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Protective effects of piperine against copperascorbate induced toxic injury to goat cardiac mitochondria in vitro Mousumi Dutta,ab Arnab Kumar Ghosh,a Prachi Mishra,c Garima Jain,c Vinod Rangari,c Aindrila Chattopadhyay,b Tridib Das,d Debajit Bhowmickd and Debasish Bandyopadhyay†*a Piperine, the main alkaloid of black pepper, Piper nigrum Linn., is an important Indian spice used in traditional food and medicine in India. In the present study, we investigated the antioxidant activities of piperine against copper-ascorbate induced toxic injury to mitochondria obtained from a goat heart, in vitro. Incubation of isolated cardiac mitochondria with copper-ascorbate resulted in elevated levels of lipid peroxidation and protein carbonylation of the mitochondrial membrane, a reduced level of mitochondrial GSH and altered status of antioxidant enzymes as well as decreased activities of pyruvate dehydrogenase and the Kreb's cycle enzymes, altered mitochondrial morphology, mitochondrial swelling, di-tyrosine level and mitochondrial DNA damage. All these changes were found to be ameliorated when the cardiac mitochondria were co-incubated with copper-ascorbate and piperine, in vitro. Piperine, in our in vitro experiments, was found to scavenge hydrogen peroxide, superoxide anion free radicals, hydroxyl radicals and DPPH radicals, in a chemically defined system, indicating that this

Received 25th April 2014 Accepted 29th June 2014

compound may provide protection to cardiac mitochondria against copper-ascorbate induced toxic injury through its antioxidant activities. The results of this study suggest that piperine may be considered

DOI: 10.1039/c4fo00355a

as a future therapeutic antioxidant and may be used singly or as a co-therapeutic in the treatment of

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diseases associated with mitochondrial oxidative stress.

Introduction Piper nigrum family Piperaceae is a climbing vine native to Southern India and Sri Lanka and is extensively cultivated there and elsewhere in tropical regions. Its dried mature fruits have long been used in ayurveda and siddha systems of medicine as one of the ingredients along with other drugs. Piperine is a main constituent present in these mature fruits which is a transstereoisomer of 1-piperoylpiperidine. It is also known as (E,E)-1piperoylpiperidine and (E,E)-1-[5-(1,3-benzodioxol-5-yl)-1-oxo2,4-pentdienyl] piperidine. Piperine is the alkaloid responsible for the pungency of black pepper and long pepper, along with chavicine (an isomer of piperine). It is used as an antia

Department of Physiology, Oxidative Stress and Free Radical Biology Laboratory, University of Calcutta, University College of Science and Technology, 92, APC Road, Kolkata 700 009, India. E-mail: [email protected]; Tel: +91-9433072066

b

Department of Physiology, Vidyasagar College, Kolkata 700 006, India

c

S.L.T. Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya (Central University), Koni, Bilaspur-495 009, CG, India d

Acharya Prafulla Chandra Sikhsha Prangan, University of Calcutta, JD-2, Sector-III, Salt Lake City, Kolkata 700098, India † Principal Investigator, Centre with Potential for Excellence in Particular Area (CPEPA), University of Calcutta, University College of Science and Technology, 92, APC Road, Kolkata 700 009, India.

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inammatory and antimalarial agent in India. It is also used in the treatment of leukemia as well.1–3 Recent medical studies have shown that it is helpful in increasing the absorption of certain vitamins, selenium and beta-carotene.4 It also increases the body's natural thermogenic activity.5 Morocco uses it to treat weight loss and leukemia. Indonesia uses it to reduce or prevent headache and fever, as a treatment for snake poisoning, and to treat epilepsy.6,7 Recently, many bacterial pathogens have become resistant to existing antibiotics due to their indiscriminate use in the treatment of infectious diseases.8 Piperine was found to act as a scavenger of reactive oxygen species (ROS) at low concentrations.9 It has the ability to enhance the drug bioavailability by inhibiting drug metabolism and it is known to have insecticidal activity against mosquitoes and ies.10,11 Externally, it is used as a rubefacient and as a local application for relaxed sore throat and some skin disorder. It has antimicrobial and antimutagenic activities.12,13 The aqueous and ethanolic extract of black pepper has antioxidant and radical scavenging properties and inhalation of black pepper oil increases the reexive swallowing movement.14,15 The anticancer effect of piperine may be attributed to the inhibition of NF-kB, c-Fos, ATF-2 and CREB activities, suppression of angiogenesis by inhibiting Akt phosphorylation, or blockade of the production of pro-inammatory cytokines and the activity of

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matrix metallo-proteinases that are likely to promote tumor growth and metastasis.16–18 Piperine also inhibits P-glycoprotein-mediated transport and CYP3A4-mediated drug metabolism, thus increasing the efficacy of antitumor agents.19 It has also been reported that piperine has an inhibitory effect on the proliferation of human prostate cancer cells via induction of cell cycle arrest and autophagy and also inhibits PMA-induced cyclooxygenase-2 expression through down-regulating NF-kB, CEBP and AP-1 signaling pathways in murine macrophages.20,21 Piperine along with curcumin ameliorates benzo(a)pyrene induced DNA damage.22 Piperine possesses anti-depression like activity and cognition enhancing effect. Therefore piperine, especially at low dose, has been shown to be the potential alternative to improve brain function.23 This molecule also has a chemopreventive effect in DMBA-induced hamster buccal pouch carcinogenesis.24 But to date there is no report available, to the best of our knowledge and belief, about the protective effect of piperine against mitochondrial oxidative stress. Herein, we provide evidence, perhaps for the rst time, that piperine has the ability to protect against copper-ascorbate induced toxic injury to goat heart mitochondria, in vitro, and antioxidant mechanism(s) may be responsible for such protections.

Materials and methods Chemicals Thiobarbituric acid (TBA), eosin, nicotinamide adenine dinucleotide (NAD), Direct Red-80, 2,2-dithiobis-nitro benzoic acid (DTNB), xanthine, xanthine oxidase (XO), cytochrome C, nitro blue tetrazolium (NBT), and glutathione (GSH) were obtained from Sigma, St. Louis, MO, USA. Hematoxylin, hydrogen peroxide (H2O2) and dimethyl sulfoxide (DMSO) were obtained from Merck Limited, Delhi, India. The superoxide dismutase (SOD) 1, SOD 2, glutathione-S-transferase (GST) and glutathione reductase (GR) were also obtained from Sigma, St. Louis, MO, USA. All other chemicals used including the solvents were of analytical grade and were obtained from Sisco Research Laboratories (SRL), Mumbai, India, Qualigens (India/Germany), SD ne chemicals (India), Merck Limited, Delhi, India. Isolation and extraction of piperine from the dry fruit of Piper nigrum Dried ripe fruits of black pepper, P. nigrum, were defatted with petroleum ether (60–80
C) in a Soxhlet extractor for 24 h. The extract was dried and further extracted with ethyl alcohol (95%) for 48 h. The total ethyl alcohol extract was cooled and ltered to remove ne particles if necessary, and concentrated under reduced pressure to yield the total alcohol extract in 2.5% yield. The concentrated solution was kept in an ice bath, and water was added dropwise (about 30 mL was required) to precipitate piperine. Piperine was collected on a sintered glass funnel. It was further recrystallized from acetone : hexanes (3 : 2) to afford pale yellow crystals of piperine. The thin layer chromatographic study of isolated piperine along with the authentic primary standard has shown a single spot of Rf 0.52 (Fig. 1A–C),

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Fig. 1 (A) Thin layer chromatography and number of spots of piperine, (B) chemical structure of piperine and (C) Rf value of piperine from Piper nigrum fruits.

in solvent system toluene–ethyl acetate (7 : 3) when spread with Dragendorff's reagent. This preparation of piperine is 99% pure and it is authenticated by the presence of single spot when it was spread with Dragendorff's reagent. No other components are present in the extract. Determination of antioxidant properties of piperine Hydroxyl radical scavenging activity. A hydroxyl radical was generated in sodium phosphate buffer (0.05 mM, pH 7.4) with 1 mM ascorbate and 0.2 mM Cu2+ for 60 minutes in the presence and absence of DMSO (500 mM) and different concentrations of piperine in a volume of 1 mL to determine the hydroxyl radical scavenging activity of piperine in an in vitro system. The reaction was terminated in each case by the addition of 0.1 mM EDTA. Methanesulnic acid (MSA) formed during incubation was measured by the method of Babbs and Steiner (1990)25 as modied by Bandyopadhyay et al., (2004).26 Superoxide anion free radical (O2c) scavenging activity. The superoxide anion free radical (O2c) scavenging activity was studied by following the rate of epinephrine oxidation in alkaline pH at 480 nm.27 The reaction mixture had, in a volume of 1 mL, 50 mM Tris–HCl buffer (pH 10), 0.6 mM epinephrine and different concentrations of piperine. The increase in absorbance due to the formation of the adrenochrome was followed for 7 minutes spectrophotometrically and the activity was calculated from the linear part in the absence and presence of piperine. The involvement of O2c was checked with standard SOD. Hydrogen peroxide (H2O2) scavenging activity. The hydrogen peroxide (H2O2) scavenging activity was assayed by studying the breakdown of H2O2 at 240 nm spectrophotometrically.28 The reaction mixture contained 50 mM phosphate buffer (pH 7.4), 1 mM H2O2 and 62.5 mM, 125 mM, 250 mM and 500 mM piperine in a nal volume of 3 mL. DPPH free radical scavenging activity. The DPPH free radical scavenging activity of each sample was determined according to

