Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Involvement of NFκB in the antirheumatic potential of Chenopodium album L., aerial parts extracts Sumit K. Arora 1, Prakash R. Itankar n, Prashant R. Verma 2, Ashish P. Bharne 3, Dadasaheb M. Kokare 4 University Department of Pharmaceutical Sciences, Department of Pharmacognosy and Phytochemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Amravati Road, Nagpur 440033, Maharashtra, India

art ic l e i nf o

a b s t r a c t

Article history: Received 19 October 2013 Received in revised form 24 April 2014 Accepted 19 May 2014

Ethnopharmacological relevance: Chenopodium album L. (C. album) is commonly known as Bathua in Hindi (Family: Chenopodiaceae). Traditionally, the plant is used as a laxative, diuretic, sedative and the infusion of the plant is used for the treatment of rheumatism. However, no scientific validation is available on the antirheumatic potential of the plant. In the present investigation, role of NF kappa B (NFκB) in the antiarthritic potential of extracts of aerial parts of Chenopodium album was explored and evaluated. Methods: The defatted aerial parts of Chenopodium album were successively extracted with ethylacetate, acetone, methanol and 50% methanol to study their antioxidant capacity followed by antiarthritic potential using Complete Freund's adjuvant (CFA) induced arthritis model in rats. The polyphenol, flavonoid and flavanone contents of different extracts were quantified and correlated with their antioxidant capacity, antiarthritic activity and NFκB inhibition potential. Results: The experimental data indicated that the acetone extract of Chenopodium album (ACCA) has shown significant reduction in rat paw edema (80.13%) at dose level of 200 mg/kg per oral in 21 days of this study. On 22nd day, hematological and biochemical parameters were estimated and it was observed that the altered hematological parameters (Hb, RBC, WBC and ESR), biochemical parameters (Serum creatinine, total proteins and acute phase proteins) and loss in body weight in the arthritic rats were significantly brought back to near normal level by the ACCA extract. In order to assess possible mechanism of ACCA extract for its antiarthritic activity, NFκB inhibition assay was carried out by immunohistochemistry. The polyphenolic and flavonoid content of different extracts were in the range of 14.56 7 0.21–42.00 7 0.2 mg (gallic acid equivalent/g extract) and 2.20 70.003–7.3370.5 mg (rutin equivalent/g extract) respectively. Conclusion: The antiarthritic activity possessed by ACCA extract can be correlated directly to its antioxidant potential, high flavonoidal content achieved by successive extraction and its capacity to inhibit the NFκB protein, as proven by immunohistochemistry study. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Chenopodium album DPPH Nitric oxide Complete Freund's adjuvant NFκB Immunohistochemistry

1. Introduction Rheumatoid arthritis (RA) is a chronic disease of the joint, where pro-inflammatory mediators are believed to be responsible n Corresponding author. Tel.: þ 91 712 2550324; mobile: þ 91 9766191818; fax: þ91 712 2500355. E-mail addresses: [email protected] (S.K. Arora), [email protected] (P.R. Itankar), [email protected] (P.R. Verma), [email protected] (A.P. Bharne), [email protected] (D.M. Kokare). 1 Mobile: þ 918793484246. 2 Mobile: þ 919423100313. 3 Mobile: þ 919970309402. 4 Mobile: þ 919850318502.

for the cartilage destruction and bone erosion that characterizes the disease (Kingsley and Panayi, 1997). These pro-inflammatory molecules are regulated by the Nuclear factor Kappa B (NFκB) family of transcription factors (Pahl, 1999). Much evidence indicates a pivotal role for NFκB in the etiology of RA. Nuclear localization of subunits of NFκB (p50 and p65) has been shown to be significantly increased in synovial tissue of RA patients (Han et al., 1998). Similarly, it was demonstrated that fibroblast-like synoviocytes from RA synovium contain constitutively active NFκB and spontaneously produce large quantities of Interleukin-6 (IL-6) consistent with the clinical data. Increased NFκB binding activity has been demonstrated in the synovium of rodents following the development of collagen induced arthritis (CIA), adjuvant induced arthritis (AIA) and streptococcal cell wall-induced arthritis.

http://dx.doi.org/10.1016/j.jep.2014.05.026 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: Arora, S.K., et al., Involvement of NFκB in the antirheumatic potential of Chenopodium album L., aerial parts extracts. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.026i

