Phenolic profile and Enzyme Inhibitory activities of Almond (Terminalia catappa) leaf and Stem bark

ABSTRACT

Almond (Terminalia catappa) is a species used in Nigerian folkloric medicine because of its antihypertensive property. In this study, the acetylcholinesterase, arginase, phosphodiesterase-5 and angiotensin-1-converting enzyme inhibition and antioxidant properties of the phenolic-rich extracts from almond leaf and stem bark were determined. The chemical composition was evaluated using high-performance liquid chromatography. The total phenol and flavonoid contents were estimated and the antihypertensive potential was estimated by the determination of acetylcholinesterase, arginase, phosphodiesterase-5 and angiotensin-1-converting enzyme inhibition in vitro. Three assays were used for the evaluation of antioxidant properties of the extracts. Eleven and eight phenolic compounds were detected in leaf and stem bark respectively. The phenolic-rich extracts showed remarkable enzyme inhibition activity in concentration dependent manner, with high antioxidant activities. Almond leaf and stem bark can serve as potential natural source for the development of a novel antihypertensive agent.

KEYWORDS:

• Terminalia catappa
• antioxidants
• antihypertensive agent
• phenolic-rich
• HPLC

Introduction

In this dispensation, high blood pressure (BP), also called hypertension, is one of the most common causes of early death in symptom-free adults. Uncontrolled hypertension increases the risk of serious health issues and is considered a major risk factor for stroke, heart attack or myocardial infarction, ischemia, blindness, erectile dysfunction and kidney disease.^[1] The increase in blood pumps from the heart and the narrowing of the arteries normally lead to hypertension. Moreover, there is a direct link between systolic BP and hemorrhagic stroke.^[2] Longevity and prevalence of contributing factors such as obesity, physical inactivity and unhealthy diet have made hypertension to be an increasingly common health problem worldwide.^[3] Over expression of renin angiotensin aldosterone system (RAAS) is one of major causative factors in the development of hypertension.^[4] Angiotensin-1-converting enzyme (ACE) plays a prominent role in RAAS, by converting the precursor angiotensin I to angiotensin II which is a system vasoconstrictor, the peptide responsible for the mechanisms which increase the blood pressure. Inhibition of ACE is a promising way of controlling the over activation of RAAS. In addition, free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in the pathogenesis of several human diseases including atherosclerosis, diabetes mellitus, chronic inflammation, hypertension, among others.^[5] Recently, interest has increased considerably in finding naturally occurring antioxidants for use in food and/or pharmaceutical applications, which can protect human body from free radicals and retard the progress of many chronic human diseases.^[6]

Inspite of the availability of several antihypertensive agents, which successfully reduce blood pressure in many hypertensive subjects, numerous patients remain unresponsive to treatment and are left with severely high BP.^[7] For instance, hypertension is not effectively managed in approximately 10% of populations who are compliant with prescriptive therapeutics.^[8] The available synthetic antihypertensive agents in clinical practice are not also effective in all cases in addition to the well-known severity of the disease itself.^[9] Moreover, these agents are characterized by high cost, lack of definitive curative regimen, and combination therapy, which hinders drug adherence and increase the risk of side effects as well as drug-drug interactions.^[10] These observations are indications that new agents and/or approaches are still needed to manage hypertension.

Herbs have been used as medical treatments since the beginning of human existence and some derivatives (e.g., quinine, reserpine and cardiac glycosides) have become mainstays of human pharmacotherapy.^[10] Terminalia catappa belongs to family Combretaceae and its generic name originated from Latin word “terminalis”. It is a tree commonly grown in tropical and subtropical regions because of its edible, ornamental and medicinal purposes.^[11] Terminalia catappa is a valuable remedy for several common ailments. Terminalia catappa have found to possess various pharmacological properties, such as anti-stress, anti-oxidant.^[11] The leaves, barks and roots are used in traditional medicine for various purposes which have been greatly attributed to the presence of phenolic compounds such as catechin, protocatechuic acid, prenylated benzoic acid, 2-prenylated benzoic acid, and 2-prenyl-4-O-β-D-glucopyranosyl-oxy-4-hydroxybenzoic acid in the stem, root and leaf part of the almond plant.^[12] However, despite its traditional usage in the management of hypertension and cardiovascular diseases, there is limited or no information regarding its effectiveness and mode of action. Therefore, this study was aimed to assess the claim of its involvement in folkloric medicine in hypertension management, and most especially to assess its effect on some enzymes relevant to hypertension in vitro.

