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R E S E A R C H A R T I C L E Open Access
Anti-Hyperuricemic, Anti-Inflammatory and
Analgesic Effects of Siegesbeckia orientalis L.
Resulting from the Fraction with High
Phenolic Content
Thuy Duong Nguyen
1
, Phuong Thien Thuong
2
, In Hyun Hwang
3
, Thi Kim Huyen Hoang
1
, Minh Khoi Nguyen
2
,
Hoang Anh Nguyen
1*
and MinKyun Na
4*
Abstract
Background: The medicinal plant Siegesbeckia orientalis L. has been commonly used for the treatment of acute
arthritis, rheumatism, and gout in Vietnam. However, pharmacological research of this plant associated with gout
has not been reported. Anti-hyperuricemic and anti-inflammatory effects were evaluated and observed for the
crude ethanol extract (CEE) of S. orientalis. Retention of these biological properties was found in a n-butanol-soluble
fraction (BuOH fr.) of the extract, and therefore further biological and chemical investigations were undertaken on
the BuOH fr. to support the medical relevance of this plant.
Methods: The aerial part of S. orientalis was obtained in the mountainous region of Vietnam. The crude ethanol
extract (CEE) and its BuOH fr. were prepared from the plant materials. Anti-hyperuricemic activities of the CEE and
BuOH fr. were tested in vivo using the model oxonate-induced hyperuricemia rats through determination of serum
uric acid levels and inhibitory effects on xanthine oxidase (XO) in the rat liver. Anti-inflammatory activities of the
BuOH fr. were also evaluated in vivo using carrageenan-induced paw edema and urate-induced synovitis in rats.
Active components of the BuOH fr. were characterized by comparison of HPLC retention time (t
R
) and
spectroscopic data (UV,
1
H–NMR) with those of reference compounds.
Results: The CEE of S. orientalis displayed anti-hyperuricemic activity, and the BuOH fr. was found to be the most
active portion of the extract. Further in vivo studies on this fraction showed 31.4% decrease of serum uric acid
levels, 32.7% inhibition of xanthine oxidase (XO), 30.4% reduction of paw edema volume, symptomatic relief in
urate-induced synovitis and significant analgesic effect at the dose of 120 mg/kg, as compared to the
corresponding values of the control groups. Chemical analysis of the BuOH fr. revealed high phenolic content,
identified as caffeic acid analogues and flavonones.
Conclusions: This study suggested that anti-hyperuricemic and anti-inflammatory mechanism of S. orientalis is
related to XO inhibitory effect of the phenolic components. Our findings support the use of this plant as the
treatment of gout and other inflammatory diseases.
Keywords: Siegesbeckia orientalis, Anti-hyperuricemic activity, Anti-inflammatory activity, Analgesic activity, Xanthine
oxidase, Caffeic acid analogues, Flavonones
* Correspondence: anh90tk@yahoo.com;mkna@cnu.ac.kr
1
Department of Pharmacology, Hanoi University of Pharmacy, 13 Le Thanh
Tong, Hoan Kiem, Hanoi, Vietnam
4
College of Pharmacy, Chungnam National University, Yuseong-gu, Daejeon
34134, Republic of Korea
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Nguyen et al. BMC Complementary and Alternative Medicine (2017) 17:191
DOI 10.1186/s12906-017-1698-z
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
The plant Siegesbeckia orientalis L. (syn. S. glutinosa), a
member of Asteraceae, is widely distributed in Vietnam
and other South-East Asian countries [1, 2]. The aerial
part of this plant has been used for treating various in-
flammatory diseases, such as neurasthenia, insomnia, im-
petigo, furuncle, wound, and burn [2]. Being known as a
Vietnamese indigenous medicine “hy-thiem”,thisplant
has been applied in a series of traditional remedies for the
treatment of acute arthritis, rheumatism, inflammation,
and especially gout and pain [1, 3, 4]. Although biological
potential of S. orientalis has been previously investigated,
the interest was limited to anti-inflammatory and anal-
gesic activities of either whole herbal extract [5] or kirenol
[2], which is an ent-pimarane type diterpene identified
from this plant and commonly encountered from the
same genus Aster [6–8]. Furthermore, a recent in vitro
and in vivo study on the anti-inflammatory mechanism of
S. orientalis demonstrated that its ethanol extract sup-
presses mitogen-activated protein kinases (MAPKs)- and
NF-κB-dependent pathways [9]. Given that inflammatory
response is a key step in the onset of gout symptoms [10],
anti-inflammatory effects were thought to be responsible
for traditional utilization of S. orientalis as a part of symp-
tomatic treatment of this disorder.
