Potent Anti-Inflammatory Effect of Root of Sidaguri (Sida rhombifolia L) on Rat Periapical Lesion Model

Abstract

Sidaguri (S. rhombifolia L) is one of the most important species of medicinal part in Indonesia as anti-inflammation. To clarify this, we investigated the anti-inflammatory effect of the root of S. rhombifolia on rat periapical lesion model. The incisivus teeth were drilled to expose the dental pulp to the oral cavity and apical lesions were induced with LPS isolated from Porphyromonas gingivalis. Pour plate method was used to determine the bacterial CFU of gingival crevicular fluid (GCF) and C-reactive protein (CRP) serum was examined by enzyme-linked immunosorbent assay. The results showed that the root of S. rhombifolia could not reduce the total bacteria of GCF; however, it could reduce the level of CRP compared to negative control (p<0.05). In conclusion, S. rhombifolia has anti-inflammatory potency on rat periapical lesion.

Full text

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International Journal of Toxicological and Pharmacological Research 2016; 8(6); 412-415
ISSN: 0975-5160
Research Article
*Author for Correspondence: maria_tanumiharja@yahoo.com
Potent Anti-Inflammatory Effect of Root of Sidaguri (Sida rhombifolia
L) on Rat Periapical Lesion Model
Maria Tanumihardja1*, Nurhayaty Natsir1, Indrya K Mattulata1, Lukman M2
1Department of Endodontics, Dental Faculty, Hasanuddin University, South Sulawesi, Makassar, Indonesia.
2Sekolah Tinggi Ilmu Farmasi (STIFA) Makassar, Perintis Kemerdekaan Street KM 13.7, South Sulawesi, Makassar.
Available Online: 25th December, 2016
ABSTRACT
Sidaguri (S. rhombifolia L) is one of the most important species of medicinal part in Indonesia as anti-inflammation. To
clarify this, we investigated the anti-inflammatory effect of the root of S. rhombifolia on rat periapical lesion model. The
incisivus teeth were drilled to expose the dental pulp to the oral cavity and apical lesions were induced with LPS isolated
from Porphyromonas gingivalis. Pour plate method was used to determine the bacterial CFU of gingival crevicular fluid
(GCF) and C-reactive protein (CRP) serum was examined by enzyme-linked immunosorbent assay. The results showed
that the root of S. rhombifolia could not reduce the total bacteria of GCF; however, it could reduce the level of CRP
compared to negative control (p<0.05). In conclusion, S. rhombifolia has anti-inflammatory potency on rat periapical
lesion.
Keywords: anti-inflammation, Sida rhombifolia, periapical lesion.
INTRODUCTION
According to data of Indonesian Health Profile year 2009,
pulpal and periapical diseases were the 8th rank of the 10th
outpatients at hospital in Indonesia. This situation
increased and become the 7th in year 20101,2. Pulpal and
periapical diseases are inflammatory diseases caused by
colonized bacteria in the root canal system. Mixed bacteria
are usually found in the root canal such as
Peptostreptococcus micros (35%), Fusobacterium
necrophorum (23.3%), Fusobacterium nucleatum
(11.7%), Prevotella intermedia/nigrescens (16.7%),
Porphyromonas gingivalis (6.7%) and Porphyromonas
endodontalis (5%)3. Inflammation of pulpal and periapical
diseases is a pathophysiological response to irritants that
leads to accumulation of plasmatic fluid and immune cells
in the apical area of teeth. Complex events and mediators
involved in the inflammatory reaction can be induced
which maintain or aggravate the diseases4. Currently
available anti-inflammatory drugs have problems during
their clinical use and development of newer anti-
inflammatory drugs with lesser side effects is necessary5,6.
In recent years, Indonesian government promotes various
herbs for the treatment as part of traditional medicines
which has been applied in some primary health care since
year 2009. For dental treatment, a wide variety of plants
are commonly used by people as analgesics; clove oil,
ginger, and Andrographis paniculata, as antibacterial;
onion, curcuma, as anti-inflammatory; lemongrass, ginger,
noni7. Some other plants have also been used for tooth
ache such as jatropha, gambir, and sidaguri7-9, however
Sidaguri has not been further explored. Sidaguri (Sida
rhombifolia), a genus of flowering plants of mallow family
Malvaceae, has been studied, and each part of the plants
could be used for relieving various symptoms of the body.
However only some people use the root of Sidaguri for
tooth ache10. Previous study using agar diffusion method
showed the ethanol extracts of roots of Sidaguri has an
antibacterial activity against Enterococcus faecalis, while
no inhibition zone against Actinomyces spp was noted. In
addition, anti-inflammation test using plethysmometer on
carrageenan-induced rat of the ethanol extracts of root of
Sidaguri showed significant anti-inflammatory effect
compared to negative control11. Further studies on animal
model are recommended for optimal results. This study
aimed to examine the anti-inflammatory effect of root of
Sidaguri on rat periapical lesion model.
MATERIALS AND METHODS
Plant collection and extraction
The roots of Sidaguri were collected from their natural
habitat in Bone, South Sulawesi. Firstly, they were
identified and determined, then the roots were cleaned,
sun-dried, grounded into powder and extracted using
reflux method with ethanol 96%. The extracts were
evaporated using rotary evaporator and dried extracts were
kept in vacuum desiccator.
Animal model
Healthy male Wistar rats of 2-3 month-age were chosen
(180-200 gr) and housed in the same environment, adapted
for one week prior to treatment, fasting for 18 hours, water
and food et libitum. The rats were intra-peritoneal-
anesthetized with Ketamine-HCl (Pfizer) (80mg/kgBW)12,
fixed at retraction board, and the lower tooth was drilled
with steel bur ¼ (Emeco, Vanadium, Germany) to expose

