Journal of Ethnopharmacology 150 (2013) 1071–1079

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Anti-inflammatory and antinociceptive effects of Chinese medicine

SQ gout capsules and its modulation of pro-inflammatory cytokines
focusing on gout arthritis
Nandani Darshika Kodithuwakku 1, Min Pan 1, Yi-lin Zhu, Yan-yan Zhang,
Yi-dong Feng, Wei-rong Fang n, Yun-man Li n
State Key Laboratory of Medicines, Department of Physiology, China Pharmaceutical University, Mailbox 207, # 24, TongJiaXiang,
Nanjing 210009, PR China

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Shuang-Qi gout capsule is a traditional Chinese medicine prescription,
Received 20 June 2013 which has been used in the treatment of joint pain, inflammation and gout arthritis. This study evaluates
Received in revised form anti-inflammatory and antinociceptive effects of Shuang-Qi gout capsule and its modulation of pro-
11 September 2013
inflammatory cytokines with special reference to gout arthritis.
Accepted 7 October 2013
Available online 22 October 2013
Materials and methods: Anti-inflammatory effect of Shuang-Qi gout capsule was investigated bymice tail-
flick response, acetic acid induced writhing response, Xylene-induced auricle inflammation and the hind
Keywords: paw volume of the monosodium urate (MSU) crystal induced rats with different time durations.
Anti-inflammation To investigate the effects on gout arthritis, ankle joint of rats induced by MSU crystals and assessed
Antinociceptive effect
for edema and histopathological changes. In vitro, prepared serum was incubated with urate crystal
SQ gout capsule
induced HUVE cells and the release of TNF-α and IL-1β determined by ELISA.
Gout arthritis
Pro-inflammatory cytokines Results: Shuang-Qi gout capsule showed significant and dose dependent anti-inflammatory effect via
reducing edema and pain, throughout all the models. The high dose of Shuang-Qi gout capsule and
Indomethacin significantly attenuated the edema. Histopathological results showed that high and
medium dose of Shuang-Qi gout capsule and Indomethacin reduced gouty joint inflammatory features,
while the high dose of Shuang-Qi gout capsule showed a better therapeutic effect. High and medium
dose of Shuang-Qi gout capsule significantly reduced the release of TNF-α and IL-1β (po0.05).
Conclusion: Shuang-Qi gout capsule can effectively inhibit the inflammation, analgesia, through the
modulation of emission of pro-inflammatory cytokines and the curative effect is dose dependent.
Conversely, these MSU induced in vivo and in vitro studies of Shuang-Qi gout capsule suggest that,
Shuang-Qi gout capsule may be a potential agent for treatment in gouty arthritis.
& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Many ethno botanic traditions provide a rich repertory of
medicinal plants or extracts that are used all around the world
for many diseases and symptoms including pain and inflamma-
tion. Even though well-established treatments are available for
inflammation and severe pain, such as Non Steroidal Anti-
inflammatory drugs (NSAIDs), analgesic drugs, current trend is
more interested in herbal drugs due to their safety and efficacy
(Wen et al., 2005).
Shuang-Qi (SQ) gout capsule encompasses six traditional Chi-
nese medicines, including Phragmites communis Trin. (Lú gēn,
n
Corresponding authors. Tel./fax: þ86 2583271173.
E-mail addresses: weirongfang@163.com (W.-r. Fang),
yunmanlicpu@hotmail.com (Y.-m. Li).
1
These two authors contributed equally to this work.
rhizome of Reed), Berchemia floribunda (wall) Brongn (Tiě bāo jīn,
root of Linate Supplejack), Mallotus apelta (Lour.) Muell.-Arg (Bái
bèi yè gēn, Root of White-back leaf Mallotus), Schefflera arboricola
Hayata. (Qī yè lián, dry stem of Dwarf Umbrella tree), Cinnamo-
mum camphara (L.) Presl (Bīngpiàn, resin from the plant-Borneol)
and Panax notoginseng (Burk. F.H. Chen) (Sānqī, the root of Radix
Notoginseng) (Chan et al., 2012). Phragmites communis is generally
indicated for fever anti-inflammation while Schefflera arboricola
can cure rheumatoid arthritis, rheumatism, joint pain, fever, sore
throat etc. Cinnamomum camphara is indicated for rheumatism,
pain, fever and inflammation. Similarly Berchemia floribunda, and
Mallotus apelta are also used for inflammation, fever, and arthritis.
Panax notoginseng has the effect of heamostasis and relieving
edema to lessen pain. According to traditional medicinal view, it is
said to act on the qi channels, which are originating from the heart
and kidney in the body. Prescription of SQ gout capsule is also
found to be effective clinically for inflammation, pain, fever and

