Anti-arthritic and antioxidant activity of leaves of Alstonia scholaris Linn. R.Br.

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European Journal of Integrative Medicine 3 (2011) e83–e90

Original article

Anti-arthritic and antioxidant activity of leaves of Alstonia scholaris Linn. R.Br. Sinnathambi Arulmozhi a,∗ , Papiya Mitra Mazumder b , Lohidasan Sathiyanarayanan c , Purnima Ashok d a

Department of Pharmacology, Bharati Vidyapeeth University, Poona College of Pharmacy, Erandwane, Pune 411 038, Maharashtra, India b Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835 215, India c Department of Pharmaceutical Chemistry, Bharati Vidyapeeth University, Poona College of Pharmacy, Erandwane, Pune 411 038, Maharashtra, India d Department of Pharmacology, KLES’s College of Pharmacy, Rajaji Nagar, Bangalore 560 010, Karnataka, India Received 29 December 2010; received in revised form 9 April 2011; accepted 11 April 2011

Abstract Aim of the study: Alstonia scholaris (Family: Apocynaceae) is a medicinal plant which is indicated for the treatment of various diseases including arthritis in folklore medicine. The purpose of this study is to investigate the antiarthritic activity and in vivo antioxidant role of A. scholaris leaves in animal models. Materials and methods: The ethanol extract of A. scholaris leaves (EEAS) was tested against Freund’s Complete Adjuvant (FCA) induced arthritic rats. Arthritis assessment and body weight were measured daily till day 28 whereas nociceptive threshold was measured once in 2 days. On day 28, the animals were anaesthetized, synovial fluid withdrawn and leukocyte concentration was determined. The animals were sacrificed, synovial tissue was extracted and estimated for the myeloperoxide, malonaldehyde, glutathione, glutathione peroxidase and superoxide dismutase. Effect of EEAS on ethanol and sodium salicylate induced gastropathy was also studied. Results: EEAS significantly decreased the arthritis which was evident with arthritis index, body weight and leukocyte infiltration. EEAS significantly reduced gastric lesion indices and gastric juice secretion. It also significantly decreased the levels of lipid peroxidation and myeloperoxide in the articular tissue, whereas it significantly increased the antioxidant enzymes glutathione, glutathione peroxidase and superoxide dismutase. Conclusion: The present study is suggestive that EEAS has prominent antiarthritic activity which may be attributed to its analgesic, antiinflammatory, immunosuppressant and antioxidant activities. © 2011 Elsevier GmbH. All rights reserved. Keywords: Alstonia scholaris; Antiarthritic; Antioxidant; Antigastropathy

Introduction Alstonia scholaris Linn. R.Br., belongs to the family of Apocynaceae and is native to India. It grows wild throughout in deciduous, evergreen forests and even in plains. Bark of A. scholaris possesses a spectrum of pharmacological activity, ranging from bitter, astringent, thermogenic, laxative, antipyretic, anthelmintic to galactogoguic and cardiotonic properties, therefore used in fever, malarial fever, abdominal disorder,

∗

Corresponding author. Tel.: +91 9371085077. E-mail address: [email protected] (S. Arulmozhi).

1876-3820/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.eujim.2011.04.019

dyspepsia, leprosy, skin diseases, asthma, bronchitis, cardiopathy, etc. [1,2]. An antimalarial Ayurvedic preparation, Ayush-64 containing A. scholaris is marketed [3]. Folklore use include application of milky juice of leaves on wounds, ulcers, and for rheumatic pain, as well mixed with oil and applied for earache [1]. Extracts of A. scholaris is reported to possess several pharmacological activities that include antiplasmodial activity [4], antimutagenic effect [5], immunostimulatory effect [6], hepatoprotective activity [7], antidiabetic and antihyperlipidemic activities [8]. Echitamine, an indole alkaloid extracted from bark was found to exhibit anticancer activity [9]. The plant is reported to relieve rheumatic pains in folklore medicine [1]. The leaves of A. scholaris have illustrated antinoci-

