Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2019, Article ID 3612481, 17 pages
https://doi.org/10.1155/2019/3612481
Research Article
In Vitro Anti-Inflammatory and In Vivo Antiarthritic Activities of
Aqueous and Ethanolic Extracts of Dissotis thollonii Cogn.
(Melastomataceae) in Rats
Stephanie Flore Djuichou Nguemnang,1 Eric Gonzal Tsafack,1 Marius Mbiantcha ,1
Ateufack Gilbert ,1 Albert Donatien Atsamo ,2 William Yousseu Nana,1
Vanessa Matah Marthe Mba,1 and Carine Flore Adjouzem1
1
Laboratory of Animal Physiology and Phytopharmacology, Department of Animal Biology, Faculty of Science,
University of Dschang, PO. Box 67, Dschang, Cameroon
2
Laboratory of Animal Physiology, Faculty of Science, University of Yaounde I, PO Box 812, Yaoundé, Cameroon
Correspondence should be addressed to Marius Mbiantcha; mbiantchamarius@yahoo.fr and Ateufack Gilbert;
ateufack2000@yahoo.fr
Received 15 July 2019; Accepted 1 November 2019; Published 15 November 2019
Academic Editor: Jae Youl Cho
Copyright © 2019 Stephanie Flore Djuichou Nguemnang et al. This is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Dissotis thollonii Cogn. (Melastomataceae) is a tropical plant widely used in traditional Cameroonian medicine to relieve and treat
many pathologies. It is widespread in the western region where it is used to treat typhoid fever, gastrointestinal disorders, and
inflammatory diseases. The purpose of this study is to scientifically demonstrate the anti-inflammatory and antiarthritic properties
of the aqueous and ethanolic extracts of the leaves of Dissotis thollonii. The anti-inflammatory properties were evaluated in vitro by
inhibition tests for cyclooxygenase, 5-lipoxygenase, protein denaturation, extracellular ROS production, and cell proliferation;
while antiarthritic properties were evaluated in vivo in rats using the zymosan A-induced monoarthritis test and the CFA-induced
polyarthritis model. This study shows that aqueous and ethanolic extracts at a concentration of 1000 μg/ml inhibit the activity of
cyclooxygenase (47.07% and 63.36%) and 5-lipoxygenase (66.79% and 77.7%) and protein denaturation (42.51% and 44.44%).
Similarly, both extracts inhibited extracellular ROS production (IC50 � 5.74 μg/ml and 2.96 μg/ml for polymorphonuclear leukocytes, 7.47 μg/ml and 3.28 μg ml for peritoneal macrophages of mouse) and cell proliferation (IC50 � 16.89 μg/ml and 3.29 μg/
ml). At a dose of 500 mg/kg, aqueous and ethanolic extracts significantly reduce edema induced by zymosan A (69.30% and
81.80%) and CFA (71.85% and 79.03%). At the same dose, both extracts decreased sensitivity to mechanical hyperalgesia with
69.00% and 70.35% inhibition, respectively. Systemic and histological analyzes show that both extracts maintain the studied
parameters very close to normal and greatly restored the normal architecture of the joint in animals. Dissotis thollonii would
therefore be a very promising source for the treatment of inflammatory diseases.
1. Introduction
Rheumatoid arthritis (RA) is a chronic, disabling, and
progressive autoimmune disease in which chronic proliferative synovitis and synovial inflammation arde observed
with significant bone destruction and cartilage destruction
resulting in significant joint damage and reduced functionality [1–3]. This pathology can evolve very quickly in an
individual and affect several parts of the body that become
inflamed or extremely painful particularly affecting the elderly, but also individuals with degenerative bone disorder
or immune system dysfunction [4]. This pathology, which
can also occur as a result of the immune system attacking the
synovial membrane, is accompanied by swelling, stiffness,
pain, and a reduction or loss of joint function [4]. During the
establishment and development of rheumatoid arthritis,
many inflammatory mediators play a key role in bone destruction and inflammation of the synovial membrane,
2
including tumor necrosis factor (TNF-α), interleukin-1β,
interleukin-6, nitric oxide (NO), prostaglandins, reactive
oxygen species (ROS), platelet-activating factor, leukotrienes, enzymes (lipoxygenases, cyclooxygenases (COX-1 and
COX-2), and phospholipases) [5, 6].
Animal models of zymosan A-induced monoarthritis and
Freund’s complete adjuvant-induced polyarthritis (CFA) are
widely used in research for the activity of many pharmacological substances on rheumatoid arthritis. In fact, the injection of zymosan into the knee joint of the rats causes
erosive synovitis with increased vascular permeability, neutrophil infiltration, and formation of edemas and exudates in
the acute phase, and then in the chronic phase, infiltration of
macrophages and lymphocytes, pannus formation, and fibroblastic reaction characteristic of chronic rheumatoid synovitis [7, 8]. CFA is an immunogenic adjuvant consisting of
a suspension of Mycobacterium tuberculosum or Mycobacterium butyricum killed by heat. When injected at the base of
the animal’s tail, it causes the development of polyarthritis
that evolves in a two-phase cycle of time: the first phase
appears in a few hours and disappears after 3 to 5 days and
manifests itself by an acute local inflammatory reaction, and
then the second phase appears after two weeks and corresponds to a chronic systemic reaction [9–11]. This polyarthritis is not primarily aimed at the knee joint, and it can
affect the general state of the animal body; it is a real systemic
disease resulting in inflammation of the distal joints of the
limbs, vertebrae, lesions of the genitourinary tract, gastrointestinal tract, eyes, nose, ears, skin, and anorexia accompanied by significant weight loss [9, 11]. In addition, the
pathology will persist, and other symptoms will appear,
namely, joint deformity, synovitis, synovial hyperplasia,
capsular fibrosis, angiogenesis, pannus formation, cartilage
destruction, bone erosion, inflammation of the bone marrow,
resorption of bone matrix, and ankylosis [12].
The severity and persistence of rheumatoid arthritis
require long-term management with anti-inflammatory
drugs. Nevertheless, these anti-inflammatory drugs have for
the most part risks of toxicity for long-term use, which
seriously limits their use. Current research in the management of rheumatoid arthritis is turning to a new generation
of substances capable of selectively inhibiting TNF alpha
and/or cyclooxygenase (COX-2) and having no major side
effects [13]. Recent interest in alternative treatments for
arthritis favors the use of traditional medicine although
scientific evidence of efficacy for most cases is lacking.
