Original research paper
Pomegranate seed oil: Effect on 3-nitropropionic acid-induced neurotoxicity in PC12 cells and elucidation of unsaturated fatty acids composition
Pro of
Bushra N. Al-Sabahi 1, Majek O. Fatope 1, Musthafa Mohamed Essa2,3, Selvaraju Subash2,3, Saleh N. Al-Busafi 1, Fatma S. N. Al-Kusaibi 1, Thamilarasan Manivasagam 4 1
ed
Department of Chemistry, Natural Products Research Laboratory, College of Science, Sultan Qaboos University, Al-Khod, Muscat, Oman, 2Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al-Khod, Muscat, Oman, 3Ageing and Dementia Research Group, Sultan Qaboos University, Al-Khod, Muscat, Oman, 4Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar, India
Un
cor rec t
Background: Seed oils are used as cosmetics or topical treatment for wounds, allergy, dandruff, and other purposes. Natural antioxidants from plants were recently reported to delay the onset or progress of various neurodegenerative conditions. Over one thousand cultivars of Punica granatum (Punicaceae) are known and some are traditionally used to treat various ailments. Aim: The effect of pomegranate oil on 3-nitropropionic acid- (3-NP) induced cytotoxicity in rat pheochromocytoma (PC12) neuronal cells was analyzed in this study. Furthermore, the analysis of unsaturated fatty acid composition of the seed oil of pomegranate by gas chromatography-electron impact mass spectrometry (GC-MS) was done. Results: GC-MS study showed the presence of 6,9-octadecadiynoic acid (C18:2(6,9)) as a major component (60%) as 4,4-dimethyloxazoline derivative. The total extractable oil with light petroleum ether by Soxhlet from the dry seed of P. granatum was 4–6%. The oil analyzed for 48.90 ± 1.50 mg gallic acid equivalents/g of oil, and demonstrated radical-scavenging-linked antioxidant activities in various in vitro assays like the DPPH (2,2-diphenyl-l-picrylhydrazyl, % IP = 35.2 ± 0.9%), ABTS (2,2’-azino-bis-3-ethylene benzothiozoline-6sulfonic acid, % IP 2.2 ± 0.1%), and β-carotene bleaching assay (% IP = 26 ± 3%), respectively, which could be due the possible role of one methylene interrupted diynoic acid system for its radicalscavenging/antioxidant properties of oil. The oil also reduced lipid peroxidation, suppressed reactive oxygen species, extracellular nitric oxide, lactate/pyruvate ratio, and lactase dehydrogenase generated by 3-NP- (100 mM) induced neurotoxicity in PC12 cells, and enhanced the levels of enzymatic and nonenzymatic antioxidants at 40 μg of gallic acid equivalents. Conclusion: The protective effect of pomegranate seed oil might be due to the ability of an oil to neutralize ROS or enhance the expression of antioxidant gene. Keywords: Acetylenic fatty acid, Pomegranate seed oil, Oman, Lipid peroxidation, Neuro-protection, Anti-oxidant, 3-nitropropionic acid
Highlights • The oil offered protection to 3-nitropropionic acidinduced neurotoxicity in rat pheochromocytoma (PC12) neuronal cells. Correspondence to: Musthafa Mohamed Essa, Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, Al-Khod 123, Muscat, Oman. Email: [email protected]
© W. S. Maney & Son Ltd 2014 DOI 10.1179/1476830514Y.0000000155
• The seed oil of Punica granatum is rich in unsaturated fatty acid acyl glycerols of which C18:2(6,9) diynoic acid derivative was the most abundant. • The oil demonstrated better antioxidant activity in invitro radical-scavenging assays. • Superior antioxidant capacity of oil was attributed to the presence of one methylene interrupted diynoic acid system.
Nutritional Neuroscience
2014
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Al-Sabahi et al.