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AIA causes activation of hypothalamus pituitary adrenal (HPA) axis which ultimately leads to glucocorticoid secretion, a factor responsible for regulation of cytokine (Chesnokova and Melmed, 2000; Eskandari et al., 2003). It is already reported that the cytokines causes the activation of NFκB. In addition, novel therapies for RA are based on addressing and correcting the dysregulation of these neural and neuroendocrine pathways. The number of antirheumatic agents including glucocorticoids, sulfasalazine, gold salts, leflunomide and aspirin are inhibitors of NFκB which explain the part of their anti-inflammatory effects (Tak and Firestein, 2001). Above evidences suggest that NFκB signaling pathway are potential targets for the development of novel RA therapeutics. Chenopodium album L., (C. album) (family: Chenopodiaceae) is an annual shrub widely grown in Asia, Africa, Europe and North America. It is commonly known as Bathua (in Hindi), pigweed, fat hen or lamb-quarters. The plant is used in folk medicine in different parts of the world as diuretic, laxative, sedative, hepatoprotective and antiparasitic. The leaves possesses anthelmentic, antiphlogistic, antirheumatic, mildly laxative and odontalgic properties, applied as wash or poultice to bug bites, sunstroke, rheumatic joints and swollen feets (Kokanova-Nedialkova et al., 2009). Additionally, decoction of its aerial parts mixed with alcohol was used in the rheumatism (Gupta et al., 2008). However, no scientific validation is available on the antirheumatic potential of the plant. Phytochemical analysis revealed the presence of phenolic glycoside cinnamic acid (Nahar and Sarker, 2005), sinapic acid, ferulic acid and their derivatives, quercetin and kaempferol glycosides (Kowalewski, 1997), xyloside, cinnamic acid amide alkaloid, chenoalbicin (Cutillo et al., 2003). In view of the above folklore claim, it was thought worthwhile to evaluate the effect of extracts of aerial parts of Chenopodium album in Complete Freund's adjuvant (CFA) induced RA in rats. Furthermore, to explore the mechanism of action, modulation of NFκB in hypothalamic brain nucleus is also studied employing immunohistochemistry using antibody against p65 subunit of NFκB.

2. Material and methods 2.1. Chemicals and standard drugs NFκB p65 Antibody (A) (cat. No. Sc-109 Santa Cruz Biotechnology, USA) was obtained from NCCS, Pune as a generous gift. Indomethacin and CFA reagent were obtained from Sigma Aldrich chemicals, Powai, Mumbai respectively.

2.2. Plant material and preparation of extract The aerial parts were collected from Ramtek region in the month of August, authenticated by Dr. (Mrs.) Alka Chaturvedi, Department of Botany, R.T.M. Nagpur University, Nagpur. A voucher specimen has been deposited in the Herbarium of Department of Botany, with collection number RA 9576. The aerial parts of Chenopodium album were dried under shade and pulverized to a coarse powder. The powdered crude material (1 kg) was defatted with petroleum ether and then extracted successively with ethyl acetate, acetone and methanol using Soxhlet extractor followed by cold maceration (7 days) with 50% methanol. The extracts were concentrated using rotary vacuum evaporator to yield ethyl acetate extract (EACA, yield: 3.9% w/w), acetone extract (ACCA, yield: 4.79% w/w), methanolic extract (MECA, yield: 13.58% w/w) and 50% methanolic extract (HACA, yield: 12.76% w/w). These extracts were subsequently subjected to phytochemical and pharmacological screening.

2.3. Phytochemical screening (Harbone, 1976; Stahl, 1969) The extracts were screened for the presence of different phytochemicals by employing thin layer chromatographic (TLC) techniques. Thin layer plates precoated with silica gel G (Merck, 0.25 mm thickness) were used. Development was carried out with different solvent systems such as ethyl acetate:methanol:water (100:13.5:10, v/v/v), ethyl acetate:formic acid:acetic acid:water (100:11:11:26, v/v/v/v), chloroform:methanol:water (70:30:4, v/v/v), toluene:ethyl acetate:diethylamine (70:20:10, v/v/v) and ethyl acetate:methanol: water:acetic acid (65:15:15:10, v/v/v/v). After development of chromatogram in the solvents, the plates were dried and sprayed with Dragendorff's, AlCl3, hydroxylamine-ferric chloride, ninhydrin and antimony trichloride reagents for the detection of alkaloids, flavonoids, lactones/esters, protein/amino acids, unsaturated sterols and triterpenes respectively. Detection of anthraquinones, saponins, tannins, carbohydrate and/or glycosides was carried out using KOH, anisaldehyde-sulphuric acid, ferric chloride and naphthoresorcinol reagent respectively. Visualization was carried out under visible and UV light (λ: 366 nm). The important secondary metabolites such as total polyphenol, flavonoid and flavonone were quantified in EACA, ACCA, MECA and HACA extracts respectively. 2.4. Determination of total polyphenol compounds (TP), flavonoids (TFA), total flavanones (TFO) and degree of polymerization 2.4.1. Determination of total polyphenol compounds (TP) Total polyphenol content was measured using the Folin– Ciocalteu colorimetric method (Singleton et al., 1999). Briefly, 0.1 ml of test sample was mixed with 1 ml of diluted Folin– Ciocalteu's phenol reagent (1:10 with distilled water; 0.2 N), to which 1 ml of saturated sodium carbonate (75 g/l) solution was added after 3 min and adjusted to 10 ml with distilled water. The reaction mixture was kept in the dark for 90 min and the absorbance was read at 725 nm (Shimadzu UV–VIS spectrophotometer 1600) against standard blanks prepared in the same way without the Folin–Ciocalteu's phenol reagent. Gallic acid was used as a reference for constructing the standard curve (10–100 mg/ml). The results were expressed as mg of gallic acid equivalents (GAE)/g of extract. All determinations were performed in triplicate. 2.4.2. Determination of flavonoids (TFA) Flavonoid content was determined by the aluminum chloride method (Stanojevic et al., 2009). Briefly, to 1 ml of test solution (1 mg/ml), 1.5 ml of 95% alcohol, 0.1 ml of 10% aluminum chloride hexahydrate (AlCl3  6H2O), 0.1 ml of 1 M sodium acetate (CH3COONa) and 2.3 ml of distilled water was added. After incubation at room temperature for 40 min, absorbance of the reaction mixture was measured at 435 nm against corresponding blank, prepared in the same manner without adding AlCl3. Rutin was used as a reference standard and results were expressed as mg of rutin equivalents (RE)/g of extract. All determinations were performed in triplicate. 2.4.3. Determination of total flavanones (TFO) The modified 2,4-dinitrophenylhydrazine (DNPH) method was used for determination of flavanones (Nagy and Grancai, 1996). Naringin was used as the reference standard. Twenty milligrams of naringin was dissolved in methanol and then diluted to 500, 1000 and 2000 μg/ml. One milliliter of each of the diluted standard solutions was reacted separately with 2 ml of 1% DNPH reagent and 2 ml of methanol at 50 1C for 50 min. After cooling at room temperature, the reaction mixture was mixed with 5 ml of 1% KOH in 70% methanol and incubated at room temperature for 2 min. Then 1 ml of the mixture was taken, mixed with 5 ml of methanol and centrifuged at 1000g for 10 min to remove the precipitate.