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2013.10.016
1072 N. Darshika Kodithuwakku et al. / Journal of Ethnopharmacology 150 (2013) 1071–1079
gout arthritis (Province(Ed.), 2009; State Pharmacopoeia Comm-
ission of People's Republic of China, 2010). According to Traditional
Chinese Medicine concepts, this combination is recommended for
clearing the heat, removing dampness, detoxification, promoting
blood circulation, inflammation, dispelling wind and relieving
pain. However, there are not sufficient scientific evidences on
their combined effect and possible mechanisms underlying the
effectiveness of these plant materials.
Gout arthritis has been a common inflammatory arthritis due to
its rising prevalence and incidence over the world for the last few
decades. Recently, the prevalence rate of gout in China is found to be
increasing especially in coastal areas (Miao et al., 2008). Gout is
characterized by recurrent attacks of acute arthritis which is caused
by deposition of monosodium urate crystals within joints, tendons
and the periarticular tissues as a result of persistent hyperuricemia
(Richette and Bardin, 2010). Patients experience a nociceptive pain
during the acute attacks of gout and the affected joint, most often
being the big toe shows the cardinal features of inflammation.
If remain untreated, it will result in permanent disability of the
affected joint (Pluta et al., 2012).
Acute inflammatory features are the collective effects of potent
pro-inflammatory stimuli including xylene, acetic acid, monoso-
dium urate (MSU) crystals etc. (Buzzi Fde et al., 2010). Some
studies have emphasized that induction of MSU crystals triggers
the pro-inflammatory reaction to produce cytokines, interleukin-6,
1β (IL-6, IL-1β) and tumor necrosis factor-α (TNF-α) in neutrophil
recruitment (Hamburger et al., 2011). These pro-inflammatory
mediators, which are produced in both the synovial fluid and
the synovial membrane, trigger membranolysis, which progresses
to joint damage (Feng et al., 2010). Nociceptive effects are
also found to be due to the stimulation of peripheral or central
nociceptive receptors via cytokines and other inflammatory
mediators followed by thermal or chemical stimuli (Julius and
Basbaum, 2001).
The present study was designed, to investigate the anti-
inflammatory reaction and analgesic effect of Chinese herbal
medicine SQ gout capsule focusing on gout arthritis. To get an
insight of the possible mechanisms of anti-inflammatory effect,
in vitro study was performed with regard to suppression of pro-
inflammatory mediators. The Tong Feng Shu tablet was used as the
positive control, a traditional Chinese medicine that is indicated
for gout arthritis in common clinical practice (Approval number as
a Chinese medicine: 720080410).
2. Materials and methods
2.1. Reagents and materials
Phragmites communis Trin., Berchemia floribunda (wall) Brongn,
Mallotus apelta (Lour.) Muell.-Arg, Schefflera arboricola Hayata.,
Cinnamomum camphara (L.) Presl and Panax notoginseng were
purchased from JiangSu Medicine Company (Nangjing, China)
and authenticated by Prof. Qin min Jian in School of Chinese
Herbal Medicine, China Pharmaceutical University and all the
voucher specimens were deposited in the Herbarium of School
of Traditional Chinese Medicine, China Pharmaceutical University.
Uric acid sodium salt and Indomethacin, were purchased from
Sigma (St. Louis, MO, USA). Colchicine was purchased from
Kunming Pharmaceutical Holdings Co Ltd. Tong Feng Shu tablets
were purchased from Hunan Kangpu Pharmaceutical Co. Ltd.
Xylene and acetic acid were purchased from Nanjing Chemical
Reagent Co., Ltd. (PR China). ELISA Kits for human TNF-α and h
IL-1β were purchased from the Hysen Hung Industrial Co., Ltd.
Shanghai (PR China).
Prepared SQ gout capsules were kindly provided by the China
Pharmaceutical University. Tong Feng Shu was brought from Hunan
Comm Scope Pharmaceutical Co., Ltd. (batch no.: 20101102).
2.2. Preparation of SQ gout capsule
First, the crude extract (12.3 g) was extracted with 50% ethanol
two times for 6 h to yield equivalent of 1 g Schefflera arboricola
Hayata (Qī yè lián) drug. Berchemia floribunda (Tiě bāo jīn) (1 g)
was obtained from 21.1 g of plant material extracted in 80%
ethanol 3 times for 9 h. Phragmites communis Trin. (Lú gēn) (6 g)
was obtained as a total water extract while Mallotus apelta (Lour.)
Muell.-Arg (Bái bèi yè gēn) (1 g) was extracted in 80% ethanol six
times for 9 h from 26.0 g of crude drug. All the extractions were
maintained between 70 and 80 1C.
The individual extracts and inspissations of each material (Con-
centration was done by using rotatory evaporator) obtained were
mixed properly, a fine powder of the Panax notoginseng (Sānqī)
(1.2 g), and excipients were then added, and spray dried. This was
then sifted through a 24 mesh size and dried at 50 1C. After which a
fine powder of Cinnamomum camphara (Bing pian) (0.6 g) was added,
mixed and sifted again through 24 mesh. Predetermined amounts of
the obtained powder was filled into the capsules. The quality of each
capsule was controlled by measuring the amount of oleanolic acid
and Ginseng saponinin (ginsenoside Rg1 (C42H72O14), Ginseng
saponins Rb1 (C54H92O23) and 37 saponins R1 (C47H80O18))by
using HPLC analysis as the total amount 0.42 mg and 3.3 mg,
respectively, (Commission, 2010a, 2010b). Moreover, TLC was carried
out for quality standards for each component by comparing with the
existing prescription.
2.3. Preparation of drugs
SQ gout capsule powder was ground, dispersed in 0.5% CMC-Na
to make a homogeneous suspension of 45 mg/ml, 22.5 mg/ml and
11.3 mg/ml, as the high, medium and low dose respectively.
Indomethacin, Tong Feng Shu, and colchicines were also freshly
dispersed in 0.5% CMC-Na. The administered volumes of drugs
were calculated based on 20 ml/kg body weight for mice and rats
all through the study, which was measured prior to each drug
administration. Doses of SQ gout capsule were determined based
on the preliminary studies of the drug with the series of different
concentrations. Doses for other drugs and the doses for the mice
and rats were decided according to the dose conversion proce-
dures in accordance with the Pharmacopoeia and the study of
Reagan Shaw (Chinese Pharmacopoeia Committee, 2005; Reagan-
Shaw and Ahmad, 2008).
2.4. Animals
ICR mice (20.0 72.0 g) were purchased from Yangzhou Uni-
versity Medical Center [Jiangsu Province, China]. Male Spargue-
Dawley (SD) rats (200.0 7 20.0 g) were purchased from Zhejiang
Province Experimental Animal Center (Zhejiang Province, China).
They were acclimatized at least for 1 week before being used
for experiments. Animals were housed 5 per cage (320 cm 
180 cm  160 cm) under a normal 12-h/12-h light/dark schedule
with the lights on at 07:00 a.m. They were housed at room
temperature (2272 1C) with relative humidity (5575%), and stan-
dard chow and water were provided ab libitum for the duration of
study. All the procedures and animal care were performed in
accordance with the Provision and General Recommendation of