Xanthine oxidase is an enzyme converting xanthine and
hypoxanthine into uric acid. A high level of serum uric
acid (hyperuricemia) is a well-known major cause of gout,
and this metabolic syndrome is closely related to inflam-
matory responses [10]. Deposition of monosodium urate
crystals in a joint could lead to an acute inflammatory
pain. Phytochemical studies of S. orientalis identified vari-
ous secondary metabolites, which include sesquiterpe-
noids[11,12],diterpenoids[6–8, 13], and caffeic acid and
rutin [14]. It is notable that in vitro xanthine oxidase (XO)
inhibitory activities of caffeic acid and its analogues were
reported previously [14–16], while rutin exhibited the
anti-hyperuricemic effect in mice mediated by XO inhib-
ition in vivo, but not in vitro [17, 18]. Our preliminary
screening also confirmed that the ethanol extract of S.
orientalis was a potent inhibitor of XO among more than
300 Vietnamese medicinal plants. Therefore, it was sup-
posed that S. orientalis could have dual role in treatment
of gout which related to both hypouricemic and anti-
inflammatory activity. Based on a literature search, kirenol
was suggested to be the main active compound which was
responsible for the anti-inflammatory activity of S.
orientalis [2]. To our knowledge, this compound, however,
has not been observed for biological activities with regard
to XO inhibition. Important active constituents involved
in XO inhibition activity of S. orientalis therefore remain
to be determined.
The present study evaluates anti-hyperuricemic and
anti-inflammatory effects of S. orientalis extract using
well-established animal models. Taking into consider-
ation both anti-inflammatory and XO inhibitory effects,
we focused on flavonoids and other phenolic compounds
which are extensively studied and well-known antioxi-
dants as potential phytochemical agents for treating
diseases mediated by free radicals, including inflamma-
tion and gout [19, 20].
Methods
Chemicals and reagents
All the chemicals and reagents used for in vivo tests
were of biological grade purchased from Sigma Aldrich
(St Louis, MO, USA): xanthine 99–100% (Cat.
XO626-25G; Lot#/Batch# 097 K5307), carrageenan
(C1013-100G; Pcode 100,160,665); uric acid (> = 99%,
crystalline, U2625); oxonic acid potassium salt (97%;
156,124-100G); xanthine oxidase, from bovine milk
(X1875-50UN; 1,000,877,910). Solvents for extraction
and fractionation were of industrial grade purchased
from a licensed chemical company in Hanoi, Vietnam,
and used without purification.
Plant material
The aerial parts of Siegesbeckia orientalis L. (Asteraceae)
were collected in the mountainous region of Hoa Binh
province, in the North of Vietnam. The plant was au-
thenticated by Prof. Tran Van On, Department of Bot-
any, Hanoi University of Pharmacy. A voucher specimen
(VDL-HT01) has been deposited at the Herbarium of
the Department of Pharmaceutical Analysis and
Standardization, Vietnam National Institute of Medicinal
Materials, Hanoi, Vietnam.
Preparation of extraction and fractionation
The dried plant material (5 kg) was extracted three times
with 96% ethanol (EtOH) at room temperature for 9
days. The solution was filtered and combined, and the
organic solvent was removed under reduced pressure to
give a crude ethanol extract (CEE, 180 g). A part of the
CEE (150 g) was suspended in water (1 L) and then suc-
cessively partitioned with n-hexane (Hex), ethyl acetate
(EtOAc), and n-butanol (BuOH) (each 1 L). The organic
solvents and water were evaporated to yield Hex- (44 g),
EtOAc- (33 g), and BuOH-soluble (30 g) fractions, and
the remaining water soluble fraction (31 g).
Animals
The adult male Wistar rats (8 weeks old, weighing
140 ± 10 g) were obtained from Animal Facilities,
Research Center for Medicine and Pharmacy, Vietnam
Military Medical University (Hanoi, Vietnam). Animals
were used and processed according to the suggested eth-
ical guidelines for the care of laboratory animals [21],
and the experimental protocols in this study were
Nguyen et al. BMC Complementary and Alternative Medicine (2017) 17:191 Page 2 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
approved by the Scientific and Ethical Committee of
Hanoi University of Pharmacy (156/DHN-QD). The rats
were acclimatized at least 7 days to adapt to their envir-
onment before any experimental manipulation. They
were housed in 612 × 345 × 216 mm cages (Tecniplast
2000P) in an animal room (Department of Pharmacol-
ogy, Hanoi University of Pharmacy) with a temperature
of 24–26 °C, humidity of 55–60%, regular 12/12 h light/
dark cycle (7:00 a.m. –7:00 p.m.), and access to standard
laboratory diet and tap water freely until used for experi-
ments. General health status of the rats was monitored
on alternate days, and no adverse events were recorded
during the housing period. At the beginning of each ex-
periment, body weight of the animals ranged from 180
to 220 g. All the samples from animals subjected to the
treatments were included in the data analysis.
