Angiotensin‐1‐converting enzyme inhibition, antioxidant activity, and modulation of cerebral Na+/K+ ATPase by free phenolics of African locust bean (Parkia biglobosa )
Abstract
Abstract
Aims
To investigate the antioxidant activities and effects of free phenols (FPPB) and bound phenols (BPPB) of
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 (
Conclusion
The effects of
Phenolic fingerprinting, antioxidant activities and effects of free phenols (FPPB) and bound phenols (BPPB) of Parkia biglobosa leaves on some neuro‐cardiovascular enzymes were evaluated. On the overall, FPPB showed better antioxidant activity than BPPB and compared well with reference standards. FPPB also inhibits angiotensin‐1‐converting enzyme (ACE) and augments cerebral Na+/K+ ATPase. The effects of P. biglobosa on the enzymes are thus related to its constituent phenols and the documented hypotensive effect of the plant could proceed via ACE inhibition mechanism.
1INTRODUCTION
Many pathological conditions have a significant relationship with oxidative stress. This reveals the importance of antioxidants because they are proven antidotes to the damaging effects of free radicals which are responsible for oxidative stress.1 Advocacy for the use of natural antioxidants in place of synthetic ones is becoming increasingly stronger due to the toxicities associated with the latter.1, 2 Polyphenols constitute an important group of natural antioxidants in plants that could help prevent or mitigate the effects of oxidative stress on the body. Apart from 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
The therapeutic mechanisms of
2MATERIALS AND METHODS
2.1Chemicals
2,2‐Diphenlyl‐1‐picrylhydrazyl (DPPH), 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and 6‐hydroxy‐2,5,7,8‐tetramethychroman‐2‐carboxylic acid (Trolox) were obtained from Sigma (St. Louis, MO, USA). Analytical grade phenolic standards (gallic acid, catechin, chlorogenic acid, caffeic acids, epigallocatechin/gallate, rutin, quercetin, kaempferol) and solvents (methanol, acetic acid) were purchased from Merck KGaA, Darmstadt, Germany. Potassium persulfate (K2S2O8), ascorbic acid, and trichloroacetic acid (TCA) were obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. All other reagents and chemicals were of analytical grade.
2.2Plant material
The collection of fresh leaves of
2.3Extraction of free phenolics
2.3.1Extraction of free phenols ofP biglobosa (FPPB)
2.3.2Extraction of bound phenols ofP biglobosa (BPPB)
R1 was hydrolyzed directly with NaOH (4 M, 20 mL) at room temperature with shaking (1 hour, 40 rpm). Concentrated hydrochloric acid was added to acidify the mixture to pH 2 before extracting 6 times with 200 mL of ethyl acetate. BPPB was obtained by pooling the ethyl acetate fractions together and evaporating the solution to dryness at 45°C under vacuum.14
2.4HPLC‐DAD fingerprinting ofP biglobosa phenolics
HPLC analysis of phenolics in FPPB and BPPB was carried out as previously reported.13 A phenomenex C‐18 column (4.6 mm × 150 mm) packed with 5‐μm diameter particles was employed to carry out the chromatographic analyses. The mobile phase was 2% acetic acid in water (A) and methanol (B). The composition gradient was as follows: 5% of B for 2 minutes and 25%, 40%, 50%, 60%, 70%, and 100% B at 10, 20, 30, 40, 50, and 60 minutes, respectively. The phenolic extracts (free and bound) were dissolved in methanol (10 mg/mL), and the flow rate and injection volume were 0.7 mL/minute and 50 μL, respectively. Comparison of the retention time and UV absorption spectrum with those of the commercial standards allows for the identification of phenolic compounds in the extracts. Prior to use, all the samples and mobile phase were filtered through 0.45‐μm membrane filter (Millipore) and then degassed by an ultrasonic bath. Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range of 0.020 to 0.250.
2.5Animals
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.
2.6Antioxidant assays
2.6.1Total 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).
2.6.2DPPH radical scavenging activity
DPPH radical‐scavenging activity of
2.6.3Reducing property
The ferric reducing property of both FPPB and BPPB was determined as previously described9 using the synthetic antioxidant, butylated hydroxytoluene (BHT) as a reference compound.
2.6.4Anti‐hemolytic effect
The anti‐hemolytic activity of the phenolic extracts was determined as described by Yang et al.15 Blood (5 mL) was taken from healthy volunteers (n = 3) and centrifuged at 1500 rpm for 3 minutes. The resulting red blood cell pellets were washed thrice with sterile phosphate buffered saline (PBS) solution (pH 7.4) and then diluted to 20% cell suspension with PBS. To approximately 250 μL of resulting red blood cell in a test tube, 250 μL of FPPB and BPPB (0, 5, 10, 15, 20, and 25 μg/mL final concentrations) and 125 μL of HgCl2 (5 μM) were added. The mixture was incubated at 37°C for 3 hours in a water bath. Thereafter, 1 mL of PBS was added, and the mixture centrifuged at 2000 g for 10 minutes. The absorbance of the supernatant was read at 540 nm. For the control, the extract was replaced with PBS. Ascorbic acid (0, 10, 15, 20, and 25 μg/mL final concentrations) was used as reference standard.
