Anti-Inflammatory Activity of Peptic–Tryptic Cowpea ( Vigna Unguiculata ) Β-Vignin Hydrolysate in In Vitro Membrane Stabilization and Formaldehyde-Induced Inflammation Models

BUHREC536/20. Acute Oral Toxicity Testing The median lethal dose (LD50) was determined using the limit dose test of the up-and-down procedure according to the Organization for Economic Co-operation and Development Test Guideline 425. A single dose of the cowpea hydrolysate was administered via oral gavage (between 2000 mg/kg and 5000 mg/kg b.w.) to 5 rats sequentially at 48 h intervals. Animals were fasted overnight prior to dosing, and their fasted body weights were determined. Following administration, the rats were observed continuously for 14 days for signs of toxic symptoms (weakness, loss of appetite, difficulty in movement, reaction to noise) and mortality. In Vivo Formaldehyde-Induced Inflammation Model The in vivo anti-inflammatory evaluation was carried out following the method described by Agnel & Shobana (2012), with modifications by Anyasor et al. (2019). Thirty rats were randomly divided into six groups of five animals each and treated orally for 7 days according to the following design: • Group I (Normal Control): Administered 0.2 mL of 0.9% NaCl saline solution. • Group II (Inflamed Control): Administered 0.2 mL of 0.9% NaCl saline solution + sub-plantar formalin induction. • Group III (Standard Group): Administered 10 mg/kg b.w. diclofenac sodium + sub-plantar formalin induction. • Group IV (Low-Dose Test Group): Administered 100 mg/kg b.w. cowpea hydrolysate + sub- plantar formalin induction. • Group V (Mid-Dose Test Group): Administered 300 mg/kg b.w. cowpea hydrolysate + sub- plantar formalin induction. • Group VI (High-Dose Test Group): Administered 500 mg/kg b.w. cowpea hydrolysate + sub- plantar formalin induction. On the first and third days of treatment, 0.02 mL of 2% (v/v) formaldehyde was injected into the sub-plantar region of the left hind paw of the rats to induce localized arthritis/inflammation. Linear paw thickness was measured using a micrometer screw gauge 30 min before formal induction and every 24 h for 7 consecutive days. The percentage inhibition of inflammation was calculated as stated below: % Inhibition of inflammation = (1 −Vt Vc) × 100 Where Vt is the mean paw edema in the treated groups while Vt is the mean paw edema in the control group Haematological and Liver Transaminase Assays On day 8, blood samples were collected via cardiac puncture using 5 mL hypodermal syringes and transferred into EDTA and heparinized tubes. Whole blood samples collected in EDTA tubes were immediately analyzed using an automated hematology analyzer (Swelab Alfa 3-Part Analyzer, Boule Medicals, Sweden) to quantify White Blood Cells , lymphocyte percentages (Lymph%), granulocyte percentages (Gran%), Eosinophil-Monocyte-Basophil fractions, and platelet counts. Blood samples in heparinized tubes were centrifuged at 3500 rpm for 5 min to separate plasma. Plasma activities of alanine aminotransferase and aspartate aminotransferase were measured spectrophotometrically using commercial diagnostic kits (Randox Laboratories, United Kingdom) to monitor liver membrane stability. Statistical Analysis Data analysis was performed using GraphPad Prism (Standard Version 6.0). Inter-group variations were evaluated using One-Way Analysis of Variance , followed by post-hoc comparisons to separate distinct means. Results are expressed as the mean of replicates ± standard error of the mean , with statistical significance defined at p < 0.05. Results and Discussion Acute Toxicity Study Single oral administration of the cowpea hydrolysate at doses ranging from 2000 to 5000 mg/kg body weight produced no mortality during the 14-day observation period. No obvious signs of toxicity, including weakness, loss of appetite, difficulty in movement, or other abnormal behavioural responses, were observed. The estimated oral LD50 was therefore greater than 5000 mg/kg body weight under the conditions of the study. Thin Layer Chromatography TLC screening verified the progressive degradation of cowpea β-vignin. Intact β-vignin (Lane A) showed minimal migration, remaining concentrated near the baseline origin because of its long chain structure and high molecular