Protective Effects of Nephelium lappaceum Rind Extract against Collagen-induced Arthritis in Dark Agouti Rats
Gan Seng Chiew,
Pathogenic mechanisms of arthritis are studied using Collagen-Induced
Arthritis (CIA) animal models. Plant derived antioxidants are known to reduce
the inflammatory response in CIA. The aim of the study was to assess the protective
efficacy of Nephelium lappaceum ethanol extract against Collagen-Induced
Arthritis (CIA) in dark agouti rats. Arthritis was induced with 4 mg kg-1
of collagen in complete Freund's adjuvant. CIA rats were orally treated with
100 and 200 mg kg-1 per oral of N. lappaceum from day 25-50.
Changes in body weight, joint thickness, C-reactive protein were recorded and
immunohistochemistry for matrix metalloproteinase-13 (MMP-13) and tissue inhibitor
of matrix metalloproteinase-1 (TIMP-1) was done. N. lappaceum (100 and
200 mg kg-1) significantly reduced (p<0.05) the arthritis-induced
changes in body weight and paw edema. There was a significant reduction (p<0.05)
in the C-reactive protein in the treatment groups. A significant reduction (p<0.05)
in the arthritis-induced histopathological changes was seen after treatment
with N. lappaceum. Treatment with N. lappaceum showed dose dependent
effects on MMP-13 and TIMP-1 levels. N. lappaceum rind extract significantly
suppressed the physiological, biochemical and histopathological changes produced
during collagen-induced arthritis in dark Agouti rats. N. lappaceum extract
supplementation may be beneficial in preventing the tissue damage and inflammatory
conditions in arthritis.
to cite this article:
Shashi Kumar, Srikumar Chakravarthi, Gan Seng Chiew, Thavamanithevi Subramaniam, Umadevi Palanisamy, Ammu Radhakrishnan and Nagaraja Haleagrahara, 2012. Protective Effects of Nephelium lappaceum Rind Extract against Collagen-induced Arthritis in Dark Agouti Rats. Journal of Biological Sciences, 12: 385-392.
July 11, 2012; Accepted: October 18, 2012;
Published: December 27, 2012
Rheumatoid Arthritis (RA) is a chronic inflammatory disease that mainly targets
the synovial tissue, cartilage and subchondral bone. Patients will suffer from
joint inflammation, joint destruction and motor disability. Progressive and
erosive destruction of the peripheral joints will lead to severe motor disability
leading to decreased quality of life (Kumar et al.,
2009). The hallmark of RA is a persistent inflammation of synovial membrane,
pannus formation and cartilage thinning which ultimately leads to joint deformities
(Choy and Panayi, 2001; Navarro-Cano
et al., 2003).
Antioxidant micronutrients are presumed to play an important role in protecting
against the tissue damage caused by Reactive Oxygen Species (ROS). Antioxidants
provision in suppression of cytokines and collagenase expression induced by
Tumor Necrosis Factor (TNF)-α has been demonstrated in RA patients (Halliwell
et al., 1988; Mahajan and Tandon, 2004; Sato
et al., 1996) which suggests additional mechanisms of protection
against RA. The reports about the modulation of pro-inflammatory cytokine expressions
by diet derived antioxidants have lead to the discussion that antioxidants have
the potential to protect against rheumatoid arthritis. But there are very few
researches available about the anti-inflammatory and antioxidant role of antioxidants
from the plants in rheumatoid arthritis (Buchanan et
al., 1991; Mangge et al., 1999; Panush,
1991; Shukla et al., 2008).
Rambutan (Nephelium lappaceum), a tropical fruit of the Sapindaceae
family is native to Malaysia. This seasonal tropical fruit is an important commercial
crop in Asia (Dembitsky et al., 2011). The rind
of N. lappaceum, habitually discarded, is having extremely high anti-oxidant
(Palanisamy et al., 2008; Thitilertdecha
et al., 2010), antibacterial (Martinez-Castellanos
et al., 2009; Thitilertdecha et al., 2008),
anti-hyperglycemic and free radical scavenging activities (Palanisamy
et al., 2011). Ethanolic N. lappaceum rind extract possess
high free radical-scavenging activity and low pro-oxidant capability (Dembitsky
et al., 2011; Palanisamy et al., 2011).
The current study was designed to explore the anti-inflammatory effect of N.
lappaceum rind extract on Collagen-Induced Arthritis (CIA) in rats. We hypothesize
that the ethanol extract of N. lappaceum has significant anti-inflammatory
effect and protects against arthritis-induced histopathological changes in Dark
MATERIALS AND METHODS
Drugs and chemicals: Type II collagen, Complete Freunds
Adjuvant (CFA) and all other analytical chemicals used for the experiment were
obtained from Sigma-Aldrich (St. Louis, MO, USA).