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IJTPR, Volume 8, Issue 6, December 2016- January 2017 Page 413
the dental pulp to the oral cavity. Periapical inflammation
was induced with LPS isolated from Porphyromonas
gingivalis. To enhance the inflammation, LPS was injected
through the cavity entrance and sulcular gingival. The rats
were kept for 3 weeks following evaluation.
Treatment
Suspension of Sidaguri extracts in Na-CMC 1% was
evenly distributed. The extracts were given orally using
oral cannula with each of doses 0.6; 1.2; and 2.4 g/kgBW,
every day for 3 weeks. The body weight, quantity of meal
consumed, and the activities were examined every day.
Determination of CFU bacteria with pour plate method
Number of bacterial load (CFU) on animal model was
evaluated using pour plate method. The samples with
bacterial contained, mixed with agar medium and colony
of bacteria in teeth of animal model was taken with cotton
button, then inserted into the tube.
Evaluation of CRP level
CRP level of the collected blood was determined using
ELISA method according to the procedures in manual kit.
Statistical Analysis
All the grouped data were statistically evaluated,
hypothesis testing methods included one-way analysis of
variance followed by least significant difference test. P
values of less than 0.05 were considered to indicate
statistical significance. All the results were expressed as
mean ± SD for five experiments in each.
RESULTS AND DISCUSSION
Malvaceae is a cosmopolitan family of herbs, shrub and
trees revealed that most of the plants belonging to this
family are medicinally important as they contain
biologically active compounds like S. rhombifolia, locally
known as Sidaguri in Indonesia. It has a long history for its
Figure 1: Number of cultured bacteria on animal model following treatment with Sidaguri extracts in various
concentration.
Figure 2: Anti-inflammatory activities of various dosage of root extracts of Sidaguri on animal model following 14-
day treatment.
Sidaguri 0,6 g/kgBB
Sidaguri 1,2 g/kgBB
Sidaguri 2,4 g/kgBB
K-diclofenac
Negative control
CFU/mL
0
50
100
150
200
250
Sidaguri 0,6 g/kgBB
Sidaguri 1,2 g/kgBB
Sidaguri 2,4 g/kgBBK-diclofenac
Negative control
Concentration of CRP (ng/mL)
0
1
2
3
4
5
6

