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Role of phenolics from Spondias pinnata bark in amelioration of iron overload induced hepatic damage in Swiss albino mice
© The Author(s). 2016
Received: 10 March 2016
Accepted: 14 July 2016
Published: 26 July 2016
Crude Spondias pinnata bark extract was previously assessed for its antioxidant, anticancer and iron chelating potentials. The isolated compounds gallic acid (GA) and methyl gallate (MG) were evaluated for their curative potential against iron overload-induced liver fibrosis and hepatocellular damage.
In vitro iron chelation property and in vivo ameliorating potential from iron overload induced liver toxicity of GA and MG was assessed by different biochemical assays and histopathological studies.
MG and GA demonstrated excellent reducing power activities but iron chelation potential of MG is better than GA. Oral MG treatment in mice displayed excellent efficacy (better than GA) to significantly restore the levels of liver antioxidants, serum markers and cellular reactive oxygen species in a dose-dependent fashion. Apart from these, MG exceptionally prevented lipid peroxidation and protein oxidation whereas GA demonstrated better activity to reduce collagen content, thereby strengthening its position as an efficient drug against hepatic damage/fibrosis, which was further supported by histopathological studies. Alongside, MG efficiently eliminated the cause of liver damage, i.e., excess iron, by chelating free iron and reducing the ferritin-bound iron.
The present study confirmed the curative effect of GA and MG against iron overload hepatic damage via their potent antioxidant and iron-chelating potential.
An imbalance in pro-oxidant/antioxidant homeostasis eventually gives rise to the physical condition known as “oxidative stress”. An irregular surge in oxidants such as reactive species of oxygen (ROS) and nitrogen (RNS) is generated endogenously through mitochondrial respiration as well as through exogenous oxidizing agents including ionizing radiation, heavy metals, and hypoxia. These oxidants come in contact with different biological molecules such as proteins, lipids and DNA, thereby disrupting them and causing maladies such as cancer, atherosclerosis, cardiovascular disease, liver injury, aging and inflammatory diseases . Previous research has indicated that redox-active metals such as iron also play a major role in ROS over production, mainly through highly reactive hydroxyl radicals by undergoing redox cycling . Conversely, liver executes the normal metabolic balance of the body as well as biotransformation, detoxification and excretion of several toxic by-products. Increasing evidence has indicated that oxidative stress and its associated damage designate a common association between different forms of chronic liver injuries and hepatic fibrosis  due to iron overload . Thus, any substance that is capable of providing protection against metal toxicity by trapping free radicals or by chelating metal ions that would consequently terminate the chain reaction of free radicals acts as an antioxidant . In addition to endogenous antioxidant systems, frequent consumption of food that is rich in natural antioxidants also exhibits increased resistance to oxidative stress by altering the redox environment and is associated with a lower risk of many oxidative stress-related diseases.
Several phenolics and flavonoids from natural resources effectively scavenge different free radicals through their relevant iron chelating capabilities. Previous reports suggested that these types of compounds could also be used to treat iron-induced liver toxicity . Crude Spondias pinnata (Linn. f.) Kurz (Fam. Anacardiaceae) extract was previously studied for its in vitro and in vivo antioxidant and iron chelating potential, which also demonstrated the presence of significant amounts of phenolic and flavonoid compounds [7, 8]. These previous studies prompted us to isolate phenolic compounds from Spondias pinnata bark and evaluate their ameliorating effect on iron overload-induced hepatotoxicity and hepatic fibrosis in mice.
Bathophenanthroline sulfonate disodium salt, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) and N-(1-Naphthyl) ethylenediamine dihydrochloride (NED) were procured from Sisco Research Laboratories Pvt. Ltd, India. 1-chloro-2,4-dinitrobenzene (CDNB), Dimethyl-4-aminobenzaldehyde, and N,N- dimethyl-4-nitrosoaniline ammonium iron (II) sulfate hexahydrate ((NH4)2Fe(SO4)2, 6H2O) were obtained from Merck, Mumbai, India. Guanidine hydrochloride and Iron-dextran was purchased from Sigma-Aldrich, USA. Cipla Ltd., India provided Desirox (Generic name-Deferasirox). All of the other used reagents were of molecular biology grade and were obtained from reputable suppliers.