Materials and methods

Sample collection

Almond leaf and stem bark were collected from Botanical Garden, Federal University of Technology, Akure, Nigeria during dry season (January, 2017). Identification and authentication of the plant was done in the Department of Biology, Federal University of Technology, Akure and a voucher specimen was deposited at the Departmental Herbarium. The fresh plant materials were dried to a constant weight and ground to powder using Laboratory blender.

Sample extraction

Twenty grams of the powdered plant material was extracted by maceration at room temperature using distilled water with plant/solvent ratio of 1:5. After 48 h, it was filtered with Whatman no 1 filter paper. The filtrate was then freeze-dried in a lyophilizer. The lyophilized samples were transferred into a vial and kept in refrigerator (−4°C) prior to subsequent analysis.

Chemicals and reagents

Phosphodiesterase-5 (PDE-5, from S. frugiperda) was obtained from Merck Millipore. Hippuryl-l-histidyl l-leucine (HHL), para-nitrophenylphenylphosphonate, 1,1-diphenyl-2-picrylhydrazyl (DPPH), acetylthiocholine iodide, 5,5ʹ-dithiobio-(2-nitrobenzoic acid) (DTNB), acetylcholinesterase (AChE, from electric eel, type VI-S), arginase (from bovine liver) and angiotensin-1-converting enzyme (ACE, from rabbit lung) were obtained from Sigma–Aldrich Co. (Steinheim, Germany), potassium ferricyanide from BDH Chemicals Ltd. (Poole, England); trichloroacetic acid, ascorbic acid, ferric chloride (FeCl₃) and methanol from Merck KGaA (Darmstadt, Germany). All other chemicals and reagents used were of analytical grade and glass distilled water was used throughout.

Phosphodiesterase-5 inhibition assay

The phosphodiesterase-5 inhibitory assays were carried out by Kelly and Butler^[13] method with slight modification. Briefly, solution containing the enzyme (concentration 40 µg/ml) with various concentration of extract in Tris-HCl buffer (pH 8.0) containing 1 mM CaCl₂ and 50 mM NaCl was incubated at 37°C for 10 min. Thereafter, 200 µl of substrate (5 mM p-nitrophenyl phenylphosphonate) was added. The intensity of p-nitrophenol produced was measured spectrophotometrically at 405 nm. The control experiment was carried out without addition of extract. Phosphodiestrease-5 inhibitory property of the extract was subsequently calculated and expressed as percentage inhibition.

Arginase inhibition assay

Arginase inhibibtion assay was performed according to the method of Kaysen and Strecker.^[14] In brief, a solution containing enzyme (concentration of 30 μg/ml) and various concentration of extracts were pre-incubated for 5 min in 0.01 mM Tris-HCl buffer (pH 7.5) containing 0.05 mM MnCl₂. The reaction was initiated by addition of 50 mM L-arginine followed by incubation at 37°C for 10 min. The reaction was terminated by adding Ehlrich’s reagent (p-dimethylaminobenzaldehyde in 3.6 N HCl). The amount of urea produced was measured spectrophotometrically at 450 nm. The control experiment was carried out without addition of extract. Arginase inhibitory property of the extract was subsequently calculated and expressed as percentage inhibition.

