Chapter 6: Antiarthritic activity of cultured mycelium of Volvariella volvacea
6.1. Introduction Arthritis is a very common medical condition estimated to affect around seven million people in the UK. However, it is not a single disease but comes in around 200 different forms. The term essentially mea an inflammation of the joints. This inflammation makes the joints painful, stiff and swollen and in severe cases can significantly restrict movement. Arthritis includes osteoarthritis, rheumatoid arthritis, gout, systemic lumpus, erythematosus, juvenile rheumatoid arthritis, bursitis, lyme arthritis, carpel tunnel diseases and other disorders. But the two most common forms are osteoarthritis and rheumatoid arthritis. Osteoarthritis is caused by the breakdown of protective tissue called cartilage in the joints. Inflammation results when the unprotected bones of the joint begin to rule together. Rheumatoid arthritis is a condition caused by inflammation of the living (Synovium) of the joints. In some people, it also affects other parts of the body, including the blood, the lungs and the heart. Adjuvant arthritis in rats exhibits many similarities to human rheumatoid arthritis. Noteroidal anti-inflammatory drugs (NSAIDs) are widely used clinically to treat inflammatory diseases and rheumatoid arthritis. The other main categories of treatment are COX-2 specific inhibitory, combination therapies, pain reliever s analgesics steroids, disease-modifying antirheumatic drugs (DMARD) and biologic respoe modifiers. Free radicals are continuously generated in almost all aerobic cells (Fridovich, 1983; Cheeseman et al., 1993). If they are not scavenged by the compreheive array of endogenous antioxidant mechanisms, they lead to the tissue damage and loss of function in a numbers of tissues and orga (Halliwell, 1994; Elkhatib, 1997), The most reactive oxygen species in biological system are superoxide anion (. O2 - ), hydroxyl (. OH) and hydrogen peroxide (H2O2) (Batteli et al., 1973). Oxygen free radicals such as superoxides, hydroxyl radicals and related oxygen species such as hydrogen peroxide and singlet oxygen are 88
involved in the pathogenesis of many inflammatory reactio such as rheumatoid arthritis (Biemond et al., 1984). Many cellular defence mechanisms are recruited agait the toxic effects of these radicals in inflammation including serum sulph hydryl groups (SH-gps) (Lorber et al., 1975), ceruloplasmin (CP) (Goldstein et al., 1979), albumin (Gutteridge, 1986) and blood glutathione (GSH) (Lamds, 1971). It is well known that these oxygen free radicals have deleterious effects on bio membranes due to the formation of lipid peroxides (Lunec et al., 1981). Lipid-peroxide formatio as well as altered levels of endogenous, glutathione peroxidase and reduced glutathione have been coidered as indirect in vivo evidence for the participation of free radicals in the progression of arthritis. A number of medicinal properties have been reported to have significant antiinflammatory activity and some of them have been demotrated to possess antiarthritic activity (Meera et al., 2009). The effect of aqueous ethanolic extract of cultured mycelia of V. volvacea agait the free radicals generated during Freund s complete adjuvant induced arthritis in rats was examined and the results are presented in this chapter. 6.2. Materials and method 6.2.1. Preparation of the extract Aqueous-ethanolic extract of V. volvacea mycelium was prepared as described in section 3.2.2. 6.2.2. Animals Male Wistar rats weighing 250-350 g were employed for antiarthritic studies. 89
6.2.3. Determination of anti - arthritic activity using Freund s complete adjuvant (FCA) induced arthritis model Male Wistar rats (weighing 250-350g) were divided into six groups each coisting of six animals. Arthritis was induced in all groups except normal group by the intradermal injection of 0.1ml of Fruend s Complete Adjuvant (FCA) (Genei, Bangalore) into the sub planar region in the right hind paw (New bould, 1963; Gokhale et al., 2002). Group 1 : Normal male Wistar rats without any adjuvant inoculation/treatment Group 2: Group 3: Control Injected with Fruend s complete adjuvant Standard Diclofenac was administered, orally at a dosage of 10mg/kg body weight in 1ml distilled water. Group 4: V. volvacea mycelial extract were administered orally at a dosage of 100mg/kg body weight in 1ml distilled water. Group 5: V. volvacea mycelial extract were administered orally at a dosage of 250mg/kg body weight in 1ml distilled water. Group 6: V. volvacea mycelial extract were administered orally at a dosage of 500mg/kg body weight in 1ml distilled water. Diclofenac and the extract were administered orally one hour prior to Freund s complete adjuvant (FCA) injection. The oral administration of the drug was continued once daily for 12 days. The changes in paw thickness was assessed by the measurement of the right hind paw volume of all arthritis animals by using vernier calipers just before drug administration and after adjuvant inoculation. The paw thickness is measured at every three days after adjuvant inoculation until 22 nd day. 90
