Bioactive compounds, anti-inflammatory, anti-nociceptive and antioxidant potentials of ethanolic leaf fraction of Sida linifolia L. (Malvaceae)

2.7.1 Sample preparation

This study employed the methods outlined by Buss and Butle (2010) and Kelly and Nelson (2014). Graded amounts of ethanol (15 mL) and 50 % (m/v) potassium hydroxide (10 mL) were added into a test tube containing 1 g of the test sample. The mixture was allowed to stand in a water bath for 60 min at 60 °C. Thereafter, the resultant mixture was dispensed into a separatory funnel. The tube was washed successively with ethanol (20 mL), cold water (10 mL), hot water (10 mL), and hexane (3 mL) and collectively transferred into the funnel. After washing thrice with 10 mL of 10 % (v/v) aqueous solution of ethanol, drying with anhydrous sodium sulfate and solvent evaporation was performed. The resultant sample was dissolved in 1000 µL of pyridine, and 200 µL of it, was transferred to the vial for analysis.

2.7.2 GC-FID quantification

Phytochemical analysis of the test sample was performed on a BUCK M910 Gas chromatography Machine coupled with a flame ionization detector. The RESTEK 15-meter MXT-1 1 (15 m × 250 µm × 0.15 µm) column was used. Helium 5.0 pas was the carrier gas with a flow rate of 40 mL/min. The injector temperature was 280 °C with a splitless injection of 2 µL of sample and a linear velocity of 30 cm/s. The oven operated initially at 200 °C and was heated to 330 °C at a rate of 3 °C min⁻¹ which was maintained for 5 min. The detector was operated at a temperature of 320 °C. Quantification of the phytochemicals was done by evaluating the ratio of the area and mass of internal standard and the area of the identified phytochemicals. The concentration of the different phytochemicals was expressed in µg/g (which is equivalent to µg/mL and ppm).

2.8.1 Membrane stabilization tests

The preparation of red blood cells (RBC) suspension followed the Gandhisan et al. (1991) method with minor modifications. Blood samples were drawn from healthy human volunteers who had not taken any NSAIDs for at least three weeks before the collection date. The blood collection was done with tubes containing sodium oxalate. The whole blood was transferred into an equal volume of Alsever solution (0.8 % sodium citrate, 0.42 % NaCl, 2 % dextrose, and 0.5 % citric acid) and allowed to stand for 24 h at 4 °C. After then, the mixtures were centrifuged at 3000 × g for 10 min, and the packed cells volume was reconstituted to a 40 % (v/v) RBC suspension using phosphate-buffered saline (10 mM, pH 7.4), at room temperature (30 °C).

2.8.2 Inhibition of platelet aggregation assay

This study adopted the Born and Cross (1963) method with slight modifications . The principle of the test lies in CaCl₂ induction of platelet aggregation which is inversely proportional to the absorbance of the mixture at 520 nm. Essentially, the inhibitory effect of pharmacological agents on platelet aggregation correlates positively with the absorbance of the mixture after the addition of CaCl₂. Blood samples drawn from healthy human volunteers into EDTA bottles were centrifuged for 10 min at 3000 × g and the resultant supernatant diluted (twice) in normal saline was adopted as the platelet-rich plasma (PRP). A known volume (0.2 mL) of PRP was transferred into sets of three test tubes containing 1 mL of various concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) of the leaf fraction or reference drug (aspirin) in normal saline. The vehicle (normal saline) was used to raise the volume of the reaction mixtures to 2.2 mL. A test tube containing 2 mL normal saline and 0.2 mL PRP served as the control. Incubation at 30 °C for 5 min was performed after which CaCl₂ (0.4 mL of 1.47 %) was added to the reaction mixtures. The blanks contained the leaf fraction or reference drug void of PRP. All The experiment was done in triplicates. The change in absorbance of the mixture (at 520 nm) at 2 min intervals for 8 min was recorded, and the mean % inhibition was evaluated.