Nevertheless, several herbs, used in a care program and a
very effective preventive medicine, can act individually and/
or in synergy to reduce chronic joint inflammation (osteoarthritis and/or rheumatoid arthritis) [14–16]. To reach the
total health care coverage of the world’s population, traditional medicine is considered by WHO to be the most effective means since about 25% of modern prescription drugs
are more or less obtained from plants [17, 18].
Comprising about 163 genera, the family of Melastomataceae which are mainly pantropical plants include
more than 4,300 species so many of them are known for their
effectiveness in traditional medicine as antihepatitic, antihypertensive,
anti-inflammatory,
antihyperglycaemic,
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antioxidant, hemostatic, and antidiarrheal [19–24]. Dissotis
thollonii (D. thollonii) is one of many species of the family of
Melastomataceae used in traditional medicine in Cameroon
to treat typhoid fever, gastrointestinal disorders, inflammatory diseases, and sinusitis [25–27]. The leaves are
recommended for the treatment of ulcers and gastrointestinal disorders. A previous study showed that D. thollonii
significantly inhibited fluid accumulation in intestine induced by prostaglandin E2 [28]. Based on recent work by
Ateufack et al. [29], this plant has antidiarrheic and antibacterial properties and then has several secondary metabolites
including
tannins,
flavonoids,
sterols,
anthraquinones, phenols, and polyphenols. In addition, the
work of TadjouaTchoumbou et al. [24] showed that this
plant significantly inhibited leukocyte migration in peritoneal fluid, intracellular ROS production, proliferation of
Hela cell lines, and TNF-α production. Tala et al. [27]
showed that aqueous and ethanolic extracts were devoid of
toxicity after 28 days of daily treatment. Similarly, Nono
et al. [30] showed the antimicrobial and antioxidant
properties of this plant. Several compounds have already
been isolated from this plant, among which 3,3′-diomethylellagic acid 4′-O-β-D-xylopyranoside, 3-4′-O-β-Darabinopyranoside, casuarinine, betulinic acid, β-sitosterol3-O-D-glucopyranosyl-6′-mirystate, cellobiosylsterol, β-sitosterol, β-sitosterol-3-O-β-D-glucopyranoside, arjunolic
acid, 3,3′-diomethylellagic acid, ellagic acid, and 3,3′-diomethylellagic acid 4′-O-β-D-glucopyranoside [30]. Although this plant is traditionally used to relieve many
disorders of the body, no information or scientific report to
our knowledge has been found in the literature relative to its
antiarthritic properties. In our continuous search for bioactive extracts from plants used in traditional Cameroonian
medicine [31, 32], and in order to support and improve the
traditional use of D. thollonii, we undertook to carry out the
present study on in vitro anti-inflammatory activities and in
vivo antiarthritic activities of the leave extracts of D.
thollonii.
2. Materials and Methods
2.1. Plant Material and Extraction. The plant material, referenced to the national herbarium of Cameroon under the
number N° 133292/SRF Cam, in the name of D. thollonii
(Melastomotaceae) was used in this study. The fresh leaves
were harvested in the town of Dschang (western Cameroon),
dried in the shade, and then crushed into a fine powder. In
order to prepare the aqueous extract, 500 g of powder was
mixed into 500 ml of distilled water during 72 hours and
filtrated (Whatman paper No. 4); the filtrate obtained was
evaporated at 40°C to give the aqueous extract (8.2% yield).
The same weight of dried powder plant was mixed into
500 ml of ethanol for 72 hours and then filtered. The filtrate
was concentrated with a rotary evaporator set at 96°C to give
the ethanolic extract with 9.6% yield.
2.2. Phytochemical Assay of D. thollonii Extracts. The different extracts were subjected to chemical screening in order
Evidence-Based Complementary and Alternative Medicine
to detect the presence of the main groups of compounds
following the principles stated by Matos [33].
2.3. Triterpenes and Steroids: Lieberman–Burchard Test.
In a tube containing 3 ml of MeOH, 0.1 g of extracts was
dissolved, and then 0.2 ml of each of the following reagents
was added: chloroform, glacial acetic anhydride, and concentrated H2SO4. The mixture was observed to look for the
appearance of the greenish-blue or purple-pink color
characteristics of the presence of sterols and triterpenes,
respectively.
2.4. Phenols. The tube contained 3 ml of ethanol; 0.1 g of
extracts was dissolved, and then three drops of 10% iron III
chloride were added. The solution was then observed to
observe for the appearance of the blue-violet or greenish
coloration which characterizes the presence of the phenols.
2.5. Tannins. 5 ml of MeOH was introduced in the tube, and
0.1 g of extracts was dissolved. The solution was then added
with 5 drops of 0.5% sulfuric acid, and the mixture was
observed to detect the green or blue-black color, indicating
the presence of tannins.
2.6. Flavonoids: Shinoda Test. In a tube containing 3 ml of
MeOH, 0.1 g of extracts was dissolved, and the mixture was
then treated with 0.05 g of magnesium chloride chips and 3
drops of concentrated H2SO4. The flavonoids were highlighted by the appearance of the following colorings: orange
for flavones, red for xanthones, and pink for flavonols.
2.7. Anthocyanings. In a test tube containing 0.1 g of extracts, 5 drops of concentrated hydrochloric acid were introduced. The solution was then observed to look for the
appearance of red color indicating the presence of
anthocyanins.
2.8. Saponins. The presence of saponins is generally materialized by the formation of a stable foam after stirring a
solution. Thus, a solution of 5 ml of distilled water and 5 ml
of each extract was vigorously shaken to check for the
presence or absence of saponins in each extract of our plant.
2.9. Anthraquinone. The presence of free anthraquinones
and/or anthraquinone derivatives in a mixture is indicated
by a pink, violet, or red coloration in the lower phase
(ammoniacal phase) of the mixture after stirring. Thus, two
methods made it possible to verify the presence of this class
of compound in our various extracts:
(i) Stirring a solution prepared in the following manner:
extract (3 ml) and benzene (3 ml), followed by filtration and then 5 ml ammonia (10%) in the filtrate.
(ii) A mixture of extract (3 ml) and sulfuric acid (3 ml) is
boiled and filtered while hot and benzene (3 ml) is
added to the filtrate followed by stirring. After
3
separation of the benzene layer, ammonia prepared
at 10% (3 ml) was added.