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ceptive, anti-inflammatory activities [10,11]. Based on the above perspective, the present study is designed to investigate the antiarthritic property of A. scholaris. Materials and methods Collection and authentication of plant The leaves of A. scholaris (Family: Apocynaceae) were collected in the months of September–October 2009 from the hills of Sawantwadi, Maharashtra, India. The plant material was taxonomically identified by Dr. P.S.N. Rao, Botany Survey of India (BSI), Pune and the voucher specimen AS-1 is retained in the herbarium of BSI, Pune for future reference. Preparation of ethanolic extract of leaves of A. scholaris The dried powdered leaves (500 g) were defatted using petroleum ether and subjected to extraction in a Soxhlet apparatus by using ethanol. The solvent was removed from the extract under reduced pressure to obtain a semisolid mass and vacuum dried to yield solid residue (5.24% (w/w) ethanol extract). The extract (EEAS) showed positive tests for alkaloids, tannins, saponins, glycosides, triterpenoids and flavonoids. Chemicals and reagents Freund’s Complete Adjuvant (FCA) (Sigma Aldrich, USA) and Indomethacin (Medico Remedies Pvt. Ltd., India) were used. Other chemicals and reagents used for the study were of analytical grade and procured from approved organizations. Animals Female Sprague Dawley rats weighing between 150 and 250 g were used for the present study. The animals were maintained under standard environmental conditions and were fed with standard pellet diet and water ad libitum. The study was approved by Institutional Animal Ethics Committee (Reg. No. 626/02/a/CPCSEA). CPCSEA guidelines were adhered to during the maintenance and experiment. Acute toxicity studies Acute toxicity study was carried out for the ethanol extract [12]. The extract suspended in water with 2% (w/v) Tween 80 in the dose of 2 g/kg body weight was orally administered to overnight-fasted, healthy rats (n = 3). The animals were observed continuously for 24 h for mortality. Induction of experimental arthritis and treatment protocol The animals were divided into six groups of six animals each as follows:

Group I – Vehicle control, 2% (w/v) Tween 80, p.o. (nonarthritic) Group II – Arthritic control, 2% (w/v) Tween 80, p.o. Group III – Arthritic standard treated, 10 mg/kg indomethacin, p.o. Group IV – Arthritic EEAS 100 mg/kg, p.o. Group V – Arthritic EEAS 200 mg/kg, p.o. Group VI – Arthritic EEAS 400 mg/kg, p.o. Arthritis was induced in all the groups except vehicle control by injection of 0.1 ml of Freund’s Complete Adjuvant (FCA) in the subplantar region of the left hind paw on day 1 [13]. Once the signs of arthritis set in, the treatment was given to the respective groups once daily orally

Arthritis assessments Evaluation of joint inflammation was performed by a blinded independent observer with no knowledge of the treatment protocol. The severity of the arthritis in each paw was quantified daily by a clinical score measurement from 0 to 4 as follows: 0 – no macroscopic signs of arthritis (swelling or erythema), 1 – swelling of one group of joints (namely, wrist or ankle joints), 2 – swelling of two groups of swollen joints, 3 – swelling of three groups of swollen joints, 4 – swelling of the entire paw. The maximum score for each rat was 16 [14]. The change in body weight was also monitored.

Measurement of nociceptive threshold The nociceptive threshold was determined using an analgesiometer (Ugo Basile, Italy) once in 2 days. The mechanical pressure stimulus was applied on the center of the hind paw and was increased at a rate of 32 g/s. The pressure intensity (g) that caused the escape reaction was defined as the nociceptive threshold [15].

Determination of leukocyte concentration in synovial fluid Leukocyte concentration in the synovial fluid was determined on day 28. The animals were anaesthetized, the skin overlying the anterior aspect of the left knee was incised and a 26-gauge hypodermic needle was inserted into the synovial cavity. This arrangement facilitates the infusion of 250 ␮l 0.9% (w/v) saline into the synovial cavity over a period of 2 min. A second hypodermic needle (26 gauge), which served as an outflow cannula, was inserted into the synovial cavity approximately 3 mm from the infusion needle. Five minutes after infusion of all the saline, 200 ␮l of fluid was withdrawn from the synovial cavity over a period of 2 min. The concentrations of total leukocytes, lymphocytes and monocytes/macrophages were estimated from the synovial fluid in a Coulter Counter (Serwell Laboratories, Bangalore) [16].

