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Indonesian Journal of Biotechnology, December, 2011 Ability of Curcuminoid from Curcuma domestica Val. in Reducing the
Secretion of Reactive Oxygen Intermediates by Synovial Fluid Monocytes
in Patients with Osteoarthritis
Nyoman Kertia¹,2*, Ahmad Husain Asdie¹,2, Wasilah Rochmah¹,2, and Marsetyawan3 1Department of Internal Medicine, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, 2Department of Internal Medicine, Dr Sardjito Hospital, Yogyakarta, Indonesia3Department of Histology and Cell Biology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Abstract
Increasing the secretion of reactive oxygen intermediates (ROI) by monocytes in the synovial fluid is an indicator to determine the severity of joint inflammation. Previous studies have shown that curcumin inhibit the osteoarthritis progression with its ability to inhibite the activity of the nitric oxide synthase (NOS) enzyme from macrophages. In this prospective randomized open end blinded evaluations = PROBE study, 80 patients with knee osteoarthritis were eligable. The subject were devided in to two group: group who received 3 x 30 mg of curcuminoid from Curcuma domestica Val. extract (curcuminoid group) and group who received 3 x 25 mg of diclofenac sodium (diclofenac group) as comparison. The treatment was for 4 weeks time. The secretion of ROI by sinovial fluid monocytes was calculated by scoring the amount of formazan formation after neutral red staining in nitrobleu tetrazolium reduction assay. The result of this study showed that the secretion of ROI by synovial fluid monocytes was significantly decreased in both groups (p <0.001) respectively. There was no significant difference in decreasing of ROI secretion of synovial fluid monocytes between both treatment groups (p = 0.92).
Keywords : curcuminoid, diclofenac sodium, reactive oxygen intermediates, monocyte, osteoarthritis
Introduction
joint disease found in people. The disease is a major cause of musculoskeletal disorders largest cause of physical disability after ischemic heart disease for over 50 years of synovial fluid of affected joints (Ratiner et age. This disease causes loss of working h and al., 2001). In OA there is imbalance between high cost of treatment (Dieppe, 2008). The synthesis and degradation of joint cartilage, prevalence of OA increases with increasing is initiated by mechanical loading which resulting in chondrocyte metabolism disorders, production of proteolytic enzymes Corresponding author:
such as matrix metalloproteinase (MMP) and Nyoman KertiaDepartment of Internal Medicine, Faculty of damage of joint cartilage. The occurrence of Medicine, Universitas Gadjah Mada, Yogyakarta, multiple microfractur cause degradation and depletion of vulnerable joints, changes in joint architecture and the formation of osteophyte. If it happen, the osteoarthritis progression rhyzome extract in inhibit the ROI secretion of will continue. Changes in architecture of the synovial fluid monocytes in osteoarthritis. joint resulting in joint mechanics change that cause increase pressure on the joints, further Materials and Methods
Research design
and inflammation of the joints (Berenbaum, 2008). In the elimination process of foreign end blinded evaluation (PROBE) study.
antigens or when interacting with stimuli such as cytokines, the oxidative burst Materials
of monocytes showed an increased capacity to produce ROI in large quantities. Reactive oxygen intermediates play an important role washed, cut into pieces with a thickness of 1-2 in the process of inflammation and tissue mm and dried with drying cupboard for 24 h at 40 0 C to obtain maximum water content The study of anti-inflammatory activity ethanol and then macerated (soaked) for 24 powerful anti-inflammatory activity than h, then filtered with a Buchner funnel (with the other components (Parslow & Bainton, vacuum pressure). Collected filtrate was evaporated at 45 0 C in vacuum condition. For activity of phospholypase, lypoxigenase, the determination of curcuminoids curcumin standard solution used with varying degrees prostaglandin, thromboxane, Nitric Oxide, of concentration. Extract obtained was diluted to 100 mg/mL and touched on silica gel GF interferon, TNF-α and IL-12. In people with 254 plates and then eluted with a mobile the stiffness, swollen joints and walking v/w. Detection of spots is done by ultraviolet light 254 nm and 365 nm. The scan was then operative inflammation (Joe et al., 2004). performed by Thin Layer Chromatography scanner. The level of curcumin, desmetoxi synovial fluid determine the severity of joint inflammation. Two important mediators components of curcuminoids was calculated produced by monocytes in the synovial fluid by linear regression method. The extract was in initiate the inflammatory process and entered in the capsuls which the dose of 30 mg curcuminoid. The other capsules contain and reactive oxygen intermediates (ROI). Increasing of ROI secretion by monocytes in the synovial fluid will follow by Research subjects