Please cite this article as: Arora, S.K., et al., Involvement of NFκB in the antirheumatic potential of Chenopodium album L., aerial parts extracts. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.026i

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The supernatant was collected and adjusted to 25 ml. The absorbance of the supernatant was measured at 495 nm. All the test samples were similarly treated with 2,4-DNPH for determination of flavanones. The mean of three readings was used and the data was expressed as mg of naringin equivalents (NE)/g of extract. 2.4.4. Degree of polymerization Degree of polymerization was estimated by the ratio between the TP and TFA contents (Jesus et al., 2008). 2.5. in vitro antioxidant studies The in vitro antioxidant studies of the extracts were determined by their ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical. Percentage inhibition was expressed in terms of IC50 value calculated by the linear regression method. 2.5.1. 2,2-diphenyl-1-picrylhydrazyl (DPPH method) The antioxidant activities of the extracts were assessed by the method described previously (Scherer and Godoy, 2009). Briefly, test samples or standard solutions (0.1 ml) at various concentrations (final concentration 0.001–2 mg/ml) were added to 4.9 ml of 50 μM of buffered DPPH (pH 5.5) solution. The absorbance was measured at 517 nm against the corresponding test and standard blank. Ascorbic acid was used as a reference standard and results were expressed as mg of ascorbic acid equivalents (AA)/g of extract. All determinations were made in triplicates. 2.5.2. Nitric oxide radical scavenging method (NO method) NO was generated from sodium nitroprusside and measured by the Greiss reaction (Marcocci et al., 1994). The reaction mixture containing 4 ml of sodium nitroprusside (5 mM), 1 ml phosphate buffer saline (pH 7.4) and 1 ml of test sample or standard solution at various concentrations (final concentration 0.001–2 mg/ml) was incubated at 25 1C for 150 min. After incubation, 1 ml of the reaction mixture containing nitrite was removed, mixed with 4 ml of Griess reagent [0.1% w/v NEDA þ sulfanilic acid (0.33% w/v; 1:1)] and allowed to stand for 30 min. A red-violet colored chromophore was formed in diffused light. The absorbance of these solutions was measured at 540 nm against the corresponding blank solution in triplicates and results were expressed as mg of ascorbic acid equivalent (AA)/g of extract. 2.6. Evaluation of anti-arthritic activity 2.6.1. Animals Male Wistar albino rats (175–200 g) were obtained from animal house of our institute. The animals were fed a standard pellet diet and water ad libitum. They were maintained in a controlled environment and temperature (22 7 5 1C with 12-h of light/dark cycle). All experimental protocols were approved by the institutional animal ethical committee (10/2010/CPCSEA). 2.6.2. Acute toxicity studies (OECD, 2000) Rats were divided into test and control groups (n ¼5). The test group was given an increasing oral dose (1, 3 and 5 g/kg) of EACA, ACCA, MECA and HACA extracts respectively. The rats were allowed food and water ad libitum and were kept under regular observation for symptoms of mortality and behavioral changes for the period of 48 h. 2.6.3. Induction of experimental arthritis Arthritis was induced by the method as described previously (Tijani, 2008). Briefly, rats were injected with 100 ml of CFA (contained heat killed Mycobacterium tuberculosis in sterile paraffin oil;

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10 mg/ml) intradermally into left hind paw. The injection day was regarded as day zero.

2.6.4. Experimental design Arthritic animals were divided into 10 groups comprising of 6 animals in each group. Group I served as control (CFA 100 mg, sub planter), group II served as standard (CFA, 100 mgþ indomethacin, 10 mg/kg; p.o.) and group III–X served as EACA, ACCA, MECA and HACA extract (CFA, 100 mgþ extract) at the dose level of 100 and 200 mg/kg; p.o. respectively. The drug treatment was started from day 0 and continued till 14 days. The thickness of contralateral hind paw before the injection and that of left hind paw at 0, 4, 8, 14 and 21 days after the administration of extracts was measured by using Vernier caliper. The magnitude of inflammatory response was evaluated by increase in hind paw thickness after CFA injection. However, the body weight changes were observed every day. On 22nd day, at the end of the experiment, blood was collected in plain and EDTA containing tubes for biochemical and hematological examination respectively.