Effect of PGO on 3-NP-induced cytotoxicity in PC12 cells
Materials and methods
Seed oils are used as cosmetics or topical treatment for wounds, allergy, and dandruff1,2 and have acylglycerol of saturated and unsaturated fatty acids as major components. Polyunsaturated fatty acids are an essential part of human nutrition considering that the ratio of ω6/ω3 in a healthy human diet should not exceed 10:1. Some polyunsaturated fatty acids have antiinflammatory,3 antifouling,4 and superoxide scavenging5 properties. Linoleic acid, an unsaturated fatty acid present in some seed oils, is often formulated into skin care products to moisturize the skin, enhance the healing process of sunburns or dermatoses, and to treat acne.6 In this paper, the unsaturated fatty acid composition, antioxidant and neuroprotective potential of the seed oil of Punica granatum are of interest. Natural antioxidants from plants were recently reported to delay the onset or progress of various neurodegenerative conditions.7,8 Over one thousand cultivars of P. granatum ( pomegranate) are known9 and some are traditionally used to treat urinary infection, kidney disorder,10 cardiovascular diseases, atherosclerosis, and thyroid dysfunction.11 The seed oil has antioxidant properties12,13 and some polyunsaturated fatty acid acyl glycerols were isolated previously from chloroform extract of fermented seeds of P. granatum. 14 Hartman et al.15 and Braidy et al.16 demonstrated the neuroprotective effect of pomegranate juice extracts in human primary neurons with Parkinson’s disease-like neurotoxicity. Substances with antioxidant properties can protect cells from collateral damage by reactive oxygen species (ROS) such as hydroxyl radical (OH·), superoxide anion radical (O2·−), hydrogen peroxide (H2O2), and singlet oxygen (1O2).17 The damage of proteins, DNA, and cell membrane18 by ROS can lead to premature wrinkly skin, cell death,19 cardiovascular diseases, and cancer. Typical antioxidants found in seed oils include, tocopherols, carotenoids, polyunsaturated fatty acids, flavonoids, and polyphenolic compounds,20,21 but to our knowledge, the radicalscavenging activity of oil has not been linked to the presence of methylene gap between unsaturated fatty acids; and the health benefit of pomegranate seed oil in 3-nitropropionic acid (3-NP) induced cytotoxicity in rat pheochromocytoma (PC12) neuronal cells is unknown. One research direction in our laboratory is to find seed oils which can relieve oxidative stress and be formulated into skin care products. The objective of this study was to structurally analyze the unsaturated fatty acids of the seed oil of P. granatum and to find if a link exists between unsaturated fatty acid moieties and radical-scavenging potential of the seed oil of P. granatum.
Absorbance of testing samples at designated wave lengths was recorded on Shimadzu UV-1800 spectrophotometer. Gas chromatography-electron impact mass spectrometry (GC-MS) analysis was performed on a Perkin Elmer Claus 600 GC System, fitted with a Rtx-5MS capillary column (low polarity phase; cross bond® biphenyl dimethyl polysiloxane; 30 m × 0.25 mm i.d. × 0.25 μm film thickness; maximum temperature, 350°C), coupled to Perkin Elmer Clarus 600C MS. Ultra-high purity helium (99.9%) was used as a carrier gas at a constant flow rate of 1.0 ml/minute. Samples were injected sample in aliquots of 1 μl with a split ratio of 10:1. The injection, transfer line, and ion source temperatures were 290, 290, and 290°C, respectively. The ionizing energy was 70 eV. Electron multiplier voltage was obtained from auto tune. All data were obtained by collecting the full-scan mass spectra within the range 40–550 amu. The oven temperature program was 60°C at a rate of 3°C/minute to 280°C hold for two minutes. The gas chromatogram of the total 4,4dimethyl oxazoline derivatives of fatty acids (DMOX) of the oil is shown in Fig. 1.
ed
Pro of
Introduction
Plant materials
Un
cor rec t
The fruits of P. granatum grown in Jabel Al Akhder were purchased from Nizwa Souq in October 2012. The seeds were removed from the fruits and allowed to ferment at room temperature for 2 weeks, draining off the fluid produced periodically. The seeds free of the soft parts were dried in the hot room for a week, ground and extracted with petroleum ether (40–50°C) by Soxhlet for 6 hours, and evaporated on a rotatory apparatus at 40°C.
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Nutritional Neuroscience
2014
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Chemicals
Linoleic acid, linoleic acid methyl ester, gallic acid (3,4, 5-trihyroxybenzoic acid), 2-amino-2-methylpropanol (AMP), 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) were obtained from Sigma (Sigma-Aldrich Ltd, Pool, Dorset, UK); Folin–Ciocalteu’s reagent and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Fluka Biochemika; and butylated hydroxyanisole (BHA) and Tween-40 were purchased from Kanto Chemical Co. Inc. Petroleum ether (30–40°C) and chloroform were purified by distillation before use.
