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Attenuation of inflammatory mediators, oxidative stress and toxic risk evaluation of Aporosa lindleyana Baill bark extract Yakub Ali, Mohammad Sarwar Alam, Hinna Hamid, Asif Hussain, Chetna kharbanda, Sameena Bano, Syed Nazreen, Saqlain Haider

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Journal of Ethnopharmacology

Received date: 7 December 2013 Revised date: 11 June 2014 Accepted date: 16 July 2014 Cite this article as: Yakub Ali, Mohammad Sarwar Alam, Hinna Hamid, Asif Hussain, Chetna kharbanda, Sameena Bano, Syed Nazreen, Saqlain Haider, Attenuation of inflammatory mediators, oxidative stress and toxic risk evaluation of Aporosa lindleyana Baill bark extract, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2014.07.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Attenuation of inflammatory mediators, oxidative stress and toxic risk evaluation of Aporosa lindleyana Baill bark extract Yakub Alia, Mohammad Sarwar Alama, Hinna Hamida, Asif Hussainb, Chetna kharbandaa, Sameena Banoa, Syed Nazreena, Saqlain Haidera *a

Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New

Delhi-110062, India. b

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard

University), New Delhi-110062, India. Correspondence to Prof. Mohammad Sarwar Alam, Jamia Hamdard, New Delhi-110062, Email ID. [email protected] [email protected] Phone: +91 11 26059688(5555), Fax: +91 11 26059663

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Abstract Ethnopharmacological relevance: Traditionally, Aporosa lindleyana Baill. has been used against various ailments viz. jaundice, fever, headache, seminal loss and insanity. The present study aims to evaluate the anti-inflammatory and anti-oxidant activity of the ethanolic extract of Aporosa lindleyana Baill. bark and its fractions. Method: The anti-inflammatory activity of ethanolic extract of Aporosa lindleyana Baill. bark and its various fractions at doses of 200 mg/kg and 300 mg/kg b.w. has been carried out by carrageenan induced hind paw edema method. To establish the probable mechanism of action, TNF-α and NO levels have been estimated by ELISA method and the effect of active fraction on COX-2 and NF-κB expressions has been evaluated. The effect on the levels of anti-oxidative enzymes (CAT, SOD & GPX) by the ethanolic extract and its fractions has also been investigated. Furthermore, peptic ulcer and hepatotoxic risk evaluation has also been carried out at three times higher dose than that used in inflammatory in vivo model. Results: Among the extract and its various fractions tested for anti-inflammatory activity, the methanolic fraction at a dose of 300 mg/kg showed significant inhibition in paw edema by 73% as compared to Indomethacin which showed 77% inhibition after 5h. The same dose of methanolic fraction also caused significant reduction in TNF-α (59.27%) and NO concentration (57.12%) while Indomethacin showed inhibition of 63.91% and 60.12%, respectively. The active methanolic fraction was also found to inhibit the expression of NF-κB and COX-2 induced by carrageenan. Histological studies showed that the ethanolic extract and its fractions did not cause any damage to the stomach as well as to liver. Moreover, the active fractions also decreased lipid peroxidation levels and increased the antioxidant enzyme activities (SOD, CAT, GPX).

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Conclusion:

The results of present study demonstrated that significant anti-inflammatory

activity of methanolic fraction of A. lindleyana may be attributed to the modulation of proinflammatory mediators. Same fraction was also found to be effective against oxidative stress as it was found to elevate the levels of anti-oxidative enzymes. It can therefore be concluded that the methanolic fraction could be explored as a disease modifying agent against inflammation and oxidative stress. Keywords: Anti-inflammatory, Anti-oxidant, Aporosa lindleyana, TNF-α, NO, ulcerogenic

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1. Introduction Inflammation plays a decisive role in immune surveillance and responses to therapy. It has also been evident during last decade that the chronic inflammatory microenvironment is a substantive requirement for the development and progression of chronic diseases like tumor, rheumatoid arthritis, asthma, psoriasis and dermatitis (Karin, 2006). Since the oxidative stress plays a pivotal role in pathogenesis of inflammation, prevention against oxidative stress might also result in preventing the progression of related diseases. Oxidative stress is a consequence of discrepancy between the production of reactive oxidative species (ROS) and antioxidants in a system. Antioxidants have the ability to alleviate disease by scavenging ROS and by reducing resultant oxidative stress (Oday et al., 2012). From ancient times, several medicinal plants and their products are being used for treatment of various ailments. Aporosa lindleyana Baill, commonly called kotili in Kerala and kodali in Tamil, belongs to the family Euphorbiaceae (Kirtikar and Basu, 1993). This plant is traditionally being used against various ailments viz. jaundice, fever, headache, seminal loss, insanity and excessive thirst (Chopra et al., 1992; Kirtikar and Basu, 1987, Anonymous, 1985). It is a branched, evergreen, glabrous tree, grown in southern part of India and Sri Lanka. The antimicrobial and analgesic activity of Aporosa lindleyana bark has been well reported (Lingadahalli et al., 2008). The root of this plant has been reported to exhibit hypoglycemic (Jayakar and Suresh, 2003), anti-oxidant (Shrishailappa et al., 2005), antiviral and diuretic (Venkataraman et al., 2010) activities. Keeping in view the ethnopharmacological use of this plant, the present study has been carried out. The aim of this study is to evaluate the antiinflammatory and anti-oxidant potential of the ethanolic extract of bark of A. lindleyana and its various fractions. 2. Materials and Methods 4   

