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Theaflavin, chief flavanoid of black tea protect adjuvant induced rheumatoid arthritis in animal models*

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  • GlaxoSmithKlime

Abstract and Figures

Objectives: Aim of the study was to evaluate the anti arthritic activity of theaflavin (TF), chief flavonoid of black tea in rheumatoid arthritis animal model. Materials and Method: Rheumatoid arthritis (RA) was induced by Freund's complete adjuvant. Male albino Wistar rats (120±10g) were used and divided into following groups: Gr1- Sham control, Gr2 -Arthritis control, Gr 3- Standard drug, Gr 4- TF treated (0.01mg 100gm-1 ip x14 days), Gr5- TF treated (0.05mg 100gm-1 ip x14 days). Anti-arthritic activity of TF was examined through physical, urinary, serum, synovial fluid parameters, histological structure, X-ray of joints and bone minerals content. All animal experiments were approved by Institutional animal ethical committee. Results were expressed in terms of mean±SEM (n=6) and level of significance determined through one way ANOVA (P<0.05). Results: Ankle and paw diameter significantly restored in TF treated group. Urinary markers hydroxyproline, glucosamine, pyridoline and deoxypridoline levels were significantly restored in TF treated groups. Serum enzymes (ACP/ALP), cytokines (osteocalcin, IL6, TNFα, IL 10, IL 12), synovial fluid cytokines, antioxidant markers, bone ash minerals (Ca++, P and Na) were restored significantly in TF treated group. Histological and Xray studies showed partial restoration in TF treated groups. Conclusion: Findings showed that TF possess distinct anti arthritic activity in experimental arthritis models.
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RESEARCH ARTICLE
Anti arthritic activity of theaflavin (TF), chief flavonoid
of black tea against adjuvant induced rheumatoid arthritis
in experimental animal models
Poulami Datta &Sanghamitra Mukherjee &
Subir Chandra Dasgupta &Aparna Gomes &
Antony Gomes
Received: 27 July 2013 /Accepted: 8 December 2013
#Institute of Korean Medicine, Kyung Hee University 2013
Abstract Rheumatoid arthritis is nowadays major problem
among the aged old people in the society. During the past few
decades there has been a dramatic increase in the use of natural
herbal products for the treatment of this disease. Theaflavin (TF)
is the chief flavonoid of black tea and possess anti oxidant, anti
inflammatory property. The aim of the study was to evaluate the
anti-arthritic activity of TF, chief flavonoid of Black tea on
experimental arthritic animal model. Rheumatoid arthritis was
induced in animal models by Freunds complete adjuvant
(FCA). Male albino Wister rats (120± 10 g) were used and they
were divided into 5 groups: Group 1: Sham control; Group 2:
Arthritis control, Group 3: Standard (Indomethacin), Group 4:
TF treated (low dose), Group 5: TF treated (high dose). Anti-
arthritic activity of TF was examined through urinary, serum,
synovial fluid parameters, bone ash parameters, histological,
radiological studies of joints, SEM studies of joint architecture
and cell cycle analysis. It has been observed that urinary param-
eters changed significantly in arthritic group as compared with
sham control and the change was significantly restored in TF
treated and standard drug treated groups. Serum enzymes, se-
rum cytokines, synovial cytokines, bone ash minerals levels
were restored significantly in TF treated groups as compared
with arthritic groups. Histological, radiological and SEM studies
of the joint/bone architecture showed restoration of the structur-
al architecture of the arthritic joint/bone after TF treatment. TF
treatment significantly arrested the cell cycle of white blood
cells at Go/G1 phase. The findings showed that TF possess
distinct anti-arthritic activity in animal model and further studies
are warranted on the mechanism of action.
Keywords Black tea .Theaflavin .Flavonoid .Rheumatoid
arthritis .Experimental arthritis
Introduction
Rheumatoid arthritis (RA) is a chronic immune inflammatory
joint disease characterised by symmetrical, destructive and
deforming polyarthritis affecting small and large synovial
joints with associated disturbances and multisystem involve-
ment. There is a slight female preponderance in a ratio of 2: 3
female per one male. It has been reported that approximately
25 % of persons of 55 yrs of age or older have knee pain and
almost half of them having symptomatic osteoarthritis, that
leads to physical disabilities or loss of functional capacity and
reduce quality of life (Lawrence et al. 1999).