Chinese Experimental Animals Administration Legislation and were
approved by the Science and Technology Department of Jiangsu
Province and also by the Animal Care and Use Committee of China
Pharmaceutical University.
 N. Darshika Kodithuwakku et al. / Journal of Ethnopharmacology 150 (2013) 1071–1079
2.5. The effect of SQ gout capsule on mice-tail flick response caused
by hot water
According to the work of Davies et al. (1946), 60 ICR male mice
were randomly divided into 6 groups: control (0.5% CMC-Na),
Indomethacin (8 mg/kg), Tong Feng Shu (600 mg/kg), and SQ gout
capsule at high (450 mg/kg), medium (225 mg/kg) and low (113 mg/
kg) dose (n¼10). All the mice were administered by oral gavage once
daily for 7 consecutive days. Tail-flick latency assessment was done
half an hour after the final drug administration. The lower end of the
mouse's tail (5 cm) was submerged in 5571 1C hot water and
constant heat intensity was maintained. When the mouse flicked
its tail in response to the noxious stimuli of the heat, stopwatch was
stopped. The same procedure was carried out to get the basic pain
thresh hold prior to the drug administration on the 1st day, in order
to calculate the pain increase ratio and the tail-flick latency in t-test.
Pain increase ratio was calculated using the following formula:
Pain increase ratio ð%Þ ¼ ðTail  flick latency of each group
=Tail  flick latency of control groupÞ  1  100%:
2.6. The effect of SQ gout capsule on mice writhing response caused
by acetic acid
Acetic acid was used to induce intraperitonium in mice to
assess the writing response (Collier et al., 1968). Thirty male and
thirty female mice were randomly divided into 6 groups, as
mentioned in text 2.5. All the mice were administered orally once
daily for seven consecutive days. Thirty minutes after the final
drug administration, acetic acid (0.7%) was injected intraperitone-
ally (10 ml/kg) in all the mice. Intensity of writhing response of
each mouse was determined by cumulative counting of writhings
that occurred over a period of 15 min to calculate the analgesic
rate. Analgesic ratio was calculated using the following formula:
Analgesic ratio ð%Þ ¼ 1
 ðNumber of writhing response of each group
=Number of writhing response of control groupÞ  100%
2.7. The effect of SQ gout capsule on xylene-induced mice auricle
inflammation
Assessment of anti inflammatory effect was done by using
xylene induced ear edema in mice (Cao et al., 1992). In brief, 30
male mice and 30 female mice were randomly divided into
6 groups as above (2.5). All the mice were administered with the
respective drugs by gavage once daily for seven consecutive days.
Thirty minutes after the final administration, 0.2 ml of Xylene was
applied to the anterior and posterior surfaces of the right auricle of
each mouse. The left ear was considered as control. After 30 min of
xylene application, mice were sacrificed by cervical dislocation
and both left and right ears were removed. Circular sections were
excised from both ears by a cork borer (diameter 7 mm) and
swelling degree was assessed by the weight differences between
the left and right ear of the same mouse. The swelling inhibition
ratio was calculated using the formula:
Swelling inhibition ratio ð%Þ ¼ 1
 ðear swelling degree of each group
=ear swelling degree of control groupÞ  100%
2.8. The effect of SQ gout capsule on MSU crystal induced arthritis
in rats
Urate crystal was prepared according to the method of Coderre
and Wall (1987).Seventy male SD rats, weighing 180–220 g, were
1073
randomly divided into7 groups: control (0.5% CMC-Na), model
(0.5% CMC-Na), colchicine (0.15 mg/kg), Tong Feng Shu (300 mg/
kg), and SQ gout capsule high (225 mg/kg), medium (113 mg/kg)
and low dose (57 mg/kg) groups (n¼ 10).
All the animals were administered orally, once daily for 14
consecutive days. Thirty minutes after the final administration,
animals were injected intra-articularily under anesthesia with
0.1 ml of normal saline and 0.1 ml of MSU crystal suspension at
the medial side of the right ankle joint of the hind limb for control
group and all other groups respectively. Joints were observed to
ensure whether all the volume of drug had been administered
properly. All the procedures were conducted according to the
methods of Coderre and Wall (1987) with few modifications.
Swelling was measured by the volumetric method with the
immersion of the right ankle of each mouse in water up to
0.5 mm above the right ankle joint hourly for 5 h. Results were
calculated as changes from the baseline measurements by using
the below mentioned formula. Then the animals were sacrificed by
cervical dislocation, and their synovial membranes were isolated
and fixed in 10% formalin. Synovial specimens were embedded in
paraffin, sectioned, and examined via staining to assess the
histopathological changes of synovial tissue. Paw swelling ratio
and swelling inhibition ratio were calculated using the following
formula:
Paw swelling ratio ð%Þ ¼ ðPaw volume of each group
=Paw volume of each groupÞ  1  100%
Swelling inhibition rate ð%Þ ¼ 1  ðSwelling inhibition degree
each group=swelling inhibition degree of control groupÞ  100%
2.9. The effects of SQ gout capsule on secretion of TNF-α and IL-1β
in urate induced human umbilical vein endothelial cells (HUVECs)
2.9.1. Preparation of urate solution
Urate solution was prepared according to Inokuchi with a few
modifications (Inokuchi et al., 2008). In brief, after sterilization by
heating at 120 1C for 2 h, 5 mg of MSU was dissolved in 0.1 ml of
DMSO and then 50 ml of serum free sterile culture medium
(DMEM) (pH 7.2  7.4 Mol/L) was added. Then, the solution was
homogenized by using an ultrasonic homogenizer and finally,
obtained 100 mg/ml of uric acid solution.
2.9.2. Preparation of serum
Fourty two male Sprague Dawley (SD) rats were randomly
divided into 7 groups: control (normal saline), acute gout model
(100 μg/ml of urate solution), Indomethacin (4 mg/kg), Tong Feng
Shu (300 mg/kg), and SQ gout capsule, high (225 mg/kg), medium
(113 mg/kg) and low dose (57 mg/kg) groups (n ¼ 10). Drugs were
administered intragastrically daily once, for 1 week. Before the last
drug administration on the 7th day all the mice were fasted for
12 h. One hour after the last administration, blood samples from
the rats were obtained and germ-free serums were diluted with
the culture solution (DMEM) to get the 20% serum, (Perera et al.,
2012) under sterile condition to prepare the serums which con-
tained drug metabolites (Bochu et al., 2005; Qin et al., 2007).
2.9.3. In vitro study
HUVE cells were properly cultured in 96-well microplates,
divided into control group, model group, and Indomethacine
group, Tong Feng Shu group and SQ gout capsule in high, medium
and low dose group. DMEM (100 mg/ml) was added to the control
group and 100 mg/ ml of urate solution was added to all other
groups. Prepared serums were added to each corresponding well
1074 N. Darshika Kodithuwakku et al. / Journal of Ethnopharmacology 150 (2013) 1071–1079

