Background
There is increasing awareness of potential health benefits of naturally occurring phytochemicals from plants. Herbs have long been used for a large range of nutritional and medicinal purposes. In fact, herbs were the basis for nearly all medicinal therapy until synthetic drugs were developed in the nineteenth century [
1]. Bioactive molecules from natural sources could have fewer side effects than synthetic ones. The established inverse relationship between intake of plant-derived foods and mortality from age-related degenerative diseases such, as cancer, diabetes, emphysema, cardiovascular diseases and brain dysfunction [
2] has been attributed to their antioxidant property. Thus, antioxidant principles from natural sources can provide a multifaceted approach to modulate oxidative imbalance found in human degenerative diseases. Electron leakage from the mitochondrial electron transport chain (ETC) during cellular respiration accounts for about 2% of oxygen consumed being converted to superoxide anion (O
2
.-) and this is thought to be the major source of ROS generation in somatic cells [
3] with the rate of O
2
.- production being dependent on mitochondrial potential [
4]. Mitochondrial ROS production under certain conditions is capable of overwhelming the endogenous antioxidant defence mechanisms, resulting in oxidative stress, with a grave implication in numerous pathological conditions and contributes to retrograde redox signaling from the organelle to the cytosol and nucleus. Early ROS production in the mitochondria could be detected using dichlorodihydrofluorescein (DCFH) derivatives which localize to the mitochondrial matrix [
5] and is sensitive to H
2O
2 which is immediately generated on spontaneous or catalyzed dismutation of superoxide anion by mitochondrial SOD. The resulting H
2O
2 is capable of diffusing out of the mitochondria into the cytoplasm.
Of particular therapeutic significance, polyphenolics appear to play a significant role as antioxidants in the protective effect of medicinal plants [
6] and have become the focus of current nutritional and therapeutic interest. Generally, antioxidant compounds like phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydroperoxide of lipid hydroxyl and thus inhibit the oxidative mechanisms involved in the progression of degenerative diseases [
7]. These natural antioxidants have been shown to have access to metabolic processes and are capable of interrupting free radical-mediated reactions by donating hydrogen from the phenolic hydroxyl groups to free radicals [
8]. They also have the aptitude to scavenge oxygen-nitrogen derived free radicals by donating hydrogen atom or an electron, chelating metal catalysts and activating antioxidant enzymes [
8,
9]. Plant phytochemicals have a multifunctional nature. It is, therefore, necessary that in vitro antioxidant investigations combine radical scavenging and lipid peroxidation inhibitory effects in order to arrive at a solid conclusion on the total antioxidant potential of phytochemicals and plant products.
Parkia biglobosa (Jacq.) Benth., commonly known as ‘African locust bean’, is a plant used extensively in West Africa for timber, food and medicine. It was largely prescribed in traditional medicine for its multiple medicinal virtues in tropical Africa. A decoction of the bark, root and leaves is used in treating toothaches, leprosy, hypertension and fevers [
10]. The phenolic constituents [
11] and hypotensive effect of the leaf extract was earlier reported [
12]. However, the mechanism underlying the hypotensive effect is still a subject of investigation. Also, there is a paucity of information on the antioxidant activity and the effect of the leaf on mitochondrial redox status. In the present study, we have assessed the antioxidant activity, angiotensin-converting enzyme inhibition and effect of the aqueous-methanolic extract of
P. biglobosa leaf on isolated mitochondrial integrity.
Methods
Chemicals
2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) or ABTS and dichlorofluorescin diacetate (DCFHDA) were obtained from Sigma (St. Louis, MO, USA). 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox) and 2,2-diphenlyl-1-picrylhydrazyl (DPPH) were obtained from Fluka, Buchs, Switzerland. Potassium persulfate (K2S2O8), ethylenediaminetetraacetic acid (EDTA), ascorbic acid, 2- deoxy-2-ribose, trichloroacetic acid (TCA), and quercetin were obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Hydrogen peroxide, Ferrous sulfate, potassium hexacyanoferrate, Folin-Ciocalteu reagent, sodium carbonate (Na2CO3) and butylated hydroxytoluene (BHT) were obtained from Merck, Mumbai, India. Thiobarbituric acid (TBA) was obtained from Loba Chemie, Mumbai, India.