The aim of the management of rheumatism is to reduce
pain and to minimize the change that occurs during arthritis
development. Physiotherapy, physical exercise and analgesics
are often prescribed by the rheumatologists. Non steroidal anti
inflammatory drugs (NSAIDs) like aceclofenac, diclofenac
are the first line of defence against arthritis. NSAIDs act
through cyclo oxygenase (COX 1 and COX 2) inhibition that
This paper has been partly published as conference abstract in Planta
Medica (Vol. 78 Issue:11 Page: PI105, 19.07.2012)
P. Datta :A. Gomes (*)
Laboratory of Toxinology & Experimental Pharmacodynamics,
Department of Physiology, University of Calcutta, 92, A P C Road,
Kolkata 700 009, India
e-mail: agomescu@gmail.com
S. Mukherjee :S. C. Dasgupta
Postgraduate Department of Zoology, Maulana Azad College,
Kolkata 700 013, India
A. Gomes
Drug Development/Diagnostics & Biotechnology Division, Indian
Institute of Chemical Biology, 4 Raja S C Mullick Road,
Kolkata 700 032, India
Orient Pharm Exp Med
DOI 10.1007/s13596-013-0144-0
inhibit prostaglandins. COX 1 inhibition causes GI tract irri-
tation by causing increased secretion of gastric acid, dimin-
ished bicarbonate secretion which ultimately results in nausea,
dyspepsia, etc. Prostaglandins serve to dilate the afferent
arteriole but due to NSAIDs blocking of this prostaglandin-
mediated effect cause renal failure, unopposed constriction of
the afferent arteriole and decreased renal perfusion pressure.
COX 1 plays an important role in the formation of mediators
that increase the ability of platelets aggregation. By inhibition
of COX 1, ability of platelet aggregation is decreased and clot
formation gets disrupted (Warden 2010). Since these new
therapies and drugs have certain limitations so for the past
few decades there has been a dramatic increase in the use of
natural products/plants for the treatment of this disease be-
cause of their less toxicity, cost factor etc.
Therapeutic value of Tea (Camellia sinensis) has been
recognized in various systems of traditional medication for
the treatment of different diseases and ailments. Gomes et al.
2013 reported that black tea extract has immunomodulatory
activity against immunocompetent and immunodeficient ex-
perimental rodents. Aqueous black tea extract possess anti
arthritic activity in experimental animals (Datta et al. 2012).
Flavonoids have been known to be present in several plants
and have several actions like anti viral, anti inflammatory, anti
sclerotic, anti thrombogenic, anti ulcer etc. The flavonoids
play an important role against adverse effects of NSAIDs by
inhibiting the neutrophil degranulation and by reducing the
release of arachidonic acid (Patel 2008). Earlier, it has been
shown that black tea constitutes 603.28 mg of theaflavin per
100gm of black tea, which is the chief flavonoid of black tea.
For the preparation of black tea, the freshly leaves are allowed
to weather until their moisture content is reduced to about
55 % of the original leaf weight, which result in the concen-
tration of the polyphenols in the leaves. The weathered leaves
are then rolled and crushed, initiating fermentation of the
polyphenols. During these processes, the catechins are con-
verted to theaflavin and thearubigin (Ahmad et al. 2000). The
fermentation process results in oxidation of simple polyphe-
nols to more complex condensed polyphenols to give black
and oolong teas their characteristic colour and flavour.
Theaflavin has been reported to be the active constituent of
black tea and have anti oxidant property, inhibits tumor
growth and cancer suppression (Wang 2006). Black tea poly-
phenol (theaflavin) downregulates MMP-2 in human melano-
ma cell line A375 by involving multiple regulatory molecules
has been established (Sil et al. 2010). It was shown that
theaflavin attenuates ischemia-reperfusion injury in a mouse
fatty liver model (Luo et al. 2012). It has been reported that TF
also protect nigral dopaminergic neurons against chronic
MPTP/probenecid induced Parkinsons disease has been report-
ed (Anandhan et al. 2012). Theaflavin showed preventive
effects against mouse type IV allergy (Yoshino et al. 2010)
and possess anti inflammatory activity (Aneja et al. 2004).
The present study aims to evaluate the anti arthritic role of
theaflavin (TF) against experimental arthritis in male albino rats.
