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J Acupunct Meridian Stud 2013;--(-):--e--

Available online at www.sciencedirect.com

Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com

- RESEARCH

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In Vitro Antioxidant Activity and Effect of Parkia biglobosa Bark Extract on Mitochondrial Redox Status Kayode Komolafe 1,2, Tolulope Mary Olaleye 1, Olaposi Idowu Omotuyi 3, Aline Augusti Boligon 2, Margareth Linde Athayde 2, Akintunde Afolabi Akindahunsi 1, Joao Batista Teixeira da Rocha 2,* 1

Department of Biochemistry, School of Sciences, Federal University of Technology, PMB 704, Akure, Nigeria 2 Department of Chemistry, Biochemical Toxicology, Federal University of Santa Maria, Santa Maria, Brazil 3 Department of Molecular Pharmacology and Neuroscience, Nagasaki University, Japan Available online - - Received: Oct 29, 2012 Revised: Jul 28, 2013 Accepted: Jul 30, 2013 KEYWORDS antioxidant; extract; HPLC; mitochondria; Parkia biglobosa; phenolics

Abstract Aqueous-methanolic extract of Parkia biglobosa bark (PBB) was screened for its polyphenolic constituents, in vitro antioxidant activity, and effect on mitochondria redox status. The in vitro antioxidant activity was assessed by using the scavenging abilities and the reducing powers of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and ABTS 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt radical cation; ABTS) against Fe3þ. Subsequently, the ability of PBB to inhibit lipid peroxidation induced by FeSO4 (10 mm) and its metal-chelating potential were investigated. The effects of the extract on basal reactive oxygen species (ROS) generation and on the mitochondrial membrane potential (DJm) in isolated mitochondria were determined by using 20 , 70 -dichlorodihydrofluorescin (DCFH) oxidation and safranin fluorescence, respectively. PBB mitigated the Fe(II)-induced lipid peroxidation in rat tissues and showed dose-dependent scavenging of DPPH (IC50: 98.33  10.0 mg/mL) and ABTS. (trolox equivalent antioxidant concentration, TEAC value Z 0.05), with considerable ferric-reducing and moderate metal-

* Corresponding author. Department of Chemistry, Biochemical Toxicology, Federal University of Santa Maria, Santa Maria, Brazil. E-mail: [email protected] (K. Komolafe). Copyright ª 2013, International Pharmacopuncture Institute pISSN 2005-2901 eISSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2013.08.003

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K. Komolafe et al. chelating abilities. PBB caused slight decreases in both the liver and the brain mitochondria potentials and resulted in a significant decrease (p < 0.001) in DCFH oxidation. Screening for polyphenolics using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) revealed the presence of caffeic acid, gallic acid, catechin, epigalocatechin, rutin, and quercetin. These results demonstrate for the first time the considerable in vitro antioxidant activity and favorable effect of PBB on mitochondria redox status and provide justification for the use of the plant in ethnomedicine.

1. Introduction Parkia biglobosa (Jacq) Benth, commonly called the African locust bean, is a perennial deciduous tree with dark green and alternate bipinnate leaves [1]. The tree is known as Igi iru or Igba iru among the Yoruba people of southwestern Nigeria, where the seeds are fermented to make a strongsmelling, tasty food rich in protein, popularly called Iru. P. biglobosa tree is largely prescribed in traditional medicine for its multiple medicinal virtues, and the bark and seeds are prescribed for the treatment of arterial hypertension [2]. The medicinal values of plants lie in their component phytochemicals, which produce definite physiological actions on the human body [3]. The presence of phytochemicals such as saponins, tannins, phenolics, and cardiac glycosides in P. biglobosa was reported [4]. Many of the therapeutic actions of phytochemicals are ascribed to their biologically active constituents with powerful antioxidant activities [5]. Polyphenolics appear to play a significant role as antioxidants in the protective effect of plant-derived foods and medicine [6] and have become the focus of current nutritional and therapeutic interest. Antioxidants are vital substances that possess the ability to protect the body from oxidative stress induced by free radicals [7]. Antioxidant principles from natural sources provide multifacetedness in their multitude and magnitude of activities and provide enormous scope in correcting oxidative imbalance. A number of biochemical reactions in the human body involve the generation of reactive oxygen species (ROS), and mitochondria are an important source of ROS within most mammalian cells [8,9] even under normal conditions. Excessive ROS not effectively eliminated by the antioxidant defense system stimulates oxidative damage by attacking lipids, carbohydrates, proteins, and DNA, and resulting in oxidative stress that leads to various disorders and diseases [10]. This ROS production contributes to mitochondrial damage in a range of pathologies and is also important in redox signaling from the organelle to the rest of the cell [8]. The mitochondrial membrane potential can be used as an indicator of mitochondrial health and oxidative state because this measure of ion transport reflects metabolic activity and integrity of the mitochondrial membrane [11]. There is documentation on the phytochemical constituents and antibacterial activity of the leaf and stem bark of plants [12], but little is known about its in vitro antioxidant activity and the effect on the mitochondrial integrity. In the current study, we have assessed the antioxidant activity of aqueous-methanolic extract of P. biglobosa bark (PBB)