2.7Assay of enzymes of neurological and cardiovascular relevance
2.7.1Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities
Activities of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8; BChE) were determined using the spectrophotometric method described by Ellman et al16 with slight modifications. Fresh whole brains of male albino rats were harvested and homogenised in 10 parts of cold 60 mM Tris‐HCl, pH 7.4 (1/10, w/v). The homogenate was centrifuged at 3000 g for 10 minutes to obtain a clear supernatant. Exactly 100 μL of sodium phosphate buffer (100 mM, pH 7.5) containing 10 mM DTNB, 10 μL of FPPB/BPPB (5, 10, 25, 50 μg/mL), and 10‐μL supernatant were added in a 96‐well microplate (SpectraMax M2e Multi‐Mode Reader, USA) and incubated for 5 minutes at 25°C. The addition of 20 μL of acetylthiocholine iodide or S‐butyrylthiocholine iodide (8 mM) initiated the reaction. The hydrolysis of the iodides was monitored at 412 nm by following the enzyme‐catalyzed formation of the yellow 5‐thio‐2‐nitrobenzoate anion, as a result of the reaction of DTNB with thiocholines.
2.7.2Na+/K+‐ATPase activity
Na+/K+‐ATPase activity was evaluated in freshly prepared whole brains of male albino rats incubated with FPPB or BPPB as described by Wyse et al.17 The brains were harvested and homogenised in 10 parts of cold Tris‐HCl buffer (10 mM, pH 7.4) and centrifuged (3000 g, 10 minutes) to obtain a clear supernatant. The assay mixture consisted of 50 μL of Na+/K+‐ATPase substrate buffer (pH 7.4) (containing in mM, 30 Tris‐HCl, 0.1 EDTA, 50 NaCl, 5 KCl, and 6 MgCl2), 50 μL of phenolic extract (FPPB/BPPB—5, 10, 20 μg/mL), 50 μL of supernatant (50 μg protein) with or without ouabain (1 mM, 50 μL). The addition of 50‐μL adenosine triphosphate to a final concentration of 3 mM initiated the reaction and the mixture was further incubated for 30 minutes at 37°C before terminating the reaction with 70 μL of TCA 50% (w/v). Quantification of the released inorganic phosphate (Pi) was done by using a reaction mixture containing 100 μL of ammonium molybdate (50 mM), 40 μL of the reaction mixture from first grid and 10 μL of ascorbic acid (8%). A calibration curve of inorganic phosphate was prepared using different concentrations (0, 4, 8, 10, 20, 40 nMol) of NaH2PO4 (1 mM). The specific Na+/K+‐ATPase activity (nmol of Pi/mg of protein/minute) was obtained by subtracting the ouabain‐insensitive activity from the overall activity (in the absence of ouabain).
2.7.3Angiotensin‐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, 20 The tissue was homogenised in cold 125 mM Tris buffer, pH 8.3 (1/10, w/v) and centrifuged at 4°C for 10 minutes at 4000 g to yield a low‐speed supernatant. The reaction mixture contains 40‐μL Tris‐HCl buffer (125 mM, pH 8.3), enzyme source (50 μL), and 10‐μL phenolic extracts/drug (FPPB/BPPB—10, 25, 50 μg/mL; ramipril—0.1, 0.5, 1.0 μM). This was incubated at 37°C for 15 minutes. Thereafter, ACE substrate, HHL (8.3 mM; 150 μL), was added and further incubated for 30 minutes at the same temperature in an orbital shaker (40 rpm) before terminating the reactions with 1 M HCI (1 mL). The cleavage product of the action of ACE on HHL, hippuric acid, was extracted from the acidified solution into ethyl acetate (1 to 2 mL) by vortex mixing for 15 seconds. After centrifugation (3000 g, 5 minutes), aliquot of each ethyl acetate layer (l mL) was obtained, and the fractions were evaporated by heating at 120°C for 30 minutes. The hippuric acid so obtained was re‐dissolved in 1‐mL distilled water, and the concentration was calculated from its absorbance at 228 nm.
2.8Statistical 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. The significance level was set at P < 0.05. Statistical analysis, graphing, and EC50/IC50 determinations were done using Graph Pad Prism (ver.5.0a).