weight. The peptic intermediate (Lane B) demonstrated enhanced upward migration, indicating initial breakdown. The final peptic-tryptic digest (Lane C) left no residual protein footprint at the origin line, confirming effective cleavage into smaller peptide fragments. Furthermore, subsequent TLC evaluation of the filtrate (Lane D) confirmed the successful removal of large macromolecular contaminants following passage through the 0.22 μm membrane filter, yielding a clarified pool of mobile, low-molecular-weight peptides. Figure 1: Thin layer chromatography showing the hydrolytic effect of pepsin and trypsin on β-vignin. Legend: A – undigested β-vignin; B – peptic β-vignin digest; C – peptic-tryptic β-vignin digest. Figure 2: Thin layer chromatography showing the efficacy of syringe filter in the partial purification of peptic-tryptic digest of β- vignin. Legend: B – peptic β-vignin digest; C – crude peptic-tryptic β-vignin digest; D – partially purified β-vignin. Heat-induced hemolysis The cowpea hydrolysate demonstrated concentration-associated membrane-stabilizing activity in the heat-induced haemolysis assay. The percentage inhibition of erythrocyte haemolysis ranged from 15.23 ± 0.41% to 37.86 ± 1.09% over the tested concentration range of 50–400 μg/mL. Diclofenac sodium produced inhibition ranging from 23.45 ± 0.71% to 45.26 ± 1.09%. Diclofenac sodium showed significantly greater inhibition than the hydrolysate at the tested concentrations (p < 0.05). 50 100 200 300 400 0 10 20 30 40 50 Diclofenac sodium (Standard) Hydrolysate Concentration (ug/mL) % Stabilization a a a a a Hypotonicity-induced heamolysis The hydrolysate exhibited concentration-dependent membrane-stabilizing activity in the hypotonicity-induced haemolysis assay. Between 100 and 400 μg/mL, membrane stabilization ranged from 18.53 ± 0.06% to 24.08 ± 0.08%, compared with 22.44 ± 0.082% to 25.17 ± 0.11% for diclofenac sodium. At 500 μg/mL, the hydrolysate produced 25.87 ± 0.08% membrane stabilization, while diclofenac sodium produced 25.43 ± 0.07%. The difference between the two treatments at 500 μg/mL was not statistically significant. Figure 2: Effect of peptic–tryptic cowpea β-vignin hydrolysate and diclofenac sodium on heat-induced haemolysis of human erythrocytes at different 100 200 300 400 500 0 10 20 30 Diclofenac sodium (Standard) Hydrolysate Concentration (ug/mL) % Stabilization a a a b a Formaldehyde-Induced Paw Oedema Oral administration of cowpea hydrolysate at 100, 300, and 500 mg/kg significantly inhibited formaldehyde-induced paw oedema compared with the untreated inflamed control group (p < 0.05). The effect increased with dose. At 100 mg/kg, the hydrolysate produced 15.44% inhibition, corresponding to a paw-oedema value of 1.33 ± 0.26 mm. At 300 mg/kg, inhibition increased to 25.32%, with paw oedema of 0.67 ± 0.25 mm. At 500 mg/kg, the highest inhibition was observed at 36.81%, with paw oedema of 0.29 ± 0.03 mm. Diclofenac sodium at 10 mg/kg produced 23.33% inhibition, with a paw-oedema value of 0.95 ± 0.35 mm. Thus, the 500 mg/kg hydrolysate produced a greater percentage inhibition of paw oedema than the diclofenac-treated group in the reported endpoint. Figure 3: Effect of peptic–tryptic cowpea β-vignin hydrolysate and diclofenac sodium on hypotonicity-induced haemolysis of human erythrocytes 1 2 3 4 5 6 7 0 1 2 3 4 Days Change in Paw Thickness Normal control(untreated) 10mg/kg b.w diclofenac sodium 100mg/kg b.w hydrolysate 300mg/kg b.w hydrolysate 500mg/kg b.w hydrolysate 0% 15.44% 23.33% 25.32% 36.81% Plasma Alanine Aminotreansferase activity Plasma ALT activity in the untreated inflamed group was 3.56 ± 0.07 U/L, compared with 2.904 ± 0.21 U/L in the normal control group. The 100 mg/kg hydrolysate-treated group had an ALT value of 3.72 ± 0.22 U/L, which did not differ significantly from the untreated inflamed group. Treatment with 300 and 500 mg/kg hydrolysate significantly reduced ALT activity to 2.65 ± 0.23 U/L and 2.43 ± 0.35 U/L, respectively, compared with the untreated inflamed control group (p < 0.05). The decrease occurred in a dose-dependent manner. Figure 4: Change in paw edema by different doses of peptic-tryptic β-vignin hydrolysate in formaldehyde inflamed rats Normal Untreated(control) DS (10mg/kg b.w) CH (100mg/kg