in vitro and its effect on the redox status of mitochondria isolated from the liver and brain of rats. In addition, the phenolic composition of the extract was characterized by reverse phase HPLC coupled to a diode array detector (DAD).

2. Materials and methods 2.1. Chemicals Thiobarbituric acid (TBA), malonaldehyde bis-(dimethyl acetal; MDA), 20 ,70 -dichlorofluorescein-diacetate (DCFHDA), ethylene glycol tetraacetic acid (EGTA), and 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Plant material Fresh bark of P. biglobosa was collected from a private farm in Isua-Akoko, Akoko South East Local Government Area of Ondo State, Nigeria, in January 2011. Botanical identification and authentication was carried out at the herbarium Forestry Research Institute (FHI) Ibadan, Oyo state, Nigeria and assigned the voucher number 109603.

2.3. Preparation of aqueous-methanolic extract of PBB The bark was air-dried 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 hours. The filtrate was concentrated using a rotary evaporator and then subjected to freeze-drying. The residue was kept at 20  C for future use. In each case, extract was reconstituted in water to give specific concentrations (in mg/mL or mg/mL) prior to use.

2.4. Total antioxidant activity Total antioxidant activity was determined by the 3ethylbenzothiazoline-6-sulfonic acid diammonium salt radical cation (ABTS) test described by Re et al [13]. The ability of the test sample to scavenge ABTS.þ radical cation was compared to that of the trolox standard. To determine the trolox equivalent antioxidant concentration (TEAC), the percentage inhibition of absorbance was calculated and plotted as a function of the concentration of standard or

Please cite this article in press as: Komolafe K, et al., In Vitro Antioxidant Activity and Effect of Parkia biglobosa Bark Extract on Mitochondrial Redox Status, Journal of Acupuncture and Meridian Studies (2013), http://dx.doi.org/10.1016/j.jams.2013.08.003

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sample. The gradient of the plot for the sample was thereafter divided by the gradient of the plot for trolox.

2.10. Inhibition of membrane lipid peroxidation in vitro

2.5. Reducing power

The ability of PBB to inhibit membrane lipid peroxidation in tissues was determined by quantifying the level of thiobarbituric acid reactive substances (TBARS) as described by Puntel et al [16]. Briefly, 20 mL of PBB (25e250 mg/mL) and prooxidant agent (100 mm Fe2þ) were added to 100 mL of supernatant (S1) from rat brain, liver, or heart in Tris-HCL buffer (10 mM; pH 7.4). Twenty mL of distilled water was added to the control tubes instead of PBB. The reaction mixture was incubated at 37  C in a water bath. Color reaction was developed by adding 200 mL of 8.1% sodium dodecyl sulfate (SDS) to the reaction mixture. This was subsequently followed by the addition of 500 mL of acetic acid/HCl buffer (1.34M; pH 3.4) and 500 mL 0.6% thiobarbituric acid (TBA). The mixture was incubated at 100  C for 1 hour. TBARS produced were measured at 532 nm and the absorbance was compared with malondialdehyde (MDA) standard curve.