Antioxidant activity of oil by DPPHradical The free radical-scavenging activity of the plant extract was estimated using a slight modification of DPPH radical-scavenging protocol reported by Chen et al.22 Two microliters of (100 μm DPPH) solution (3.8 mg in 100 ml ethanol) was mixed with 2.0 ml of 1.0 mg/ ml of ethanolic oil solution. The reaction mixture was incubated in the dark for 30 minutes and the
Figure 1
Effect of PGO on 3-NP-induced cytotoxicity in PC12 cells
Pro of
Al-Sabahi et al.
The gas chromatogram showing the DMOX of fatty acid mixture obtained from PGO.
( polyoxyethylene sorbian monopalmitate) (200 mg). Chloroform was removed on a rotary evaporator and 50 ml of oxygenated distilled water was added with vigorous shaking to form emulsion A. Three microliters of emulsion A was transferred into test tubes containing 0.20 ml of ethanol solutions of oil (concentration of stock solution was 2.0 mg/ml). A control without antioxidant, containing 0.2 ml of ethanol and 3.0 ml of emulsion was prepared. A second emulsion B consisting of 20 mg of linoleic acid, 200 mg of Tween 40, and 50 ml oxygenated water was also prepared. Three microliters of emulsion B was used to zero the spectrophotometer. The absorbance of all samples was measured at 470 nm immediately (t=0) and at 15- minute intervals for 120 minutes. The reaction mixture was carried out at 50°C using BHA and gallic acid as positive control. The percentage inhibition of discolorations of β-carotene was calculated from the expression:
cor rec t
ed
absorbance was recorded at 517 nm against the blank. For the control, 2.0 m of (100 μm DPPH) solution in ethanol was mixed with 2.0 ml of ethanol and the absorbance of the solution was recorded after 30 minutes. The assay was carried out in triplicates using gallic acid and BHA as positive control. DPPH radical-scavenging activities were calculated and expressed as a percentage using the following expression. Percentage inhibition of DPPH radical (%IP) = 1 − At=30 /At=0 × 100
where At=30 is the absorbance of the sample after 30 minutes; At=0 : is the absorbance of the control. The results of the assay are shown in Fig. 2.
Antioxidant activity of oil by β-carotene bleaching method
Un
The antioxidant activity of the oil was determined according to a modified version of the β-carotene bleaching method by Singh et al.13 The β-carotene (0.5 mg) was dissolved in chloroform (0.2 ml) and mixed with linoleic acid (20 mg) and Tween 40
Figure 2 tests.
Percentage inhibition of discolorations of β − carotene(%IP) = [1 − At=15 /At=0 ] × 100
Radicals inhibiting activity of the PGO assessed by using DPPH, ABTS discoloration, and β-carotene bleaching assay
Nutritional Neuroscience
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Effect of PGO on 3-NP-induced cytotoxicity in PC12 cells
where At=15is the absorbance of the sample after 15minutes; At=0 is the absorbance of the control. The results of the assay are shown in Fig. 2.
Antioxidant activity of oil by ABTS+ discoloration method
Percentage inhibition of ABTS radical (%IP) = 1 − At=30 /At=0 × 100
Cells and media The pheochromocytoma cell line (PC12) was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in growth medium (RPMI 1640 medium, 10% (v/v) donor horse serum, 5% fetal bovine serum) in a 75 cm2 culture flask. Cells used for the experiments were seeded at a density of 4.5 × 105 cells per well on a six-well cell culture plate and cell differentiation was achieved in 4 days in a differentiation medium (RPMI 1640 medium, 1% donor horse serum, 100 ng NGF/ml, 50 ng cAMP/ml).