simultaneously and all the groups were incubated at 37 1C in a 5% 3.2. The effect of SQ gout capsule on mice writhing response caused
CO2 humidified atmosphere for 12 h. by acetic acid

As shown in Fig. 2, compared with the control group, both the

2.9.4. Cell viability assay
Survival rate of cells in each group was analyzed by the
conventional MTT assay. After the 12th hour of the above men-
tioned (2.9.3) procedures of the cell culture study, 20 ml of MTT
solution (5 mg/ml) was added to the each well and cultured for
another 4 h. Then supernatant was collected and the formozone
crystals were dissolved in DMSO. Optical density was read at
570 nm absorbance using a microplate reader.
2.9.5. Assessment of excretion of TNF-α and IL-1β
HUVE cells were cultured in 96-wells microplates and grouped
as control group, model group, and Indomethacin group, Tong
Feng Shu group and SQ gout capsule in high, medium and low
dose group. Urate solution and corresponding drugs were incu-
bated and maintained all the conditions and time durations as
mentioned in 2.9.3. After the 12th hour of drug administration, as
mentioned above (2.9.3), supernatant was collected and centri-
fuged. The production of TNF-α and IL-1β were determined by
enzyme-linked immunosorbent assay (ELISA) to infer the degree
Indomethacin group and the group of SQ gout capsule at 450 mg/
kg (high dose) significantly decreased the cumulative number of
writhing caused by acetic acid (po 0.01), with the analgesic rates
of 42.61% and 36.97% respectively (Table 2). Tong Feng Shu group
also decreased the number of writhing times caused by acetic acid
(p o0.05), with the analgesic rate of 20.77% (Table 2).
3.3. The effect of SQ gout capsule on xylene-induced mice auricle
inflammation
As shown in Fig. 3, comparing with the control group, the
Indomethacin group, Tong Feng Shu group and the group of SQ gout
capsule at 450 mg/kg and 225 mg/kg dose significantly inhibited the
swelling of mice auricle caused by Xylene (po0.01), the inhibition
Table 1
The effect of SQ gout capsule on pain increase threshold ratio of mice tail flick
response caused by hot water.
Groups Dose(mg/kg) Pain threshold increase ratio (%)

of cell injury.
Control 00
Tong feng shu 600 27.07
SQ (H) 450 28.82
2.10. Statistical analysis SQ(M) 225 23.14
SQ(L) 113 14.01
All data were expressed as the mean7standard error (Mean7SE.)
Control group (Con), Tong feng shu (Grt), SQ gout capsule at high dose (450 mg/kg);
and statistical analysis was performed using one-way analysis SQ (H), SQ gout capsule at middle dose (225 mg/kg);SQ(M), SQ gout capsule at low
of variance (ANOVA) followed by post hoc analysis po0.01 and dose (113 mg/kg); SQ(L).].
po0.05 were considered statistically significant.

3. Results

3.1. The effect of SQ gout capsule on mice tail flick response caused
by hot water

As shown in Fig. 1, compared with the control group, the group

of SQ gout capsule at 450 mg/kg dose could significantly increase
the incubation period of tail flick response (p o0.01), with the pain
threshold increase rate was 28.82% (Table 1). Both Tong Feng Shu
group and the group of SQ gout capsule at 225 mg/kg dose also
increased the incubation period of tail flick response (po 0.05), the
pain threshold increased rates were 27.07% and 23.14% respec-
tively (Table 1). Fig. 2. The effect of SQ gout capsule on mice writhing response caused by acetic
acid (Mean 7SE n¼ 10), npo 0.05, nnp o 0.01 compared with Control group. [Con-
trol group (Con), Indomethacin group (Ind), Tong Feng Shu group (Grt), SQ gout
capsule at high dose (H) (450 mg/kg), SQ gout capsule at middle dose (M) (225 mg/
kg), SQ gout capsule at low dose (L) (113 mg/kg)].

Table 2
The effect of SQ gout capsule on mice writhing response caused by acetic acid
(n ¼10).