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the method described by Dutta et al. (2013).29 A solution of 0.1 mM DPPH in methanol was prepared. The initial absorbance of the DPPH in methanol was measured spectrophotometrically at 515 nm. 40 mL of piperine solution was added to 3 mL of methanolic DPPH solution. The change in absorbance at 515 nm was measured aer 30 min. The antiradical activity (AA) was determined using the following formula: AA% ¼ 100  ((Abssample  Absempty sample)  100)/Abscontrol

Preparation of goat heart mitochondria (caprine heart mitochondria) Goat heart mitochondria were isolated according to the procedure of Dutta et al. (2013) with some modications.29 A goat heart was purchased from a local Kolkata Municipal Corporation approved meat shop. Aer collection it was brought into the laboratory in a sterile plastic container kept in ice. Then, the heart tissue was cleaned and cut into pieces. Five grams of tissue was placed in 10 mL of sucrose buffer [0.25 (M) sucrose, 0.001 (M) EDTA, 0.05 (M) Tris–H2SO4 (pH 7.8)] at 25
C. Then the tissue was blended for 1 minute at low speed by using a Potter Elvenjem glass homogenizer (Belco Glass Inc., Vineland, NJ, USA), aer which it was centrifuged at 1500 rpm for 10 minutes. The supernatant was poured through several layers of cheesecloth and kept in ice. Then it was centrifuged at 4000 rpm for 5 minutes. The supernatant obtained was further centrifuged at 14 000 rpm for 20 minutes. The supernatant obtained was discarded and the pellet was resuspended in sucrose buffer and was stored at 20
C for further use. Each experiment was repeated three times with the mitochondria prepared from a fresh batch of heart. Incubation of mitochondria with copper-ascorbate The incubation mixture containing mitochondrial membrane protein (1.6 mg mL1), 50 mM potassium phosphate buffer (pH 7.4), and 0.2 mM Cu2+ and 1 mM ascorbic acid in a nal volume of 1.0 mL was incubated at 37
C in an incubator for 1 hour. The reaction was terminated by the addition of 40 ml of 35 mM EDTA.30 Protection of Cu2+-ascorbate-induced toxic injury to mitochondria by piperine The goat heart mitochondria were co-incubated with copperascorbate and four different concentrations of piperine. Aer incubation, the intact mitochondria were used for the examination of oxidative stress biomarkers including lipid peroxidation level, reduced glutathione and protein carbonyl content and the activities of antioxidant enzymes, Kreb's cycle enzymes, mitochondrial swelling, DNA damage and di-tyrosine level were determined. Determination of mitochondrial intactness by using Janus green B stain Aer incubation, the mitochondrial sample was diluted 1 : 200 by using 50 mM phosphate buffer (pH 7.4). Then, the mitochondria were spread and dried on a slide to prevent being

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washed away while staining. Aer that a few drops of Janus green stain were put on the slide and le for 5–10 min for staining. Aer 5 min of staining, the mitochondria were rinsed once with distilled water so that complete stain was not gone and a diluted stain remained. Then the mitochondria were mounted in a drop of distilled water with a cover slip and imaged with a laser scanning confocal system (Zeiss LSM 510 META, Germany) and the stacked images were captured. The digitized images were then analyzed using an image analysis system (ImageJ, NIH Soware, Bethesda, MI) and the intactness of mitochondria of each image was measured and expressed as the % uorescence intensity. Measurement of reactive nitrogen species (RNS) in mitochondria Nitric oxide concentrations in the incubated goat cardiac mitochondria were measured spectrophotometrically at 548 nm according to the method of Fiddler (1977) by using Griess reagent (1879).31,32 The reaction mixture in a spectrophotometer cuvette (1 cm path length) contained 100 mL of Griess Reagent, 700 mL of the sample (i.e., incubated mitochondrial suspension) and 700 mL of distilled water. The nitric oxide concentration was expressed as mM per mg protein. Determination of activities of respiratory complex enzymes under coupling and uncoupling conditions to assess the mitochondrial status The NADH–cytochrome C oxidoreductase activity was measured spectrophotometrically by following the reduction of oxidized cytochrome C at 565 nm according to the method of Goyal et al. (1995).33 1 ml of assay mixture contained, in addition to the enzyme, 50 mM phosphate buffer, 0.1 mg BSA, 20 mM oxidized cytochrome C and 0.5 (M) NADH. The activity of the enzyme was expressed as units per min per mg tissue protein. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as an uncoupler (0.1 mM). The cytochrome C oxidase activity was determined spectrophotometrically by following the oxidation of reduced cytochrome C at 550 nm according to the method of Goyal et al. (1995).33 One mL of assay mixture contained 50 mM phosphate buffer, pH 7.4, 40 mM reduced cytochrome C and a suitable aliquot of the enzyme. The enzyme activity was expressed as units per min per mg tissue protein. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as an uncoupler (0.1 mM). The ATP synthase activity, measured in the direction of ATP hydrolysis (ATPase activity), was determined by the continuous spectrophotometric assay of Rosing et al. (1975)34 except that 2 mM EGTA replaced EDTA in the reaction medium. Aliquots of sonicate (20–40 mg in 20 ml sample volume) were added to the reaction medium containing 60 mM sucrose, 50 mM triethanolamine–HCl, 50 mM KCl, 4 mM MgCl2, 2 mM ATP, 2 mM EGTA, 1 mM KCN, pH 8.0 (KOH) with 100 mM NADH, 5 units per mL pyruvate kinase and 5 units per mL lactate dehydrogenase. The total volume in the cuvette was 1 mL. The linear reaction was followed for 2 min at 340 nm and 37
C. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as an uncoupler (0.1 mM).

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Biochemical analysis Measurement of mitochondrial lipid peroxidation (LPO) level, reduced glutathione (GSH) and protein carbonyl (PCO) content. The lipid peroxides in the incubated mitochondria were determined as thiobarbituric acid reactive substances (TBARS) according to the method of Buege et al. (1978) with some modications as adopted by Bandyopadhyay et al. (2004).35,36 The incubated mitochondria were mixed with the thiobarbituric acid–trichloroacetic acid (TBA–TCA) reagent with thorough shaking and heated for 20 min at 80
C. The samples were then cooled to room temperature. The absorbance of the pink chromogen present in the clear supernatant aer centrifugation at 8000 rpm for 10 min at room temperature was measured at 532 nm using a UV-VIS spectrophotometer (BioRad, Hercules, CA, USA). The values were expressed as nmol of TBARS per mg protein. The GSH content (as acid soluble sulydryl) was estimated by its reaction with DTNB (Ellman's reagent) following the method of Sedlak et al. (1968)37 with some modications according to Bandyopadhyay et al. (2004).36 Incubated mitochondria were mixed with Tris–HCl buffer, pH 9.0, followed by DTNB for color development. The absorbance was measured at 412 nm using a UV-VIS spectrophotometer to determine the GSH content. The values were expressed as nmol GSH per mg protein. The protein carbonyl content was estimated by DNPH assay.38 0.25 mL of incubated mitochondrial suspension was taken in each tube and 0.5 mL DNPH in 2.0 M HCl was added to the tubes. The tubes were vortexed every 10 min in the dark for 1 h. Proteins were then precipitated with 30% TCA and centrifuged at 2000 rpm for 10 min. The pellet was washed carefully three times with 1.0 mL of ethanol : ethyl acetate (1 : 1, v/v). The nal pellet was dissolved in 1.0 mL of 6.0 M guanidine HCl in 20 mM potassium dihydrogen phosphate (pH 2.3). The absorbance was determined spectrophotometrically at 370 nm. The protein carbonyl content was calculated using a molar absorption coefficient of 2.2  104 M1 cm1. The values were expressed as nmol per mg protein. Measurement of the activities of Mn-superoxide dismutase (Mn-SOD), glutathione reductase (GR) and glutathione peroxidase (GPx) of goat cardiac mitochondria. The manganese superoxide dismutase (Mn-SOD or SOD2) activity was measured by the pyrogallol autooxidation method.39 To 50 ml of the mitochondrial sample, 430 ml of 50 mM Tris–HCl buffer (pH 8.2) and 20 ml of 2 mM pyragallol were added. An increase in absorbance was recorded at 420 nm for 3 min using a UV/VIS spectrophotometer. One unit of enzyme activity is 50% inhibition of the rate of autooxidation of pyragallol as determined by the change in absorbance per min at 420 nm. The enzyme activity was expressed as units per min per mg protein. The glutathione reductase (GR) assay was carried out according to the method of Krohne-Ehrich et al. (1977).40 The assay mixture in the nal volume of 3 mL contained 50 mM phosphate buffer, 200 mM KCl, 1 mM EDTA and water. The blank was set with this mixture. Then, 0.1 mM NADPH was added together with a suitable amount of incubated

