytochemical analysis using standard procedures [18].
Gas Chromatography–Mass Spectrometry Analysis of methanol crude extract of T. superba.
The GC–MS analysis of bioactive compounds from the different extracts was done using Agilent Technologies GC systems with GC-7890A/MS-5975C model (Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-5MS column (30 m in length × 250 μm in diameter × 0.25 μm in thickness of film). Spectroscopic detection by GC–MS involved an electron ionization system that utilized high-energy electrons (70 eV). Pure helium gas (99.995%) was used as the carrier gas with a flow rate of 1 mL/min. The initial temperature was set at 50–150 °C with an increasing rate of 3 °C/min and a holding time of about 10 min. Finally, the temperature was increased to 300 °C at 10 °C/min. One microliter of the prepared 1% of the extracts diluted with respective solvents was injected in a spitless mode. The relative quantity of the chemical compounds present in each of the extracts was expressed as a percentage based on the peak area produced in the chromatogram.
Quantitative DPPH radical scavenging assay
The ability of the extract to scavenge 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radicals was assessed according to the modified method used by Okolo and Orisakwe [53]. The percentage inhibition of DPPH radical scavenging activity was calculated based on the following equation:
$$\% \;{\text{ inhibition }} = \, 100\% \, \times \, \left( {A_{{\text{o}}} - \, A_{{\text{s}}} /A_{{\text{o}}} } \right)$$
where Ao is the absorbance of the control and As is the absorbance of the test sample.
DPPH radical scavenging property was quantified using a regression line of best fit where the abscissa represents the concentration, and the ordinate represents the percentage of inhibitory activity for three replicates.
Quantitative hydroxyl ion (OH
•
) scavenging assay
Hydroxyl radical scavenging activity of the extractives was determined by the method of Rahman et al. [60]. The generation of hydroxyl radical was instituted by the Fe3+-ascorbate-EDTA-H2O2 system (Fenton reaction). The assay principle is based on the quantification of the 2-deoxy-D-ribose degradation product, which forms a pink chromogen upon heating with TBA at low pH. The reaction mixture contained 0.8 mL of phosphate buffer solution (50 mmol/L, pH 7.4), 0.2 mL of extractives/standard at different concentration (12.5–100 μg/mL), 0.2 mL of EDTA (1.04 mmol/L), 0.2 mL of FeCl3 (1 mmol/L), and 0.2 mL of 2-deoxy-D-ribose (28 mmol/L). The mixtures were maintained at 37 °C in a water bath, and the reaction was started by adding 0.2 mL of ascorbic acid, AA (2 mmol/L), and 0.2 mL of H2O2 (10 mmol/L). After incubation for 1 h, 1.5 mL of cold thiobarbituric acid, TBA (10 g/L) was added to the reaction mixture followed by 1.5 mL of HCl (25%). The mixture was heated at 100 °C for 15 min and then cooled down with ice water. The absorbance of the solution was measured at 532 nm with a spectrophotometer. The hydroxyl radical scavenging capacity was evaluated with the inhibition of the percentage of 2-deoxy-D-ribose oxidation on hydroxyl radicals. The percentage of hydroxyl radical scavenging activity was calculated according to the following formula:
$$\% {\text{ hydroxyl radical scavenging activity }} = \, [A_{0} - { (}A_{1} - A_{2} {)] } \times { 1}00/A_{0}$$
where A0 is the absorbance of the control without a sample.
A1 is the absorbance after adding the sample and 2-deoxy-D-ribose.
A2 is the absorbance of the sample without 2-deoxy-D-ribose.
The percentage inhibition was plotted against concentration, and the experiment was repeated three times at each concentration.
Ferrous reducing antioxidant capacity assay
The ferrous reducing antioxidant capacity (FRAC) of the sample was evaluated by the method of Rahman et al. [60]. The Fe2+ is measured by measuring the formation of Perl’s Prussian blue at 700 nm. 0.25 mL samples/standard solution at different concentration (12.5–100 μg/mL), 0.625 mL of potassium buffer (0.2 M) and 0.625 mL of 1% potassium ferricyanide, [K3Fe (CN)6] solution were added into the test tubes. The reaction mixtures were incubated in a water bath for 20 min at 50 °C to complete the reaction. Then, 0.625 mL of 10% trichloroacetic acid (TCA) solution was added to the test tubes. The total mixture was centrifuged at 3000 rpm for 10 min, after which 1.8 mL of supernatant was withdrawn from the test tubes and mixed with 1.8 mL of distilled water and 0.36 mL of 0.1% ferric chloride (FeCl3) solution. The absorbance of the solution was measured at 700 nm using a spectrophotometer against blank. A typical blank solution contained the same solution mixture without plant extracts/standard and was incubated under identical conditions. The absorbance of the blank solution was measured at 700 nm. Increased absorbance of the reaction mixture indicates increased reducing capacity. The experiment was carried out in triplicate.
Animal husbandry
Eight-week-old albino rats and mice of both sexes were obtained from the animal facility of the Department of Pharmacology and Toxicology, University of Nigeria, Nsukka, Enugu State-Nigeria. The rats and mice were of the weight ranging from 150 to 200 g and 17–25 g, respectively. The animals were kept differently in steel cages to acclimatize within the facility and allowed free access to clean water and food ad libitum. They were kept in a well-ventilated room with 12/12-h light/dark conditions and at room temperature. Animal experiments were conducted in compliance with the National Institute of Health Guide for Care and Use of Laboratory Animals (Pub. No. 85-23, revised 1985), and per the University of Nigeria, Nsukka Ethics Committee established rules on the use of laboratory animals (PHARM/01/072).