Plant collection, isolation and standardization: The extract was standardized
using Geraniin as described previously (Palanisamy et
al., 2011). N. lappaceum was obtained from Kuala Lumpur, Malaysia
from 2009-2010 and plants were authenticated by the herbarium of the Forest
Research Institute of Malaysia (FRIM), Malaysia. Plants (1 kg) were cleaned
and dried at 40°C in the oven and then, powdered using the Fritsch dry miller.
Ethanol extraction (1:15; w/v) was carried out at room temperature in an orbital
shaker. The suspension obtained was filtered using a 114 Whatman filter paper
and filtrate was collected. Ethanol filtrate was concentrated using a rotary
evaporator to yield a dark brown ethanol extract of rambutan rind.
Determination of total phenolic content: Total phenolics were determined
based on a colorimetric oxidation and reduction reaction using the Folin-Ciocalteu
method (Palanisamy et al., 2008, 2011).
Aliquot of the extracts (1 mL) was added to 5 mL of Folin-Ciocalteu reagent.
After 3 min, 4 mL of 7.5% Na2CO3 solution in water was
added to the mixture and the content was thoroughly mixed. The absorbance at
765 nm was read after 1 h. Blank consisted of Folin-Ciocalteu reagent (5 mL),
ethanol/distilled water (1 mL) and 7.5% Na2CO3 solution
(4 mL). A linear dose-response regression curve was generated using absorbance
reading of gallic acid at the wavelength of 765 nm. The calibration curve using
gallic acid was obtained in the same manner as above except that the absorbance
was read after 30 min. Results were expressed as milligrams of gallic acid equivalent
per gram of dry weight (mg GAE/g DW) of extracts (Palanisamy
et al., 2008).
Experimental animals: The autoimmune arthritis is mediated by sex hormones
and is associated with a female preponderance for development of arthritis (Holmdahl,
1995; Van den Berg, 2009). Inbred female Dark Agouti
(DA) rats of 6-8 weeks of age (200-220 g) were maintained at room temperature
(24±2°C), with a 12 h light-dark cycle, relative humidity 60-70%
and allowed food and water ad libitum (Tudave et
Research protocol approval: All the experimental procedures were according
to internationally approved ethical guidelines for the care of laboratory animals
and the study got the approval from Institutional research and ethics committee.
Collagen-induced arthritis (CIA) and drug administration: Collagen-Induced
Arthritis (CIA) in rats was induced as previously described, with minor modification
(Brand et al., 2007). Approximately 4 mg kg-1
of type II collagen in complete Freunds adjuvant was administered intradermally
in the tail, 2 cm distal to the base of the tail for each rat (Tudave
et al., 2011). On day 21 after the primary immunization, the rats
were randomly divided into five groups (n = 6):
Group 1: Control
Group 2: Arthritis control
Group 3: 100 mg kg-1 N. lappaceum extract
Group 4: 200 mg kg-1 N. lappaceum extract
Group 5: 300 mg kg-1 glucosamine
These rats were orally administered with N. lappaceum extract and glucosamine
once a day from day 25-50.
Body weight and paw thickness measurement: The severity of arthritis
was assessed by measuring changes in paw edema and joint thickness using a digital
caliper. The body weight changes of the rats were also measured during the study
Biochemical analysis: All the rats were sacrificed 24 h after the last
day of treatment. Approximately 5 mL of blood samples were collected by cardiac
puncture. Blood was immediately centrifuged and plasma was separated and stored
(80°C) until further analysis. From the plasma, alanine transaminase
(AST), creatinine, Blood Urea Nitrogen (BUN) and total protein assay was done
using commercially available kits. C-Reactive Protein (CRP) levels in plasma
were determined using Millipore®
Rat C-Reactive Protein ELISA kit (CYT294) in accordance with the manufacturer's
instructions. The CRP concentration of each of the samples calculated based
on the standard curve obtained.
Histopathological analysis: Paw joints were collected at the end of
experiment and fixed in 10% (v/v) neutral formalin. The joints were trimmed
into 5-6 mm thickness, decalcified and the tissue was processed and embedded
into paraffin blocks. The slides, stained with haematoxylin and eosin (H and
E) were examined under a Nikon Eclipse 80β (CF160) bright field microscope.
Immunohistochemistry for MMP-13 and TIMP-1: The paraffin embedded sections
were deparaffinized with xylene and rehydrated in a gradient of ethanol. Endogenous
peroxidase activity was quenched with 0.5% H2O2 in ethanol
for 5 min. Immunohistochemistry was performed with the avidin-biotin peroxidase
complex (ABC) technique. After being washed with Tris Buffer Saline (TBS), antigen
retrieval was performed manually by using a hot water bath immersion of 75°C
for an hour with Target retrieval solution (S1699, Dako) of pH 9.0 for 45 min.