The Fe3þ-reducing power of the extract was determined as described by Oyaizu [14] with a slight modification. Briefly, 0.5 mL of PBB (25e200 mg/mL), the reference butylated hydroxytoluene, BHT (5e50 mg/mL), or the appropriate solvent (distilled water or ethanol) were mixed with 0.5 mL phosphate buffer (0.2M, pH 6.6) and 0.5 mL potassium hexacyanoferrate (0.1%), followed by incubation at 50  C in a water bath for 20 minutes. Also, 0.5 mL of trichloroacetic acid (TCA,10%) was added to terminate the reaction. The upper portion of the solution (1 mL) was mixed with 1 mL of distilled water and 0.1 mL FeCl3 solution (1%) was added. The reaction mixture was left for 10 minutes at room temperature and the absorbance was measured at 700 nm against an appropriate blank solution.

2.6. DPPH radical scavenging activity of extract 2.11. Isolation of fresh rat brain mitochondria DPPH radical-scavenging activity of PBB and the reference compound (ascorbic acid) was determined as described by Hatano et al. [15]. The capacity of extracts to scavenge the lipid-soluble 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, which results in the bleaching of the purple color exhibited by the stable DPPH radical, is monitored at an absorbance of 517 nm.

2.7. Fe2D chelation assay The ferrous ion chelating activity of extract was evaluated by a standard method [16]. EDTA was used as a positive control.

2.8. Animals Male Wistar rats (3 months old), weighing between 270 g and 320 g, from the University breeding colony (Animal House holding, Universidade Federal de Santa Maria, UFSM, Brazil) were kept in cages with free access to food and water in a room with controlled temperature (22  C  3  C) and in 12-hour light/dark cycle with lights on at 7:00 AM. The animals were maintained and used in accordance to the guidelines of the Brazilian association for laboratory animal science (Cole ¸˜ ao Ani´gio Brasiliero de Experimentac mal; COBEA).

2.9. Preparation of tissue homogenates Rats were sacrificed by decapitation on the day of experiment and rapidly dissected. Whole brain, liver, and heart were excised, placed on ice, and weighed. Tissues were immediately homogenized in cold 10 mM TriseHCl, pH 7.4 (1/10, w/v). The homogenate was centrifuged for 10 minutes at 4000 g to yield a pellet that was discarded and a low-speed supernatant (S1) that was used in the experiments.

Brain and liver mitochondria were isolated as previously described by Brustovetsky and Dubinsky [17,18] with minor modifications. Wistar rats were killed by decapitation. Both organs were rapidly removed and placed on an ice-cold isolation buffer containing 225 mM mannitol, 75 mM sucrose, 1 mM 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 minutes at 2000 g. Next, the supernatant was centrifuged for 10 minutes at 12,000 g. The pellet was resuspended in isolation buffer II containing 225 mM mannitol, 75 mM sucrose, 1 mM EGTA, and 10 mM HEPES pH 7.2 and centrifuged at 12,000 g for 10 minutes. Finally, the last supernatant was discarded, and the pellet was resuspended and maintained in buffer III (sucrose 100 mM, KCl 65 mM, Kþ-HEPES 10 mM, and EGTA 50 mM pH 7.2) to a protein concentration of 0.5 mg/mL for subsequent analyses.

2.12. Determination of ROS production in brain and liver mitochondria ROS production in brain and liver mitochondria was measured using the oxidant sensing fluorescent probe, 20 ,70 -dichlorofluorescein diacetate (DCFHeDA) as described by Wagner et al [19]. An aliquot (5 mL) of the isolated mitochondria (approximately 50 mg protein) was added to 3 mL of buffer III containing 5 mM succinate. The reaction medium was exposed to 10 mL of PBB (25 mg/mL, 50 mg/mL, or 100 mg/mL) or 10 mL of distilled water (for control; indicated 0 mg/mL PBB). After 10 seconds, 10 mM DCFHeDA (prepared in ethanol) was added to the mixture and the fluorescence intensity emission arising from the oxidized fluorescent derivative (DCF) was measured over a 300-second period using a spectrofluorimeter (RF-5301 Shimadzu, Kyoto, Japan).

