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Int J Med Res. 2011; 1(3): 163-169

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ISSN: 0976-8971

International Journal of Medicobiological Research

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Research article

Evaluation of Anti-Inflammatory Activity on Justicia genadarussa Burm.f. Leaf Extract in Adjuvant Induced Arthritic Rats

Manickam Chinna , Ponnuswamy Renuka Devi1, Mohamad Froze Fazaludeen2

1

Biotechnology centre, Anna University of Technology, Coimbatore, Mettupalayam road, Jothipuram post, Coimbatore-641047. Tamil Nadu, India. 2University of Hail, Kingdom of Saudi Arabia, Hail. Corresponding author: [email protected]

Article information

Keywords: Justicia gendarussa, Anti-inflammatory, Lipid peroxide, Antioxidants, Paw volume

ABSTRACT

The present investigation has aimed to evaluate the anti-inflammatory activity of Justicia gendarussa in freund's complete adjuvant induced arthritic rats. The methanolic leaf extract of Justicia gendarussa was administered orally at a limit dose of 300 mg/kg b.w. for 14 days to the experimental rats. The anti-inflammatory activity of Justicia gendarussa leaves was evaluated by paw volume measurement and its ability to stabilize lipid peroxide level in experimental rats. The activities of enzymatic antioxidants such as SOD, CAT, GPx, GR and GST and the levels of non-enzymatic antioxidants such as GSH and vitamin C were also measured in hemolysate and liver. The paw volume and lipid peroxide level of hemolysate and liver in arthritic rats was significantly reduced to near normal level by administration of methanolic leaf extract of Justicia gendarussa. The decreased level of enzymatic antioxidants activities and non-enzymatic antioxidants levels were reverted to normal levels when methanolic extract given to arthritic rats. This study also found that there were no changes in lipid peroxide and antioxidant levels in extract administered normal rats. The preliminary phytochemical analysis of methanolic extract showed the presence of many biologically active phytochemicals such as flavonoids, alkaloids, phenolic compounds, saponins, glycosides, and tannins and these compounds might be responsible for the anti-inflammatory properties found in the present study. The possible anti-inflammatory mechanism of the Justicia gendarussa leaf extract may be through its free radical scavenging activity, its stabilizing action on lipid peroxide and increase antioxidants levels. Thereby, methanolic leaf extract of Justicia gendarussa preventing the spread of inflammation. species (ROS) which include superoxide (O2-), hydrogen peroxide (H2O2) and hydroxyl (OH·) radicals which increase the cell membrane permeability initiated by oxidation of lipids (formation of lipid peroxide) on the membrane. This results in capillary dilation causing redness and pain, and increased vascular permeability allows plasma to escape into the surrounding tissue giving rise to edema.[3-4] Other evidence suggested that ROS activate activated proteins-1 (AP-1), nuclear factor-kappa B (NF-B) and mitogen activated protein kinase family through oxidation of critical cysteine residue present in that signaling proteins, which are involved in pathogenesis of

Received on: 08.07.2011 Revised on: 15.07.2011 Accepted on: 21.07.2011 1. INTRODUCTION

Rheumatoid arthritis (RA) is a chronic joint inflammatory disease that affects more than 1% of the general population and is more prevalent among women's and in developed countries.[1] RA is characterized by proliferation of synovial cells and infiltration of the joints by a various inflammatory cells that result in the formation of pannus tissue, which invades and destroys adjacent cartilage and bone.[2] The etiopathogenesis of RA is still unknown; however, experimental data suggest the inflammatory cells produce wide variety of reactive oxygen

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Manickam Chinna, et al.,/Int J Med Res. 2011; 1(3):163-169 RA.[5-8] The activated NF-B increase expressions of both nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) which lead to pain, swelling and destruction of cartilage (9) and activated AP-1 increase expression of MMPs which also leads to destruction of cartilage at joint in RA condition.[10]