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Myeloperoxidase analysis Myeloperoxidase activity was analysed as an index of neutrophil infiltration in the synovial tissue. MPO closely correlates with the number of neutrophils present in the tissue [17]. Synovial tissue samples were separated from rat joints and were first homogenized in a solution containing 20 mM potassium phosphate buffer, pH 7.0, to 1:10 (w/v) and then centrifuged for 30 min at 20,000 g and 4 ◦ C. The supernatant of each sample was discarded and the resulting pellet was added to a buffer solution consisting of 0.5% hexadecyltrimethylammonium bromide dissolved in 50 mM potassium phosphate buffer, pH 6, containing 50 ␮l of protease and phosphate inhibitor. Samples were then sonicated for 1 min and centrifuged for 30 min at 20,000 × g and 4 ◦ C. An aliquot of the supernatant was allowed to react with a solution of o-dianisidine dihydrochloride (0.167 mg/ml) and 0.0005% hydrogen peroxide. The rate of change in absorbance was measured spectrophotometrically at 405 nm (Shimadzu UV-Vis 1700). Myeloperoxidase activity has been defined as the concentration of enzyme degrading 1 ␮mol of peroxide/min at 37 ◦ C and was expressed as U/g of protein [18]. Measurement of TBARS Rats were sacrificed on day 28 by excess ether. The thiobarbituric acid-reactive substance was measured as a marker of lipid peroxidation in the articular cartilage. The homogenized tissue was added to 1.5 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 20% acetate buffer (pH 3.5) and 1.5 ml of 0.8% TBA (thiobarbituric acid) solution. The mixture was heated at 95 ◦ C for 1 h. After cooling, 5 ml of n-butanol–pyridine (14:1) was added for extraction and the absorbance of n-butanol–pyridine layer at 532 nm (Shimadzu UV-Vis 1700) was measured for determination of TBA reactive substance [19]. Determination of glutathione An aliquot of articular tissue homogenate supernatant (0.4 ml) was added to dark polyethylene tube containing 1.6 ml of 0.4 M Tris–EDTA buffer, pH 8.9. After vortex-mixing, 40 ␮l of 10 mM dithiobisnitrobenzoic acid in methanol was added. The samples were vortex-mixed again and the absorbance was read at 412 nm after 5 min (Shimadzu UV-Vis 1700). The values of unknown samples were drawn from a standard curve plotted by assaying different known concentrations of glutathione (GSH). The amount of GSH was expressed as ␮mol/g of protein [14]. Measurement of superoxide dismutase (SOD) activity Total SOD activity was measured by determining the ability to inhibit the auto-oxidation of pyrogallol. The rate of auto-oxidation was determined by measuring increases in the absorbance at 420 nm. Reaction mixture containing 0.2 mM pyrogallol in 50 mM Tris–cacodylic acid buffer (pH 8.5) and 1 mM diethylene triamine penta acetic acid was incubated for

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90 s at 25 ◦ C. One unit of SOD activity is defined as the amount of the enzyme required to inhibit the rate of pyrogallol autooxidation by 50% [19]. Measurement of glutathione peroxidase (GPx) The reagents of H2 O2 , 1 mM GSH (glutathione), 0.2 mM NADPH and the homogenized articular cartilage were added to 0.1 M Tris–HCl buffer solution (pH 7.2) and reacted at 25 ◦ C for 5 min. NADPH consumed by the reduction of the oxidized form of glutathione was determined by measuring absorbance at 340 nm (Shimadzu UV-Vis 1700) and glutathione peroxidase (GPx) activity was calculated. Enzyme activity is quoted as the units of NADPH oxidized nmol/1 mg protein/min [19]. Induction of gastropathy The animals were administered with 4 ml/kg of ethanol and 200 mg/kg of sodium salicylate once a day for 14 days. To prevent normal healing, saline (0.9% NaCl solution) was orally administered for 7 days [20]. After the last administration of ethanol and sodium salicyate, indomethacin (10 mg/kg) and EEAS (100, 200 and 400 mg/kg) were orally administered once a day for 7 days. Vehicle control group was administered with saline for 21 days and cimetidine (100 mg/kg) served as standard. Effect of EEAS on gastric juice secretion After 24 h of fasting, animals were anaesthetized with ether and then the pylorus was ligated and then sealed up after the sample solutions had been placed in the duodenal tract. Four hours after the sealing-up of the abdomens, the rats were anaesthetized with ether, the stomachs were excised, and gastric juices were collected. They were centrifuged at 3000 rpm, and then the gastric volumes, pH and acidities were measured and total acid output was calculated. Acidity and total acid output were determined by titration versus 0.05 N sodium hydroxide using phenolphthalein as indicator [20]. Effect of EEAS on gastric lesions The excised stomach was fixed in 2% (v/v) formalin solution for 10 min. After incising the greater curvature, extent of gastric damage in the glandular region was defined as the gastric lesion index [21]. Statistical analysis The data were subjected to ANOVA followed by Newman–Keul’s multiple comparison test [13]. The values of p < 0.05 were considered statistically significant.

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Fig. 1. Effect of EEAS on articular indexes of FCA induced arthritic animals. Values are expressed as mean ± SEM, n = 6 in each group; statistical analysis by one-way ANOVA followed by Newmans–Keul multiple comparison test using Graphpad Instat software; *p < 0.05, **p < 0.01, ***p < 0.001 compared to arthritic control.

Results

Fig. 2. Effect of EEAS on body weights of FCA induced arthritic animals. Values are expressed as mean ± SEM, n = 6 in each group; statistical analysis by one-way ANOVA followed by Newmans–Keul multiple comparison test using Graphpad Instat software; *p < 0.05, **p < 0.01, ***p < 0.001 compared to arthritic control.