Acetylcholinesterase inhibition assay

The acetylcholinesterase (AChE) inhibition of the plant samples was assessed by colorimetric method.^[15] The AChE activity was determined in a reaction mixture containing 200 μl of a solution of AChE (0.415 U/ml in 0.1 M phosphate buffer, pH 8.0), 100 μl of a solution of 5,5′­dithio­bis(2­nitrobenzoic) acid (3.3 mM) in 0.1 M phosphate buffer solution (pH 7.0) containing NaHCO₃ (6 mM), extract dilutions (0–100 μl), and 500 μl phosphate buffer (pH 8.0). After incubation for 20 min at 25°C, acetylthiocholine iodide (100 μL of 0.05 mM water solution) was added as the substrate, and AChE activities were determined by JENWAY UV­Visible spectrophotometer from the absorbance changes at 412 nm for 3.0 min at 25°C. The enzyme inhibitory activities were expressed as percentage inhibition.

Angiotensin-i-converting enzyme (ACE) inhibition assay

The inhibition of ACE activity of the extract was determined according to the described method of Cushman and Cheung.^[16] Different concentrations of the extract and 50 μl ACE stock solution (4 mU/ml) were preincubated at 37°C for 15 min. Thereafter, enzymatic reaction was initiated by adding 150 μl of 8.33 mM ACE substrate [hippuryl-l-histidyl l-leucine (HHL)] in 125 mM of Tris-HCl buffer (pH 8.3) to the reaction mixture and incubated at 37°C for 30 min. The reaction was stopped by adding 250 μl of 1 M HCl. The hippuric acid (Bz-Gly) produced by the reaction was extracted with 1.5 ml ethyl acetate. The mixture was then centrifuged to separate the ethyl acetate layer, after which the 1 ml of the ethyl acetate layer was transferred to a clean test tube and evaporated to dryness. The residue was re-dissolved in distilled water and its absorbance was measured at 228 nm.

Measurement of the free radicals scavenging capacity

The 1,1-diphenyl–2 picrylhydrazyl (DPPH) radical scavenging capacity of the Almond leaf and stem bark extract was determined using the method of Gyamfi et al..^[17] In addition, ABTS* radicals scavenging capacity of the extract was determined by the method previously described by Re et al..^[18] Trolox was used as standard.

Ferric reducing power

The ferric reducing power of the extracts was evaluated using method of Oyaizu,.^[19] Ascorbic acid was used as standard and results are expressed in mg ascorbic acid equivalent per gram of dry sample (mgAAE/g).

Determination of total phenolic and flavonoid contents

The total phenolic and flavonoid contents of the extracts were determined through Folin-Ciocalteu and aluminium chloride colorimetric methods as described by Singleton et al.^[20] and Meda et al.^[21] respectively. Total phenolic contents are expressed as mg of gallic acid per gram of dry sample (mgGAE/g), while total flavonoid contents of the extracts are expressed as mg of quercetin per gram of dry sample (mgQUE/g).

Quantification of phenolic compounds by HPLC-DAD

Samples at a concentration of 12 mg/ml were injected by means of a model SIL-20A Shimadzu Auto sampler. Separations were carried out using Phenomenex C₁₈ column (4.6 mm x 250 mm x 5 μm particle size). The mobile phase was water with 1% formic acid (v/v) (solvent A) and HPLC grade methanol (solvent B) at a flow rate of 0.6 ml/min and injection volume 40 μl. The composition gradient was: 5% solvent B reaching 15% at 10 min; 30% solvent B at 25 min, 65% solvent B at 40 min and 98% solvent B at 45 min, followed by 50 min at isocratic elution until 55 min. At 60 min the gradient reached the initial conditions again, following the method described by Duarte et al.^[22] with slight modifications. The sample and mobile phase were filtered through 0.45 μm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use. Stock solutions of standards references were prepared in the methanol: water (1:1, v/v) at a concentration range of 0.025 – 0.300 mg/ml. Quantifications were carried out by integration of the peaks using the external standard method, at 254 nm for gallic acid, 280 nm for catechin, 327 nm for chlorogenic acid and caffeic acid; and 365 for rutin, apigenin, kaempferol and luteolin. The chromatography peaks were confirmed by comparing its retention time with those of reference standards and by DAD spectra (200 to 600 nm). Calibration curve for gallic acid: Y = 13278x + 1192.5 (r² = 0.9998); catechin: Y = 10957x + 1083.4 (r² = 0.9999); kaempferol: Y = 11635x + 1403.9 (r² = 0.9996); chlorogenic acid: Y = 12735x + 1354.0 (r² = 0.9995); caffeic acid: Y = 11458x + 1191.8 (r² = 0.9999); apigenin: Y = 14183x + 1194.1 (r² = 0.9997); luteolin: Y = 12950x + 1362.3 (r² = 0.9994); ellagic acid: Y = 12631x + 1095.8 (r² = 0.9999); quercetin: Y = 13256x + 1272.5 (r² = 0.9995) and rutin: Y = 13791x + 1428.5 (r² = 0.9999). All chromatography operations were carried out at ambient temperature and in triplicate.