increasing of joint inflammation (Chainani, 2003). Production of nitric oxide and oxygen radicals in chondrocytes are affected by the biological process of osteoarthritis and Mada/Dr. Sardjito Hospital - Yogyakarta. The rheumatoid arthritis (Mazzetti et al., 2001). The study population were patients with knee OA, results of previous studies have shown that registered and were still controlled in Dr. Sardjito curcumin inhibits the osteoarthritis process General Hospital. Random selection was done among 168 patients to find 80 patients who eligible in this research. The subjects have no arthritis other than osteoarthritis, had no abnormalities of liver, kidney or bone differentiated into 4 groups. Without secreted marrow function, did not have any history of ROI (no formazan deposits in the cytoplasm, gastritis, peptic or duodenal ulcer, no history given the value 0), which secreted a little amount of hypersensitivity to diclofenac sodium and reaches a quarter of the cytoplasm, given the medication or other anti-inflammatory drugs, value 1), which secreted ROI in large quantities and agree to follow the study with signed the (formazan sedimentation between a quarter to three-quarter of the cytoplasm, given the value 2) and which secreted ROI in a very large assignment was done so that the sample was quantities (more than three-quarter formazan divided into treatment groups and control sedimentation of the cytoplasm, given the groups. The treatment was for 4 weeks period. The treatment group were given 3 x 30 mg curcuminoid from Curcuma Val. rhyzome extract, while the control group were given 3 x 25 mg diclofenac sodium. Assessment of the treatment results was performed before and after 4 weeks treatment by scoring the formazan sedimentation in the cytoplasm of monocytes. Data analysis was conducted on the amount of ROI secretion by synovial fluid monocytes before and after treatment. Laboratory examination
synovial fluid was taken from the affected knee. The culture of monocytes by PMA stimulation was performed. By nitroblue tetrazolium (NBT) reduction assay, the formazan sedimentation was able to assess. Since the formazan is the Figure 1. Secretion of ROI by synovial fluid end result of the reaction between the NBT and ROI, the amount of ROI secretion by synovial fluid monocytes were calculated by scoring the formazan sedimentation in the cytoplasm researchers test the value of kappa to determine of monocytes. Monocytes culture was added intraobserver and interobserver consistency by 500 ul NBT solution containing 125 ng/mL of the ROI value secreted by monocytes in PMA and incubated at 37 o C and 5% CO for the synovial fluid. Observation was done 60 min. Cells was then washed 3 times with PBS, dried at room temperature and fixed 40 monocytes then calculated the value of in absolute methanol for 30 sec and painted intraobserver and interobserver consistency. with neutral red solution for 15 min and then In assessing the effectiveness of the treatments washed with distilled water then dried at room temperature and ready to be observed. The each preparate before starting the treatmen cytoplasm of monocytes without secreted ROI was pale, while that secreted ROI looks blue insoluble formazan sedimentation as a result Statistical analysis
of ROI and NBT reaction. Based on the amount of formazan sedimentation the monocytes were Results and Discussions
Number of subjects in the diclofenac group osteoarthritis were eligible and willing to participate in this study. Subjects were divided randomly into two groups: the treatment group treatment were 2.25 ± 0.23 in the curcuminoid (named curcuminoid group) were given 3 x 30 mg curcuminoid from Curcuma domestica group. Reactive oxygen intermediates play Val. rhyzome extract, while the control group (named diclofenac group) were given 3 x 25 mg inflammation and tissue damage. Exposure of ROI to normal cells will cause a variety of pathological changes leading to cell and who participated in the study were 39 patients tissue damage for example in pathogenesis consisting of 15 men and 24 women. The mean of osteoarthritis (Ahmed et al., 2005; Shah age was 64.05 ± 8.83 years. The duration et al., 2005). Monocytes from patients with suffering of osteoarthritis was 41.23 ± 32.60 rheumatoid arthritis release increase amount months. The body mass index was 26.28 ± of reactive oxygen intermediates. Reactive 3.62 kg / m 2. Five subjects were excluded oxygen intermediates play an important role from the study with the reasons: 1 Subject in the process of cartilage degradation in the drank piroxicam, 1 subject experienced ureter colic due to urinary tract stones, 1 subject had through lipid peroxidation in chondrocytes hematuria due to tumor of vesica urinaria, 1 (Arora et al., 2000). In this study the ROI subject stopped the curcuminoid treatment as secretion by synovial fluid monocytes was his family request and 1 subject experienced not significantly different between the two an acute exacerbation of chronic obstructive pulmonary disease. Number of subjects in the curcuminoid group who finished the study was 34 patients, comprising 11 men and 23 women.
