Vol. 11 (4) April (2016) Res. J. Biotech Research Journal of Biotechnology Effect of methanolic root extract of Blepharispermum subsesssile DC in controlling arthritic activity Das Soni and Sureshkumar P.* Department of Biotechnology, BIT-Campus, Anna University, Tiruchirappalli – 620 024, INDIA *sureshbiotech2003@yahoo.co.in; drsureshbiotech2003@gmail.com arthritic activity. Proteinases have also been implicated in RA reactions. Neutrophiles are known to be rich source of proteinases carrying in their lysosomal granules. It was previously reported that leukocytes proteinases play an important role in the development of tissue damage during inflammatory reactions and a significant level of protection was provided by proteinase inhibitors36. Abstract The aim of the study was to evaluate the in vitro and in vivo anti-arthritic potential in the roots of Blepharispermum subsessile DC (MEBS). In vitro models such as HRBC membrane stabilization (Human red blood cell), inhibition of protein denaturation and proteinase inhibitory activity was performed for the assessment of anti-arthritic activity. Three different in vivo models of arthritis namely Freund’s Complete Adjuvant (FCA)-, formaldehydeand carragennan-induced models were also used in this study. The MEBS significantly inhibited the HRBC (95.00±0.03), protein denaturation (94.22±0.04) and proteinase inhibitory activity (85.44±0.45). The various biochemical parameters (Hb, Total WBC count, RBC count, ESR and Creactive protein) histopathological analysis and radiographic examinations studies substantiated the antiarthrtic potential of MEBS. Although there are various types of drugs used for the treatment of RA such as steroids (glucocorticoids), auranofin, methotrexate (DMRDS), adalimumab, anakinra, tocilizumab (biological agents), azathioprine, cyclosporinA and cyclophosphamide (immunosuppressive agents), they cannot be used for longer period of time because of their potential toxic effects and high cost44. As a consequence, researchers are now searching for alternatives therapeutics agents. As a part of this search, significant attention has been focused on plant based drugs that are used in traditional medicine because those drugs elicit fewer side effects and are inexpensive39,41. Herbal medicine can also act as synergically to cure health ailments42. In addition, recent interest in alternative treatments for arthritis is gaining momentum but lack of scientific validation in herbal medicine is a lacuna. Keywords: MEBS, in vitro activity, Freund’s complete adjuvant, formaldehyde, carragennan, anti-arthritic activity. Introduction One such plant that is widely used in Indian traditional medicines but that has not been studied in a well-controlled experimental trial till date is the species of B.subsessile DC. Its different parts have been used for various health ailment treatments by local medical practitioners. B.subsessile DC belongs to the family of Compositae or Asteraceae which includes ten species. It is a rare shrub of India, found in dry deciduous forest of Madhya Pradesh, Maharashtra, Karnataka, Odisha and Tamilnadu1. It is endangered in Odisha11.The root of B.subsessile DC has anti-arthiritic activity27 and leaf juice is used as “eye drop” in various ophthalmic diseases. It is also used in different ailments like diarrhea17, skin diseases6, backache40 and irregular menstruation45. Rheumatoid arthritis (RA) is a chronic, progressive, disabling autoimmune disease characterized by systemic inflammation of joints, damaging cartilage and bone around the joints25. It is characterized by persistent synovitis, systematic inflammation and auto antibodies production33. Of all the rheumatoid disorders, RA remains a formidable disease, being capable of producing severe crippling deformities and functional disabilities19. The disorder is more common in women and in elderly persons. Its etiology is unknown although the main risk factors include genetic factors and smoking26. Lysosomal enzymes released during inflammation of joints produce tissue injury by damaging the macromolecules and causing lipid peroxidation of membranes. HRBC is analogous to the lysosomal membrane and its stabilization implies that the extract may as well stabilize lysosomal membranes8. The production of auto antigens in certain arthritic diseases like RA