2.7. Immunohistochemical evaluation Arthritic animals were divided into 3 groups comprising of 6 animals in each group. Group I served as Control (CFA 100 mg, sub planter), group II served as standard (CFA, 100 mgþindomethacin 10 mg/kg; p.o.) and group III served as ACCA extract treated (CFA, 100 mgþ extract, 200 mg/kg; p.o.). In AIA arthritis, chronic activation of the HPA axis is seen on 7–21 days after adjuvant injection and found to be maximum on 14th day (Sarlis et al., 1992). Thus, on 14th day, when the swelling appears on right hind paw, animals from each group were perfused transcardially. Brain of animals from each group (n¼5) were isolated for immunohistochemical profiling of NFκB in Paraventricular nucleus (PVN) with NFκB p65 antibody using the streptavidin–biotin–peroxidase method (Dandekar et al., 2009). Briefly, the rats were deeply anesthetized with thiopentone sodium (65 mg/kg, i.p.), perfused transcardially with heparinized phosphate-buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed, post-fixed in the same fixative for overnight and then cryoprotected in 30% sucrose solution in PBS at 4 1C for 3–4 days. After that, brain was embedded in polyvinyl–pyrrolidone, serially sectioned in the coronal plane at 30 μm thickness using a cryostat (Leica, CM1850) at  28 1C and collected in PBS. Sections were washed three times (10 min each) in PBS and incubated in rabbit polyclonal antibody against NFκB p65 diluted in PBS (1:1000) (Cat. No. sc-109, Santa-Cruz Biotechnology, USA) containing 2% normal horse serum, 0.3% Triton X-100, 0.2% Kodak PhotoFlo solution and 0.08% sodium azide for 48 h at 4 1C on shaker. The sections were washed in PBS and incubated with biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA; 1:100) for 2 h at room temperature. This serves as a bridge between primary antibody and the avidine peroxidase complex. Followed by three washings with PBS, the sections were incubated in ExtrAvidin-peroxidase conjugate (Sigma, St. Louis, MO, USA; 1:100) for 45 min at room temperature. For visualization of immunoreactions product, the sections were incubated for 3 min in a chromogenic substrate solution containing 0.03% hydrogen peroxide and 3-amino-9-ethyl-carbazole (AEC, Sigma). This chromogenic substrate reacts with the peroxidase reagent in the presence of the substrate hydrogen peroxide to form a colored precipitate. Reddishbrown precipitate indicated the presence of antigen (NFκB p65) in the sections. The sections were washed with double distilled water and mounted in glycerol–gelatin.

Please cite this article as: Arora, S.K., et al., Involvement of NFκB in the antirheumatic potential of Chenopodium album L., aerial parts extracts. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.026i

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2.7.1. Morphometric analysis The area occupied by NFκB-immunoreactive cells in the PVN was evaluated using microscopic images from predetermined areas in the sections. The images (  480) were analyzed using Leitz-LaborLux S microscope, DFC 450 C video camera system (Leica Microsystems, Germany) and Leica-Qwin Standard software (version 3). The method has already been standardized in our laboratory. The area (um2) covered by NFκB immunoreactivity in the cells was estimated from the coronal sections passing through the PVN from different treatment group of rats. The images of immunoreactive cells were digitized, the background was considered as threshold and areas occupied by immunostained cells were measured based on individual pixel intensity in all groups. The immunoreactive cells above the threshold were filled with overlaid color and the area of the color overlay was automatically obtained using Leica-Qwin standard software. Five measurements from predetermined fields of the PVN on both sides of each brain were taken. The data from all the animals in each group were pooled separately and the mean 7SEM was calculated. 2.8. Statistical analysis All the data concerning antiarthritic study, in vitro antioxidant activity, changes in blood parameters and body weight were expressed as mean 7SD. Statistical differences between the treatments and the controls were tested by two-way analysis of variance (ANOVA) followed by Bonferronis multiple comparison post-hoc tests using the “GraphPad-Prism” statistic computer program. The immunohistochemical data were analyzed by unpaired t-test. A difference in the mean values of p o0.05 or less was considered statistically significant.

expressed in rutin equivalents (RE), was found to be in the decreasing order of ACCA 4HACA 4EACA 4MECA. The flavanone content (mg/g) determined by the regression equation of the calibration curve (y¼ 0.006x þ0.020, r2 ¼0.995) and expressed in naringin equivalents (NE), was observed in the decreasing order of HACA 4MECA 4ACCA 4EACA. To ascertain the complexity of the extract, degree of polymerization was calculated. The highest polymerization was observed in MECA extract and lowest in ACCA extract. 3.3. in vitro antioxidant activities Antioxidant activity of the plant extracts were determined by two different methods. In DPPH assay, MECA extract has shown most potent activity, while in NO assay, EACA extract has shown the potent antioxidant activity. Our results revealed that, amongst the extracts of Chenopodium album plant, the MECA extract which was low in flavonoid and polyphenol content has shown lowest IC50 value (13.85 mg/ml) followed by the flavonoid rich ACCA extract (18.72 mg/ml) in DPPH assay. On the other hand EACA extract which was least in polyphenol and total flavonoid content has shown potent antioxidant activity in NO assay with IC50 value of 498.41 mg/ml followed by ACCA extract having IC50 value of 527.40 mg/ml. These results were in opposition to many published data where measured antioxidant capacity depends on the polyphenol content. While, HACA extract has shown least potent activity in both the assay (Table 1). Table 2 Effect of different extracts of Chenopodium album on % inhibition of paw thickness in adjuvant induced arthritic rats. Treatment1;#

3. Results

% Inhibition of paw thickness Day 4

3.1. Phytochemical screening Indomethacin (10 mg/kg; p.o.) EACA (100 mg/kg; p.o.) EACA (200 mg/kg; p.o.) ACCA (100 mg/kg; p.o.) ACCA (200 mg/kg; p.o.) MECA (100 mg/kg; p.o.) MECA (200 mg/kg; p.o.) HACA (100 mg/kg; p.o.) HACA (200 mg/kg; p.o.)