Methods
Chemicals
Anesthetic ether (Narsonspharma, India); Freunds complete
adjuvant(Sigma, USA); Disodium hydrogen phosphate, DPX,
Paradimethyl amino benzaldehyde, Sodium carbonate, Sodium
hydroxide (SRL, India); ELISA CINC-1, TNF-α,IL6,PGE2,
IL-1β, MMP 1, C reactive protein kit (R & D, USA);Eosin,
Formaldehyde, Hematoxylin, Indomethacin (Qualigen, India);
Methyl prednisolone (Pfizer, India); Potassium dihydrogen
phosphate, Sodium chloride, Sodium potassium tartarate,
Xylene (Merck, India); Theaflavin (80 % theaflavins and
theaflavin gallates basis Sigma, USA); Propidium iodide &
Ribonuclease (BD Sciences, USA).
Experimental animals
Wistar male albino rats (120±10 g) were procured from the
approved animal breeders and housed in standard polypropyl-
ene cages at controlled temperature (25± 2 °C), with light
conditions (12 h light and dark cycle) and relative humidity
(65±5 %). The animals were provided with pellet diet
(Ashirbad & Company, Chandigarh, India), green vegetables,
gram and water ad libitum. The experiments were conducted
according to the departmental animal ethics committee for the
purpose of control and supervision of experiments on animals.
All animal experiments were approved by the Institutional
animal ethics committee.
Development of arthritis model
Rheumatoid arthritis was induced by injecting (sub-plantar)
0.1 ml emulsion of Freunds complete adjuvant (FCA) in olive
oil (1:1 v/v) at a concentration of 0.25 mg/ml heat killed
Mycobacterium tuberculosis emulsion into the right hind
footpad of Wistar male albino rats on day 0. Olive oil was
injected in the contra-lateral footpad (Newbould 1963)as
control. Theaflavin was dissolved in distilled water and used
for treatment.
Treatment ScheduleOn the 1st day after induction of
arthritis 30 animals were divided into the following groups,
Group 1: Sham control, Group 2: Arthritic control, Group 3:
Standard, Group 4: TF treated (low dose), Group 5: TF treated
(high dose). Animals of Group 3 treated with standard anti
P. Datta et al.
arthritic drug Indomethacin orally (p.o) (2.5 mg/kg/p.o. ×
5 days alternately). Animals of Group 4 and Group 5 were
treated with TF, 0.1 ml and 0.5 ml (0.1 mg and 0.5 mg/kg)
intraperitoneally (i.p) for 14 days for the study of experiment.
Group 1 and Group 2 animals received distilled water intra-
peritoneally for 14 days. Animals of all the groups were
provided with normal diet and water ad libitum. After the
treatments, animals were placed in special urine collection
cage with water load (5 ml/100gm/p.o). After18 h urine was
collected under light liquid paraffin. On the next day, synovial
fluid was collected aseptically from the joint of the lightly
anesthetized rats by a syringe using 26 gauge needle. Blood
samples from all the animals were collected and serum was
separated. Ankle joints/femur bone were collected for histo-
logical, SEM studies and bone mineral content.
Biochemical analysis of urine/serum/synovial fluid
Urinary hydroxyproline and glucosamine (Elson and Morgan
1933) were measured spectrophotometrically (Neuman and
Logan 1950). Urinary pyridoline and deoxy pyridoline were
measured with ELISA kit manuals of the manufacturer
(Cusabio). Rat serum alkaline and acid phosphatase was mea-
sured spectrophotometrically using p-nitro phenyl as a sub-
strate (Mitchell et al. 1970). Serum C-reactive protein,
osteocalcin, IL-12, MMP-1, Cathepsin-K and synovial fluid
markers TNF-α, IL-6 were measured with ELISA kit manuals
of the manufacturer (R& D).
Bone minerals estimation
Bone (Ankle joint) ash was prepared in a muffle furnace at
700 ° C for 6 h. Bone extract was prepared in 0.1 N HCl. Bone
minerals (Ca, P &Na) were measured by using atomic absorp-
tion spectrophotometer (Varian, USA, Model No.
AA575ABQ).
Histological studies
Joints (ankle) of the rats were collected, fixed in 10 % buffered
formalin for 24 h and decalcified in Osteomol for 3 days. The
tissues were then dehydrated in graded (50100 %) ethanol
followed by clearing in Xylene. Paraffin (5658 °C) embed-
ding was done at 58±1 °C for 4 h, followed by paraffin block
preparation. Paraffin sections of 5 μwere cut using a rotary
microtome (Weswox Optik, India). Paraffin sections were
deparafinised with xylene, stained with hematoxylin-eosin,
followed by mounting in DPX with a cover slip.
Histological changes were observed with a bright field micro-
scope (Motic, Germany) and photographs were captured with
Motic software (Motic Images Plus 2.0 software).