Effect of EEAS on clinical signs of arthritis Animals began to show evidence of clinical inflammation in one or more hind paws from day 12. The first manifestation of disease was erythema of one or more ankle joints followed by involvement of the metatarsal and interphalangeal joints. In this study, the arthritic rat joints exhibited the typical swelling patterns of rat polyarthritis. The EEAS treatment was initiated at the onset stage of polyarthritis development (day 12). The articular index of the arthritic control group rapidly increased from day 12 which indicated that the polyarthritic symptoms had certainly developed. During the initial phase of EEAS treatment (day 12–17), the articular indexes of the EEAS treated groups showed little difference with those of arthritic control group. However, after this phase, the indexes started to decrease and finally reached 50% of those of the arthritic control group (Fig. 1). Effect of EEAS on body weight In the first two weeks, there was increment in body weight, which was similar in all groups, and no significant difference observed. After day 12, a significant loss in body weight was observed in the arthritic control rats compared with the control rats, which was continued till the end of the experiment. Treatment with EEAS ameliorated the decrease in body weight (Fig. 2). Nociceptive threshold Hyperalgesia induced by adjuvant inoculation has been assessed by measuring the nociceptive threshold. The nocicep-

Fig. 3. Effect of EEAS on nociceptive threshold of FCA induced arthritic animals. Values are expressed as mean ± SEM, n = 6 in each group; statistical analysis by one-way ANOVA followed by Newmans–Keul multiple comparison test using Graphpad Instat software; *p < 0.05, ***p < 0.001 compared to arthritic control.

tive threshold decreased in all the adjuvant induced groups from day 2 to 14 compared to the vehicle control. EEAS treatment significantly increased the nociceptive threshold compared to the arthritic control, which remained low till the end of the experiment (Fig. 3).

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Table 1 Effect of EEAS on leukocyte infiltration. Treatment

Total leukocytes × 103 cells

Lymphocytes × 103 cells

Monocytes × 103 cells

Vehicle control Arthritic Control Standard Indomethacin 10 mg/kg EEAS(100 mg/kg) EEAS(200 mg/kg) EEAS(400 mg/kg)

1.280 ± 0.018 275.66 ± 9.30a 5.38 ± 0.18***

0.018 ± 0.008 6.32 ± 0.098a 1.02 ± 0.070***

0.148 ± 0.002 0.584 ± 0.012a 0.266 ± 0.006***

100.82 ± 2.64*** 90.00 ± 0.58*** 72.32 ± 0.94***

5.54 ± 0.062*** 4.96 ± 0.08*** 4.20 ± 0.072***

0.516 ± 0.008*** 0.425 ± 0.008*** 0.290 ± 0.006***

Values are expressed as mean ± SEM, n = 6 in each group; statistical analysis by one-way ANOVA followed by Newmans–Keul multiple comparison test using Graphpad Instat software. a p < 0.001 compared to vehicle control. *** p < 0.001 compared to arthritic control.

Effect of EEAS on leukocyte infiltration Total leukocyte concentration in the fluid of the synovial cavity was determined on day 28. Total leukocyte count of EEAS and standard treated groups of rats was significantly lower (p < 0.001) than the count in the arthritic control group of rats (Table 1). Differential counts of the infiltrated leukocytes show that the EEAS treatment significantly (p < 0.001) lower counts of lymphocytes and monocytes/macrophages compared to the arthritic control (Table 1). Indomethacin treatment also resulted in significantly (p < 0.001) lower counts of lymphocytes and monocytes/macrophages (Table 1). Myeloperoxidase analysis Very low myeloperoxidase activity was observed in vehicle control group which was in contrast to the elevated myeloperoxidase level in the arthritic control group. However, treatment with EEAS decreased neutrophil accumulation by reducing myeloperoxidase activity in the synovial tissue of the joints. This decrease in myeloperoxidase activity was similar in all the EEAS treated groups (Table 2). Assessment of malonaldehyde (MAL) Determination of malonaldehyde in the articular cartilage was performed to estimate free-radical damage to biological membranes (Table 2). Low level of malonaldehyde was seen in the vehicle control group at the end of the experiment while there was a significant increase in malonaldehyde in the arthritic control group. Treatment with EEAS significantly (p < 0.001) decreased malonaldehyde concentrations by inhibiting lipid peroxidation in the cartilage tissue. GSH assay The concentration of GSH was evaluated to estimate endogenous defenses against hydrogen peroxide formation. Table 2 shows the changes in GSH content in the joint articular cartilage in the experimental groups. In vehicle control rats, GSH level ranged between 5 and 7.2 ␮mol/g of protein. In contrast, a marked decrease in GSH concentration (2–3 ␮mol/g of pro-