Evaluation of anti-hyperuricemic effect
Drug administration
Each test sample of CEE and BuOH fraction was
suspended in 0.5% sodium carboxymethylcellulose
(CMC-Na). Food, but not water, was withdrawn from
the animals 2 h prior to drug administration. Based on
the Vietnamese traditional usage of “hy-thiem”,ratswere
given the CEE at 300, 600, 1200 mg/kg and the BuOH
fraction at 120 mg/kg once a day for five consecutive days.
Rats in the negative control group were orally adminis-
trated with 0.5% CMC-Na only, while those of the positive
control group were given allopurinol at 10 mg/kg.
Animal hyperuricemia model
Hyperuricemia of rats was induced by potassium
oxonate, an uricase inhibitor [22]. In brief, potassium
oxonate was suspended in 0.5% CMC-Na. One hour be-
fore administration of the test samples, rats were intra-
peritoneally injected with the freshly prepared potassium
oxonate suspension at the dose of 250 mg/kg to increase
their serum uric acid levels. Whole blood samples were
collected from the tail vein of the rats 1 h after the final
administration of tested compounds. Blood was allowed
to clot for approximately 1 h at room temperature and
then centrifuged at 3500 rpm for 5 min to obtain the
serum, which was stored at −20 °C until used.
Determination of blood uric acid levels
Serum uric acid levels were determined by the phospho-
tungstic acid method [23].
Enzyme preparation from rat liver
Rat liver was rapidly excised and homogenized in an ice-
cold 50 mM potassium phosphate buffer (pH 8.0). The
homogenate was centrifuged at 3000×gfor 10 min at 4 °C,
and then lipid layer was carefully removed. The resulting
supernatant was further centrifuged at 10000×gfor
60 min at 4 °C. The supernatant obtained from the final
centrifugation was used to detect XO activity.
Assay for XO inhibition in rat liver
Enzyme activity of XO was assayed by monitoring uric
acid formation using the spectrophotometric method as
described elsewhere [24]. Briefly, a reaction was started by
adding 100 μL of the supernatant to a phosphate buffer
solution (pH 7.5) containing 0.12 mM xanthine and
0.192 mM EDTA. The mixture (total 5.0 mL) was incu-
bated for 30 min at 37 °C, and the reaction was terminated
by the addition of 1 M HCl (0.5 mL). The production of
uric acid was determined by measuring UV absorbance at
290 nm. The XO activities were expressed as mmol of
produced uric acid per minute per gram protein. Protein
concentration was determined by the Lowry method [25]
using bovine serum albumin as a standard.
Evaluation of anti-inflammatory effect
Drug administration
Rats were randomly divided into 3 groups (n= 8 for
each): negative control, positive control, and test sample
(BuOH fraction). Indomethacin, a nonsteroidal anti-
inflammatory drug, was used as a reference compound
for the positive control group. Each sample of BuOH
fraction and indomethacin was dissolved/suspended in
0.5% CMC-Na, and orally given to rats at the dose of
120 mg/kg and 10 mg/kg body weight, respectively. The
rats in the negative control group were orally adminis-
trated with 0.5% CMC-Na only. Volume of administra-
tion was identical for all rats in three groups.
Carrageenan-induced rat paw edema
Paw edema test was performed according to the previ-
ously described carrageenan-induced method [26] with
some modification. Briefly, 1 h after the drug administra-
tion, paw edema was induced by injecting 0.1 mL of 1%
(w/v) carrageenan in buffer saline into the plantar sur-
face of the right hind paw in all rats. The paw was
marked in order to immerse it to the same extent in the
measurement chamber. Volume of the rat paws was
measured using a plethysmometer (Ugo Bisile, Italy) im-
mediately before the carrageenan subplantar injection
and at intervals of 1, 3, and 5 h. The assessment of paw
volume was always performed in double blind and by
the same operator. Indomethacin was administered p.o.
as a reference drug. The increased percentage of edema
was calculated as follows:
Increased edema %ðÞ¼
Volume at the end of 3hmLðÞ
Basal paw volume mLðÞ 100
The percentage inhibition of edema for each group
was calculated by the following formula
Nguyen et al. BMC Complementary and Alternative Medicine (2017) 17:191 Page 3 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
P%Inhibition of edemaðÞ¼
Ec−Et
Ec 100
Ec = % Edema of the negative control group, and Et = %
Edema of the treatment group.