Plant material
Fresh leaves of Parkia biglobosa were collected in Isua-Akoko, Ondo State, Nigeria. Botanical identification and authentication was carried out by Dr. Ugbogu A.O and Mr. Shasanya O.S at the herbarium of the Forestry Research Institute (FRIN) Ibadan, Oyo state, Nigeria where a voucher specimen (no 109603) was deposited.
The leaves were air-dried for 28 days at room temperature and ground to fine powder using a blender. A 500 g sample of the powdered material was macerated in 1200 mL of a mixture of methanol and water (4:1) for 48 h. This was filtered and concentrated to a small volume to remove the entire methanol using rotary evaporator. The concentrated extract was then lyophilized. The residue was kept at −20 °C for future use. Extract yield was approximately 11%.
Animals
Male Wistar rats (±3 months old), weighing between 270 g and 320 g, from the University breeding colony (Animal House Holding, UFSM, Brazil) were kept in cages with free access to foods and water in a room with controlled temperature (22 °C ± 3) and in 12-h light/dark cycle with lights on at 7:00 a.m. The animals were maintained and used in accordance to the guidelines of the Brazilian Association for Laboratory Animal Science.
Preparation of tissue homogenates
Rats were sacrificed by decapitation on the day of experiment and rapidly dissected to harvest the whole brain and liver which were then placed on ice and weighed. Tissues were immediately homogenized in ten volumes of cold (4 °C) Tris–HCl (10 mM, pH 7.4). The homogenate was centrifuged for 10 min at 4000 g to yield a pellet that was discarded and a low-speed supernatant that was used in thiobarbituric acid reactive substances (TBARs) quantification.
In vitro antioxidant/radical scavenging and metal ion chelating activities assays
Total antioxidant activity
Total antioxidant activity was determined by the ABTS test described by Re et al. [
13]. The ABTS
.+ radical cation was generated by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and incubating for 12–16 h in the dark at room temperature. The absorbance of the ABTS
.+ solution was equilibrated to 0.70 (± 0.02) by diluting with distilled water. ABTS
.+ solution (1 ml) was mixed with 10 μl of PBE dissolved in distilled water (0, 10, 25, 50, 100 and 150 μg/ml final concentration) or Trolox standard dissolved in deionized water (0, 1, 2.5, 5.0, 7.5, and 10 μg/ml final concentration). The absorbance was measured at 734 nm after 6 min. All experiments were carried out in replicates. The percentage inhibition of absorbance was calculated and plotted as a function of the concentration of standard and sample to determine the Trolox equivalent antioxidant concentration (TEAC). To calculate the TEAC, the straight line gradient of the plot for the sample was divided by that of Trolox (Additional file
1: Figure S1).
DPPH radical scavenging activity of extract
DPPH radical-scavenging activities of
P. biglobosa extract and reference compound (Ascorbic acid) were determined as described by Batool et al. [
14]. The capacity of extracts to scavenge the lipid-soluble 2, 2- diphenyl-1-picrylhydrazyl (DPPH) radical, which results in the bleaching of the purple colour exhibited by the stable DPPH radical, could be monitored at 517 nm.
Briefly, PBE (0, 10, 25, 50, 100, and 250 μg/ml) or the reference compound, ascorbic acid (0, 10, 20, 30, 40 and 50 μg/ml) was added to an ethanol solution of DPPH (0.03 mM). The mixture was shaken and left to stand at room temperature for 30 min. The absorbance of the resulting solution was measured spectrophotometrically at 517 nm. The radical scavenging activity was calculated as a percentage of DPPH
.
discolouration.