tein) was found in the joint articular cartilage of arthritic control group. Treatment with EEAS (200, 400 mg/kg, p < 0.01, 0.001, respectively) significantly inhibited the decrease in GSH levels. The maximum effect was obtained in EEAS (400 mg/kg) treated group (Table 2). GPx assay GPx, which cleaves hydrogen peroxide and lipid peroxide, is generally considered to be a free radical scavenging enzyme. In vehicle control animals, GPx activity ranged between 38 and 43 nmol/mg of protein. The GPx was found to be considerably lowered (18–24 nmol/mg of protein) in the arthritic control rats. Treatment with EEAS (200, 400 mg/kg, p < 0.01, 0.001, respectively) significantly increased the GPx activity (Table 2). SOD activity SOD activity was evaluated to estimate endogenous defenses against superoxide anions. Table 2 shows the articular cartilage content of SOD in the experimental groups. In control animals, SOD activities ranged between 8 and 9 U/mg of protein. In contrast, a significant decrease (2.8–3.6 U/mg of protein) in this antioxidant was seen in arthritic control rats. Administration of EEAS significantly EEAS (200, 400 mg/kg, p < 0.01, 0.001, respectively) limited the decline in SOD. Effect of EEAS on gastric lesions The control group treated with ethanol–sodium salicylate had a lesion index of 74.66 ± 2.29. EEAS treated groups and cimetidine treated groups produced a significant (p < 0.001) decrease in lesion index at 100, 200 and 400 mg/kg, whereas indomethacin treatment significantly (p < 0.001) increased the lesions (Table 3). Effect of EEAS on gastric juice secretion Table III shows the effect of EEAS on gastric secretion in the rat. EEAS treatment significantly decreased gastric secretion volumes, total acid output, and gastric juice acidity. However, no

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Table 2 Effect of EEAS on Lipid peroxidation and Antioxidant enzymes. Treatment

Malonaldehyde (nmol/mg of protein)

Vehicle control 4.805 ± 0.1240 Arthritic control 13.32 ± 0.4944a standard 8.00 ± 0.2582*** Indomethacin 10 mg/kg EEAS(100 mg/kg) 10.33 ± 0.4216*** EEAS(200 mg/kg) 9.00 ± 0.5164*** EEAS(400 mg/kg) 8.16 ± 0.3073***

Glutathione (␮mol/g of protein)

Glutathione peroxidase (nmol/mg of protein)

Superoxide (U/mg of protein)

Myeloperoxide (U/g of protein)

6.324 ± 0.1260 2.50 ± 0.1342a 5.56 ± 0.3703***

41.526 ± 0.2760 20.50 ± 0.8851a 33.0 ± 0.8944***

8.410 ± 0.1330 3.20 ± 0.1155a 5.633 ± 0.0954***

0.152 ± 0.0084 1.66 ± 0.098a 0.656 ± 0.0640***

2.70 ± 0.1915 3.60 ± 0.0988** 4.10 ± 0.2049***

22.33 ± 0.9545 24.33 ± 0.3333** 29.5 ± 0.7188***

3.166 ± 0.1745 3.93 ± 0.049** 4.233 ± 0.098***

0.9533 ± 0.0500*** 0.56 ± 0.0766*** 0.543 ± 0.0414***

Values are expressed as mean ± SEM, n = 6 in each group; statistical analysis by one-way ANOVA followed by Newmans–Keul multiple comparison test using Graphpad Instat software. a p < 0.001 compared to vehicle control. ** p < 0.01, *** p < 0.001 compared to arthritic control. Table 3 Effect of EEAS on gastric lesions and gastric juice secretion. Treatment

Lesion index (mm)

Vehicle control Indomethacin (10 mg/kg) Cimetidine (100 mg/kg) EEAS (100 mg/kg) EEAS (200 mg/kg) EEAS (400 mg/kg)

74.66 ± 2.290 92.33 ± 3.20c 11.33 ± 2.10***,c

pH 1.25 ± 0.0342 1.16 ± 0.0210

Acidity (mEq/l)

Total acid output

7.2 ± 0.188 7.61 ± 0.066a

107.66 ± 3.27 134.83 ± 6.069c

606.33 ± 4.24 612.66 ± 5.99c

1.95 ± 0.1258***,c

36.33 ± 3.48***,c

1.133 ± 0.033

6.63 ± 0.080***,b

91.33 ± 5.554***,b

51.33 ± 2.765***,c

1.23 ± 0.033

6.18 ± 0.080***,c

44.5 ± 2.705***,c

1.33 ± 0.050

5.26 ± 0.066***,c

72.66 ± 0.980***

2.6 ± 0.115***,c

Volume (ml)

