Hepatoprotective Potential of Caesalpinia crista against Iron-Overload-Induced Liver Toxicity in Mice
The present study was carried out to evaluate the ameliorating effect of Caesalpinia crista Linn. (CCME) extract on iron-overload-induced liver injury. Iron overload was induced by intraperitoneal administration of iron dextran into mice. CCME attenuated the percentage increase in liver iron and serum ferritin levels when compared to control group. CCME also showed a dose-dependent inhibition of lipid peroxidation, protein oxidation, and liver fibrosis. The serum enzyme markers were found to be less, whereas enhanced levels of liver antioxidant enzymes were detected in CCME-treated group. In presence of CCME, the reductive release of ferritin iron was increased significantly. Furthermore, CCME exhibited DPPH radical scavenging and protection against Fe2+-mediated oxidative DNA damage. The current study confirmed the hepatoprotective effect of CCME against the model hepatotoxicant iron overload and the activity is likely related to its potent antioxidant and iron-chelating property.
Liver is one of the largest organs in the human body and the main site for intense metabolism and excretion. It is involved with almost all the biochemical pathways to growth, fight against disease, nutrient supply, energy provision, and reproduction . Hepatic damage is associated with distortion of these metabolic functions  and, sometimes, resulting in serious health problems. Hepatotoxicity is the most common finding in patients with iron overloading as liver is mainly the active storage site of iron in our body . Iron, the most important transition element of the body, is found in functional forms in haemoglobin, myoglobin, the cytochromes, enzymes with iron sulphur complexes, and other iron-dependent enzymes . Although an optimum level of iron is always maintained by the cells to balance between essentiality and toxicity, in some situations it is disrupted, resulting in iron overload which is associated to the oxidative stress induced disorders including anemia, heart failure, liver cirrhosis, fibrosis, diabetes, arthritis, depression, impotency, infertility, and cancer . In all iron-overload-induced diseases, iron removal by iron chelation therapy is an effective life-saving strategy. The currently available iron-chelating agents used clinically are deferoxamine, 1,2-dimethyl-3-hydroxypyrid-4-one (deferiprone, L1), and deferasirox. However, such compounds show several side effects and limitations [6, 7] that direct towards the finding of a more effective and safe drug [8, 9] which may rise the therapeutic benefits for patients.
Phytoconstituents including phenolics and flavonoids are most important representatives to offer alleviation of hepatic ailments. It has been found that most of them are effective antioxidants [10, 11] and iron chelation is very important part of their antioxidant activity . Thus, search for crude drugs of the plant origin with antioxidant activity has become a central focus of study of hepatoprotection. Caesalpinia crista Linn. (syn. C. bonducella[L.]Roxb.) (family-Fabaceae) is a large scandent prickly shrub widely distributed throughout the tropical and subtropical regions of Southeast Asia. In India, it is commonly known as Katikaranja or Natakaranja and used in different system of traditional medication for the treatment of diseases and ailments of human beings. Traditionally, in Ayurveda, various plant parts such as leaves, stem, root, seed, and oil are used as febrifugal, periodic, tonic, and vesicant for the treatment of gynaecological disorders, skin diseases, constipation, piles, and ulcers . Phytochemical investigations of this plant have revealed the presence of several cassane- and norcassane-type diterpenes [14–18]. This plant has profound medicinal use and reported to have adaptogenic , anthelmintic , anti-inflammatory , antipyretic and analgesic [22, 23], antimalarial , antiamyloidogenic , antibacterial , antifilarial , antitumor , anticonvulsant , nootropic , immunomodulatory , hepatoprotective , anxiolytic , antidiabetic, and hypoglycemic activity . Previously, 70% methanol extract of Caesalpinia crista (CCME) leaf has shown in vitro antioxidant and iron-chelating property and was found to be a rich source of phenolic and flavonoid compound . Therefore, the present study was undertaken to assess whether the damage caused to liver by iron overload can be normalised by administration of CCME in mice.