Reducing power
The Fe
3+- reducing power of the extract was determined as described by Oyaizu [
15] with a slight modification. Different concentrations (0.0–200 μg/mL) of the extract (0.5 mL) were mixed with 0.5 mL phosphate buffer (0.2 M, pH 6.6) and 0.5 mL potassium hexacyanoferrate (0.1%), followed by incubation at 50 °C in a water bath for 20 min. After incubation, 0.5 mL of TCA (10%) was added to terminate the reaction. The upper portion of the solution (1 mL) was mixed with 1 mL distilled water, and 0.1 mL FeCl
3 solution (1%) was added. The reaction mixture was left for 10 min at room temperature and the absorbance was measured at 700 nm against an appropriate blank solution. All tests were performed three times. A higher absorbance of the reaction mixture indicated greater reducing power. Butylated hydroxytoluene (BHT) was used as a positive control.
Fe2+ chelation
The ferrous ion chelating activity of extract was evaluated by a standard method [
16] with minor changes. The reaction was carried out in Tris-HCL buffer (0.1 M, pH 7.5). Briefly, various concentrations (0–200 μg/mL) of plant extract were added to 100 μM ferrous sulfate solution. The reaction mixture was incubated for 30 s, before the addition of 1, 10-Phenanthroline (0.25%
w/
v). The absorbance was subsequently measured at 510 nm in a spectrophotometer. EDTA was used as a positive control.
Hydroxyl radical scavenging
This was assayed by a standard method [
17]. Deoxyribose is degraded by hydroxyl radicals with the release of thiobarbituric acid (TBA) reactive materials. The assay was based on the generation of hydroxyl radical by the Fe
2+-H
2O
2 system (the Fenton reaction) and quantification of the degradation product of 2-deoxyribose by condensation with TBA.
The reaction mixture contained 120 μl of 2-deoxy-2-ribose (3 mM); 80 μl of potassium phosphate buffer (50 mM, pH 7.4); 80 μl of FeSO4 (100 μM); 80 μl of H2O2 (1.0 mM) and 40 μl of PBE (0, 25, 50, 100, 150, and 200 μg/ml) of the test sample and distilled water to make up a final volume of 1 ml. After incubation for 1 h at 37 °C, 0.5 ml of the reaction mixture was added to 1 ml of 2.8% (w/v) TCA, then 1 ml of 1% aqueous TBA was added. The mixture was incubated at 90 °C for 15 min. After cooling, the absorbance was measured at 532 nm against an appropriate blank solution. The percentage inhibition was evaluated by comparing the test and blank solutions.
Evaluation of membrane lipid peroxidation
Quantification of thiobarbituric acid reactive substances (TBARs) production, an index of biological membrane peroxidation, was determined as described by Puntel et al. [
16]. Briefly, 20 μL of PBE (50–250 μg/mL) and prooxidant agent (100 μm Fe
2+) were added to 100 μL of rat liver or brain tissue homogenate in Tris-HCL buffer (10 mM; pH 7.4). The reaction mixture was incubated at 37 °C in a water bath. Color reaction was developed by adding 200 μL of 8.1% sodium dodecyl sulfate (SDS) to the reaction mixture. This was subsequently followed by the addition of 500 μL of acetic acid/HCl buffer (1.34 M; pH 3.4) and 500 μL 0.6% thiobarbituric acid (TBA). The mixture was incubated at 100 °C for 1 h. TBARs produced were measured at 532 nm and the absorbance was compared with a malondialdehyde (MDA) standard curve.