Please cite this article in press as: Komolafe K, et al., In Vitro Antioxidant Activity and Effect of Parkia biglobosa Bark Extract on Mitochondrial Redox Status, Journal of Acupuncture and Meridian Studies (2013), http://dx.doi.org/10.1016/j.jams.2013.08.003

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2.13. Measurement of mitochondrial membrane potential (Djm) Mitochondrial membrane potential was estimated by fluorescence changes of safranine [20]. 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 (1.5M) added after 10 seconds. The fluorescence was monitored for 200 seconds after which 10 mL of PBB (25 mg/mL, 50 mg/mL, or 100 mg/mL) or distilled water (for control) was added and allowed for additional 150 seconds. Finally 2,4-dinitrophenol, DNP (100mM) 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 (Djm) was obtained by the difference between the fluorescence intensity prior to and after DNP addition.

2.14. Protein estimation Protein concentrations of the tissue and mitochondrial homogenates were measured by the method described by Lowry et al. [21] using BSA as standard.

2.15. Statistical analysis Experiments conducted in replicates were expressed as means  standard error of the mean (SEM). Data on mitochondrial ROS production was analyzed using two-way analysis of variance followed by Bonferroni posttest. Unless otherwise stated, other data were analyzed using oneway analysis of variance followed by Dunnett post hoc test. The significance level was set at p < 0.05.

K. Komolafe et al. 50 mL, and wavelength 254 nm for gallic acid, 280 nm for catechin and epigallocatechin, 325 nm for caffeic acid, and 365 nm for quercetin and rutin. All the samples and mobile phase were filtered through 0.45-mm membrane filter (Millipore; Sigma-Aldrich (St. Louis, MO, USA)) and then degassed by ultrasonic bath prior to use. All chromatography operations were carried out at ambient temperature and in triplicate.

3. Results 3.1. DPPH free-radical scavenging property The extract caused a concentration-dependent decrease in DPPH radical with an IC50 value of 98.52  1.0 versus 14.6  0.8 for ascorbic acid (Fig. 1).

3.2. Total antioxidant activity The total antioxidant activity of aqueous-methanolic extract of PBB was calculated from the decolorization of ABTSþ. The presence of the extract or trolox standard decreased the intensity of the absorbance of ABTS radical in a dose-dependent manner (Fig. 2). The TEAC value of the extract was 0.05  0.001.

3.3. Reducing power The transformation of Fe3þ to Fe2þ in the presence of PBB and the reference compound BHT was used to measure the reductive capability (Fig. 3). PBB showed concentrationdependent reducing property with the highest absorbance of 0.89  0.04 at 200 mg/mL final concentration. At 50 mg/ mL concentrations, the absorbance for PBB was 0.40  0.05 versus 0.95  0.007 for the synthetic antioxidant BHT.

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HPLC-DAD was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector, and LC solution 1.22 SP1 software. Reverse phase chromatography analyses were carried out under gradient conditions using a Phenomenex C18 column (4.6 mm  150 mm) obtained from Phenomenex (Torrance, CA 90501-1430, USA) and packed with 5-mmdiameter particles. The mobile phase was solvent A Z water/ acetic acid (99:1 v/v) and solvent B Z acetonitrile. The gradient program was started with 13% of B until 10 minutes and changed to obtain 20%, 30%, 50%, 70%, 20%, and 10% B at 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, and 80 minutes, respectively. Aqueous-methanolic bark extract of P. biglobosa was analyzed after dissolving in ethanol at a concentration of 10 mg/mL. Identification of compounds was performed by comparing their retention time and UV absorption spectrum with those of the commercial standards. The flow rate was 0.5 mL/min, injection volume

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Figure 1 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) radical scavenging activity of Parkia biglobosa bark extract (PBB) and the reference compound, ascorbic acid. Results are mean  standard error of the mean of three parallel measurements performed in triplicates. ))) p < 0.001 from 0 mg/ mL PBB/ascorbic acid (control).

Please cite this article in press as: Komolafe K, et al., In Vitro Antioxidant Activity and Effect of Parkia biglobosa Bark Extract on Mitochondrial Redox Status, Journal of Acupuncture and Meridian Studies (2013), http://dx.doi.org/10.1016/j.jams.2013.08.003

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Figure 2 Total antioxidant activity of Parkia biglobosa bark extract (PBB) and the reference, trolox. Results are mean  standard error of the mean of three parallel measurements performed in triplicates. p < 0.001 from 0 mg/mL PBB/trolox.