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mitochondria (as the source of enzyme) into the cuvette. The reaction was initiated with 1 mM oxidized glutathione (GSSG). The decrease in NADPH absorption was monitored spectrophotometrically at 340 nm. The specic activity of the enzyme was calculated as units per min per mg protein. The glutathione peroxidase (GPx) activity was measured according to the method of Paglia et al. (1967) with some modications as adopted by Dutta et al. (2014).41,42 The assay system contained, in a nal volume of 1 mL, 0.05 M phosphate buffer with 2 mM EDTA, pH 7.0, 0.025 mM sodium azide, 0.15 mM glutathione, and 0.25 mM NADPH. The reaction was started by the addition of 0.36 mM H2O2. The linear decrease of absorbance at 340 nm was recorded using a UV/VIS spectrophotometer. The specic activity was expressed as units per min per mg protein. Measurement of the activities of pyruvate dehydrogenase and some of the Kreb's cycle enzymes. The pyruvate dehydrogenase (PDH) activity was measured spectrophotometrically according to the method of Chretien et al. (1995)43 with some modications by following the reduction of NAD+ to NADH at 340 nm using 50 mM phosphate buffer, pH 7.4, 0.5 mM sodium pyruvate as the substrate and 0.5 mM NAD+ in addition to the enzyme.41 The enzyme activity was expressed as units per min per mg protein. The isocitrate dehydrogenase (ICDH) activity was measured according to the method of Duncan et al. (1979)44 by measuring the reduction of NAD+ to NADH at 340 nm with the help of a UV-VIS spectrophotometer. One mL assay volume contained 50 mM phosphate buffer, pH 7.4, 0.5 mM isocitrate, 0.1 mM MnSO4, 0.1 mM NAD+ and the suitable amount of incubated mitochondria as the source of enzyme. The enzyme activity was expressed as units per min per mg protein. The alpha-ketoglutarate dehydrogenase (a-KGDH) activity was measured spectrophotometrically according to the method of Duncan et al. (1979)44 by measuring the reduction of 0.35 mM NAD+ to NADH at 340 nm using 50 mM phosphate buffer, pH 7.4 as the assay buffer, incubated mitochondria as the source of enzyme and 0.1 mM a-ketoglutarate as the substrate. The enzyme activity was expressed as units per min per mg protein. The succinate dehydrogenase (SDH) activity was measured spectrophotometrically by following the reduction of potassium ferricyanide [K3Fe (CN)6] spectrophotometrically at 420 nm according to the method of Veeger et al. (1969) with some modications.45 One mL assay mixture contained 50 mM phosphate buffer, pH 7.4, 2% (w/v) BSA, 4 mM succinate, 2.5 mM K3Fe(CN)6 and a suitable aliquot of the incubated mitochondria as the source of enzyme. The enzyme activity was expressed as units per min per mg protein. Measurement of di-tyrosine uorescence intensity. Emission spectra of di-tyrosine, a product of tyrosine oxidation, were recorded in the range of 380 to 440 nm (5 nm slit width) at an excitation wavelength of 325 nm (5 nm slit width).46 Emission spectra (from 425 to 480 nm, 5 nm slit width) of lysine conjugated with LPO products were recovered at an excitation wavelength of 365 nm (5 nm slit width). Excitation spectra (from 325 to 380 nm, 5 nm slit width) were recorded at 440 nm (5 nm slit width).47

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Measurement of mitochondrial swelling. Mitochondrial swelling was assessed by measuring the changes in absorbance of the suspension at 520 nm (D) by spectrophotometry according to Halestrap et al. (1990).48 The standard incubation medium for the swelling assay contained 250 mmol L1 sucrose, 0.3 mmol L1 CaCl2 and 10 mmol L1 tris (pH 7.4). Mitochondria (0.5 mg protein) were suspended in 3.6 mL of phosphate buffer. 1.8 mL of this suspension was added to both sample and reference cuvette and 6 mmol L1 succinate was added to the sample cuvette only, and at 520 nm wavelength, changes in absorption were recorded continuously at 25
C for 10 min. Swelling of mitochondria was evaluated according to the decrease in values of absorption at 520 nm. Determination of mitochondrial (mt) DNA damage with agarose gel electrophoresis. The incubated mitochondria with or without copper-ascorbate and copper-ascorbate plus piperine and piperine only were lysed with 3 mL of TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0) containing 0.5% SDS and 0.3 mg mL1 of proteinase K overnight at 37
C. Mitochondrial DNA was isolated using extraction with 1 M NaCl for 10 min at room temperature and puried twice with chloroform/isoamyl alcohol, 24 : 1. Then, the samples were precipitated and dissolved in TE buffer, and the DNA, thus obtained gave an average 260/280 absorbance ratio of 2–2.5. The obtained DNA samples were then mixed with 6 loading dye and resolved in 0.8% agarose gel. The gel was stained with ethidium bromide and DNA bands were detected using a Gel-Doc apparatus (Biorad, Hercules, CA). Scanning electron microscopy The mitochondrial suspension (250 ml) was centrifuged, and the supernatant was removed. The pellet was xed overnight with 2.5% glutaraldehyde. Aer washing three times with PBS, the pellet was dehydrated for 10 min at each concentration of a graded ethanol series (50, 70, 80, 90, 95 and 100%). The pellet was immersed in pure tert-butyl alcohol and was then placed into a 4
C refrigerator until the tert-butyl alcohol solidied. The frozen samples were dried by placing them into a vacuum bottle. Mitochondrial morphology was evaluated by scanning electron microscopy (SEM; Zeiss Evo 18 model EDS 8100). Estimation of protein The protein content of the isolated mitochondria was determined by the method of Lowry et al. (1951).49 Statistical evaluation Each experiment was repeated at least three times. Data are presented as mean  S.E. The D'Agostino and Pearson omnibus normality test was applied to all data sets. The signicance of mean values of different parameters between the treatment groups was analyzed using one way post hoc tests (Tukey's HSD test) of analysis of variances (ANOVA) aer ascertaining the homogeneity of variances between the treatments. Pairwise comparisons were done by calculating the least signicance. Statistical tests were performed using Microcal Origin version 7.0 for Windows.

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Results ROS scavenging activity of piperine in vitro The hydroxyl radical (cOH) scavenging ability of piperine was studied in an in vitro standard model system using Cu2+ and ascorbic acid where cOH was generated. Fig. 2A indicates that piperine directly scavenged cOH in a concentration dependent manner exhibiting about 84.38% (P # 0.001) scavenging activity at a concentration of 2 mg mL1. The superoxide anion (O2c) free radical scavenging ability of piperine was studied by following the rate of superoxide mediated epinephrine oxidation (Fig. 2B). Increasing concentrations of piperine altered the rate of superoxide mediated epinephrine oxidation indicating the O2c scavenging ability of this molecule. About 57.35% (P # 0.001) scavenging activity was observed at a concentration of 2 mg mL1 of piperine which is statistically highly signicant. The H2O2 scavenging activity of the piperine, if any, was also tested in vitro by studying the breakdown of H2O2 at 240 nm. Fig. 2C clearly indicates that piperine possesses the ability to scavenge H2O2, in vitro in a dose-dependent manner. The DPPH free radical has been widely accepted as a model compound to evaluate the antioxidant abilities of various samples. This assay indicated that piperine has dose-dependent DPPH radical scavenging activity. Fig. 2D shows that piperine exhibited 0.7835  0.002% (P # 0.001) inhibition of the DPPH activity at the dose of 2 mg mL1, which is highly signicant.

Effect of piperine on the intactness of mitochondria Fig. 3(A)–(D) depict a signicant decrease in the mitochondrial intactness following the incubation of mitochondria with copper-ascorbate (64.29%, P # 0.001 vs. control). This decreased level of mitochondrial intactness was found to be signicantly protected from being altered (1.94 fold compared to the copper-ascorbate-incubated group, P # 0.001) when the mitochondria were co-incubated with copper-ascorbate and piperine (2 mg mL1), indicating the ability of piperine to protect the mitochondria against copper-ascorbate induced changes in mitochondrial swelling which may be due to oxidative stress.

