RESEARCH ARTICLE
An Antioxidant Extract of the Insectivorous Plant Drosera burmannii Vahl. Alleviates IronInduced Oxidative Stress and Hepatic Injury in Mice Nikhil Baban Ghate☯, Dipankar Chaudhuri☯, Abhishek Das, Sourav Panja, Nripendranath Mandal* Division of Molecular Medicine, Bose Institute, P 1/12, Scheme—VIIM, Kolkata, West Bengal, India ☯ These authors contributed equally to this work. * [email protected]
Abstract
OPEN ACCESS Citation: Ghate NB, Chaudhuri D, Das A, Panja S, Mandal N (2015) An Antioxidant Extract of the Insectivorous Plant Drosera burmannii Vahl. Alleviates Iron-Induced Oxidative Stress and Hepatic Injury in Mice. PLoS ONE 10(5): e0128221. doi:10.1371/journal.pone.0128221 Academic Editor: Krishnendu Acharya, University of Calcutta, INDIA Received: December 18, 2014 Accepted: April 24, 2015 Published: May 26, 2015 Copyright: © 2015 Ghate et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Dr. Abhishek Das is grateful to the Department of Biotechnology (DBT), Govt. of India for the support of Research Associateship.
Free iron typically leads to the formation of excess free radicals, and additional iron deposition in the liver contributes to the oxidative pathologic processes of liver disease. Many pharmacological properties of the insectivorous plant Drosera burmannii Vahl. have been reported in previous studies; however, there is no evidence of its antioxidant or hepatoprotective potential against iron overload. The antioxidant activity of 70% methanolic extract of D. burmannii (DBME) was evaluated. DBME showed excellent DPPH, hydroxyl, hypochlorous, superoxide, singlet oxygen, nitric oxide, peroxynitrite radical and hydrogen peroxide scavenging activity. A substantial iron chelation (IC50 = 40.90 ± 0.31 μg/ml) and supercoiled DNA protection ([P]50 = 50.41 ± 0.55 μg) were observed. DBME also displayed excellent in vivo hepatoprotective activity in iron-overloaded Swiss albino mice compared to the standard desirox treatment. Administration of DBME significantly normalized serum enzyme levels and restored liver antioxidant enzymes levels. DBME lowered the raised levels of liver damage parameters, also reflected from the morphological analysis of the liver sections. DBME also reduced liver iron content by 115.90% which is also seen by Perls’ staining. A phytochemical analysis of DBME confirms the presence of various phytoconstituents, including phenols, flavonoids, carbohydrates, tannins, alkaloids and ascorbic acid. Alkaloids, phenols and flavonoids were abundantly found in DBME. An HPLC analysis of DBME revealed the presence of purpurin, catechin, tannic acid, reserpine, methyl gallate and rutin. Purpurin, tannic acid, methyl gallate and rutin displayed excellent iron chelation but exhibited cytotoxicity toward normal (WI-38) cells; while DBME found to be non-toxic to the normal cells. These findings suggest that the constituents present in DBME contributed to its iron chelation activity. Additional studies are needed to determine if DBME can be used as a treatment for iron overload diseases.
Competing Interests: The authors have declared that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0128221 May 26, 2015
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Antioxidant and Hepatoprotective Activity of an Insectivorous Plant
Introduction Free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), play a significant role in the early onset of oxidative stress and are capable of damaging biologically relevant molecules, such as proteins, nucleic acids and plasma membrane lipids [1]. Antioxidants can interrupt the chain reaction cycle (oxidation process) via different mechanisms, such as chelating metals that catalyze the formation of free radicals and scavenging the free radicals. Therefore, antioxidants are vital for the human body due to their ability to combat oxidative damage [2]. Iron is a metal that is needed by all mammalian cells for growth and survival [3]; however, its extreme deposition can increase oxidative stress in the liver and lead to further injuries, such as hepatocellular necrosis [4], inflammation [5], fibrosis [6,7] and cancer [8]. The human body is largely dependent on the liver for the facilitation of many vital biochemical pathways that manage growth, nutrient supply, energy provision, reproduction and defense [9]. Liver damage (hepatotoxicity) caused by iron overload hinders these processes and can result in serious health problems [10]. Iron removal by chelation therapy is an effective life-saving strategy for nearly all the aforementioned iron overload-induced diseases. Several synthetic iron chelating agents, such as deferoxamine, 1,2-dimethyl-3-hydroxypyrid-4-one (deferiprone, L1) and deferasirox, are available for clinical use; however, these drugs possess several undesirable side effects [11,12]. Thus, the scientific community continues to search for a raw material or isolated natural product that can act as an antioxidant and iron chelator without adverse effects. Drosera burmannii Vahl. (family Droseraceae) is an acaulescent insectivorous herb commonly known as sundew that belongs to one of the largest genera of carnivorous plants, with over 105 species. This herb is distributed throughout the Indian subcontinent as well as China, Australia and West Africa, and it is reported to possess rubefacient properties [13]. Moreover, antifertility [14], anticonvulsant [15] and antitumor activities in mice [16] were reported in the alcohol and aqueous extracts of D. burmannii. Different species of Drosera contain several medicinally active compounds, including 1,4-naphthoquinones such as plumbagin [17], hydroplumbagin glucoside [18], flavonoids (kaempferol, myricetin, quercetin and hyperoside) [19] and rossoliside (7-methyl-hydrojuglone-4-glucoside). Previous literature reports that several species of Drosera are used in various traditional and homeopathic treatments. Some of the species of this family are used for the treatment of cough in Asia. This plant was also listed in French Pharmacopoeia in 1965 for the treatment of chronic bronchitis, asthma and Whooping cough [20]. The present study aimed to assess the in vitro antioxidant and in vivo hepatoprotective properties against iron-overload-induced liver toxicity in Swiss albino mice.