Figure 4 Effect of Parkia biglobosa bark (PBB) extract and the reference compound EDTA on phenantroline-Fe2þ complex formation. Results are mean  standard error of the mean of three parallel measurements performed in triplicates. ) p < 0.05 from 0 mg/ml PBB/EDTA. ))) p < 0.001 from 0 mg/ mL PBB/EDTA.

3.5. Inhibition of Fe(II) induced lipid peroxidation 3.4. Iron chelating potential PBB showed inferior but dose-dependent Fe(II) chelating activity with slightly over 40% chelation at 200 mg/mL extract concentration compared with that of the standard EDTA with about 55% chelation at 50 mg/mL concentration (Fig. 4).

Treatment of tissue homogenates with FeSO4 (10 mM) caused the accumulation of lipid peroxides as manifested by up to 80%, 85%, and 77% increase in MDA content of the liver, heart, and brain, respectively, when compared to the respective basal homogenates. Incubation of tissues in the presence of extract resulted in statistically significant

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Figure 5 Inhibition of Fe(II)-induced lipid peroxidation in rats’ liver, brain, and heart by Parkia biglobosa bark extract. Results are mean  standard error of the mean of three parallel measurements performed in triplicates. ) p < 0.05 from 0 mg/ml PBB. )) p < 0.01 from 0 mg/ml PBB. ))) p < 0.001 from 0 mg/mL PBB.

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decrease (p < 0.001) and dose-dependent inhibition of lipid peroxides accumulation caused by toxicant in rat tissue (Fig. 5).

minutes; 3.98%; peak 3), epigallocatechin (tR Z 41.29 minutes; 4.15%; peak 4), rutin (tR Z 55.03 minutes; 0.52%; peak 5) and quercetin (tR Z 65.01 minutes; 2.37%; peak 6; Fig. 8 and Table 1).

3.6. Mitochondrial ROS generation

4. Discussion

Basal mitochondrial ROS generation in the liver and brain typified by the fluorescent DCF production was significantly reduced in the presence of 50 mg/mL and 100 mg/mL PBB when compared to the controls (Fig. 6A and B).

The therapeutic actions of most medicinal plants can be linked to their biologically active constituents with powerful antioxidant activities. The effect of antioxidants on the stable DPPH radical scavenging is due to their hydrogen-donating ability. It may be postulated that P. biglobosa reduces the radical to the corresponding hydrazine when it reacts with hydrogen donors in the antioxidant principle [22] (Fig. 1). As revealed in Fig. 2, P. biglobosa bark extract produced a concentration-dependent reduction of the radical cation to ABTS. The results were compared with those obtained using trolox and the TEAC value of 0.05 demonstrates the extract exhibits considerable antioxidant activity by the scavenging of the cation radical. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. As shown in Fig. 3, PBB possesses considerable reducing

3.7. Mitochondrial membrane potential PBB caused a slight decrease in the membrane potential of both liver and brain mitochondria. The decrease was statistically significant (p < 0.05) at 100 mg/mL concentration in mitochondria from both tissues (Fig. 7A and B).

3.8. HPLC screening HPLC fingerprinting of PBB revealed the presence of gallic acid (tR Z 12.78 minutes; 0.81%; peak 1), caffeic acid (tR Z 23.97 minutes; 4.73%; peak 2), catechin (tR Z 33.17

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Figure 6 Effect of Parkia biglobosa bark (PBB) extract on reactive oxygen species (ROS) production in mitochondria isolated from rat liver (A) and brain (B). Mitochondria were incubated in a medium containing buffer III (100 mM sucrose, 65 mM KCl, 10 mM KþHEPES and 50 mM ethylene glycol tetraacetic acid; pH 7.2), 5 mM succinate and PBB (25 mg/mL, 50 mg/mL or 100 mg/mL) or 10 mL of distilled water (for control; indicated 0 mg/mL PBB) for 10 seconds. The reaction was initiated by the addition of 20 ,70 -dichlorofluorescein diacetate (DCFH-DA) and the fluorescence intensity emission arising from the oxidized fluorescent derivative (DCF) was measured over a 300-second period. Results are presented as mean  standard error of the mean of three experiments performed in triplicates using independent mitochondrial preparations. Data analysis was done by two-way analysis of variance, followed by Bonferroni posttests (p < 0.05 was considered statistically significant). (A) 50 mg/mL PBB: p < 0.001 from 0 mg/mL PBB; 100 mg/mL: p < 0.01 from 0 mg/mL PBB. (B) 50 and 100 mg/mL PBB: p < 0.001 from 0 mg/mL PBB. Please cite this article in press as: Komolafe K, et al., In Vitro Antioxidant Activity and Effect of Parkia biglobosa Bark Extract on Mitochondrial Redox Status, Journal of Acupuncture and Meridian Studies (2013), http://dx.doi.org/10.1016/j.jams.2013.08.003