2.8.3 Protease inhibition assay

This study employed the method outlined by Sakat et al. (2010) with minor modifications. Graded volume (2 mL) of the reaction mixture consisted of 1 mL of various concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) of the leaf fraction or reference drug (ASP) and 0.06 mg trypsin dissolved in Tris HCl buffered solution (20 mM, pH 7.4). Incubation at 37 °C for 20 min and treatment with 70 % perchloric acid (2 mL) to halt the reaction was performed, after which the resultant cloudy suspension was centrifuged at 3000 × g for 10 min. Thereafter, the absorbance (Abs) of the supernatant was read at 210 nm (blanking with Tris buffer). The experiment was done in triplicates, and the inhibitory effect of the leaf fraction or standard drug at varying concentrations on protease activity compared with control was estimated with the formula as thus; $$\%\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{P}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}=1-\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{f}\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{f}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$

2.8.4 Inhibition of phospholipase A₂ assay

This study adopted the protocol described by Vane (1971) with slight modifications. The activity of phospholipase A₂ (PLA₂) is implicated in several inflammatory cascades leading to inflammation. Activated PLA₂ rips out membrane phospholipids (including arachidonic acid, the precursor molecule for the synthesis of several vasoactive molecules) and liberates hemoglobin (in erythrocyte) into the extracellular milieu. The ability of pharmacological agents to inhibit phospholipase A₂ activity on the cell membrane (for erythrocyte is directly proportional to the amount of oxidized hemoglobin liberated into the medium which can be estimated spectrophotometrically at 418 nm) correlates with their anti-inflammatory potentials. Briefly; isolation of the enzyme, PLA₂ was from a pure culture of the Aspergillus niger strain, while red cells suspension obtained from a volume (5 mL) of whole blood drawn from healthy human volunteers served as the substrate for PLA₂. Two sets of test tubes (labeled Blank and Test) contained equal volumes of RBC suspension (0.2 mL), CaCl₂ (0.2 mL), and phosphate-buffered saline (1 mL). A known volume (1 mL) of boiled and free enzymes was added to Blank and Test tubes, respectively. Thereafter, 1 mL of various concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) of leaf fraction or prednisolone (in phosphate-buffered saline) were dispensed separately into test tubes. The control test tube contained red cell suspension, CaCl₂, and free enzyme. Incubation at 30 °C for 1 h and centrifugation at 3000 × g for 10 min was performed on the mixtures after which, the supernatant absorbance of the mixtures was recorded at 418 nm. The peak % enzyme activity and inhibition were estimated using the formulae as follows; $$\%\mathrm{M}\mathrm{a}\mathrm{x}\mathrm{i}\mathrm{m}\mathrm{u}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$ $$\%\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=100-\%\mathrm{m}\mathrm{a}\mathrm{x}\mathrm{i}\mathrm{m}\mathrm{u}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{z}\mathrm{y}\mathrm{m}\mathrm{e}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}$$

2.8.5 Albumin denaturation assay

This study employed the method outlined by Mizushima and Kobayashi (1968) with minor modification. The reaction mixture contained varying concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) of the leaf fraction or reference drug (ASP) and 1 % bovine albumin leaf fraction in distilled water. The mixture pH was adjusted occasionally by adding a few drops of 1 N HCl. Incubation at 30 °C for 20 min followed by heating in a water bath for 30 min at 57 °C, then cooling to room temperature was performed, after which the turbidity of the reaction mixture was estimated at 660 nm spectrophotometrically. The test was done in triplicates, and the percentage inhibitory effect of the leaf fraction or standard drug at varying concentrations on albumin denaturation compared with control were estimated with the formula as thus; $$\%\mathrm{p}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=1-\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{f}\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{f}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$

2.9.1 Total antioxidant capacity (TAC)

This study adopted the Prieto et al. (1999) phosphomolybdenum method. The principle of the study lies in the propensity of antioxidant agents to reduce Mo⁶⁺ to Mo^5+, which results in green phosphate/Mo⁵⁺complex formation in an acid medium. The experiment mixture contains a graded volume (0.1 mL) of the leaf fraction or gallic acid at various concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) mixed with a known volume (1 mL) of the reagent solution [prepared by introducing 0.6 M sulfuric acid into tubes containing 28 mM sodium phosphate and 4 mM ammonium molybdate]. Incubation of the resultant mixtures for 90 min at 95 °C, was done, after which the test tubes were allowed to cool to room temperature, and the mixture absorbance was read at 695 nm in a spectrophotometer. Blanking was done with 0.1 mL methanol mixed with 1 mL of the reagent solution. The calibration curve was plotted by adding various concentrations of gallic acid into tubes containing methanol. The free radical scavenging activity was expressed as gallic acid-gram equivalent. The percentage free radical scavenging activity of the leaf fraction was evaluated using the formula as thus; $$\%\mathrm{S}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=1-\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{f}\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{f}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$

The IC₅₀ value, the concentration of the sample that scavenged 50 % of the initial concentration of the free radical in the mixtures was also evaluated graphically.