2.1. Collection and identification of plant material The bark of A. lindleyana was collected from district Thiruvananthapuram of Kerala during the month of May and was identified by Dr. Sunita Garg, taxonomist, NISCAIR, CSIR, New Delhi (Voucher Number: 2013/2223/04). 2.2. Plant preparation and ethanolic extract The bark of plant was air dried under shade (25-35°C with 45-60% relative humidity) and powdered. The powdered plant material was then extracted using ethanol (95% v/v) in a Soxhlet apparatus and concentrated under vacuum. The yield of the ethanolic extract was 9.42% (w/w). The ethanolic extract was fractionated by different solvents of increasing polarity using petroleum ether, chloroform and methanol. The yields of petroleum ether, chloroform and methanolic fractions were 1.1% w/w, 4.2% w/w and 6.5% w/w, respectively. 2.3. Animals Albino Wistar rats of either sex (130-160 g) were obtained from Central Animal House, Hamdard University, New Delhi. The animals were kept in cages at the room temperature and fed with food and water ad libitum. Before starting the experiment, the animals were fasted overnight. The experiments were performed in accordance with the rules of Institutional Animals Ethics Committee (registration number- CPCSEA 863) 2.4. Drugs and Chemicals Indomethacin, carrageenan, carboxymethylcellulose, Griess reagent, acetonitrile (HPLC grade), trichloroacetic acid and thiobarbituric acid were purchased from Sigma-Aldrich Chemicals Pvt. Limited, Bangalore, India. 2.5. Phytochemical screening

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The ethanolic extract of A. lindleyana was subjected to phytochemical analysis for determining the presence of alkaloids, phenols, lipids, flavonoids, saponins, sterols, tannins, carbohydrates and terpenoids (Wagner et al., 1984). 2.6. RP-HPLC profiling Reverse Phase High Performance Liquid Chromatography (RP-HPLC) of ethanolic extract of A. lindleyana was carried out using C-18 reverse phase HPLC column (250 x 4.6mm) with low pressure gradient. The sample (0.1g) was dissolved in 10ml methanol/water (1:1) and filtered. The column was eluted using acetonitrile (A) and 1% orthophosphoric acid (B) for 40min after loading 10μL of the sample. Gradient elution was carried out by changing concentration of A from 30% to 90% at a flow rate of 1.0 ml/min and the chromatograph was recorded at 254nm. Toxicity Study The selected albino Wistar rats of either sex were used to determine the dose of the 95% ethanolic extract of A. lindleyana. The animals were fasted overnight before the start of the experiment and were divided into six groups containing five rats in each. The Karbers method (Kharbanda et al., 2014) was used to determine the dose. Carboxymethylcellulose (1%w/v) was used as vehicle to suspend the extract and administered orally. The other groups received the extract in one of the following doses– 100, 200, 300, 400, 500, 600, 700, 800, 900 & 1000 mg/kg. The animals were observed continuously for first four hours for behavioral changes after dosing and for mortality at the end of 24, 48 and 72h. No mortality was seen even after 72 h. This experiment indicated that the ethanolic extract is safe up to a single dose of 1000 mg/kg b.w. 2.7. Anti-inflammatory activity

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Anti-inflammatory activity was carried out by Carrageenan-induced hind paw edema method (Winter et al., 1962; Kumar et al., 2010; Haider et al., 2011, 2012). Two groups of five rats each were given 0.5% carboxymethylcellulose solution and Indomethacin (20mg/kg/b.w.) which served as control and standard, respectively. Rest of the groups was administered with ethanolic extract and its various fractions orally at doses of 200 and 300mg/kg b. w. A freshly prepared solution of Carrageenan (1.0% in sterile 0.9% NaCl solution) in a volume of 0.1 ml was injected subcutaneously into the subplantar region of the right hind paw of rats after 1 h of administration of the test extract and fractions. Right hind paw volume was measured at 3 h and 5 h after Carrageenan injection with the help of a digital plethysmometer. The percent anti-inflammatory activity was calculated according to the formula given below: % Anti-inflammatory Activity = [VC -Vt /VC] x 100 where, Vt represents the mean increase in paw volume in rats treated with test extracts and VC represents the mean increase in paw volume in control group of rats. 2.8. TNF-α Assay Overnight fasted rats were divided into 11 groups of 6 rats in each group. Standard (Indomethacin, 20mg/kg b.w.) and test samples of extract and its fractions (200 & 300 mg/kg b.w.) suspended in vehicle (1% CMC in water in volume 10ml/kg) were administered orally to respective groups. Control (Carrageenan control), standard and test groups received a subplantar injection of Carrageenan (0.1ml of 1% suspension in normal saline) in the right hind paw. The right hind paw of each rat was cut at the level of the calcaneous bone after 5 h of dosing. Paws were washed in saline and gently centrifuged at 4000 rpm for 30 min in order to recover oedemantous fluid. The fluid recovered was then filtered through Millipore cut-off filter (10,000 mol. wt.) to remove traces of blood cells if any (Millipore, Bedford, MA, U.S.A.). The level of 7   