Radiological analysis of bone knee joints
Animals were lightly anesthetized with anesthetic ether. Radio
photographs of the hind leg joints of rats were taken with a X-
ray instrument with computerized radiographic systems
(Siemens, Multifalse Stain Plus). The film focus distance was
60 inches and the machine was operated at 43 kV peak, 2 mA.
The X-ray image of the joint of each rat was evaluated for
radiographic changes. As reported previously the radiological
alterations were noted according to the severity of the swelling
of the soft tissue around the joints, periarticular bone resorption,
periarticular bone erosion and narrowing of the joint space.
Scanning electron microscopy (SEM) study of the bone
(femur)
Femur bone was sectioned transversely by fine hacksaw blade
and the bone chips were fixed (10 % buffered formalin for
1 week). Then the bone chips were coated in gold with
Hitachi-E-1010 (ion sputter) for 30 s. The gold coated proc-
essed bone was then analysed at 25 KV accelerating voltage
by scanning electron microscope (hitachi-S-3400 N, 15.0KV)
at magnification 500×, 1,000× by.
TF induced cell cycle arrest
Blood was collected by cardiac puncture of Wistar male
albino rats using heparinised vials. The white blood cells were
pelleted down by centrifuging at 1,000 r.p.m for 20 min
followed by washing with lysis solution. Then the cells were
harvested and washed with PBS, fixed with 70 % ethanol and
kept at 4 °C for 30 mins. Again the cells were washed with
PBS by spining in a centrifuge. The cells were treated with
ribonuclease and Propidium iodide. Flow cytometric analysis
was then done using FACS Verse double laser cytometer
(Becton Dickinson, USA).
Statistical analysis
Data were expressed in terms ofmean ± SEM (n=6). The data
were subjected to one way analysis of variance (ANOVA)
followed by Tukeys test using Origin 8 software to establish
statistical significance.
Results
Effect of TF on urinary parameters
Urinary hydroxyproline and glucosamine level of arthri-
tis control group Gr 2 rats showed an increase as
Anti arthritic activity of theaflavin (TF)
compared with the control group Gr 1 rats (by respec-
tively 52.41 and 70.11 %). TF (0.1 mg and 0.5 mg/kg×
14 days, i.p) treated Wistar male albino rats Gr 4 and
Gr 5 showed a decrease in urinary hydroxyproline and
glucosamine level (by respectively 40.25 and 54.11,
54.22 and 65.32 %); whereas the standard drug,
Indomethacin (2.5 mg/kg/p.o× 5 days alternate) treated
Gr 3 rats showed decrease in hydroxyproline and glu-
cosamine level as compared with arthritis control Gr 2
rats (by respectively 48.83 and 53.83 %) (Fig. 1).
Urinary pyridinoline and deoxypyridinoline level of arthri-
tis control group Gr 2 rats showed an increase as compared
with the control Gr 1 rats (by respectively 34.63 and 61.53 %).
TF (0.1 mg and 0.5 mg/kg × 14 days, i.p) treated Wistar male
albino rats groups ie, Gr 4 and Gr 5 showed a decrease in the
urinary pyridinoline and deoxypyridinoline level (by respec-
tively 19.85 and 47.51, 50.47 and 75.71 %) whereas the
standard drug treated Gr 3 rats showed a decrease as compared
with the arthritis control Gr 2 rats (by respectively 25.83 and
54.28 %) (Table 1).
Effect of TF on serum enzymes
Serum acid phosphatase and alkaline phosphatase activ-
ity of arthritis control Gr 2 rats showed an increase as
compared with control Gr 1 rats (by respectively 40.76
and60.19%).TF(0.1mgand0.5mg/kg×14days,i.p)
treated Wistar male albino rats Gr 4 and Gr 5 rats
showed decrease in serum ACP activity (by respectively
39.9 and 44.26, 15.9 and 24.94 %) whereas standard
drug treated Gr 3 rats showed decrease as compared
with arthritis control Gr 2 rats (by respectively 31.97
and 48.52 %) (Fig. 2).
Effect of TF on serum osteocalcin, C-reactive protein,
cytokines level
Serum osteocalcin level of arthritis control Gr 2 rats showed
decrease as when compared with control Gr 1 rats (by respec-
tively 61.10 %). TF (0.1 mg and 0.5 mg/kg × 14 days, i.p)
treated Wistar male albino rats Gr 4 and Gr 5 rats showed
decrease in serum osteocalcin level whereas standard drug
treated Gr 3 rats showed decrease as compared with the arthritis
control Gr 2 rats (by respectively 50.18 and 95.90, 55.41 %).