2.9.2 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical-scavenging assay

This study employed the method outlined by Liyana-Pathirana and Shahidi (2005). The test principle lies in the ability of antioxidant agents to donate an electron pair to DPPH radicals (resulting in the reduction of their distinctive purple colour to colorless). Briefly; 200 μL of 0.1 mM DPPH solution (in methanol) was dispensed into sets of test tubes containing 100 μL of various concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) of the leaf fraction or butylated hydroxytoluene (BHT) (standard). The mixtures thus formed were allowed to incubate at 37 °C for 15 min. The mixture's absorbance was registered at 517 nm with a spectrophotometer. The experiment was done in triplicates. The calibration curve was plotted by adding various concentrations of BHT into tubes containing methanol. The results of the assay were expressed as BHT-gram equivalent; the percentage free radical scavenging activity of the leaf fraction was estimated using the formula above;

2.9.3 Ferric reducing antioxidant power (FRAP) assay

This test adopted the Sahreen et al (2014) method. The principle of the test lies in the propensity of antioxidant agents to reduce Fe³⁺ to Fe²⁺ in an acid medium. Briefly; graded volume (2.0 mL) of the leaf fraction or standard drug (ascorbic acid) at various concentrations were dispensed into different test tubes containing 2.0 mL of 10 mg/L (0.1 % (w/v) potassium ferricyanide and 2.0 mL of 0.2 M, phosphate-buffered solution of pH 6.6. Incubation for 20 min at 50 °C in a water bath, followed by the introduction of 2.0 mL of 100 mg/L (10 % (w/v) trichloroacetic acid solution into the reaction mixtures, was performed. Thereafter, graded volume (2.0 mL) of the resultant mixture was dispensed into 0.4 mL of 0.1 % (w/v) ferric chloride (FeCl₃.6H₂0) solution, containing 2.0 mL of distilled water. After standing for 10 min, the absorbance of the mixture was recorded at 700 nm with a spectrophotometer. The calibration curve was plotted with various concentrations of ascorbic acid. The results of the assay were expressed as ascorbic acid-gram equivalent. The percentage scavenging activity of the leaf fraction and standard drug on the free radicals was evaluated using the formula above.

2.9.4 Nitric oxide free radical scavenging assay

This test adopted the Marcocci et al. (1994) method. Generation of nitric oxide radicals was done with sodium nitroprusside, and the Griess reaction was used to evaluate the degree of inhibition exerted by the test samples. Briefly; varying concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) the leaf fraction or reference (ascorbic acid) was dispensed into graded volume (5.0 mL) of phosphate-buffered sodium nitroprusside (5 mM) solution. Incubation of the resultant mixture for 20 min at 25 °C was performed. An equal volume of the buffer treated in a like manner void of the test solution served as the control. Standing for 5 h, a graded volume (0.5 mL) of Griess solution (2 % Ophosphoric acid, 0.1 % naphthyl ethylenediamine dihydrochloride, and 1 % sulfanilamide) was added to the mixtures, thereafter, the mixture absorbance was recorded at 546 nm with a spectrophotometer. The calibration curve was plotted with various concentrations of ascorbic acid. The results were expressed as the quantity in gram-equivalent of ascorbic acid. The total antioxidant capacity (in %) of the leaf fraction compared to the reference drug was estimated with the formula above.