2. Material and Methods
Iron dextran and guanidine hydrochloride were purchased from Sigma-Aldrich, USA. Trichloroacetic acid (TCA), nitro blue tetrazolium (NBT), reduced nicotinamide adenine dinucleotide (NADH), phenazinemethosulfate (PMS), ferrozine, glutathione reduced, bathophenanthrolinesulfonate disodium salt, Thiobarbituric acid (TBA), and 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) were obtained from Sisco Research Laboratories Pvt. Ltd, Mumbai, India. Hydrogen peroxide, ammonium iron (II) sulfatehexahydrate [(NH4)2Fe(SO4)26H2O], 1-chloro-2,4-dinitrobenzene (CDNB), chloramine-T, hydroxylamine hydrochloride, Dimethyl-4-aminobenzaldehyde, and 2,4-dinitro phenylhydrazine (DNPH) were obtained from Merck, Mumbai, India. Ferritin was purchased from MP Biomedicals, USA. Streptomycin sulphate was obtained from HiMedia Laboratories Pvt. Ltd, Mumbai, India. The standard oral iron-chelating drug, desirox, was obtained from Cipla Ltd., Kolkata, India.
2.2. Plant Material
The leaves of Caesalpinia crista (CC) were collected from the Bankura district of West Bengal, India. It was identified and authenticated by the Central Research Institute (Ayurveda), Kolkata, India, and a voucher specimen (CRHS 121/08) was deposited there.
2.3. Preparation of Plant Extract
The leaves of CC were dried at room temperature for 7 days, finely powdered and used for extraction. The powder (100 g) was mixed with 500 ml methanol : water (7 : 3) using a magnetic stirrer for 15 hours, then the mixture was centrifuged at 2850 ×g and the supernatant was decanted. The extraction was repeated again with the precipitated pellet. The supernatants were collected, concentrated in a rotary evaporator and lyophilized. The dried extract, denoted as CCME was stored at −20°C until use.
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. The animals were provided with normal laboratory pellet diet and water ad libitum. Institutional animal ethics committee (IAEC) approved all experiments performed and care of the animals was taken as per the guidelines of the committee for the purpose of control and supervision of experiments on animals (CPCSEA), Ministry of Environment and Forest, Government of India.
2.5. In Vitro Study
2.5.1. DPPH Radical Scavenging
The free radical scavenging activity of CCME was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) using a standard method . Briefly, the reaction mixture contains 0.05 ml of 1 mM DPPH solution, 0.5 ml of 99% ethanol, and 0.45 ml of sample and standard ascorbic acid at different concentrations. The solution was rapidly mixed and the reduction of DPPH was measured by reading the decrease in absorbance at 517 nm. All tests performed six times. Ascorbic acid was used as a reference compound.
2.5.2. Oxidative DNA Damage
pUC18 plasmid DNA was used for DNA protection study by CCME, according to a previously described method  with minor modifications. In Hepes buffer (pH 7.2, 100 mM), FeSO4 solution (750 μM), CCME of varying doses (0–30 μg/ml), DNA (0.5 mg/ml), and water were added to make an initial reaction mixture. Finally, H2O2 solution (7.5 mM) was added to start the reaction. Desferal was used to stop the reaction after 10 min. 25 μl of each reaction mixture was loaded in 1% agarose gel. After migration, the gel was stained with ethidium bromide and visualized in a UV transilluminator. The DNA bands were quantified through densitometry and the following formulae were used to calculate the percentage of protection: where, SC is the supercoiled; OC is the open circular; 1.4 is the correction factor The ability of the plant extract to protect the DNA supercoil can be expressed by the concentration of sample required for 50% protection, designated as the [P]50 value.
2.6. In Vivo Study
2.6.1. Experimental Design
Thirty-six mice were divided into six groups containing six mice in each group. One group served as blank (B) and received normal saline only. The other five groups were given five doses (one dose every two days) of 100 mg/kg b.w. each, of iron dextran saline (i.p). One iron dextran group (C) received normal saline and other four groups were orally administered with 50 mg/kg b.w. (S50), 100 mg/kg b.w. (S100), 200 mg/kg b.w. (S200) plant extract, and 20 mg/kg b.w. desirox (D), respectively, for three consecutive 7 day periods, started from the day after the first iron dextran injection.