Groups Dose(mg/kg) Analgesic ratio (%)

Control
Indomethacin 8 42.61
Tong feng shu 600 20.77
SQ(H) 450 36.97
SQ (M) 225 15.14
Fig. 1. The effect of SQ gout capsule on mice tail-flick response caused by hot water SQ (L) 113 13.03
(Mean7 SE, n¼ 10), npo 0.05, nnp o 0.01 vs. Control group. Control group (Con),
Tong Feng Shu (Grt), SQ gout capsule at high dose (450 mg/kg) (H), SQ gout capsule SQ gout capsule at high dose: SQ(H), SQ gout at middle dose:SQ(M), SQ gout
at middle dose (225 mg/kg) (M), SQ gout capsule at low dose (113 mg/kg) (L). capsule at low dose:SQ (L).
 N. Darshika Kodithuwakku et al. / Journal of Ethnopharmacology 150 (2013) 1071–1079
Fig. 3. The effect of SQ gout capsule on Xylene-induced mice auricle inflammation
(Mean 7SE, n¼ 10), npo 0.05, nnp o0.01 compared with Control group. [Control
group (Con), Indomethacin group (Ind), Tong Feng Shu group (Grt), SQ gout
capsuleatat high dose (H) (450 mg/kg), SQ gout capsule at middle dose (M)
(225 mg/kg), SQ gout capsule at low dose (L) (113 mg/kg)].
Table 3
The effect of SQ gout capsule on Xylene-induced mice auricle arthritis.
Groups Dose(mg/kg) Inhibition ratio (%)
Control
Indomethacin 8 39.51
Tong feng shu 600 29.59
SQ(H) 450 39.89
SQ (M) 225 31.74
SQ (L) 113 19.57
SQ gout capsule at high dose: SQ(H), SQ gout capsule at middle dose:SQ(M), SQ
gout capsule at low dose:SQ (L).
ratios were 39.51%, 29.59%, 39.89% and 31.74% respectively (Table 3).
The group of SQ gout capsule at low dose (113 mg/kg) also inhibited
the swelling of mice auricle (po0.05) and its inhibition ratio was
19.57% (Table 3).
3.4. The effect of SQ gout capsule on arthritis caused by sodium
urate in rats
The anti-arthritic effect of the SQ gout capsule was assessed by
measuring the swelling of MSU induced right hind paw. Compared
to the control group, the paw swelling rate of the model group was
increased in an hour (po 0.05), and significantly increased within
2–4 h (p o0.01). Compared with the model group, colchicine
group significantly decreased the degree of inflammation in MSU
crystal induced rat paw swelling by the 5th hour (p o0.01). During
the 3rd to 5th hour period Tong Feng Shu and SQ gout capsule at
225 mg/kg and 113 mg/kg doses significantly decreased the
inflammation degree of rat paw swelling (po 0.01), while it was
less significant during the 1st and 2nd hour (p o0.05). SQ gout
capsule in the low dose group significantly decreased the inflam-
mation degree of rat paw swelling during 4th and 5th hour
(p o0.01) and less significant in the 3rd hour (po 0.05).
3.5. Effect of SQ gout capsule on MSU induced rat ankle joint lesion:
assessment through histopathological analysis of synovial
membranes (Fig. 5)
Histopathological assessment was done by evaluating inflam-
matory cell infiltration, synovial inflammatory features such as
edema, cell intact, the degree of the epithelial cell necrosis and
exudates in arthritic joints.
1075
Ankle Joints induced by MSU crystals in model group showed
acute inflammation in the synovial lining of the joint, as well as
extensive local infiltration of leucocytes into the joint cavities and
epithelial cell necrosis. In the control group, there were no visible
inflammatory features. In the colchicine group, ankle joint synovial
tissue was not intact, and synovial membrane epithelial cells are
necrosis, while fiber membrane was visible. There was no exudant in
intracavity but inflammatory cells infiltrate. Edema was reduced
significantly. At the high dose (225 mg/kg), the middle dose (113 mg/
kg) of SQ gout capsule group and in the Tong Feng Shu group,
synovial membrane epithelial cells were necrosis, while fiber mem-
brane was visible. There was no exudant in intracavity but inflam-
matory cells infiltrate, but at the high dose (225 mg/kg) synovial
tissue was not intact, at the middle dose (113 mg/kg)and in the Tong
Feng Shu group it was basically intact. Edema was also reduced
significantly in both groups.
At the low (57 mg/kg) of SQ gout capsule group, ankle joint
synovial tissue was basically intact, but synovial membrane
epithelial cells were necrosis, while fiber membrane was visible.
There was no exudant in intracavity but inflammatory cells
infiltrate with edema.
In brief, inflammatory features were substantially higher in the
model group. Even though there were synovial membrane epithe-
lial cell necrosis, significant diminution of inflammatory features
were displayed in the all treatment groups except for the low dose
(57 mg/kg) of the SQ gout capsule.
3.6. The effects of SQ gout capsule on emission of TNF-α and IL-1β in
human umbilical vein endothelial cells (HUVECs) stimulated by urate
3.6.1. Cell viability and MTT assay
The cell viabilities of HUVE cells were observed under an
inverted microscope (10  10) and through MTT assay. Compared
to model group, Indomethacin group and SQ gout capsule at high
dose (225 mg/kg) group improved cell viability. However, Tong
Feng Shu group, SQ gout capsule at middle (113 mg/kg) and low
dose (57 mg/kg) groups had no significance difference. It was
proved by the MTT assay.
In the MTT assay OD570 nm (Table 5) significantly declined in
sodium urate crystals induced HUVE cells. Compared to the normal
group, OD value was significantly different (po0.01, po0.05) in the
acute-gout model group, which meant that the model was successful.
Compared to the acute-gout model group, the value of OD570 nm of
the Indomethacin group, Tong Feng Shu group and SQ gout capsule
at high (225 mg/kg) and middle (113 mg/kg) dose group were
significantly increased (po0.01, po0.05 respectively). Results sug-
gest that, the reference herbal drug and the medial dose (113 mg/kg)
of the SQ gout capsule have equal effect on cell viability. High dose
(225 mg/kg) of the SQ gout capsule has better effect on cell toxicity
than the medium dose (113 mg/kg).
3.6.2. The effects of SQ gout capsule on survival rate of HUVECs
stimulated by urate (n ¼6)
Stimulation of HUVE cells by MSU markedly elevated the TNF-α
and IL-1β production. Compared to the model group, TNF-α and
IL-1β levels of the Indomethacin group, Tong Feng Shu group and
SQ gout capsule at high (225 mg/kg) and middle dose (113 mg/kg)
groups were decreased significantly (po 0.01) after 12 h of the
treatment. High dose (225 mg/kg) of the SQ gout capsule showed
approximately equal potential with Indomethacin, the most com-
mon non steroidal anti-inflammatory drug used in the treatment
of acute gout arthritis. While Tong Feng Shu, reference herbal drug
and the medium dose (113 mg/kg) of SQ gout capsule demon-
strated the equal effect which are less significant.
1076 N. Darshika Kodithuwakku et al. / Journal of Ethnopharmacology 150 (2013) 1071–1079
4. Discussion
Intense pain during the acute attack of gout is one of the most
irritating symptoms of gout arthritis. There are many methodol-
ogies that can be elicited the antinociceptive effect. In order to
assess the antinociceptive effect of the SQ gout capsule, we used
the tail-flick method as the thermal and the acetic acid induced
writhing as the chemical method, which are the most frequent
methods in antinociceptive assessment (Rose, 2008).
Antinociceptive effect can be caused due to centrally or
peripherally acting analgesic effect of the drug. Since the tail-
flick test evaluates the central acting analgesic effect (Richardson
and Vasko, 2002) and writhing test using acetic acid basically