2.10.1 Carrageenan-induced rat paw edema test

This test was performed according to the protocol described by Winter et al. (1962). Thirty minutes post-administration, fresh carrageenan (0.1 mL, 0.01 g/mL) solution was injected into the subplantar region of the left hind paw of mice. The volume of the paw was measured using a digital Plethysmometer device. In effect, the animal paw is immersed in a particular water cell containing water; the resistance of which changes as a result of the animal paw's immersion. The change in resistance (which corresponds to the volume displacement) is measured in milliliters and displayed on the Plethysmometer's electronic monitor. The volume displacement change was measured at time zero of the carrageenan injection into the mice's hind paw and every 1 h thereafter for 5 h. The difference in volume displacement following phlogistic agent administration and zero time volume displacement of the induced paw (At – Ao) was used to calculate the average edema in each period (Anosike et al., 2012). Evaluation of the % inhibition of paw edema was done using the formula as thus; $$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}(\%)\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{d}\mathrm{e}\mathrm{m}\mathrm{a}=\frac{(\mathrm{A}\mathrm{t}-\mathrm{A}\mathrm{o})\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-(\mathrm{A}\mathrm{t}-\mathrm{A}\mathrm{o})\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}}{(\mathrm{A}\mathrm{t}-\mathrm{A}\mathrm{o})\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$

• Here; (At – Ao) = instanteneous change in mouse paw edema volume.

• Ao = time zero mouse paw edema volume.

• At = various time intervals mouse paw edema volume.

2.10.2 Egg albumin-induced rat paw edema test

This test was performed according to the method described by Okokon and Nwafor (2010). The egg albumin edema model is a useful inflammation model for screening anti-inflammatory agents. Increased vascular permeability and swelling of the paw are signs of induced edema. The amplitude and extent of the inflammation cascades are reduced when the attendant edema is inhibited, indicating anti-inflammatory activities. One hour after various treatments, 0.1 mL of undiluted fresh egg albumin was injected (i.p.) into the peritoneal cavity of the right hind paw of mice. Changes in the mice right hind paw volume was recorded starting at time zero of the experiment and every-one hour after the injection with egg albumin, for 5 h (Anosike et al., 2012). In each interval, the average edema and % paw edema inhibition were assessed.

2.10.3 Acetic acid-induced writhing test

This test was performed according to the method outlined by Mbagwu et al. (2007), with minor modifications. The acetic acid writhing model has been used to evaluate analgesic and anti-inflammatory drugs. Acetic acid is injected into the peritoneal cavity of animals to induce writhing, indicative of pain. A stretch, extension of the hind limbs, abdominal contraction so that the animal trunk touches the floor, or twisting of the trunk are all signs of pain (Koster et al., 1959). The analgesic activity of the test compounds is determined by their propensity to decrease writhing frequency. Sixty minutes post-treatment, the animals were injected (i.p.) 0.6 % (v/v) (10 mL/kg bw) acetic acid. Thirty minutes, post-acetic acid injection, the number of writhes (characterized by abdominal constrictions) was recorded for 30 min at 5 min intervals, and the average responses of mice pretreated with varying dosages of the leaf fraction were compared with the control and standard group. The % inhibition of writhing represents the anti-nociceptive activity of the vehicle/reference drug/fraction. The % inhibition of writhing was calculated using the expression as follows; $$\%\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{w}\mathrm{r}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{i}\mathrm{n}\mathrm{g}=\frac{\mathrm{M}\mathrm{W}\mathrm{N}\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)-\mathrm{M}\mathrm{W}\mathrm{N}(\mathrm{T}\mathrm{e}\mathrm{s}\mathrm{t})}{\mathrm{M}\mathrm{W}\mathrm{N}\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}\times 100$$ where MWN = Mean number of writhing.

2.10.4 Formalin-induced arthritis test

This test was performed following the procedure described by Adeyemi and Akindele (2007), with minor modifications. Thirty minutes post-oral administration of the various treatment regimes, formalin (0.02 mL of 1 % v/v) solution was injected (i.p.) into the subplantar region of the right hind paw of mice. The amount of time (in sec.) spent by the mice licking and biting the injected paw, indicative of pain, was recorded for each animal. The formalin test is said to be biphasic. The first phase is characterized by a nociceptive response, which usually peaks 5 min after injection, while the second phase, indicated by a neurogenic and anti-inflammatory response peaks 15–30 min post-formalin injection. The mice responses of the first (0–5 min) and second phases (15–30 min) were recorded after formalin injection.