Statistical analysis

All the analyses were carried out in triplicates. The results are expressed as mean ± SD. Statistical comparisons were investigated by one-way ANOVA followed by Duncan’s post-hoc test for multiple comparisons with control. Statistical analyses were performed using Statistical package for social sciences (SPSS) 16.0 software. Furthermore, the IC₅₀ values were calculated using non-linear regression analysis.

Results and discussion

Enzyme inhibition assays

The role of renin-angiotensin system (RAS) in regulating blood pressure cannot be underestimated. Renin catalyzes the conversion of inactive angiotensinogen to angiotensin I, it is thereafter converted to a potent system vasoconstrictor (angiotensin II) by angiotensin-1-converting enzyme (ACE). Elevated ACE activity has been linked with hypertension as it degrades bradykinin, a system vasodilator.^[23] However, inhibition of ACE activity could be of immense benefit in lowering blood pressure and in the overall management of hypertension. In this present study, the ACE inhibitory property of almond leaf and stem bark was carried out and result revealed that extracts inhibited ACE in concentration-dependent manner (Fig. 1A). However, stem bark extract showed a higher inhibitory effect against ACE activity than leaf extract as shown by IC₅₀ values (Table 2). Several synthetic ACE inhibitors are currently in use and have been proved to be effective in the management of hypertension.^[24] Nevertheless, adverse effects and cost implications associated with the use of synthetic ACE inhibitors increase the search for naturally-occurring ACE inhibitors. These naturally-occurring substances have been shown to be readily available with little or no adverse effect.^[23] Previous findings have highlighted the antihypertensive and cardio-protective properties of some medicinal plants and plant foods.^[23,25–28] Almond leaf and stem bark inhibit ACE activity, and could therefore serve as natural and alternative therapy in the management of hypertension. However, ACE inhibitory potential of almond leaf and stem bark could be related to the inherent phytochemicals especially phenolic acids and flavonoids.

One of important strategies for treatment of hypertension is the inhibition of phosphodiesterase-5. Phosphodiesterase-5 (PDE-5) inhibitors act by inhibiting PDE-5, a key metabolic enzyme of intracellular cyclic guanosine monophosphate (cGMP) in pulmonary vasculature. cGMP serves as intracellular second messenger for nitric oxide (NO) and natriuretic peptide (NP) signaling and initiates most of muscle relaxation especially of pulmonary circulation.^[29] Almond leaf and stem bark extracts inhibited PDE-5 in a concentration-dependent manner (Fig. 1B). However, leaf extract showed higher inhibitory effect against PDE-5 activity (IC₅₀ = 238.44 µg/ml) than stem bark extract (IC₅₀ = 273.43 µg/ml) (Table 2). Recently, it was shown that PDE-5 activity was found to be increased in hypertensive diseases, thereby elevating the cGMP hydrolysis and thereby causing impairment of cGMP-mediated pulmonary vasodilation.^[30] Several in vitro studies have screened some medicinal plants for PDE-5 inhibitory potentials.^[31-33] In addition, findings revealed that inhibition of PDE-5 have been shown to reverse the acute pulmonary vasoconstriction and thereby attenuate the development of hypertension in animal models.^[34] Inhibition of PDE-5 by the extracts of almond leaf and stem bark could serve as therapeutic approach in the management of hypertension, and this could be associated with inherent polyphenols which have been previously described to possess PDE-5 inhibitory effects.^[35]

Figure 1. A. angiotensin-1-converting enzyme (ACE), B. phosphodiesterase-5 (PDE-5), C. acetylcholinesterase (AChE) and D. arginase inhibitory activities of almond leaf and stem bark extracts.