2.6.2. Sample Collection and Tissue Preparation
Mice were fasted overnight after the experiment ended on the 21st day. They were anesthetized with ethyl ether and blood was collected by cardiac puncture. After the clotting of blood samples, sera were separated using cooling centrifuge and store at −80°C until analysis. The liver was dissected out and rinsed with ice-cold saline to eliminate the blood cells; half of them were cut, weighed, and homogenized in 10 volume of 0.1 M phosphate buffer (pH 7.4) containing 5 mM EDTA and 0.15 M NaCl, and centrifuged at 8000 g for 30 min at 4°C. The supernatant was collected and used for the assay of enzyme activities, protein oxidation, levels of hydroxyproline content, and lipid peroxidation products. A standard graph of BSA was prepared to estimate the protein concentration in the homogenate by Lowry method . The other half of the liver samples were weighed and digested with equivolume (1 : 1) mixture of sulphuric acid and nitric acid and their iron content were analysed.
2.6.3. Serum Markers
Alanine amino transferase (ALAT), aspartate amino transferase (ASAT), and billirubin in serum samples were measured using the commercial kits of Merck, Mumbai, India. Serum alkaline phosphatase (ALP) was estimated using the kit supplied by Sentinel diagnostics, Italy.
2.6.4. Antioxidant Enzymes
Superoxide dismutase (SOD) was assayed by measuring the inhibition of the formation of blue colored formazan at 560 nm . Catalase (CAT) activity was measured by following the decomposition of H2O2 over time at 240 nm . Glutathione-S-transferase (GST) was determined based on the formation of GSH-CDNB conjugate and increase in the absorbance at 340 nm . A spectrophotometric method was used to measure reduced glutathione (GSH) level at 412 nm .
2.6.5. Biochemical Parameters
The lipid peroxide levels in liver homogenates were measured in terms of thiobarbituric acid reactive substances (TBARS) as an index of malondialdehyde accumulation . As a marker of protein oxidation, protein carbonyl contents were estimated spectrophotometrically by DNPH method . Briefly, 50 μl streptomycin sulphate (10% w/v) was added to 450 μl homogenate samples and then centrifuged at 2800 g for 15 minutes. The supernatant (200 μl) was incubated with the same volume of 10 mM DNPH in 2 M HCl at room temperature for 20 mins. After the reaction was completed, 10% cold TCA was added to precipitate the proteins and the precipitates were washed with ethyl acetate-ethanol mixture (1 : 1) for three times to remove unreacted DNPH. The final protein pellet was dissolved in 1 ml of 6 M guanidine hydrochloride solution and the absorbance was measured at 370 nm, using the molar extinction coefficient of DNPH, ε = 2.2*10−4 M−1 cm−1. The measurement of hydroxyproline content in the liver allows the actual quantitation of collagen content which is an important marker of liver fibrosis. Liver samples were hydrolyzed in 6 M HCl and hydroxyproline was measured by Ehrlich’s solution . A standard curve ( = 0.9907) of 4-hydroxy-L-proline was prepared and results were calculated after taking absorbances at 558 nm. Total hydroxyproline content in each sample was multiplied by a factor of 7.69 to determine the collagen content . Results are expressed as milligrams of collagen per liver (wet weight).
2.6.6. 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. A paraffin-embedding technique was carried out and sections were taken at 5 μm thickness, stained with hematoxylin and eosin, and examined microscopically for histopathological changes.
2.6.7. Liver Iron and Serum Ferritin
Liver iron was measured according to a formerly reported colorimetric method . Samples were incubated with bathophenanthrolinesulfonate for 30 min at 37°C and absorbances were read at 535 nm. Serum ferritin levels were measured using enzyme-linked immunosorbent assay kit (from Monobind Inc., USA) according to the manufacturer’s instructions.
2.6.8. Iron Release from Ferritin
As previously described, iron reduction and release was determined spectrophotometrically . The ferrous chelator, ferrozine, was used as a chromophore for this assay. The reaction mixture (3 ml final volume) contained 200 μg ferritin, 500 μM ferrozine, in 50 mM pH 7.0 phosphate buffer. Reaction was induced by the addition of 500 μl plant extracts of different concentrations and alteration in absorbance was measured continuously at 560 nm for 20 min. A cuvette containing ferritin, ferrozine, and phosphate buffer but lacking plant extract was used as the reference solution.