3.4.1 Membrane stabilizing effect of ELFSL

The membrane-stabilizing effect of ELFSL is shown in Table 3. From the result, ELFSL demonstrated excellent erythrocyte membrane stabilizing activity against hypotonicity- and heat-induced hemolysis in a concentration-dependent manner. The maximum membrane-stabilizing activities (63.19 ± 0.07 and 60.20 ± 0.71 %), exhibited by ELFSL against hypotonicity- and heat-induced hemolysis, respectively, were produced with the lowest dose (1.0 mg/mL) of the fraction; however, ASP showed more stabilizing potential (76.35 ± 0.48 and 76.69 ± 0.20 %), respectively, at a similar concentration (1.0 mg/mL). Our result agrees with Kumar et al. (2014), who reported effective membrane-stabilizing activity of extract from Sida cordata. The susceptibility of the lysosomal membrane to damage by injurious agents is critical in the modulation of inflammatory response. Injury on the lysosome may result in excessive release of proteolytic enzymes, which further exacerbates the inflammatory events (Chaitanya et al., 2011; Bukhari et al., 2013). Since, the erythrocyte membrane is an excellent model of the lysosomal membrane (Yoganandam et al., 2010), the ability of fraction to stabilize red cells membrane implies its lysosomal membrane-stabilizing potential (Anosike et al., 2012; Labu et al., 2015).

3.4.2 Effect of ELFSL on platelet aggregation

The inhibitory effect of ELFSL on platelet aggregation is shown in Table 3. The ELFSL displayed excellent inhibitory potential on CaCl₂-induced platelet aggregation in a concentration-dependent manner and was at par with the reference drug (aspirin). The peak inhibitory effect (60.60 ± 0.19 %) of the fraction was produced at the highest concentration (1.0 mg/mL). However, ASP demonstrated more potent (P < 0.05) anti-platelet aggregatory effect (76.35 ± 0.48 %) at similar concentration (1.0 mg/mL). The inhibitory effect of the leaf fraction on platelet aggregation may have followed the suppression of phospholipase A2 and cyclooxygenase activities, essential for the biosynthesis of thromboxane A2 (TXA2), a powerful platelet aggregator. When synthesized, TXA2 mediates platelet aggregation by increasing intracellular Ca2 + concentrations, facilitating the exocytosis of platelet granules and subsequent release of adenosine diphosphate (ADP) critical in platelet aggregation (Wang et al., 2021). The aggregation of platelets (thrombocytes) in the sight of injury initiates blood clotting and arrests bleeding; however, this facilitates the adhesion and diapedesis of leukocytes into the infected tissues and enhances endothelial responses to a variety of inflammatory stimuli (Hosseinzadegan and Tafti, 2017). Perhaps, the anti-platelet aggregatory action of ELFSL could also be related to a subsequent decrease in P-selectin and histamine activities, which, with platelet degranulation and aggregation, mediates an increase in vascular permeability and leucocyte extravasations (Zarbock et al., 2006). Platelet aggregation is associated with the pathophysiology of several diseases, including myocardial infarction, stroke, embolism, and other vascular thromboses (Fabre and Gurney, 2010). Therefore, the ability of the plant fraction to inhibit platelet aggregation suggests its antithrombotic and anti-inflammatory potentials. Our result correlates with Bahar et al. (2013) and Khan et al. (2014), which reported significant thrombolytic activity of Sida acuta and Sida rhombifolia, respectively.