may be due to in vivo denaturation of proteins which involves alteration in electrostatic, hydrogen, hydrophobic and disulphide bondings16. Previous study has shown only 5 chromene compounds isolated from MEBS2. The present study was undertaken to evaluate in vitro and in vivo anti-arthritic activities of MEBS. Material and Methods Plant Material: B. subsessile DC was collected from Gurudangar Reserve forest (Odisha) in October, 2011. The plant was identified by the Botanical Survey of India, (BSI) Coimbatore, Tamilnadu, India and the voucher specimen So, by controlling the production of auto antigen through inhibiting denaturation of protein in RA can lead to anti- 65 Vol. 11 (4) April (2016) Res. J. Biotech Research Journal of Biotechnology was kept in the institutional herbarium for future reference (BSI/SRC/5/23/2011-12/Tech.1250). 3 min. After cooling, add 2.5 ml of phosphate buffer to the above solutions. The absorbance was measured using UVVisible spectrophotometer at 416 nm30. The control represents 100% protein denaturation. The results were compared with Diclofenac sodium. The percentage inhibition of protein denaturation can be calculated as indicated below: Drugs and Chemicals: All the chemicals and reagents used were of highest analytical grade except the drug, Diclofenac sodium was purchased from Sigma-Aldrich, USA. Preparation of the Plant Extract: The dried powdered roots of the plant material were extracted separately with methanol using Soxhlet apparatus for 48 h. The solvent was distilled at lower temperature under reduced pressure and concentrated on water bath to get the crude extract which is stored in desiccators for future use47. Percentage Inhibition = [100 - (optical density of test solution – optical density of product control) ÷ (optical density of test control) × 100]. Proteinase Inhibitory Activity: The test was performed according to the modified method of Oyedepo et al.38 The reaction mixture (2 ml) was containing 0.06 mg trypsin, 1 ml 20 mM Tris HCl buffer (pH 7.4) and 1 ml test sample of different concentrations. The mixture was incubated at 37°C for 5 min and then 1 ml of 0.8% (w/v) casein was added. The mixture was incubated for additional 20 min. 2 ml of 70% perchloric acid was added to terminate the reaction. Cloudy suspension was centrifuged and the absorbance of the supernatant was read at 210 nm against buffer as blank. The experiment was performed in triplicate. The percentage inhibition of proteinase inhibitory activity was calculated: In vitro anti-arthritic activity Membrane Stabilization: The membrane stabilization of HRBC membrane was performed using the method of hypo tonicity membrane lysis43. Blood was collected (2ml) from healthy volunteers and was mixed with equal volume of sterilized Alsevers solution (2% dextrose, 0.5% citric acid and 0.42% NaCl in distilled water) and centrifuged at 3000 rpm. The packed cells were washed with isosaline solution and a 10% v/v suspension was prepared with normal saline and kept at 4°C undisturbed before use. Different concentrations of MEBS extract (50, 100, 250, 500, 1000 and 2000 μg/ml) in normal saline, diclofenac sodium as standard (50, 100, 250, 500, 1000, 2000 μg/ml) and control (distilled water to produce 100 % hemolysis) were separately mixed with 1ml phosphate buffer [pH 7.4, 0.15 M], 2 ml hypo saline [0.36 %], 0.5 ml of 10 % v/v. Percentage inhibition = 100–[(optical density of test solution)÷(optical density of control)× 100]. In vivo anti-arthritic activity Experimental Animals: Male albino wistar rats weighing approximately 170–200 g were used for the studies. The animals were housed in cages in a group of six under standard laboratory conditions (12/12 h light/dark cycle at 25ºC). The rats were fed with commercial rat diet and water ad libitum. The Institutional Animal Ethics Committee approved the experimental protocols (CPCSEA-3618.8.2012). Care of animals was taken according to CPCSEA guidelines. HRBC suspension was added to above said preparations. All the assays were incubated at 37oC for 30 min and centrifuged at 30000 rpm centrifuged for 20 min and haemoglobin content of supernatant solution was estimated spectrometrically at 560 nm28. The percentage inhibition of membrane stabilization can be calculated as: Percentage inhibition = 100–[(optical density of test