The phytochemical screening of EACA extract has shown the presence of tannins, sterol and flavonoids. ACCA extract has shown the presence of proteins, tannins and flavonoids. However, MECA and HACA extracts has shown the presence of proteins, carbohydrates, tannins, saponin and flavonoids. 3.2. Determination of total polyphenol, flavonoid, flavanones and degree of polymerization The content of polyphenol (mg/g) determined by the regression equation of the calibration curve (y¼ 0.005xþ 0.075, r2 ¼0.998) and expressed in gallic acid equivalents (GAE), was observed in the decreasing order of HACA4 ACCA4 MECA4EACA. The content of flavonoids (mg/g) determined by the regression equation of the calibration curve (y¼0.018x  0.001, r2 ¼0.990) and

nnn

33.03 3.500 25.19 11.42 34.35 8.337 24.50 – –

Day 8 nnn

54.40 11.50 46.93nn 19.88 57.94nn 17.19 48.03nn 3.49 9.19n

Day 14 nnn

69.94 25.50 65.21nn 34.26 65.64nn 23.97 59.31 8.30 53.14nn

Day 21 86.78nnn 31.60 72.62 40.60nnn 80.00nnn 33.35 68.13 25.34nnn 64.38nnn

The data are expressed in mean 7 S.E.M. n¼6 in each group. EACA, ethyl acetate extract of Chenopodium album; ACCA, acetone extract of Chenopodium album; MECA, methanolic extract of Chenopodium album; HACA, 50% methanolic extract of Chenopodium album. # All groups received CFA 100 mg, (Sub Planter) at day zero and individual treatments as per the groupings. n Represents statistical significance vs. arthritic control (po 0.05). nn Represents statistical significance vs. arthritic control (p o0.01). nnn Represents statistical significance vs. arthritic control (p o 0.001).

Table 1 Total polyphenol, flavonoid, flavanone, total flavonoid content degree of polymerization and antioxidant potential of different extracts of Chenopodium album. Sr. Extracts Total polyphenol content no. (TP) (GAE mg/g of extract) 1. 2. 3. 4.

EACA ACCA MECA HACA

14.56 7 0.47 30.53 7 0.72 25.53 7 0.55 42.007 0.36

Flavonoid content (TFA) Flavanone content (TFO) Total flavonoid (RE mg/g of extract) (NE mg/g of extract) content (TF)1;#

Degree of Antioxidant activity (IC 50 polymerization values in lg/ml) DPPH⋯NO

1.534 7 0.34 6.3737 0.02 1.4987 0.27 6.025 7 0.01

9.491 4.792 17.04 6.97

0.6717 0.01 0.9627 0.23 1.0117 0.01 1.0377 0.870

2.209 7.335 2.509 7.062

42.34⋯498.41 18.72⋯527.40 13.85⋯778.02 46.69⋯792.58

Results are mean7 SD of three replicates: GAE, RE and NE: gallic acid, rutin and naringin equivalents, respectively. The estimation of the degree of polymerization is calculated by the ratio between TP and TFA. EACA, ethyl acetate extract of Chenopodium album; ACCA, acetone extract of Chenopodium album; MECA, methanolic extract of Chenopodium album; HACA, 50% methanolic extract of Chenopodium album. #

Total flavonoid content is determined by adding flavonoid content with flavanone content.

Please cite this article as: Arora, S.K., et al., Involvement of NFκB in the antirheumatic potential of Chenopodium album L., aerial parts extracts. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.026i

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reduction in rat paw thickness with 53.14% and 64.38%, respectively (Table 2).

3.4. Evaluation of anti-arthritic activity Acute toxicity studies revealed the non-toxic nature of plant extracts. There was no lethality or toxic reactions upto a dose level of 5000 mg/kg, p.o. Anti-arthritic activity has shown that, amongst the different extracts, ACCA extract and indomethacin have shown a significant reduction in rat paw thickness, while comparing with the arthritic control. Results obtained from the experiment revealed that, the extracts have shown antiarthritic activity in the decreasing order of ACCA4 EACA4 MECA4 HACA. ACCA at the dose of 200 mg/kg p.o. extract shows 34.35%, 57.94%, 65.64% and 80.00% inhibition of paw thickness on 4th, 8th, 14th and 21st day of study, respectively, which was comparable to standard drug indomethacin (10 mg/kg; p.o.). While HACA extract did not show any reduction in rat paw thickness on the 4th day in both selected dose and marginally lowered the thickness on 8th day of treatment. However, on 14th and 21st day of study, the HACA extract at dose level 200 mg/kg (p.o.) has shown significant