3RESULTS
3.1Polyphenolic composition of free and bound phenolic extracts ofP biglobosa
HPLC fingerprinting of free and bound phenolic extracts of
Compounds | Free Phenolics (FPPB) | Bound Phenolics | ||
---|---|---|---|---|
mg/g | % | mg/g | % | |
Gallic acid | 10.47 ± 0.01 | 1.04 | 6.15 ± 0.02*** | 0.61 |
Catechin | 29.85 ± 0.03 | 2.98 | 32.70 ± 0.01*** | 3.27 |
Chlorogenic acid | 15.36 ± 0.02 | 1.53 | 11.84 ± 0.05** | 1.18 |
Caffeic acid | 30.29 ± 0.04 | 3.02 | 30.51 ± 0.03*** | 3.05 |
Epigallocatechin | 2.87 ± 0.01 | 0.28 | 11.90 ± 0.04*** | 1.13 |
Epigallocatechin gallate | 12.73 ± 0.02 | 1.27 | 19.65 ± 0.01*** | 1.96 |
Rutin | 6.85 ± 0.01 | 0.68 | 18.17 ± 0.02*** | 1.81 |
Quercetin | 17.10 ± 0.03 | 1.71 | 14.25 ± 0.03*** | 1.42 |
Kaempferol | 5.53 ± 0.02 | 0.55 | 2.30 ± 0.01*** | 0.23 |
Abbreviations: BPPB, bound phenolic extract of
Results are presented as the mean ± standard deviation of replicate measurements (n = 3). Data analysis was done using Student t test.
**P < 0.01,
***P < 0.001 compared with the corresponding property of FPPB.
3.2Reducing property, antihemolytic effect, and relative superior radical scavenging activity ofP biglobosa free phenolics
FPPB and BPPB caused concentration‐dependent, significant (P < 0.01/P < 0.001) scavenging of both ABTS and DPPH radicals (Figure 2A,B). The free phenolic extract showed higher potency than the bound phenolic extract (Table 2) as reflected in their respective EC50 values for the scavenging of DPPH (3.82 ± 0.2 vs 10.22 ± 0.1 μg/mL) and ABTS (4.06 ± 0.3 vs 24.07 ± 2.1 μg/mL) radicals as well as the TEAC (0.46 ± 0.003 vs 0.23 ± 0.02 μg/mL). The ferric reducing effect of both phenolic extracts was concentration dependent (Figure 2C). FPPB (36%) and BPPB (40%) exhibited highest anti‐hemolytic potential at 15 μg/mL final concentration but with a reduction in their capacity to prevent hemolysis at higher concentrations (20 and 25 μg/mL). The anti‐hemolytic activity of the reference ascorbic acid was also statistically significant at 10 μg/mL (P < 0.001), 15 and 20 μg/mL (P < 0.01) concentrations, whereas at a higher concentration (25 μg/mL), there was a significant hemolysis of the red blood cells (Figure 2D).
Sample Parameter | EC50 (μg/mL) | TEAC | |
---|---|---|---|
DPPH | ABTS | ||
FPPB | 3.82 ± 0.2 | 4.06 ± 0.3 | 0.46 ± 0.003 |
BPPB | 10.22 ± 0.1 | 24.07 ± 2.1 | 0.23 ± 0.02 |
Trolox | ‐ | 4.44 ± 0.08 | ‐ |
Ascorbic acid | 12.58 ± 0.4 | ‐ | ‐ |
Results are mean ± SD (n = 3).
Abbreviations: BPPB, bound phenolic extract of
3.3Effects of FPPB and BPPB on enzymes of neurological and cardiovascular relevance
FPPB, BPPB, and the reference, catechin, produced no inhibitory effect on cerebral acetylcholinesterase in vitro. Butyrylcholinesterase activity was, however, only increased at the highest concentration (50 μg/mL) evaluated for both phenolic extracts (Figure 3A,B). Increase in cerebral Na+/K+ ATPase activity produced by free phenolics of
Sample | IC50 (μg/mL) |
---|---|
FPPB | 15.35 ± 4.0 |
BPPB | 46.85 ± 3.3 |
Ramipril | 0.173 ± 0.04 |
Results are mean ± SD (n = 3).
Abbreviations: BPPB, bound phenolic extract of
4DISCUSSION
The hydroxyl groups of polyphenols often make them capable of contributing directly to antioxidative actions.21 Although the bioavailability of polyphenols is generally low, antioxidant phytochemicals are still able to elicit pharmacological responses because very low in vivo concentrations (nM range) are actually required for therapeutic effects.1, 22 The antioxidant and radical scavenging effects of many of the constituent phenolics of
It could be postulated that
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 HgCl2‐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
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
5CONCLUSION
It is concluded that the antioxidant activity, modulation of cerebral ATPase activity, and inhibition of ACE activities by
CONFLICTS OF INTEREST
There are no conflicts of interest to declare by authors.
ACKNOWLEDGEMENT
This work was supported by the Brazilian National Council for Scientific and Technological Development, (CNPq) and the Academy of Sciences for the Developing World, (TWAS) [grant number: 3240256075].
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