solution)÷(optical density of control)]×100 Acute Toxicity Test: To study the toxic effect of methanolic root extract of B. subsessile DC on rats, the appearance, the general behavioural pattern, mortality and histological analysis were performed. The symptoms, mortality were observed in rats for up to 14 days following the administration of MEBS at single dosage of 2000 mg/kg body weight. The behavioural patterns of animals were observed for first 6 h and followed by 14 h after the administration. At the end of toxic test, rats were killed and spleen was sent for histological examination24. Inhibition of Protein Denaturation: Test solution (0.5ml) consists of 0.45ml of Bovine serum albumin (5% w/v aqueous solution) and 0.05ml of test (plant extract) solution. Test control solution (0.5ml) consists of 0.45ml of bovine serum albumin (5% w/v aqueous solution) and 0.05ml of distilled water. Product control (0.5ml) consists of 0.45ml of distilled water and 0.05 ml of test solution. Standard solution (0.5ml) consists of 0.45ml of Bovine serum albumin (5% w/v aqueous solution) and 0.05ml of Diclofenac sodium33. Various concentrations (250, 500, 1000 and 2000 μg/ml) of plant extracts (test solution) and diclofenac sodium (standard) were taken. All the above solutions were adjusted to pH 6.3 using 1N HCl. Dosage Fixation: Oral administration of the MEBS for group of six rats in graded doses of 0.25, 0.5, 1.0, 1.5 and 2.0g/kg of body weight dissolved in dimethly sulfoxide : methanol : distilled water (25% : 15%: 60%) was performed. Earlier, the rats were fasted overnight with free access to water. LD50 obtained from this experiment was 1000mg/kgb.w./rat. Dose selection was performed by The samples were incubated at 370C for 20 min and the temperature was increased to keep the samples at 570C for 66 Vol. 11 (4) April (2016) Res. J. Biotech Research Journal of Biotechnology taking 1/10th of the lethal dose. The dose of the MEBS selected for our experiment was therefore 100mg /kg rat. Indomethacin was used as a standard drug against which to compare the efficacy of MEBS. The dose of indomethacin was selected as 25mg/kg rat by using a human-rat conversion factor. (Vc-Vt) x 100/Vc where Vc is the mean paw edema of the control group (II) and Vt is the mean paw edema of the test groups (III and IV)3. Biochemical assays: Biochemical changes with respect to Hemoglobin (Hb) concentration, total WBC count, RBC count, ESR and C- reactive protein was observed in the FCA induced arthritic rats after 28 days of treatment. The Hb content was estimated by the method of Drabkin and Austin7. Red blood cell (RBC) and white blood cell (WBC) counts were estimated according to the method of Chesbrough and McArthur13 in an improved Neubauer chamber. Estimations of erythrocyte sedimentation rates (ESR) were carried out by the method of Westergren18. Creactive protein levels were estimated using the ELISA kit obtained from Alpha Diagnostics Intl., USA. Experimental design Freund’s complete adjuvant (FCA)-induced arthritis: Rats were subdivided into four groups of six rats in each group and the experiments were performed for 28 days. Group I – Normal rats administered vehicle (normal saline); Group II – Arthritic rats administered with normal saline; Group III - Arthritic rats administered with Indomethacin 25mg/kg and Group IV - Arthritic rats administered the MEBS 100mg/kg. The rats were made arthritic by subcutaneous injection of 0.1 ml 1 % FCA into the sub-plantar side of the left hind paw of rats from all groups except normal control group37. Thirty mins before FCA injection and thereafter, on each day animals from different groups received respective treatments. Paw volume was measured on 0th, 8th, 14th, 21st and 28th days of experimental periods by using Plethysmometer (UGO Basile, Italy). The mean changes in paw edema with respect to initial paw volume were calculated on respective days. At the end of the 28th day, rats were euthanized by cervical dislocation and serum samples were collected for further biochemical assays. Histopathological examination of spleen: On 28thday, after the final scan, animals were anesthetized by CO2 inhalation and euthanized by cervical dislocation and spleens were fixed in 10% neutral buffered formalin. Then it was decalcified prior to embedding in paraffin. Sections were stained