The increase in paw thickness in control / treated group, (Pc/Pt) = Pt Po Where, Pt = Paw thickness of time t, Po = Initial paw thickness. Percentage of inhibition = (Pc Pt) x 100 Pc Where, Pc = Increase in paw thickness of control, Pt = Increase in paw thickness of treated. At the end of the experimental period i.e., on 22nd day, the animals were killed under anaesthesia and blood samples were collected directly from heart and non-coagulated (heparin) blood was used for determination of antioxidant enzyme activities and reduced glutathione level. Serum was separated and used for determining the MDA level. SOD (section 3.2.5.9), CAT (section 3.2.5.10), GPx (section 3.2.5.11), GSH (section 3.2.7) were estimated in erythrocyte lysate and MDA in serum was determined as described in section 3.2.8. The whole blood was used for analyzing Hb content using Drabkin s reagent (section 3.2.12). 6.3. Results 6.3.1. Freund s complete adjuvant (FCA) induced arthritis model Aqueous ethanolic extract of V. volvacea, at a concentration of 100, 250 and 500 mg/kg body weight when administrated orally, showed 37.68%, 63.29% and 80.68% inhibition of acute inflammation respectively. Standard drug diclofenac (10mg/kg body weight) when administered orally showed 82.61% inhibition of the inflammation (Table 6.1). The changes in right hind paw oedema volume during 22 days after inoculation of Freund s complete adjuvant showed a biphasic inflammatory respoe. An immediate acute phase was detected at day post inoculation followed by a delayed chronic phase that reached a maximum at 22 nd day. In 91
V. volvacea extract treated groups, after the post inoculation phase, the paw thickness was found to be decreased without leading to a chronic inflammation phase. The result thus indicates that the aqueous ethanolic extract of V. volvacea possess significant activity agait Freund s complete adjuvant induced arthritis in rats (Figure 6.1). The activities of SOD, CAT and GPx in erythrocytes were lowered in the control arthritic rats when compared to the normal rats. The activities of all the enzymes were restored to normal level in the extract treated groups (Table 6.2). The SOD activity in the control group of animals was 1.23 ± 0.46 U/ml of haemolysate, which increased to 2.62 ± 0.66 U/ml of haemolysate by treatment with 500 mg/kg b.wt of mushroom extract (Fig. 6.2). The activity of GPx in blood was increased to 4.31 ± 0.43 U/ml of haemolysate after treatment with 500 mg/kg b.wt of mushroom extract (Fig. 6.3). The activity of catalase in arthritic rats was 68.08 ± 11.85 K/g Hb while in the 500 mg/ kg b.wt treated group it was 103.08 ± 15.74 K/g Hb (Fig. 6.4). Treatment with the extract significantly enhanced the glutathione antioxidant system such as GSH. The activity of GSH in the extract (500 mg/kg b.wt) treated animals was 34.00 ± 9.89 n mol/ml of haemolysate (Fig. 6.5). The effect of extract on lipid peroxidation in the serum of the control and treated groups is given in the figure 6.6. FCA administration to rats enhanced the serum MDA level. Administration of different doses of V. volvacea extract could effectively reduce the serum lipid peroxidation. 6.4. Discussion The term arthritis covers more than 200 diseases and conditio affecting joints, the surrounding tissues and other connective tissues. Arthritis is a very common medical condition, estimated to affect around seven million people in the UK. Adjuvant arthritis shares many features with human rheumatoid 92
arthritis. It involves most of the joints and associated tissues. Lesio similar to rheumatoid arthritis in joints can be induced by Freund s complete adjuvant in rat s footpad. Injectio of complete Freund s adjuvant in to the rat paw induce inflammation as primary lesion with a maximum after 3 to 5 days. Secondary lesio occur after a delay of approximately 11 to 12 days which are characterized by inflammation of non-injected sites (hindleg, forepaws, ears, nose and tail), a decrease of weight and immune respoes. During the last decade, coiderable attentio have been focused on the involvement of O2 free radicals (OFR) in various diseases. Free radicals have long been implicated in changes in connective tissues on inflammation and arthritis. Active oxygen molecules such as superoxide and hydroxyl radicals have been demotrated to play important role in the inflammation process produced by ethanol, CCl4 or carrageenan (Yoshikawa et al., 1983, Halliwell and Parihar, 1984; Yuda et al., 1991). Several report have shown that during