evaluates the peripheral acting analgesic effect, (Deng et al., 2013)
we applied the both methods to verify not only antinociceptive
effect but also the mechanism of action of antinociceptive effect of
the SQ gout capsule. Recent studies have shown that a variety of
stimuli flow through different pathways that provide clues about
the mechanism of the nociceptive effect (Santos et al., 2005).
Tail-flick test assesses the efficacy of centrally acting analgesics
(Richardson and Vasko, 2002) and it is supposed to modulate
brainstem signals via the spinal reflex (Le Bars et al., 2001). In the
tail-flick test, vigorous removal of the tail can be observed by
application of radiating heat to the tail of the rat. This reaction
time is recorded and the extension of this time duration was
construed as an antinociceptive effect. In this study, when animals
were subjected to nocieceptive thermal stimuli, compared to the
normal group, intragastric administration of both high dose
(450 mg/kg) and medium dose (225 mg/kg) of the SQ gout capsule
and the positive herbal drug, Tong Feng Shu significantly increased
the duration of response. Low dose (113 mg/kg) of the SQ gout
capsule also increased the duration of response less significantly
(p o0.05), divulge that the SQ gout capsule has the centrally acting
analgesic effect in a dose dependent manner (Fig. 1 and Table 1).
These data are consistent with the study of M6G by Samantha and
Smith which demonstrated the nociceptive effect using the hot
plate and tail-flick methods (South and Smith, 1998) and the study
of Quinpirole by Roane et al. demonstrated the same effects
(Roane et al., 1998).
Though acetic acid induced writhing test is not specific, it is
known to be eliciting both central and peripheral analgesic effects.
Even though it is most commonly used to assess the peripheral
analgesic effect (Deng et al., 2013) and competent in acquiring
antinociceptive effects which are waning in other methods such
as the hot plate method (Bentley et al., 1981). The abdominal
responses triggered by intraperitoneally-induced acetic acid,
which provokes the cyclooxygenase, lipooxygenase products and
increase the excretion of other inflammatory mediators such as
bradykinin, prostaglandins and some cytokines such as IL-8, IL-1β,
and TNF-α. Residential peritoneal macrophages and mast cells are
mainly responsible for secretion of these cytokines (Bentley et al.,
1981; Vale et al., 2003).
In our study, both the high dose (450 mg/kg) of SQ gout capsule
and Indomethacin demonstrate the significant antinociceptive
effect in the writhing test (Fig. 2, Table 2), suggests that it may
suppress the intervention of chemosensitive nociceptors such as
vanilloid receptors that promote inflammatory pain via the central
nervous system or it may inhibit the peripheral receptors (Ikeda
et al., 2001; Vale et al., 2003). These results were equivalent to
effect of Acetyle salisilic acid and tramadole (Perez et al., 2012).
However, further investigation is needed to elucidate the actual
magnitude of the antinociceptive activity of the SQ gout capsule to
interpret the possible mechanism of the drug.
Anti-inflammatory effect of SQ gout capsule was evaluated
using different inflammatory models such as xylene induced ear
edema model and MSU induced models. We implemented MSU
induced model to verify the anti-inflammatory effect against the
gout arthritis.
Xylene induced ear edema is one of the most frequently used
model for inflammatory effect to determine the anti-inflammatory
effect and provide favorable evidences in the screening of anti-
inflammatory agents (Richardson and Vasko, 2002). Therefore, we
applied this methodology to evaluate the anti-inflammaotry effect
of SQ gout capsule.
MSU induction through intra-articular injection in animal
models demonstrated equivalent symptoms as those found in
clinical gout, suggesting that, this model has good predicting
values for clinical efficacy of gout arthritis at the research level
(Getting et al., 2002). Therefore, based on the methods of Coderre
we verified the anti-inflammatory effect of SQ gout capsule using
MSU induced gout models since we were expecting to find out the
efficacy of SQ gout capsule on gout arthritis. We employed the
HUVEC cells to investigate the anti-inflammatory mechanism
of SQ gout capsule through the expression of proinflammatory
cytokines.
Therefore, herein we discuss the underlying mechanism of
anti-inflammatory effect of the SQ gout capsule using xylene
induced ear edema model and MSU induced gout arthritis model.
When xylene is applied on ear to get the ear edema model,
xylene agitates the sensory neurons that act on immune cells
which express the pro-inflammatory mediators which partially
associate with substance P and produce neurogenic inflammation
which appears with edema, erythmea etc. (Richardson and Vasko,
2002). Substance P is widely spread in the nervous system and
functions as a neurotransmitter or neuro-modulator. However,
when stimulated peripherally, it is supposed to be responsible for
neurogenic inflammation. Conversely, xylene induced inflamma-
tory reaction is followed by innate immunity reaction of the skin, a
cytotoxicity reaction of activated T cells and then leukocytes
infiltration which results in edema, thickening and heaviness of
the ear (Saint-Mezard et al., 2004).
Taken together, our results showed obvious suppression of the
swelling (Fig. 3) implying the antiphlogistic effect of SQ gout
capsule (Parveen et al., 2007). We can also suggest that the SQ
gout capsule may have anti-inflammatory effect of the neurogenic
inflammation. Generally, Indomethacin is a peripherally acting
drug that inhibits only the late phase of the norciceptive effect and
this late phase is related to inflammatory pain proving its anti-
inflammatory effect. In this study high dose (450 mg/kg) of the SQ
gout capsule is showing the equivalent effect as Indomethacine
signifying the anti-inflammatory effect of SQ gout capsule (Fig. 3
and Table 3) These results are equivalent to studies demonstrating
the anti-inflammatory effect of extractions of Traditional Chinese
medicinal plant (Parveen et al., 2007) and Tibetan folk medicinal
plants (Zhang et al., 2008).
As mentioned above to evaluate anti-inflammatory effect of SQ
gout capsule on gout arthritis we used the MSU crystals induced
rat's ankle joint and then evaluated the amelioration of edema and
the histopathological changes of the synoviyal membrane of the
ankle joint. Inflammatory mechanism of induced phlogistic agents
are considered to be biphasic. During the first 1–2 h the inflam-
matory reaction is stimulated by histamine or serotonin, and then
in the 3rd hour prostaglandins and lysosome reactions take place
to form the edema (Franzotti et al., 2000). In addition, pro-
inflammatory cytokines such as TNF-α and IL-1 are released with
the stimulation of resident macrophage after 1 h of the induction
of inflammatory stimuli to the joint cavity which triggers the
inflammatory reaction. During the first hour synovial neutrophil
and other leukocytes infiltrate to the site of stimuli and then in the
2nd hour they are gradually attenuated (Cremasco et al., 2008).
According to our data, from the 2nd–5th hour swelling in the
model group was significantly increased. From the 3rd hour
 N. Darshika Kodithuwakku et al. / Journal of Ethnopharmacology 150 (2013) 1071–1079 1077