with haematoxylin/eosin to assess inflammation/cellular infiltration within the spleen. Images were captured with an Olympus Provis light microscope, USA and sections were also digitized with a microscope slide scanner, USA. Formaldehyde-induced arthritis: Animals were randomly divided into four groups of six animal in each group (n=6). Group I – Normal rats administered vehicle (normal saline); Group II – Formaldehyde rats administered with normal saline; Group III –Formaldehyde rats administered with Indomethacin 25mg/kg and Group IV - Arthritic rats administered the MEBS (100mg/kgb.w.). Rats were injected with 0.1ml 2 % (v/v) of formaldehyde solution in the planar surface of the left foot on the first and third day of the test. Drug treatment started from the initial day i.e. from the day of formaldehyde injection (0th day) and continued till 10th day as performed in the FCA model. The rat paw volume was recorded daily by using Plethysmometer20. Radiographic examination: Radiographic examinations were performed on 28th day of FCA injection by digital Xrays (Agfa-CR-30, USA) to determine deformities in joints, bones, cartilages and soft tissues of hind paws. Statistical Analysis In vitro anti-arthritic activity: Results obtained were analyzed using one-way analysis of variance (ANOVA). Difference between means were accepted as significant at P<0.05. Results were presented as mean±SEM. In vivo anti-arthritic activity: The results were analyzed using One way Analysis of Variance (ANOVA) followed by appropriate Post-hoc tests (Bonferroni test for FCA model, Dunnett test for Formaldehyde and Carrageenan models) using SPSS 17.0. The values are expressed as means ± SEM and p < 0.05 was considered statistically significant. Carrageenan- induced acute inflammation: Rats of group II, III and IV were injected with 1 ml of 1% carrageenan w/v in normal saline into the right hind paw while the rats of group I were injected with normal saline alone and served as normal control. Rats of groups I with distilled water and II were administered with distilled water orally half an hour before paw injection while rats of groups III and IV received orally with Diclofenac 25 mg/kg and MEBS 100mg/kg respectively. Paw volumes was measured plethysmographically from 0 to 4 h at every one hour interval. The paw edema at any point of time was calculated as the difference from the initial paw volume of that rat. In all three arthritis induction model, percentage inhibition of paw edema was calculated by the formula: Results In vitro anti-arthritic activity Membrane stabilization method: MEBS showed significant in vitro anti-arthritic activity compared to the standard drug diclofenac sodium. The percentage of stabilization was found to be 95±0.003 and 97.5±0.74 for MEBS extract and standard respectively. The activity of MEBS was concentration dependent because with the 67 Vol. 11 (4) April (2016) Res. J. Biotech Research Journal of Biotechnology increasing concentration, the activity also increased (Table 1). radiographic examinations from the FCA-induced arthritis control group revealed severe soft tissue swelling, narrowing of the joint spaces and destruction of the bones and cartilages in the ankle joint (Figures 2B and 2C) as compared to the normal control group (Figure 2A). The administration of MEBS has appreciably prevented the soft tissue swelling and the destruction of the ankle joints (Figure 2D). Inhibition of protein denaturation: The MEBS exhibited maximum inhibition of protein denaturation 94.22±0.04 at 2000µg/ml and its effect was compared with standard antiarthritic drug. Diclofenac sodium showed the maximum inhibition 97.93±0.70 at the same concentration (Table 2). Proteinase inhibitory activity: The MEBS and diclofenac sodium showed significant percentage of proteinase inhibitory activity 85.44±0.45 and 87.66±0.55 respectively (Table 2). Formaldehyde and carrageenan-induced arthritis models: In both models, we only checked the effect of MEBS on rat paw volume. Table 6 and 7 depict changes in paw volume in formaldehyde and Carrageenan-induced arthritis models respectively. The development of edema in the paw of the rat after injection of formaldehyde (0.1ml, 2% w/v) is due to the release of histamine, serotonin and the prostaglandin like substances at the site of injection9,23. The maximum