3.5. Determination of changes in hematological and biochemical parameters The changes in hematological parameters in adjuvant induced arthritic rats are shown in Fig. 1. The RBC and hemoglobin count significantly decreased and WBC count and ESR increased in arthritic rats as compared to normal rats. The treatment with standard and extracts has significantly brought back the altered hematological changes in both developing and developed phases of CFA induced arthritis. The results of total and individual protein levels are presented in Fig. 2. In AIA rats, there was significant decrease (po0.001) in total protein, albumin levels and A/G ratio, but significant (po0.001) increase in globulin level as compared to normal group. On treatment with all the extracts of Chenopodium album and indomethacin,

Normal Control Std (10mg/kg) EACA (200 mg/kg) ACCA (200 mg/kg) MECA (200 mg/kg) HACA (200 mg/kg)

20

*** ***

15

** 10

***

*** 5

*** ***

*** ***

**

0 RBC (millons/mm 3 )

WBC (1000/mm 3 )

ESR (mm/hr)

Hb (g/dl)

Hematological Parameters Fig. 1. Effect of Chenopodium album extracts on hematological parameter in adjuvant induced arthritic rats. On 22nd day of adjuvant induction, hematological parameters were estimated. The altered hematological parameters (Hb, RBC, WBC and ESR) in the arthritic rats were significantly brought back to near normal level by the treatment with standard drug Indomethacin and plant extracts. The bars values are shown as the mean 7SEM of five measurements. The data were analyzed by Bonferroni posts-test using the “GraphPad-Prism” statistic computer program. *P o 0.05, **Po 0.01, ***P o 0.001 vs. CFA control. EACA, ethyl acetate extract of Chenopodium album; ACCA, acetone extract of Chenopodium album; MECA, methanolic extract of Chenopodium album; HACA, 50% methanolic extract of Chenopodium album.

10 9 8 7

** **

****

6 5

* **

*** *

Normal Control Std (10mg/kg) EACA (200 mg/kg) ACCA (200 mg/kg) MECA (200 mg/kg) HACA (200 mg/kg)

4 3 2 1 0 Creatinine (mg/dl)Protein (g/dl)

Albumin (g/dl) Globulin (g/dl)

A/G Ratio

Biochemical Parameters Fig. 2. Effect of Chenopodium album extracts on biochemical parameter in adjuvant induced arthritic rats. On 22nd day of adjuvant induction, hematological parameters were estimated. The altered biochemical parameters (creatinine, albumin, globulin and A/G ratio) in the arthritic rats were significantly brought back to near normal level by the treatment with standard drug Indomethacin and plant extracts. The bars values are shown as the mean 7 SEM of five measurements. The data were analyzed by Bonferroni posts-test using the “GraphPad-Prism” statistic computer program. *p o 0.05, **p o0.01, ***p o 0.001 vs. CFA control. EACA, ethyl acetate extract of Chenopodium album; ACCA, acetone extract of Chenopodium album; MECA, methanolic extract of Chenopodium album; HACA, 50% methanolic extract of Chenopodium album.

Please cite this article as: Arora, S.K., et al., Involvement of NFκB in the antirheumatic potential of Chenopodium album L., aerial parts extracts. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.026i

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Fig. 3. Photomicrographs showing NFκB -immunoreactive cells (arrows) in the paraventricular nucleus of hypothalamus. Single injection of CFA activated the expression of NFκB in hypothalamic paraventricular nucleus (PVN) at day 14 post-injection (A). Treatment with standard drug (Indomethacin) (B), and extract ACCA (C) showed significant reduction in the NFκB immunoreactivity. The morphometric analysis of NFκB-immunoreactivity is represented in bar graph (D). The outline of the transverse section through brain indicates the regions of the PVN at the co-ordinates  1.80 mm with respect to bregma (Paxinos G, 1998) from which the measurements were collated (square, not to scale). 3 V, third ventricle; AHC, central part of the anterior hypothalamus; Rch, retrochiasmatic nucleus. Scale bar ¼200 mm. The bar values are shown as the mean7 SEM of five measurements from predetermined fields of the PVN on both the sides of each brain (n¼ 5/group). The data were analyzed by unpaired t-test. *p o 0.05, **po 0.01 vs. CFA control.

it was observed that all these hematological and biochemical changes were restored back near to normal.

3.6. Effect on body weight In the present study, it was evident that, there was a close relationship between the extent of joint inflammation and the degree of weight loss. In the first week after adjuvant injection, the arthritic rats showed marked weight loss, followed by normal weight gain in the subsequent weeks, whereas the Chenopodium album extracts and indomethacin treated groups did not show any weight loss. Based on the results, ACCA extract was selected for further immunohistochemical evaluation of NFκB.

3.7. Immunohistochemical evaluation and morphometric analysis In order to assess possible mechanism of ACCA extract for its antiarthritic activity, NFκB inhibition assay was carried out by immunohistochemistry. Results obtained from experiment revealed that, ACCA extract significantly reduced the expression of NFκB in the PVN of hypothalamus and this inhibition was found to be comparable with that of standard drug indomethacin. In the present investigation, the morphometric analysis of the area occupied by NFκB immunoreactive cells in the PVN of hypothalamus was evaluated using microscopic images of the brain sections. Changes in the immunohistochemical profile of the NFκB -immunoreactive cells in the PVN of standard and extract treated rats were recorded. A significant reduction in the NFκB -immunoreactive cells was observed in the ACCA extract treated rats (49.82%; po 0.001) as compared to control animals after 14 days of

Please cite this article as: Arora, S.K., et al., Involvement of NFκB in the antirheumatic potential of Chenopodium album L., aerial parts extracts. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.026i

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treatment (Fig. 3). This study also provides evidence on the involvement of NFκB and HPA axis in AIA models of RA.