TNF-α was determined using a commercially available ELISA kit (e-Bioscience, San Diego, CA.) according to the manufacturer’s instruction (Syed et al., 2013). 2.9. NO assay Nitrite assay was done using Griess reagent by the reported method of Green et al., 1982 with some modifications (Rehman et al, 2013). In brief, 100 μl of Griess reagent (1:1 solution of 1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediaminedihydrochloride in water) was added to 100 μl of PMS incubated for 5-10 min at room temperature protected from light. Purple/magenta color began to form immediately. Absorbance was measured at 546 nm, nitrite concentration was calculated using a standard curve for sodium nitrite, and nitrite levels were expressed as l mol/mg protein. 2.10.

Determination of antioxidant enzyme activity in paw tissue

For all antioxidant enzyme assays, dosing pattern was kept same as that for anti-inflammatory activity. After 5 h, the treated animals were sacrificed and carrageenan induced paw tissue has been taken for performing biochemical assay. 10% homogenate of paw tissue was centrifuged at 10,000 rpm for 10 min at 4oC to obtain post mitochondrial solution (PMS). 2.10.1. Catalase (CAT) activity Catalase (CAT) activity was carried out by previously reported method (Claiborne, 1985). The reaction mixture consisted of 1.95 ml phosphate buffer (0.1 M, pH 7.4), 1.0 ml hydrogen peroxide (0.019 M) and 0.05 ml of tissue post mitochondrial solution (PMS, 10 %) in a final volume of 3 ml. Absorbance was recorded at 240 nm. Catalase activity was calculated as nmol H2O2 consumed per min per mg protein. 2.10.2 Superoxide dismutase (SOD) activity

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The Superoxide dismutase (SOD) activity was measured by the reported method (Marklund & Marklund, 1974). The reaction mixture consisted of 2.87 ml Tris–HCl buffer (50 mM, pH 8.5), pyrogallol (24 mM in 10 mM HCl) and 100 µl of tissue 10% PMS making a total volume of 3 ml. The absorbance of SOD was measured at 420 nm and was expressed as units/mg protein. One unit is defined as the enzyme activity that inhibits auto-oxidation of pyrogallol by 50%. 2.10.3 Glutathione peroxidase activity The Glutathione peroxidase (GPX) activity was measured by previously reported the method (Jollow et al., 1974). In this method, 1.44 ml phosphate buffer (0.1 M, pH 7.4), 0.1 ml EDTA (1 mM), 0.1 ml sodium azide (1.0 mM), 0.05 ml GR (1 eu/ml), 0.05 ml GSH (1.0 mM), 0.1 ml NADPH (0.2 mM), 0.01 ml H2O2 (0.25 mM) and 0.1 ml PMS (10%) was mixed in a total volume of 2.0 ml. The absorbance was read at 340 nm. The enzyme activity was calculated as nmol NADPH oxidized/min/mg protein with the help of the molar extinction coefficient of 6.22x103 M-1 cm-1. 2.11. Ulcerogenic risk evaluation The ethanolic extract and its various fractions were further evaluated for their ulcerogenic effects. The ulcerogenic studies (Vogel and Vogel, 1997) were carried out after oral administration of test extracts and standard drug at a dose of 900 & 60 mg/kg b.w., respectively which is three times of the dose used for anti-inflammatory activity. Control rats were orally administered suspension of 1% carboxymethylcellulose only. Animals were sacrificed 5 h after administration of standard and test samples. 2.12. Lipid peroxidation content The reported method was followed for evaluating lipid peroxidation (LPO) content (Ohkawa et al., 1979). The gastric mucosa was scraped with two glass slides and weighed (100 mg) and 9   