76.5 ± 0.8851***,c 68.33 ± 1.200***,c

128 ± 4.532***,c 541.66 ± 14.380***,c 456 ± 7.882***,c 422.33 ± 7.088***,c

Values are expressed as mean ± SEM, n = 6 in each group; statistical analysis by one-way ANOVA followed by Newmans–Keul multiple comparison test using Graphpad Instat software. a p < 0.05. b p < 0.01, c p < 0.001 compared to vehicle control. *** p < 0.001 compared to arthritic control.

significant change in pH was observed by the EEAS treatment, though cimetidine (standard) increased the pH. Discussion Adjuvant induced arthritis of rat is a typical animal model, widely used for the studies of rheumatoid arthritis, autoimmune diseases, and inflammation. This model has been used to examine and evaluate newly developed anti-arthritic drugs. Freund’s Complete Adjuvant is a reagent frequently used to induce rheumatoid arthritis in animal models [22]. In this model the affected articulations are infiltrated by blood-derived cells, mainly neutrophils, macrophages and dendritic cells [23]. In response to activation, these cells generate ROS released in large amounts surrounding tissue. This released ROS overcomes endogenous antioxidant defenses and induces impairment and destruction of the affected joint constituents such as synovial fluid, cartilage and other articular constituents [24]. The tissues are damaged by the overproduction of reactive oxygen species [25,26]. One of the several approaches for the treat-

ment of rheumatoid arthritis is to employ antioxidants [27]. A. scholaris is a medicinal plant reported to relieve rheumatic pains in folklore medicine [1] which exhibits antinociceptive, antiinflammatory activities of leaf extract of A. scholaris [10] with an additional antioxidant property [12]. In the present study, the EEAS treatment showed anti-arthritic effect, in regards to the inflammatory parameters of adjuvant induced arthritis. It significantly decreased the inflammation when compared to arthritic control as evident by articular index (Fig. 1). The analgesic effect of EEAS in rats with adjuvant induced arthritis was marked as demonstrated by nociceptive threshold (Fig. 3), which further confirms the antinociceptive, anti-inflammatory activities of leaf extract of A. scholaris [10]. Arthritic symptoms of adjuvant-induced arthritic rats were alleviated by EEAS treatment including the recovery of weight loss in EEAS treated group (Fig. 2). The effectiveness of EEAS in reducing the FCA induced inflammation was associated with significant reduction of total leukocyte migration as well as lymphocytes and monocytes/macrophages migration from the blood into the synovial cavity (Table 1). These inflammatory

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cells are major contributors to the initiation and maintenance of the immune response [29,30]. The concentration of these inflammatory cells was significantly greater in the arthritic control group compared to the vehicle control. EEAS treatment significantly decreased the concentration of inflammatory cells and thereby decreased the immune response. The rationale behind the immunosuppression may be the anti-inflammatory activity, which in turn blocks the maintenance of immune response [29,30] and also inhibition of hypersensitivity reaction at higher doses by EEAS as illustrated earlier [6]. Further, the myeloperoxidase assay demonstrated a strong decrease in infiltration of polymorphonuclear cells in the synovial tissue of joints in EEAS treated group (Table 2). Lipid peroxidation is a critical mechanism of the injury that occurs during rheumatoid arthritis, which is often measured by analysis of tissue malonaldehyde [31,32]. The large amount of malonaldehyde in arthritic control group is consistent with the occurrence of damage mediated by free radicals. Treatment with EEAS produced a significant attenuation of malonaldehyde. The decrease in neutrophil accumulation by EEAS treatment might be due to the inhibition of lipid peroxidation (Table 2) and the consequent decrease in the chemotactic decrease of peroxide [31]. The production of oxygen free radicals that occurs with the development of arthritis in the articular cartilage leads to decreased GSH and SOD levels as a consequence of their consumption during oxidative stress and cellular lysis [33,34], which is evident by decreased levels of GSH, GPx and SOD in arthritic control group. EEAS treatment blunted the depletion of GSH, GPx and SOD (Table 2), probably by competing for scavenging of free radicals, which in turn resulted in recuperation of antioxidant enzyme levels. The present study also revealed that EEAS reduced gastric lesion indices and gastric juice secretion in rats with gastropathy induced by ethanol and sodium salicylate (Table 3). These observations confirm the merits of anti-arthritic natural product in avoiding the harmful effects of synthetic anti-inflammatory drug which makes EEAS to be potential candidate for longterm administration as an anti-arthritic agent. It is reported that many natural antioxidants inhibit the gastropathy caused by oxidative stress [35,36]. The presence of potential antioxidant moieties like flavonoids, alkaloids and triterpenoids in alcoholic extract of A. scholaris has been reported [37] and the in vitro antioxidant activity of EEAS has been established [28]. These findings suggest that the anti-inflammatory, immunosuppressant and antioxidant activities of EEAS may be the possible reason behind the observed antiarthritic activity. EEAS is also gastroprotective which is a point of distinct advantage when considering the chronic administration in arthritic medication. Hence, it is worthwhile to isolate and elucidate the bioactive principles that are responsible for the anti-arthritic activity that is underway. Conclusion The antiarthritic and anti-oxidant effects of ethanolic extract of leaves of A. scholaris Linn. R.Br., is mediated through the anti-inflammatory, immunosuppressant and antioxidant activi-