4. Discussion The results reveal that extracts of plant Chenopodium album possess significant antioxidant and antiarthritic activity. In order to study the relationship between antioxidant and antiarthritic activity, secondary metabolites present in Chenopodium album plant were determined. Flavones, flavonols and isoflavones form complexes only with aluminum chloride, while flavanones strongly react only with 2,4-dinitrophenylhydrazine, so the contents determined by the two methods were added up to obtain the total flavonoid content. It has been observed that flavonoid and flavanone content represent only 14.34% (w/w) and 2.47% (w/w) of total polyphenol in HACA extract. Similar pattern is observed in all the other extracts, suggesting that the extracts are very complex and contain many other polyphenols (such as chalcones, isoflavones, phenolic acids, etc.). Thereby, degree of polymerization of the polyphenols present in the samples is high. To ascertain the complexity of extract, degree of polymerization was calculated as it may also affect the solubility and steric accessibility of molecule which in turn have effect on the antioxidant and antiarthritic activity of extracts. On the other hand in DPPH assay, antioxidant activity was observed in the order of MECA4 ACCA 4EACA 4 HACA (Table 1). It was observed that EACA extract has shown higher antioxidant potential than the HACA extract having higher amount of polyphenols and flavonoid may be by virtue of its sterol content and also the other secondary metabolites. However, in NO radical scavenging activity, nearly inverse pattern of antioxidant activity was observed for the extracts. The more non-polar extracts (EACA and ACCA) have shown most potent scavenging activity than the polar extract. The higher antioxidant potency of polar extract in DPPH assay than NO assay can be explained, as both DPPH and NO assays measure the free radical scavenging activity but DPPH is carried out in an organic environment (an alcohol for example), while NO assay requires a buffered aqueous solution. Although the NO assay is carried out in an aqueous buffer, the NO molecule itself is a lipophilic species, hence have higher compatibility to such compounds that could both disperse well in the buffer and interact with the free radical. These results are in conformation with previous findings that antioxidant capacity of an extract depends greatly on the methodology used, specifically, the oxidant and the oxidizable substrate used in the measurement (Cao and Prior, 1998). The obtained antioxidant result also confirms that measured antioxidant capacity does not depend upon the polyphenol content alone. At the same time, there are also many reports on the ambiguous or even adverse relationships between polyphenols as a whole and the antioxidant capacity (Bolling et al., 2010; Chew et al., 2009; Gregoris and Stevanato, 2010; Sun and Ho, 2005). Thus, it is evident that successive extraction has resulted in segregation of compounds with high antioxidant potential. This would suggest that either only some polyphenol actually act as antioxidant or there is also a substantial contribution of nonphenolic compounds. Among the extracts, ACCA extract was effective in both developing (initial stages) and developed phases of arthritis. It is also clear from the study that not only the polyphenol and flavonoid present in the ACCA extract but some other phytoconstituents also accounts for its synergistic effect during the initial days of treatment. These activity can be attributed to presence of some flavonoidal aglycones, including quercetin which is reported to block both the cyclooxygenase and lipoxygenase pathways of the arachidonate cascade at relatively high concentrations, while at lower concentrations only the lipoxygenase pathway is blocked

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(Di Carlo et al., 1999). While, the HACA extract rich in polyphenols and flavonoid was effective only in developed phase of arthritis. This can be explained as most of the flavonoids are present in glycosidic form which are water soluble and are effective at developed phase of arthritis. While, compared to EACA and ACCA extracts, HACA and MECA extracts contain saponins as additional phytoconstituents that may also account for their antiarthritic activity in the later stages of arthritis. Similarly, EACA, ACCA and MECA extract has also shown the significant antioxidant activity compared to HACA extract. Also, the free radical scavenging properties contributes to their anti-inflammatory actions (Comalada et al., 2006). Pronounce antiarthritic effect of MECA and EACA extract than HACA extracts may be attributed to its increased degree of polymerization. Park et al. reported that monomeric, dimeric and trimeric flavonoids (polymerized flavonoid) displayed a selective activity in terms of TNF-α secretion, NO production and NFκB dependent gene expression in RAW 264.7 macrophages. In addition, he suggestted that the degree of polymerization of flavonoids seems to be important in determining the mechanism(s) by which flavonoids may exert their immunomodulatory and antiinflammatory activity. Monomers and dimers inhibited NO production, TNF-α secretion and NFκB dependent gene expression induced by interferon γ, whereas, the trimeric procyanidin enhanced these parameters. It is also evident from previous studies that with increase in degree of polymerization of extract, cytotoxic, anti-inflammatory and immunomodulatory activity increases (Park et al., 2000). On the other hand, it is also reported that individual composition of polymerized flavonoid (monomeric, dimeric, trimeric and oligomer) also plays an important role in determining its effect on biological activity. Due to increased molecular size, absorption of polymeric flavonoids across the intestinal epithelium requires preliminary degradation to smaller and low molecular weight compounds. Procyanidin dimers and trimers, but not oligomer averaging 7 units, are capable of translocating across the small intestinal epithelium (Heim et al., 2002). Thus, upto a certain degree of polymerization of the extract the biological activity increases and hence it is interesting to consider this aspect in relation to the observed effects in this study. Reduction in expression of NFκB pathways by ACCA extract, may be attributed to the presence of important secondary metabolites such as polyphenols, flavonoids, saponins and proteins which are in agreement with previously published studies (Laladhas et al., 2010). Previous studies have shown that polyphenols exert their antiinflammatory activity by modulating NFκB activation and acting at multiple steps of the activation process (Rahman et al., 2006). Moreover, a recent ex vivo study demonstrated that quercetin inhibited TNFα-induced expression of the pro-inflammatory cytokines interferon- inducible protein 10 and macrophage inflammatory protein-2 in primary murine small intestinal epithelial cell. In lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treated BV-2 microglia, quercetin suppressed NO production and inducible nitric oxide synthase (iNOS) gene transcription by reducing activation of I kappa B kinase (IKK), NFκB, activating protein-1, signal transducer and activator of transcription-1 (STAT 1) and interferon regulatory factor1 (IRF-1). In addition, quercetin inhibited DNA binding activity of NFκB in a dose-dependent manner (Chen et al., 2005). Thus the treatment of arthritis with the ACCA extract significantly suppressed the expression of NFκB, which may be attributed to the synergistic action of the phytoconstituents present in it, thereby justifying their significant role in arthritic conditions.

