Chemicals

ABTS – 2, 2-azinobis (3- ethylbenzothiazoline-6-sulfonic acid); BHT- Butylated hydroxytoluene; BSA – Bovine serum albumin; DNPH – Dipenylhydrazine; DPPH – 2, 2-diphenyl-1-picrylhydrazyl; DTNB – 5, 5-dithiobisnitrobenzoic acid; HCl – Hydrochloric acid; EDTA- Ethylenediamine tetraacetate disodium salt; FeCl₃ – Ferric chloride; HEPES – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; H2O2 – hydrogen peroxide; NaCl – Sodium chloride; SDS – Sodium dodecyl sulphate; TBA – Thiobarbituric acid and K2Cr₂O₇ – Potassium dichromate. All the chemicals used in the experiment were purchased from Sigma Aldrich, USA.

Plant material and extraction

Curculigo orchoides Gaertn. rhizomes were collected from Mysore and properly authenticated by the Department of Botany, University of Mysore, Mysore, India (Voucher specimen no. AND8024). Rhizomes were shade dried and powdered using lab mill grinder and screened with sieve no 40. The rhizome powder (100 g) was defatted with diethyl ether for 24 h and the filtrate was suspended in 500 ml of 50% ethanol for 2 days on shaker at 200 rpm. This mixture was filtered using Whatman no.1 filter paper, and then concentrated under reduced pressure then, lyophilized and stored at -20^oC for further analysis. The yield of the Curculigo orchoides CO extract was 6.12% of dry rhizome.

Determination of in vitro antioxidant activities

In vitro antioxidant activity of CO was evaluated for DPPH, nitric oxide, hydrogen peroxide and ABTS radical scavenging activities. Free radical scavenging activities were determined by spectropho-tometric method (Kumar et al., 2013a,b). Percent inhibition was calculated as follows: [A_cont- A_sample]/A_cont x 100, where A_cont is the absorbance of the control and A _sample is the absorbance of sample. Ferrous reducing antioxidant power assay (FRAP) was measured for the colored product of ferrous tripyridyltriazine complex at an absorption maximum of 593 nm and expressed in units of µmol Fe(II) (Kumar et al., 2013a,b).

Experimental procedure

Animal studies were conducted according to the institute animal ethical committee regulations approved by the Committee for the Purpose of the Control and Supervision of Experiments on Animals (CPCSEA) with the IAEC approval no.28/1999/CPCSEA. Male Wistar albino rats (150–200 g) were selected from the stockpile colony, Defense Food Research Laboratory (DFRL), Mysore, India and were housed in an acryl fibre cage in a temperature controlled room (23 ± 2^oC). Animals were maintained at 12 h light/dark cycle with standard pellet diet and water was provided ad-libitum.

Experimental design

Thirty six animals were randomly divided into 6 groups: control group received distilled water (Con); positive control group received silymarin (Syl, 60 mg/kg) (Pradhan and Girish, 2006); treatment groups received 25, 50 and 100 mg of C. orchoides (CO 25, CO 50, and CO 100 mg/kg, respectively). K2Cr2O7 (30 mg/kg) was administered by oral gavage to all animals except control group for a period of 28 days. Negative control group received only K2Cr2O7 (Cr VI). Dosage selection and mode of administration of both K2Cr2O7 and CO were based on previous studies (Susa et al., 1996). Animals were sacrificed under mild anaesthesia; serum was separated and stored at -20^oC for further biochemical assays. The tissue samples were excised, washed with normal saline and stored in -80 ^oC for further biochemical assays. Washed tissues were fixed immediately in 10% formalin solution for histopathology studies.

Assessment of serum biochemical markers

Serum analyses of various liver marker enzymes like alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) were estimated using commercially available kits (AGAPEE, India).

Estimation of TBARS

Lipid peroxidation was determined by thiobarbutric acid reactive species (TBARS) and expressed as malondialdehyde (MDA mmol/cm/g) (Garcia et al., 2005). Liver tissues were homogenized in phosphate buffer (pH 7.0); Trichloroacetic acid (TCA) (10%), 0.5 ml and 2 ml of TBA mixture were added to tissue homogenate (0.5 ml). The TBA mixture contained TBA (0.35%), SDS (0.2%), FeCl₃ (0.05 mM) and BHT in glycine- HCl buffer (100 mM, pH 3.6). The above reaction mixture was boiled at 100^oC for 30 min and then allowed to cool. The mixture was centrifuged at 8000 rpm for 10 min and the absorbance was measured at 532 nm.

Protein carbonyl assay

Protein carbonyl content was estimated by Dalle et al. (2003) with slight modifications. Samples (10 ml) were diluted 1:40 with phosphate-buffered saline (PBS) containing sodium phosphate (10 mM; pH 7.4) and NaCl (0.14 M), and centrifuged twice at 14,000 rpm. Cold trichloroacetic acid (20%; w/v) was used to precipitate the protein portion. A solution of 10 mM DNPH in 2 N HCl was added to the protein pellet to get a final protein concentration of 1-2 mg/ml. Samples were allowed to stand in the dark at room temperature for 1 h by vortexing every 10 min and then precipitated with 10% TCA. The protein pellets were washed with 20% TCA (two times) and ethanol/ethyl acetate (1:1, v/v; three times) to remove free DNPH. Samples were then suspended in 6 M guanidine hydrochloride at 37^oC for 15 min. Carbonyl content was determined from the absorbance at 366 nm using a molar absorption coefficient of 22,000 M ^-1cm^_1.