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Figure 7 Effect of Parkia biglobosa bark (PBB) extract on membrane potentials of isolated liver (A) and brain (B) mitochondria. The reaction mixture contained 3 mL of buffer III (100 mM sucrose, 65 mM KCl, 10 mM Kþ-HEPES and 50 mM ethylene glycol tetraacetic acid; pH 7.2,) and an aliquot of the isolated mitochondria (approximately 500 mg protein) in a spectrofluorimeter. The reaction was started by the addition of safranine (67 mM) and followed by succinate (1.5M) after 10 seconds. Fluorescent intensity was monitored for 200 seconds prior to adding 10 mL of PBB (25 mg/mL, 50 mg/mL, or 100 mg/mL) or distilled water (for control). 2,4dinitrophenol, (DNP; 100 mM) was added after 350 seconds and the changes in fluorescence recorded. The potential difference (Djm) was obtained by the difference between the fluorescence intensity prior to and after DNP addition. Results are presented as mean  standard error of the mean of three assays performed in triplicates using independent mitochondrial preparations. y Statistical significance in Djm of both liver and brain mitochondria; p < 0.05 from 0 mg/mL PBB (control) at 100 mg/mL PBB.

properties as demonstrated by its ability to reduce Fe3þ to Fe2þ, suggesting that the polyphenolics present in the leaves could act as reductones by donating electrons to free radicals and terminating the free radical mediated chain reactions [23]. The propensity for metal chelation, particularly iron and copper, supports the role of

polyphenols as preventive antioxidants in terms of inhibiting transition metal-catalyzed free radical formation because it reduces the concentration of the transition metal that catalyzes lipid peroxidation [24]. As shown in Fig. 4 and as expected of polyphenols [25], the plant extract does not work as well as the standard EDTA;

Table 1

Phenolic composition of Parkia biglobosa bark.

Compounds

Parkia biglobosa mg/g

Gallic acid Caffeic acid Catechin Epigallocatechin Rutin Quercetin

Figure 8 Representative high performance liquid chromatography profile of Parkia biglobosa bark.

8.15 43.70 39.84 41.52 5.23 23.71

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a

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0.81 4.73 3.98 4.15 0.52 2.37

Results are expressed as mean  standard deviations (SD) of three determinations. aee Averages followed by different letters differ by Tukey test at p < 0.001.