rat paw edema was observed on 6th day in formaldehyde-induced rat. The maximum inhibition of paw edema in formaldehyde-induced arthritis was observed at day 10 for MEBS which may be due to the antiinflammatory potential of MEBS (Table 6). In Carrageenan-induced model, the maximum rat paw edema was observed in 3rd hour when compared to control rat. MEBS inhibited (Table 7) Carrageenan-induced paw volume about 43.9%, 40.4%, 32.4% and 30% at 4th h, 3rd h, 2nd h and 1st h respectively. Acute toxicity study: The toxic effects of MEBS on the appearance and the general behavioural pattern of rats were shown in table 3. No toxic symptoms or mortality were observed in any animal which lived up to 14 days after the administration of extract. The behavioural patterns of animals did not display any significant changes in behavior, skin effects, breathing, impairment in food intake and water consumption, postural abnormalities and hair loss. In the extract treated group, rapid heartbeat was observed in first 6 h but it became normal and this may due to the stress of receiving the oral administration of the extract. Histological Analysis: Histopathology of normal control rats spleen showed no inflammation, inflammatory cell proliferation (Fig. 1A). FCA treated rats showed cartilage destruction, decreased synovial space and severe inflammation (Fig. 1B). Indomethacin treated rats showed minor cartilage destruction, synovial space thinning and vascular proliferation (Fig. 1C). MRBS treated showed significant protection against cartilage destruction, synovial thinning and vascular proliferation (Fig. 1D). Discussion Stabilization of lysosomal membrane is important in limiting the anti-inflammatory response. The lysosomal enzymes released during inflammation produce various disorders. The extra cellular activity of these enzymes is said to be related to acute or chronic inflammation31. MEBS extract exhibited membrane stabilization effect by inhibiting hypotonicity-induced lysis of HRBC membrane. The inhibition of hypotonicity-induced HRBC membrane lysis i.e. stabilization of HRBC membrane was taken as a measure of the anti inflammatory activity of MEBS extract. Denaturation of tissue protein is one of the well documented causes of inflammatory and arthritic diseases. Steroids can decrease inflammation and reduce the activity of immune response 46. With the increasing concentrations of the MEBS extract, the proteinase inhibitory activity was found to be decreased and exhibited enhanced protection. Thus, our in vitro study demonstrates that the MEBS extract was capable of controlling the membrane lysis, inhibiting denaturation of protein and proteinase inhibitory activity, revealing a potential of therapeutic benefit of MEBS extract. Radiographic examination: The results of radiographic examination of rats from the arthritis control group revealed severe soft tissue swelling, narrowing of the joint spaces, and destruction of the bones and cartilages in the ankle joint (Fig. 2B and C) as compared to the normal control group (Fig. 2A). The administration of MRBS extract (100 mg/kg) has markedly prevented the soft tissue swelling and the destruction of the ankle joints (Fig. 2D). In vivo anti-arthritic activity Freund’s complete adjuvant arthritis (FCA) model: First, the rat paw edema was observed in FCA model, the maximum rat paw edema was observed in the 3rd week following FCA injection. It was reduced by MEBS which was highly significant at 0.55±0.02 (P < 0.005) (Table 4) following 4th week of administration. Then, we measured biochemical parameters in blood of all experimental groups (Table 5). After the administration of MEBS (100mg/kg) to the FCA-injected rats (group IV) for 28 days, there was a significant decrease in the levels of WBC, ESR and Creactive protein and a concomitant increase in the levels of Hb and RBC when compared to FCA injection alone. The Adjuvant-induced arthritis of rat is a typical animal model, widely used for the studies of rheumatoid arthritis, autoimmune diseases and inflammation5. This model has been used to examine and evaluate newly developed antiarthritic drugs. Among various adjuvants, FCA is a reagent frequently used to induce Rheumatoid Arthritis in animal models. In this model, eosinophil infiltration, synovitis and 68 Vol. 11 (4) April (2016) Res. J. Biotech