inflammatory joint diseases, such as adjuvant-induced arthritis, phagocytes accumulate in the joints and produce superoxide radicals and hydrogen peroxide, which in the presence of traces of iron salts found in synovial fluids interact to form the highly reactive hydroxyl radical (Babior, 1978; Gutteridge et al., 1983). Granulocytes are strongly increased in number in this disease they produce large amounts of O2 and H2O2 during phagocytosis of immune complexes and other materials. Oxygen free radicals and H2O2 are closely involved in the pathogenesis of rheumatoid arthritis (Winrow et al., 1993) and are respoible for a major part of the joint destruction (Biemond et al., 1984). The most reactive oxygen species in biological system are superoxide anion (O2 ), hydroxyl radical (.OH) and hydrogen peroxide (H2O2) (Batteli et al., 1973). It has been reported that inflammatory stimuli trigger an oxidative burst, resulting in the formation of several ROS, namely, superoxide anion, hydrogen peroxide, hydroxyl radical and singlet oxygen. Once generated, free 93
radicals provoke deleterious effects on various cellular targets, foremost among which are membrane lipids, leading to an exteive process of lipid peroxidation (Symo et al., 1988). In the present study, lipid peroxides formation as well as altered levels of endogenous scavengers was taken as an indirect in vivo evidence for the participation of free radicals in the progression of arthritis. Endogenous free radicals scavengers are either enzymes or non enzymes and exist in both the aqueous and membrane compartment of the cells. The superoxide dismutase (SOD) enzyme selectively eliminates free radicals by converting it to hydrogen peroxide. That can in turn destroy by CAT or GPx reactio. Catalase enzyme reacts with H2O2 to form water and molecular oxygen. The glutathione peroxidase enzyme intercepts the propagation of lipid peroxidation (ROOH and H2O2) using GSH. Thereby protecting mammalian cells agait oxidative damage. GPx is a detoxifying enzyme, changing peroxides to water. Increment in the level of lipid peroxides in synovial fluid, serum and tissue of arthritis animals as well as patients suffering from arthritis has been demotrated (Symo et al., 1998; Lunec et al., 1981; Yoshikawa et al., 1985). Furthermore aggravation of arthritis was shown to be associated with enhancement of lipid peroxidation (Symo et al., 1988). Superoxide dismutase can change superoxide anion radical to hydrogen peroxide (Free, 1980). Administration of aqueous-ethanolic extract of V. volvacea to arthritic rats caused a significant increase in SOD activity. This increase in enzyme activity appears to be protective agait the extracellular oxygen free radicals (Marklund et al., 1987). GPx protects the cell agait damage resulting from the increased disease associated peroxides by enhancing the GPx catalysed peroxide destruction (Kasama et al., 1998). Glutathione peroxides are also detoxifying enzymes, changing the peroxides to water (Lawrence and Burk, 1976). The administration of the mushroom extract in arthritis rats caused a 94
significant increase in GPx activities. Catalase is hemoprotein. The main function of catalase is to detoxify hydrogen radicals (Chance et al., 1979). Catalase activity is significantly increased in the groups treated with the mushroom extract. In this study the blood GSH content of the control group was reduced coiderably compared to the healthy untreated group. The large difference points to the high coumption of GSH to counteract free radicals produced during chronic arthritis. The administration of V. volvacea mushroom mycelial extract to arthritic rats caused a significant elevation in GSH level. Thus, the results indicate that the in vivo antioxidant activity of this mushroom extract might be, at least in part, an important component of its anti-inflammatory properties. Results of the present investigation reveal that V. volvacea mushroom mycelia extract possess pronounced anti-inflammatory properties in the immunologically mediated inflammatory respoe induced by inoculation of Freund s complete adjuvant in rats. The results thus suggest the therapeutic uses of V. volvacea for the treatment of arthritis. 95
Table 6.1. Effect of V. volvacea on F C A induced arthritic rats Group Treatment (mg/kg b.wt) Initial paw thickness (cm) Final paw thickness (cm) Increase in paw thickness (cm) Percentage of inhibition (%) Control - 0.408 ± 0.062 0.580 ± 0.022 0.173 ± 0.043 - Standard 10 0.400 ± 0.008 0.430 ± 0.014 0.030 ± 0.014*** 82.61% V. Volvacea 100 0.413 ± 0.009 0.520 ± 0.018 0.108 ± 0.026** 37.68% V. Volvacea 250 0.430 ± 0.030 0.493 ± 0.015 0.063 ± 0.029*** 63.29% V. Volvacea 500 0.427 ± 0.021 0.460 ± 0.010 0.030 ± 0.011*** 80.68% Values are Mean ± SD, n=6., ***P<0.001, **P<0.01, with respect to control.