3. In Vitro Anti-Inflammatory Assays
3.1. Inhibition of Protein Denaturation. To evaluate the antiinflammatory effects of the extracts, the protocol described
by Padmanabhan and Jangle [34] and Elias and Rao [35] was
used with small modifications. A volume of 1 ml of extracts
(aqueous and ethanolic) or of diclofenac sodium at different
concentrations (100, 200, 500, and 1000 μg/ml) was
homogenised with 1 ml of aqueous solution of bovine serum
albumin (5%) and incubated at 27°C for 15 minutes. The
mixture of distilled water and BSA constituted the control
tube. Denaturation of the proteins was caused by placing the
mixture in a water bath for 10 minutes at 70°C. The mixture
was cooling inside the ambient room temperature, and the
activity each mixture was measured at 660 nm. Each test was
done three times. The following formula was used to calculated inhibition percentage:
% inhibition �
absorbance of control − absorbance of sample
× 100.
absorbance of control
(1)
4. Assay of Cyclooxygenase and
5-Lipoxygenase Inhibition
4.1. Lymphocyte Culture Preparation. RPMI 1640 (HIMEDIA) added with inactivated fetal calf serum, penicillin, and
streptomycin was used for culture of human
peripheral lymphocytes, and cell proliferation was induced
by phytohemagglutinin (HIMEDIA). After filtration (using
0.2 micron cellulose acetate, Sartorios), plasma was added
(1 × 106 cells/ml) and incubated for 72 hours, and the culture
was activated by the addition of lipopolysaccharide (1 μl) and
incubated again for 24 hours. Extracts (aqueous and ethanolic) and ibuprofen were added at a final concentration of
100, 200, 500, and 1000 μg/ml, incubated for 24 hours, and
centrifuged for sedimentation at 6000 rpm for 10 min. After
removal of the supernatant, cell lysis buffer was added
(50 μl), and the mixture was again centrifuged at 6000 rpm
for 10 min. The anti-inflammatory test was performed
according to the method used by Viji and Helen [36].
4.2. Assay of Cyclooxygenase. A mixture of glutathione, trisHCl buffer, enzyme, and hemoglobin was used to make the
assays. After addition of arachidonic acid and TCA (10% in
1N HCl, 0.2 ml), the mixture was incubated at 37°C (20
minutes). The TBA (0.2 ml) was added to the contents which
were then heated (for 20 minutes in boiling water), and after
cooling, the mixture was centrifuged (1000 rpm, 3 min) and
the supernatant was used to measure COX activity at 632 nm
[36].
4.3. Assay of 5-Lipoxygenase. In 4 ml of nonoxygenated
water, linoleic acid (70 mg) and an equal weight of interpolation were dissolved and pipetted, followed by sodium
4
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hydroxide (0.5 N) and water without oxygen (25 mL) was
added. The final solution was divided into small portions of
0.5 ml each and rinsed with nitrogen and frozen. A quartz
cuvette (25°C) optical path of 1 cm made it possible to carry
out the reaction. The OD measured at 234 nm was performed
with a mixture of tris buffer (2.75 ml, pH 7.4), sodium linoleate (0.2 ml), and enzyme (50 ml) [36]. The following
formula was served to determine percent inhibition:
% inhibition �
abs control − abs sample
× 100.
abs control
(2)
4.4. Chemiluminescence Assay. The human blood samples
used in this work were received from a donor following the
procedure accepted by the Independent Ethics Committee,
ICCBS, University of Karachi, No : ICCBS/IEC-008-BC2015/Protocol/1.0. The blood donors were informed that it
should be used for an experimental study.
4.5. Isolation of Human Polymorphoneutrophils (PMNs).
Aseptically, a volume of 10 ml of venous blood was taken
from a very healthy 33-year-old volunteer donor and then
introduced into a tube with anticoagulant (heparin).
Neutrophils were isolated following the protocol described
by hypoflat Ficoll density gradient centrifugation [37]. For
this, in an empty tube of 45 ml, the whole blood, HBSS, and
LSM (lymphocyte separation medium) were introduced at
equal volume, and after 30 min latency, the supernatant of
this mixture was then removed and introduced into a 15 ml
tube previously containing 5 ml of LSM; then, the tube was
centrifuged at 400 g for 20 minutes (room temperature).
After removal of the supernatant, 1 ml of distilled water was
introduced into the tube (for lysis of red blood cells); after a
duration of 1 minute, the HBSS (2x) (1 ml) was also introduced (to stop the lysis). After adding 5 ml of HBSS
again, the tube was centrifuged for 10 minutes at 4°C
(300 g), and 1 ml of HBSS was added after removal of the
supernatant and the tube was kept in ice. The trypan blue
technique was used to assess viability while the hemocytometer counted the cells. For each test, the cell concentration used was 1 × 106 cells/ml.
4.6. Peritoneal Macrophage Isolation from Mice. One milliliter of FBS was injected (intraperitoneally) into NMRI mice
weighing an average of 22 g, and these animals were kept and
observed for 3 days; then, they underwent cervical dislocation for sacrifice. The RPMI medium (10%, 10 ml) was
introduced into the peritoneal cavity, after 2 minutes of
massage, the skin of the abdomen was removed, and the
peritoneal cavity was exposed. Using a syringe, the RPMI
medium injected into the peritoneum and containing the
macrophages was removed, introduced into a tube,
centrifuged (400 g, 20 minutes, 4°C), the supernatant was
removed, 5 ml incomplete RPMI was supplemented, the
tube was further centrifuged (300 g, 10 minutes, 4°C), and
then RPMI/HBSS (1 ml) was supplemented. The trypan blue
technique was used to assess viability, while the
hemocytometer counted the cells. For each test, the cell
concentration used was 1 × 106 cells/ml [38, 39].
For the chemiluminescence assay, the modified protocol
of Mbiantcha et al. [40] was used. In white plates (96 wells),
25 μl of PMNs cells, whole blood or macrophages, 25 μl of
extracts (aqueous or ethanolic), or ibuprofen were mixed.
While the well without extracts represent the control and
received only cells and HBSS++. After 20 minutes of incubation, each well obtained 25 μl of luminol and 25 μl of
PMA, which made it possible to obtain a total volume of
100 μl in each well. After completing this test, the results were
expressed in RLU (relative light units), and the following
formula allowed to calculate the percentage of inhibition:
inhibition(%) �
(RLUcontrol − RLUsample)
× 100. (3)
RLUcontrol
4.7. T-Cell Proliferation Assay [41]. 96-well white plates were
used for this test. In each well, were introduced the extracts
(2, 10 and 50 μg/ml), prednisolone (diluted in RPMI (5%)),
50 μl of T lymphocytes at a concentration of 2 × 106 cells/ml,
50 μl of PHAL at a concentration of 7.5 μg/ml (phytohemagglutinin-L). Wells considered as negative controls received only 550 μl of cells and 150 μl of RPMI (5%), whereas
those considered as positive controls received 50 μl of cells,
50 μl of PHA, and 100 μl of RPMI (5%). The plates were then
incubated (3 days, 37°C, CO2 (5%)), 25 μl of 0.5 μCi/well
(methyl 7 3H) thymidine was used to pulse the cultures,
followed by a second incubation for 18 hours, and then the
cells were harvested (using a fiberglass filter). The level of
thymidine integrated in the cells was determined using a
counter (LS65000 liquid scintillation). The following formula allowed to calculate the percentage of inhibition using
CPM (counts per minute):
inhibitory activity(%) �
(CPMcontrol − CPMsample)
× 100.