Research Journal of Biotechnology damage of cartilage were evident15. Anemia is most commonly noted in patients with chronic arthritis21. The result of MEBS extract showed profound antiinflammatory activity against formaldehyde induced edema14. The carrageenan-induced edema has been described as also biphasic event, a rapid early phase (up to 0 to 2 h) triggered by the concerted release of histamine, bradykinin, 5-hydroxytryptamine, or cyclooxygenase products and a more sustained late phase (2 to 5 h) regulated by neutrophil infiltration and sustained production of arachidonic metabolites (prostanoids) primarily by cyclooxygenase or nitric oxide from inducible nitric oxide synthase12,14. Hence, in the present study the paw volume in all models revealed that MEBS showed significant inhibition of paw edema, indicating antiinflammatory effect of the extract. The two most common explanations are gastrointestinal blood loss due to arthritis medications and also bone marrow changes in patients with inflammatory arthritis which prevented the release of iron for the incorporation into red blood cells4,35. The anemic condition persisted was associated with decreased level of RBC count and Hb and an increased level of WBC count and C-reactive protein indicating an infectious and inflammatory condition32. MEBS extract showed a significant recovery from the FCA-induced anemia. Subsequently, we analyzed the paw volume using FCA, formaldehyde and Carrageenan induced models. Paw swelling is one of the major factors in assessing the degree of inflammation and curative efficacy of drugs10. The edema induced by adjuvants is highly sensitive to NSAIDs and has been accepted as a useful indicator for identifying the new anti-inflammatory molecules. Histopathological studies suggested that in the FCA treated group of rats eosinophill infiltration, synovitis and damage of cartilage are evident. Treatment with MEBS extract significantly inhibited inflammatory cells, synovitis and also maintained the integrity of cartilage tissues. Treatment with indomethacin successfully inhibited all these inflammatory processes. Finally, radiographic studies showed that the FCA injected groups of animals were affected by deformation and abnormalities in the toes. Treatment with MEBS extract restored not only the normal architecture but also maintained the integrity of cartilage tissue and joints. Adjuvant arthritis in rats exhibited many similarities to human rheumatoid arthritis22. The percentage inhibition of paw edema on 21st day is significantly higher in MEBS than indomethacin treated rats suggesting the effect of MEBS inhibitory effect on the prostaglandin – mediated inflammatory pathway29. Activation of polymorphonuclear neutrophils is primary immunological response to invading pathogens. Conclusion In conclusion, the present study indicates that MEBS extract exhibits strong anti-arthritic activity and it may be due to the presence of high phenolic content, flavonoids and terpenoids in MEBS extract. Further, the potent bioactive compounds will be screened, isolated and identified for their anti-arthritic activity in our future studies. The nociceptive effect of formaldehyde is biphasic, an early neurogenic component followed by a later tissue mediated response48. Acute inflammation induced by formaldehyde result from cell damage prevented the production of endogenous mediators such as histamines, serotonins, prostaglandins and bradykinins. Table 1 Effect of MEBS on HRBC membrane stabilization Conc. (μg/ml) B. subsessile DC 50 100 250 500 1000 2000 % Membrane Stabilization % Membrane Stabilization Diclofenac sodium B. subsessile DC 68.9±1.22 79.4±0.35 77.4±1.23 82.4±1.28 87.5±1.33 97.5±0.74 56.4±0.18 80.6±0.30 85.5±0.37 91.5±0.45 93.5±0.40 95±0.003 69 Vol. 11 (4) April (2016) Res. J. Biotech Research Journal of Biotechnology Table 2 Effect of MEBS extract on Protein denaturation and proteinase inhibitory activity Conc.