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. Bynitroblue 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 independentt – 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.
Felson, D. T., 2008 Osteoarthritis in Fauci,
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