Angiotensin‐1‐converting enzyme inhibition, antioxidant activity, and modulation of cerebral Na+/K+ ATPase by free phenolics of African locust bean (Parkia biglobosa)

Abstract Aims To investigate the antioxidant activities and effects of free phenols (FPPB) and bound phenols (BPPB) of Parkia biglobosa leaves on some enzymes of neuro‐cardiovascular relevance. Methods and Results HPLC‐DAD fingerprinting of FPPB and BPPB, and the antihemolytic, radical (1,1‐diphenyl‐2 picrylhydrazyl, DPPH; 2,2‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid), ABTS) scavenging and ferric reducing antioxidant properties of extracts, were assessed. In addition, the effects of the phenolics on angiotensin‐1‐converting enzyme (ACE), cerebral acetylcholinesterase/butyrylcholinesterase (AChE/BuChE), and Na+/K+ATPase were determined in vitro. FPPB was more potent than BPPB in terms of ABTS (EC50:4.06 ± 0.3 vs 24.07 ± 2.1 μg/mL) and DPPH (EC50:3.82 ± 0.2 vs 10.22 ± 0.1 μg/mL) radicals scavenged, respectively. The free phenolic extract was a better DPPH. scavenger than ascorbic acid (EC50 = 12.58 ± 0.4 μg/mL; DPPH reference) and compared well with Trolox (EC50:4.44 ± 0.08 μg/mL; ABTS reference). The anti‐hemolytic effect of FPPB (36%) and BPPB (53%) was highest at 15 μg/mL but lower than that recorded for ascorbic acid (67% at 10 μg/mL). Even though FPPB (IC50 = 15.35 ± 4.0 μg/mL) and BPPB (IC50 = 46.85 ± 3.3 μg/mL) showed considerably lower ACE‐inhibitory effect than ramipril (IC50:0.173 ± 0.04 μg/mL), both extracts demonstrated dose‐dependent, significant (p < 0.01/p < 0.05) inhibition of the enzyme. FPPB increased cerebral Na+/K+ATPase activity but neither phenolic extract affects cerebral AChE/BuChE activities. HPLC‐DAD revealed catechin, caffeic acid, and quercetin, respectively, as the major phenolics (mg/g) in FPPB (29.85, 30.29, and 17.10) and BPPB (32.70, 30.51, and 19.25). Conclusion The effects of P biglobosa on ACE and cerebral ATPase are related to its constituent phenolics. ACE inhibition could be an important mechanism underlying the documented hypotensive effect of the plant.

their roles in attenuating oxidative stress, plant phenolics could also provide other health benefits related to or distinct from their antioxidant property. For instance, the widely documented inhibition of angiotensin-converting enzyme (ACE) by plant extracts and/or plant-derived compounds was said to be largely dependent on the phenolic constituents, including flavonoids. [3][4][5][6] One plausible mechanism of ACE inhibition by phenolics, notably the flavonoids, is the generation of chelates capable of forming complexes within the active centre of ACE thereby inactivating the enzyme. 3,6 Phenolic compounds exist as either free, solvent extractable form or covalently bound to the plant matrix in plant cells. Extraction of the latter into water or aqueous/organic solvents mixtures is virtually impossible. 7 It is pertinent to give considerable attention to the bound forms, which constitutes 4% to 57% of total phenolic in plants, to prevent underestimating the antioxidant contents, activities as well as the therapeutic efficacy of different medicinal plants. 8 Parkia biglobosa (Jacq.) Benth., commonly known as "African locust bean," is a tropical tree in West Africa popular for its uses as food and medicine. In Southwestern Nigeria, the seeds are fermented to make Iru-a strong smelling and tasty soup condiment rich in protein. 9 Ethnomedicinally, the plant has wide applications in the treatment of hypertension and fevers 10 in tropical Africa. The crude hydromethanolic extract of the leaf was reported to lower blood pressure, 11 exhibit cardioprotective effect against doxorubicin toxicity 12 and protect against neurotoxic agents in rat brain hippocampal slices. 13 Preliminary phytochemical investigation of P biglobosa leaf extract revealed the presence of polyphenols, saponins, cardiac glycosides, terpenoids, and alkaloids, 12 while further fingerprinting of the phenolics gave caffeic acids, quercetin, and catechin derivatives as the principal antioxidant phytochemicals in the crude extracts (CEs) of the leaf and bark of the plant. 9,13 The therapeutic mechanisms of P biglobosa are still not well understood, and little or no information is available on the effects of its component phenolics on key enzymes of cardiovascular and neurological relevance. The present study, therefore, sought to compare the antioxidant property of the free and bound phenolics of P biglobosa and evaluate their effects on angiotensin-1 converting enzyme as well as cerebral acetylcholinesterase, butyrylcholinesterase, and Na+/K+-ATPase in vitro. All other reagents and chemicals were of analytical grade.