This lower-than-average temperature was used to prevent excessive destruction
of the sample tissues (Lee et al., 2008). The
sections were blocked with peroxidase-blocking solution (S2023, Dako) at room
temperature for 30 min and then incubated overnight at 4°C with the anti-TIMP1
(ab1827) and anti-MMP13 (ab3208) antibodies (Abcam), respectively. After washing
with TBS three times, they were incubated with HRP-conjugated secondary antibodies
(K0609, Dako) at 37°C for 30 min. After washing with TBS three times, all
sections were visualized with diaminobenzidine (DAB) (K3467, Dako). The slides
were counterstained with Mayers hematoxylin and mounted with coverslip.
Statistical analysis: All the results were expressed as Mean±SEM.
Results were analyzed by one-way analysis of variance (ANOVA) followed by Tukeys
test (all pair wise multiple comparison procedure). A value of p<0.05 was
considered statistically significant.
Changes in paw thickness: The macroscopic sign of severe arthritis at
50th day included swelling, redness deformity and ankylosis in the hind paw
and ankle joints. The symptoms of arthritic control rats showed significant
difference (p<0.05) as compared to the hind paw of normal rats. The arthritic
rats treated with N. lappaceum (100 mg kg-1), N. lappaceum
(200 mg kg-1) and glucosamine (300 mg kg-1) showed no
significant changes in the joints when compared to arthritis alone group (Fig.
1). A significant increase (p<0.05) in the hind paw thickness was observed
in the arthritic control rats from 0 to 50th day during the development of arthritis
(Fig. 2). N. lappaceum treatment depicted a significant
reduction in paw thickness compared to arthritic control at 50th day. The arthritic
rats treated with N. lappaceum (200 mg kg-1) also showed more
significant reduction (p<0.05) in paw thickness during the development of
arthritis as compared 100 mg kg-1 group (Fig. 2).
Changes in the body weight: Rats that developed arthritis showed reduction
in body weight during first three weeks, found to be almost similar in all the
groups of rats. However, after three weeks, the body weight of arthritic control
rats was declined significantly (p<0.05) compared to its normal counterpart.
|| Hind-limbs distal inter-phalangeal joints of representative
rat groups (a) Normal rat, (b) Arthritis (c) N. lappaceum treated
200 mg kg-1 and (d) N. lappaceum treated 100 mg kg-1
||Changes in paw thickness in normal, arthritis, N. lappaceum
(100 and 200 mg kg-1) and glucosamine (300 mg kg-1)
treated rats. Data are expressed as Mean±SE of six rats per group.
*p<0.05: Control vs. other groups, groups _p<0.05: Arthritis
vs. other groups
||Changes in body weight in normal, arthritis and N. lappaceum
(100 and 200 mg kg-1) and glucosamine (300 mg kg-1)
treated rats, data are expressed as Mean±SE of six rats per group,
*p<0.05: Control vs. other groups *p<0.05: Arthritis vs. other groups
N. lappaceum (100 mg kg-1 and 200 mg kg-1) treated
arthritic rats showed a significant increment (p<0.05) in their body weight
as compared to arthritic control (Fig. 3).
Biochemical changes: There were no significant differences in the AST,
BUN, Creatinine and total proteins analysed between the controls and treated
groups. There was a significant increase (p<0.05) in the CRP levels in the
arthritis rats than controls. The concentration of C-reactive protein was found
to be significantly reduced (p<0.05) in N. lappaceum treated as well
as the glucosamine treated groups (Fig. 4). Glucosamine treatment
group had shown more significant decrease in the CRP levels (p<0.05).
Histopathological analysis of joints: There was extensive proliferation
of synovial cells, resulting in pannus formation and infiltration of mononuclear
cells and neutrophils to the sub synovial region, with damage to the articular
surfaces and discontinuity in the cartilage in arthritis rats. All groups except
the baseline normal control showed varying levels of pathological changes (Fig.
5a). Severe arthritic and degenerative changes were observed, which showed
reduction in higher dosage group (N. lappaceum 200 mg kg-1)
(Fig. 5b, d). The degree of reduction of
morphological changes was more in glucosamine (300 mg kg-1) treated
group (Fig. 5e). There was healthy regenerative synovium and
areas of fibrosis, angiogenesis and fibroblasts proliferation in the treatment
||Plasma C-Reactive Protein (CRP) levels in arthritis and treated
groups, *p<0.05: Control vs. other groups, p<0.05: Arthritis
vs. other groups
|| Histopathological analysis of joint morphology and immunohistochemistry
for MMP-13 and TIMP-1 (200X) of (a) Normal control, (b) Arthritis, (c)
N. lappaceum 100 mg kg-1, (d) N. lappaceum 200 mg
kg-1 and (e) Glucosamine 300 mg kg-1 treated rats
Immunohistochemistry of MMP-13 and TIMP-1: Immunohistochemical expression
of MMP-13 and TIMP-1 were observed in the sections under light microscopy and
graded for positivity. IHC analysis indicated that the joint specimens from
arthritic control rats strongly stained for MMP-13, which is a key contributor
to the destruction of cartilage, however treatment with N. lappaceum
reduced the expression of MMP-13 in inflammatory articular cartilage (Fig.