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Kiliani, and Molish test), phenols (ellagic acid and phenol test), saponins (foam and haemolysis test), sterols (LibermanBurchard and Salkowski test) and tannins (gelatin test) were carried out according to Dey et al.,[18] and Evans,[19] method. The quantitative estimation of polyphenols (Folin-Ciocalteu's asay) and flavonoids (aluminium chloride colorimetric assay) were Normally, ROS are effectively detoxified by antioxidants carried out according to Marinova et al.,[20] method. such as Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx), Glutathione reductase (GR), 2.4. Free radical scavenging activity Glutathione-S-Transferase (GST), Glutathione (GSH) and Vitamin C.[11] However, in conditions of RA there is a decreased The DPPH and H2O2 scavenging activity of the extracts and production of the body's endogenous antioxidants. This, phytochemical fractions were measured by simple UV therefore, leads to the overproduction of free radicals causing spectroscopic method. The absorbance of 0.004% DPPH and oxidative stress.[12] A treatment with antioxidants decrease ROS 30mM H2O2 solution were recorded at 514nm and 230nm generation, restore the endogenous antioxidants activity and respectively with or without phytochemical fractions. The reverting joint destruction. As consequences, antioxidants appear percentage of scavenging activity of methanolic leaf extract was to be promising agents for treatment of RA. calculated by using following formula The percentage of scavenging activity of MLE was calculated by Indian traditional medicine system used Justicia gendarussa using following formula leaves to treat fever, hemiplegia, rheumatism, arthritis, headache, earache, muscle pain, respiratory disorders, and digestive trouble.[13-14] In recent experimental and clinical data Where, A0 is the absorbance of the control and A1 is the have provided evidence for hepatoprotective and anti-arthritic [15-16] absorbance of sample Adjuvant induced activity of leaves of Justicia gendarussa. by intradermal injection of freund's complete adjuvant (FCA) is an accepted experimental model of chronic inflammation that 2.5. Animal Study exhibits several pathological changes similar to those occurring 2.5.1. Experimental animals in RA.[17] Hence, the present study was undertaken to evaluate anti-inflammatory activity of methanolic leaf extract (MLE) of The anti-inflammatory study was conducted on male Wistar justicia gendarussa by analysis of paw volume, lipid peroxide albino rats (100-150g). The rats were housed in large spacious polypropylene cages, they were given food, and water supplied and various antioxidant parameters in AIA. by Hindustan Lever Ltd., Mumbai, India under the trade name Gold Mohur rat feed. The rats were acclimatized for 1 week 2. MATERIALS AND METHODS before beginning of the experiment. 2.1. Plant collection The Justicia gendarussa leaves were collected from Kanjicode 2.5.2. Acute toxicity test hills, Kerala, India, in November 2009 and authenticated by Dr. Arumugaswamy, Professor and Department of botany from the Rats were divided into two groups of three each. Rats were kept for fasting for overnight providing only water then first group Kongunadu Arts and Science College, Coimbatore, India. received 0.2% CMC (carboxyl methyl cellulose), this group acts as a vehicle control and second group received 3000 mg/kg b.w. 2.2. Preparation of leaf extract of MLE suspended in 0.2% CMC, this group acts as a test group. Leaves of Justicia gendarussa was air dried at room Immediately after the dose, Rats were continuously watched for temperature, coarsely powdered and extracted with absolute mortality and behavioral responses for 48 hr and once daily till th th methanol by soxhlet apparatus for 20 hr. The resulting extract the 14 day. Dose selection was performed by taking 1/10 of was concentrated and equal volume of double distilled water was the lethal dose. added and incubated for 24 hr. After the incubation period, chlorophylls were precipitated and removed by filtration and the 2.5.3. Experimental design and anti-inflammatory studies resulting filtrate was evaporated in a rotary evaporator at a maximum temperature of 45 C. The residue was stored in a Animals were divided into four groups of six animals each. Group I served as control rats receiving 0.2% CMC, Group II refrigerator at 4-5oC, until use. served as arthritic rats induced by subcutaneous injection of 0.1 ml of freund's complete adjuvant (10 mg heat killed 2.3. Phytochemical analysis mycobacterium tuberculosis per ml of paraffin oil) into the planter surface of the left hind paw. Group III served as extract The MLE was subjected to qualitative estimation of phytochemicals such as alkaloids (Wagner and Dragendorff's treated arthritic rats receiving orally 300 mg/kg b.w. of MLE test), flavonoids (Shinoda and NaOH test), glycosides (Keller- suspended in 0.2% CMC after 14 days from the day adjuvant