| Animals
Male albino rats (Wistar strain) weighing between 270 and 320 g were used for this study. They were kept in cages and provided food and water ad libitum and maintained in a room with controlled temperature (22°C ± 3) with 12-h light/dark cycle. The use and maintenance of animals were in line with the guidelines of the Brazilian Association for Laboratory Animal Science.
2.6 | Antioxidant assays 2.6.1 | Total antioxidant activity The ABTS test was employed to measure the total antioxidant activity as described previously. 9 The Trolox equivalent antioxidant capacity (TEAC) was obtained by calculating the percentage inhibition of absorbance and plotting it as a function of the concentration of standard and sample. Division of the straight line gradient of the plot for the sample by that of Trolox gave the TEAC (graphs not shown).

| DPPH radical scavenging activity
DPPH radical-scavenging activity of P biglobosa extract and reference compound (ascorbic acid) were determined using a standard method. 9 The ability of both FPPB and BPPB to scavenge the DPPH radical was monitored at 517 nm.

| Reducing property
The ferric reducing property of both FPPB and BPPB was determined as previously described 9 using the synthetic antioxidant, butylated hydroxytoluene (BHT) as a reference compound.

| Anti-hemolytic effect
The anti-hemolytic activity of the phenolic extracts was determined as described by Yang et al. 15 Blood (5 mL

| Angiotensin-converting enzyme (ACE) inhibition assay
The assay was based on the hydrolysis of N-hippuryl-His-Leu hydrate (HHL) by the ACE as described by Cushman and Cheung. 18 The lung capillaries are the major repositories of ACE. 4 Freshly removed rat lungs were therefore used as the enzyme source in the present study according to previous reports. 19

| Statistical analysis
Values are expressed as mean ± SEM/SD of replicate measurements (n = 3). Unless otherwise stated, statistical evaluation was done using 1-way analysis of variance followed by Dunnett's Multiple Range Test.

| DISCUSSION
The hydroxyl groups of polyphenols often make them capable of contributing directly to antioxidative actions. 21 Although the  It could be postulated that P biglobosa phenolics reduce the stable DPPH radical to the corresponding hydrazine following reaction of the radicals with hydrogen donors in the antioxidant principle. 9 The radical scavenging activity and reducing potential of FPPB, when compared with the reference compounds in the present study, revealed the promising profile of the free phenolics of P biglobosa as a potent antioxidant source.
The erythrocytes are among the most abundant cells in the body.
Oxidative stress and erythrocyte membrane alterations may be responsible for hemolysis, with deleterious consequences. 27 Both FPPB and BPPB could protect against HgCl 2 -provoked lysis of human erythrocytes at a specific concentration range. The observed decrease in the antihemolytic effect of the extracts and ascorbic acid at higher concentrations could be due to the prooxidative tendency of phenolics and antioxidant molecules, including ascorbic acid, at concentrations above a particular threshold. 28,29 Phenolic compounds have been speculated to contribute towards lowering the incidence of neurodegenerative diseases. 30 Of considerable importance to neuronal functions are the acetylcholinesterase and Na + /K + -ATPase enzymes. Some cases of psychiatric disorders are known to involve a disruption in ion homoeostasis and are often characterised by decreased Na + /K + -ATPase activity. 31 In the present study, increase in cerebral Na + /K + -ATPase activity produced by the free phenolics of P biglobosa is in line with what was obtained for the CE of the plant 13 and might be associated with the antioxidant properties of the component phenolics, because Na + /K + -ATPase is very sensitive to oxidative stress conditions. 32 It was reported that prior to the formation of pathologic lesions observed in transgenic models and Alzheimer disease cases, impairment in brain function with regards to cognition and memory usually occurs as a result of oxidative stress, which induces a decrease of Na + /K + -ATPase and other signal transduction proteins. 33 Acetylcholinesterase and butyrylcholinesterase inhibitors are viable therapeutic targets in Alzheimer's disease, which is characterised by a cholinergic deficit. 34 In the present study, P biglobosa phenolics caused no inhibition of cerebral acetylcholinesterase. This further supports the proposition of a different mechanism of neuroprotection possibly related, albeit partly, to the potential ability of the phenolics to prevent oxidative stress-related damage of neurones. 13 Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase that plays a vital role in the regulation of vascular tone. ACE functions by converting the inactive peptide angiotensin I into angiotensin II, which increases blood pressure by its vasoconstrictive effect and promotes sodium and water retention in the body. 35 Several studies have demonstrated that phenolic compounds, isolated from different plants, can inhibit ACE activity and reduce blood pressure. 4,5,35 The present study presents novel information on ACE inhibitory effect of P biglobosa phenolics. Free hydroxyl groups of phenolic compounds could chelate the active zinc ions in ACE, thereby inactivating the enzyme often through competitive inhibition. 36 It could thus be speculated that the earlier reported hypotensive effect of the CE of P biglobosa leaf 11 is due to the interaction of its constituent phenolics with ACE.

| CONCLUSION
It is concluded that the antioxidant activity, modulation of cerebral ATPase activity, and inhibition of ACE activities by P biglobosa are related to its constituent phenolics. The latter finding could explain the biochemical rationale behind the use of the plant in the management of hypertension in traditional medicine.

CONFLICTS OF INTEREST
There are no conflicts of interest to declare by authors.