Angiotensin-converting enzyme (ACE) inhibition assay
The assay was based on the hydrolysis of N-hippuryl-His-Leu hydrate (HHL) by the angiotensin-converting enzyme as described by Cushman and Cheung [
18]. Briefly, the enzyme source was prepared with freshly removed rat lung. The tissue was homogenised in cold 125 mM Tris buffer, pH 8.3 (1/10,
w/
v) and centrifuged at 4 °C for 10 min 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 extracts/drug (PBE- 25, 50, 100 μg/mL; ramipril- 0.04, 0.2, 0.4 μg/mL). This was incubated at 37 °C for 15 min. Thereafter, ACE substrate, HHL (8.3 mM; 150 μl) was added and further incubated for 30 min at the same temperature in a shaker. The enzymic reactions were terminated by addition of 1 ml of 1 M HCI. The hippuric acid formed by the action of the angiotensin-converting enzyme on HHL was extracted from the acidified solution into l-2 ml of ethyl acetate by vortex mixing for 15 s. After centrifugation at 3000
g for 5 min, an l ml aliquot of each ethyl acetate layer was transferred into a clean tube. The ethyl acetate fractions were evaporated by heating at 120 °C for 30 min in a Temp-Blok module heater. The hippuric acid was re-dissolved in 1 ml distilled water and the amount formed was determined from its absorbance at 228 nm wavelength.
Isolation of hepatic mitochondria
Liver mitochondria were isolated as previously described [
19]. Wistar rats were killed by decapitation and the liver tissues were rapidly removed and placed on ice-cold isolation buffer containing 225 mM mannitol, 75 mM sucrose, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 0.1% bovine serum albumin (BSA; free fatty acid) and 10 mM HEPES pH 7.2. The tissues were then homogenized and the resulting suspension centrifuged for 7 min at 2,000Xg. Next, the supernatant was centrifuged for 10 min at 12,000Xg. The pellet was re-suspended in isolation buffer II containing 225 mM mannitol, 75 mM sucrose, 1 mM EGTA and 10 mM HEPES pH 7.2 and centrifuged at 12,000Xg for 10 min. Finally, the last supernatant was discarded, and the pellet was re-suspended and maintained in buffer III (sucrose 100 mM, KCl 65 mM, K
+-HEPES 10 mM and EGTA 50 μM pH 7.2) to a protein concentration of 0.5 mg/mL for subsequent analyses. Protein concentration was measured by the method described by Lowry et al. [
20] using bovine serum albumin (BSA) as standard.
Measurement of mitochondrial membrane potential (∆ψm)
Mitochondrial membrane potential was estimated by fluorescence changes of safranine recorded by spectrofluorimeter [
19]. The cuvette inside the spectrofluorimeter contains 3 mL of buffer III to which an aliquot (30 mL) of the isolated mitochondria (approximately 500 mg protein) was added. The reaction was started by the addition of safranine (67 mM) and succinate (1.5 M) added after 10 s. The fluorescence was monitored for 200 s after which 10 μL of extract/drug (PBE: 25, 50, 100 μg/mL; catechin: 1, 5, 10 μg/mL) or distilled water (for control) was added and allowed for additional 150 s. Finally 2,4-dinitrophenol, DNP (100 mM, 30 μL) was added to uncouple oxidative phosphorylation and inhibit adenosine triphosphate production. The change in fluorescence was recorded by a RF-5301 Shimadzu spectrofluorimeter (Kyoto, Japan) operating at excitation and emission wavelengths of 495 nm and 586 nm, respectively, with slit widths of 3 nm. The potential difference (∆Ψm
) was obtained by the difference between the fluorescence intensity prior to and after DNP addition.
Evaluation of reactive species (RS) formation with DCH (dichlorofluorescein-reactive species (DCH-RS)
RS levels were measured using the oxidant sensing fluorescent probe, 2′,7′-dichlorofluorescein diacetate (DCHF–DA) [
21]. The oxidation of DCHF–DA to fluorescent dichlorofluorescein (DCF) was determined at 488 nm for excitation and 525 nm for emission. An aliquot of 5 μL (50 μg/protein) of the homogenate of the isolated mitochondria was added to 3 mL of buffer III (containing 5 mM succinate). The reaction medium was exposed to PBE (25, 50, 100 μg/mL) or catechin (1, 5, 10 μg/mL) with or without Ca
2+ (80 μM) or SNP (150 μM). After 10 s, DCHF–DA (10 μM, in absolute ethanol) was added to the mixture and the fluorescence intensity from DCF was measured for 300 s. Values of mitochondrial membrane potential (∆ψm) were expressed as percent of control.