Materials and Methods Chemicals 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was procured from Roche diagnostics, Mannheim, Germany. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was obtained from Fluka, Buchs, Switzerland. Potassium persulfate (K2S2O8), 2-deoxy2-ribose, ethylene diammine tetraacetic acid (EDTA), ascorbic acid, trichloroacetic acid (TCA), mannitol, nitro blue tetrazolium (NBT), reduced nicotinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS), sodium nitroprusside (SNP), 1,10-phenanthroline, sulphanilamide, N-(1-Naphthyl)ethylenediamine dihydrochloride (NED), L-histidine, lipoic acid, sodium pyruvate, quercetin, ferrozine glutathione reduced, bathophenanthrolinesulfonate disodium salt and 5,50 -dithiobis-2-nitrobenzoic acid (DTNB) were obtained from Sisco Research Laboratories Pvt. Ltd, Mumbai, India. HPLC grade acetonitrile, ammonium acetate,
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hydrogen peroxide, potassium hexacyanoferrate, Folin-ciocalteu reagent, sodium carbonate, mercuric chloride, potassium iodide, anthrone, vanillin, thiourea, 2,4-dinitrophenylhydrazine (DNPH), sodium hypochlorite, aluminum chloride, xylenol orange, butylated hydroxyltoluene (BHT), N,N- dimethyl-4-nitrosoaniline ammonium iron (II) sulfatehexahydrate [(NH4)2Fe (SO4)26H2O], 1-chloro-2,4-dinitrobenzene (CDNB), chloramine-T, hydroxylamine hydrochloride and Dimethyl-4-aminobenzaldehyde were procured from Merck, Mumbai, India. 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferritin, methyl gallate, tannic acid, rutin, gallic acid, (+) catechin and curcumin were obtained from MP Biomedicals, France. Catalase, reserpine, streptomycin sulfate and sodium bicarbonate were obtained from HiMedia Laboratories Pvt. Ltd, Mumbai, India. Evans blue was purchased from BDH, England. D-glucose was procured from Qualigens Fine Chemicals, Mumbai. Diethylenetriaminepentaacetic acid (DTPA) was obtained from Spectrochem Pvt. Ltd, Mumbai, India. Thiobarbituric acid (TBA) was obtained from Loba Chemie, Mumbai, India. Iron-dextran and guanidine hydrochloride was purchased from Sigma-Aldrich, USA. The standard oral iron chelating drug, desirox, was obtained from Cipla Ltd., Kolkata, India.
Ethics A sample of the insectivorous plant Drosera burmannii Vahl. was collected in January 2014 from public areas adjoining villages in the Bankura district in the state of West Bengal, India. These areas are not within a National Park/Reserve Forest/Govt. protected area, and only verbal permission from village headmen was obtained before collection. The conservation status of this insectivorous plant was classified using the International Union for Conservation of Nature (IUCN) World Conservation Union guidelines (1994). The status was ‘LC; Least concern’ as per IUCN red list criteria. The material collected for this study was sampled on a very limited scale and therefore had negligible effects on broader ecosystem functioning. All animal experiments (Swiss albino mice) were performed in strict accordance with the recommendations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India (Bose Institute Registration. No. 95/1999/CPCSEA). The protocol was approved by the Institutional Animal Ethics Committee, Bose Institute. All surgery was performed under ethyl ether anesthesia, and all efforts were made to minimize suffering.
Animals Male Swiss albino mice (20 ± 2 g) were purchased from Chittaranjan National Cancer Institute (CNCI), Kolkata, India and were maintained under a constant 12-h dark/light cycle at an environmental temperature of 22 ± 2°C. Mice were provided a normal laboratory pellet diet and water ad libitum. The condition of the animals were monitored every 6-h after the treatment and there were no unintended animal deaths during the experimental procedures.