CPMcontrol
(4)
5. In Vivo Antiarthritis Assays
5.1. Animals. Female Wistar rats (3 to 4 months old and 150
to 200 g) were used for these tests. The animals were breeded
from the faculty of science (laboratory of animal physiology
and phytopharmacology) of the University of Dschang
(Cameroon). They were raised under normal conditions
(19–23°C, 12 hour light) with water and access without diet.
The experimental procedures have been approved by the
local Ethics Committee and are in accordance with the
guidelines for the study of pain in awake animals, published
by the NIH (publication no. 85-23, “Principles of Animal
Protection,” Laboratory, and Study of Pain, Ministry of
Scientific Research and Technology, which adopted the
European Union Guidelines on Animal Care and Experimentation (EWC 86/609).
5.2. Treatment Regimen. Animals were grouped by their
weight into different cages, and in each group, they were
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identified by the tail with a number using a marker pen. In
each test, 42 female rats were divided into 7 groups (6 rats
each): group 1 (healthy control) received no treatment and no
injection of zymosan A (Sigma Chemical Co., St. Louis,
Missouri, USA) or CFA (Sigma Chemical Co., St. Louis,
Missouri, USA), group 2 (arthritic control group) received the
solution of DMSO (5%) + PBS with injection of zymosan A or
CFA, group 3 (positive control) received diclofenac (5 mg/kg)
with injection of zymosan A or CFA, groups 4 and 5 received
the aqueous extract of D. thollonii (250 and 500 mg/kg) with
injection of zymosan A or CFA, and groups 6 and 7 received
the ethanolic extract of D. thollonii (250 and 500 mg/kg) with
injection of zymosan A or CFA. All treatments were administered orally 1 hour before the induction of zymosan A or
CFA (day 0), and then the animals were treated daily for 5
days for zymosan A-induced monoarthritis and 35 days for
polyarthritis induced by the CFA.
5.3. Monoarthritis Induced by Zymosan. In this test, the
protocol of Mbiantcha et al. [31] has been used with some
modifications. One hour after oral administration of the
various treatments, the thiopental is injected into each
animal (0.1 ml/100 g, intraperitoneal route), and then zymosan A (0.3 ml, 0.9% v/v NaCl) was injected in the knee
joint using a digital caliper (Mitutoyo, Japan); before the oral
treatment, the thickness of the injected joint was measured,
and then 1, 2, 3, 4, 5, 6, 24, 48, 72, 96, and 120 h after the
injection of zymosan A. On the fifth day after taking the last
parameters, the animals were again anesthetized, and the
injected knee joint was incised and preserved in a formalinPBS mixture (10%). The general scheme of method in histology was followed for histological analysis.
5.4. Polyarthritis Induced by CFA. The modified protocol of
Mbiantcha et al. [40] was used. One hour after administration of each treatment, the animals were anesthetized by
inhalation of ether vapor, and then 100 μl of CFA (10 mg/ml)
was injected into the tail vein, and then the animals were
returned to well-labeled cages and observed.
The severity of arthritis was assessed by measuring the
thickness of the hind leg joint using a digital caliper (days 0, 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35), the
pain sensitivity threshold using an analgesimeter (days 2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34), and the
arthritic score every day with certain criteria (score
0 � normal state, score 1 � edema on one foot or nodule, score
2 � two feet with edema or paw and nodule tail, score 3 � two
feet with edema and nodosity of the tail or three feet with
edema, and score 4 � three feet with edema and nodosity of
tail or edema of four feet) and body weight every week.
At day 36, the animals were anesthetized with thiopental
(0.1 ml/100 g body weight), the blood was removed by
catheterization of the abdominal artery and introduced into
two different types of tubes; tubes containing EDTA as
anticoagulant for analysis of hematological parameters by
standard laboratory method and tubes containing no anticoagulant were centrifuged (4900 rpm, 5 minutes), serum
was collected to evaluate ALT, AST, ALP, MDA, creatinine,
5
total protein, and stress parameters (NO, MAD, SOD,
catalase, and glutathione). The weights of various organs
such as liver, kidneys, thymus, and spleen were weighed, and
knee joints of all animals were collected and stored in a
formalin-PBS mixture (10%) for histological analysis.
5.5. Statistical Analysis. All in vitro test data indicate
mean ± standard deviation in triplicate while for the in vivo
test, the data are presented as an average of 6 animals ± SEM.
Differences between groups were assessed by ANOVA (one
way and two way) followed by Bonferroni posttest. Significant differences were considered at p < 0.05. a, b, and c or α,
β, and λ denote significant differences with respect to the
healthy control group and/or the arthritic control group.
6. Results
6.1. Chemical Composition. Several groups of chemical
compounds have been demonstrated in extracts of D. thollonii. It can be seen from this table that the ethanolic extract
contains all the test compounds with the exception of anthocyanins and triterpenes, whereas the aqueous extract
contains only flavonoids, phenols, and polyphenols (Table 1).
7. In Vitro Anti-Inflammatory Activity
7.1. Inhibition of Protein Denaturation. For the results of this
study, aqueous and ethanolic extracts effectively inhibit
protein denaturation (albumin) caused by heat. Table 2
shows significant inhibition (p < 0.001) of 42.51% and
44.44%, respectively, for the aqueous extract and the ethanolic extract at a concentration of 1000 μg/ml, whereas
diclofenac sodium produced 89.19% inhibition.
7.2. Cyclooxygenase Inhibitory Assay. Evaluation of cyclooxygenase activity determined the effect of both extracts on
prostaglandin production. The results show that, at 1000 μg/
ml, the aqueous extract, the ethanolic extract, and ibuprofen
significantly (p < 0.001) inhibit the activity of cyclooxygenase with 47.07%, 63.36%, and 97.88%, respectively
(Table 2).