3.4.3 Effect of ELFSL on protease activity

The inhibitory effect of ELFSL on protease activity is shown in Table 3. The ELFSL exhibited a significant (P < 0.05) degree of inhibition on protease activity that correlates positively with concentration. The maximum inhibition (64.33 ± 0.17 %) exerted by ELFSL occurred at the highest concentration (1.0 mg/mL). However, ASP showed significantly (P < 0.05) greater degree of protease inhibition (66.44 ± 0.13 %) at similar concentration (1.0 mg/mL). The observed inhibitory effect of the leaf fraction on protease activity may be attributable to its rich composition in phytochemicals known to inhibit protease activity, such as catechin, kaempferol, and other flavonoids. Studies have proposed the involvement of disproportionate release of proteases by immune cells or in lysosomal leakage in several inflammatory disorders (Bermúdez-Humarán et al., 2015; Enechi et al., 2019). Neutrophils being the first line of defense during an infection or tissue injury, upon activation and degranulation, secrete serine proteases such as proteinase 3, cathepsin G, and elastase into the extracellular environment, which degrade extracellular matrix proteins such as collagen, fibronectin, and elastin, facilitating leucocytes adhesion and diapedesis into the infected tissues (Pham, 2006; Korkmaz et al., 2008; Jakimiuk et al., 2021). Inhibition of proteases as a pharmacological target in treating various human diseases has proven to be a successful strategy in recent drug discoveries (Copeland et al., 2007). Elastase inhibitors are used to treat cancers, rheumatoid arthritis, and pulmonary disorders, among other ailments (Reboud-Ravaux, 2001; Tundis et al., 2015). Plants synthesize several protease inhibitors to protect them from insects, pest infestation, and herbivores (Srikanth and Chen, 2016). Flavonoids (flavan-3-ols, flavones, and flavanones) and other phenolics with two adjacent phenol groups are said to have inhibitory effects on trypsin-like serine proteases at the micromolar potency (Xue et al., 2017). As a result, the ability of ELFSL to efficiently block these hydrolytic enzymes could be due to its rich flavonoid composition.

3.4.4 Effect of ELFSL on phospholipase A₂ activity

The result of the inhibitory effect of ELFSL on phospholipase A₂ activity is shown in Table 3. The ELFSL also inhibited phospholipase A₂ in a concentration-dependent manner. The maximum inhibition (60.20 ± 0.71 %) occurred at the highest concentration (1.0 mg/mL). However, the reference drug (prednisolone) exhibited significantly (P < 0.05) greater phospholipase A₂ inhibition (67.11 ± 1.18 %) at similar concentration (1.0 mg/mL). These depict a close resemblance in the mechanism of action between the leaf fraction and the reference drug (prednisolone), a typical steroidal anti-inflammatory drug. The leaf fraction may have acted by inhibiting the enzyme activity and preventing the release of arachidonic acid from the cell membrane lipid bilayers, which is essential for the biosynthesis of eicosanoids (Coutinho and Chapman, 2011). Steroidal anti-inflammatory drugs, such as corticosteroids and other glucocorticoids, have a similar mechanism of action; the ability to inhibit phospholipase A₂ (Patel and Savjani, 2015). Moreover, an appreciable amount of steroids was detected in the leaf fraction, which may be attributable to the observed bioactivities. Our result correlates with that of Eze and Nwodo (2016).

3.4.5 Effect of ELFSL on protein denaturation

The result of the inhibitory effect of ELFSL on protein denaturation is shown in Table 3. The protein denaturation inhibition assay is a useful tool for determining the propensity of pharmacologic agents to reduce inflammation (Meredith, 2005; Enechi et al., 2019). The result showed that ELFSL exhibited an effective inhibitory effect on albumin denaturation in a concentration-dependent manner. The maximum inhibitory effect (56.57 ± 1.01 %) occurred at the highest concentration (1.0 mg/mL) of ELFSL. However, at similar concentration (1.0 mg/mL), ASP showed significantly (P < 0.05) higher inhibition (69.73 ± 0.47 %). The result suggests the ability of ELFSL to inhibit inflammation akin to the reference drug. Proteins lose their native structure and biological functions when they are denatured. This event usually results in increased recruitment of leukocytes into the infected tissues intending to eliminate the damaged proteins. However, this defensive response sometimes results in the indiscriminate destruction of healthy tissues by the immune cells (Ogbu et al., 2019). The ability of the fraction to inhibit protein denaturation suggests its anti-inflammatory properties. Our result correlates with that of (Shankar et al. 2021), who reported similar activities with Sida acuta ethanolic leaf extract.