Table 1. Total Phenolic content, flavonoid content and ABTS radical scavenging ability of almond leaf and bark.

Parameter	Leaf	Stem bark
Total phenol (mgGAE/g)	62.50 ± 1.84^b	37.30 ± 1.05^a
Total flavonoid (mgQUE/g)	7.80 ± 1.32^b	3.44 ± 0.51^a
ABTS radical scavenging ability (mmolTEAC/g)	0.17 ± 0.002^b	0.09 ± 0.001^a

Values represent mean±standard deviation of replicate readings.

Values with the same superscript along the same row are not statistically different at p < 0.05.

Table 2. IC₅₀ values for DPPH radical scavenging ability and arginase, phosphodiesterase-5 (PDE-5), acetylcholinesterase (AChE) and angiotensin-1-converting enzyme (ACE) inhibitory activities of almond leaf and stem bark.

Parameter	Leaf	Stem bark	Positive Control
DPPH* scavenging ability (mg/ml)	0.49 ± 0.05^a	0.57 ± 0.09^b
Arginase inhibition (µg/ml)	51.65 ± 2.57^b	53.91 ± 4.03^b	0.78 ± 0.21^a
AChE inhibition (µg/ml)	265.39 ± 5.79^b	303.53 ± 4.99^c	2.35 ± 0.22^a
PDE-5 inhibition (µg/ml)	238.44 ± 3.57^b	273.43 ± 4.03^c	2.68 ± 0.10^a
ACE inhibition (µg/ml)	64.74 ± 1.57^c	57.29 ± 2.03^b	0.20 ± 0.05^a

Results are expressed as mean ± standard deviations (SD) of three determinations. Values with the same alphabet along the same row are not significantly different (p < 0.05). N^ѡ-hydroxy-L-arginine (NOHA) was used as positive control for arginase, Prostigmine for acetylcholiesterase, Sildenafil citrate for phosphodiesterase-5 and Lisinopril for angiotensin-1-converting enzyme.

Acetylcholinesterase (AChE) has been reported to play a crucial role in regulating number of vital functions such as learning and cerebral blood flow which reveals the level of importance of acetylcholine as a neurotransmitter target for examining cerebrovascular diseases related hypertension.^[36] Acetylcholine is a neurotransmitter produced at the synaptic gap. Deficient cholinergic functions and neurotransmitter disturbances among others have been identified as pathological features in many disorders.^[36] This study evaluated the capacity of Almond leaf and stem bark as inhibitor of acetylcholinesterase, an important enzyme in the breakdown of acetylcholine. The study revealed that both almond leaf and stem bark extracts showed concentration-dependent inhibition of AChE (Fig. 1C). The results (Table 2) showed that leaf extract had a higher inhibitory activity (IC₅₀ = 265.39 µg/ml) when compared with stem bark (IC₅₀ = 303.53 µg/ml). It is within the interest of our study to show that this paramount activity of almond leaf and stem bark may apparently be attributed to their high content of polyphenols such as caffeic acid and chlorogenic acid, which have been shown to possess inhibitory property against AChE.^[37,38]