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Manickam Chinna, et al.,/Int J Med Res. 2011; 1(3):163-169 injection by gastric intubation and was terminated on 28th day. Group IV served as drug control rats receiving 300 mg/kg b.w. of MLE suspended in 0.2% CMC for 14 days by gastric intubation. Inflammation was assessed by measuring the left hind paw volume on 0, 4, 8, 12, 16, 20, 24and 28th day using digital plethysmometer. 2.5.4. Experimental procedure After the 28th day, rats were sacrificed by cervical decapitation. Blood was collected with EDTA and centrifugation at 1500 ×g (4°C) for 10 min. the red cells were washed three times with phosphate buffered saline (pH: 7.2) and the packed cells were hemolysed by adding an equal volume of cold distilled water. The liver was dissected out immediately and washed with icecold saline solution. The 10% of liver tissue homogenate was prepared in tris­HCl (0.1 M, pH 7.4) buffer by using tissue homogenizer. After centrifugation of homogenate at 18000×g for 15 min at 4°C, the supernatant was extracted, and kept at -20°C in advance of assays. 2.5.5. Biochemical assays The level of lipid peroxidation estimated by Ohkawa et al.,[23] method, the enzymatic antioxidant activities such as superoxide dismutase estimated by (SOD) Marklund et al.,[24] method, catalase estimated by (CAT) Sinha[25] method, glutathione peroxidase estimated by (GPx) Rotruck et al.,[26] method, glutathione reductase estimated by (GR) Carlberg et al.,[27] method, glutathione-S-transferase estimated by (GST) Habig et al.,[28] method and the non-enzymatic antioxidant such as glutathione estimated by (GSH) Moron et al.,[29] method and vitamin C estimated by Omaye et al.,[30] method. 3. RESULTS AND DISCUSSION 3.1. Prelimanry phytochemical analysis. The results of qualitative estimation of phytochemical in MLE are depicted in table1. The estimated level of polyphenol and flavonoids in MLE are 253.7±28.3 and 142.3±9.4 µg/mg respectively. Eva Guillamon et al.,[31] have reported that the Flavonoids are polyphenol subclass, it exhibited antiinflammatory activity through several mechanisms includes antioxidative and free radical scavenging activities, regulation of cellular activities of inflammation-related cells, modulation of the activities of arachidonic acid metabolism enzymes (phospholipase A2, cyclooxygenase, lipoxygenase) and nitric oxide synthase, modulation of the production of other proinflammatory molecules and modulation of proinflammatory gene expression. 3.2. Free radical scavenging activity Purple color of stable DPPH free radicals decolorized when accepting electron from antioxidant molecules, which can be quantitatively measured from changes in absorbance at 517nm. The scavenging or break down of H2O2 by MLE can be

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measured from change in absorbance at 230 nm. The IC50 values of DPPH and H2O2 scavenging ability of MLE are 54.59±9.3 and 326.3±13.7 µg/ml respectively. These results explained that polyphenol composition in MLE showed the higher redox potentials which allow them to act as reducing agents and hydrogen donors.[32] Table. 1. Secondary metabolites detected in MLE of Justicia gendarussa. Name of secondary metabolites Alkaloids Flavonoids Glycosides Phenols Saponins Name of the test Wagner test Dragendorff's test Shinoda Test NaOH test Keller-Kiliani test Molish test Ellagic acid test Phenol test Foam test Haemolysis test MLE ++ ++ ++ + + + ++ +++ ++ +

3.3. Animal Studies 3.3.1. Acute toxicity study of methanolic leaf extract of Justica gandarussa. Acute toxicity studies of JG extract at limited dose of 3000mg/kg b.w. showed no mortality and no adverse effects in animals up to 14 days, indicating that extract was nontoxic in rat up to an oral dose of 3000 mg/kg b.w. In anti-inflammatory studies, the experimental rats were administered with1/10th of limit dose. 3.3.2. Anti-inflammatory activity of methanolic leaf extract (MLE) in AIA rat model. The FCA arthritis develops acute local inflammatory reaction within 24 hr that subside after 8­10 days and then develop secondary lesions, which is characterized by inflammation at non-injected sites, and further increase in paw volume in FCA injected hind leg.[33] The determination of degree of inflammation in hind paw is very useful and sensitive method for evaluating anti-inflammatory activity and therapeutic effect of treatment. In the present study, it was found that the paw volume reached its maximum on the 14th day in adjuvant induced arthritic rats. This increased paw volume endured for the next 14 days in Group II rats. A significant reduction in paw volume was found in the methanolic leaf extract treated (group III) rats as compared to group II. The results are illustrated in (Figure. 1.). The decreased activity of antioxidants and increased production of ROS in an arthritic rat is evidence for paw swelling,[3-4] these consequence was suppressed in extract treated arthritic rats through detoxification ROS by increasing antioxidants activities in inflamed tissues.