2.7. Statistical Analysis
All data are reported as the mean ± SD of six measurements. Statistical analysis was performed using KyPlot version 2.0 beta 15 (32 bit) and Origin professional 6.0. Comparisons among groups were made according to pair t-test. In all analyses, a P-value of <0.05 was considered significant.
3. Results and Discussion
3.1. In Vitro Study
3.1.1. DPPH Scavenging
The bleaching of DPPH indicates the free radical scavenging capacity of CCME. Figure 1 demonstrated the DPPH radical scavenging activity of the extract in comparison to the standard. The IC50 values of the sample and standard were 14.2 ± 0.63 μg/ml and 5.27 ± 0.27 μg/ml, respectively. The results clarify the antioxidant activity of CCME as it is an effective DPPH free radical scavenger.
3.1.2. DNA Damage
The protective effect of CCME against Fe2+-dependent oxidative DNA damage of pUC18 plasmid was demonstrated in Figure 2. The results showed the dose-dependent protection of extract with a [P]50 value of 9.44 ± 0.76 μg/ml. The significant reduction in the formation of nicked DNA and increase in supercoiled DNA in the presence of the CCME reveals its excellent iron-chelating activity.
3.2. In Vivo Study
3.2.1. Serum Markers
The serum enzymes are very important adjuncts to clinical diagnosis of diseases and tissue injury. Hepatic injury by iron results in the leakage of cellular enzymes into the bloodstream, resulting in augmented levels of serum ALAT, ASAT, ALP, and bilirubin . Increase in the levels of serum enzymes, namely, ALAT (136.24%), ASAT (145.69%), ALP (149.21%), and billirubin (265.48%) as shown in Table 1 clearly signifies iron-induced liver damage. Oral administration of CCME markedly reduced the elevated levels of serum enzymes and billirubin of iron-overloaded mice to approach the normal control values.
3.2.2. Antioxidant Enzymes
Cells are armed with a stock of endogenous antioxidant defence machinery to protect them. These include enzymes such as SOD, CAT, and GST or compounds such as GSH . In excess, iron is a major cause of oxidative stress and lowers the levels of SOD (77.04%), CAT (52.25%), GST (70.02%), and GSH (32.36%), whereas treatment with CCME arrested the iron-induced depletion of these enzymes dose-dependently (Table 2). Thus, CCME significantly mended the levels of antioxidant enzymes and helping revival from hepatic damage.
3.2.3. Biochemical Parameters
The enhanced lipid peroxidation has been proposed as an initial step by which iron causes structural and functional alterations in cell integrity . The present result showed that 84% increment of lipid peroxidation in liver homogenates of iron-injected mice than blank was significantly reduced by 29%, 32%, and 41% in mice fed with S50, S100, and S200, respectively, (Figure 3). Protein oxidation is another outcome in iron-overload-induced hepatic damage. The iron-mediated oxidative modification of protein leads to their degradation and carbonyl formation , which was confirmed by elevated level of protein carbonyl (147%) from liver samples in iron-overloaded mice compared to normal mice. The results of current study clearly establish that CCME efficiently reduced the carbonyl content 11%, 20%, and 55% with gradual increase of concentration (S50, S100, and S200) (Figure 4). The increase in collagen content (197%) of iron-intoxicated mouse was a significant indicator of liver fibrosis. The excessive iron deposition in liver resulted in iron-catalysed oxidative stress contributing to the pathogenesis and progression of liver fibrosis . The collagen content in iron-overloaded mice was found 15.56 ± 1.09 mg/liver compared to 5.23 ± 1.37 mg/liver of normal mice. The upsurge of collagen content was gradually reduced to 15.38 ± 1.2 mg/liver, 12.17 ± 1.29 mg/liver, and 8.95 ± 1.45 mg/liver in CCME treated mice (S50, S100, and S200, resp.), signifying the hepatic fibrosis inhibitory potency of the plant extract (Figure 5).