b.w) CH (300mg/kg b.w) CH (500mg/kg b.w) 0 1 2 3 4 5 Plasma alanine aminotransferase activity (U/I) Treatment groups a a b b b Plasma Aspartate Aminotransferase activity AST activity increased from 0.99 ± 0.16 U/L in the normal control group to 1.30 ± 0.04 U/L in the untreated inflammation group. Treatment with the cowpea peptic–tryptic hydrolysate produced a dose-related reduction in AST activity. The values were 1.35 ± 0.05 U/L, 0.91 ± 0.04 U/L, and 0.69 ± 0.09 U/L at doses of 100, 300, and 500 mg/kg body weight, respectively. The diclofenac- treated group recorded 0.85 ± 0.10 U/L. The 300 and 500 mg/kg hydrolysate groups showed significantly lower AST activity compared with the untreated inflammatory group (p < 0.05), with Figure 5: Effects of peptic–tryptic cowpea β-vignin hydrolysate on plasma ALT activity in normal and formaldehyde-inflamed rats DS – diclofenac sodium CH – cowpea hydrolysate the 500 mg/kg dose producing the lowest AST value. Normal Untreated (control) DS (10mg/kg b.w) CH (100mg/kg b.w) CH (300mg/kg b.w) CH (500mg/kg b.w) 0 1 2 Plasma aspartate aminotransferase activity (U/I) Treatment groups a a b b a Haematological analysis The result in Table 4.2 shows that 100 and 300mg/kg b.w cowpea peptide hydrolysate treated animals induced with formaldehyde had (p<0.05) a significant difference in their WBC, lymph and gran counts while the 500mg/kg b.w cowpea peptide hydrolysate treated animals showed no significant difference when compared with the untreated control group. Futhermore, only the 100mg/kg b.w cowpea peptide hydrolysate treated animals had (p<0.05) a significant difference in the EMB counts. 300mg/kg b.w cowpea peptide hydrolysate treated animals had (p<0.05) a significant difference in the PLT counts. However, at p<0.05, there was a significant difference in the WBC and PLT counts and no difference in the lymph and gran counts between 10mg/kg b.w diclofenac sodium treated animals Figure 6: Effects of peptic–tryptic cowpea β-vignin hydrolysate on plasma AST activity in normal and formaldehyde-inflamed rats. DS – diclofenac sodium CH – cowpea hydrolysate and the untreated control group. RBC, HCT and HGB did not differ significantly from the untreated inflamed control. Paramete rs Normal Untreate d (control) Diclofenac 10mg/kg Hydrolysa te 100mg/kg Hydrolysat e 300mg/kg Hydrolysat e 500mg/kg WBC 10^3/μL 7.16±1.02 11.21±2.0 5 8.275±1.96 7±1.54 8.367±1.76 10±2.14 Lymph % 33±2.68 36.8±3.74 39.75±3.94 30.8±1.59 23.33±2.03 44.67±8.41 Gran % 65.2±3.01 59.8±4.16 58.5±3.61 67.6±1.91 75±3.22 53.67±9.21 EMB (10^9/L) 2±0.77 3.2±0.73 1.75±0.85 1.6±0.51 2±1.53 1.67±0.88 RBC 10^12/L 7.34±0.24 7.13±0.31 6.72±0.61 7.696±0.30 7.843±0.32 6.99±0.30 HCT % 39.52±1.7 5 38.4±0.75 36.825±3.95 41.04±1.72 41.067±2.1 7 40.4±3.07 HGB g/dL 12.96±0.6 0 12.92±0.2 6 12.2±1.18 13.94±0.77 14.07±0.55 12.93±0.84 PLT 10^9/L 618.4±58. 29 337±121. 47 691.75±208. 20 454±16.73 530.33±96. 88 482.33±10. 48 White blood cell , lymphocytes (Lymph%), granulocyte (Gran%), red blood cell , haematocrit (HCT %), haemoglobin , platelet and Eosinophil, monocyte and basophil The present study demonstrates that peptic–tryptic hydrolysis of cowpea β-vignin generated a hydrolysate with measurable anti-inflammatory activity in both in vitro and in vivo models. These findings are significant because this biological protection was demonstrated using complementary experimental approaches: the stabilization of human erythrocyte membranes under chemically or physically induced stress and the systematic suppression of formaldehyde- induced paw oedema in Wistar rats. Erythrocyte membrane stabilization provides a reliable preliminary indication of systemic membrane-protective activity (Shenoy et al., 2010). Exposure of erythrocytes to hypotonic conditions or elevated temperatures disrupts membrane structural integrity, altering ion channels and promoting cellular haemolysis (Sakat et al., 2010). Compounds capable of stabilizing the erythrocyte membrane interface can prevent this lysis, minimizing the release of intracellular components. This assay serves as an effective analogue for investigating lysosomal membrane stability in vitro (Shinde et al., 1999). Preventing the extracellular leakage of lysosomal constituents such as acid hydrolases and proteases