Plant extract preparation D. burmannii was authenticated by the Botanical Survey of India, Kolkata, India. Samples were sorted, cleaned of substratum and shadow dried for extraction. The dried sample (100 g) was then powdered and stirred using a magnetic stirrer with 70% methanol in water (1000 ml) for 15 hours. The mixture was then centrifuged at 2,850 g, and the supernatant was decanted. The process was repeated by adding more solvent to the precipitated pellet. The supernatants from the two phases were mixed, concentrated in a rotary evaporator at 40°C, lyophilized and labeled as DBME. The dried extract was stored at -20°C until use.
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In vitro study Total antioxidant activity and reducing power. The total antioxidant capacity of DBME was evaluated by an ABTS•+ radical cation decolorization assay in comparison to a trolox standard [21] and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay [22]. The reducing power of DBME was determined using a previously described method [21]. Reactive oxygen species (ROS) scavenging activity. The ROS scavenging ability of DBME was determined using multiple stable ROS radical scavenging assays, such as hydroxyl, superoxide, hypochlorous radical, singlet oxygen and hydrogen peroxide assays by standard procedures [21]. Reactive nitrogen species (RNS) scavenging activity. The RNS scavenging activity of DBME was determined using nitric oxide and peroxynitrite radical scavenging assays [21]. Iron chelation assay. The Fe2+ chelating activity of DBME, catechin, methyl gallate, purpurin, reserpine, rutin and tannic acid was evaluated according to standard methods [23]. HEPES buffer (20 mM, pH 7.2), DBME (0–120 μg/ml) and a positive control EDTA (0–20 μg/ ml) were separately added to a 12.5 μM ferrous sulfate solution, and 75 μM ferrozine was added to start the reaction. The mixture was shaken vigorously and left standing for 20 min at room temperature. Next, the absorbance was measured at 562 nm. All tests were performed six times. DNA protection assay. DNA protection was studied using supercoiled pUC18 plasmid DNA according to previously described methods [24] with minor modifications. In HEPES buffer, (pH 7.2, 13 mM), a FeSO4 solution (15 μM), DBME of varying doses (0–100 μg), DNA (1 μg) and water were added to produce an initial reaction mixture. Next, an H2O2 solution (0.0125 mM) was added to start the reaction. After 10 min, the reaction was stopped by adding desferal (0.2 mM) followed by a loading buffer. Each reaction mixture (20 μl) was loaded in a 1% agarose gel. After migration, the gel was stained with ethidium bromide and visualized with a UV transilluminator. The DNA bands were quantified using densitometry, and the following formulas were used to calculate the protection percentage: % SC ¼ ½1:4 X SC=ðOC þ ð1:4 X SCÞÞ X 100 and % protection ¼ 100 X ½ðcontrol SC chelator SCÞ=ðcontrol SC no chelator SCÞ 1; where SC = supercoiled; OC = open circular; 1.4 = correction factor. The ability of the plant extract to protect DNA supercoils can be expressed as the [P]50 value, which is defined by the concentration of sample required for 50% protection. Ferritin iron release assay. This assay was performed according to a previously described method [25]. The release of ferritin iron was measured using the ferrous chelator ferrozine as a chromophore. The reaction mixture contained 200 μg ferritin and 500 μM ferrozine in 50 mM phosphate buffer with a pH of 7.0. The reaction was started by the addition of DBME at different concentrations (100–500 μg), and the change in absorbance was measured continuously at 560 nm for 20 min. A cuvette containing ferritin, ferrozine and phosphate buffer but no plant extract was used as the reference solution.