3.2.4. Histopathological Study
Histological observations are performed along with the level of various biochemical parameters in circulation to mark the extent of hepatic damage. The liver sections of normal mice showed normal cell morphology with well-preserved cytoplasm, prominent nucleus, and well-brought-out central vein (Figure 6(a)). Iron dextran control mice showed various degrees of pathological changes including hepatocellular necrosis, ballooning degeneration, and loss of cellular boundaries (Figure 6(b)). In contrast, the liver sections taken from CCME-treated mice showed lessening of the pathogenesis and revealed marked reduction in hepatic injuries (Figures 6(c), 6(d), and 6(e)). Figure 6(f) exhibited the improved histology of liver sections taken from desirox-treated group. However, these observations indicate the in situ hepatoprotective evidence of the extract.
3.2.5. Liver Iron and Serum Ferritin
The elevated (142%) liver iron content was found in iron-overloaded mouse compared to normal mouse. Administration of CCME reduced the iron level 17%, 26%, and 45% with the effect of increased dose (S50, S100, and S200, resp.) (Figure 7). The decrease in liver iron deposition induced by CCME treatment supports its iron-chelating potency which was established previously . Body’s iron level is positively correlated with ferritin, a ubiquitous intracellular protein that stores iron in a nontoxic form and also helps prevent iron from mediating oxidative damage to cell constituents . The increased level of ferritin is generally noticed in iron-overload-induced liver toxicity which substantially reduced as treated with CCME dose-dependently (Figure 8).
3.2.6. Reductive Release of Ferritin Iron and Its Correlation with Reducing Power
Within cell, ferritin served as the storage protein for excess iron in ferric state. In case of iron overload, various iron chelators are administered to attenuate the situation, but, most of these chelators have limited binding activity for ferric iron as well, as iron in ferritin is not properly accessed to them. So, iron chelation therapy is dependent on the reductive release of ferritin iron, which is achieved by supplemented addition of a reducing agent such as ascorbate to increase the availability of storage iron to chelators . Figure 9 showed the reductive release of ferritin iron by CCME, that was measured with a ferrous complex of ferrozine [Fe(ferrozine)3]2+. Control experiments without CCME produced negligible amounts of [Fe(ferrozine)3]2+, whereas, after dose dependant addition of CCME the [Fe(ferrozine)3]2+ complex formation was increased significantly with time. However, reducing property of an iron chelator should definitely increase the efficiency of iron chelation therapy to treat iron overload. Previous results had shown the reductive ability of CCME  as well as in the present study, a significant (P < 0.001) positive correlation (R = 0.9571) between reducing power and (%) iron released from ferritin has been well established (Figure 10).
From the present study, it might be concluded that CCME has protective effect against iron-overload-induced liver toxicity as evidenced by biochemical and histopathological studies. CCME may exert its hepatoprotective activity by upregulating antioxidant enzymes and chelating iron to excrete form the body. The findings suggest its benefit in pathological sequence of iron-overload-linked liver disease and can be used as promising hepatoprotective agent.
Cipla Ltd., Kolkata, India is acknowledged for providing desirox as reference iron-chelating drug for this study. The authors would also like to thank Mr. Ranjit Kumar Das and Mr. Pradip Kumar Mallick for technical assistance in sample preparation and handling of lab wares and animals in experimental procedures.
F. M. Ward and M. J. Daly, “Hepatic disease,” in Clinical Pharmacy and Therapeutics, R. Walker and C. Edwards, Eds., pp. 195–212, Churchill Livingstone, New York, NY, USA, 1999.View at: Google Scholar
P. L. Wolf, “Biochemical diagnosis of liver disease,” Indian Journal of Clinical Biochemistry, vol. 14, no. 1, pp. 59–90, 1999.View at: Google Scholar
D. A. Papanastasiou, D. V. Vayenas, A. Vassilopoulos, and M. Repanti, “Concentration of iron and distribution of iron and transferrin after experimental iron overload in rat tissues in vivo: study of the liver, the spleen, the central nervous system and other organs,” Pathology Research and Practice, vol. 196, no. 1, pp. 47–54, 2000.View at: Google Scholar