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ties and the effects may be attributed to the components such as flavonoids and triterpenoids present in the EEAS. EEAS is also gastroprotective which is a point of distinct advantage when considering the chronic administration in arthritic medication. However, further studies are underway to isolate the lead molecule(s) responsible for the activity and also to pinpoint on the mechanism of action of the same. Authors All research done by the authors. Financial support None. Conflict of interest None. Acknowledgements The authors would like to acknowledge Dr. K.R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth University, Pune, India and Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi, India for providing the necessary facilities to carryout the study. References [1] Nadkarni AK. Dr. K.M. Nadkarni’s Indian Materia Medica, vol. 1. Bombay, India: Popular Prakashan; 1976. p. 80–3. [2] Kirtikar KR, Basu BD. Indian Medicinal Plants, vol. 1. Allahabad, India: Lalit Mohan Basu; 2002. p. 111–4. [3] Versha P, Ghosh B, Anroop B, Ramanjit M. Antimicrobial activity of Alstonia scholaris leaf extracts. Indian Drugs 2003;40:412–3. [4] Keawpradub N, Kirby GC, Steele JCP, Houghton PJ. Antiplasmodial activity of extracts and alkaloids of three Alstonia species from Thailand. Planta Medica 1999;65:690–4. [5] Lim-Sylianco CY, Jocano AP, Linn CM. Antimutagenicity of twenty Philippine plants using the micronucleus test in mice. Philippine Journal of Science 1990;117:231–5. [6] Iwo MI, Soemardji AA, Retnoningrum DS, Sukrasno UUM. Immunostimulating effect of pule (Alstonia scholaris L. R. Br., Apocynaceae) bark extracts. Clinical Hemorheology and Microcirculation 2000;23:177–83. [7] Lin SC, Lin CC, Lin YH, Supriyatna S, Pan SL. The protective effect of Alstonia scholaris R. Br. on hepatotoxin-induced acute liver damage. American Journal of Clinical Medicine 1996;24:153–64. [8] Saraswathi V, Ramamoorthy N, Subramaniam S, Mathuram V, Gunasekaran P, Govindasamy S. Inhibition of glycolysis and respiration of sarcoma-180 cells by echitamine chloride. Chemotherapy 1998;44:198–205. [9] Arulmozhi S, Mazumder PP, Sathiyanarayanan L, Thakurdesai PA. Antidiabetic and anti-hyperlipidemic activity of leaves of Alstonia scholaris Linn. R. Br. European Journal of Integrative Medicine 2010;2:23–32. [10] Arulmozhi S, Mazumder PM, Ashok P, Hulkoti B, Sathiyanarayanan L. Antinociceptive and anti-inflammatory activities of Alstonia scholaris Linn. R. Br. Pharmacognosy Magazine 2007;3:106–11. [11] Shang JH, Cai XH, Feng T, Zhao YL, Wang JK, Zhang LY, et al. Pharmacological evaluation of Alstonia scholaris: anti-inflammatory and analgesic effects. Journal of Ethnopharmacology 2010;129(2):174–81.