Serum C-reactive protein level of arthritis control Gr 2 rats
showed an increase as compared with control Gr 1 rats
(by 85.36 %). TF (0.1 mg and 0.5 mg/kg × 14 days, i.p)
treated Wistar male albino rats Gr 4 and Gr 5 rats showed a
decrease in serum C reactive protein level whereas standard
drug treated Gr 3 rats showed decrease as compared with the
arthritis control Gr 2 rats (by respectively 6.79 and 24.09,
25.25 %).
Serum IL 12 level of arthritis control Gr 2 rats showed an
increase as compared with control Gr 1 rats (by respectively
85.36 %). TF (0.1 mg and 0.5 mg/kg × 14 days, i.p) treated
Wistar male albino rats Gr 4 and Gr 5 rats showed a decrease
in serum IL 12 level whereas standard drug treated Gr 3 rats
showed decrease as compared with the arthritis control Gr 2
rats (by respectively 4.73 and 24.09, 25.25 %) (Table 1).
Effect of TF on synovial fluid cytokines level
Synovial fluid cytokines IL-6 and TNF-αlevel of arthritis
control Gr 2 rats showed an increase as compared with
control Gr 1 rats (by respectively 31.29 and 42.76 %). TF
(0.1 mg and 0.5 mg/kg × 14 days, i.p) treated Wistar male
albino rats Gr 4 and Gr 5 rats showed a decrease in serum IL
Fig. 1 Effect of TF (0.1 mg and
0.5 mg/kg B.W × 14 days, i.p) on
urinary markers of arthritis
induced rats. Data represent as the
mean ± SEM (n= 6). Statistical
analysis was done with one-way
ANOVA followed by Tukeys
test. *(p<0.05), **(p<0.01),
***(p<0.001) considered as
significant, (Gr.1 vs Gr.2, Gr.2 vs
Gr.3 Gr.4/Gr.5). Gr.1: Control,
Gr.2: Arthritis control, Gr.3:
Standard drug (indomethacin),
Gr.4 and 5: TF (0.1 mg and
0.5 mg/kg B.W × 14 days, i.p)
treated
P. Datta et al.
6andTNFαlevel whereas standard drug treated Gr 3 rats
showed decrease as compared with the arthritis control Gr 2
rats (by respectively 54.33 and 65.75, 19.58 and 39.26,
44.62 and 34.17 %) (Table 1).
Effect of TF on bone ash parameters
Bone calcium, phosphate and sodium content of arthritis
control Gr 2 rats showed a decrease as compared with control
Gr 1 rats (by respectively 58.45, 31.45 and 27.91 %). TF
(0.1 mg and 0.5 mg/kg × 14 days, i.p) treated Gr 4 and Gr 5
rats showed an increase in calcium, Phosphate and sodium
content whereas standard drug treated Gr 3 rats showed in-
crease as compared with the arthritis control Gr 2 rats
(by respectively 12.01 and 21.17, 17.00 and 18.07, 4.22 and
8.45, 56.27, 14.98, 9.54 %) (Table 2).
Effect of TF on MMP1level and serum cathepsin K
Serum MMP1 and Cathepsin K level of arthritis control group
Gr 2 rats showed an increase as compared with the control Gr
1 rats (by respectively 56.94 and 62.30 %). TF (0.1 mg and
0.5 mg/kg × 14 days, i.p) treated Gr 4 and Gr 5 showed a
decrease in the levels whereas as the standard drug treated Gr
3 rats showed a decrease as compared with the arthritis control
Gr 2 rats (by respectively 43.12 and 55.87, 50.34 and 61.63,
57.12 and 58.37 %) (Fig. 3).
Effect of TF on bone SEM studies, joints histological
and radiological studies
In the SEM studies, in the arthritic group of rats there was
disturbance in the collagen fibril network and pore formation
also took place. Theaflavin treatment partially restored the
micro architecture of the bone as compared to the arthritic
group of rats (Fig. 4).
In histological studies, arthritis control Gr 2 rats showed that
the spaces between the joints were decreased and destruction of
the synovial membrane occurred. But TF treated Gr 5 rats
showed partial restoration of the architectural structure of joints.
Radiological studies showed that in arthritic control
Gr 2 rats the space in between the synovial joints were
decreased which were partially restored after theaflavin
treatment (Fig. 5).