Carrageenan-induced mechanical hyperalgesia
Mechanical hyperalgesia was examined in an inflamma-
tory pain rat model by measuring the withdrawal thresh-
olds of hind paw to an increasing pressure stimulus,
using an analgesymeter (model 37,215; Ugo Basile, Italy).
Paw withdrawal thresholds were measured in naive ani-
mals prior to the intraplantar injection of carrageenan
into the hind paw. The cut-off was set at 250 g and the
endpoint was taken as paw withdrawal or vocalization.
Withdrawal thresholds were measured before (predose)
and up to 3 h after drug or vehicle administration (post-
dose). In all cases, testing was done in blind. Reversal of
mechanical hyperalgesia was calculated according to the
following formula [27]:
%Reversal ¼Postdose threshold−Predose threshold
Naive threshold−Predose threshold 100
Carrageenan-induced thermal hyperalgesia
Carrageenan-induced thermal hyperalgesia was assessed
in separate groups of animals. The measurement of the
nociceptive response to a thermal stimulus used a hot-
plate test with the temperature adjusted to 51 ± 1 °C
[28]. The withdrawal latencies of the hind paw were
measured. Hyperalgesia to heat was defined as a de-
crease in the Δlatency (sec), calculated by the difference
of latency times between the carrageenan- and non-
injected paw (naive). BuOH fraction was administered
orally at the dose of 120 mg/kg one hour before the
carrageenan injection, and the positive control group
received indomethacin (10 mg/kg, p.o.).
Urate-induced synovitis
One hour after the drug administration, rats were anesthe-
tized and injected with 100 μL of sodium urate crystals
into one knee. The animals were allowed to walk on a
metal grid after 18–24 h. A scoring system is adopted in
which inflammatory symptoms ranging from tenderness,
limping, occasional 3-legged gait to complete 3-legged gait
are scored from 1+ to 4+ [29]. BuOH fraction was admin-
istered orally at the dose of 120 mg/kg 1 h before and
20 h after the urate injection, and the positive control
group received indomethacin (10 mg/kg, p.o.).
Statistical data analysis
Data are expressed in mean ± standard error of the
mean (SEM) from eight animals per group. Statistical
analysis was performed by one-way analysis of variance
(ANOVA). Dunnett’s multiple range test was performed
to determine significant differences among means.
Kruskal-Wallis test was used to analyze non-parametric
data, followed by Mann- Whitney U test if applicable.
The values of p< 0.05 were considered to be statistically
significant.
Determination of total phenolic content
Total phenolic content was measured using the
Folin-Ciocalteau method as described previously [25].
Characterization of phenolic compounds
Phenolic compounds in the BuOH fraction were analyzed
by a Shimadzu HPLC system equipped with a DAD de-
tector and C
18
column (250 × 4.6 mm; 5 μm), using a gra-
dient of MeOH (solvent A) and 0.1% phosphoric acid in
water (solvent B) as mobile solvents. The following HPLC
method was used: flow rate (0.6 mL/min); 0–5 min (10%
A); 5–7 min (10–25% A); 7–20 min (25% A); 20–40 min
(25–50% A); 40–55 min (50% A); 55–63 min (50–80% A);
63–68 min (80–100% A). The compounds isolated were
identified by comparison of their retention time (t
R
)and
spectroscopic data (UV and
1
H- and
13
C–NMR) with
those of reference compounds [25].
Results
Effect of CEE on serum uric acid levels in rats
The CEE at 300, 600, and 1200 mg/kg were orally ad-
ministered for 5 days on oxonate-induced hyperuricemia
rats, and serum uric acid levels were measured by the
phosphotungstic acid method. As presented in Table 1,
compared with the uric acid value of the normal rats
(116.17 μmol/L), the value of the control rats
(218.66 μmol/L) was significantly increased by the injec-
tion of potassium oxonate (p< 0.01). Administration of
the CEE enabled lowering the uric acid levels of hyper-
uricemic rats to normal ranges at 600 and 1200 mg/kg
(p< 0.05), while no effect was observed at dose of
300 mg/kg. The positive control, allopurinol at dose of
10 mg/kg, displayed more extensive hypouricemic activ-
ity, which significantly reduced the serum uric acid level
to 101.69 μmol/L (p< 0.01).