onwards, the high dose (225 mg/kg) and the middle dose (113 mg/
kg) of SQ gout capsule significantly reduced the edema suggesting
that SQ gout capsule is dose dependently (Fig. 4 and Table 4)
showing anti-inflammatory effect against prostaglandin and lyso-
somal reactions. Even though it initiates to react against the
histamine and serotonin effects, compared to 3rd hour, initiation
of anti-inflammatory effect at the 2nd hour was less significant.
Usually opioids are centrally acting drugs that are acting on both
phases, but the drugs that are acting peripherally such as indo-
metacin, aspirin, and dexamethasone only attenuate the late phase
Fig. 4. The effect of SQ gout capsule on arthritis caused by sodium urate. Swelling
degree of rat paw up to the ankle joint. (Mean7 SE, n ¼10) np o0.05, nnp o 0.01-
compared to Model group.[Control group (Con), Model group (Mod), Colchicine
(Col), Tong Feng Shu group (Grt), SQ gout capsule at high dose (225 mg/kg)
(SQGC-H), SQ gout capsule at middle dose (113 mg/kg) (SQGC-M), SQ gout capsule
at low dose (57 mg/kg) (SQGC-L)].
Table 4
The effect of SQ gout capsule on arthritis caused by sodium urate in rats.
(Deng et al., 2013). However, our data imply that the SQ gout
capsule may have a considerable effect in the both phases.
Histopthological analysis and the MSU induced HUVE cells also
provide the proof for the anti-inflammatory effects and mechan-
ism of action of the SQ gout capsule. (Fig. 5 and Table 5). Compared
to model group, synovial specimens of the SQ gout capsule group
showed marked reduction in edema, less leukocyte infiltration and
intact tissues acquaint the anti-inflammatory effect of SQ gout
capsule (Fig. 5). All these facts disclose that SQ gout capsule is
reducing the leukocyte infiltration. Consequently we hypothesized
that SQ gout capsule may perform its action by inhibiting the
proinflammatory cytokines.
Recently both experimental and clinical studies revealed that
IL-1β plays a major role in gout. Furthermore, TNF-α plays a critical
role in the development of gout arthritis (di Giovine et al., 1991).
IL-1β, stimulates the leukocyte infiltration and can activate every
cell type such as endothelial cells, leukocytes, fibroblasts etc.
TNF-α also stimulates the leukocyte, endothelial cell adhesion
and reacts in the acute inflammatory process. Precisely, both
IL-1β and TNF-α play a critical role in developing acute inflamma-
tory features of gout flares (Punzi et al., 2012). Considering the
results that we obtained and evidence of functions of these pro-
inflammatory cytokines, we designed our study to discover the
possible inflammatory mechanism of the SQ gout capsule via
evaluating the inhibition of TNF-α and IL-1β secretion in urate
induced HUVE cells.
According to the studies of Haskard and Landis (2002) and as
discussed in the studies of Yagnik et al. (2004) MSU induced
endothelial cells could activate the endothelial cell adhesion
Table 5
The effects of prepared serum on survival rate of HUVECs stimulated by urate
(Mean7 SD, n¼ 6)
Groups Serum concentration (%) OD570 nm Cell viability (%)