Table 1 Effect of TF (0.1 mg and 0.5 mg/kg B.W × 14 days, i.p) on
urinary markers, serum cytokines and synovial fluid cytokines of arthritis
induced rats. Data represent as the mean ± SEM (n=6). Statistical
analysis was done with one-way ANOVA followed by Tukeystest.
*(p<0.05), **(p<0.01), ***(p< 0.001) considered as significant,
(Gr.1 vs Gr.2, Gr.2 vs Gr.3 Gr.4/Gr.5). Gr.1: Control, Gr.2: Ar-
thritis control, Gr.3: Standard drug (indomethacin), Gr.4 and 5: TF
(0.1 mg and 0.5 mg/kg B.W × 14 days, i.p) treated
Group of animals Urinary parameters (pg/ml) Serum cytokines (pg/ml) Synovial fluid cytokines (pg/ml)
Pyridoline Deoxypyridoline Osteocalcin IL 12 C-reactive protein IL 6 TNF α
Gr 1 13.02±0.51 1.30±0.06 13.78±1.01 6.15±0.30 20.69±1.16 62.22±2.04 39.66±0.86
Gr 2 17.53±0.48** 2.10± 0.07** 5.36± 0.78*** 11.40±1.81*** 42.21±1.47*** 81.07±3.07*** 56.62±2.50***
Gr 3 13.99±0.58** 0.96± 0.03** 8.33± 1.42*** 7.27±0.40*** 31.55±0.86** 44.89±3.76*** 37.27± 1.03***
Gr 4 14.05±0.38** 1.04±0.033 ** 8.05±0.68*** 10.86±1.84*** 39.34±0.81** 37.02 ±2.08*** 45.53±4.00***
Gr 5 9.20 ±0.41*** 0.51±0.02** 11.30± 0.76*** 5.72±1.61*** 32.04±1.46** 27.76±1.82*** 34.39± 2.96***
Fig. 2 Effect of TF (0.1 mg and
0.5 mg/kg B.W × 14 days, i.p) on
serum enzymes of arthritis
induced rats. Data represent as the
mean ± SEM (n= 6). Statistical
analysis was done with one-way
ANOVA followed by Tukeys
test. *(p<0.05), **(p<0.01),
***(p<0.001) considered as
significant, (Gr.1 vs Gr.2, Gr.2 vs
Gr.3 Gr.4/Gr.5). Gr.1: Control,
Gr.2: Arthritis control, Gr.3:
Standard drug (indomethacin),
Gr.4 and 5: TF (0.1 mg and
0.5 mg/kg B.W × 14 days, i.p)
treated
Anti arthritic activity of theaflavin (TF)
Effect of TF on WBC cell cycle arrest
In the present study WBC collected from TF (0.5 mg/kg body
weight) treated animals showed WBC cell cycle arrest at the
Go/G1 which was greater than the standard drug (TF induced
showed 5.14 % and standard drug showed 2.96 % arrest) as
compared with the arthritis control WBC (Fig. 6).
Discussion
The tea polyphenols especially the catechins are primarily
responsible for the curing property of green tea. However
during the production of black tea a significant part of the
catechins is converted to the theaflavins (comprising of
theaflavin-3-gallate, theaflavin-3-gallate, and theaflavin-3, 3-
digallate) and thearubigins by the activity of a polyphenol
oxidase. Theaflavin enriched black tea extract has shown its
anti inflammatory properties against muscle soreness
(Arent et al. 2010). Hydroxyproline, a non essential amino
acid is present in all collagen types of bone matrix (Harris
1989; Felson 2006). After breakdown of collagen hydroxy-
proline was released into the extracellular fluid and the level of
hydroxyproline increase in the urine. But hydroxyproline is
not specific to bone collagen and is also derived from diet.
Since hydroxyproline is absorbed through GI tract, the intake
of collagen rich food was restricted for at least 24 h before
urine sample collection. In the present study increased urinary
hydroxyproline level observed in the arthritis control but TF
treatment showed significant restoration in the urinary hy-
droxyproline level in both the models. Another marker, glu-
cosamine, signifies degradation of cartilage matrix glycopro-
tein by glycohydrolase activity. Experimentally induced ar-
thritic rats showed significant increase in urinary glucosamine
level which was decreased significantly by TF treatment.
Pyridinoline (PYD) and deoxypyridinoline (DPD) are colla-
gen cross-links, and their urinary concentrations have been
proved to be accurate markers of bone resorption. After bone
degradation, PYD and DPD are released in the circulation and
excreted directly into urine without further systemic metabo-
lism. Pyridinium-based cross-links are an important part of the
extracellular collagen fibrils in most connective tissue types.