Cellular viability test
The cellular viability test was based on the estimation of mitochondrial activity in living cells25 on a 96-well plate by quantitative colorimetric assay in which MTT was reduced to formazan by mitochondrial respiratory enzymes. PC12 cells were pre-treated with different concentrations of oil (20–100 μg of GAE), and then exposed to 100 mm of 3-NP for 24–48 hours. After the medium was removed, the cells were incubated with 0.25 mg/ml MTT for 4 hours at 37°C. The reaction was stopped by adding dimethyl sulfoxide. The amount of MTT formazan product was determined by measuring absorbance in a microplate reader at a testing wavelength of 570 nm and a referencing wavelength of 630 nm. The best dose of oil (40 μg of GAE,) was chosen for the other cell-based assays. The results are shown in Fig. 3.
ed
The experiment was carried out using free radical discoloration assay developed by Re et al.23 The ABTS+chromophore was prepared by reacting ABTS solution 7 mm (0.19201 g in 50 ml distilled water) with 2.45 mm (0.03342 g in 50 ml H2O) persulfate (K2S2O8). The mixture was allowed to stand for 15 hours in the dark at room temperature. The solution was diluted with ethanol to obtain absorbance of (0.7±0.2) at 734 nm. The oil was dissolved in ethanol to produce a concentration of 2.0 mg/ml. An aliquot of 20 μl of ethanolic test solution of each sample was mixed with 2.0 ml of ABTS+ reagent and the absorbance taken at 5-minute intervals. BHA and gallic acid were used as positive control. The percentage inhibition of ABTS radical was calculated from the expression:
was incubated at room temperature for 30 minutes. The absorption was measured at 765 nm. The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram of oil.
Pro of
Al-Sabahi et al.
cor rec t
where At=30 is the absorbance of the sample after 30 minutes; At=0 is the absorbance of the control. The results of the assay are shown in Fig. 2.
Determination of total phenolic contents
The total phenolic content of oil was determined according to Folin–Ciocalteu procedure.24 One microliter of Folin–Ciocalteu’s reagent and 0.8 ml 7.5% (w/v) Na2CO3 were added to 0.2 ml of seed extract dissolved in ethanol (2.0 mg/ml). After shaking, the mixture
Un
Extracellular lactate dehydrogenase activity as a measurement for cytotoxicity The release of lactate dehydrogenase (LDH) into culture supernatant correlates with the amount of cell death and membrane damage, providing an accurate measure of cellular toxicity. LDH activity was assayed using a standard spectrophotometric technique. The results are shown in Table 1.
Intracellular pyruvate and lactate measurement Determination of lactate and pyruvate in the cell lysate was done, using Bio vision USA kits and expressed as lactate over pyruvate ratio. The results are shown in Table 1. Figure 3 Cell viability assay using MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reductions assay to its insoluble formazan. Briefly, cells were seeded in 96-well plate following the kit specifications. The data presented as the mean±SEM of three independent experiments performed in triplicates. *P ≤ 0.05.
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Determination of extracellular nitric oxide Determination of extra cellular nitric oxide (NO) in the culture supernatant was done, using kits from Bio Vision (California, USA) and the results are expressed in relative fluorescent units shown in Table1.
Al-Sabahi et al.
Effect of PGO on 3-NP-induced cytotoxicity in PC12 cells
Table 1 Effect of oil on 3-NP-induced cytotoxicity in PC12 cells Parameters
Control
3-NP
40 μg of GAE+3-NP
40 μg of GAE
LDH (IU/l/mg protein) ROS (RFU) RNS (RFU) L/P ratio MDA (% control) SOD (% control) GSH (% control) GPx (% control)
25.01 ± 0.77a 15.00 ± 0.47a 34.01 ± 1.06a 1.490 ± 0.05a 21.00 ± 0.65a 94.79 ± 4.53a 97.02 ± 2.47a 98.02 ± 3.05a
190.0 ± 5.90b 78.01 ± 2.43b 67.01 ± 2.01b 2.700 ± 0.08b 64.01 ± 1.99b 38.01 ± 2.12b 36.51 ± 1.14b 54.21 ± 1.69b
55.01 ± 1.71c 40.01 ± 1.24c 52.01 ± 1.62c 2.100 ± 0.07c 46.01 ± 1.43c 63.01 ± 1.96c 60.41 ± 1.88c 71.01 ± 2.21c
29.01 ± 0.90a 17.00 ± 0.53a 31.01 ± 0.96a 1.610 ± 0.05a 23.00 ± 0.72a 97.02 ± 3.01a 95.52 ± 4.29a 96.02 ± 2.96a
All data from three different assessments in triplicates. RFU, relative fluorescent unit. Values not sharing common superscript differs significantly (P < 0.05) – DMRT.