7.3. 5-Lipoxygenase Inhibitory Assay. The evaluation of the
activity of 5-lipoxygenase was used to study the effect of the
various extracts on the production of leukotrienes. This table
shows that the aqueous and ethanolic extracts, as well as
ibuprofen, have a significant inhibitory effect (p < 0.001) on
the activity of 5-lipoxygenase with 66.79% inhibition,
77.48% and 95.31%, respectively (Table 2).
7.4. Effect of D. thollonii on Production of Intracellular ROS
and T-Cell Proliferation. Table 3 presents the results for
extracellular ROS production and T-cell proliferation. PMA
is used to activate ROS and luminol as a developer. The
ethanolic extract of D. thollonii significantly inhibited the
release of ROS in whole blood with an IC50 of 4.98 μg/ml. In
the presence of polymorphonuclear, an inhibition was
6
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Table 1: Phytochemical profile of Dissotis thollonii.
Extracts
Aqueous extract
Ethanolic extract
1
−
−
2
−
+
Phytochemical compounds
4
5
6
+
−
−
+
+
3
−
+
7
+
+
8
+
+
9
−
−
− : absent; +: present; 1: triterpenoids; 2: sterols; 3: tannins; 4: flavonoids; 5: anthocyanings; 6: anthraquinones; 7: phenol/polyphenol; 8: saponins; 9: cardiac
glycosides.
Table 2: Effect of aqueous and ethanolic extracts of Dissotis thollonii on protein denaturation, cyclooxygenase inhibition, and 5-lipoxygenase inhibition.
Compounds
Diclofenac
Ibuprofen
Aqueous extract
Ethanolic extract
Dose (μg/ml)
100
200
500
1000
100
200
500
1000
100
200
500
1000
100
200
500
1000
Protein denaturation
74.41
76.72
81.48
89.19
—
—
—
—
27.33
29.26
33.83
42.51
30.87
32.79
37.30
44.44
Inhibition (%)
Cyclooxygénase
—
—
—
—
83.46
89.90
93.39
97.88
24.72
34.14
40.70
47.07
28.14
40.89
53.55
63.36
5-lipoxygénase
—
—
—
—
82.03
88.63
92.96
95.31
31.26
45.60
58.17
66.79
36.12
48.24
64.11
77.48
The percentage values were obtained using various concentrations of test compounds, and readings are presented as mean of triplicates.
Table 3: IC50 value of aqueous and ethanolic extracts of Dissotis thollonii on human whole blood evaluated by luminal-amplified
chemiluminescence.
Aqueous extract
Ethanolic extract
Ibuprofen
Prednisolone
WB
>100
4.89 ± 0.49
12.71 ± 0.13
—
Oxidative burst (IC50 (μg/ml))
PMNs
5.74 ± 0.65
2.96 ± 0.12
12.53 ± 0.21
—
MQ
7.47 ± 2.68
3.28 ± 0.09
13.28 ± 1.07
—
T-cell proliferation (IC50 (μg/ml))
16.89 ± 2.55
3.29 ± 1.96
—
<3.10
The IC50 values were obtained using various concentrations of test compounds, and readings are presented as mean ± SD of triplicates. WB: whole blood;
PMNs: polymorphonuclear leukocytes; MQ: mice peritoneal macrophages.
observed with an IC50 of 5. 74 μg/ml and an IC50 of 2.96 μg/
ml with aqueous and ethanolic extracts. These extracts again
inhibited the ROS produced by the macrophages with an
IC50 of 7.47 μg/ml for the aqueous extract and an IC50 of
3.28 μg/ml for the ethanolic extract.
With respect to T-cell proliferation, both extracts
showed significant antiproliferative property. Table 3 shows
that the aqueous extract has an antiproliferative activity with
an IC50 of 16.86 μg/ml, while the ethanolic extract is 3.29 μg/
ml and that of prednisolone is less than 3.10 μg/ml.
8. In Vivo Antiarthritis Assays
8.1. Effect of D. thollonii Treatment on Monoarthritis Induced
by Zymosan A in Rats. After injection of zymozan A in
articulation of rats, knee joint results in the significant growth
(p < 0.001) in the articular diameter, which is maximal from
the first hour and still from the fifth day after the administration of zymosan A (Figure 1). Ethanolic and aqueous
extracts of D. thollonii leaves (250 and 500 mg/kg) significantly (p < 0.05) inhibited zymosan A-induced edema. The
inhibition of edema caused by the injection of the zymosan A
was significant (p < 0.01) from the 3rd hour with a percentage
inhibition of 52.90% for the ethanolic extract (500 mg/kg) and
from the 4th hour with the aqueous extract (33.30%, p < 0.05,
500 mg/kg) and diclofenac (36.30%, p < 0.01, 5 mg/kg). The
maximum and significant inhibitory effect (p < 0.001) was
observed at the 5th day for the ethanolic extract (81.80%,
500 mg/kg), at the 5th day for the aqueous extract (69.30%,
500 mg/kg), and at day 3 for diclofenac (53.10%, 5 mg/kg).
Evidence-Based Complementary and Alternative Medicine
7
10
λ
λ
9
λ
λ
λ
λ
Joint diameter (mm)
λ
λ
λ
8
λ
λ
λ
λ λ
λ λ
λ λ
λ
λ
λ
7
λ
λ
λ
λ
λa
λb
λb
βc
λ
λ
λ
λ
λ
λ
λb
αc
λ
λ
λ
λ
λ
λ
λ
λ
λb
λc
λ
λb
βc
λ
λ
λ
λ
λc
λb
λ
αc
λb
c
λc
βc
λc
αc
c
c
6
5
0
1
2
3
4
Healthy control
Arthritic control
Diclofenac 5 mg/kg
5
6
Time (hour)
AE 250mg/kg
AE 500mg/kg
24
48
72
96
120
EE 250mg/kg
EE 500mg/kg
Figure 1: Effect of aqueous (AE) and ethanolic (EE) extracts of Dissotis thollonii on joint diameter in zymosan A-induced monoarthritis. Values
are expressed as mean ± SEM for six animals and analyses by two-way ANOVA followed by Bonferroni post hoc test, αp < 0.05, βp < 0.01, and
λ
p < 0.001 when compared with the healthy control and ap < 0.05, bp < 0.01, and cp < 0.001 when compared with the arthritis control.
Figure 2 shows the histological analysis of the knee joints
taken after injection of zymosan A. The architecture of the
joint has a normal appearance in the nonarthritic control
group; when in arthritic control, there is an erosion of the
synovial membrane, a very large joint space, and erosion of
bone and articular cartilage. However, treatment with
aqueous and ethanolic extracts of D. thollonii prevented the
destruction of the articular architecture of treated animals.