In vivo experiments were performed abiding by the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India with due approval from the Institutional Animal Ethics Committee, Bose Institute (Registration. No. 95/1999/CPCSEA). Male Swiss albino mice (20 ± 2 g) were obtained from the Chittaranjan National Cancer Institute (CNCI), Kolkata, India and were acclimated under a constant 12 h light/dark cycle at 22 ± 2 °C. The animals were fed on general laboratory diet and water ad libitum. Experimental animals were taken care every 6 h during the treatment period and it was observed that there was no unwanted animal death. All surgeries were done using ethyl ether as anesthetic (by inhalation in a ethyl ether saturated chamber inside a fume hood), taking utmost care to reduce suffering.
S. pinnata bark was collected from the villages of Bankura district, West Bengal, India and the plant was authenticated by Dr. Jayaram Hazra, Director, Central Research Institute of Ayurveda (CRIA), Kolkata, India. The herbarium was submitted at CRIA, Kolkata with an accession no of CRHS 111/08.
Extraction and isolation of phyto-compounds
Different compounds were isolated following the method of Chaudhuri, et al. . Briefly, the S. pinnata stem bark was cut into small pieces, dried, ground into powder, and extracted with 70 % methanol and water. The lyophilized extract was re-extracted successively with hexane, chloroform, ethyl acetate and water. All of the fractions were concentrated in reduced pressure, and the ethyl acetate fraction was further purified through silica gel column chromatography. Dichloromethane and methanol elution yielded four compounds namely SPE1, SPE2, SPE3 and SPE4. Structures of the bioactive compounds were analysed using different spectroscopic methods such as EIMS (JEOL JMS-700, Germany), FT-IR spectra recorded in KBr (Perkin Elmer, USA) and different nuclear magnetic resonance (NMR) experiments including 1H and 13C with a Bruker-500 MHz NMR Spectrometer (Germany).
In vitro reducing power and iron chelation potential of the isolated compounds
Fe3+-reducing power of the compounds was determined by a standard method  where ascorbic acid was used as standard. Various concentrations (0–1.0 mg/ml) of the compounds were tested and their absorbance was measured at 700 nm against an appropriate blank. The Fe2+ chelating ability was determined as described earlier, and the result was expressed as inhibition percentage . Briefly, in HEPES buffer (20 m mol/l, pH 7.2) medium, test samples (0–120 μg/ml) and positive control EDTA (0–20 μg/ml) were separately added to a 12.5 μ mol/l ferrous sulfate solution and 75 μ mol/l ferrozine was added to start the reaction. After rapid vortexing the mixture was left at room temperature for 20 min and absorbance was taken at 562 nm (OD). The percentage of inhibition of ferrozine-Fe2+ complex formation was determined by the formula- % of inhibition = [(Control OD-Sample OD)/Control OD] × 100. All tests were performed six times.
In vivo hepato-ameliorating activity
Experimental design and tissue preparation
In total, nine groups of mice were randomly prepared consisting of six mice per group. Among them, one group received normal saline only, which was labeled as a blank (B); however, other groups were intoxicated with five doses of 100 mg/kg b.w. iron-dextran (one dose every alternative days) by intraperitoneal injection (ip). Of the groups, one iron-dextran group (C) was treated orally with only saline, and other groups were treated orally with 2 mg/kg b.w. gallic acid (GA) (R2), 2 mg/kg b.w. methyl gallate (MG) (S2), 4 mg/kg b.w. GA (R4), 4 mg/kg b.w. MG (S4), 8 mg/kg b.w. GA (R8), 8 mg/kg b.w. MG (S8) test samples and 20 mg/kg b.w. desirox (D) for 21 consecutive days starting the day following the first iron-dextran injection. All experimental animals were sacrificed on 22nd day under mild anesthesia (ethyl ether), and cardiac puncture was performed to collect blood and serum was separated and stored at −80 °C. After collecting the blood, the liver was quickly excised, cleaned thoroughly with cold phosphate buffer saline (PBS) to remove the remaining blood and cut into three sections. The major liver portion was dissected and homogenized using 10 volumes of 0.1 M phosphate buffer (pH 7.4) supplemented with 0.15 M NaCl and 5 mM EDTA and centrifuged for 30 min at 8000 g in the cold. The clear homogenate (supernatant) was collected and the protein concentration was quantified by Folin-Lowry method , where BSA was used as a standard; the remaining supernatant was then stored at −80 °C. Second liver fragment was treated with a mixture of nitric acid and sulfuric acid (1:1) to analyze the iron content. The last portion was processed for histopathological examinations.