Status of reactive nitrogen species (RNS) Nitric oxide (NO), one of the reactive nitrogen species (RNS), is a molecular mediator of many physiological processes including vasodilatation, inammation, thrombosis, immunity and neurotransmission. The level of NO in mitochondria in the copper-ascorbate incubated group was found to be increased signicantly (Fig. 4) when compared to the control group by 66.47% (P # 0.001). However, a dose-dependent protection of the level of NO was observed when the cardiac mitochondria were co-incubated with copper-ascorbate and increasing concentrations of piperine. At 2 mg mL1, piperine was found to maximally protect the level of mitochondrial NO from being altered (48.70% protection, P # 0.001).

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Fig. 2 (A) Hydroxyl radical, (B) superoxide anion, (C) hydrogen peroxide and (D) DPPH radical scavenging activity of piperine; CuAs00 : copperascorbate-incubated mitochondria for ‘0’ minutes, i.e. control; CuAs600 : copper-ascorbate-incubated mitochondria for ‘60’ minutes; P0.25–2 ¼ group incubated with piperine at the dose of 0.25–2mg mL1 respectively; the values are expressed as mean  S.E.; *P # 0.001 compared to control and #P # 0.001 compared to CuAs00 .

Assessment of the mitochondrial status by determining activities of respiratory complex enzymes under coupling and uncoupling conditions Fig. 5A shows a signicant decrease in cardiac mitochondrial ATP synthase activity following the incubation of mitochondria with CCCP (39.97%, P # 0.001 vs. control without CCCP). But there were no signicant changes in cardiac mitochondrial NADH cytochrome C oxidoreductase and NADH cytochrome C oxidase activities following the incubation of mitochondria with CCCP. Fig. 5B reveals highly signicant decreases in the activities of NADH cytochrome C oxidoreductase, NADH cytochrome C oxidase and ATP synthase (52.87%, 54.56% and 32.03%, respectively, P # 0.001 vs. control group) following incubation of mitochondria with copper-ascorbate. The activities of these enzymes were found to be signicantly protected from being decreased when the mitochondria were co-incubated with copper-ascorbate and piperine (1.81 fold, 1.95 fold and 75.21% protection, respectively; P # 0.001 compared to the copperascorbate-incubated group, at the dose of 2 mg mL1). Fig. 5C reveals that in the case of CCCP pre-incubation of mitochondria caused signicant decreases in the activities of NADH cytochrome C oxidoreductase, NADH cytochrome C oxidase and ATP synthase (59.17%, 60.67% and 49.25%, respectively, P # 0.001 vs. control group) following incubation of mitochondria with copper-ascorbate. The activities of NADH

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cytochrome C oxidoreductase and NADH cytochrome C oxidase were found to be signicantly protected from being decreased when the mitochondria were co-incubated with copper-ascorbate and piperine (2.04 fold and 3.04 fold protection, respectively; P # 0.001 compared to the copper-ascorbate-incubated group, at the dose of 2 mg mL1) but the activity of ATP synthase was not found to be signicantly protected when the mitochondria were co-incubated with copper-ascorbate and piperine at the dose of 2 mg mL1 (27.75% protection; P # 0.001 compared to the copper-ascorbate-incubated group). Effect of piperine on the oxidative stress biomarkers Fig. 6A shows a signicant increase in cardiac mitochondrial LPO level following the incubation of mitochondria with copper-ascorbate (3 fold, P # 0.001 vs. control). This elevated level of lipid peroxidation products was found to be signicantly protected from being increased (73.53% compared to the copper-ascorbate-incubated group, P # 0.001) when the mitochondria were co-incubated with copper-ascorbate and piperine (2 mg mL1), indicating the ability of piperine to protect the mitochondria against oxidative stress induced due to copperascorbate. On the other hand, a signicant decrease was observed in cardiac mitochondrial GSH content following the incubation of mitochondria with copper-ascorbate (45.77%, P # 0.001 vs. control). The GSH content was found to be signicantly

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Fig. 3 Changes in intactness of mitochondria. (A–C) Janus green B stained (40 magnification) and (D) graphical representation of changes of mitochondrial intactness; CuAs ¼ copper-ascorbate incubated group; P0.25–2 ¼ group incubated with piperine at the dose of 0.25–2 mg mL1 respectively; CuAs-P0.25–2 ¼ group co-incubated with copper-ascorbate and piperine at the dose of 0.25–2 mg mL1 respectively; the values are expressed as mean  S.E.; #P # 0.001 compared to control values and *P # 0.001 compared to copper-ascorbate incubated values.

Fig. 4 Protective effect of piperine against copper-ascorbate-induced increase in nitric oxide concentration in goat cardiac mitochondria. CON

¼ control group; CuAs ¼ copper-ascorbate incubated group; P0.25–2 ¼ group incubated with piperine at the dose of 0.25–2 mg mL1 respectively; CuAs-P0.25–2 ¼ group co-incubated with copper-ascorbate and piperine at the dose of 0.25–2 mg mL1 respectively; The values are expressed as mean  S.E.; #P # 0.001 compared to control values and *P # 0.001 compared to copper-ascorbate incubated values.

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Fig. 5 (A) Effect of CCCP on the activities of NADH cytochrome C oxidoreductase, NADH cytochrome C oxidase, ATP synthase; #P # 0.001 compared to control values without CCCP. Effect of piperine against copper-ascorbate-induced decrease in activities of NADH cytochrome C oxidoreductase, NADH cytochrome C oxidase, ATP synthase in goat cardiac mitochondria (B) in the absence of CCCP and (C) in the presence of CCCP; CuAs ¼ copper-ascorbate treated group; P0.25–2 ¼ group incubated with piperine at the dose of 0.25–2 mg mL1 respectively; CuAsP0.25–2 ¼ group co-incubated with copper-ascorbate and piperine at the dose of 0.25–2 mg mL1 respectively; The values are expressed as mean  S.E.; #P # 0.001 compared to control values and *P # 0.001 compared to copper-ascorbate incubated values.

protected from being decreased (1 fold from the copper-ascorbate-treated group, P # 0.001) when the mitochondria were coincubated with copper-ascorbate and piperine (2 mg mL1) (Fig. 6B). The protein carbonyl assay showed a signicant increase in cardiac mitochondrial protein carbonyl content following the incubation of mitochondria with copper-ascorbate (1 fold, P # 0.001 vs. control). This elevated level of protein carbonyl content was found to be signicantly protected from being increased (79.24% compared to the copper-ascorbate-incubated group, P # 0.001) when the mitochondria were co-incubated with copper-ascorbate and piperine (2 mg mL1) (Fig. 6C).

when the mitochondria were co-incubated with copper-ascorbate and piperine (P # 0.001 compared to the copper-ascorbateincubated group, at the dose of 2 mg mL1). Fig. 7C further depicts a highly signicant decrease (50.15%, P # 0.001 vs. control group) in the activity of GR following incubation of mitochondria with copper-ascorbate. The GR activity was found to be signicantly protected from being decreased when the mitochondria were co-incubated with copper-ascorbate and piperine (P # 0.001 compared to the copper-ascorbate-incubated group, at the dose of 2 mg mL1). The results indicate that piperine may have an inuence on the GSH biosynthesis.

Effect of piperine on the activities of antioxidant enzymes

Effect of piperine on the activities of pyruvate dehydrogenase and some of the Kreb's cycle enzymes

Fig. 7A reveals a highly signicant increase (6 fold, P # 0.001 vs. control group) in the activity of Mn-SOD following incubation of mitochondria with copper-ascorbate. The activity of this enzyme was found to be signicantly protected from being increased when the mitochondria were co-incubated with copper-ascorbate and piperine (92.27% protection; P # 0.001 compared to the copper-ascorbate-incubated group, at the dose of 2 mg mL1). Fig. 7B also reveals a highly signicant decrease (31.67%, P # 0.001 vs. control group) in the activity of GPx following incubation of mitochondria with copper-ascorbate. The GPx activity was found to be signicantly protected from being decreased

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The succinate dehydrogenase (SDH) activity was found to be signicantly decreased when the goat heart mitochondria were incubated with copper-ascorbate (52.31%, P # 0.001 vs. control). The enzyme activity was found to be signicantly protected from being decreased when the mitochondria were co-incubated with copper-ascorbate and piperine (2 fold protection, P # 0.001 compared to the copper-ascorbate-incubated group, at 2 mg mL1 dose) (Fig. 8A). Piperine alone had no effect on the SDH activity. Incubation of the cardiac mitochondria with copper-ascorbate decreased the isocitrate dehydrogenase activity (40.09%,

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Fig. 6 Protective effect of piperine against copper-ascorbate-induced increase in (A) lipid peroxidation level, (C) protein carbonylation level and decrease in (B) reduced glutathione level in goat cardiac mitochondria; CON ¼ control group; CuAs ¼ copper-ascorbate treated group; P0.25–2 ¼ group incubated with piperine at the dose of 0.25–2 mg mL1 respectively; CuAs-P0.25–2 ¼ group co-incubated with copper-ascorbate and piperine at the dose of 0.25–2 mg mL1 respectively; The values are expressed as mean  S.E.; #P # 0.001 compared to control values and *P # 0.001 compared to copper-ascorbate incubated values.