homogenized in 1.8 ml of 1.15% ice cold KCl solution. One milliliter of suspension medium was taken from the supernatant, 0.5 ml of 30% trichloroacetic acid followed by 0.5 ml of 0.8% thiobarbituric acid reagent were added to it. The tubes were covered with aluminum foil and kept in a shaking water bath for 30 min at 80°C. After 30 min, tubes were taken out and kept in ice cold water for 10 min. These were then centrifuged at 3000 rpm for 15 min. The absorbance of supernatant was read at 540 nm at room temperature against the blank on UV. 2.13. Hepatotoxicity studies For this study, a dose of 900 mg/kg b.w. of the ethanolic extract and its fractions were administered orally (three times the dose used for anti-inflammatory activity). The standard drug indomethacin was administered at 60 mg/kg b.w. dose. A group of healthy rats served as control and was administered with1% carboxymethylcellulose. The rats were sacrificed after 5h of the administration of the test sample and standard drug and their liver specimens were removed. The specimens were stored in 10 % formalin solution. (Lambert et al., 2010) 2.14. Immunohistochemistry The liver tissues were fixed in formalin and embedded in paraffin. Sections of 5 µm thickness were cut onto poly-lysine coated glass slides. Sections were deparaffinized three times (5 min) in xylene followed by dehydration in graded ethanol and finally rehydrated in running tap water. For antigen retrieval, sections were boiled in 10mM citrate buffer (pH 6.0) for 5-7 min. Sections were incubated with hydrogen peroxide for 15 min to minimize non-specific staining and then rinsed three times (5 min each) with 1X PBST (0.05% Tween-20). Blocking solution was applied for 10 min, then sections were incubated with diluted (1:100) primary anti-bodies, purified rabbit polyclonal anti-NF-κB antibody (BioLegend) and rabbit polyclonal anti-COX-2 antibody (Bio Vision), overnight at 4oC in humid chamber. Further processing was done according to the 10   

instructions of Ultra Vision plus Detection System Anti-Polyvalent, HRP/DAB (Ready-To-Use) staining kit (Thermo scientific system). The peroxidase complex was visualized with 3,3’diaminobenzidine (DAB). Lastly the slides were counterstained with haematoxylin, cleaned in xylene, dehydrated with ethanol and after that DPX mounting microscopic (BX 51 Olympus) analysis was done at 40x magnification. 2.15. Statistical analysis Data was analyzed by one way ANOVA followed by Dunnett’s ‘t’ test (n = 5), *p < 0.05, **p < 0.01 & ns p > 0.05 significant in comparison to control. 3. Results 3.1. Phytochemical screening and RP-HPLC profiling Preliminary chemical tests of ethanolic extract confirmed the presence of alkaloids, flavonoids and triterpenoids. RP-HPLC performed on the ethanolic extract of A. lindleyana showed numerous peaks in the chromatogram with the retention time ranging between 0 to 40 min at the wavelength of 254 nm. Two major peaks appearing at retention time 25.69 and 27.11 min were identified as stigmasterol and β-sitosterol, respectively. The chromatogram also displayed many peaks before the retention time of 12.00 min (Fig.1) which suggested that highly polar phytoconstituents are present in the ethanolic extract. RP-HPLC profile thus generated can be used as a fingerprint to authenticate the identity of the plant material. 3.2. Anti-inflammatory Activity The results of anti-inflammatory activity of the ethanolic extract and its various fractions of A. lindleyana bark are shown in table 1. Among the tested fractions, at a dose of 300mg/kg, the methanolic fraction showed comparable activity (73% inhibition) to that of Indomethacin (77% inhibition). It was observed that chloroform fraction and ethanolic extract showed moderate 11   

activity whereas the petroleum ether fraction did not show any significant anti-inflammatory activity as compared to standard drug. 3.3. TNF-α Level The effect of ethanolic extract and its various fractions on the level of TNF-α are shown in Fig. 2. The amount of TNF-α in the serum of the rats treated with methanolic fraction at a dose of 300mg/kg b.w. was reduced by 59.27% as compared to that of the control group. The percentage reduction seen in case of methanolic fraction treated group (300mg/kg b.w.) was comparable to that of Indomethacin (63.91%). The rest of the fractions also exhibited inhibitory activity in TNF-α production. Inhibition was significant with chloroform fraction and ethanol extract at dose of 300 mg/kg b.w. However, the petroleum ether fraction was not seen to be much effective in inhibiting TNF-α level. 3.4. Nitrite level Effect of ethanolic extract of bark of A. lindleyana and its various fractions on nitrite levels are shown in Fig. 3. The level of nitrite in the serum of the rats treated with methanolic fraction at a dose of 300mg/kg b.w. was reduced by 57.12% as compared to that of the control group. The percentage reduction seen in case of methanolic fraction treated group (300mg/kg b.w.) was comparable to Indomethacin (60.12%). The rest of the fractions also exhibited inhibitory activity in nitrite levels. Inhibition was significant with chloroform fraction and ethanol extract at dose of 300 mg/kg b.w. However, the petroleum ether fraction was not seen to be much effective in inhibiting the level of nitrites. 3.5. Anti-oxidant enzyme activity The results of CAT, SOD, and GPX activities in rat paw edema are shown in Table 2. It can be deduced from the results that carrageenan decreased the activities of CAT, SOD and GPX in the 12   