All data were obtained from at least three different measurements, unless otherwise stated. Data are presented as the mean±SEM and statistical analyses were performed using one-way analysis of variance followed by the Tukey test and P
Pomegranate seed oil: Effect on 3-nitropropionic acid-induced neurotoxicity in PC12 cells and elucidation of unsaturated fatty acids composition
Pro of
Bushra N. Al-Sabahi 1, Majek O. Fatope 1, Musthafa Mohamed Essa2,3, Selvaraju Subash2,3, Saleh N. Al-Busafi 1, Fatma S. N. Al-Kusaibi 1, Thamilarasan Manivasagam 4 1
ed
Department of Chemistry, Natural Products Research Laboratory, College of Science, Sultan Qaboos University, Al-Khod, Muscat, Oman, 2Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al-Khod, Muscat, Oman, 3Ageing and Dementia Research Group, Sultan Qaboos University, Al-Khod, Muscat, Oman, 4Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar, India
Un
cor rec t
Background: Seed oils are used as cosmetics or topical treatment for wounds, allergy, dandruff, and other purposes. Natural antioxidants from plants were recently reported to delay the onset or progress of various neurodegenerative conditions. Over one thousand cultivars of Punica granatum (Punicaceae) are known and some are traditionally used to treat various ailments. Aim: The effect of pomegranate oil on 3-nitropropionic acid- (3-NP) induced cytotoxicity in rat pheochromocytoma (PC12) neuronal cells was analyzed in this study. Furthermore, the analysis of unsaturated fatty acid composition of the seed oil of pomegranate by gas chromatography-electron impact mass spectrometry (GC-MS) was done. Results: GC-MS study showed the presence of 6,9-octadecadiynoic acid (C18:2(6,9)) as a major component (60%) as 4,4-dimethyloxazoline derivative. The total extractable oil with light petroleum ether by Soxhlet from the dry seed of P. granatum was 4–6%. The oil analyzed for 48.90 ± 1.50 mg gallic acid equivalents/g of oil, and demonstrated radical-scavenging-linked antioxidant activities in various in vitro assays like the DPPH (2,2-diphenyl-l-picrylhydrazyl, % IP = 35.2 ± 0.9%), ABTS (2,2’-azino-bis-3-ethylene benzothiozoline-6sulfonic acid, % IP 2.2 ± 0.1%), and β-carotene bleaching assay (% IP = 26 ± 3%), respectively, which could be due the possible role of one methylene interrupted diynoic acid system for its radicalscavenging/antioxidant properties of oil. The oil also reduced lipid peroxidation, suppressed reactive oxygen species, extracellular nitric oxide, lactate/pyruvate ratio, and lactase dehydrogenase generated by 3-NP- (100 mM) induced neurotoxicity in PC12 cells, and enhanced the levels of enzymatic and nonenzymatic antioxidants at 40 μg of gallic acid equivalents. Conclusion: The protective effect of pomegranate seed oil might be due to the ability of an oil to neutralize ROS or enhance the expression of antioxidant gene. Keywords: Acetylenic fatty acid, Pomegranate seed oil, Oman, Lipid peroxidation, Neuro-protection, Anti-oxidant, 3-nitropropionic acid
Highlights • The oil offered protection to 3-nitropropionic acidinduced neurotoxicity in rat pheochromocytoma (PC12) neuronal cells. Correspondence to: Musthafa Mohamed Essa, Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, Al-Khod 123, Muscat, Oman. Email: [email protected]
© W. S. Maney & Son Ltd 2014 DOI 10.1179/1476830514Y.0000000155
• The seed oil of Punica granatum is rich in unsaturated fatty acid acyl glycerols of which C18:2(6,9) diynoic acid derivative was the most abundant. • The oil demonstrated better antioxidant activity in invitro radical-scavenging assays. • Superior antioxidant capacity of oil was attributed to the presence of one methylene interrupted diynoic acid system.
Nutritional Neuroscience
2014
VOL.
0
NO.
0
1
Al-Sabahi et al.