In vivo study Experimental design. Mice were divided into six groups of six mice each. One group was labeled as blank (B) and received normal saline. The other five groups received five intraperitoneal injections of iron-dextran at a dose of 100 mg/kg b.w. (one dose every two days). One iron-dextran group (C) was administered normal saline, and the other four groups were orally treated with either 50 mg/kg b.w. (S50), 100 mg/kg b.w. (S100), 200 mg/kg b.w. (S200) DBME
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or 20 mg/kg b.w. desirox (D) for 21 days beginning on the day following the first irondextran injection. Sample collection and tissue preparation. After treatment, mice were fasted overnight on the 21st day and anesthetized with ethyl ether. Blood was collected by cardiac puncture. Serum from the blood samples was separated using a cooling centrifuge and stored at -80°C until analysis. The liver was washed with ice-cold saline and divided into three parts. One major portion of the liver was cut, weighed and homogenized in 10 volumes of 0.1 M phosphate buffer (pH 7.4) containing 5 mM EDTA and 0.15 M NaCl. The sample was centrifuged at 8,000 g for 30 min at 4°C. The protein concentration in the supernatant was estimated according to Lowry's method using BSA as a standard. The remaining samples were stored at -80°C until further analysis. Another portion of the liver was weighed and digested using an equivolume mixture of sulfuric acid and nitric acid for iron content analysis. A final liver sample was used for histopathological studies. Serum markers and ferritin levels. Alanine amino transferase (ALAT), aspartate amino transferase (ASAT), and bilirubin levels were measured in serum samples using commercial kits from Merck, Mumbai, India. Serum alkaline phosphatase (ALP) was estimated using a kit supplied by Sentinel diagnostics, Italy. The serum ferritin level was measured using an enzymelinked immunosorbent assay (Monobind Inc., USA) according to the manufacturer’s instructions. Antioxidant enzymes. Superoxide dismutase (SOD) [26], catalase (CAT) activity [27], glutathione-S-transferase (GST) [28] and reduced glutathione (GSH) levels [29] were assayed according to previously reported methods. Lipid peroxidation products, protein carbonyl content, hydroxyproline and liver iron content The lipid peroxide levels [30], protein carbonyl content, hydroxyproline content [31] and liver iron levels [32] were measured in samples according to standardized methods. Histopathological analysis. The liver samples were excised, washed with normal saline, and processed separately for histological study. Initially, the material was fixed in 10% buffered neutral formalin for 48 h. The samples were then paraffin-embedded, and sections with a 5-μm thicknesses were stained with hematoxylin and eosin (morphological examination), Perls’ Prussian blue dye (iron content) and Masson’s trichrome stain (liver fibrosis). The stained sections were examined for histopathological changes under a light microscope.
Phytochemical and high performance liquid chromatography (HPLC) analyses of DBME The analysis of resident phytochemicals, including alkaloids, carbohydrates, flavonoids, glycosides, phenols, saponins, tannins, terpenoids, anthraquinones and triterpenoids, in the extract was completed using standard qualitative and quantitative methods as previously described [33,34]. For HPLC analysis, standard stock solutions (10 μg/ml) were prepared in mobile phase for PRME, purpurin, catechin, tannic acid, reserpine, methyl gallate and rutin. All the samples were filtered through a 0.45-μm polytetrafluoroethylene (PTFE) filter (Millipore) to remove any particulate matter. The analysis was performed using a HPLC-Prominence System RF10AXL (Shimadzu Corp.) equipped with a degasser (DGU-20A5), quaternary pump (LC20AT), auto-sampler (SIL-20A) and detectors of reflective index (RID-10A), fluorescence (RF10AXL) and diode array (SPD-M20A). A 20 μl aliquot of each sample and standard was injected and analyzed in triplicate. Gradient elution consecutive mobile phases of acetonitrile and 0.5 mM ammonium acetate in water at a flow rate of 1 ml/min for 65 min through the column (ZIC-HILIC) was maintained at 25°C. The detection was completed at 254 nm.
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WST-1 cytotoxicity assay The human lung fibroblast (WI-38) cell line was purchased from the National Centre for Cell Science (NCCS), India. Cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin G, 50 μg/ml gentamycin sulfate, 100 μg/ml streptomycin and 2.5 μg/ml amphotericin B. The cell line was maintained in a CO2 incubator at 37°C in a humidified atmosphere containing 5% CO2. Cell proliferation and cell viability were quantified using the WST-1 Cell Proliferation Reagent, Roche diagnostics, according to previously described methods [35]. For this experiment with pure compounds (purpurin, catechin, tannic acid, reserpine, methyl gallate and rutin), a 2 mg/ml aqueous solution with 0.2% DMSO is used; in such a way that DMSO concentration in the cell culture media did not exceed 4 x 10–4%, thus being non-toxic to the cells. Briefly, WI-38 cells (1 × 104 cells/well) were treated with DBME, purpurin, catechin, tannic acid, reserpine, methyl gallate or rutin at doses ranging from 0–120 μg/ml for 48 hours in 96-well culture plates. After treatment, 10 μl of the WST-1 cell proliferation reagent was added to each well followed by 2 hours of incubation at 37°C. Cell proliferation and viability were quantified by measuring the absorbance at 460 nm using a microplate ELISA reader MULTISKAN EX (Thermo Electron Corporation, USA).