rat paw by 39.49%, 35.68% and 43.58%, respectively, as compared to control group. The ethanolic extract of A. lindleyana and its fractions increased the activities of CAT, SOD and GPX as compared to carrageenan group. The results indicated that among all fractions and ethanolic extract, the methanolic fraction increases the activities of CAT, SOD and GPX by 73.30%, 75.28% and 74.14% at a dose of 300mg/kg when compared to standard drug ascorbic acid which increase the activities by 80.23%, 79.32% and 82.38%, respectively. 3.6. Ulcerogenic activity The results of the histopathological studies are shown in Fig. S1. When compared with Indomethacin, ethanolic extract and its fractions did not cause any gastric ulceration and disruption of epithelial cells at the given oral dose. Stomach wall of indomethacin treated group at low power (10x) photomicrograph showed damage of the mucosa and sub mucosa. High power (40x) photomicrograph of same section showed some loss of epithelial cells from the superficial and deep layer of the mucosa whereas test group animals revealed no surface epithelial damage and sub mucosal damage. 3.7. Lipid peroxidation The results of lipid peroxidation activity were measured as nmol of MDA per 100 mg of gastric mucosa tissue and are shown in fig 4. The level of lipid peroxidation in the carrageenan induced animals was significantly increased. The elevation in lipid peroxidation was also seen in groups treated with indomethacin and petroleum ether fraction but not as pronounced as in case of carrageenan treated group. However, the level of lipid oxidation was greatly reduced in the presence of ethanolic extract and its various fractions. Again, the methanolic fraction caused significant reduction in lipid peroxidation in comparison to standard group. 3.8. Hepatotoxicity study 13   

From the photomicrograph obtained from hepatotoxicity study, it was observed that the chloroform fraction, methanol fraction and ethanolic extract did not cause any damage to the liver as compared to indomethacin which caused significant inflammation to the portal and centrizonal area and sinusoidal dilation. However, the petroleum ether fraction exhibited slight inflammation in the regions of portal and sinusoidal vein (Fig S2). 3.9. Immunohistochemistry Hepatic expressions of COX-2 and NF-κB have been shown in the figures 5a and 5b, respectively. The intensity of brown colour in the animals treated with carrageenan only (group II) clearly indicated more number of cells having COX-2 and NF-κB expression as compared to group I containing healthy rats. Treatment with standard drug Indomethacin in the group III and active methanolic fraction in the group IV reduced the number of cells showing expression of these proteins as compared to group II. However the active methanolic fraction reduced slightly more number of cells as compared to group III standard. 4. Discussion Carrageenan induced rat paw edema has been a well established biphasic inflammatory model to investigate the anti-inflammatory potential of a drug. The first phase involves the release of histamine, serotonin and kinins whereas the second phase is related to the production of prostaglandins and slow reacting substances which peak at > 4h (Manfred F. et al., 1985). In the present study, the methanolic fraction has shown comparable inhibition of inflammation to the standard drug Indomethacin whereas the other fractions have shown moderate activity at 5 h. Therefore it could be expected that the ethanolic extract as well as fractions of A. lindleyana might be inhibiting the production of several mediators of inflammation like TNF-α, NF-κB, COX-2 etc. TNF-α is a proinflammatory mediators and its inhibition may prove effective in 14   

healing inflammation (Schimmer et. al., 2001). From the results of TNF-α assay, it was found that the ethanolic extract of A. lindleyana and its various fractions decreased the level of TNF-α. The methanolic fraction showed prominent attenuation in TNF-α level, which was comparable to the standard drug Indomethacin. The evaluation has also been carried out on the production of nitrite oxide (NO), another important mediator in the pathogenesis of various inflammatory diseases (Rehman et al., 2013). The results showed that carrageenan induced group markedly increased the nitrite levels indicating that the induction with carrageenan clearly facilitated the process of inflammation while this effect was quite controlled by the ethanolic extract of A. lindleyana and its various fractions. Among all the fractions, the methanolic fraction was observed to be the most effective in inhibiting the production of nitrite levels. A proinflammatory enzyme cyclooxygenase (COX) is involved in the process of inflammation (Dubois et. al 1998). Its two major forms, COX-1 and COX-2 are mechanistically similar but different in their expression in genes. The expression of COX-1 involves normal homeostasis of prostaglandins while the expression of COX-2 can be induced by various stimuli like growth factors, cytokines and oncogenes (Smith et al., 2000). Therefore, the suppression in the activity of COX-2 enzyme would inhibit the development of inflammation. The findings of the current study demonstrated that the methanolic fraction which exhibited significant anti-inflammatory activity also inhibited carrageenan induced hepatic expression of COX-2. NF-κB also plays a pivotal role as a redox sensitive transcription factor for inflammation. It is a constituent in the cytoplasm of resting cell. On exposure to different stimuli, it gets translocated into the nucleus where it mediates the transcription of different proinflammatory mediators, cytokines, chemokines, and adhesion molecules (Baeuerle and Baltimore, 1996; Chao et al., 2010).  The