Groups Dose (mg/kg) Swelling inhibition ratio (%) Control 20 0.63 7 0.04 100
Model 20 0.20 7 0.04 31.7
1h 2h 3h 4h 5h Indomethacin 20 0.42 7 0.05 67.1
Tong feng shu 20 0.30 7 0.05 48.1
Colchicine 0.15 20.66 30.74 42.87 46.24 45.68 SQ (H) 20 0.36 7 0.04 57.5
Tong Feng shu 300 12.11 18.22 38.88 39.47 35.82 SQ (M) 20 0.30 7 0.04 48.0
SQ(H) 225 18.44 18.07 41.62 40.59 39.15 SQ (L) 20 0.217 0.03 32.9
SQ (M) 113 16.62 18.23 38.86 32.76 32.32
SQ (L) 57 9.66 3.47 22.86 26.64 25.28 Control (normal saline), acute gout model (100 μg/ml of urate solution), Indo-
methacin (4 mg/kg), Tong Feng Shu (300 mg/kg), and SQ gout capsule, high
Swelling inhibition ratio of the MSU induced gout arthritis in rats. SQ gout capsule (225 mg/kg) (SQ-H), medium (113 mg/kg) (SQ-M)and low dose (57 mg/kg)
at high dose: SQ(H), SQ gout capsule at middle dose:SQ(M), SQ gout capsule at low (SQ –L) were used to prepare the relevant serum. Yi Shen Juan Bi
dose:SQ (L).

Fig. 5. Effect of SQ gout capsule on MSU induced rat ankle joint lesion. [Control group (Con), Model group (Mod), Colchicine group (Col), Tong Feng Shu group (Grt), SQ gout
capsule at high dose (225 mg/kg) (SQ-H), SQ gout capsule at middle dose (113 mg/kg) (SQ-M), SQ gout capsule at low dose (57 mg/kg) (SQ-L)].
1078 N. Darshika Kodithuwakku et al. / Journal of Ethnopharmacology 150 (2013) 1071–1079
Fig. 6. The effects of SQ gout capsule on secretion of TNF-α and IL-1β in HUVECs
stimulated by urate (Mean 7 SE, n¼ 6), npo 0.05, nnpo 0.01 compared with Model
group, #p o0.05, ##p o0.01 compared with Control group. [Control group (Con),
Model group (Mod), Indomethacin group (Ind), Gout relaxing tablet group (Grt), SQ
at high dose (225 mg/kg) (SQ-H), SQ at middle dose (113 mg/kg) (SQ-M), SQ at low
dose (57 mg/kg) (SQ-L)].
molecules which can be activated the acute inflammatory path-
ways and activate these proinflammatory cytokines including the
IL-1β and TNF-α. Therefore, as demonstrated by Chapman et al.
(1997), HUVE cells could show the significant evidence in expres-
sion of IL-1β and TNF-α with the stimulation of MSU solution.
Thus, to demonstrate the suppression of IL-1β and TNF-α by SQ
gout capsule, MSU induced HUVE cells could provide sufficient
competence.
In this study we used some methodologies in serum pharma-
cology, since it is supposed to be more scientific and more
appropriate in traditional medicine prescriptions than conven-
tional pharmacology in which crude powdered drug is directly
applied in the cell culture studies (Bochu et al., 2005). Based on the
crude powdered drugs of the SQ gout capsule, we prepared serum
from the rats that were treated with the corresponding drugs and
incubated to the urate induced HUVE cell culture preparations.
Stimulation of cells by urate exacerbate the pro-inflammatory
cytokines, especially TNF-α and IL-1β and causes cellular damage.
MTT assay and the cell viability observed through microscope
demonstrated that SQ gout capsule has protective effect against
the urate solution induced cellular damage (Table 5). Results of the
ELISA for TNF-α and IL-1β also revealed that SQ gout capsule
suppressed the production of TNF-α and IL-1β. It is disclosed that
the SQ gout capsule may demonstrate its anti-inflammatory
mechanism via inhibiting the activation of TNF-α and IL-1β
(Fig. 6). Even though the type of cells that are applied for the
evaluation is different, Scopoletin, (Yao et al., 2012), inducible
nitric oxide synthase (Ju et al., 2011), Withania A (Sabina et al.,
2008) are also showing the similar effects.
However, we have suggested many possible mechanisms of
antinociceptive and anti-inflammatory effect of our SQ gout
capsule, further investigations are essential to confirm these
possible mechanisms. At present we can confirm that, SQ gout
capsule can suppress inflammatory effect via inhibiting the secre-
tion of TNF-α and IL-1β.
Conversely, though there are remarkable anti-inflammatory
and antinociceptive drugs existing, such as NSAIDs, side effects
are more common among those drugs (Wen et al., 2005). On the
other hand, it would be very interesting that one drug has both of
these effects and the same drug could ameliorate the MSU induced
gout arthritis. Considering the data obtained in our study SQ gout
capsule may occupy that position in the management of MSU
induced inflammation and pain.
5. Conclusion
SQ gout capsule can effectively inhibit the inflammation,
nociceptive effect, through the modulation of expression of pro-
inflammatory cytokines, TNF-α and IL-1β and the curative effect is
dose dependent. Considering all the results of the study it can be
concluded that high dose (450 mg/kg in mice) of the SQ gout
capsule has a better therapeutic effect while lower dose(113 mg/kg
in mice) has lesser effect. SQ gout capsule could attenuate the
inflammatory reaction and the nociceptive effect of MSU induced
models. Consequently, these MSU induced in vivo and in vitro
studies of SQ gout capsule suggest that, SQ gout capsule may be a
potential agent for treatment in gouty arthritis.
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