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enzymes in arthritic conditions were reverted back close to the normal condition when MLE given to arthritic rats. The superoxide molecule can react with NO forms a highly toxic peroxynitrite, which play important role in pathogenesis of inflammation.[37] The SOD and CAT are prime enzymes providing defense against superoxide. The SOD can catalyze the dismutation of toxic superoxide into O2 and H2O2 and then CAT decompose the H2O2to H2O and O2. As the consequence of these enzyme actions can decrease lipid peroxide level and provide protective effect against oxidative damage of cells.[38-39] The results of present study show that the decreased activates of SOD and CAT were restored in MLE of Juticia gendarussa given arthritic rats.

Figure. 1. Changes in the paw volumes of rats in-group II (FCA-induced arthritic control), and group III (FCA-induced arthritic rats treated with J. gendarussa extract). All values expressed as mean±SD, n=6 animals in each groups. **-highly significant (P< .005 for the comparison of Groups I with of groups III with Group I &II on the 28th day 28th day), *- low significant (P< .005 for the comparison of groups III with Group I &II on the 28th day on the 28th day).

The GPx and GST enzymes plays important protective role against oxidative damage by breakdown of H2O2 and lipid peroxide with the help of substrate GSH. The GSH also has ability to reduce oxidized glutathione (GSSG) is catalyzed by the widely distributed glutathione reductase (GR). Thus, the level of GSH influences activities of GPx, GST and GR enzymes (4042). In our results, the decreased activities of GPx, GST, and GR were found in arthritic rats due to minimal level of GSH. In 3.3.3. In vivo anti-oxidant activity of methanolic leaf extract MLE, treated rats the activities of GPx, GST, and GR were (MLE) in AIA rat model increased with heighted level GSH. The effect of MLE on lipid peroxide level, activity of enzymatic antioxidants (SOD, CAT, GPx, GST) and concentration of non enzymatic antioxidants (GSH, Vitamin C) in hemolysate and liver tissue homogenate of various experimental rats were depicted as mean ± stranded deviation in (Table. 2.). A report by Lenton et al.,[43] pointed out vitamin C can augment GSH in human lymphocytes, thereby it inhibit lipid peroxidation. Levine et al.,[44] also reported that the vitamin C could regenerate the vitamin E from the -tocopheroxyl radical, which is formed from as a result of the inhibition of lipid peroxidation by vitamin E. The possible anti-inflammatory mechanism of vitamin C is inactivation of NF-B during inflammation process, independently of its antioxidant activity.[45] As a result, the use of vitamin C as antioxidant is their ability to detoxify oxygen free radicals and other molecules, which are involved in pathogenesis of rheumatoid inflammation and act as a cofactor in collagen synthesis, the main protein in joint tissue and bone. Flavonoids defend vitamin C from being destroyed in the body by oxidation, via reducing dehydroascorbic acid to ascorbate.[46] Thus, due to flavonoid content of MLE increase vitamin C in arthritic rats. Treatment with methanolic leaf extract of Justicia gendarussa decreased the lipid peroxide by increasing the activities of enzymatic and non-enzymatic antioxidants, It has been reported that flavonoids are good inhibitor of arachidonic acid peroxidation.[47]

In Adjuvant induced arthritis, macrophage and lymphocytes are activated due to structural mimicry between mycobacterium and cartilage proteoglycans in rat. Thus activated inflammatory cell produce large amount of ROS and hence inhibit activity of antioxidant enzyme, which leads to increased production of lipid peroxide.[34-35] This report correlates with the present investigation. The lipid peroxide was significantly increased in arthritic rats (group II). This heightened level of lipid peroxide was decreased by extract in arthritic rats. This effect might be due to presence of flavonoids in the methanolic extract. This result also correlates with Klopman et al.,[36] reports. They were reported that the inhibition of lipid peroxidation might be due to the free radical scavenging activity of flavonoids. The present study also reports that declined activities of antioxidant

Table. 2. Effects of Juticia gendarussa on biochemical parameters in liver and hemolysate of control and experimental rats. BIOCHEMICAL PARAMETERS Lipid peroxide Liver homogenate Hemolysate (µmol of MDA/mg protein) GROUP I 0.045±0.01 0.773±0.12 GROUP II 0.303±0.04** 2.19±0.19** GROUP III 0.116±0.02 0.983±0.18 GROUP IV 0.03±0.008 0.68±0.098