Fig. 7 Protective effect of piperine against copper-ascorbate-induced increase in (A) Mn-SOD activity and decrease in (B) glutathione peroxidase and (C) glutathione reductase activities of goat cardiac mitochondria; CON ¼ control group; CuAs ¼ copper-ascorbate incubated group; P0.25–2 ¼ group incubated with piperine at the dose of 0.25–2 mg mL1 respectively; CuAs-P0.25–2 ¼ group co-incubated with copper-ascorbate and piperine at the dose of 0.25–2 mg mL1 respectively; The values are expressed as mean  S.E.; #P # 0.001 compared to control values and *P # 0.001 compared to copper-ascorbate incubated values.

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Fig. 8 Protective effect of piperine against copper-ascorbate-induced decrease in (A) succinate dehydrogenase, (B) isocitrate dehydrogenase,

(C) a-ketoglutarate dehydrogenase and (D) pyruvate activities of goat cardiac mitochondria; CON ¼ control group; CuAs ¼ copper-ascorbate incubated group; P0.25-2 ¼ group incubated with piperine at the dose of 0.25–2 mg mL1 respectively; CuAs-P0.25–2 ¼ group co-incubated with copper-ascorbate and piperine at the dose of 0.25–2 mg mL1 respectively; The values are expressed as mean  S.E.; #P # 0.001 compared to control values and *P # 0.001 compared to copper-ascorbate incubated values.

P # 0.001 vs. control). The activity of the enzyme was found to be completely protected from being decreased when mitochondria were co-incubated with copper-ascorbate and piperine at the dose of 2 mg mL1 (1 fold protection, P # 0.001 compared to the copper-ascorbate-incubated group) (Fig. 8B). However, piperine alone had no effect on the isocitrate dehydrogenase activity. Fig. 8C also shows that incubation of cardiac mitochondria with copper-ascorbate signicantly decreased the alpha keto glutarate dehydrogenase activity (60.07%, P # 0.001 vs. control). This enzyme was found to be able to generate ROS during its catalytic function, which is regulated by the NADH/NAD+ ratio.50 The activity of a-KGDH was found to be signicantly protected from being decreased when the mitochondria were co-incubated with 2 mg mL1 dose of the piperine (2 fold protection, P # 0.001 compared to the copper-ascorbate-incubated group). However, piperine alone had no effect on the a-KGDH activity. Fig. 8D reveals that the incubation of the mitochondria with copper-ascorbate decreased the cardiac pyruvate dehydrogenase (PDH) activity (45.48%, P # 0.001 vs. control). When the mitochondria were co-incubated with copper-ascorbate and piperine, the activity of this enzyme, however, was found to be signicantly protected compared to the activity observed in the copper-ascorbate-incubated group (1 fold, P # 0.001 compared to the copper-ascorbate-incubated group, at the dose of 2 mg mL1). Piperine alone had no effect on the PDH activity.

associated with declining mitochondrial function, and with numerous diseases and aging.51,52 Accumulation of mtDNA damage likely leads to organellar dysfunction, and therefore is biologically relevant.53 Incubation of cardiac mitochondria caused damage to mtDNA. However, when the mitochondria were co-incubated with copper-ascorbate and piperine (at 2 mg mL1 dose), the mtDNA damage was found to be almost completely protected (Fig. 9A). Mitochondrial swelling Aer the mitochondrial sample was added to the reaction buffer (at pH 7.2) or 0.3 mmol L1 of CaC12, the mitochondrial absorbance at 520 nm declined, indicating mitochondrial swelling due to alteration in osmotic pressure. The extent of decrease in absorbance in mitochondria incubated with copper-ascorbate was found to be lower compared to the control group (Fig. 9B), demonstrating that incubation with copper-ascorbate caused mitochondrial dysfunction. The absorbance was found to be signicantly increased when the goat heart mitochondria were co-incubated with copperascorbate and piperine (at a dose of 2 mg mL1) compared to mitochondria incubated with copper-ascorbate only. This indicates that piperine has the potential to improve the impaired mitochondrial function. Status of di-tyrosine uorescence intensity

Mitochondrial (mt) DNA damage Detection of mtDNA damage is an important indicator of mitochondrial stress. Increased mtDNA damage has been

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The effect of the free radical-generating system (in this case the copper-ascorbate system) on protein structure was examined by measuring di-tyrosine uorescence. That Food Funct., 2014, 5, 2252–2267 | 2261

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Protective effect of piperine against copper-ascorbate-induced (A) mitochondrial DNA damage, (B) decrease in mitochondrial swelling and (C) increase in di-tyrosine level in goat cardiac mitochondria. CON ¼ control group; CuAs ¼ copper-ascorbate incubated group; P2 ¼ group incubated with piperine at the dose of 2 mg mL1; CuAs-P2 ¼ group co-incubated with copper-ascorbate and piperine at the dose of 2 mg mL1 The values are expressed as mean  S.E.; #P # 0.001 compared to control values and *P # 0.001 compared to copper-ascorbate incubated values. Fig. 9

copper-ascorbate induced oxidative stress has a direct effect on the oxidation level of amino acid is evident from increased di-tyrosine formation (76.64% increase, P # 0.001 compared to the control group) (Fig. 9C) as observed using uorimetric analysis of this amino acids' basal auto-uorescence. Coincubation of cardiac mitochondria with copper-ascorbate and piperine (at the dose of 2 mg mL1) was found to protect these molecules from losing their original conguration as indicated by the recovered auto-uorescence level for dityrosine formation (29.43% protection, P # 0.001 compared to the CuAs-incubated group). Piperine, by itself, has no effect on the di-tyrosine uorescence of cardiac mitochondria.

Scanning electron microscopy Fig. 10 shows the changes brought about in cardiac mitochondrial surface following incubation with copper-ascorbate and studied through scanning electron microscopy. The gure shows a perforated surface with convoluted membranes. Moreover, the mitochondria were found to be markedly contracted, with large membrane blebs covering its surface. However, when the cardiac mitochondria were co-incubated with copper-ascorbate and piperine (at 2 mg mL1), the changes on the mitochondrial surface were found to be signicantly protected from being taken place.

Scanning electron micrograph (6000) of the mitochondrial surface. Arrow heads indicate the perforated surface of mitochondria. CuAs ¼ copper-ascorbate incubated group; P2 ¼ group incubated with piperine at the dose of 2 mg mL1; CuAs-P2 ¼ group co-incubated with copper-ascorbate and piperine at the dose of 2 mg mL1. Fig. 10

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Discussion Piperine, an amide alkaloid obtained from the dried mature fruits of Piper nigrum, is the rst and until now the most potent bioenhancer discovered.54 The bioenhancing activity of piperine and the concept of bioactivity enhancement using piperine both were discovered and scientically validated in 1979 in India.54 It was studied successfully to reduce the dose of the drug and cost of the treatment.55 Collaborative studies conducted by Cadila Labs Ltd. at RRL, Jammu, led to successful launching of the well known anti-TB drug Rifampin (200 mg) along with bioenhancer piperine (10 mg) under the trade name ‘Risorine’, in 2009. In the above case, Rifampin's conventional dose of 450 mg has been reduced to 200 mg with the same bioavailability.56 Piperine brings about its bioenhancing activity by inhibition of drug efflux pump (peptidoglycan LD-carboxypeptidase Pgp2) and by inhibiting enzymes such as CYP1A1, CYP1B1, CYP1B2, CYP1E1 and CYP3A4. All the drugs metabolized by these enzymes are inuenced by bio-enhancer piperine.57 All categories of drugs like cardiovascular, respiratory, CNS, GIT, anticancer, immunomodulatory drugs, antibiotics, several other classes of drugs and nutraceuticals are greatly inuenced by piperine. It is interesting to note that piperine brings about its bioenhancing effect at a dose of 10 mg in all formulations irrespective of the dose of combination drug. Reactive oxygen species (ROS) are generated due to redox imbalance in tissues,58 which is responsible for tissue damage by modication of lipids, proteins and nucleic acids. In our in vitro experimental system, copper-ascorbate was used as an inducer of oxidative stress in the mitochondria isolated from goat cardiac tissue. Cu2+ in the presence of ascorbate at low concentrations (0.2–2.0 mM) generates cOH. Ascorbate by virtue of its metal reducing capacity generates cOH together with H2O2 in the presence of Cu2+.59,60 Ascorbate + Cu2+ + O2 + 2H+ / Dehydroascorbate + Cu+ + H2O2 Cu+ + H2O2 / Cu2+ + OHc + OH