Serum ferritin and liver iron levels
Manufacturer’s instructions were followed to quantify serum ferritin levels using an ELISA kit from Monobind Inc., USA. Liver iron content was quantified using a previously reported method . Briefly, samples were mixed with bathophenanthroline sulfonate and incubated at 37 °C for 30 min, and absorbance was recorded using a spectrophotometer (Shimadzu, UV-2401PC, Japan) at 535 nm.
In vitro ferritin iron release
Iron reduction and release were determined using ferrozine, a spectrophotometric reagent for iron, as previously described . Briefly, the reaction was initiated by adding different concentrations (100–500 μg/ml) of test compounds in 50 mM phosphate buffer (pH 7.0) containing 200 μg ferritin and 500 μM ferrozine, and the absorbance change was measured for 20 min at 560 nm. The reaction mixture excluding the test samples was used as a reference.
Aspartate amino transferase (ASAT), alanine amino transferase (ALAT), and bilirubin levels in serum were evaluated using commonly available kits from Merck, India. Similarly, alkaline phosphatase (ALP) levels were measured by a kit from Sentinel diagnostics, Italy.
Measurement of ROS levels in liver, spleen homogenate and serum
ROS levels in liver and spleen tissue homogenate and serum were estimated using 2,7-dichlorofluorescein diacetate (DCFDA) following the method of Rashid et al.  with slight modifications. Briefly, the sample containing 50 μg protein was mixed with 1 ml assay buffer (130 mM KCL, 5 mM MgCl2, 20 mM NaH2PO4, 20 mM Tris HCl, 30 mM Glucose and 10 μM DCFDA) and incubated at 37 °C for 15 min in the dark. DCF formation was recorded at the excitation wavelength of 488 nm and emission wavelength of 523 nm for 2 min using a fluorescence spectrometer (Hitachi, Model F4500) that had been equipped with a FITC filter.
Evaluation of liver damage and fibrosis
Thiobarbituric acid reactive substances (TBARS) quantities were measured to evaluate the levels of lipid peroxidation . Protein oxidation levels were resolved by estimating protein carbonyl contents . Collagen content, an important marker of liver fibrosis, was evaluated using previously described methods . Collagen content in each sample was determined by multiplying 7.69 by overall hydroxyproline content .
Excised liver samples were cleaned with saline and fixed for two days in 10 % buffered neutral formalin. Sections (5 μm thick) were paraffin-embedded and stained with hematoxylin and eosin (morphological examination), Perls’ Prussian blue dye (iron content) and Masson’s trichrome stain (liver fibrosis). Stained sections were checked microscopically for histopathological changes.
All of the data were reported as the mean ± SD of six measurements. Statistical analysis was performed using KyPlot version 2.0 beta 15 (32 bit) and Microsoft excel 2010. The relationships between the groups were evaluated using a paired t-test for in vitro study and one way ANOVA followed by Tukey's post hoc tests for in vivo study, and a p value of <0.05 was considered to be statistically significant.