8.2. Effect of D. thollonii Treatment on Polyarthritis Induced by
CFA in Rats
8.2.1. Effect of D. thollonii Treatment on Joint Diameter.
After the injection of CFA into the tail, the diameter of the
joint increased significantly on the 11th day after injection
(Figure 3). Aqueous and ethanolic extracts (500 mg/kg)
bring out the significative (p < 0.001) reduction of joint
diameter from day 13 with inhibition percentages of 54.34%
and 65.48%, respectively, compared with the negative
control. The inhibitory effect of different extracts remained
significant during the days of experimentation. The maximum significant activity (p < 0.001) is observed at day 23 for
diclofenac (60.03%, 5 mg/kg), at day 23 for the aqueous
extract (71.85%, 500 mg/kg), and at the 31st day for the
ethanolic extract (79.03%, 500 mg/kg).
8.2.2. Effect of D. thollonii on Mechanical Nociceptive Pain
Threshold. The induction of arthritis with CFA in rats
generates the hypersensitivity threshold to mechanical pain
which increased to day 10 still the end of experimentation
(34th day) with negative control. The continuous administration of different treatments significantly protected
(p < 0.05, p < 0.01, and p < 0.001) animals against mechanical pain which was observed from the 10th day until
the end of treatment. The maximal inhibitory activity is
observed on the 12th day with the aqueous extract (500 mg/
kg, p < 0.001, 69.00%), and on the 34th day with the ethanolic extract (500 mg/kg, p < 0.001, 70.35 %) and at day 34
with diclofenac (5 mg/kg, p < 0.001, 42.05 %). However,
there is little improvement observed with the aqueous extract (250 mg/kg) with respect to mechanical pain sensitivity
threshold (Figure 4).
8.2.3. Effect of D. thollonii on Arthritic Score. The arthritic
score does not materialize in normal controls, but in the
groups that received CFA, it is very well materialized. Arthritic control groups are more affected from 11 until the end
of treatment (Figure 5). Nevertheless, animals treated with
different extracts showed a decrease in arthritic score values
until the end of treatment. At the dose of 500 mg/kg,
ethanolic extract has more activity than the aqueous extract
8
Evidence-Based Complementary and Alternative Medicine
B
A
C
(a)
(b)
(c)
(d)
(e)
Figure 2: Histopathological analysis of ankle joints stained with H&E. (a) Healthy control showing normal structure with small joint space;
(b) Arthritic control showing very large joint space (A), erosion of articular cartilage (B), and bone erosion (C), (c) diclofenac 5 mg/kg
treated, (d) aqueous extract (AE) 500 mg/kg treated, and (e) ethanolic extract (EE) 500 mg/kg treated showing a decrease in joint space,
erosion of the synovial membrane and a reduction of erosion of articular cartilage.
because it has not only delayed the onset of arthritis materialization, but has also reduced the physical value of
materialization. However, animals treated with diclofenac
and ethanolic extract presented a significant (p < 0.001)
reduction in the physical value of this materialization at the
end of treatment.
8.2.4. Effect of D. thollonii on Body Weight and Organ
Weight. In the arthritic control group, body weight decreased
progressively and became significant (p < 0.01) from week 2 on
positive group animals. In animals treated with the ethanolic
extract (500 mg/kg), the change in body weight was significant
(p < 0.05) at week 3 and persisted throughout treatment
compared with arthritic control group animals. In animals
from different groups treated with aqueous extract (500 and
250 mg/kg), with ethanolic extract (250 mg/mg), and with
diclofenac (5 mg/kg), the change in body weight did not occur
and was not significant (p < 0.05) throughout treatment
(Figure 6).
Results showed that weights of the liver, spleen, and
kidney increased significantly (p < 0.01), and the weight of
thymus decreased significantly (p < 0.01) in all control animals arthritic compared with the healthy control group.
Recovery of organ weight balance was observed with continuous administration of the different extracts in arthritic
animals compared with negative control (Figure 7).
8.2.5. Effect of D. thollonii Extracts on Hematological
Parameters. The results of the change in hematological
parameters are shown in Table 4. In animals in the arthritic
Evidence-Based Complementary and Alternative Medicine
9
λ
130
λ
λb
λ
λc
λb
115
λ
β
α
105
λc
λc
βc
λc
βc
λa
λc
λc
λc
λc
λc
λb
λc
λc
λa
λc λc
λc
110
λ
λ
λ
120
λ
λ
λ
λ
125
Joint diameter (% increase)
λ
λ
λc λc
λc
λc
αc
αc
αc
13
15
17
19
21
Period (week)
λc λc
λc
λc
λc
λc λc
λc
λc
λc
λc
βc
βc
λc
λc
λc
λc
λc
βc
λc
λc
βc
c
λc
λc λc
λc
λc
λc
βc
αc
βc
33
35
100
95
1
3
5
7
9
11
Healthy control
Arthritic control
Diclofenac 5 mg/kg
23
25
27
29
AE 250 mg/kg
AE 500 mg/kg
31
EE 250 mg/kg
EE 500 mg/kg
Figure 3: Effect of aqueous (AE) and ethanolic (EE) extracts of Dissotis thollonii on change in joint diameter in CFA-induced arthritis. Values
are expressed as mean ± SEM for six animals and analyses by two-way ANOVA followed by Bonferroni post hoc test, αp < 0.05, βp < 0.01, and
λ
p < 0.001 when compared with the healthy control and ap < 0.05, bp < 0.01, and cp < 0.001 when compared with the arthritic control.
120
110
100
λc
90
λc
Sensibility (%)
80
λλc
λb
λb λc λc
70
λa
λc
60
λ
50
λc
λc λc
λc
λc
λc
λc
λc λc
λc
λc λc
λc λc
λc
λc
λc
λc
λc
λ
λ
λ
16 18 20
Period (week)
22
24
λc λc
λc
λc λc
λc
λc
λ
λc
λc
λc λc
λc
λc
λc
λc
λc
λc λc
λc λcλc
λc
λc
λc λc λc
λc λc λc
λc λc
λc
λc
λc
λc λc
40
30
λc
λ
λ
20
λ
λ
λ
λ
λ
λ
28
30
32
34
10
0
1
2
4
6
8
10
12
14
Healthy control
Arthritic control
Diclofenac 5 mg/kg
AE 250mg/kg
AE 500mg/kg
26
EE 250mg/kg
EE 500mg/kg
Figure 4: Effect of aqueous (AE) and methanolic (EE) extracts of Dissotis thollonii on mechanical hyperalgesia in CFA-induced arthritis.