The hydroxyl radical induces mitochondrial damage by impairment of mitochondria functionalities which sensitizes cells to oxidative challenges. In the past few years, there has been growing interest in the involvement of reactive oxygen species (ROS) in several pathological situations.36,61,62 ROS produced in vivo as well as in vitro include superoxide radical (O2c), hydrogen peroxide (H2O2) and hydroxyl radicals (cOH).36,61 H2O2 and O2c can interact in the presence of certain transition metal ions (Cu2+ and/or Fe3+) to yield a highly-reactive oxidizing species, the hydroxyl radical (cOH).62 The role of antioxidants in the maintenance of health and chemoprevention of disorders and diseases has received great attention.63 As a result of the participation of oxidative processes in the onset and development of degenerative diseases, much attention has been paid to the antioxidant properties of foods rich in polyphenols.64 Literature reviews

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conrm that different dietary compounds from plant foods affect mitochondria functionalities in different in vitro and in vivo models.65 cOH is an extremely reactive oxygen based free radical in biological systems and has been implicated as highly damaging species in free radical pathology.66 The superoxide anion (O2c) free radical is known to be very harmful to cellular components as a precursor of the more reactive oxygen species, contributing to the tissue damage and various diseases. The DPPH test showed the ability of the test compound to act as a free radical scavenger. The DPPH assay method is based on the ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH), a stable free radical, to decolorize in the presence of antioxidants.67 DPPH, a protonated radical, has characteristic absorbance maxima at 517 nm, which decreases with the scavenging of the proton radical. This property has been widely used to evaluate the free radical scavenging activity of natural antioxidants.68 The stable free radical DPPH has been widely used to test the free radicalscavenging ability of various dietary antioxidants.69 Piperine shows hydroxyl radical, superoxide anion free radical, H2O2, and DPPH radical scavenging activity. This indicates that piperine is capable of scavenging ROS in vitro. Piperine has been demonstrated previously, in in vitro experiments, to protect against oxidative damage by inhibiting or quenching free radicals and reactive oxygen species and inhibiting lipid peroxidation.9 Piperine was also found to act as a hydroxyl radical scavenger at low concentrations, but at higher concentrations, it activated the Fenton reaction resulting in increased generation of hydroxyl radicals. It acts as a powerful superoxide anion free radical scavenger with an IC50 of 1.82 mM. A 52% inhibition of lipid peroxidation was observed at a dose of 1.4 mM with an IC50 of 1.23 mM.9 Our study revealed that in the case of copper-ascorbate-incubated mitochondria, in vitro, piperine acts as a hydroxyl radical scavenger and superoxide anion free radical scavenger with IC50 values of 1.19 mg mL1 and 1.74 mg mL1 respectively. Our results are close to what has been observed by previous researchers in other models of oxidative stress. The results further conrm the antioxidant activity of piperine studied previously.9,14,15,70 So, piperine can act as a good protective regimen against mitochondrial toxic injuries causing various diseases. We have investigated the possibility of using a color reaction as a measure of viable mitochondria. Ideally, a colorimetric assay for cells or mitochondria should utilize a colorless substrate that is modied to a colored product by any living cells or organelles but not by dead cells or dead mitochondria. The tetrazolium ring is cleaved in active mitochondria and so the reaction occurs only in living mitochondria.71 Cooperstin and Lazarow (1953)72 have shown that when a super vitally stained cell is treated with 0.001 (M) KCN, the JG-B is regenerated, and when the cyanide is removed JG-B is reactive. The cytochrome oxidase system, an enzyme within mitochondria, prevents the reduction of JG-B. This enzyme system is oxygen dependent and cyanide sensitive.73 The study of mitochondrial respiratory parameters provides an important tool to understand mitochondrial physiology and the potential role of mitochondrial pathologies in cellular damage. Mitochondrial uncoupling can arise from oxidative

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damage of the mitochondrial membrane as induced by increased ROS concentrations.74 Return of protons to the mitochondrial matrix either through ATP synthase (coupled respiration) or proton leak mechanisms (uncoupled respiration) can decrease the proton motive force (PMF) and thereby acutely decrease ROS emission. CCCP is a chemical uncoupler which can have effects only on the activity of ATP synthase. Our study provided the evidence that when mitochondria are incubated with CCCP it can only decrease the activity of ATP synthase but not the activities of NADH cytochrome C oxidoreductase and NADH cytochrome C oxidase. This indicates that mitochondria remained viable under both coupling and uncoupling conditions. Heavy metals are also known to affect respiratory chain complexes and there is absolute substrate specicity.75 The impairment of electron transfer through NADH: ubiquinone oxidoreductase (complex I) and ubiquinol: cytochrome C oxidoreductase (complex III) may induce superoxide formation. Mitochondrial production of ROS is thought to play an adverse role in many pathologic disorders. When the mitochondria were co-incubated with copper-ascorbate and different doses of piperine then it was observed that piperine can protect the activities of NADH cytochrome C oxidoreductase and NADH cytochrome C oxidase from being decreased due to copper-ascorbate but the activity of ATP synthase was not protected in the presence of CCCP. So, from our study it is established that copper-ascorbate is an inhibitor but not the uncoupler and piperine can protect the mitochondria from the inhibitory effect of copper-ascorbate but not the uncoupling effect of CCCP. Lipid peroxidation products are not produced on purpose and inhibition of lipid peroxidation by antioxidants should be benecial for maintenance of health and reducing disease risk.76 In addition, a relationship between copper metabolism and the intracellular availability of glutathione has been dened.77 Moreover, copper-ascorbate may induce oxidative stress by enhancing tissue LPO and by altering the antioxidant system in the organs. The copper-ascorbate induced mitochondrial damage is due to generation of oxidative stress as is evident from elevated levels of LPO and protein carbonyl content and a decreased tissue level of GSH. Piperine was found to be effective in decreasing the lipid peroxidation level of the cardiac mitochondria. Oxidation of proteins can generate stable as well as reactive products that can generate additional radicals on reaction with transition metal ions. Most oxidized proteins are functionally inactive and are rapidly removed; some gradually accumulate and contribute to damage.78 Copper along with higher concentration of ascorbic acid induces severe cardiac toxicity. The mechanism underlying this cardiac toxicity in mitochondria is accelerated ROS production mediated by copper-ascorbate, thereby ensuing oxidative stress.79 The stress condition is all the more aggravated when the GSH pool starts getting depleted. Copper-ascorbate is also found to have the potential to modulate antioxidant and redox enzymes (like SOD, GPx and GR). In addition, it enhanced GSH depletion along with NADPH oxidation, compromised the GPx and GR activities, dismantled redox homeostasis, compromised antioxidant defense and enhanced oxidative stress.80 This

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enhanced ROS/RNS along with compromised antioxidants proved deleterious for cellular macro-molecules like proteins, lipids and DNA causing signicant damage to them. In our studies, we found that there has been considerable decrease in activities of pyruvate dehydrogenase and the Kreb's cycle enzymes like isocitrate dehydrogenase, alpha-keto glutarate dehydrogenase and succinate dehydrogenase following incubation of cardiac mitochondria, in vitro with copperascorbate. The activities of all these enzymes were protected from being altered when cardiac mitochondria were co-incubated with piperine. These enzymes are sensitive to reactive oxygen species (ROS) and the decrease in the activities of these enzymes could be critical in the metabolic deciency induced by oxidative stress.81,82 It has been demonstrated that the principal site of ROS production by cadmium (Cd2+) seems to reside in complex III.83 The impairment of electron transfer through complex I and complex III may induce superoxide anion free radical formation.84 The electron transfer chain of mitochondria is also a well-documented source of H2O2. Copper-ascorbate induces an imbalance in the mitochondrial steady state that allows the induction and effects of oxidative stress.29 In our present study, incubation of cardiac mitochondria with copperascorbate decreased the activities of some of the Kreb's cycle enzymes including succinate dehydrogenase which is associated with structural alteration in cardiac mitochondria29 as is evident from our studies by scanning electron microscopy. The activities of these enzymes were found to be protected when the cardiac mitochondria were co-incubated with copper-ascorbate and piperine. This strongly indicates that piperine is able to protect mitochondria from copper-ascorbate induced injury by itself being a quencher of reactive oxygen species. Mitochondrial respiration and its regulation by nitric oxide (NO) are important in the heart for several reasons. NO interacts with the mitochondrial respiratory chain by different mechanism(s).85 It has been shown that pH changes and high calcium contents can cause mitochondrial swelling, which is detected by a reduction in mitochondrial absorbance at certain wavelengths (520 nm). It is well established that energized mitochondria supplemented with high Ca2+ concentration swell in the presence of inorganic phosphate. Compared with the copper-ascorbate incubated group, the absorbance of co-incubated mitochondria with copper-ascorbate and piperine (2 mg mL1) was signicantly increased, indicating that piperine improved the impaired cardiac mitochondrial functions. This effect may be due to blockage of calcium entrance which appears to be due to inhibition of the calcium channels. The high intra-mitochondrial concentration of calcium prevents the electron transport of the respiratory chain and then oxidative phosphorylation, or activates the principal enzymes responsible for ROS generation.86 It seems appropriate that piperine enters into a competition with calcium in preventing its binding at the level of the transition pore and by consequential inhibition of swelling and apoptosis which was also established by our morphological study of mitochondria by scanning electron microscopy. It has been demonstrated previously that H2O2 exposure induces differential cell toxicity and oxidative DNA damage in stressed mitochondria through different mechanisms.87