Effect of CEE and BuOH fraction on serum uric acid levels
in rats
A part of CEE (150 g) was fractionated, and obtained
30 g of the BuOH-soluble portion. The CEE (600 mg/kg)
and BuOH fraction (120 mg/kg) were further tested for
hypouricemic activity in an animal model, and were orally
administered for 5 days on oxonate-induced hyperurice-
mia rats, and the serum uric acid levels were measured by
the phosphotungstic acid method. As presented in Table
2, the CEE and BuOH fr. of S. orientalis reduced uric acid
levels. Of the samples tested, the BuOH fr. exhibited
Nguyen et al. BMC Complementary and Alternative Medicine (2017) 17:191 Page 4 of 9
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notable hypouricemic activity by lowering the serum uric
acid level by 31.4% at dose of 120 mg/kg (p<0.01).
Effect of BuOH fraction on XO activity in rats
Detailed investigation for hypouricemic activity of the
BuOH fraction was subsequently carried out with
in vivo test for liver XO activity in rats. As shown in Fig.
1, the values observed for XO activity in the control and
BuOH fr. (120 mg/kg p.o.) groups were 2.08 ± 0.20 and
1.40 ± 0.14 mmol/min per mg protein, respectively. It
means that an in vivo inhibition of XO by 32.7% was ob-
served for the BuOH fr. as compared to the control
group (p< 0.05). In the same experiment, allopurinol
inhibited XO activity (41.8% inhibition) at the dose of
10 mg/kg, showing slightly more potent activity than the
BuOH fr.
Effect of BuOH fraction on paw edema in rats
Anti-inflammatory activity of the BuOH fraction was
evaluated using a carrageenan-induced paw edema
model. Intraplantar injection of carrageenan (1% w/v)
markedly increased paw volume of the rats, reaching
its maximal effect after 3 h (Fig. 2). Treatment with
the BuOH fr. (120 mg/kg) reduced the volume of
paw edema by 30.4% (p< 0.05), as compared to the
maximum volume measured before the treatment.
Indomethacin (10 mg/kg) served as a positive control,
displaying inhibitory activity by reducing 44.6% paw
edema.
Table 1 Inhibitory effect of crude ethanol extract (CEE) from S.
orientalis on serum uric acid levels in rats
Group treatment Dose
(mg/kg)
Number Serum uric acid
levels (μmol/L)
Inhibition
(%)
Normal 116.17 ± 8.71
Control 8 218.66 ± 16.82
##
CEE 300 8 174.49 ± 12.31 20.2
600 8 159.16 ± 16.36* 27.2
1200 8 158.18 ± 14.07* 27.7
Allopurinol 10 8 101.69 ± 15.98** 53.5
Hyperuricemic rats were induced by a potassium oxonate injection 1 h before
the last drug administration. The CEE at 300, 600 and 1200 mg/kg and
allopurinol at 10 mg/kg were orally administrated once a day for five
consecutive days. The control and normal groups were orally administered
with 0.5% CMC-Na. The serum uric acid levels of rats were measured by the
phosphotungstic acid method. Values are displayed as mean ± SEM
*p< 0.05, **p< 0.01 vs. hyperuricemic rats (control);
#p< 0.05, ##p< 0.01 vs. normal rats
Table 2 Inhibitory effect of crude ethanol extract (CEE) and
BuOH fraction (BuOH fr.) from S. orientalis on serum uric acid
levels in rats
Group treatment Number Dose
(mg/kg)
Serum uric acid
levels (μmol/L)
Inhibition
(%)
Normal 8 114.54 ± 11.23
Control 8 222.38 ± 24.58
##
CEE 8 600 163.58 ± 12.94* 26.4
BuOH fr. 8 120 152.47 ± 11.68** 31.4
Allopurinol 8 10 115.28 ± 11.13** 48.2
Hyperuricemic rats were induced by a potassium oxonate injection 1 h before
the last drug administration. The CEE at 600 mg/kg, the BuOH fr. at 120 mg
and allopurinol at 10 mg/kg were orally administrated once a day for five
consecutive days. The control and normal groups were orally administered
with 0.5% CMC-Na. The serum uric acid levels of rats were measured by the
phosphotungstic acid method. Values are displayed as mean ± SEM
*p< 0.05, **p< 0.01 vs. hyperuricemic rats (control);
#p< 0.05, ##p< 0.01 vs. normal rats
Fig. 1 Xanthine oxidase (XO) enzyme from rat liver was treated with