5. Conclusion It can be concluded from the study that, extracts of plant Chenopodium album possess significant antioxidant and antiarthritic

Please cite this article as: Arora, S.K., et al., Involvement of NFκB in the antirheumatic potential of Chenopodium album L., aerial parts extracts. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.026i

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activity. Immunohistochemistry studies revealed the prominent antiarthritic activity of the ACCA extract, mainly because of its capacity to inhibit the NFκB protein. Thus, through these preclinical studies, the selected plant Chenopodium album is found beneficial on the treatment of arthritis and NFκB induced diseases. The ACCA extract also provides a new insight in the treatment of arthritis with the enrichment of secondary metabolites. It is therefore re-envisaged that, the biomolecules of this plant species can be a good source of phytomedicine in the prophylaxis, management and cure of NFκB induced diseases including arthritis. Acknowledgments The authors are thankful to the Council of Scientific and Industrial Research, New Delhi, India for providing financial support. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.05.026. References Bolling, B.W., Dolnikowski, G., Blumberg, J.B., Chen, C.Y.O., 2010. Polyphenol content and antioxidant activity of California almonds depend on cultivar and harvest year. Food Chemistry 122, 819–825. Cao, G., Prior, R.L., 1998. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clinical Chemistry 44, 1309–1315. Chen, J.C., Ho, F.M., Pei-Dawn Lee, C., Chen, C.P., Jeng, K.C., Hsu, H.B., Lee, S.T., Wen Tung, W., Lin, W.W., 2005. Inhibition of iNOS gene expression by quercetin is mediated by the inhibition of IkappaB kinase, nuclear factor-kappa B and STAT1, and depends on heme oxygenase-1 induction in mouse BV-2 microglia. European Journal of Pharmacology 521, 9–20. Chesnokova, V., Melmed, S., 2000. Leukemia inhibitory factor mediates the hypothalamic pituitary adrenal axis response to inflammation. Endocrinology 141, 4032–4040. Chew, Y.-L., Goh, J.-K., Lim, Y.-Y., 2009. Assessment of in vitro antioxidant capacity and polyphenolic composition of selected medicinal herbs from Leguminosae family in Peninsular Malaysia. Food Chemistry 116, 13–18. Comalada, M., Ballester, I., Bailon, E., Sierra, S., Xaus, J., Galvez, J., de Medina, F.S., Zarzuelo, A., 2006. Inhibition of pro-inflammatory markers in primary bone marrow-derived mouse macrophages by naturally occurring flavonoids: analysis of the structure-activity relationship. Biochemical Pharmacology 72, 1010–1021. Cutillo, F., D'Abrosca, B., DellaGreca, M., Di Marino, C., Golino, A., Previtera, L., Zarrelli, A., 2003. Cinnamic acid amides from Chenopodium album: effects on seeds germination and plant growth. Phytochemistry 64, 1381–1387. Dandekar, M.P., Singru, P.S., Kokare, D.M., Subhedar, N.K., 2009. Cocaine- and amphetamine-regulated transcript peptide plays a role in the manifestation of depression: social isolation and olfactory bulbectomy models reveal unifying principles. Neuropsychopharmacology 34, 1288–1300. Di Carlo, G., Mascolo, N., Izzo, A.A., Capasso, F., 1999. Flavonoids: old and new aspects of a class of natural therapeutic drugs. Life Sciences 65, 337–353. Eskandari, F., Webster, J.I., Sternberg, E.M., 2003. Neural immune pathways and their connection to inflammatory diseases. Arthritis Research and Therapy 5, 251–265.

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Please cite this article as: Arora, S.K., et al., Involvement of NFκB in the antirheumatic potential of Chenopodium album L., aerial parts extracts. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.05.026i