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8 however, the decrease in concentration-dependent color formation in the presence of the extract indicates PBB has iron chelating activity. In the current work, we have assessed the inhibitory effects of crude extract against iron- induced lipid peroxidation in the homogenates of isolated rat liver, brain, and heart (Fig. 5) to provide information on what lipid or other substrate is protected. The toxicity of Fe(II) proceeds via the Fenton reaction, in which iron catalyses a one-electron transfer reaction that generates ROS, such as the OH.e from H2O2. The current study demonstrated that PBB exerts concentration-dependent and significant inhibition of ironinduced peroxidation in the tissue homogenates tested (Fig. 5). This is suggestive of potential protectivity of PBB against toxicities resulting from potential overload of iron in cardiac, hepatic, and cerebral tissues [26,27]. HPLC-DAD analysis is advantageous over total phenolics content as determined by the Folin-Ciocalteu method, because it provides more precise information of individual compounds. The major polyphenols in PBB are caffeic acid and the flavonoids catechin and epicatechin (Table 1). Typical phenolics that possess antioxidant activity are known to be mainly phenolic acids and flavonoids. The free radical-scavenging activity of flavonoids is attributed to their hydrogen-donating ability. The phenolic groups of flavonoids serve as a source of readily available H atoms such that the subsequent radicals produced can be delocalized over the flavonoid structure [28]. Catechins are a type of efficient chain-breaking antioxidants under in vitro conditions because they are capable of efficiently scavenging lipid free radicals, hydroxylradicals, and DPPH radicals, and chelate the iron ion [29]. The catechins and flavonol quercetin exhibit considerable antioxidant activity in the lipophilic phase and are capable of protecting lowdensity lipoprotein from toxicant-mediated oxidation albeit with a varying degree of effectiveness [30]. Such properties have been suggested to explain the relationship between cardioprotection and the increased consumption of flavonoids from dietary sources such as onions, apples, tea, and red wine [31]. For metal chelation, the two points of attachment of transition metal ions to the flavonoid molecule are the o-diphenolic groups in the 30 ,40 -dihydroxy positions in the B ring, and the ketol structures 4-keto, 3- hydroxy or 4-keto and 5-hydroxy in the C ring of the flavonols. Some antioxidant activities in herbs may be attributable to other unidentified substances or to synergistic interactions among constituents. In the current study, we investigated the effect of PBB on basal ROS generation in mitochondria isolated from brain and liver of rats. Mitochondria in cells are the major producers of ROS that can result in the oxidative stress that contributes to mitochondrial damage in a range of pathologies [8,32]. PBB showed a protective effect by attenuating ROS production in the brain and liver mitochondria (Fig. 6) and caused partial depolarization in membrane potential of mitochondria isolated from both the liver and brain (Fig. 7). The mechanisms by which a partial mitochondrial depolarization elicited by preconditioning with isoflurane or by treatment with the uncoupling agent DNP protected cardiomyocytes from oxidative stress was recently demonstrated [33]. The DJm-dependent attenuation of excess ROS production by mitochondria appears to play a

K. Komolafe et al. central role in the adaptation to oxidative stress by anesthetic-induced preconditioning, which induces a delay in oxidative stress-triggered mitochondrial permeability transition pore opening [34], thereby increasing cell survival [33]; similar mechanisms may also be involved in neuroprotection. Cardioprotection can be mediated by mitochondrial uncoupling proteins and agents known to depolarize mitochondria [35], indicating the importance of DJm regulation. It can be speculated that the constituent phenolics of PBB play an important role in the observed mitigation of mitochondrial ROS formation, taking into consideration the well-established antioxidant properties of these phytochemicals. Catechins and caffeic acid, which are major phenolics in PBB, were reported to exhibit a protective effect on the mitochondria via various mechanisms including attenuation of ROS generation [36e38]. Flavonoid extracts from medicinal plants could also attenuate toxicanteinduced mitochondrial ROS formation and oxidative damage [39]. Based on the findings in the current study and others [37,39], it might be inaccurate to attribute the mild mitochondrial depolarization propensity by PBB to specific phytoconstituents. We recently discovered that mild depolarization of mitochondrial Djm by P. biglobosa leaf extract is independent of catechin, one of its major phenolic constituents [40]. In this regard, identifying the active molecules involved is subject to further studies. Notwithstanding the observed effect of PBB on mitochondrial redox status, especially considering that the extract is a complex mixture of phytochemicals, in vivo studies are necessary to define the concentrations that could exert biological effects. Apparently for most polyphenols, however, sufficient absorption occurs to reach biologically relevant concentrations in the bloodstream [41,42]. In conclusion, the results suggest that PBB exhibits considerable antioxidant activity in vitro and favorable effects on mitochondria redox status that may be attributed to its predominant phenolics. Thus, the beneficial outcome from the use of P. biglobosa herbal preparations could be additionally ascribed to the confirmed antioxidant properties of the plant.

Acknowledgments The authors would like to acknowledge the financial support from the Brazilian National Council for Scientific and Technological Development (CNPq) and the Academy of Sciences for the Developing World (TWAS).

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Please cite this article in press as: Komolafe K, et al., In Vitro Antioxidant Activity and Effect of Parkia biglobosa Bark Extract on Mitochondrial Redox Status, Journal of Acupuncture and Meridian Studies (2013), http://dx.doi.org/10.1016/j.jams.2013.08.003

In vitro antioxidant activity and effect of Parkia biglobosa bark extract on mitochondrial redox status.

Aqueous-methanolic extract of Parkia biglobosa bark (PBB) was screened for its polyphenolic constituents, in vitro antioxidant activity, and effect on...
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