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present study showed that the active methanolic fraction strongly suppressed the activation of NF-κB in rats by carrageenan which in turn interrupted the various transcriptional processes. Since oxidative stress is involved in triggering proinflammatory cytokines and mediators (TNFα, NO, COX-2 and NF-κB), the reduction in the levels of TNF-α, NO and in the expression of COX-2, NF-κB can also be taken as the consequence of diminishing effect on oxidative stress (Alessandro et al., 2007). Several assays have been carried out to study the effect of the ethanolic extract and its various fractions on the activities of various antioxidative enzymes such as SOD, CAT and GPX. These enzymes are directly involved in free radical scavenging and therefore in attenuating oxidative stress. From the results, it could be implied that the protective effects of methanolic fraction of A. lindleyana might be attributed to its potential to elevate the activity of antioxidant enzymes during inflammation. Major side effects associated with currently available non steroidal anti-inflammatory drugs (NSAIDs) are gastric ulceration and hepatotoxicity (Gil, 2002). Therefore, there is a need to evaluate the efficacy of any new anti-inflammatory treatment with respect to their peptic and hepatic tolerance. In the present work, the ethanolic extract of A. lindleyana and its various fractions were evaluated for their ulceration and hepatotoxic risk at three times higher dose than that used in the evaluation of the in vivo anti-inflammatory activity. Again, it has been found that the active methanolic fraction did not show any gastric ulceration as well as hepatotoxicity. Gastric ulceration is also associated with cellular membrane disruption and destabilizing via lipid peroxidation. It has already been reported that the process of lipid peroxidation proceeds by free radical chain reaction which is scavenged through enzymatic and non enzymatic antioxidants. Our results are in complete agreement with the earlier observations indicating that the methanolic fraction significantly brought down the level of LPO as compared to carrageenan 16   

group. Furthermore, the decreased expression of COX-2 and NF-κB in treated groups suggested that the hepatic tolerance has increased after treatment than in carrageenan induced control group. Preliminary phytochemical screening of the ethanolic extract of A. lindleyana has shown the presence of alkaloids, flavonoids and triterprnoids. These phytoconstitutions have already been reported for several biological activities (Huss et al., 2002) and might be responsible for biological activity of ethanolic extract of A. lindleyana bark and its fractions. 5. Conclusion It could be concluded that the methanolic fraction of ethanolic extract of A. lindleyana bark exhibited significant anti-inflammatory and anti-oxidant activity. The methanolic fraction attenuated the level of proinflammatory mediators like TNF-α, NO, NF-κB, COX-2 and elevated the activity of anti-oxidative enzymes (SOD, CAT & GPX). Therefore, the bark of A. lindleyana might provide a potent agent against inflammation and oxidative stress. Acknowledgements The authors are thankful to Dr. G. N. Qazi, Vice Chancellor, Jamia Hamdard for providing the necessary facilities to the Department of Chemistry. Yakub Ali is also thankful to Hamdard National Foundation for providing the financial assistance.

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References Alessandro, F., Floriana, M., Concetta, T., Fortunato, C., Carmela, L., 2007. Chronic inflammation and oxidative stress in human carcinogenesis. International Journal of Cancer. 121, 2381-2386. Anonymous, 1985. The Wealth of India, vol. I, CSIR, New Delhi, India, p. 327. Baeuerle, P.A., Baltimore, D., 1996. NF-Kappa B, Ten year after. Cell. 87, 13-20. Carlberg, I., Mannervik, B., 1975. Glutathione level in rat brain. Journal of Biological Chemistry. 250, 5475-5480. Chao, W.T, Daquinag, A.C, Asheroft, F., Kanz, J., 1990. Type I phosphatidylinositol phosphate kinase beta regulates focal adhesion disassembly by promoting beta1 integrin endocytosis. Molecular Cell Biology. 190, 247-262. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1992. Glossary of Indian Medicinal Plants. CSIR, New Claiborne, A., 1985. Catalase activity. In: Greenwald, R.A. (Ed.), CRC Handbook of 559 Methods in Oxygen Radical Research. CRC, Boca Raton, RA. 283–284. Gil, Ã., 2002. Polyunsaturated fatty acids and inflammatory diseases. Biomedicine and Pharmacotherapy. 56, 388–396. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Analytical Biochemistry. 126, 131–138. Haider, S., Nazreen, S., Alam, M.M., Hamid, H., Alam, M.S., 2012. Antiinflammatory and antinociceptive activities of Platanus orientalis and its ulcerogenic risk evaluation. Journal of Ethnopharmacology. 143, 236-240.

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Huss, U., Ringbom, T., Perera, P., Bohlin, L., Vasänge, M., 2002. Screening of ubiquitous plant constituents for COX-2 inhibition with a scintillation proximity based assay. Journal of Natural Product. 65, 1517-1521. Jayakar, B., Suresh, B., 2003. Antihyperglycemic and hypoglycemic effect of A. lindleyana. Journal of Ethnopharmacology. 84, 247-249. Jollow, D.J., Mitchell, J.R., Zampaglione, N., Gillette, J.R., 1974. Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology. 11, 151-169. Karin, M., 2006. Nuclear factor-kappaB in cancer development and progression. Nature 441, 431–436. Kharbanda, C., Alam, M.S., Hamid, H., Bano, S., Haider, S., Nazreen, S., Ali, Y., Javed, K., 2014. Trapa natans L. root extract suppresses hyperglycemic and hepatotoxic effects in STZ induced diabetic rat model. Journal of Ethnopharmacology. 151, 931–936. Kirtikar, K.R., Basu, B.D., 1987. Indian Medicinal Plants, vol. III. International Book Distributors, Book Sellers and Publishers, Dehradun, India, p. 2251. Kirtikar, K.R., Basu, B.D., 1993. Indian Medicinal Plants. International Book Publisher, Dehradun, p. 225-/226. Kumar, D., Agarwal, R.C., Bhati, S.K., Kumar, A., 2010. Synthesis of newer substituted thiadiazolyl and pyrazolyl phenothiazines as potent anti-inflammatory agents. Oriental Journal of Chemistry. 26, 497-508. Lambert, J.D.M., Kennett, J., Sang, S., Reuhl, K.R.J., Jihyeung , J., & Yang, C.S., 2010. Food and Chemical Toxicology. 48, 409-416.