Unlike other connective tissues, bone is continuously
remodelled and therefore forms the main source of urinary
cross-links. Furthermore, the ratio of PYD to DPD in urine is
approximately the same as in adult human bone (3.5:1),
further supporting bones as the predominant source of urinary
PYD and DPD (Nur et al. 2010). Arthritic models showed
increased levels of PYD and DPD but TF treatment signifi-
cantly decreased their levels. These findings signify that TF
treatment may provide protection against cartilage degrada-
tion. A marked increase in the serum ACP and ALP levels
occurred in adjuvant induced arthritis in experimental rats
(Gomes et al. 2010). In the present study, TF treatment
Table 2 Effect of TF (0.1 mg and 0.5 mg/kg B.W × 14 days, i.p) on bone
mineral content of arthritis induced rats. Data represent as the mean ±
SEM (n=6). Statistical analysis was done with one-way ANOVA follow-
ed by Tukeys test. *(p<0.05), **(p<0.01), ***(p<0.001) considered as
significant, (Gr.1 vs Gr.2, Gr.2 vs Gr.3 Gr.4/Gr.5). Gr.1: Control, Gr.2:
Arthritis control, Gr.3: Standard drug (indomethacin), Gr.4 and 5: TF
(0.1 mg and 0.5 mg/kg B.W × 14 days, i.p) treated
Group of
animals
Calcium Phosphorus Sodium
(mg/gm of bone) (mg/gm of bone) (mg/gm of bone)
Gr 1 497.1 ±6.9 122.7± 5.3 19.7± 0.7
Gr 2 206.5 ±5.9*** 84.1 ±2.1*** 14.2± 0.3**
Gr 3 322.7 ±5.3*** 96.7 ±2.4*** 15.5± 0.5**
Gr 4 234.7 ±5.7*** 98.4 ±1.9*** 14.8± 1.1**
Gr 5 250.22±5.9*** 99.3±1.5*** 15.4± 0.4**
***
***
**
**
**
**
**
**
Fig. 3 Effect of TF (0.1 mg and
0.5 mg/kg B.W × 14 days, i.p) on
bone mineral content of arthritis
induced rats. Data represent as the
mean ± SEM (n=24). Statistical
analysis was done with one-way
ANOVA followed by Tukeys
test. *(p<0.05), **(p<0.01),
***(p<0.001) considered as
significant, (Gr.1 vs Gr.2, Gr.2 vs
Gr.3 Gr.4 Gr. 5). Gr.1: Control,
Gr.2: Arthritis control, Gr.3:
Standard drug (indomethacin), Gr
4 and 5: TF(0.1 mg and 0.5 mg/kg
B.W × 14 days, i.p) treated
P. Datta et al.
significantly decreased the serum ACP and ALP levels. This
could therefore be ascribed to the restoration of lysosomal
membrane integrity brought about by TF treatment. Datta
et al. 2012 also reported that aqueous black tea extract could
decrease the activity of these serum enzymes and the cyto-
kines level in experimental animals.
It has been found that Theaflavin inhibits LPS-induced
IL-6, MCP-1, and ICAM-1expression in bone marrow-
derived macrophages through the blockade of NF-κB and
MAPK signalling pathways (Kim and Joo 2011). In the pres-
ent study it has been found that theaflavin also restore both
anti and pro inflammatory cytokines levels in serum and
synovial fluid which were increased in adjuvant induced
arthritis. Osteocalcin is secreted solely by osteoblasts and
thought to play a role in the bodys metabolic regulation.
Since osteocalcin produced by osteoblasts, it is often used as
a marker for the bone formation process. Serum osteocalcin
was considered as a specific and sensitive marker of osteo-
blastic activity and bone formation (Bullon et al. 2007). In
arthritic group of rats the osteocalcin level was significantly
decreased as compared with the control group of rats. The
osteoblastic activity was inhibited as well as osteoclastic ac-
tivity was increased in arthritis, as a result there was a decrease
in serum osteocalcin level. After treatment with theaflavin
serum osteocalcin level was significantly restored as com-
pared to the arthritic group of rats. C-reactive protein (CRP)
is an acute phase protein and a golden marker of inflammation
(Patel et al. 2001). CRP is mainly produced in the liver,
rapidly starts increasing when inflammation occurs and rap-
idly decreases when inflammation improves. Since arthritis is
an inflammatory disease the production of CRP increases in
the serum during arthritis (Holmes et al. 2010). After TF
treatment significant decrease in the CRP level has been
observed as compared with the arthritic group of rats.