Assay of SOD, CAT and GST activities

Liver tissues (100 mg) were homogenized in 50 mM phosphate buffer saline (1 ml) (pH 7.4) and 0.1 ml of tissue homogenate was used for each assay. Superoxide dismutase (SOD) activity was determined with commercially available kit (Randox, Cat no. SD. 125; Canada). The catalase activity (CAT) was determined by measuring the decay of 6 mM H2O2 solution at 240 nm by spectrophotometric degradation method (Kumar et al., 2013a,b). Glutathione S-transferase (GST) activity was assayed using the CDNB conjugate method (Habig et al., 1974).

Gene expression studies

RNA isolation and cDNA conversion Liver tissue samples were placed in a vial containing RNA later and stored at -80^oC. Total RNA was extracted from ~100 mg of tissue by TRIzol reagent (Sigma Aldrich), as described the manufacturer’s protocol. RNA samples were treated with RQ1 RNase-free DNase (Applied Bio-systems) to avoid DNA contami-nation. The concentration of total RNA was measured with spectrophotometer (NanoDrop) and the OD₂₆₀/OD₂₈₀ ratio was tested for the purity of RNA and the purity of RNA yield was also checked by electrophoresis. The first cDNA strand was synthesized by using high capacity cDNA converting kit (Applied Bio systems) in one vial procedure containing a total reaction of 20 µl having 3.2 µl of distilled H₂O, 0.8 µl of dNTP’s, 2 µl of the 10 x buffer, 2 µl of the random primers, 1 µl of RNAse inhibitor and 10 µl of RNA sample. Further the sample was incubated as per manufacturer’s protocol (Applied Bio systems).

Semi-quantative polymerase chain reaction (PCR)

Ten different genes were targeted to check the modulation of gene expressions in response to Cr(VI) induced toxicity, such as: GAPDH (glyceraldehyde 3-phosphate dehydrogenase, housekeep-ing gene), TNF-a (tumor necrosis factor), IL-1 (interlukin), Mapk1 (mitochondria associated protein kinase), Atf-1(cyclic AMP dependent transcription factor), Gadd45 (growth arrest and DNA damage inducible protein), OGG-1 (oxoguanine glycosylase), Bcl-2 (B-cell lymphoma), Bax (Bcl-2 associated protein) and Casp-1 (caspase). Primers were designed on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/nucleotide/) using the Rattus nor vegicus gene sequences. Primers were supplied by Sigma Aldrich (India), and stored at _20 _C prior to using them for the gene expression study. Forward and reverse primers of targeted genes were provided in Table 1. PCR amplification was performed as a 20 ml reaction with 6 mmol l_1 of each forward and reverse primer, 2 µl of 10 x PCR buffer, 1.5 mmol l^-1 of MgCl₂, 80 mmol l^-1of each dNTP’s and 1 unit of Taq polymerase (Sigma, India) in a thermal cycler (Eppendorf, Germany). The PCR programme consisted of initial denaturation at 94^oC for 4 min, 30 cycles of denaturation at 94^oC for 45 s, annealing at 55^oC to 60^oC (subject to primer; Table 1) for 30 s and extension at 72^oC for 8 min. PCR amplicons were resolved on 1.5% agarose gel and visualized under UV transilluminator.

Western blot analysis

Liver tissue samples were processed for SDS-PAGE followed by western blot for the analysis of antioxidant enzymes viz., superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) expression. Tissue samples were homogenized with HEPES buffer (pH 7.4) and were centrifuged at 10,000 rpm for 20 min. Total protein content was estimated from the supernatant (Lowry et al., 1951). Protein from each sample (50 mg) was separated on 12% SDS-PAGE and transferred onto a nitrocellulose membrane using an electro blotting apparatus (Bio-Rad, USA). The membrane was blocked in 5% defatted milk solution at 4^oC for 24 h, later probed by respective primary antibody (1: 1000) and incubated at room temperature for 3 h. The membrane was washed four times in PBST and one time in PBS for 15 min followed by incubation for 2 h in HRP conjugated rabbit anti-mouse secondary antibody (Sigma, India; 1: 1000). The membranes were washed again and developed using an enhanced chemiluminescent method (luminal and p-coumaric acid, Sigma). The band intensity was captured by exposing the membranes onto the X- ray film (Anand et al., 2012).

Histopathological studies

Sections of brain, lungs, liver, kidney, spleen and intestine were carried out for histopathology to know tissue architecture. Fixed tissues were embedded in paraffin, cut into 5-mm thin sections, and stained with hematoxylin and eosin for light microscopic examination

Male Wistar Albino Rats