Effect of PGO on 3-NP-induced cytotoxicity in PC12 cells
Materials and methods
Seed oils are used as cosmetics or topical treatment for wounds, allergy, and dandruff1,2 and have acylglycerol of saturated and unsaturated fatty acids as major components. Polyunsaturated fatty acids are an essential part of human nutrition considering that the ratio of ω6/ω3 in a healthy human diet should not exceed 10:1. Some polyunsaturated fatty acids have antiinflammatory,3 antifouling,4 and superoxide scavenging5 properties. Linoleic acid, an unsaturated fatty acid present in some seed oils, is often formulated into skin care products to moisturize the skin, enhance the healing process of sunburns or dermatoses, and to treat acne.6 In this paper, the unsaturated fatty acid composition, antioxidant and neuroprotective potential of the seed oil of Punica granatum are of interest. Natural antioxidants from plants were recently reported to delay the onset or progress of various neurodegenerative conditions.7,8 Over one thousand cultivars of P. granatum ( pomegranate) are known9 and some are traditionally used to treat urinary infection, kidney disorder,10 cardiovascular diseases, atherosclerosis, and thyroid dysfunction.11 The seed oil has antioxidant properties12,13 and some polyunsaturated fatty acid acyl glycerols were isolated previously from chloroform extract of fermented seeds of P. granatum. 14 Hartman et al.15 and Braidy et al.16 demonstrated the neuroprotective effect of pomegranate juice extracts in human primary neurons with Parkinson’s disease-like neurotoxicity. Substances with antioxidant properties can protect cells from collateral damage by reactive oxygen species (ROS) such as hydroxyl radical (OH·), superoxide anion radical (O2·−), hydrogen peroxide (H2O2), and singlet oxygen (1O2).17 The damage of proteins, DNA, and cell membrane18 by ROS can lead to premature wrinkly skin, cell death,19 cardiovascular diseases, and cancer. Typical antioxidants found in seed oils include, tocopherols, carotenoids, polyunsaturated fatty acids, flavonoids, and polyphenolic compounds,20,21 but to our knowledge, the radicalscavenging activity of oil has not been linked to the presence of methylene gap between unsaturated fatty acids; and the health benefit of pomegranate seed oil in 3-nitropropionic acid (3-NP) induced cytotoxicity in rat pheochromocytoma (PC12) neuronal cells is unknown. One research direction in our laboratory is to find seed oils which can relieve oxidative stress and be formulated into skin care products. The objective of this study was to structurally analyze the unsaturated fatty acids of the seed oil of P. granatum and to find if a link exists between unsaturated fatty acid moieties and radical-scavenging potential of the seed oil of P. granatum.
Absorbance of testing samples at designated wave lengths was recorded on Shimadzu UV-1800 spectrophotometer. Gas chromatography-electron impact mass spectrometry (GC-MS) analysis was performed on a Perkin Elmer Claus 600 GC System, fitted with a Rtx-5MS capillary column (low polarity phase; cross bond® biphenyl dimethyl polysiloxane; 30 m × 0.25 mm i.d. × 0.25 μm film thickness; maximum temperature, 350°C), coupled to Perkin Elmer Clarus 600C MS. Ultra-high purity helium (99.9%) was used as a carrier gas at a constant flow rate of 1.0 ml/minute. Samples were injected sample in aliquots of 1 μl with a split ratio of 10:1. The injection, transfer line, and ion source temperatures were 290, 290, and 290°C, respectively. The ionizing energy was 70 eV. Electron multiplier voltage was obtained from auto tune. All data were obtained by collecting the full-scan mass spectra within the range 40–550 amu. The oven temperature program was 60°C at a rate of 3°C/minute to 280°C hold for two minutes. The gas chromatogram of the total 4,4dimethyl oxazoline derivatives of fatty acids (DMOX) of the oil is shown in Fig. 1.
ed
Pro of
Introduction
Plant materials
Un
cor rec t
The fruits of P. granatum grown in Jabel Al Akhder were purchased from Nizwa Souq in October 2012. The seeds were removed from the fruits and allowed to ferment at room temperature for 2 weeks, draining off the fluid produced periodically. The seeds free of the soft parts were dried in the hot room for a week, ground and extracted with petroleum ether (40–50°C) by Soxhlet for 6 hours, and evaporated on a rotatory apparatus at 40°C.
2
Nutritional Neuroscience
2014
VOL.
0
NO.
0
Chemicals
Linoleic acid, linoleic acid methyl ester, gallic acid (3,4, 5-trihyroxybenzoic acid), 2-amino-2-methylpropanol (AMP), 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) were obtained from Sigma (Sigma-Aldrich Ltd, Pool, Dorset, UK); Folin–Ciocalteu’s reagent and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Fluka Biochemika; and butylated hydroxyanisole (BHA) and Tween-40 were purchased from Kanto Chemical Co. Inc. Petroleum ether (30–40°C) and chloroform were purified by distillation before use.