Acute toxicity test
The estimation of the mean lethal dose (LD50) of the ME of T. superba in mice was done using the modified method described by Lorke [44]. Firstly, nine mice were divided into three groups (n = 3), received oral administration of 10, 100 and 1000 mg/kg of METS (prepared in 3% tween 80) and were observed for 24 h for a number of deaths. At the end of 24 h, no death was recorded. Consequently, a fresh batch of mice divided into four groups (n = 1) received 1600, 2900, 3600, and 5000 mg/kg of METS in the second stage of the study and were observed for 24 h for death. Based on the result, it is believed that the extract is safe up to 5000 mg/kg because there was no physical or concealed signs and symptoms of toxic effects or any record of death for all the periods of observation of the animals.
Antibacterial activity test
The effects of METS on microorganisms were evaluated using antimicrobial activity on inflamed wound isolates. The standard bacteria sample were obtained from the pathology laboratory at a medical school in Enugu, Nigeria. The clinical wound isolates were collected in sterile swab sticks from patients before dressing the wounds and characterized based on the method [33]. Patient selection was randomly done with no consideration for gender or age. The swabs were streaked and sub-cultured three times in sterile nutrient agar plates and subsequently maintained on agar slants stored at 4 °C. The isolates were characterized and identified using gram staining, colony characterization, cetrimide agar, gelatin liquefaction, sodium chloride, and Mannitol fermentation tests [52]. An antimicrobial activity test was performed using the agar well diffusion method described by Balouiri et al. [7]. Briefly, sterile Muller Hinton agar plates were flooded with 1 × 106 cfu/ml concentration of microorganisms. Using a sterile cork borer (7 mm diameter), 6 wells were bored on the agar, and three drops of the METS (12.5, 25, 50, 100) mg/ml in 10% dimethyl sulfoxide (DMSO) were placed in the appropriate well. DMSO (10%) was used as control, while levofloxacin served as the standard drug. The plates were allowed 30 min for diffusion and incubated in an inverted form for 24 h at 37 °C.
Microbial sensitivity was determined in triplicate. After incubation, the diameter of the inhibition zone for each well was measured horizontally and vertically, and the mean was obtained. The minimum inhibitory concentration (MIC) was determined as the intercept on the concentration axis of concentration vs. the mean IZD2 plot.
Induction of carrageenan-induced rat paw edema
Twenty-five albino rats were weighed and randomly divided into five groups (n = 5) as follows:
Group I: Negative control and received oral administration of distilled water (2 ml/kg).
Group II: Positive control and received the standard drug, Indomethacin (25 mg/kg).
Group III: Treatment group and received 100 mg/kg of ME of T. superba.
Group IV: Treatment group and received 300 mg/kg of ME of T. superba.
Group V: Treatment group and received 600 mg/kg of ME of T. superba.
One hour after the treatments, 0.1 ml of 1% w/v carrageenan (phlogistic agent) in normal saline was injected into the sub-plantar region of the right hind paw of the rats, and the volume of the paw size was measured by water displacement method at times 0, 0.5, 1, 2, 3, 4, and 5 h after carrageenan injection [39].
The percent inhibition of edema was calculated using the following formula:
$${\text{Inhibition of edema }}\left( \% \right) \, = \frac{{V_{{\text{c}}} \, - \, V_{{\text{t}}} }}{{V_{{\text{c}}} }}$$
where Vc is the mean paw edema volume of control at each hour and Vt is the mean paw volume of treated animals at each hour.
After the 5th hour, paw supernatant was collected and used for the quantification of lipid peroxidation.
Quantification of lipid peroxidation and antioxidant enzyme
Paw supernatant lipid peroxidation was quantified as malondialdehyde (MDA) based on the method described by Katerji et al. [38]. The MDA level was calculated according to the method of Todorova et al. [70] and expressed as µg/ml. Superoxide dismutase (SOD) was assessed using a commercial kit (Biovision, Mountain View, CA, USA) obtained from a local representative and assayed according to the manufacturer’s protocol.
Topical edema of the mouse ear (xylene model)
The effect of the extract on acute topical edema was assessed using xylene-induced ear edema in mice. Mice were divided into four groups (n = 5). The animals were treated for 4 days. On the 4th day, topical application (5 mg/ear) of METS was applied on the anterior surface of the right ear, while xylene (0.05 ml) was instantly applied on the posterior surface of the same ear. Control animals received an equivalent volume of the vehicle (3% v/v Tween 80). The left ear was left untreated. Two hours after xylene application, animals were sacrificed and both ears were removed. Circular disks were punched out of the ear lobes using a cork borer (6 mm diameter) and weighed. The difference in the weight of disks from the right treated and left untreated ear was calculated and used as a measure of edema [65]. The level of inhibition (%) of edema was calculated using the relation:
$${\text{Inhibition }}\left( \% \right) \, = { 1}00\left[ {1 - \, \left( {R_{{\text{t}}} - \, L_{{\text{t}}} /R_{{\text{c}}} - \, L_{{\text{c}}} } \right)} \right]$$
where Rt is the mean weight of the right earplug of treated animals, Lt is the mean weight of the left ear plug of treated animals, Rc is the mean weight of the right earplug of control animals, and Lc is the mean weight of the left ear plug of control animals.
Evaluation of in vivo antioxidant activity of T. superba
Statistical analysis
Data obtained were analyzed by one- or two-way analysis of variance (ANOVA) followed by Turkey multiple comparisons post hoc test using Graphpad Prism version 5.0. The values were expressed as mean ± standard deviation (SD). p < 0.05 was considered statistically significant.