5c, d). It was seen that the groups having the worst histological
findings, showed to have the highest levels of MMP-13 and the lowest levels
of TIMP-1. Expression of MMP-13 was higher in the cartilage cells of the arthritic
group and the least in N. lappaceum (200 mg kg-1) (Fig.
5c) and glucosamine (300 mg kg-1) (Fig. 5e).
A corollary effect was seen for TIMP-1 (Fig. 5c), which showed
the lowest expression in the above group.
Classical signs of severe arthritis were observed with intradermal type II
collagen administration, including symmetrical joint involvement typically involving
the hind paws, swelling, redness and erythema over the joints (Asquith
et al., 2009; Brand et al., 2007).
The hallmark symptoms of RA include fever, fatigue and weight loss (Lee
and Weinblatt, 2001). The body weights of CIA rats gradually decreases following
immunization and it will be at the lowest after three weeks of immunization
(Trentham et al., 1977; Nagatomo
et al., 2010). Our study showed a significant loss in body weight
around 25th day in the CIA rats as compared to the normal control. N. lappaceum
(both doses) and glucosamine (300 mg kg-1) showed significant recovery
in the body weight during the last 10 days of treatment when compared to their
C-Reactive Protein (CRP) is produced in the liver under conditions of systemic
inflammation in tissue destructions such as cartilage and bone and is a useful
biomarker in the evaluation of disease progression and response to therapeutic
intervention in a number of systemic inflammatory disorders, including RA. Higher
concentration of CRP will indicate increased joint changes in arthritis (Poole
et al., 2008; Rhodes et al., 2011).
Severity of joint damage should correlate with CRP levels (Jones
et al., 2011). In our study, it is shown N. lappaceum exhibited
a greater reversal of structural damage in the joints when compared with untreated
arthritic rat. The levels of CRP quantified in our study showed that this correlation
is true for the N. lappaceum, thus, proving the nutraceutical potential
of the extract (Palanisamy et al., 2008).
Histopathological changes correlated with macroscopic observations including
changes in the paw thickness. Untreated arthritic rats showed maximum degenerative
changes and significant reductions in the joint changes were seen in the treated
groups. Observed protective effect may be due to the inhibition of inflammatory
mediators by the anti-inflammatory compounds in the extract (Asquith
et al., 2009). An ellagitannin have been described to inhibit these
mediators, primarily TNF-α, IL-1β and IL-6  which could possibly
correlate to the attenuation of histopathological changes.
Matrix metalloproteinases-13 (MMP-13) is thought to increase in degenerative
bone diseases such as RA whilst tissue inhibitor of matrix metalloproteinases
(TIMP-1) has the opposite action and it is an important factor in maintaining
the integrity of connective tissue (Uchida et al.,
2000). Our results in this study showed an inverse relationship between
MMP-13 and TIMP-1. Rats fed with the both doses of N. lappaceum extract
seemed to be the most effective in increasing TIMP-1 expression and suppressing
MMP-13 expression. Controlling MMP activity as well as the production of pro-inflammatory
cytokines is required for the effective treatment of arthritis (McInnes
and Schett, 2007). This observation provides evidence to suggest the potential
anti-inflammatory effect of N. lappaceum against collagen-induced arthritis.
The significant decline in symptoms of arthritis, paw thickness, histological
symptoms and normalization of body weight and biochemical parameters in N.
lappaceum treated arthritic rats postulates the possible anti-inflammatory
and anti-oxidant effect of N. lappaceum rind extract. These effects
may be attributed to the scavenging action of geraniin, an ellagitannin, present
in the extract against Reactive Oxygen Species (ROS) such as OH, RO and RO2
(Hagfors et al., 2003).
Thus, the present study showed that N. lappaceum rind extract is able
to diminish the physiological, histological and biochemical changes produced
during collagen-induced arthritis in Dark Agouti rats. N. lappaceum could
be regarded as one of the natural products having vast range of therapeutic
effects against collagen-induced arthritis. Further studies at the biochemical
and molecular levels on the effectiveness and mechanisms of actions of N.
lappaceum are being carried out.
This research was funded by Ministry of Health (MOH), Malaysia [NMRR Project
ID: 09-406-4108] and supported by International Medical University (IMU) research
grants [IMU 211-2010].
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