(μg/ml) % of protein denaturation % of proteinase inhibitory activity Diclofenac Methanolic Root Diclofenac Methanolic Root extract of B. subsessile extract of B. subsessile sodium sodium 50 45.67±0.90 40.77±0.85 30.88±1.18 25.38±1.26 100 56.89±0.78 51.34±0.67 38.56±0.58 33.28±0.65 250 79.34±1.30 77.82±0.25 47.73±1.40 40.56±1.44 500 85.33±0.30 83.75±0.35 55.46±0.78 58.35±0.85 1000 93.77±1.26 90.80±0.30 62.35±1.36 67.65±1.25 2000 97.93±0.70 94.22±0.04 87.66±0.55 85.44±0.45 Table 3 General appearance and behavioral observations for control and treated groups Observation Control group Test group 6h 14 h 6h 14 h Skin and fur Normal Normal Normal Normal Eyes Normal Normal Normal Normal Mucous membrane Normal Normal Normal Normal Behavioural patterns Normal Normal Rapid heart beat Normal Salivation Normal Normal Normal Normal Lethargy Normal Normal Normal Normal Sleep Normal Normal Normal Normal Diarrhea Normal Normal Normal Normal Table 4 Effect of MEBS extraction rat paw volume in FCA-induced arthritis model MeanPaw volume ( ml) ± SEM Groups 0 Week 1st Week 2nd Week 3rd Week 4th Week Group I 0.35±0.26 0.34±0.025 0.34±0.028 0.36±0.028 0.34±0.027 Group 0.40±0.27 1.46±0.17### 1.76±0.16### 2.05±0.13### 1.90±0.13### II Group 0.41±0.26 0.78±0.17** 1.13±0.33** 1.48±0.13** 0.78±0.04** III Group 0.35±0.22 0.65±0.12** 0.86±0.003** 1.23±0.002** 0.55±0.02** IV Values are expressed as mean ± SEM (n=6), ### = P<0.001 ( in comparison to control group (group 1),** = P<0.005 ( in comparison to FCA group (group 2) (One-way ANOVA followed by Bonferroni test). Table 5 Effect of MEBS extracts on various biochemical parameters Parameters Group I Group II Group III Group IV RBC (cells/cu.mm) 4.56±0.08 3.49±0.15 3.71±0.12 4.24±0.12 WBC(million/cu.mm) 6.68±0.06 13.64±0.11### 9.55±0.13*** 7.02±0.06*** Hb (gm %) 13.18±1.17 8.77±0.18### 10.89±0.21*** 12.63±0.19*** ESR (mm/hr) 3.18±0.06 10.67±0.37### 7.53±0.60*** 3.43±0.06 CRP(µg/ml) 172.87±4.40 427.72±27.5### 260.90±7.10*** 211.47±12.83*** Values are expressed as mean ± SEM (n=6). ### = P < 0.001 in comparison to Control group, *** = P<0.001.as compared with FCA group. (Student’s unpaired “t” test). 70 Vol. 11 (4) April (2016) Res. J. Biotech Research Journal of Biotechnology Table 6 Effect of MEBS extraction rat paw volume in Formaldehyde induced arthritis model Groups 0 Week Day3 Day6 Day10 Group I 0.87±0.02 0.89±0.014 0.87±0.17 0.85±0.009 Group 0.90±0.021 1.35±0.010### 1.88±0.02### 1.76±0.018### II Group 0.84±0.011 1.12±0.004** 1.20±0.003** 1.05±0.002** III Group 0.86±0.009 1.26±0.003** 1.30±0.002** 1.16±0.001** IV Values expressed as Mean ± SEM, ### = P<0.001 (in comparison to control group (group 1), ** = P<0.005 ( in comparison to Formaldehyde group (group 2). One way ANOVA followed by Dunnet’s test) Table 7 Effect of MEBS extraction rat paw volume in Carrageenan induced arthritis model Mean Paw volume ( ml) ± SEM at different time intervals (Percentage Inhibition in parenthesis) Groups 0hr 1 hr 2 hr 3 hr 4 hr Group I 0.36±0.33 0.48±0.030 0.60±0.036 0.60±0.057 0.41±0.030 Group II 0.43±0.12 0.80±0.38 1.08±0.87 1.36±0.12# 1.16±0.11# # Group 0.45±0.13 0.60±0.027 0.73±0.021* 0.88±0.020* 0.75±0.015* III (25%) (32.4%) (35.29%) (35.34%) * * * * Group 0.43±0.14 0.56±0.019 0.73±0.018 0.81±0.016 0.65±0.05* IV (30%) (32.4%) (40.4%) (43.9%) Values expressed as Mean ± SEM, # = P<0.05 ( in comparison to control group (group 1), *= P<0.05 ( in comparison to Carrageenan group (group 2). One way ANOVA followed by Dunnet’s test). Fig. 1: Effect of MEBSextract on histopathology of spleen inFCA induced arthritic rats: A) Normal control group; B) Arthritis control group; C) Indomethacin group and D) MRBS extract (100 mg/kg). 71 Vol. 11 (4) April (2016) Res. J. Biotech Research Journal of Biotechnology Fig. 2: Effect of MEBS extraction radiographic examinations of ankle joint in FCA induced arthritic rats: A) Normal control group; B) Arthritis control group; C) indomethacin group and D) MEBS extract (100 mg/kg). 6. Arunachal S., Science on the Periphery Enriches Mainstream Science, But At What Cost? The case of Ethnobotany, Les Sciences Hors D’ occidentau Xxe Siecle, Central Electrochemical Research Institute, Karaikudi (India), 6, 42 (1996) Acknowledgement The authors are thankful to Dr.S.Mohanty, Professor and Head, Department of Pharmacology; Dr. S. Biswal, Assistant Professor, Dr. R. Samal, Assistant Professors, Department of Pharmacology and Mr. D. Bisoi, Lab Assistant, Department of Pharmacolgy, S.C.B Medical College, Cuttack, India for providing the facilities and friendly support to carry out the study. 7. Austin J.H. and Drabkin D.L., Estimation of Haemoglobin, J. Biol. Chem., 112, 67 (1935) 8. 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