In vitro iron chelation and reducing power potential
In vivo studies
Serum ferritin and liver iron levels
Iron release from ferritin
The effect of GA and MG on serum marker enzymes (ALAT, ASAT and ALP) and Bilirubin in iron overloaded mice
14.42 ± 1.91
14.42 ± 1.91
62.46 ± 6.32
62.46 ± 6.32
84.62 ± 3.48
84.62 ± 3.48
1.58 ± 0.10
1.58 ± 0.10
64.60 ± 4.32 X3
64.60 ± 4.32 X3
234.91 ± 8.30 X3
234.91 ± 8.30 X3
252.63 ± 4.92 X3
252.63 ± 4.92 X3
3.15 ± 0.18 X3
3.15 ± 0.18 X3
2 mg/kg b.w
42.96 ± 1.16 X3Y3
36.93 ± 0.93 X3Y3
145.45 ± 5.89 X3Y3
141.51 ± 11.57X3Y3
181.90 ± 4.95 X3Y3
173.41 ± 2.93 X3Y3
2.88 ± 0.39 X3
3.04 ± 0.25 X3
4 mg/kg b.w
32.17 ± 2.16 X3Y3
29.09 ± 0.59 X3Y3
114.78 ± 8.20 X3Y3
110.28 ± 6.55 X3Y3
145.10 ± 6.46 X3Y3
132.32 ± 3.56 X3Y3
2.47 ± 0.24 X3Y3
2.11 ± 0.16 X3Y3
8 mg/kg b.w
23.22 ± 0.95 X3Y3
20.25 ± 0.90 X2Y3
83.56 ± 4.86 X3Y3
77.93 ± 13.48 X1Y3
118.69 ± 3.79 X3Y3
103.34 ± 5.88 X3Y3
2.10 ± 0.12 X3Y3
1.94 ± 0.13 X3Y3
19.85 ± 0.79 X3Y3
19.85 ± 0.79 X3Y3
88.06 ± 10.37 X3Y3
88.06 ± 10.37 X3Y3
114.97 ± 6.13 X3Y3
114.97 ± 6.13 X3Y3
1.72 ± 0.14 Y3
1.72 ± 0.14 Y3
Biochemical parameters of liver damage
Free radicals and iron metal are essential entities of aerobic life and modulate various physiological functions . In iron-overloaded liver, iron reacts with the cellular hydrogen peroxide to generate hydroxyl radical (Fenton reaction) which in turn initiate the propagation of various ROS. This situation leads to oxidative stress which damage in liver tissue mostly via lipid peroxidation of biological membranes . However, the preventive system of our body is inadequate to handle excess free radicals; therefore, external antioxidant iron chelator supplements are essential to maintain healthy physiological conditions. An effective remedial strategy should act in a dual character by decreasing the oxidation rate; one method is sequestering and chelating the stored iron in cells , and the other is a radical trap (i.e., antioxidant activity). Though the development of some synthetic antioxidants and iron chelating drugs has thrived recently, they are not yet widely accepted as therapeutic agents due to several side effects and disadvantages. Thus, there is a growing demand to develop more effective and safe therapeutic approaches to chelate catalytically active cytosolic iron  and concurrently protect cells from excess free radicals. Several bio resources have already been reported as natural antioxidants with iron chelating potentials [26, 27].
Results of in vitro iron chelation and reducing power property of the isolated compound from ethyl acetate fraction suggest that on SPE3 and SPE4 do possessed both the activities where other two compounds failed to show any activity. So, the structure of SPE3 and SPE4 was elucidated and found to be gallic acid (GA) and methyl gallate (MG). MG exhibited better iron chelation than GA although their basic structure is alike. So, this iron chelation result further supported the findings of Yang et al.  who suggested that Fe(III) in Hepes buffer, pH 7.4 interacted with three molecules of MG and produced a Fe(III)–MG3 complex with a stable octahedral geometry (binding constant- K is in the range of 1 × 1034/M to 1 × 1036/M) and chelate-free iron better than GA.
Both the compounds, GA and MG have demonstrated excellent in vitro iron chelation anf reducing power potency, a study on the in vivo ameliorating effect of GA and MG in iron overloaded liver toxicity and liver fibrosis in mice was constructed. Hemochromatosis was created by intraperitoneal injection of iron-dextran. This process will not hamper intestinal iron absorption by the test compounds, which ultimately leads to iron overload in liver and serum .