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[12] OECD Guidelines –“Guidance document on acute oral toxicity testing” (2001) Series on testing and assessment No. 24, organisation for economic co-operation and development, OECD Environment, health and safety publications, Paris (http://www.oecd.org/ehs) [accessed 12th January 2007]. [13] Purnima Ashok, Rajani GP, Arulmozhi S, Basavaraj Hulkoti, Desai BG, Rajendran R. Anti-inflammatory and Anti-ulcerogenic effect of Crotalaria juncea Linn. in albino rats. Iranian Journal of Pharmacology and Therapeutics 2006;5(2):141–4. [14] Campo MG, Avenoso A, Campo S, Ferlazzo AM, Altavilla D, Calatroni A. Efficacy of treatment with glycosaminoglycans on experimental collageninduced arthritis in rats. Arthritis Research and Therapy 2003;5:R122–31. [15] Miura T, Okazaki R, Yoshida H, Namba H, Okai H, Kawamura M. Mechanisms of analgesic action of neurotropin on chronic pain in adjuvantinduced arthritic rat: roles of descending noradrenergic and serotonergic systems. Journal of Pharmacological Sciences 2005;97:429–36. [16] Levy ASA, Simon O, Shelly J, Gardener M. 6-Shogaol reduced chronic inflammatory response in the knees of rats treated with complete Freund’s adjuvant. BMC Pharmacology 2006;6:12. [17] Lefkowitz DL, Gelderman M.P., Fuhrmann SR, Grahams Starnes JD, Lefkowitz SS, Bollen A, et al. Neutrophil myeloperoxidase–macrophage interactions perpetuate chronic inflammation associated with experimental arthritis. Clinical Immunology 1999;91:145–55. [18] Mullane KM, Kraemer R, Smith B. Myeoloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischaemic myocardium. Journal of Pharmacological Methods 1985;14:157–67. [19] Nam J-H, Jung H-J, Choi J, Lee K-T, Park H-J. The anti-gastropathic and anti-rheumatic effect of Niga-ichigoside F1 and 23-hydroxytormentic acid isolated from the unripe fruits of Rubus coreanus in a rat model. Biological and Pharmaceutical Bulletin 2006;29:967–70. [20] Dai S, Ogle CW. A simple method for the production of peptic ulceration in the rat. Life Sciences II 1973;12:505–12. [21] Mizui T, Dodeuchi M. Effect of polyamines on acidified ethanol-induced gastric lesions in rats. Japanese Journal of Pharmacology 1983;33:939–45. [22] Choi J, Lee KT, Jung HJ, Park HS, Park HJ. Anti-rheumatoid arthritis effect of the Kochia scoparia fruits and activity comparison of momordin lc, its prosapogenin and sapogenin. Archives of Pharmaceutical Research 2002;25:336–42. [23] VanderBorght A, Geusens P, Raus J, Stinissen P. The autoimmune pathogenesis of rheumatoid arthritis: role of autoreactive T cells and new immunotherapies. Seminars in Arthritis and Rheumatism 2001;31:160–75.

[24] Bauerova K, Bezek A. Role of reactive oxygen and nitrogen species in etiopathogenesis of rheumatoid arthritis. General Physiology and Biophysics 1999;18:15–20. [25] Aghdassi E, Allard JP. Breath alkanes as a marker of oxidative stress in different clinical conditions. Free Radical Biology and Medicine 2000;28:880–6. [26] Li G, Liu Y, Tzeng N, Cui G, Block ML, Wilson B, et al. Protective effect of dextromethorphan against endotoxic shock in mice. Biochemical Pharmacology 2005;69:233–40. [27] Darlington LG, Stone TW. Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders. British Journal of Nutrition 2001;85:251–69. [28] Arulmozhi S, Mazumder PM, Ashok P, Sathiyanarayanan L. In vitro antioxidant and free radical scavenging activity of Alstonia scholaris Linn. R. Br. Iranian Journal of Pharmacology and Therapeutics 2007;6(2):191–6. [29] Mapp PI, Grootveld MC, Blake DR. Hypoxia, oxidative stress and RA. British Medical Bulletin 1995;51:419–36. [30] Weyand CM. The role of T cells in RA. Arthritis and Rheumatism 2000;43:I122–33. [31] Wills ED. Evaluation of lipid peroxidation in lipid and biological membranes. In: Snell K, Mullock B, editors. Biochemical toxicology: a practical approach. Oxford: IRL Press; 1987. p. 127–52. [32] Jira W, Spiteller G, Richter A. Increased levels of lipid oxidation products in low density lipoproteins in patients suffering from rheumatoid arthritis. Chemistry and Physics of Lipids 1997;87:81–9. [33] Kizilntuc A, Cogalgil S, Cerrahoglu L. Carnitine and antioxidants levels in patients with rheumatoid arthritis. Scandinavian Journal of Rheumatology 1998;27:441–5. [34] Hassan MQ, Hadi RA, Al-Rawi ZS, Padron VA, Stohs SJ. The glutathione defense system in the pathogenesis of rheumatoid arthritis. Journal of Applied Toxicology 2001;21:69–73. [35] Soybel DI, Modlin IM. Implications of sustained suppression of gastric acid secretion. The American Journal of Surgery 1992;163: 613–22. [36] Mercer DW, Kirshner MS, Ritchie WP, Dempsey DT. Topical prostaglandin E2 and isoproterenol reduce bile acid-induced gastric mucosal injury in shocked rats. Journal of Surgical Research 1994;56: 184–91. [37] Khan MR, Omoloso AD, Kihara M. Antibacterial activity of Alstonia scholaris and Leea tetramera. Fitoterapia 2003;74:736–40.