Arginase, a metalloprotein enzyme has been implicated to play a crucial role in heart and kidney function. This study shows that both extracts (almond leaf and stem bark) inhibited arginase activity in a concentration-dependent manner (Fig. 1D). However, there was no significant difference in their arginase inhibitory activities (Table 2). Increased arginase activity has been linked to several vascular difficulties including atherosclerosis, erectile dysfunction and hypertension.^[39,40] Elevated arginase activity can impair endothelium-dependent vasorelaxation by lowering L-arginine availability to endothelium nitric oxide synthase (eNOS), thereby decreasing NO production and uncoupling eNOS function.^[41] Interestingly, downregulation of arginase has been shown to increase NO production and vasorelaxant responses to acetylcholine in hypertensive and high-fat diet animal models.^[42] Convincingly, the inhibitory property of these extracts on arginase could be a function of their inherent polyphenols. Flavonoids, a typical class of polyphenol have been reported to possess ability to form hydrogen bond and hydrophobic interactions with some amino acids at the active site of arginase, thereby inhibiting its activities.^[43,44]

Antioxidant and free radicals scavenging ability of extracts

Fig. 2A depicts the dose dependent DPPH radical scavenging ability of almond leaf and stem bark extracts as expressed as a percentage of the ratio of the decrease in absorbance of the test solution to that of DPPH solution without plants extracts. The results showed that both the extracts had a propensity in quenching DPPH free radicals, as indicated by the geometric increase in radical scavenging ability of the extracts. The IC₅₀ values of the extracts are presented in Table 2. Almond leaf extract had a higher DPPH radical scavenging ability (IC₅₀ = 0.49 mg/ml) than stem bark extract (IC₅₀ = 0.57 mg/ml). In addition, ABTS radical scavenging ability of the extracts is presented in Table 1. However, almond leaf extract had a higher ABTS radical scavenging ability (0.17 mmolTEAC/g) when compared with stem bark extract (0.09 mmolTEAC/g). It is interesting to note that almond leaf extract showed higher radicals scavenging activity also had higher total phenol and flavonoid contents. This finding agrees with earlier report that established the correlation between the phenolic contents of some medicinal plants and their radicals scavenging ability.^[45] It is a known fact that overproduction of free radicals is the basis of pathogenesis of a number of diseases^[46], and that intake of natural antioxidants can prevent the associated health problems caused by oxidative stress.^[47]

Figure 2. A. DPPH radical scavenging ability B. ferric reducing antioxidant power of almond leaf and stem bark extracts.

Ferric reducing power

The ferric reducing antioxidant power (FRAP) of almond leaf and stem bark is presented in Fig. 2B. Both samples have strong reducing power, but leaf extract had higher reducing ability (47.73 mgAAE/g) when compared with stem bark (39.55 mgAAE/g). FRAP detects compounds with oxidation-reduction potentials, the potential of the Fe³⁺-TPTZ product. Unlike other radical scavenging assays, FRAP is an entirely electron transfer reaction rather than hydrogen atom observed in most reactions.^[48] In this study, there is a correlation between the phenolic contents and ferric reducing power of the extracts. This finds agreement with earlier study carried out by Frankel et al..^[49]

Polyphenolic composition

Composition of phenolic compounds in aqueous almond leaf and stem bark extracts was determined quantitatively by high-performance liquid chromatography (HPLC) analysis. The analysis revealed the presence of gallic acid (t_R = 9.97 min; peak 1), catechin (t_R = 16.39 min; peak 2), chlorogenic acid (t_R = 21.47 min; peak 3), caffeic acid (t_R = 25.03 min; peak 4), ellagic acid (t_R = 31.20; peak 5), epicatechin (t_R = 32.71 min; peak 6), rutin (t_R = 39.98 min; peak 7), quercitrin (t_R = 44.15 min; peak 8), isoquercitrin (t_R = 47.83 min; peak 9), quercetin (t_R = 52.64 min; peak 10) and kaempferol (t_R = 59.86 min; peak 11) in almond leaf (Figs. 3A & B, Table 3). While in stem bark, the analysis revealed the presence of gallic acid (t_R = 10.27 min, peak 1), catechin (t_R = 15.83 min, peak 2), caffeic acid (t_R = 22.07 min, peak 3), ellagic acid (t_R = 23.98 min, peak 4), resveratrol (t_R = 29.65 min, peak 5), rutin (t_R = 34.51 min, peak 6), quercetin (t_R = 39.46 min, peak 7) and kaempferol (t_R = 43.01 min, peak 8) (Figs. 3 A & B and Table 3). It was evident that almond leaf and stem bark are rich source of polyphenols which validated high antioxidant, anti-phosphodiesterase-5, anti-arginase, anti-acetylcholinesterase and anti-angiotensin-1-converting enzyme activities of almond leaf and stem bark extracts. Our findings are supported by the facts that some of the identified phenolic compounds namely chlorogenic acid, caffeic acid, rutin and quercetin have already been shown to possess anti-cholinesterase and inhibitory effect on enzymes discussed in this study.^[35,50,51] Anouar et al.^[52] reported that phenolics act as antioxidants by attenuating enzymes in radical generation. In another study, Xu et al.^[53] reported that number of flavonoids affect smooth muscle contractility in response to various agonists.