Values are expressed as mean ± SEM for six animals and analyses by two-way ANOVA followed by Bonferroni post hoc test, βp < 0.01 and
λ
p < 0.001 when compared with the healthy control and ap < 0.05, bp < 0.01, and cp < 0.001 when compared with the arthritic control.
control group, platelet and WBC levels increased significantly (p < 0.001) in contrast to RBC, hemoglobin,
and of hematocrit which decreased significantly
(p < 0.001) compared with the healthy control. In treated
animals with different doses of extracts (250 and
500 mg/kg) and diclofenac (5 mg/kg), the various parameters evaluated are close to those of animals in the
healthy control group.
10
Evidence-Based Complementary and Alternative Medicine
2.5
λ
2.0
λ
λ
c
c
λ
λ
Arthritis score
1.5
λ
λ
β
β
λ
λ
1.0
β
λ
b
b
c
0.5
c
bc
c
c
c
c
0.0
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
29
31
33
35
–0.5
1
3
5
7
9
11
13
15 17 19 21
Period (week)
Healthy control
Arthritic control
Diclofenac 5 mg/kg
23
25
27
AE 250mg/kg
AE 500mg/kg
EE 250mg/kg
EE 500mg/kg
Figure 5: Effect of aqueous (AE) and ethanolic (EE) extracts of Dissotis thollonii on the arthritis score in CFA-induced arthritis. Values are
expressed as mean ± SEM for six animals and analyses by two-way ANOVA followed by Bonferroni post hoc test, λp < 0.001 when compared
with the healthy control and bp < 0.01 and cp < 0.001 when compared with the arthritic control.
115
Relative body weight (%)
110
105
λ
95
β
λ
λ
λ
α
λ
λb
λa
λa
100
λ
λ
λ
λ
λ
λ
λ λ
90
λ
λ
85
0
1
2
3
4
5
Period (week)
Healthy control
Arthritic control
Diclofenac 5 mg/kg
AE 250mg/kg
AE 500mg/kg
EE 250mg/kg
EE 500mg/kg
Figure 6: Effect of aqueous (AE) and ethanolic (EE) extracts of Dissotis thollonii on body weight in CFA-induced arthritis. Values are
expressed as mean ± SEM for six animals and analyses by two-way ANOVA followed by Bonferroni post hoc test, αp < 0.05, βp < 0.01, and
λ
p < 0.001 when compared with the healthy control and ap < 0.05 and bp < 0.01 when compared with the arthritic control.
8.2.6. Effect of D. thollonii Extracts on Biochemical
Parameters. The biochemical parameters are shown in
Table 5. Serum levels in arthritic animals show a significant
(p < 0.001) increase in AST, ALT, ALP, and creatinine and a
significant decrease (p < 0.001) of total protein relative to the
serum level of the healthy control. These different levels
Evidence-Based Complementary and Alternative Medicine
12
β
α
a
10
Organ weight (g)
11
b
8
6
4
2
β
α
β
a
β
β
a
αb
b
0
Liver
Kidney
Thymus
Spleen
AE 500 mg/kg
EE 250 mg/kg
EE 500 mg/kg
Healthy control
Arthritic control
Diclofenac 5 mg/kg
AE 250 mg/kg
Figure 7: Effect of aqueous (AE) and ethanolic extracts (EE) of Dissotis thollonii on organ weight in CFA-induced arthritis. Values are
expressed as mean ± SEM for six animals and analyses by one-way ANOVA followed by Bonferroni post hoc test, αp < 0.05 and βp < 0.01
when compared with the healthy control and ap < 0.05 and bp < 0.01 when compared with the arthritic control.
Table 4: Influence of the aqueous and ethanolic extracts of Dissotis thollonii on haematological in CFA-induced arthritis in rats.
Treatment
Healthy control
Arthritic control
Diclofenac
Aqueous extract
Ethanolic extract
Dose (mg/kg)
—
—
5
250
500
250
500
Haemoglobin (g/dl)
16.40 ± 0.73
9.43 ± 0.20λ
14.15 ± 0.62b
14.28 ± 0.67b
15.55 ± 0.59c
14.33 ± 1.02b
16.88 ± 1.32c
Hematocrit (%)
38.78 ± 1.03
25.65 ± 1.27λ
33.88 ± 2.59a
31.15 ± 1.23α
37.73 ± 1.28c
32.20 ± 1.70
38.38 ± 0.99c
Platelet (109/L)
365.25 ± 26.23
554.75 ± 21.54λ
481.75 ± 13.31β
478.74 ± 18.71β
383.50 ± 9.75c
426.00 ± 18.11c
374.75 ± 8.28c
RBC (million/μl)
7.22 ± 0.34
3.90 ± 0.37λ
5.65 ± 0.24a
5.74 ± 0.40a
6.93 ± 0.25c
6.31 ± 0.38c
7.36 ± 0.29c
WBC (109/L)
7.85 ± 0.44
13.78 ± 0.56λ
10.63 ± 0.64αa
11.85 ± 0.80λ
10.25 ± 0.41b
11.15 ± 0.48β
8.28 ± 0.42c
CFA: complete Freund’s adjuvant; RBC: red blood cell; WBC: white blood cell. Each value represents the mean ± ESM of six animals. αp < 0.05; βp < 0.01;
λ
p < 0.001 statistically significant compared with the healthy control. Each value represents the mean ± ESM of 6 animals. ap < 0.05; bp < 0.01; cp < 0.001
statistically significant compared to arthritic control.
Table 5: Effect of aqueous and ethanolic extracts of Dissotis thollonii on serum parameters in CFA-induced arthritis in rats.