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A cascade of cellular O2c production appears to be a critical determinant of selective toxicity in aggressive cancer cells,88 which is mediated by the activation of elevated NAD(P)H oxidases89 and by crosstalk with impaired mitochondrial respiration in cancer cells.90 Conversely, the resistance to H2O2induced cytotoxicity in normal cells may be attributed to a low cellular level of NAD(P)H oxidases and the normal mitochondrial function. On the other hand, a signicant level of oxidative DNA damage is induced by exogenous H2O2 in both cancer and normal cells, but is independent of cellular O2 steady-state levels. O2c accumulation may be exacerbated by a difference in the rate of O2c accumulation and conversion to H2O2 and HOc in stressed mitochondria.91 Alternatively, O2c may be metabolized promptly with other reactive species such as nitric oxide (NO). NO is shown to interact with O2c to generate peroxynitrite anions (ONOO) and nitrogen oxides,91 which could attenuate the formation of the highly reactive cHO and oxidative DNA damage in mitochondria.

Conclusions It can be concluded that piperine obtained from dried ripe fruits of black pepper; Piper nigrum, protects goat cardiac mitochondria against copper-ascorbate induced oxidative stress mediated injury via antioxidant mechanism(s). Piperine may nd its extensive use against cardio-toxic situation at a specic pharmacological dose. It may as well nd its place in alternative medicine or integrative medicinal interventions also. Until now, there has been no report on the side effects of piperine indicating that this molecule may be considered a safe antioxidant which may be used singly or in combination in situations involving oxidative stress.

Acknowledgements MD is supported under Women Scientists Scheme-A (WOS-A), Department of Science and Technology, Govt. of India. AKG is an extended SRF under DST PURSE Program of DST, Govt. of India at University of Calcutta. TD is supported by the funds from CRNN, University of Calcutta. Dr DB is supported from the funds available to him from CRNN, University of Calcutta. Dr VR is supported by the funds from All India Council of Technical Education (AICTE), New Delhi. This work is also partially supported by UGC Major Research Project Grant to Dr DB [F. no. 37-396/2009 (SR)]. Dr DB also gratefully acknowledges the award of a Major Research Project under CPEPA Scheme of UGC, Govt. of India, at University of Calcutta.

References 1 A. M. Majumdar, J. N. Dhuley, V. K. Deshmukh, P. H. Raman and S. R. Naik, Jpn. J. Med. Sci. Biol., 1990, 43, 95–100. 2 M. Anis and M. Iqbal, Fitoterapia, 1986, 57, 52–54. 3 D. P. Bezerraa, C. Pessoaa, M. O. Moraesa, E. R. Silveirab, M. A. S. Limab, F. J. M. Elmirob and L. V. Costa-Lotufoa, Z. Naturforsch., 2005, 60, 539–543.

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4 V. Badmaev, M. Majeed and E. P. Norkus, Nutr. Res., 1999, 19, 381–388. 5 M. Majeed, V. Badmaev and L. Prakash, Bioperine: Nature’s Own Thermonutrient and Natural Bioavailability Enhancer, Nutriscience Publishers, Inc., Piscataway, NJ, USA, 1998. 6 J. E. Jensen, L. Gajdzik, I. Habrel, O. Scheiner and J. Graf, J. Nutr., 1998, 128, 577–581. 7 Y. F. Bai and H. Xu, Acta Pharmacol. Sin., 2000, 21, 357–359. 8 P. Shears, Trop. Doct., 2000, 30, 114–118. 9 R. Mittal and R. L. Gupta, Expert Rev. Clin. Pharmacol., 2000, 22, 271–274. 10 C. K. Atal, A. K. Dubey and J. Singh, J. Exp. Ther., 1985, 232, 258–262. 11 M. Masakazu, N. Isamu and O. Nobuo, ACS Symposium Series 387, ed. J. T. Arnason, B. J. R. Philogene and P. Morand, American Chemical Society, New York, 1989, pp. 183–187. 12 H. J. Dorman and S. G. Deans, J. Appl. Microbiol., 2000, 88, 308–316. 13 R. El Hamss, M. Idaomar, A. Alonso-Moraga and S. A. Mu˜ noz, Food Chem. Toxicol., 2003, 41, 41–47. 14 I. Gulcin, Int. J. Food Sci. Nutr., 2005, 56, 491–499. 15 R. S. Vijayakumar, D. Surya and N. Nalini, Redox. Rep., 2004, 9, 105–110. 16 C. R. Pradeep and G. Kuttan, Int. Immunopharmacol., 2004, 4, 1795–1803. 17 C. D. Doucette, A. L. Hilchie, R. Liwski and D. W. Hoskin, J. Nutr. Biochem., 2013, 24, 231–239. 18 Y. P. Hwang, H. J. Yun, H. G. Kim, E. H. Han, J. H. Choi, Y. C. Chung and H. G. Jeong, Toxicol. Lett., 2011, 203, 9–19. 19 R. K. Bhardwaj, H. Glaeser, L. Becquemont, U. Klotz, S. K. Gupta and M. F. Fromm, J. Pharmacol. Exp. Ther., 2002, 302, 645–650. 20 D. Ouyang, L. Zeng, H. Pan, L. Xu, Y. Wang, K. Liu and K. He, Food Chem. Toxicol., 2013, 60, 424–430. 21 H. G. Kim, E. H. Han, W. S. Jang, J. H. Choi, T. Khanal, B. H. Park, T. P. Tran, Y. C. Chung and J. H. Gwang, Food Chem. Toxicol., 2012, 50, 2342–2348. 22 A. Sehgal, M. Kumar, M. Jain and D. K. Dhawan, Food Chem. Toxicol., 2011, 49, 3002–3006. 23 P. Chonpathompikunlert, J. Wattanathorn and S. Muchimapura, Food Chem. Toxicol., 2010, 48, 798–802. 24 N. Krishnakumar, S. Manoharan, R. M. Palaniappan, P. Venkatachalam and M. G. Arun Manohar, Food Chem. Toxicol., 2009, 47, 2813–2820. 25 C. F. Babbs and M. G. Steiner, Meth. Enzymol., 1990, 186, 137–147. 26 D. Bandyopadhyay, A. Chattopadhyay, G. Ghosh and A. G. Datta, Curr. Med. Chem., 2004, 11, 369–387. 27 H. P. Misra and I. Fridovich, J. Biol. Chem., 1972, 247, 3170– 3175. 28 A. K. Ghosh, E. Mitra, M. Dutta, D. Mukherjee, A. Basu, S. B. Firdaus, D. Bandyopadhyay and A. Chattopadhyay, Asian J. Pharm. Clin. Res., 2013, 6, 196–200. 29 M. Dutta, A. K. Ghosh, A. Basu, D. Bandyopadhyay and A. Chattopadhyay, Int. J. Pharm. Pharmaceut. Sci., 2013, 5, 439–447.

Food Funct., 2014, 5, 2252–2267 | 2265

View Article Online

Published on 30 June 2014. Downloaded by North Dakota State University on 22/10/2014 02:16:08.