In arthritis bone minerals specially calcium and phosphate
levels were decreased significantly as compared with control
group of rats. Bone tissue consists of mainly hydroxyapatite
crystals of calcium and phosphorus with carbonate, sodium
and potassium. At the time of bone resorption this hydroxy-
apatite crystals are broken and calcium and phosphate were
resorbed into the ECF. It has been reported that chronic
hyponatremia has been associated with arthritis, fractures
and osteoporosis (Ewout et al. 2012). In the present study
the sodium, calcium and phosphate level in the bone mineral
content was found to be significantly lower in the arthritic
group of rats. Theaflavin treatment showed significant resto-
ration of the levels of the mineral contents (sodium, calcium
and phosphate) as compared with the arthritic group of rats.
Fig. 4 Effect TF on SEM of bone
of FCA induced arthritis is Wistar
male albino rats. Magnifications
250×. 1. Pore formation in the
bones of arthritic group of rats. 2.
Reduced compactness of the bone
structure in arthritic group of
rats.3. Reduced pore formation in
higher doseof TF treated group of
rats. 4. Reduced compactness of
the bone structure in higher dose
of TF treated group of rats
Fig. 5 Effect of TF on Xray
studies of bone joints in FCA
induced Wistar male albino rats. a
Arthritis control Gr 2 rats: Altered
structure of the joints, bTF
treated high dose Gr 5 rats:
Partially recovered structure of
the joints
Anti arthritic activity of theaflavin (TF)
Rheumatoid arthritis (RA) is characterised by degradation of
cartilage and invasion of fibroblast-like synoviocytes (FLS)
into adjacent cartilage. Hirabara et al. 2013 reported that
Cathepsin K and MMP-1 level are highly expressed in case
of arthritis and thus play an important role in degradation of
the collagen also. Expression of MMP-1 and Cathepsin K has
been found to be increased during arthritis but MMP-3 and 10
were also highly expressed during arthritis (Tolboom et al.
2001). In the present study expression of MMP-1 and
Cathepsin K has been found to be increased in arthritic group
of rats but TF treatment showed significant decrease in the
expression of these two markers.
The scanning electron microscopic study showed that in
arthritis there was a disturbance in the bone collagen network
occur (Lopes et al. 2010). It has been observed that TF
treatment partially restore the changes of the bone and the
joints. The white blood cells both in clinical and experimental
studies increased during arthritis (Kyei et al. 2012). Recently it
has been reported that in arthritis the lymphocyte size and
shape were changed (Chokkalingam and Komathy 2012). The
involvement of WBC/lymphocytes in bone joint related dis-
orders are common. The lymphocytes are involved in the
release of several inflammatory mediators that are responsible
for the pro inflammatory changes of the joint areas, which in
turn are responsible for pain, inflammation in the affected
areas. One of the major targets is to reduce WBC infiltration,
pro inflammatory cytokines release. It has also been reported
that in rheumatoid arthritis fibroblast-like synoviocytes play
an important role in the initiation and progression of the
diseases. Berberine, the major constituent of Coptidis
Rhizoma, has been widely used as an antitumor and anti-
inflammation agent. Cell cycle analysis of berberine-treated
cells indicated a cell cycle arrest at the G0/G1 phase
(Tang et al. 2009). In the present study it has been observed
that TF affecting arrest of WBC cell cycle thereby may reduce
the number of WBC which in turn may decrease the release
of inflammatory mediator responsible for the signalling
cascade of arthritis inflammation/pain pathway. Further
studies in this area (using cell lines) may uncover the
inner mechanism of TF induced anti arthritic activity in
the near future.
Conclusion
It may be concluded that theaflavin (TF) possess significant
anti-arthritic activity in experimental animal models and TF
may be beneficial in bone joint related disorders. Further
detail work on TF and its mechanism of anti-arthritic
activity are warranted.
Fig. 6 Effect TF on cell cycle analysis of white blood cells of FCA induced arthritis is Wistar male albino rats. aCell cycle analysis of Arthritis group of
animals, bCell cycle analysis of TF high dose treated groups, cCell cycle analysis of standard drug treated group
P. Datta et al.
Acknowledgments Financial support for work was partially sponsored
by National Tea Research Foundation, Kolkata, India (Ref No: 141/2010).
Conflict of interest We declare that there is no conflict of interest
among the authors.
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Anti arthritic activity of theaflavin (TF)
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