3.6.1 Effect of ELFSL on carrageenan-induced edema in mice

The effect of ELFSL on carrageenan-induced edema in mice is shown in Table 5. Injection of carrageenan into the peritoneal cavity of the left hind paw of mice in the control group resulted in edema progression, which peaked (6.30 ± 0.20 mL paw volume increase) at 4 h post-phlogistic agent injection. Mice pre-treated with various doses of ELFSL showed a significant (P < 0.05) decrease in paw volume in a timely and dose-dependent manner compared with the control and was similar to that produced by the standard drug (100 mg/kg bw, po ASP). The maximum inhibitory effect (77.53 %) of ELFSL on carrageenan-induced edema occurred at the highest dose (600 mg/kg bw po, ELFSL) at the 5th hour. This effect was not significantly (P > 0.05) lower than that (77.80 %) recorded for 100 mg/kg bw (po) ASP at the same duration. The plant fraction at various doses exerted significant inhibition on all three phases of carrageenan-induced inflammation and peaked at the third phase (the 5th hour). The edema inhibition was statistically significant (P < 0.05) compared with the control and did not differ significantly (P > 0.05) from the reference drug (ASP). The ability of the fraction to inhibit the early phase of inflammation (1–2 h) in the carrageenan model suggests that it may have suppressed the release of histamine, serotonin, and bradykinin (Georgewill and Georgewill 2010; Georgewill et al., 2010). The events of the late phase are mediated by excessive activity of bradykinin protease, cyclooxygenase (COX) synthesis of prostaglandins, and the release of proteolytic enzymes from the lysosome (Wallace, 2002; Kang et al., 2008). From the result, it could be that the fraction inhibited the late phase of inflammation in this model by suppressing the release and activities of prostanoids (Ricciotti and FitzGerald, 2011). Aspirin (ASP), a typical NSAID and an excellent cyclooxygenase inhibitor capable of suppressing prostaglandin synthesis, also showed a similar inhibitory effect on the carrageenan model, suggesting a similarity in activities between the NSAID and the fraction. Our result aligns with Sutradhar et al. (2006), which reported similar activity with principles from Sida cordifolia.

3.6.2 Effect of ELFSL on egg albumin-induced mice paw edema

The effect of ELFSL on egg albumin-induced edema in mice is shown in Table 6. Intraperitoneal injection of egg albumin into the right hind paw of mice in the control group resulted in edema progression, which peaked 1 h post-induction (6.90 ± 0.10 mL increase in paw volume). However, mice pre-treated with various dosages of the leaf fraction showed a significant (P < 0.05) decrease in paw volume in a time-related and dose-dependent manner compared with the control and was akin to that of the reference drug (100 mg/kg bw, po ASP). The maximum inhibition (78.27 %) of egg albumin-induced edema occurred at the lowest dose (200 mg/kg bw, po) of ELFSL in the 5th hour. Moreover, this effect was not significantly (P > 0.05) lower than that (81.40 %) recorded for 100 mg/kg bw (po) ASP at similar time interval. Intraperitoneal injection of egg albumin into animal paws is known to provoke the release of vasoactive compounds, like histamine, and serotonin, which results in increased vascular permeability and edemogenesis. Perhaps, the fraction may have exerted its inhibitory effect on egg albumin-induced edemogenesis by inhibiting the release and vasodilatory activities of histamine and serotonin (Akindele et al., 2015). Aspirin, a powerful cyclooxygenase inhibitor, also showed a significant (P < 0.05) inhibitory effect on egg albumin-induced edema formation akin to the fractions, implying a similarity in anti-inflammatory potential between ASP and the fraction. John-Africa et al. (2013) reported similar results with Sida corymbosa leaf extract.

3.6.3 Effect of ELFSL on acetic acid-induced mice writhing

The effect of ELFSL on writhing induced in mice with acetic acid is shown in Fig. 2. Acetic acid injection into the peritoneal cavity of the control mice resulted in writhing syndrome, with 68.80 ± 1.10 writhes recorded in 30 min. The ELFSL significantly (P < 0.05) inhibited writhing syndrome in mice pre-treated with various doses of the leaf fraction in a dose-dependent fashion, compared with the control, and were comparable with the standard drug. The maximum inhibitory effect (66.54 %) exhibited by the leaf fraction occurred at the highest dose (600 mg/kg bw, po ELFSL). This did not differ significantly (P > 0.05) with the inhibition (62.20 %) demonstrated by 100 mg/kg bw ASP. Intraperitoneal injection of acetic acid in experimental animals increases vascular permeability and the levels of PGE2 and PGF2α in the peritoneal fluid, which results in writhing, indicative of pain (Bentley et al., 1983). Muscular contraction caused by acetic acid is seen as a nonselective nociceptive response because acetic acid interacts with nociceptors, sensitive to a variety of agonists, including narcotics, NSAIDs, and other CNSactive drugs such as morphine, by indirectly inducing the release of endogenous inflammatory mediators such as prostaglandin (Bighetti et al., 1999). The excellent inhibition of writhing, observed in mice treated with various dosages of ELFSL, which were at par with ASP, suggest the participation of a peripheral mechanism of anti-nociception by the fraction. Perhaps it could be that the model is associated with peripheral receptors stimulation, particularly the local peritoneal receptors expressed on the surface of the cells lining the peritoneal cavity (Zakaria et al., 2008). The NSAID, aspirin, also showed a significant (P < 0.05) anti-nociceptive effect in this model, although ELFSL was more potent at a higher dose (600 mg/kg bw, po). Our results correlate with those of Azad et al. (2017), which reported potent anti-nociceptive activities with S. rhombifolia to extract on the acetic acid model.