Antioxidant activity of oil by DPPHradical The free radical-scavenging activity of the plant extract was estimated using a slight modification of DPPH radical-scavenging protocol reported by Chen et al.22 Two microliters of (100 μm DPPH) solution (3.8 mg in 100 ml ethanol) was mixed with 2.0 ml of 1.0 mg/ ml of ethanolic oil solution. The reaction mixture was incubated in the dark for 30 minutes and the
Figure 1
Effect of PGO on 3-NP-induced cytotoxicity in PC12 cells
Pro of
Al-Sabahi et al.
The gas chromatogram showing the DMOX of fatty acid mixture obtained from PGO.
( polyoxyethylene sorbian monopalmitate) (200 mg). Chloroform was removed on a rotary evaporator and 50 ml of oxygenated distilled water was added with vigorous shaking to form emulsion A. Three microliters of emulsion A was transferred into test tubes containing 0.20 ml of ethanol solutions of oil (concentration of stock solution was 2.0 mg/ml). A control without antioxidant, containing 0.2 ml of ethanol and 3.0 ml of emulsion was prepared. A second emulsion B consisting of 20 mg of linoleic acid, 200 mg of Tween 40, and 50 ml oxygenated water was also prepared. Three microliters of emulsion B was used to zero the spectrophotometer. The absorbance of all samples was measured at 470 nm immediately (t=0) and at 15- minute intervals for 120 minutes. The reaction mixture was carried out at 50°C using BHA and gallic acid as positive control. The percentage inhibition of discolorations of β-carotene was calculated from the expression:
cor rec t
ed
absorbance was recorded at 517 nm against the blank. For the control, 2.0 m of (100 μm DPPH) solution in ethanol was mixed with 2.0 ml of ethanol and the absorbance of the solution was recorded after 30 minutes. The assay was carried out in triplicates using gallic acid and BHA as positive control. DPPH radical-scavenging activities were calculated and expressed as a percentage using the following expression. Percentage inhibition of DPPH radical (%IP) = 1 − At=30 /At=0 × 100
where At=30 is the absorbance of the sample after 30 minutes; At=0 : is the absorbance of the control. The results of the assay are shown in Fig. 2.
Antioxidant activity of oil by β-carotene bleaching method
Un
The antioxidant activity of the oil was determined according to a modified version of the β-carotene bleaching method by Singh et al.13 The β-carotene (0.5 mg) was dissolved in chloroform (0.2 ml) and mixed with linoleic acid (20 mg) and Tween 40
Figure 2 tests.
Percentage inhibition of discolorations of β − carotene(%IP) = [1 − At=15 /At=0 ] × 100
Radicals inhibiting activity of the PGO assessed by using DPPH, ABTS discoloration, and β-carotene bleaching assay
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2014
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Effect of PGO on 3-NP-induced cytotoxicity in PC12 cells
where At=15is the absorbance of the sample after 15minutes; At=0 is the absorbance of the control. The results of the assay are shown in Fig. 2.
Antioxidant activity of oil by ABTS+ discoloration method
Percentage inhibition of ABTS radical (%IP) = 1 − At=30 /At=0 × 100
Cells and media The pheochromocytoma cell line (PC12) was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in growth medium (RPMI 1640 medium, 10% (v/v) donor horse serum, 5% fetal bovine serum) in a 75 cm2 culture flask. Cells used for the experiments were seeded at a density of 4.5 × 105 cells per well on a six-well cell culture plate and cell differentiation was achieved in 4 days in a differentiation medium (RPMI 1640 medium, 1% donor horse serum, 100 ng NGF/ml, 50 ng cAMP/ml).