the BuOH fraction (BuOH fr.). The activity of XO inhibition was
evaluated by monitoring uric acid formation using the
spectrophotometric method. Allopurinol was used as a reference
agent. Values are presented as mmol/min per g protein and
mean ± SEM from 8 animals in the treatment group. * p< 0.05,
**p< 0.01 vs. control
100.0
110.0
120.0
130.0
140.0
150.0
135
Edema (%)
Time (h)
Control
Indomethacin 10 mg/kg
BuOH fr. 120 mg/kg
*
*
*
*
**
Fig. 2 The BuOH fraction (BuOH fr.) was suspended in distilled water
and orally administered. Paw edema was induced 30 min after the
subplantar injection of 1% (w/v) carrageenan in buffer saline. The
paw edema was measured before (predose) and at intervals of 1, 3,
and 5 h after the carrageenan injection. Indomethacin was used as a
reference agent. Values are expressed in percent increase of paw
before the carrageenan injection and mean ± SEM from 8 animals in
the treatment group. * p< 0.05, **p< 0.01 vs. control
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Effect of BuOH fraction on mechanical hyperalgesia in
rats
Figure 3 shows the data obtained in rats while mechan-
ical hyperalgesia was induced by carrageenan, which led
to the reduction of ipsilateral paw withdrawal thresholds
approximately from 150 g to 45–55 g after the injection
in naive animals (data not shown). Administration of the
BuOH fraction or indomethacin significantly reversed
inflammatory mechanical hyperalgesia (Fig. 3a). A max-
imal 32% reversal was observed 3 h after the administra-
tion of the BuOH fr. at dose of 120 mg/kg (p< 0.05),
while the positive control (indomethacin) produced up
to 44% reversal (Fig. 3b).
Effect of BuOH fraction on thermal hyperalgesia in rats
Carrageenan injection into the hind paw of rats induced
thermal hyperalgesia, exerting maximal effect after 3 h.
As shown in Fig. 4, the BuOH fraction remarkably in-
creased Δwithdrawal latency at dose of 120 mg/kg.
Indomethacin (10 mg/kg) also exhibited significant anti-
nociceptive activity by reducing thermal hyperalgesia.
Effect of BuOH fraction on synovitis
The inflammatory response in the leg of rats occurred
5 h after intra-synovial injection of sodium urate crystal,
and then was gradually increased during subsequent
13 h (data not shown). The hyperalgesic response was
stable in all animals for 2 h, prior to the second drug ad-
ministration. Administration of the BuOH fraction
(120 mg/kg, p.o.) alleviated urate-induced synovitis 20 h
after injection of a causative inflammatory agent
(Table 3). Indomethacin (10 mg/kg, p.o.) also
displayed anti-inflammatory effect in this model, resulting
in leg tenderness for all animals in the group.
A
B
Fig. 3 Administration of the BuOH fraction (BuOH fr.) or
indomethacin after the treatment with carrageenan significantly
reversed mechanical hyperalgesia in rats. Paw withdrawal thresholds
were measured before (predose) and at intervals of 1, 3, and 5 h
after the carrageenan injection (a). Percentage of reversal of
hyperalgesia measured 3 h after the vehicle or drug administration
(b). Values are displayed as mean ± SEM from 8 animals per group. *
p< 0.05, and ** p< 0.01 compared with vehicle
Fig. 4 Carrageenan was injected into the hind paw to produce
thermal hyperalgesia in rats. The BuOH fraction (BuOH fr.) at the oral
dose of 120 mg/kg significantly increased Δwithdrawal latency,
inhibiting the inflammatory hyperalgesia response after 3 h of the
treatment. Indomethacin (10 mg/kg) was used as a reference
compound. Values are expressed in mean ± SEM from 8 animals per
group. * p< 0.05, **p< 0.01 vs. hyperuricemic rats (control)
Table 3 Effect of BuOH fraction (BuOH fr.) on urate induced
synovitis in rats
Group Number Dose
(mg/kg p.o.)
Score
Control 8 - 3.5 (2–4)
BuOH fr. 8 120 2 (1–3)*
Indomethacin 8 10 1 (1–1)**
Inflammatory responses in the leg receiving sodium urate began 5 h after the
injection, and continued to increase during 13 h (data not shown).