L. S. Goodman and A. Gilman, The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY, USA, 11th edition, 2006.
D. R. Richardson, “The therapeutic potential of iron chelators,” Expert Opinion on Investigational Drugs, vol. 8, no. 12, pp. 2141–2158, 1999.View at: Google Scholar
F. N. Al-Refaie, B. Wonke, A. V. Hoffbrand, D. G. Wickens, P. Nortey, and G. J. Kontoghiorghes, “Efficacy and possible adverse effects of the oral iron chelator 1,2- dimethyl-3-hydroxypyrid-4-one (L1) in thalassemia major,” Blood, vol. 80, no. 3, pp. 593–599, 1992.View at: Google Scholar
G. L. Pardo-Andreu, M. F. Barrios, C. Curti et al., “Protective effects of Mangifera indica L extract (Vimang), and its major component mangiferin, on iron-induced oxidative damage to rat serum and liver,” Pharmacological Research, vol. 57, no. 1, pp. 79–86, 2008.View at: Publisher Site | Google Scholar
E. M. Williamson, Major Herbs of Ayurveda, Elsevier Health Sciences, Edinburgh, UK, 2002.
T. Kinoshita, Y. Haga, S. Narimatsu, M. Shimada, and Y. Goda, “The isolation and structure elucidation of new cassane diterpene-acids from Caesalpinia crista l. (Fabaceae), and review on the nomenclature of some Caesalpinia species,” Chemical and Pharmaceutical Bulletin, vol. 53, no. 6, pp. 717–720, 2005.View at: Publisher Site | Google Scholar
S. Awale, T. Z. Linn, Y. Tezuka et al., “Constituents of Caesalpinia crista from Indonesia,” Chemical and Pharmaceutical Bulletin, vol. 54, no. 2, pp. 213–218, 2006.View at: Google Scholar
S. Kale, G. Gajbhiye, and N. Chaudhari, “Anti-inflammatory effect of petroleum ether extract of Caesalpinia bonduc (L.) Roxb seed kernel in rats using carrageenan-induced paw edema,” International Journal of PharmTech Research, vol. 2, no. 1, pp. 750–752, 2010.View at: Google Scholar
S. Shukla, A. Mehta, P. Mehta, S. P. Vyas, S. Shukla, and V. K. Bajpai, “Studies on anti-inflammatory, antipyretic and analgesic properties of Caesalpinia bonducella F. seed oil in experimental animal models,” Food and Chemical Toxicology, vol. 48, no. 1, pp. 61–64, 2010.View at: Publisher Site | Google Scholar
M. Gupta, U. K. Mazumder, R. S. Kumar, and T. S. Kumar, “Studies on anti-inflammatory, analgesic & anti-pyretic properties of methanol extract of Caesalpinia bonducella leaves in experimental animal models,” Indian Journal of Pharmacology & Therapeutics, vol. 2, pp. 30–34, 2003.View at: Google Scholar
R. L. Gaur, M. K. Sahoo, S. Dixit et al., “Antifilarial activity of Caesalpinia bonducella against experimental filarial infections,” Indian Journal of Medical Research, vol. 128, no. 1, pp. 65–70, 2008.View at: Google Scholar
M. Gupta, U. K. Mazumder, R. S. Kumar, T. Sivakumar, and M. L. M. Vamsi, “Antitumor activity and antioxidant status of Caesalpinia bonducella against Ehrlich Ascites Carcinoma in Swiss albino mice,” Journal of Pharmacological Sciences, vol. 94, no. 2, pp. 177–184, 2004.View at: Publisher Site | Google Scholar
A. Ali, N. Venkat Rao, M. D. Shalam, T. Shivaraj Gouda, and S. M. Shantakumar, “Anticonvulsive effect of seed extract of Caesalpinia bonducella (Roxb.),” Iranian Journal of Pharmacology and Therapeutics, vol. 8, no. 2, pp. 51–55, 2009.View at: Google Scholar
S. N. Kshirsagar, “Nootropic activity of dried seed kernels of Caesalpinia crista linn against scopolamine induced amnesia in mice,” International Journal of PharmTech Research, vol. 3, no. 1, pp. 104–109, 2011.View at: Google Scholar