The excess iron stored in the liver as ferritin or hemosiderin . Thus, most procedures seek to measure liver iron levels for diagnosis of iron overload disease. In contrast, ferritin, a ubiquitous intracellular iron-binding protein, generally stores iron in a non-toxic ferric form and releases it in a controlled fashion whenever needed . Serum ferritin is a key marker that was developed as a consequence of iron overload-induced hepatic toxicity, as blood ferritin content indirectly reveals the amount of hepatic iron content. The decrease in hepatic iron deposition after MG treatment justified its in vitro iron-chelating effectiveness.
In hepatocytes, ferritin stores excess iron in the ferric state. To overcome iron overload, various readily available iron chelator drugs are administered, but many of them struggle with a narrow binding capacity for ferric iron (Fe3+). Thus, reducing agents such as ascorbic acid need to be administered as supplements to upsurge the accessibility of stored iron to chelators . From the result it was found that both MG and GA effectively release iron from ferritin which also nicely correlated with their reducing power capacity. GA possessed a greater correlation coefficient (R2 = 0.9674) than MG (R2 = 0.7872). This also validated the superior reducing power activity of GA over MG.
Living cells are equipped with an array of endogenous antioxidant enzymes such as GST, SOD, CAT and the small compound GSH, which are first line of defenses against excessive free radicals. Evaluating the levels of these antioxidant enzymes is a proper indirect way to assess pro-oxidant-antioxidant combat in tissues . SOD destroys superoxide free radicals by converting them into H2O2, whereas catalase further decomposes excess H2O2 to H2O and O2. Catalase efficiency is so commendable that it cannot be saturated by any concentration of H2O2 . The cellular GSH system is probably the most important cellular defense mechanism, which not only acts as a ROS scavenger but also regulates the intracellular redox state . The results suggested that MG restore the antioxidant enzymes much better than GA and the activity is almost equal to standard drug Desirox (20 mg/kg.b.w) but in lower concentration (8 mg/kg.b.w).
The previous phenomenon can also be further supported by the obtained ROS levels in liver spleen homogenates and serum in iron overloaded condition as well as after treatment. Surprisingly, GA exhibited much better activity than MG and desirox, which also supported its excellent antioxidant and free radical scavenging potentials. On the other hand, MG exhibited better ROS level reduction in serum of the iron overloaded mice. It is speculated that MG, which is an excellent iron chelator, assists albumin to further chelate excess iron, thereby lowering serum ROS levels. Generally, albumin represents the predominant antioxidant in plasma, which is exposed to continuous oxidative stress . Albumin concentrations increase in inflammation, and the antioxidant activities of albumin result from its ligand-binding capacities of ROS-producing metal such as copper and iron .
Levels of serum enzyme are checked in the clinical diagnosis to determine the condition of various diseases and tissue injury [38, 39]. These enzymes are predominantly found in the hepatic cell and liver damage due to excess iron leads to the release of these intracellular enzymes into the blood  as evident by increased serum parameters (Table 1). The administration of the test compounds has reduced the increased level of the serum marker enzymes almost similar to the standard desirox. This result indicated towards their healing capabilities of hepatic parenchyma and regeneration of hepatocyte as well as its functional efficiency . Hence, MG not only helped to overcome oxidative stress but also possessed better hepato-amelioration activity compared with GA.
Cell membrane lipid peroxidation is the most detrimental result of iron-induced oxidative stress mainly via hydroxyl radicals . Lipid peroxidation (LPO) and the resulting end products such as malondialdehyde (MDA) act as an important factors of liver fibrosis by activating hepatic stellate cells, resulting in increased pro-collagen α1 (I) gene expression [43–45]. This relationship between lipid peroxidation and hepatic fibrosis is well established in a variety of liver diseases including hemochromatosis, alcohol-induced liver injury and chronic hepatitis C . Subsequent to hepatic cellular damage, collagen production predominates over hepatocellular regeneration as an immediate healing response, thereby occupying the injured areas instead of destroyed hepatocytes. Collagen content is therefore considered to be a major marker of liver fibrosis and hepatotoxicity . Iron-induced liver pathogenicity also leads to the oxidation of various important structural and functional proteins and forms protein carbonyls. Thus, it serves as a marker of oxidative stress and leads to the onset/development of various diseases including cystic fibrosis and ulcerative colitis . The chelation of excess iron by MG and GA further strengthen the position of GA and MG in significantly overcoming hepatic damage/fibrosis in iron intoxicated mice, indicating the hepato-ameliorating potency of the compounds.