Hyperalgesic responses were stable in all animals 2 h prior to the second drug
administration. An anti-inflammatory agent was effective in this model. Values
are expressed in median of score range (min –max). The Kruskal-Wallis test
was performed, followed by the Mann-Whitney U test
*p< 0.05, **p< 0.01 vs. control
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Analysis of total phenolic content and identification of
phenolic compounds
Major constituents of the CEE were identified as phen-
olic compounds by analysis of the
1
H NMR data, and
the corresponding signals were also detected in the
BuOH fraction–the most active fraction of the extract.
Kirenol is an anti-inflammatory diterpene reported pre-
viously from this plant [2], but its NMR signals were not
present in the BuOH fraction. Therefore, transference of
the phenolic compounds found in the CEE into the
BuOH fraction was hypothesized to explain the
retention of the biological properties. In line with this
hypothesis, some phenolic compounds were reported to
show anti-gout and anti-inflammatory activities [24, 30–
35]. Total phenolic contents for the organic solvent
fractions (Hex, EtOAc, and BuOH fractions) and the
CEE were quantitatively calculated to be 42.5, 75.7,
173.4, and 138.1 mg/g, respectively. The BuOH fraction,
displaying the most potent biological activities, con-
tained the highest total phenolic content, consistent with
our hypothesis. The phenolic compounds were isolated
by HPLC, and their spectroscopic data (Additional file
1) were compared with those published in the literature
[14, 30]: the phenolic compounds were identified as 3-
CQA (1; 3-caffeoylquinic acid; t
R
= 17.37 min), 5-CQA
(2; chlorogenic acid; t
R
= 22.32 min), 5-CQA (3;4-
caffeoylquinic acid; t
R
= 23.10 min), caffeic acid (4,
t
R
= 27.92 min), diCQAs (5; 3,4-dicaffeoylquinic acid,
3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid;
t
R
= 43.41 min, 45.02 min, and 45.38 min, respectively),
rutin (6;t
R
= 46.86 min), quercitrin (7;t
R
= 49.01 min),
kaempferol-3-O-rutinoside (8;t
R
= 50.94 min), and
kaempferol-3-O-rhamnoside (9;t
R
= 55.63 min) (Fig. 5).
Discussion
Gout is an emerging and common metabolic disorder
closely related to hyperuricemia, the treatment of which
aims to relieve acute gouty attacks and to prevent recur-
rent gouty episodes. Therapeutic approaches for treating
gout include applications of anti-inflammatory agents
for symptomatic relief, as well as selective inhibition of
the terminal steps in uric acid biosynthesis for chronic
gout [19]. Combination of the relevant therapies, such as
lowering uric acid levels, inhibiting inflammatory re-
sponses, and modifying dietary behaviors, was suggested
for the treatment of gout [36]. Suppression of XO activ-
ity is one of the therapeutic strategies to reduce blood
uric acid levels. However, only a few XO inhibitors, e.g.,
allopurinol and febuxostat, have been clinically used
[37]. Although most of the Vietnamese traditional rem-
edies for curing gout disease contain hy-thiem [1, 3],
pharmacological research of this medicinal plant associ-
ated with the treatment of this metabolic and inflamma-
tory disorder has been underexplored.
We demonstrated in this study that CEE of
hy-thiem significantly reduced uric acid levels in
oxonate-induced hyperuricemia rats. In addition,
in vitro inhibitory activity of the CEE on XO was
observed. The most potent activity was detected for
the n-BuOH-soluble portion. Among the fractions
resulting from activity-guided fractionation, the BuOH
fraction presented XO inhibitory activity even more
potent than that of the whole crude extract (data not
shown), and showed no acute and sub-chronic tox-
icity (Additional file 2). Therefore, transference of
active components from the CEE to the BuOH frac-
tion was suggested. The BuOH fraction of hy-thiem
showed its hypouricemic effect at dose of 120 mg/kg.
Subsequent in vivo studies at the same dose also re-
vealed that the BuOH fraction remarkably inhibited
liver XO activity in rats. These observations suggested
that the hypouricemic effect of hy-thiem is caused by,
at least in part, its inhibitory potency on XO, a key
enzyme in the biosynthetic pathway of uric acid. Fur-
thermore, the BuOH fraction also displayed a notable
anti-inflammatory and antinociceptive activities in the
carrageenan-induced animal model, as shown previ-
ously with the crude extract of S. orientalis [5, 9].