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medicinal value in traditional medicine for the treatment of
gout, hypertension, diabetes, anti-tuberculosis agent,
diarrhea, and indigestion disorder13,14. This research focus
to the roots as anti-inflammation on rat periapical lesion
model induced by LPS. A model of this periapical lesion
model LPS-induced could be valuable for improving the
understanding of disease etiology and progression and
effective treatments. There are many reports regarding the
induction of periapical lesions in animals but rat models
are the most frequently used models for such studies: the
morphology of a rat molar is similar to that of a human
molar, and the genetic background of these animals is
clear, with practically no individual specificity15,16.
Antibacterial activities of Sidaguri extracts on animal
model periapical inflammation induced with LPS, Pour
Plate method
The results in Figure 1 showed the extracts of root of
Sidaguri has an effect to decrease the number of bacterial
load compared to negative control. Dosage provided is
comparable to the effect resulted although the difference is
little. Statistical analysis showed (CI: 95%) all treated
groups, either extracted Sidaguri group or positive control
group showed significant difference compared to negative
control group. It can be concluded that the extracts of roots
of Sidaguri could inhibit the growth of bacteria on animal
model periapical inflammation induced with LPS in
optimal dosage 0,6 g/kgBW.
The antimicrobial activity showed that the Sidaguri has
ability to decreases the microorganisms in GCF. Some
authors have reported the antibacterial activities of the
petroleum ether, chloroform, ethyl-acetate extracts of
Sidaguri on Gram-positive and Gram-negative bacteria17,
the antimicrobial activity attributed to the presence of
various bio-actives components such as tannins,
polyphenols, alkaloids, glycosides ((phenyl-Ethyl-D-
glucopyranoside and phytoécdysteroide), flavonoids,
steroids and saponins18,19. But in another research reported
that Sidaguri showed weak antibacterial activity against
both Gram-positive and Gram-negative test organisms20
correlated with this result that activity no dependent doses.
Anti inflammatory activities of Sidaguri extracts on
animal model periapical inflammation Induced with LPS
Porphyromonas
Periapical lesion is a local inflammatory process mediating
the destruction, triggered by bacterial or its toxic such as
LPS. Immunological marker of acute phase response is
CRP, refers to local inflammation response21.
Consequently, CRP level relationship to periodontal
disease. The level of CRP in blood rats of animal model
was evaluated using ELISA method, as seen in Figure 2.
The results of this study showed the extracts of roots of
Sidaguri have an anti-inflammatory activities and the
higher the dosage, the higher anti-inflammatory effect,
marked by decreased level of CRP. Statistical analysis
(CI:95%) showed all treated groups, either extracted
Sidaguri group or positive control group showed
significant difference compared to negative control group.
Group of dosage 2,4 g/kg BW differs significantly to the
group of dosage 1.2 g/kgBW and 0,6 g/kg BW. It can be
concluded that optimum dosage for anti-inflammatory to
inhibit CRP was 2.4 g/kgBW.
Inflammation is the first step of disease proses, inhibit this
proses can inhibit the pathogenesis of disease. Using
Sidaguri as inhibitor more possible. The stems and roots
can inhibit arthritic on rats22. The roots have scavenging
activity on 1,1-diphenyl-2picrylhydrazyl and, anti-
inflammatory on NF-Kb cell line inflammation model23.
CONCLUSION
Under the limitation of this study, it can be concluded that
the ethanol extracts of roots of Sidaguri has potent anti-
inflammatory activities but lack of antibacterial effects in
periapical inflammation induced with LPS Porphyromonas
gingivalis. Future studies need to be elucidated on the
active components of the roots of Sidaguri.
ACKNOWLEDGMENTS
The authors would like to thank the Ministry of Research,
Technology and Higher Education of the Republic of
Indonesia for funding this research.
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