from activated neutrophils is a key step in limiting tissue damage and the amplification of local inflammatory cascades (Shenoy et al., 2010). In the heat-induced haemolysis assay, the cowpea hydrolysate inhibited erythrocyte lysis by 15.23–37.86% across the tested concentration range. While this was lower than the protection shown by the reference drug, diclofenac sodium (23.45-45.26%), it confirms a clear, concentration-associated protective effect. The lower relative activity of the hydrolysate is expected; diclofenac sodium represents a pure, highly targeted pharmacologically active agent, Table 1: Effects of peptic-tryptic hydrolysate of β-vignin on the hematological parameters of rats subjected to formaldehyde-induced inflammation whereas the filtered hydrolysate is a complex mixture of diverse peptide chains with varying configurations (Chakrabarti et al., 2018). A similar pattern was observed in the hypotonicity-induced haemolysis assay, where the hydrolysate produced 18.53–24.08% stabilization between 100 and 400 μg/mL, compared with 22.44–25.17% for the reference drug. Notably, at the highest tested concentration of 500 μg/mL, the hydrolysate achieved 25.87 ± 0.08% stabilization, matching the standard diclofenac sodium group (25.43 ± 0.07%). This lack of statistical variance indicates that increasing the concentration of the hydrolysate may effectively compensate for its lower relative activity observed at lower doses. The in vivo findings provide further evidence of anti-inflammatory efficacy within the 2021–2023 experimental window. Sub-plantar administration of 2% formaldehyde into the hind paw triggers a localized inflammatory response, resulting in severe paw oedema. Oral treatment with the cowpea hydrolysate at 100, 300, and 500 mg/kg b.w. significantly reduced paw swelling relative to the untreated inflamed control. The percentage inhibition increased progressively from 15.44% at 100 mg/kg to 25.32% at 300 mg/kg, and reached 36.81% at 500 mg/kg. This dose- related response confirms that the hydrolysate exerts a predictable, biologically meaningful effect rather than producing an isolated response at a single concentration. At the 500 mg/kg b.w. threshold, the hydrolysate achieved a higher percentage inhibition of paw swelling (36.81%) than the 10 mg/kg diclofenac group (23.33%). However, this comparison must be interpreted cautiously. The two agents are chemically distinct and were administered at substantially different doses. The appropriate conclusion is that the cowpea hydrolysate demonstrates substantial anti-inflammatory efficacy in this experimental model, rather than claiming a higher absolute potency than diclofenac. The underlying molecular mechanism cannot be established conclusively from these experiments because the specific peptide sequences were not isolated. However, sequential enzymatic cleavage by pepsin and trypsin released specific amino acid sequences capable of interacting with biological targets involved in acute inflammation (de Medeiros et al., 2022). Trypsin preferentially cleaves peptide bonds at the carboxyl side of lysine and arginine residues, while pepsin targets hydrophobic or aromatic residues (Mojica & de Mejía, 2016). This combined cleavage pattern releases low-molecular-weight, amphipathic peptide fractions. Previous research indicates that such food-derived fragments can downregulate inflammatory pathways by modifying the production of nitric oxide, prostaglandins, cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines (de Medeiros et al., 2022). The biochemical markers provide additional insights into the systemic responses of the animal models. The untreated inflamed group showed an elevation in plasma ALT activity (3.56 ± 0.07 U/L) compared to the normal healthy baseline (2.904 ± 0.21 U/L), showing systemic stress from formaldehyde exposure. Oral administration of the hydrolysate at 300 and 500 mg/kg b.w. significantly reduced plasma ALT activity in a dose-dependent manner. Plasma AST levels followed a matching trend: the untreated inflamed group exhibited elevated activity (1.30 ± 0.04 U/L), which was systematically reduced in both the diclofenac sodium and 300 mg/kg hydrolysate groups. The AST value for the 500 mg/kg b.w. group was verified from the