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Lingadahalli, P.S., Hosadu, M.V., Basavanakote M.B., Vijayavittala P.V., 2008. Antimicrobial and analgesic activity of bark of A. lindleyana. International Journal of Green Pharmacy. 2, 155-157. Manfred, F., John, M.P., Dajaja, D.S., Douglas, A.K., 1985. Koenoline, a furthercytotoxic carbazole alkaloid from Murraya koenigii. Phytochemistry. 24, 3041-3043. Marklund, S., Marklund, G., 1974 Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry. 47, 469-474. Mohandas, J., Marshall, J., Duggin, G., 1984. Differential distribution of glutathione and glutathione-related enzymes in rabbit kidney. Possible implications in analgesic nephropathy. Biochemical Pharmacology. 33, 1801-1807. Oday, H., Muneeb, U. R., Mir, T., Rehan, K., Abdul, Q. K., Abdul, L., Farrah, A., Sarwat, S., 2012. Asian Pacific Journal of Cancer Prevention. 13, 4835-4844. Ohkawa, H., Ohishi N., & Yagi, K., 1997. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry. 95, 351–358. Raymond, N.D., Steven, B.A., Leslie, C., Rajnish, A.G., Lee, S.S., Leo, B.A., Van, D.P., Peter, E.L., 1988. Cyclooxgenase in biology and disease. Journal of Federation of American Societies for Experimental Biology 12, 1064-1-073. Rehman, M.U., Tahir, M., Khan, A.Q., Khan, R., Lateef, A., Oday-O-Hamiza, Qamar, W., Ali, F., Sultana, S., 2013. Chrysin suppresses renal carcinogenesis via amelioration of hyperproliferation, oxidative stress and inflammation: plausible role of NF-κB. Toxicology Letters. 216, 146-158.

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Schimmer, B.P., Parker, K.L., 2001. Adrenocorticotropic hormone; adrenocortical steroids and their synthetic analogs; inhibitors of the synthesis and actions of adrenocortical hormones. In-Hardman, J.G., Limbird, L.E., Goodman Gilman, A. (Eds.), The Pharmacological Basis of Therapeutics, tenth ed. McGraw-Hill, New York, p. 1649-1677. Shrishailappa, B., Rai, R.S., Suresh, B., 2005. Antioxidant activity of A. lindleyana root. Journal of Ethnopharmacology. 101, 180–184. Smith, D.S., Huxman, T.E, Zitzer, F.S., Charlet, N.T., Housman, C.D, Coleman, S.J., 2000. Elevated CO2 increases productivity and invasive spices success in an arid ecosystem. Letter to Nature. 408, 79-82. Syed, O., Shafiya, Y., Rafia, B., Poja, R., Mohammed, S., Surendar, S., Vinod, N., 2013. Synthesis and anti-inflammatory activity of celecoxib like compound. Journal of Enzyme Inhibition and Medicinal Chemistry. 28, 1105-1112. Venkataraman, R., Gopalakrishnan, S., Thyagarajan, S.P., 2010. Antiviral activities of A. lindleyana Baill. Annals of Biological Research. 1, 68-70. Vogel, H.G., Vogel, H.W., 1997. Drug discovery and evaluation-pharmacological assays. Springer-Verlag: Berlin Heidelberg, New York, p. 1231. Wagner, H., Bladt, S., Zgainski, E.M., 1984. Plant Drug Analysis. Springer-Verlag, Heidelberg, p. 51-92. Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenan induced edema in hind paw of rat an assay for anti-inflammatory drugs. Proceedings of Society for Expermental Biology Medicine. 111, 544-546.

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Table I. In-vivo anti-inflammatory activity of ethanolic extract of bark of A. lindleyana and its various fractions.

Group

Dose (mg/kg po)

Change in paw volume(ml) Mean + SEM

% inhibition

3hr

3hr

5hr

5hr

Carr Control

2ml/kg

1.45±0.024

1.48±0.066

-

-

Indomethacin

20

0.94±0.012**

0.90±0.023**

70

77

200

1.33±0.012ns

1.35±0.030ns

14.89

15.79

300

1.29±0.016ns

1.31±0.030ns

20.66

18.73

Chloroform fraction

200

1.03±0.016*

1.04±0.029*

55.30

56.47

300

1.03±0.026*

1.02±0.012*

56.44

58.67

Methanol fraction

200

0.98±0.012*

0.96±0.026**

62

66.94

300

0.97±0.015**

0.93±0.026**

66.18

73.00

Ethanolic Extract

200

1.08 ±0.011*

1.08±0.012*

53.89

52.34

300

1.07±0.019*

1.05±0.020*

48.59

56.47

Petroleum ether fraction

Data is analyzed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as % inhibition and mean ± SEM from five observations where * indicates p < 0.05, ** indicates p < 0.01 & ns indicates p > 0.05.