Statistical analysis
Results calculated from triplicate data were expressed as means ± standard error of means (SEM). With the exception of data on mitochondrial ROS production which was analysed using two-way ANOVA followed by Bonferroni post test, data were analyzed using one-way analysis of variance followed by Neuman-Keuls comparison of means. The significance level was set at p < 0.05. Statistical analysis, graphing and EC50/IC50 (median effective concentration/median inhibitory concentration) determination were done using Graph Pad Prism (ver.5.0a).
Discussion
Many of the therapeutic actions of phytochemicals are thought to be associated with their biologically active polyphenol components, such as flavonoids and phenolic acids, which possess powerful antioxidant activities [
22]. In the present study, the radical scavenging activity of
P. biglobosa extract was evaluated using different radical systems in vitro as it has been proposed that the efficacy and the antioxidant potency of some extracts may vary depending on the pro-oxidant used [
23]. As a principle, spectrophotometric measurement of the colour change of the DPPH solution from purple to yellow as the radical is quenched by the antioxidant gives a quantitative estimation of the antioxidant. Hence it could be proposed that reactions of antioxidant, hydrogen donors in
P. biglobosa with DPPH radicals reduce the latter to the corresponding hydrazine [
19]. A similar proposition could also be put forward for the decolourization of the ABTS radicals following exposure to PBE. Potential antioxidant activity has been reported to be concomitant with the reducing power of a plant extract.
P. biglobosa leaf extract possesses considerable reducing property as demonstrated by its concentration-dependent ability to reduce Fe
3+ to Fe
2+. In this case, the phenolics in the leaf could act as reducing agents by donating electrons to free radicals and terminating the free radical-mediated chain reactions [
24]. Iron can promote lipid peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl radicals that can perpetuate the chain reaction and via the Fenton reaction. Metal chelating agents reduce the concentration of metal ions in the Fenton-type reaction and thus would protect the system from oxidative damage through inhibition of metal-dependent processes [
25]. Understandably,
P. biglobosa extract was not as potent as EDTA in iron chelation because the metal-chelating efficiency of the phytochemicals (notably polyphenols) involved are usually lower than those of standard chelators like EDTA [
26].
In the present study, the inhibitory effects of PBE against Fe
2+and SNP-induced lipid peroxidation were assessed. Lipid peroxidation is a free radical-mediated process involving lipid-derived radicals, such as alkoxyl and peroxyl radicals, wherein oxidative damage is propagated to polyunsaturated fatty acids. The toxicity of Fe (II) proceeds via the Fenton reaction where iron catalyses one-electron transfer reactions that generate reactive oxygen species, such as the OH
.- from H
2O
2. Iron is capable of decomposing lipid peroxides leading to the generation of peroxyl and alkoxyl radicals and favouring the propagation of lipid oxidation [
25]. Sodium nitroprusside (SNP), on the other hand, has been reported to cause cytotoxicity through the release of cyanide and/or nitric oxide [
27]. The released NO is capable of causing neuronal damage in cooperation with other reactive oxygen species (ROS) notably superoxide radical to form peroxynitrite radical [
28]. The considerable inhibition of SNP-induced lipid peroxidation by PBE could be attributed to the ability of the extract to scavenge NO
˙ radical produced by the SNP, thus protecting the tissues against oxidative insults [
28].