A liver biopsy is considered to be the gold-standard method for assessment of the degree of inflammation and fibrosis alongside other biochemical tests. The liver sections stained with hematoxylin and eosin exhibited various degrees of inflammation, necrosis as well as cell wall degeneration in iron overloaded condition, but dose dependent treatment reduced the tissue damage that also supported the result of the restoration of serum enzyme and antioxidant enzyme levels. Perls’ Prussian blue staining revealed the visual confirmation of iron chelation property of the test compounds along with the liver iron content and serum ferritin content. On the other hand, Masson’s trichrome staining disclosed the reduction of liver fibrosis as the collagen content gradually decreased with the increasing doses of GA and MG, which also confirmed the anti-fibrotic effect of the fraction along with the test for collagen content. Overall results in histopathological studies signifying in situ evidence of ameliorating the effect of the iron overload-induced liver toxicity.
Excess iron plays a critical role in oxidative stress-related hepato-toxicity and liver fibrosis, which is profoundly supported by the results of the present study. Among the isolated compounds from S. pinnata bark, MG exhibited exceptional amelioration potential for hepatotoxicity mainly due to its iron chelation capacity. In contrast, GA failed to demonstrate brilliant iron chelation activity in vitro, but it displayed a pleasing amendment of oxidative stress and liver damage as it neutralized excess generated free radicals due to iron overload before they could attack any bio-entities. Thus, these two compounds act in different pathways to achieve the same goal. Although the isolated compounds showed similar effect as desirox, they proved the rationale behind the activity of the S. pinnata bark extract, which was already proven as better and safer drug than standard Desirox. These findings also suggest the beneficiary role of GA and MG in the pathological sequence of iron-overload-linked liver disease and fibrosis.
ALAT, alanine aminotransferase; ALP, alkaline phosphatase; ANOVA, analysis of variance; ASAT, aspartate aminotransferase; b.w., body weight; BSA, bovine serum albumin; CAT, catalase; CDNB, 1-chloro-2,4-dinitrobenzene; CNCI, Chittaranjan National Cancer Institute; CPCSEA, Committee for the Purpose of Control and Supervision of Experiments on Animals; CRIA, Central Research Institute of Ayurveda; DCFDA, 2,7-dichlorofluorescein diacetate; DTNB, 5,5′-dithiobis-2-nitrobenzoic acid; EDTA, ethylenediamine tetraacetic acid; EIMS, electron ionization mass spectrometry; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; FT-IR, fourier transform infrared spectroscopy; g, gram; GA, gallic acid; GSH, reduced glutathione; GST, glutathione-S-transferase; HPLC, high performance liquid chromatography; i.p, intraperitoneal; kg, kilogram; M, molar; MDA, malondialdehyde; MG, methyl gallate; mg, milligram; ml, milliliter; mM, Millimolar; NED, N-(1-Naphthyl) ethylenediamine dihydrochloride; NMR, nuclear magnetic resonance; PBS, phosphate buffer saline; RNS, reactive nitrogen species; ROS, reactive oxygen species; SD, standard deviation; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances
The authors would like to acknowledge Mr. Ranjit K. Das and Mr. Pradip K. Mallick for their technical assistance.
This study was conducted with the financial support from Council of Scientific and Industrial Research (CSIR), Govt. of India [Grant No: 27(210)/09-EMR-II Dated: 15.10.2009].
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
Conceived and designed the experiments: NM. Performed the experiments: DC, NBG, SP. Analyzed the data: DC. Contributed reagents/materials/analysis tools: NM. Wrote the paper: DC, NBG, NM. It is also confirm that all the authors have read and approve of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
The manuscript does not contain any individual person’s data in any form. So, this information is not relevant.
Ethics approval and consent to participate
Manuscripts dose not deals with the human participants, human data or human tissue. So, this information is not relevant.
In vivo experiments were performed abiding by the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Govt. of India with due approval from the Institutional Animal Ethics Committee, Bose Institute (Registration. No. 95/1999/CPCSEA).
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