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Manickam Chinna, et al.,/Int J Med Res. 2011; 1(3):163-169 SOD Liver homogenate Hemolysate Units/mg CAT Liver homogenate Hemolysate (µmol of H2O2 consumed/min/mg protein) GPx Liver homogenate Hemolysate (µM GSH consumed /mim/mg protein) GR Liver homogenate Hemolysate (nmol of GSSG reduced/min/mg protein) GST Liver homogenate Hemolysate (µmol of CDNB consumed/min/mg protein) GSH Liver homogenate Hemolysate (µg/mg protein) Viatmin C Liver homogenate Hemolysate (µg/mg protein)

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2.132±0.13 1.45±0.114

1.32±0.33** 0.46±0.117*

1.96±0.102 1.143±0.245

2.163±0.151 1.493±0.126

313.41±12.6 75.57±9.21

231.7±16.1** 46.64±3.72**

286.6±7.69 63.95±8.53

320±15.47 78.14±5.85

15.84±1.02 11.16±1.23

6.54±0.94* 5.88±1.00**

13.63±0.80 9.19±0.86

15.79±1.47 11.28±0.63

8.53±0.71 0.65±0.04

5.86±0.76** 0.36±0.04*

6.99±0.30 0.60±0.036

8.58±1.033 0.66±0.08

4.21±0.7 0.19±0.04

1.9±0.17* 0.067±0.01**

3.32±0.27 0.17±0.01

4.15±±0.34 0.193±0.047

2.12±0.187 14.28±1.53

1.32±0.09* 7.43±0.84**

1.85±0.071 13.44±0.96

2.16±0.151 14.41±1.4

1.82±0.15 3.73±0.23

1.07±0.09** 2.19±0.20**

1.68±0.11 2.89±0.24

1.88±0.114 3.64±0.39

Notes: All values expressed as mean±SD, n=6 animals in each groups. **-highly significant (P<0.005 for the comparison of Groups I with Group II, Group III &IV), *- low significant (P< .005 for the comparison of Groups I with Group II, Group III &IV). Pines A., . Perrone1 L., Bivi N., Romanello M., Damante G., Gulisano M., Kelley M.R., Quadrifoglio F., Tell G. In conclusion, the results of the present study indicate that (2005). Activation of APE1/Ref-1 is dependent on reactive Justicia gendarussa methanolic leaf extract scavenging free oxygen species generated after purinergic receptor radicals due to its polyphenolic compositions and inhibit paw stimulation by ATP. Nucleic Acids Research., 33: 4379­ edema via decrease lipid peroxide, upregulate the activity of 4394. enzymatic antioxidants and increase non-enzymatic antioxidants [6] Kaul N., Forman H.J. (1996). Activation of nfkb by the level in MLE treated AIA rats as significantly. There are no respiratory burst of macrophages. Free Radical Biology & changes in biochemical parameters in normal rats treated with Medicine., 21:401-405. MLE of Justicia gendarussa [7] Guyton K.Z., Liu Y., Gorospe M., Xu O., Nikki J. (1996). Holbrook. Activation of Mitogen-activated Protein Kinase by H2O2 role in cell survival following oxidant injury. The 5. REFERENCES journal biological chemistry., 271: 4138 ­4142. [1] Woolf A.D, Pfleger B. (2003). Burden of major [8] Denu J.M, Tanner K.G. (1998). Specific and Reversible musculoskeletal conditions. Bulletin of the World Health Inactivation of Protein Tyrosine Phosphatases by Organization., 81: 646-656. Hydrogen Peroxide: Evidence for a Sulfuric Acid [2] Firestein G.S. (2003). Evolving concepts of rheumatoid Intermediate and Implications for Redox Regulation. arthritis. Nature., 423: 356­361. Biochemistry., 37: 5633-5642. [3] Halliwell B. (1995). Oxygen radicals, nitric oxide and [9] Abramson S.B. (2008). Nitric oxide in inflammation and human inflammatory joint disease. Annals of Rheumatic pain associated with osteoarthritis. Arthritis Research & Disease., 54: 505­510. Therapy;10:1-7 [4] Symons I.S., King L.J. (2003). Inflammation, reactive [10] Vincenti1 M.P., Brinckerhoff C.E. (2002). Transcriptional oxygen species and cytochrome P450. regulation of collagenase (MMP-1, MMP-13) genes in Infammopharmacology., 11: 75­86. arthritis: integration of complex signaling pathways for the 4. CONCLUSION [5]

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