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Fig. 2

Effect of ELFSL on acetic acid induced writhing in mice, * Control = 10 mg/kg bw distilled water, n = 8; Values (analyzed with One-way ANOVA and Duncan post hoc) are presented as Mean ± SEM at P < 0.05.

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3.6.4 Effect of ELFSL on formalin-induced hind mice paw licking

The effect of ELFSL on formalin-induced edema in mice is shown in Fig. 3. In the first phase, formalin injection into the peritoneal cavity of the left hind paw of mice in the control group produced nociceptive reactions of licking and biting of inflicted paws for an extended duration of 312.01 ± 14.01 sec. However, mice pre-administered various doses of ELFSL spent significantly (P < 0.05) lesser time range (196.2 ± 5.0–205.2 ± 6.0 sec) in licking and biting the inflicted paw compared with the control and was comparable with that (181.8 ± 9.0 sec) observed in the group administered 100 mg/kg bw, po ASP. The inhibitory effect of the leaf fraction followed a dose-dependent trend with peak inhibition (50.08 %) elicited by the 600 mg/kg bw po ELFSL dosage, which was not significantly (P > 0.05) different from that (54.20 % inhibition) of the reference drug. In the second phase, the time spent by the untreated animals (control) in biting and licking the affected paw, indicative of nociception, was 306.1 ± 14.06 sec, while mice pretreated with various doses of ELFSL spent significantly (P < 0.05) lesser time range (196.2 ± 5.0–205.2 ± 6.0 sec) in paw licking. This effect was comparable to that (181.8 ± 9.0 sec) observed in the group administered aspirin. The maximum inhibitory effect (78.28 %) demonstrated by the leaf fraction occurred at the 600 mg/kg bw (po) ELFSL dose, and this did not differ significantly (P > 0.05) from that (89.90 % inhibition) obtained with 100 mg/kg bw (po) ASP. The formalin model is proposed to complement the acetic acid writhing model to distinguish between central and peripheral mechanisms of anti-nociception (Chan et al., 1983; Okokon et al., 2012). Intraperitoneal injection of formalin induces an increase in spontaneous activity of C fiber afferents and instigates distinctive and measurable responses, such as animal paw licking, indicative of pains (Akindele et al., 2015). Paw licking induced by formalin injection into mice's paw peritoneal cavity produces two distinctive phases of nociceptive reactions (Hunskaar et al., 1985). The first phase of paw licking (0–5 min) is caused by the direct interaction of formalin with certain nociceptors (Zakaria et al., 2008), while the second phase (15–30 min) is characterized by inflammatory events (Tjolsen et al., 1992). Drugs that act via the central nervous system (CNS), such as morphine, suppress both phases of nociception, while NSAIDs, such as ASP, that act via the peripheral nervous system, inhibit only the later stage (Chan et al., 1983). The formalin test suggests that ELFSL significantly (P < 0.05) inhibited nociceptive responses in both phases in mice, depicting the participation of both peripheral and central mechanisms of anti-nociception. The result of our study correlates with that of Bonjardim et al. (2011), which demonstrated similar results using Sida cordifolia leaf extract.

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Fig. 3

Effect of ELFSL on formalin-induced hind paw licking in mice, *Control = 10 mg/kg bw distilled water, n = 8; Values (analyzed with One-way ANOVA and Duncan post hoc) are presented as Mean ± SEM at P < 0.05.

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