Statistical analysis All data are reported as the mean ± SD of six measurements. The statistical analysis was performed using KyPlot version 2.0 beta 15 (32 bit) and Origin professional 6.0. Comparisons among groups were assessed with paired t-tests. In all analyses, a p value of
An Antioxidant Extract of the Insectivorous Plant Drosera burmannii Vahl. Alleviates IronInduced Oxidative Stress and Hepatic Injury in Mice Nikhil Baban Ghate☯, Dipankar Chaudhuri☯, Abhishek Das, Sourav Panja, Nripendranath Mandal* Division of Molecular Medicine, Bose Institute, P 1/12, Scheme—VIIM, Kolkata, West Bengal, India ☯ These authors contributed equally to this work. * [email protected]
Abstract
OPEN ACCESS Citation: Ghate NB, Chaudhuri D, Das A, Panja S, Mandal N (2015) An Antioxidant Extract of the Insectivorous Plant Drosera burmannii Vahl. Alleviates Iron-Induced Oxidative Stress and Hepatic Injury in Mice. PLoS ONE 10(5): e0128221. doi:10.1371/journal.pone.0128221 Academic Editor: Krishnendu Acharya, University of Calcutta, INDIA Received: December 18, 2014 Accepted: April 24, 2015 Published: May 26, 2015 Copyright: © 2015 Ghate et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Dr. Abhishek Das is grateful to the Department of Biotechnology (DBT), Govt. of India for the support of Research Associateship.
Free iron typically leads to the formation of excess free radicals, and additional iron deposition in the liver contributes to the oxidative pathologic processes of liver disease. Many pharmacological properties of the insectivorous plant Drosera burmannii Vahl. have been reported in previous studies; however, there is no evidence of its antioxidant or hepatoprotective potential against iron overload. The antioxidant activity of 70% methanolic extract of D. burmannii (DBME) was evaluated. DBME showed excellent DPPH, hydroxyl, hypochlorous, superoxide, singlet oxygen, nitric oxide, peroxynitrite radical and hydrogen peroxide scavenging activity. A substantial iron chelation (IC50 = 40.90 ± 0.31 μg/ml) and supercoiled DNA protection ([P]50 = 50.41 ± 0.55 μg) were observed. DBME also displayed excellent in vivo hepatoprotective activity in iron-overloaded Swiss albino mice compared to the standard desirox treatment. Administration of DBME significantly normalized serum enzyme levels and restored liver antioxidant enzymes levels. DBME lowered the raised levels of liver damage parameters, also reflected from the morphological analysis of the liver sections. DBME also reduced liver iron content by 115.90% which is also seen by Perls’ staining. A phytochemical analysis of DBME confirms the presence of various phytoconstituents, including phenols, flavonoids, carbohydrates, tannins, alkaloids and ascorbic acid. Alkaloids, phenols and flavonoids were abundantly found in DBME. An HPLC analysis of DBME revealed the presence of purpurin, catechin, tannic acid, reserpine, methyl gallate and rutin. Purpurin, tannic acid, methyl gallate and rutin displayed excellent iron chelation but exhibited cytotoxicity toward normal (WI-38) cells; while DBME found to be non-toxic to the normal cells. These findings suggest that the constituents present in DBME contributed to its iron chelation activity. Additional studies are needed to determine if DBME can be used as a treatment for iron overload diseases.
Competing Interests: The authors have declared that no competing interests exist.
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Antioxidant and Hepatoprotective Activity of an Insectivorous Plant
Introduction Free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), play a significant role in the early onset of oxidative stress and are capable of damaging biologically relevant molecules, such as proteins, nucleic acids and plasma membrane lipids [1]. Antioxidants can interrupt the chain reaction cycle (oxidation process) via different mechanisms, such as chelating metals that catalyze the formation of free radicals and scavenging the free radicals. Therefore, antioxidants are vital for the human body due to their ability to combat oxidative damage [2]. Iron is a metal that is needed by all mammalian cells for growth and survival [3]; however, its extreme deposition can increase oxidative stress in the liver and lead to further injuries, such as hepatocellular necrosis [4], inflammation [5], fibrosis [6,7] and cancer [8]. The human body is largely dependent on the liver for the facilitation of many vital biochemical pathways that manage growth, nutrient supply, energy provision, reproduction and defense [9]. Liver damage (hepatotoxicity) caused by iron overload hinders these processes and can result in serious health problems [10]. Iron removal by chelation therapy is an effective life-saving strategy for nearly all the aforementioned iron overload-induced diseases. Several synthetic iron chelating agents, such as deferoxamine, 1,2-dimethyl-3-hydroxypyrid-4-one (deferiprone, L1) and deferasirox, are available for clinical use; however, these drugs possess several undesirable side effects [11,12]. Thus, the scientific community continues to search for a raw material or isolated natural product that can act as an antioxidant and iron chelator without adverse effects. Drosera burmannii Vahl. (family Droseraceae) is an acaulescent insectivorous herb commonly known as sundew that belongs to one of the largest genera of carnivorous plants, with over 105 species. This herb is distributed throughout the Indian subcontinent as well as China, Australia and West Africa, and it is reported to possess rubefacient properties [13]. Moreover, antifertility [14], anticonvulsant [15] and antitumor activities in mice [16] were reported in the alcohol and aqueous extracts of D. burmannii. Different species of Drosera contain several medicinally active compounds, including 1,4-naphthoquinones such as plumbagin [17], hydroplumbagin glucoside [18], flavonoids (kaempferol, myricetin, quercetin and hyperoside) [19] and rossoliside (7-methyl-hydrojuglone-4-glucoside). Previous literature reports that several species of Drosera are used in various traditional and homeopathic treatments. Some of the species of this family are used for the treatment of cough in Asia. This plant was also listed in French Pharmacopoeia in 1965 for the treatment of chronic bronchitis, asthma and Whooping cough [20]. The present study aimed to assess the in vitro antioxidant and in vivo hepatoprotective properties against iron-overload-induced liver toxicity in Swiss albino mice.