The present study revealed that
P. biglobosa extract exerts concentration-dependent and inhibition of both iron- and SNP-induced peroxidation in liver and brain homogenates. The protections offered by
P. biglobosa suggest that the hydromethanolic extract may protect the liver and brain against toxicities resulting from a potential overload of iron and nitric oxide. Angiotensin-converting enzyme (ACE) is responsible for producing the potent vasoconstrictor and trophic angiotensin II (AII). AII increases blood pressure by its action as a potent vasoconstrictive substance and by stimulating the production of aldosterone, which promotes sodium and water retention in the body. ACE-inhibitory substances are thus desirable when the aim is to achieve lower blood pressure. In the present study, PBE demonstrated ACE- inhibitory effect, a phenomenon that could be involved in its documented hypotensive potential under experimental condition [
12]. The phytochemicals responsible for the ACE-inhibitory effect of still remains to be elucidated. Hypothetically, however, the polyphenolic constituents of the leaf could be involved. Free hydroxyl groups of phenolic compounds are important structural moieties capable of chelating the zinc ions in ACE active sites, thus inactivating the enzyme [
29]. Several flavonoids have thus been shown to demonstrate competitive inhibition towards ACE [
30]. It is noteworthy that the ACE inhibitory activity of the crude extract is much lower than that recorded for the standard drug, Ramipril. Crude extracts are known to contain various phytochemical constituents capable of interfering with one another to produce antagonistic or synergistic effects. Pure compounds, on the other hand, are devoid of negative interactions. A higher ACE-inhibitory potency could, therefore, be recorded with the pure form(s) of the phytochemical(s) responsible for the effect.
SNP is a NO donor compound that reportedly exhibited a deleterious effect on mitochondrial function and survival of synoviocytes [
31] while reducing the activity of complex IV of the mitochondrial electron transport chain (MTC) with consequent apoptotic cell death [
32]. The toxicity of NO is dependent on its reaction with the superoxide anion radical (O2
.-) which yield peroxynitrite, a highly cytotoxic ROS. Peroxynitrite is further decomposed to the hydroxyl radical which eventually leads to lipid peroxidation, protein oxidation, and DNA damage [
33]. Mitochondrial dysfunction has been suggested as the underlying mechanism of NO-mediated toxicity [
34]. In the present study, PBE and its major polyphenolic constituent, catechin attenuated SNP-induced increase in the rate of mitochondrial reactive species formation possibly due to their free radical scavenging effects.
Disruption of calcium homeostasis and free radicals generation are among the detrimental effects associated with the toxicity of some compounds [
35]. Formation of ROS by mitochondria is enhanced as a consequence of increased cytosolic calcium concentrations ([Ca2+]) [
36]. In the present study, calcium-induced reactive species formation in the mitochondria was attenuated by PBE and catechin. Catechin was selected as the reference phenolic because it was previously reported to be present in high amount in PBE [
11]. Catechins possess strong metal ion chelating potentials due to their catechol structures and have been shown to form stable complexes with ions, including Fe
2+and Ca
2+ [
37]. Therefore, the reduction of a calcium-induced surge in mitochondrial ROS production by PBE might be related to the ability of the constituent phenolics to scavenge free radicals and chelate metal ion. Mitochondrial membrane potential (∆Ψm) contributes to determining a driving force for calcium to enter the mitochondria. ROS formation in mitochondria occurs at high membrane potentials. Mild depolarization of the mitochondrial potential by PBE does not involve the constituent catechin as revealed in the present study. Some activities in herbs may be attributable to other unidentified substances or to synergistic interactions among constituents. Even though the mechanism controlling the mitochondrial membrane potential ∆Ψm in vivo are complex and not fully understood, ‘mild un-coupling’ of mitochondria are turned on in vivo to diminish the formation of ROS [
38]. Decreased ∆Ψm, due to the uncoupling of electron flow from ATP synthesis by increased proton permeability of the inner mitochondrial membrane, can reduce ROS production at complex I by decreasing NAD(P)H/NAD(P)
+ and possibly by decreasing the life span of the semiquinone radical [
39]. Such decrease in the mitochondrial membrane potential (∆Ψm) primarily attenuates mitochondrial RO production with a consequential decrease in mitochondrial Ca
2+ uptake [
40], preventing mitochondrial calcium overload and the subsequent apoptosis [
41]. This might account for the superior mitochondrial ROS mitigating property of PBE over catechin. Such mild depolarization has been attributed to the neuroprotective effect of a plant extract [
42] and the protection of cardiomyocytes from oxidative stress [
40]. In the case of PBE, further studies are warranted to identify the active molecules and the underlying mechanisms involved in its mild mitochondrial potential depolarization propensity.