Figure 3. Representative of high performance liquid chromatography profile A. almond leaf; Gallic acid (peak 1), catechin (peak 2), chlorogenic acid (peak 3), caffeic acid (peak 4), ellagic acid (peak 5), epicatechin (peak 6), rutin (peak 7), quercitrin (peak 8), isoquercitrin (peak 9), quercetin (peak 10) and kaempferol (peak 11) B. almond stem bark; Gallic acid (peak 1), catechin (peak 2), caffeic acid (peak 3), ellagic acid (peak 4), resveratrol (peak 5), rutin (peak 6), quercetin (peak 7) and kaempferol (peak 8).

Table 3. Phenolics composition of Almond leaf and stem bark extracts.

	Leaf	Stem bark
Compounds	mg/g	mg/g
Gallic acid	64.23 ± 0.01^b	1.05 ± 0.02^a
Catechin	25.18 ± 0.01^b	1.78 ± 0.03^a
Chlorogenic acid	53.47 ± 0.03
Caffeic acid	46.15 ± 0.02^b	6.93 ± 0.01^a
Ellagic acid	98.82 ± 0.03^b	5.87 ± 0.01^a
Epicatechin	21.83 ± 0.01
Rutin	45.06 ± 0.02^b	1.06 ± 0.01^a
Quercitrin	79.65 ± 0.01
Isoquercitrin	52.03 ± 0.02
Quercetin	26.71 ± 0.01^b	3.45 ± 0.02^a
Kaempferol	65.13 ± 0.01^b	1.76 ± 0.01^a
Resveratrol		5.91 ± 0.03

Results are expressed as mean ± standard deviations (SD) of three determinations. Averages followed by different letters differ by Tukey test at p < 0.05.

Total phenol and flavonoid contents

The total phenol content of almond leaf and stem bark was evaluated using gallic acid as standard. It was found to be higher in leaf extract (62.50 ± 1.84 mgGAE/g) than stem bark extract (37.30 ± 1.05 mgGAE/g). In the same vein, the total flavonoid content was evaluated using quercetin as standard. Results revealed higher flavonoid content in leaf extract (7.80 ± 1.32 mgQUE/g) when compared stem bark extract (3.44 ± 0.51 mgQUE/g) (Table 1). The study of total phenol and flavonoid contents of other medicinal plant species excluding almond leaf and stem bark have been carried out by several researchers.^[54–57]

Conclusion

This study represents the exhaustive analysis of total phenol and flavonoid contents, antioxidant, anti-acetylcholinesterase, anti-phosphodiesterase-5, anti-arginase, anti-angiotensin-1-converting enzyme and phenolic composition of almond leaf and stem bark, a medicinal plant food in Nigeria. The study revealed that almond leaf and stem bark extracts have high phenolic and flavonoid contents with remarkable antioxidant activities and potential inhibitor of some enzymes involved in hypertension. Study also revealed presence of number of polyphenols with known anti-oxidative and antihypertensive properties. These activities of almond leaf and stem bark extracts suggest their utilization for the development of alternative formulations for general health maintenance and effective antihypertensive agent.