Materials and Methods Chemicals 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was procured from Roche diagnostics, Mannheim, Germany. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was obtained from Fluka, Buchs, Switzerland. Potassium persulfate (K2S2O8), 2-deoxy2-ribose, ethylene diammine tetraacetic acid (EDTA), ascorbic acid, trichloroacetic acid (TCA), mannitol, nitro blue tetrazolium (NBT), reduced nicotinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS), sodium nitroprusside (SNP), 1,10-phenanthroline, sulphanilamide, N-(1-Naphthyl)ethylenediamine dihydrochloride (NED), L-histidine, lipoic acid, sodium pyruvate, quercetin, ferrozine glutathione reduced, bathophenanthrolinesulfonate disodium salt and 5,50 -dithiobis-2-nitrobenzoic acid (DTNB) were obtained from Sisco Research Laboratories Pvt. Ltd, Mumbai, India. HPLC grade acetonitrile, ammonium acetate,
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hydrogen peroxide, potassium hexacyanoferrate, Folin-ciocalteu reagent, sodium carbonate, mercuric chloride, potassium iodide, anthrone, vanillin, thiourea, 2,4-dinitrophenylhydrazine (DNPH), sodium hypochlorite, aluminum chloride, xylenol orange, butylated hydroxyltoluene (BHT), N,N- dimethyl-4-nitrosoaniline ammonium iron (II) sulfatehexahydrate [(NH4)2Fe (SO4)26H2O], 1-chloro-2,4-dinitrobenzene (CDNB), chloramine-T, hydroxylamine hydrochloride and Dimethyl-4-aminobenzaldehyde were procured from Merck, Mumbai, India. 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferritin, methyl gallate, tannic acid, rutin, gallic acid, (+) catechin and curcumin were obtained from MP Biomedicals, France. Catalase, reserpine, streptomycin sulfate and sodium bicarbonate were obtained from HiMedia Laboratories Pvt. Ltd, Mumbai, India. Evans blue was purchased from BDH, England. D-glucose was procured from Qualigens Fine Chemicals, Mumbai. Diethylenetriaminepentaacetic acid (DTPA) was obtained from Spectrochem Pvt. Ltd, Mumbai, India. Thiobarbituric acid (TBA) was obtained from Loba Chemie, Mumbai, India. Iron-dextran and guanidine hydrochloride was purchased from Sigma-Aldrich, USA. The standard oral iron chelating drug, desirox, was obtained from Cipla Ltd., Kolkata, India.
Ethics A sample of the insectivorous plant Drosera burmannii Vahl. was collected in January 2014 from public areas adjoining villages in the Bankura district in the state of West Bengal, India. These areas are not within a National Park/Reserve Forest/Govt. protected area, and only verbal permission from village headmen was obtained before collection. The conservation status of this insectivorous plant was classified using the International Union for Conservation of Nature (IUCN) World Conservation Union guidelines (1994). The status was ‘LC; Least concern’ as per IUCN red list criteria. The material collected for this study was sampled on a very limited scale and therefore had negligible effects on broader ecosystem functioning. All animal experiments (Swiss albino mice) were performed in strict accordance with the recommendations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India (Bose Institute Registration. No. 95/1999/CPCSEA). The protocol was approved by the Institutional Animal Ethics Committee, Bose Institute. All surgery was performed under ethyl ether anesthesia, and all efforts were made to minimize suffering.
Animals Male Swiss albino mice (20 ± 2 g) were purchased from Chittaranjan National Cancer Institute (CNCI), Kolkata, India and were maintained under a constant 12-h dark/light cycle at an environmental temperature of 22 ± 2°C. Mice were provided a normal laboratory pellet diet and water ad libitum. The condition of the animals were monitored every 6-h after the treatment and there were no unintended animal deaths during the experimental procedures.