raw records; the true value is 0.69 ± 0.09 U/L, representing the lowest treatment value. This dose-dependent reduction in circulating transaminases indicates that the cowpea hydrolysate may stabilize structural tissue membranes in vivo, mitigating enzyme leakage into the plasma during an inflammatory challenge. However, because ALT and AST variations alone do not fully establish hepatoprotection, this interpretation remains provisional until validated by future histopathological tissue examination. The hematological profiles also support the presence of an active anti-inflammatory response. The untreated inflamed control group showed a sharp increase in total WBC counts, which is consistent with the activation, proliferation, and recruitment of immune cells to sites of local tissue injury (Okoli et al., 2008). Daily oral administration of the cowpea hydrolysate lowered total WBC counts at 100 and 300 mg/kg b.w. These treatment groups also showed significant modulations in lymphocyte and granulocyte percentages, while the 100 mg/kg b.w. dose altered the EMB count. These findings demonstrate that the peptide hydrolysate can influence the systemic leukocyte response associated with formaldehyde-induced inflammation. The platelet parameters require a similarly cautious interpretation. The untreated inflamed group showed a lower platelet count than the normal group, while the 300 mg/kg b.w. hydrolysate group showed a significant difference. While platelets contribute to homeostasis, cellular signaling, and tissue repair, these experiments do not directly demonstrate a wound-healing effect. Thus, these variations are best reported as part of the overall hematological response rather than definitive proof of accelerated tissue regeneration. Importantly, red blood cell counts, hematocrit , and hemoglobin concentrations showed no significant differences from the untreated inflamed group. This preservation of erythrocyte indices suggests that the experimental peptide treatments did not produce adverse toxic effects on erythropoiesis during the study period. This safety profile is supported by the acute oral toxicity study. A single oral dose up to 5000 mg/kg b.w. caused no mortality or visible signs of clinical toxicity, establishing that the calculated LD50 is greater than 5000 mg/kg b.w. under these experimental conditions. While this confirms favorable short-term tolerability in rat models, it does not establish long-term safety profiles or clinical safety in humans. In conclusion, the in vitro membrane stabilization trends, dose- dependent inhibition of localized paw edema, modulation of leukocyte indices, and reduction of plasma transaminase activities indicate that peptic–tryptic cowpea β-vignin hydrolysate possesses clear anti-inflammatory activity. Future investigations utilizing downstream chromatographic fractionation, mass spectrometry sequencing, cytokine profiling, and tissue histology are recommended to fully resolve the specific molecular mechanisms of action. Conclusion Peptic-tryptic hydrolysis of cowpea β-vignin produced a hydrolysate with measurable anti- inflammatory activity in vitro and in vivo. The hydrolysate inhibited heat- and hypotonicity- induced erythrocyte haemolysis, indicating membrane-stabilizing activity, and significantly reduced formaldehyde-induced paw oedema in rats in a dose-dependent manner, with the highest inhibition of 36.81% observed at 500 mg/kg body weight. Treatment was also associated with reductions in plasma ALT at 300 and 500 mg/kg and changes in selected leukocyte and platelet indices, while RBC, HCT, and HGB remained relatively unaffected. Acute oral administration up to 5000 mg/kg produced no mortality or obvious signs of toxicity during the 14-day observation period. These findings support further investigation of cowpea β-vignin hydrolysate as a potential source of anti-inflammatory peptides. Future studies should isolate and characterize the active peptide fractions and determine their effects on specific inflammatory mediators and signalling pathways. Ethical Statement The animal experiments were conducted in accordance with the ethical standards established by the relevant Animal Research Ethics Committee and the Babcock University Health Research and Ethics Committee.