             

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Table.II Effects of the ethanolic extract and its various fractions of A. lindleyana and indomethacin on catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities in carrageenan (Carr) induced edema paw. Group

Dose (mg/k g po)

Control

2ml/kg 8.59±1.02

Carrageenan (Carr) Carr+ Ascorbic acid Carr+Petroleum ether fraction Carr+Chloroform fraction Carr+Methanol fraction Carr+Ethanolic extract

CAT(U/mg protein)

%

SOD(U/mg protein)

%

GPX(U/mg protein)

%

-

7.96±0.79

-

20.61±0.61

-

0.1mL

3.43±0.89ns

39.49

2.84±0.63ns

35.68

8.98±1.40ns

43.58

20mg/ kg

6.89±1.29**

80.23

6.32±0.82**

79.32

16.97±0.61**

82.38

200

3.62±0.64ns

42.22

3.08±0.42 ns

38.70

8.76±0.88 *

42.59

300

3.89±0.62 ns

45.28

3.14±0.35 ns

39.49

9.44±0.98*

45.81

200

5.10±0.20 *

59.38

4.94±0.23 *

62.07

12.37±0.53**

60.02

300

5.52±0.38**

64.27

5.20±0.42 *

65.33

13.26±0.86**

63.80

200

5.83 ±0.76**

67.84

5.73±0.42 *

71.99

14.50±0.69**

70.36

300

6.35±0.48**

73.30

5.99±0.31**

75.28

15.58±0.61**

74.14

200

4.95 ±0.38 ns

51.03

4.53±0.23 ns

56.91

10.74±0.96**

51.91

300

5.19±0.28 ns

53.50

4.58±0.25 ns

57.54

11.44±0.39**

55.51

Data is analyzed by one way ANOVA followed by Dunnett’s ‘t’ test and expressed as mean ± SEM from five observations where * indicates p < 0.05, ** indicates p < 0.01 & ns indicates p > 0.05 and % change in activities are also given.

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Figure Captions Fig.1 (A) RP-HPLC profiling of ethanolic extract of A. lindleyana Baill. (B) RP-HPLC profiling of pure β-sitosterol. (C) RP-HPLC profiling of pure sigmasterol Fig.2. In-vivo TNF-α activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions. Fig. 3. In-vivo NO activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions. Fig. 4. In-vivo LPO activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions. Fig.5a Hepatic expressions of COX-2 activation Representative photomicrographs (magnification 40x) Group I (only control), Group II (only carrageenan), Group III ( carrageenan + standard drug Indomethacein), Group IV ( carrageenan + methanol fraction). Fig.5b Hepatic expressions of NF-κB activation. Representative photomicrographs (magnification 40x). Group I (only control), Group II (only carrageenan), Group III( carrageenan + standard drug Indomethacein), Group IV ( carrageenan + methanol fraction).

24   

Fig.1 (A) RP-HPLC profiling of ethanolic extract of A. lindleyana Baill. (B) RP-HPLC profiling of pure β-sitosterol. (C) RP-HPLC profiling of pure sigmasterol

25   

26   

Fig.2. In-vivo TNF-α activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions.

Data is analyzed by one way ANOVA followed by Dunnett’s ´t´ test and expressed as percentage inhibition ± SEM from three observations; ** indicates p < 0.01, * indicates p < 0.05 & ns indicates p > 0.05.

27   

Fig. 3. In-vivo NO activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions.

Data is analyzed by one way ANOVA followed by Dunnett’s ´t´ test and expressed as percentage inhibition ± SEM from three observations; ** indicates p < 0.01, * indicates p < 0.05 & ns indicates p > 0.05.

 

28   

Fig. 4. In-vivo LPO activity of ethanolic extract of barks of A. lindleyana Baill. and its various fractions.

Data is analyzed by one way ANOVA followed by Dunnett’s ´t´ test and expressed as mean ± SEM from three observations; ** indicates p < 0.01, * indicates p < 0.05 & ns indicates p > 0.05.

29   

X-2 activattion Repressentative photomicrogr p raphs Fig. 5aa Hepatic expressionss of COX (magnificcation 40x) Group I (onnly control), Group II (onnly carrageeenan), Groupp III (carrageeenan + standarrd drug Indo omethacein), Group IV (ccarrageenan + methanol fraction).

Fig. 5b b Hepatic expressionss of NF-κ κB activatiion. Repressentative photomicrogr p raphs (magnificcation 40x). Group I (onnly control), Group II (onnly carrageeenan), Groupp III (carrageeenan + standarrd drug Indo omethacein), Group IV (ccarrageenan + methanol fraction).

30