Plant extract preparation D. burmannii was authenticated by the Botanical Survey of India, Kolkata, India. Samples were sorted, cleaned of substratum and shadow dried for extraction. The dried sample (100 g) was then powdered and stirred using a magnetic stirrer with 70% methanol in water (1000 ml) for 15 hours. The mixture was then centrifuged at 2,850 g, and the supernatant was decanted. The process was repeated by adding more solvent to the precipitated pellet. The supernatants from the two phases were mixed, concentrated in a rotary evaporator at 40°C, lyophilized and labeled as DBME. The dried extract was stored at -20°C until use.
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In vitro study Total antioxidant activity and reducing power. The total antioxidant capacity of DBME was evaluated by an ABTS•+ radical cation decolorization assay in comparison to a trolox standard [21] and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay [22]. The reducing power of DBME was determined using a previously described method [21]. Reactive oxygen species (ROS) scavenging activity. The ROS scavenging ability of DBME was determined using multiple stable ROS radical scavenging assays, such as hydroxyl, superoxide, hypochlorous radical, singlet oxygen and hydrogen peroxide assays by standard procedures [21]. Reactive nitrogen species (RNS) scavenging activity. The RNS scavenging activity of DBME was determined using nitric oxide and peroxynitrite radical scavenging assays [21]. Iron chelation assay. The Fe2+ chelating activity of DBME, catechin, methyl gallate, purpurin, reserpine, rutin and tannic acid was evaluated according to standard methods [23]. HEPES buffer (20 mM, pH 7.2), DBME (0–120 μg/ml) and a positive control EDTA (0–20 μg/ ml) were separately added to a 12.5 μM ferrous sulfate solution, and 75 μM ferrozine was added to start the reaction. The mixture was shaken vigorously and left standing for 20 min at room temperature. Next, the absorbance was measured at 562 nm. All tests were performed six times. DNA protection assay. DNA protection was studied using supercoiled pUC18 plasmid DNA according to previously described methods [24] with minor modifications. In HEPES buffer, (pH 7.2, 13 mM), a FeSO4 solution (15 μM), DBME of varying doses (0–100 μg), DNA (1 μg) and water were added to produce an initial reaction mixture. Next, an H2O2 solution (0.0125 mM) was added to start the reaction. After 10 min, the reaction was stopped by adding desferal (0.2 mM) followed by a loading buffer. Each reaction mixture (20 μl) was loaded in a 1% agarose gel. After migration, the gel was stained with ethidium bromide and visualized with a UV transilluminator. The DNA bands were quantified using densitometry, and the following formulas were used to calculate the protection percentage: % SC ¼ ½1:4 X SC=ðOC þ ð1:4 X SCÞÞ X 100 and % protection ¼ 100 X ½ðcontrol SC chelator SCÞ=ðcontrol SC no chelator SCÞ 1; where SC = supercoiled; OC = open circular; 1.4 = correction factor. The ability of the plant extract to protect DNA supercoils can be expressed as the [P]50 value, which is defined by the concentration of sample required for 50% protection. Ferritin iron release assay. This assay was performed according to a previously described method [25]. The release of ferritin iron was measured using the ferrous chelator ferrozine as a chromophore. The reaction mixture contained 200 μg ferritin and 500 μM ferrozine in 50 mM phosphate buffer with a pH of 7.0. The reaction was started by the addition of DBME at different concentrations (100–500 μg), and the change in absorbance was measured continuously at 560 nm for 20 min. A cuvette containing ferritin, ferrozine and phosphate buffer but no plant extract was used as the reference solution.