Food & Function

30 M. Dutta, A. K. Ghosh, S. Rudra, D. Bandyopadhyay, B. Guha, S. Dutta and A. Chattopadhyay, J. Cell Tissue Res., 2014, 14, 3997–4007. 31 R. M. Fiddler, J. Assoc. Offic. Anal. Chem., 1977, 60, 594–599. 32 P. Griess, Eur. J. Inorg. Chem., 1879, 12, 426–428. 33 N. Goyal and V. M. Srivastava, J. Helminthol., 1995, 69, 13–17. 34 J. Rosing, D. A. Harris, A. Kemp and E. C. Slater, Biochim. Biophys. Acta, 1975, 376, 13–26. 35 J. A. Buege and S. G. Aust, Methods Enzymol., 1978, 52, 302– 310. 36 D. Bandyopadhyay, G. Ghosh, A. Bandyopadhyay and R. J. Reiter, J. Pineal Res., 2004, 36, 195–203. 37 J. Sedlak and R. H. Lindsay, Anal. Biochem., 1968, 25, 192– 205. 38 R. L. Levine, J. A. Williams, E. R. Stadtman and E. Shacter, Methods Enzymol., 1994, 233, 346–357. 39 S. Marklund and G. Marklund, Eur. J. Biochem., 1974, 47, 469–474. 40 G. Krohne-Ehrich, R. H. Schirmer and R. Untucht-Grau, Eur. J. Biochem., 1977, 80, 65–71. 41 D. E. Paglia and W. N. Valentine, J. Lab. Clin. Med., 1967, 70, 158–169. 42 M. Dutta, D. Ghosh, A. K. Ghosh, G. Bose, A. Chattopadhyay, S. Rudra, M. Dey, A. Bandyopadhyay, S. K. Pattari, S. Mallick and D. Bandyopadhyay, Food Chem. Toxicol., 2014, 66, 262– 277. 43 D. Chretien, M. Pourrier, T. Bourgeron, M. S´ en´ e, A. R¨ otig, A. Munnich and P. Rustin, Clin. Chim. Acta, 1995, 240, 129–136. 44 M. J. Duncan and D. G. Fraenkel, J. Bacteriol., 1979, 137, 415– 419. 45 C. Veeger, D. V. DerVartanian and W. P. Zeylemaker, Methods Enzymol., 1969, 13, 81–90. 46 C. Giulivi and K. J. A. Davies, Methods Enzymol., 1994, 233, 363–371. 47 N. Dousset, G. Ferretti, M. Taus, P. Valdiguie and G. Curatola, Methods Enzymol., 1994, 233, 459–469. 48 A. P. Halestrap and A. M. Davidson, Biochem. J., 1990, 268, 153–160. 49 O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem., 1951, 193, 265–275. 50 L. Tretter and V. Adam-Vizi, J. Neurosci., 2004, 24, 7771–7778. 51 A. Y. Seo, T. Hofer, B. Sung, S. Judge, H. Y. Chung and C. Leeuwenburgh, Antioxid. Redox Signaling, 2006, 8, 529– 538. 52 A. Y. Seo, J. Xu, S. Servais, T. Hofer, E. Marzetti, S. E. Wohlgemuth, M. D. Knutson, H. Y. Chung and C. Leeuwenburgh, Aging Cell, 2008, 7, 706–716. 53 J. Lu, K. Wang, M. Rodova, R. Esteves, D. E. L. Berry, A. Craer, M. Barrett, S. M. Cardoso, I. Onyango, W. D. Parker, J. Fontes, J. M. Burns and R. H. Swerdlow, J. Alzheim. Dis., 2010, 21, 141–154. 54 C. K. Atal, I. D. M. A. Bulletin, 1979, 10, 483–484. 55 R. M. Schaubhut and C. Jones, J. Nurs. Care Qual., 2000, 14, 13–27. 56 R. K. Zutshi, R. Singh, U. Zutshi, R. K. Johri and C. K. Atal, J. Assoc. Phys. India, 1985, 33, 223–224.

2266 | Food Funct., 2014, 5, 2252–2267

Paper

57 J. Singh, R. K. Dubey and C. K. Atal, J. Pharmacol. Exp. Ther., 2002, 302, 645–650. 58 A. R. Morgan, R. L. Cone and J. M. Elgert, Nucleic Acids Res., 1976, 3, 1139–1149. 59 D. R. Blake, R. E. Allen and J. Lunec, Brit. Med. Bull. J., 1987, 43, 371–385. 60 B. Halliwell, Am. J. Med., 1991, 91, 145–225. 61 D. Bandyopadhyay and A. Chattopadhyay, Curr. Med. Chem., 2006, 13, 1187–1202. 62 O. I. Aruoma and B. Halliwell, Biochem. J., 1987, 241, 273– 278. 63 E. Niki, J. Berry Res., 2012, 1, 169–176. 64 G. Chiva-Blanch and F. Visioli, J. Berry Res., 2012, 2, 63–71. 65 T. Y. Forbes-Hern´ andez, F. Giampieri, M. Gasparrini, L. Mazzoni, J. L. Quiles, J. M. Alvarez-Suarez and M. Battino, Food Chem. Toxicol., 2014, 68, 154–182. 66 D. Harman, Ann. N. Y. Acad. Sci., 1992, 673, 126–141. 67 Y. Kumarasamy, M. Byres, P. J. Cox, M. Jaspars, L. Nahar and S. D. Sarker, Phytother. Res., 2007, 21, 615–621. 68 C. H. Jao and W. C. Ko, Fish. Sci., 2002, 68, 430–435. 69 W. Brand-Williams, M. E. Cuvelier and C. Berset, Food Sci. Technol., 1995, 28, 25–30. 70 F. M. Rauscher, R. A. Sanders and J. B. Watkins III, J. Biochem. Mol. Toxicol., 2000, 14, 329–334. 71 M. Tim, J. Immunol. Methods, 1983, 65, 55–63. 72 S. J. Cooperstin and A. Lazarow, Exp. Cell Res., 1953, 5, 82–97. 73 R. J. Jackson, K. T. Elvers, L. J. Lee, M. D. Gidley, L. M. Wainwright, J. Lightfoot, S. F. Park and R. K. Poole, J. Bacteriol., 2007, 189, 1604–1615. 74 S. Stadlmann, G. Rieger, A. Amberger, A. V. Kuznetsov, R. Margreiter and E. Gnaiger, Transplantation, 2002, 74, 1800–1803. 75 E. A. Belyaeva, S. M. Korotkov and N. E. Saris, J. Trace Elem. Med. Biol., 2011, 25, 63–73. 76 E. Niki, FEBS Lett., 2012, 586, 3767–3770. 77 S. C. Luza and H. Speisky, Am. J. Clin. Nutr., 1996, 63, 812– 820. 78 J. P. Kehrer, Toxicology, 2000, 149, 43–50. 79 J. A. Dykens and Y. Will, Drug-induced Mitochondrial Dysfunction, John Wiley and Sons, New Jersey, 2008. 80 S. H. Jo, M. K. Son, H. J. Koh, S. M. Lee, I. H. Song, Y. O. Kim, Y. S. Lee, K. S. Jeong, W. B. Kim, J. W. Park, B. J. Song and T. L. Huh, J. Biol. Chem., 2001, 276, 16168–16176. 81 L. Tretter and V. Adam-Vizi, Philos. Trans. R. Soc., B, 2005, 360, 2335–2345. 82 C. T. Sheline and L. Wei, Neuroscience, 2006, 140, 235–246. 83 S. Miccadei and A. Floridi, Chem.–Biol. Interact., 1993, 89, 159–167. 84 I. G. Kirkinezosa and C. T. Moraes, Cell. Dev. Biol., 2001, 12, 449–457. 85 S. Shiva, P. S. Brookes, R. P. Patel, P. G. Anderson and V. M. Darley-Usmar, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 7212–7217. 86 L. Jian and S. Riyi, Int. J., 2005, 46, 243–252. 87 Y. Han and J. Z. Chen, BioMed Res. Int., 2013, 2013, 1–8. 88 S. W. Chan, P. N. Nguyen, D. Ayele, S. Chevalier, A. Aprikian and J. Z. Chen, Mutat. Res., 2011, 716, 40–50.

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Paper

L. A. Liotta and E. F. Petricoin 3rd, Proteomics, 2003, 3, 1801– 1810. 91 C. Nicco, A. Laurent, C. Chereau, B. Weill and F. Batteux, Biomed. Pharmacother., 2005, 59, 169–174.

Published on 30 June 2014. Downloaded by North Dakota State University on 22/10/2014 02:16:08.

89 S. D. Lim, C. Sun, J. D. Lambeth, F. Marshall, M. Amin, L. Chung, J. A. Petros and R. S. Arnold, Prostate, 2005, 62, 200–207. 90 P. C. Herrmann, J. W. Gillespie, L. Charboneau, V. E. Bichsel, C. P. Paweletz, V. S. Calvert, E. C. Kohn, M. R. Emmert-Buck,

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