Cellular viability test
The cellular viability test was based on the estimation of mitochondrial activity in living cells25 on a 96-well plate by quantitative colorimetric assay in which MTT was reduced to formazan by mitochondrial respiratory enzymes. PC12 cells were pre-treated with different concentrations of oil (20–100 μg of GAE), and then exposed to 100 mm of 3-NP for 24–48 hours. After the medium was removed, the cells were incubated with 0.25 mg/ml MTT for 4 hours at 37°C. The reaction was stopped by adding dimethyl sulfoxide. The amount of MTT formazan product was determined by measuring absorbance in a microplate reader at a testing wavelength of 570 nm and a referencing wavelength of 630 nm. The best dose of oil (40 μg of GAE,) was chosen for the other cell-based assays. The results are shown in Fig. 3.
ed
The experiment was carried out using free radical discoloration assay developed by Re et al.23 The ABTS+chromophore was prepared by reacting ABTS solution 7 mm (0.19201 g in 50 ml distilled water) with 2.45 mm (0.03342 g in 50 ml H2O) persulfate (K2S2O8). The mixture was allowed to stand for 15 hours in the dark at room temperature. The solution was diluted with ethanol to obtain absorbance of (0.7±0.2) at 734 nm. The oil was dissolved in ethanol to produce a concentration of 2.0 mg/ml. An aliquot of 20 μl of ethanolic test solution of each sample was mixed with 2.0 ml of ABTS+ reagent and the absorbance taken at 5-minute intervals. BHA and gallic acid were used as positive control. The percentage inhibition of ABTS radical was calculated from the expression:
was incubated at room temperature for 30 minutes. The absorption was measured at 765 nm. The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram of oil.
Pro of
Al-Sabahi et al.
cor rec t
where At=30 is the absorbance of the sample after 30 minutes; At=0 is the absorbance of the control. The results of the assay are shown in Fig. 2.
Determination of total phenolic contents
The total phenolic content of oil was determined according to Folin–Ciocalteu procedure.24 One microliter of Folin–Ciocalteu’s reagent and 0.8 ml 7.5% (w/v) Na2CO3 were added to 0.2 ml of seed extract dissolved in ethanol (2.0 mg/ml). After shaking, the mixture
Un
Extracellular lactate dehydrogenase activity as a measurement for cytotoxicity The release of lactate dehydrogenase (LDH) into culture supernatant correlates with the amount of cell death and membrane damage, providing an accurate measure of cellular toxicity. LDH activity was assayed using a standard spectrophotometric technique. The results are shown in Table 1.
Intracellular pyruvate and lactate measurement Determination of lactate and pyruvate in the cell lysate was done, using Bio vision USA kits and expressed as lactate over pyruvate ratio. The results are shown in Table 1. Figure 3 Cell viability assay using MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reductions assay to its insoluble formazan. Briefly, cells were seeded in 96-well plate following the kit specifications. The data presented as the mean±SEM of three independent experiments performed in triplicates. *P ≤ 0.05.
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Determination of extracellular nitric oxide Determination of extra cellular nitric oxide (NO) in the culture supernatant was done, using kits from Bio Vision (California, USA) and the results are expressed in relative fluorescent units shown in Table1.
Al-Sabahi et al.
Effect of PGO on 3-NP-induced cytotoxicity in PC12 cells
Table 1 Effect of oil on 3-NP-induced cytotoxicity in PC12 cells Parameters
Control
3-NP
40 μg of GAE+3-NP
40 μg of GAE
LDH (IU/l/mg protein) ROS (RFU) RNS (RFU) L/P ratio MDA (% control) SOD (% control) GSH (% control) GPx (% control)
25.01 ± 0.77a 15.00 ± 0.47a 34.01 ± 1.06a 1.490 ± 0.05a 21.00 ± 0.65a 94.79 ± 4.53a 97.02 ± 2.47a 98.02 ± 3.05a
190.0 ± 5.90b 78.01 ± 2.43b 67.01 ± 2.01b 2.700 ± 0.08b 64.01 ± 1.99b 38.01 ± 2.12b 36.51 ± 1.14b 54.21 ± 1.69b
55.01 ± 1.71c 40.01 ± 1.24c 52.01 ± 1.62c 2.100 ± 0.07c 46.01 ± 1.43c 63.01 ± 1.96c 60.41 ± 1.88c 71.01 ± 2.21c
29.01 ± 0.90a 17.00 ± 0.53a 31.01 ± 0.96a 1.610 ± 0.05a 23.00 ± 0.72a 97.02 ± 3.01a 95.52 ± 4.29a 96.02 ± 2.96a
All data from three different assessments in triplicates. RFU, relative fluorescent unit. Values not sharing common superscript differs significantly (P < 0.05) – DMRT.
All data were obtained from at least three different measurements, unless otherwise stated. Data are presented as the mean±SEM and statistical analyses were performed using one-way analysis of variance followed by the Tukey test and P