subjects who participated in the study were 41 patients consisting of 12 men and 29 women. The mean age was 64.56 ± 8.86 years. The duration suffering of osteoarthritis was 40.37 ± 30.87 months. The body mass index was 26.44 ± 4.79 kg / m 2. Two subjects were excluded from the study with the reasons: 1 subject experiencing dyspepsia on the seventh day of treatment which did not improve by giving omeprazole 10 mg 1 tablet daily and Figure 2. Distribution of ROI secretion by
1 subject has no synovial fluid in aspiration. Monocytes, p = 0.200
Table 1. ROI Secretion by Monocytes Before Treatment Table 2. ROI Secretion by Monocytes Before and After Treatment secretionDescription: *Independent t-test of monocytes showed an increased capacity to monocytes before treatment spread follow produce ROI in large quantities (Donne et al., a normal curve with p = 0.200 (Figure 2). To 2006). Reactive oxygen intermediates play a analyze the differences of ROI secretion role in the process of inflammation and tissue between both group the independent t – test damage in osteoarthritis (Shah et al., 2005). In was used, because subjects in curcuminoid the production of Nitrite Oxyde (NO) and superoxide anions (O -). There are differential ROI secretion by synovial fluid monocytes roles of nitric oxide and oxygen radicals in was significantly decreased in both groups with chondrocytes affected by osteoarthritis and p <0.001 respectively (Table 2). Oxidative rheumatoid arthritis (Mazzetti et al., 2001). stress stimulate the peroxidation of lipid Curcumin inhibits the in vitro secretion of ROI membranes and able to cause a serious cells by gum fibroblasts and human submandibular gland carcinoma cells (Atsumi et al., 2005). of the cell membranes will increase tissue permeability and disrupt the normal function Apoptosis in Methylglyoxal-Treated Human of these cells. Reactive oxygen intermediates is Hepatoma G2 Cells (Chan et al., 2005). The one of the oxidative markers could be detected results of this study indicate that there was in the laboratory (Tiku et al., 2000) no significant difference in decreasing of ROI secretion by synovial fluid monocytes during antigens or when interacting with stimuli such as cytokines, the oxidative burst 3). The ability of both drugs did not differ Table 3. Change of ROI secretion in both groups Table 4. Value of number needed to be treated in both groups ROI (Target scores <1)RRR = Relative risk reduction = |CER-EER|/CERARR = Absolute risk reduction = |CER-EER|NNT = Number needed to be treated = 1/ARR significantly in reducing the secretion of ROI by monocytes synovial fluid of patients with knee osteoarthritis. It is remains to be further investigated how the mechanism of action of diclofenac sodium and curcuminoid from Carcinoma Cells. Oral Dis., 11, 236-
Curcuma domestica Val. rhyzome extract in suppressing the secretion of ROI by synovial Berenbaum, F.,2008 Osteoarthritis: Pathology and Pathogenesis in Klippel, J. H., The number needed to be treated analysis Stone, J. H., Crofford, L. J., White, P. H. of the secretion of ROI by synovial fluid (eds) Primer on The Rheumatic Diseases, monocytes get that to obtain one subject with 13th ed, pp. 229-34. Arthritis Foundation, decreasing of ROI secretion with the target score of less than 1 in the group that received Breedveld, F. C., 2004 Osteoarthritis the diclofenac sodium compared to curcuminoid Impact of a Serious Disease. J. Rheumatol., 43(1), 14-18
extract, the number of subjects needed to be longa). J. Compl. Med., 9(1), 161-168.
significantly inhibite the secretion of ROI by Chan, W. H., Wu, H. J., Hsuuw, Y. D., 2005 monocytes synovial fluid of osteoarthritic Curcuma domestica Val. rhyzome extract in Human Hepatoma G2 Cells. Ann. N.Y. inhibite the secretion of ROI by monocytes Acad. Sci., 1042, 372-378.
synovial fluid is not significantly different Dieppe, P.A., 2008 Osteoarthritis: Clinical Feature in Klippel, J. H., Stone, J. H., Crofford, L. J., White, P. H. (eds) Primer on Acknowledgements
The Rheumatic Diseases, 13th ed., pp. 224-28. Donne, I. D., Rossi, R., Colombo, R., Giustarini, director of Dr. Sardjito General Hospital for her exellent support and advice for this Clin. Chem., 52(4), 601-623.
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