Table 6.2. Effect of V. volvacea on biochemical parameters in blood and serum of arthritic rats Groups SOD (U/ml of haemolysate) GPx (U/ml of haemolysate) Catalase (K/g Hb) GSH (U/ml of haemolysate) Lipid peroxidation (nmol/ml of protein) Normal 1.95 ± 0.21 2.99 ± 0.62 71.03 ± 16.06 24.88 ± 5.72 43.51 ± 4.10 Control 1.23 ± 0.46 2.31 ± 0.12 68.08 ± 11.85 1 8.00 ± 5.79 63.36 ± 10.17 Standard 1.98 ± 0.26 3.01 ± 0.11** 101.44 ± 16.49** 27.50 ± 3.32 51.04 ± 12.18 VV 100 1.98 ± 0.48 2.75 ± 0.38 84.69 ± 7.00 21.88 ± 3.88 44.82 ± 10.22* VV 250 2.10 ± 0.76 3.02 ± 0.16** 96.21 ± 16.14* 27.00 ± 4.36 38.40 ± 7.66** VV 500 2.62 ± 0.66** 4.31 ± 0.43*** 103.08 ± 15.74** 34.00 ± 9.89*** 34.01 ± 12.02*** Values are Mean ± SD, n=6. ***P<0.001, **P<0.01, *P<0.05, P>0.05 with respect to control.
Paw thickness (cm) Figure 6.1. Effect of V. volvacea on paw oedema in arthritic rats 0.75 0.65 0.55 0.45 0.35 Control Std VV 100 VV 250 VV 500 0.25 1 4 7 10 13 16 19 22 Time period after inoculation (days) Values are Mean ± SD, n=6.
GPx (U/ml of haemolysate) SOD (U/ml of haemolysate) Figure 6.2. Effect of V. volvacea on super oxide dismutase (SOD) activity in blood 3.5 3 2.5 ** 2 1.5 1 0.5 0 NORMAL CONTROL STD VV100 VV250 VV500 Values are Mean ± SD, n=6., **P<0.01, P>0.05 with respect to control. Figure 6.3. Effect of V. volvacea on glutathione peroxidase (GPx) activity in blood 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 *** ** ** NORMAL CONTROL STD VV100 VV250 VV500 Values are Mean ± SD, n=6., ***P<0.001, **P<0.01, P>0.05 with respect to control.
GSH (U/ml of haemolysate) CAT (K/g Hb) Figure 6.4. Effect of V. volvacea on catalase (CAT) activity in blood 140 120 100 ** * ** 80 60 40 20 0 NORMAL CONTROL STD VV100 VV250 VV500 Values are Mean ± SD, n=6. **P<0.01, *P<0.05, P>0.05 with respect to control. Figure 6.5. Effect of V. volvacea on reduced glutathione (GSH) level in blood 50 45 40 35 30 25 20 15 10 5 0 *** NORMAL CONTROL STD VV100 VV250 VV500 Values are Mean ± SD, n=6., ***P<0.001, P>0.05 with respect to control.
MDA (nmol/ml of protein) Figure 6.6. Effect of V. volvacea on serum MDA level 80 70 60 50 40 30 20 10 0 * ** *** NORMAL CONTROL STD VV100 VV250 VV500 Values are Mean ± SD, n=6., ***P<0.001, **P<0.01, *P<0.05, P>0.05 with respect to control.