R. Sambath Kumar, K. Asok Kumar, and N. Venkateswara Murthy, “Hepatoprotective and antioxidant effects of Caesalpinia bonducellaon carbon tetrachloride-induced liver injury in rats,” International Research Journal of Plant Science, vol. 1, no. 3, pp. 062–068, 2010.View at: Google Scholar
A. Ali, N. Venkat Rao, M. Shalam, T. Shivaraj Gouda, J. M. Babu, and S. Shantakumar, “Anxiolytic activity of seed extract of Caesalpinia bonducella (Roxb.) in laboratory animals,” The Internet Journal of Pharmacology, vol. 5, no. 2, 2008, http://www.ispub.com/journal/the-internet-journal-of-pharmacology/volume-5-number-2/anxiolytic-activity-of-seed-extract-of-caesalpinia-bonducella-roxb-in-laboratory-animals.html.View at: Google Scholar
V. V. Rao, S. K. Dwivedi, and D. Swarup, “Hypoglycaemic effect of Caesalpinia bonducella in rabbits,” Fitoterapia, vol. 65, no. 3, pp. 245–247, 1994.View at: Google Scholar
S. Mandal, B. Hazra, R. Sarkar, S. Biswas, and N. Mandal, “Assessment of the antioxidant and reactive oxygen species scavenging activity of methanolic extract of Caesalpinia crista leaf,” Evidence-Based Complementary and Alternative Medicine, vol. 2011, Article ID 173768, 11 pages, 2011.View at: Publisher Site | Google Scholar
O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, “Protein measurement with the Folin phenol reagent,” The Journal of Biological Chemistry, vol. 193, no. 1, pp. 265–275, 1951.View at: Google Scholar
P. Kakkar, B. Das, and P. N. Viswanathan, “A modified spectrophotometric assay of superoxide dismutase,” Indian Journal of Biochemistry and Biophysics, vol. 21, no. 2, pp. 130–132, 1984.View at: Google Scholar
J. Bonaventura, W. A. Schroeder, and S. Fang, “Human erythrocyte catalase: an improved method of isolation and a reevaluation of reported properties,” Archives of Biochemistry and Biophysics, vol. 150, no. 2, pp. 606–617, 1972.View at: Google Scholar
W. H. Habig, M. J. Pabst, and W. B. Jakoby, “Glutathione S transferases. The first enzymatic step in mercapturic acid formation,” The Journal of Biological Chemistry, vol. 249, no. 22, pp. 7130–7139, 1974.View at: Google Scholar
G. L. Ellman, “Tissue sulfhydryl groups,” Archives of Biochemistry and Biophysics, vol. 82, no. 1, pp. 70–77, 1959.View at: Google Scholar
I. Bergman and R. Loxley, “Two improved and simplified methods for the spectrophotometric determination of hydroxyproline,” Analytical Chemistry, vol. 35, no. 12, pp. 1961–1965, 1963.View at: Google Scholar
K. I. Kivirikko, O. Laitinen, and D. J. Prockop, “Modifications of a specific assay for hydroxyproline in urine,” Analytical Biochemistry, vol. 19, no. 2, pp. 249–255, 1967.View at: Google Scholar
M. Barry and S. Sherlock, “Measurement of liver-iron concentration in needle-biopsy specimens,” The Lancet, vol. 1, no. 7690, pp. 100–103, 1971.View at: Google Scholar
K. B. Beckman and B. N. Ames, “The free radical theory of aging matures,” Physiological Reviews, vol. 78, no. 2, pp. 547–581, 1998.View at: Google Scholar
H. L. Bonkowsky, J. F. Healey, and P. R. Sinclair, “Iron and liver. Acute and long-term effects of iron-loading on hepatic haem metabolism,” Biochemical Journal, vol. 196, no. 1, pp. 57–64, 1981.View at: Google Scholar
P. M. Harrison, “Ferritin: an iron storage molecule,” Seminars in Hematology, vol. 14, no. 1, pp. 55–70, 1977.View at: Google Scholar
R. T. O'Brien, “Ascorbic acid enhancement of desferrioxamine induced urinary iron excretion in thalassemia major,” Annals of the New York Academy of Sciences, vol. 232, pp. 221–225, 1974.View at: Google Scholar