Finally, deposition of urate crystals, an important ini-
tiation factor in the inflammatory process of gout, has
been taken into consideration in our experiments.
The BuOH fraction was found to have
anti-inflammatory effect in the urate-induced synovitis
model, which represents acute gouty attacks, confirm-
ing that the inhibition of XO is associated with
anti-inflammatory responses [37].
Although kirenol was reported to be responsible for
the anti-inflammatory and analgesic activities of S. orien-
talis [2], it was absent in the n-BuOH-soluble fraction in
the present study. Instead, phenolic compounds were
identified as major components of the fraction. There-
fore, the anti-gout and anti-inflammatory effects of
hy-thiem were presumed to be associated with those
phenolic compounds in the BuOH fraction [32, 38]. The
major constituents in this fraction were identified as caf-
feic acid analogues (1–5) and flavonones (6–9). Many
studies have showed uricemia lowering and anti-
inflammatory activities of caffeic acid analogues [31–34]
and flavonones [24, 35], supporting our hypothesis. This
is the first report of this plant that phenolic compounds
1–9are the major constituents, showing anti-
inflammatory, analgesic, and anti-gout activities. Of
these phenolic compounds, 3-CQA (1), chlorogenic acid
(2), 4-CQA (3), di-CQA (5), quercitrin (7), kaempferol-
3-O-rutinoside (8), and kaempferol-3-O-rhamnoside (9)
were identified for the first time from this species, while
a few other phenolic metabolites including caffeic acid
(4) and rutin (6) were reported previously [14].
Nguyen et al. BMC Complementary and Alternative Medicine (2017) 17:191 Page 7 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Conclusions
This study demonstrates that crude ethanol extract and its
BuOH-soluble portion from S. orientalis (Vietnamese hy-
thiem) show anti-hyperuricemic and anti-inflammatory
activities experimentally. The active constituents respon-
sible for the biological activities were identified as phenolic
compounds. Our findings support the application of this
plant as an indigenous medicine for treating gout, which
occurs when metabolic and inflammatory disorders take
place together, by its dual pharmacological action.
Additional files
Additional file 1: Spectroscopic data for phenolic compounds from
hythiem (Siegesbeckia orientalis). (DOCX 17 kb)
Additional file 2: Report on toxicity study of BuOH fraction. (DOCX 104 kb)
Abbreviations
BuOH fr.: n-BuOH-soluble fraction; CEE: Crude ethanol extract;
CMC: Carboxymethylcellulose; CQA: Caffeoylquinic acid; DAD: Diode array
detector; HPLC: High performance liquid chromatography; NMR: Nuclear
magnetic resonance; XO: Xanthine oxidase
Acknowledgements
We are grateful to Doctor Nguyen Quynh Chi, Department of
Pharmacognosy, Hanoi University of Pharmacy for collecting and providing
plant samples.
Funding
The authors deeply acknowledge the Vietnam National Foundation for
Science and Technology Development (NAFOSTED) for providing research
grant (No 106.99–2012.90). This research was supported by the research fund
of Chungnam National University and the Priority Research Centers Program
(NRF-2009-0093815) through the National Research Foundation of Korea (NRF)
grant funded by Korean Government.
Availability of data and materials
All data and materials related to this study are included in the manuscript
and supplementary files.
Authors’contributions
PTT, NHA, and MN designed the study, and NTD carried out the experiment. NTD,
IHH, HTKH, and NMK performed data analysis and prepared the manuscript. All
authors have read and approved the final version of this manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
A
B
Fig. 5 Chromatogram (HPLC-DAD, UV 255 nm) of the BuOH fraction (a) and the corresponding chemical structures (b)
Nguyen et al. BMC Complementary and Alternative Medicine (2017) 17:191 Page 8 of 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Ethics approval and consent to participate
All experimental protocols on animals in this study were approved by the
Scientific and Ethical Committee of Hanoi University of Pharmacy (156/DHN-QD).
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Department of Pharmacology, Hanoi University of Pharmacy, 13 Le Thanh
Tong, Hoan Kiem, Hanoi, Vietnam.
2
Department of Pharmaceutical Analysis
and Standardization, National Institute of Medicinal Materials, 3B Quang
Trung, Hoan Kiem, Hanoi, Vietnam.
3
College of Pharmacy, Woosuk University,
Wanju, Jeonbuk 55338, Republic of Korea.
4
College of Pharmacy, Chungnam
National University, Yuseong-gu, Daejeon 34134, Republic of Korea.
Received: 30 August 2016 Accepted: 21 March 2017
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