In vivo study Experimental design. Mice were divided into six groups of six mice each. One group was labeled as blank (B) and received normal saline. The other five groups received five intraperitoneal injections of iron-dextran at a dose of 100 mg/kg b.w. (one dose every two days). One iron-dextran group (C) was administered normal saline, and the other four groups were orally treated with either 50 mg/kg b.w. (S50), 100 mg/kg b.w. (S100), 200 mg/kg b.w. (S200) DBME
PLOS ONE | DOI:10.1371/journal.pone.0128221 May 26, 2015
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or 20 mg/kg b.w. desirox (D) for 21 days beginning on the day following the first irondextran injection. Sample collection and tissue preparation. After treatment, mice were fasted overnight on the 21st day and anesthetized with ethyl ether. Blood was collected by cardiac puncture. Serum from the blood samples was separated using a cooling centrifuge and stored at -80°C until analysis. The liver was washed with ice-cold saline and divided into three parts. One major portion of the liver was cut, weighed and homogenized in 10 volumes of 0.1 M phosphate buffer (pH 7.4) containing 5 mM EDTA and 0.15 M NaCl. The sample was centrifuged at 8,000 g for 30 min at 4°C. The protein concentration in the supernatant was estimated according to Lowry's method using BSA as a standard. The remaining samples were stored at -80°C until further analysis. Another portion of the liver was weighed and digested using an equivolume mixture of sulfuric acid and nitric acid for iron content analysis. A final liver sample was used for histopathological studies. Serum markers and ferritin levels. Alanine amino transferase (ALAT), aspartate amino transferase (ASAT), and bilirubin levels were measured in serum samples using commercial kits from Merck, Mumbai, India. Serum alkaline phosphatase (ALP) was estimated using a kit supplied by Sentinel diagnostics, Italy. The serum ferritin level was measured using an enzymelinked immunosorbent assay (Monobind Inc., USA) according to the manufacturer’s instructions. Antioxidant enzymes. Superoxide dismutase (SOD) [26], catalase (CAT) activity [27], glutathione-S-transferase (GST) [28] and reduced glutathione (GSH) levels [29] were assayed according to previously reported methods. Lipid peroxidation products, protein carbonyl content, hydroxyproline and liver iron content The lipid peroxide levels [30], protein carbonyl content, hydroxyproline content [31] and liver iron levels [32] were measured in samples according to standardized methods. Histopathological analysis. The liver samples were excised, washed with normal saline, and processed separately for histological study. Initially, the material was fixed in 10% buffered neutral formalin for 48 h. The samples were then paraffin-embedded, and sections with a 5-μm thicknesses were stained with hematoxylin and eosin (morphological examination), Perls’ Prussian blue dye (iron content) and Masson’s trichrome stain (liver fibrosis). The stained sections were examined for histopathological changes under a light microscope.
Phytochemical and high performance liquid chromatography (HPLC) analyses of DBME The analysis of resident phytochemicals, including alkaloids, carbohydrates, flavonoids, glycosides, phenols, saponins, tannins, terpenoids, anthraquinones and triterpenoids, in the extract was completed using standard qualitative and quantitative methods as previously described [33,34]. For HPLC analysis, standard stock solutions (10 μg/ml) were prepared in mobile phase for PRME, purpurin, catechin, tannic acid, reserpine, methyl gallate and rutin. All the samples were filtered through a 0.45-μm polytetrafluoroethylene (PTFE) filter (Millipore) to remove any particulate matter. The analysis was performed using a HPLC-Prominence System RF10AXL (Shimadzu Corp.) equipped with a degasser (DGU-20A5), quaternary pump (LC20AT), auto-sampler (SIL-20A) and detectors of reflective index (RID-10A), fluorescence (RF10AXL) and diode array (SPD-M20A). A 20 μl aliquot of each sample and standard was injected and analyzed in triplicate. Gradient elution consecutive mobile phases of acetonitrile and 0.5 mM ammonium acetate in water at a flow rate of 1 ml/min for 65 min through the column (ZIC-HILIC) was maintained at 25°C. The detection was completed at 254 nm.
PLOS ONE | DOI:10.1371/journal.pone.0128221 May 26, 2015
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WST-1 cytotoxicity assay The human lung fibroblast (WI-38) cell line was purchased from the National Centre for Cell Science (NCCS), India. Cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin G, 50 μg/ml gentamycin sulfate, 100 μg/ml streptomycin and 2.5 μg/ml amphotericin B. The cell line was maintained in a CO2 incubator at 37°C in a humidified atmosphere containing 5% CO2. Cell proliferation and cell viability were quantified using the WST-1 Cell Proliferation Reagent, Roche diagnostics, according to previously described methods [35]. For this experiment with pure compounds (purpurin, catechin, tannic acid, reserpine, methyl gallate and rutin), a 2 mg/ml aqueous solution with 0.2% DMSO is used; in such a way that DMSO concentration in the cell culture media did not exceed 4 x 10–4%, thus being non-toxic to the cells. Briefly, WI-38 cells (1 × 104 cells/well) were treated with DBME, purpurin, catechin, tannic acid, reserpine, methyl gallate or rutin at doses ranging from 0–120 μg/ml for 48 hours in 96-well culture plates. After treatment, 10 μl of the WST-1 cell proliferation reagent was added to each well followed by 2 hours of incubation at 37°C. Cell proliferation and viability were quantified by measuring the absorbance at 460 nm using a microplate ELISA reader MULTISKAN EX (Thermo Electron Corporation, USA).
Statistical analysis All data are reported as the mean ± SD of six measurements. The statistical analysis was performed using KyPlot version 2.0 beta 15 (32 bit) and Origin professional 6.0. Comparisons among groups were assessed with paired t-tests. In all analyses, a p value of