Treatment
Healthy control
Arthritic control
Diclofenac
Aqueous extract
Ethanolic extract
Dose (mg/kg)
—
—
5
250
500
250
500
ALT (U/I)
47.09 ± 1.91
77.88 ± 2.07λ
63.46 ± 1.50λc
63.97 ± 1.72λc
55.14 ± 1.16αc
55.41 ± 1.84αc
46.12 ± 1.42c
AST (U/I)
173.89 ± 11.61
423.09 ± 23.51λ
250.70 ± 16.76βc
299.79 ± 11.76λc
222.59 ± 6.07c
253.30 ± 2.77βc
177.62 ± 7.62c
ALP (U/I)
90.27 ± 3.36
444.38 ± 13.69λ
240.11 ± 16.28λc
317.40 ± 17.20λc
207.33 ± 9.89λc
270.97 ± 7.43λc
143.00 ± 5.34c
Creatinine (μmol/l)
0.48 ± 0.07
1.39 ± 0.13λ
0.93 ± 0.03λc
0.98 ± 0.05λc
0.69 ± 0.03c
0.86 ± 0.3βc
0.60 ± 0.02c
Total protein (g/dl)
7.45 ± 0.24
5.90 ± 0.17β
6.65 ± 0.28
6.35 ± 0.34
7.31 ± 0.19a
7.16 ± 0.29a
7.54 ± 0.28b
CFA: complete Freund’s adjuvant; ALP: alkaline phosphatase; AST: aminotransferase; ALT: alanine aminotransferase. Each value represents the mean ± ESM
for six animals and analyses by one-way ANOVA followed by Bonferroni post hoc test, αp < 0.05, βp < 0.01, λp < 0.001 when compared with the healthy
control and ap < 0.05, bp < 0.01, cp < 0.001 when compared with the arthritic control.
improved considerably in animals treated with different
extracts (aqueous and ethanolic) and diclofenac.
8.2.7. Effect of D. thollonii Extracts on Oxidative Stress
Parameters. The results on the change in oxidative stress
parameters shown in Table 6 indicate that levels of NO and
MDA increased significantly (p < 0.01) in arthritic animals,
whereas levels of glutathione, catalase, and SOD decreased
significantly (p < 0.01) compared with the healthy control.
Animals treated with aqueous and ethanolic extracts, such as
diclofenac, tend to improve these values.
8.2.8. Histopathological Study. A histopathological study of
the knee joint showed no evidence of inflammation, bone
erosion, cartilage destruction, or cellular infiltration in the
healthy control. Animals in the arthritic control group
12
Evidence-Based Complementary and Alternative Medicine
Table 6: Effect of aqueous and ethanolic extracts of Dissotis thollonii on some parameters of oxidative stress in CFA-induced arthritis in rats.
Dose (mg/kg)
NO (μM)
Glutathion (×103 activity) Catalase (activity) MDA (×103 mol/l) SOD (activity)
—
0.048 ± 0.003
0.28 ± 0.03
52.99 ± 0.67
0.087 ± 0.021
1.93 ± 0.02
0.17 ± 0.01β
36.89 ± 0.72λ
0.138 ± 0.027
1.51 ± 0.03λ
—
0.089 ± 0.003λ
5
0.044 ± 0.001c
0.21 ± 0.01
37.72 ± 0.74c
0.112 ± 0.035
1.92 ± 0.03c
λa
β
250
0.073 ± 0.005
0.18 ± 0.02
45.33 ± 0.97
0.127 ± 0.020
1.93 ± 0.02c
Aqueous extract
500
0.064 ± 0.003αc
0.24 ± 0.01
51.45 ± 1.70c
0.109 ± 0.038
1.95 ± 0.04c
βc
c
250
0.067 ± 0.001
0.22 ± 0.01
48.95 ± 1.18
0.125 ± 0.005
1.94 ± 0.02c
Ethanolic extract
500
0.046 ± 0.003c
0.27 ± 0.01b
52.39 ± 4.09c
0.071 ± 0.020
1.95 ± 0.08c
Treatment
Healthy control
Arthritic control
Diclofenac
Each value represents the mean ± ESM for six animals and analyses by one-way ANOVA followed by Bonferroni post hoc test, αp < 0.05, βp < 0.01, and
λ
p < 0.001 when compared with the healthy control and ap < 0.05, cp < 0.001 when compared with the arthritic control.
presented joint architecture exhibiting cartilage destruction,
bone erosion, and cellular infiltration; but animals treated
with different extracts (250 and 500 mg/kg) and diclofenac
(5 mg/kg) showed a significant protection of the architecture
of the joint (Figure 8) by reducing bone erosion, cartilage
destruction, and cellular infiltration.
9. Discussion
In the present study, the results show that aqueous and
ethanolic extracts of D. thollonii have well anti-inflammatory
properties in vitro in several models such as the inhibition of
denaturation of proteins, 5-LOX, COX, and ROS. Inflammation, which is a very complex physiopathological
response, involves the production of free radicals derived
from neutrophils, NO, ROS, cytokines, and prostaglandins
during its process [42]. Protein denaturation is the process
by which proteins lose their tertiary structure and secondary
structure. Proteins denaturation is a well-documented cause
of inflammation [43]. The inflammation mechanism involves a series of events in which the metabolism of arachidonic acid plays an important role. Phenylbutazone,
salicylic acid, flufenamic acid (anti-inflammatory drugs),
etc., have shown a dose-dependent ability to thermally induced protein denaturation [44]. The pathogenesis of inflammatory diseases involves the overproduction of
substances such as prostaglandin I2, thromboxane A2,
prostaglandin E2, arachidonic acid, and leukotrienes
through two metabolic pathways, the cyclooxygenase (COX)
pathway and the 5-lipoxygenase (5-LOX) pathway [45]. In
various inflammatory and allergic disorders, COX and 5LOX are the main enzymes in the synthesis of prostanoids
and eicosanoids from polyunsaturated fatty acids. The effective reduction of chronic inflammatory conditions is
important by double inhibition of LOX and COX [46].
Substances capable of producing double inhibition of COX
and 5-LOX with consequent substantial reduction in leukotriene and prostaglandin production produce a broad
spectrum of anti-inflammatory activity and can be considered to have an excellent profile of pharmacological safety
in clinical practice [47]. The anti-inflammatory activity of
the aqueous and ethanolic extract of D. thollonii was determined using two methods, which were cyclooxygenase-2
(COX-2) and lipoxygenase (LOX) assays. All extracts tested
showed significant activities vis-à-vis both tests. The inhibitory activity of aqueous and ethanolic extracts of D.
thollonii on the denaturation of proteins, the cyclooxygenase
and 5-lipoxygenase pathways, show that these two extracts
are capable of significantly inhibiting the production of
prostaglandins and leukotrienes, which gives these two
extracts anti-inflammatory properties. These results are
justified by the fact that compounds such as betulinic acid
isolated from D. thollonii possess an in vitro inhibition
property of cyclooxygenase (COX-1 and COX-2) and leukotriene B4 formation mediated by 5-LOX [48, 49].
The injection of zymosan into the knee joint causes a
proliferative inflammatory monoarthritis resulting from the
onset of an inflammatory reaction accompanied by hypernociception, an influx of neutrophils and leukocytes and
then production by the infiltrated synovial cells and/or many
inflammatory mediators include cytokines, free radicals,
prostaglandins, and leukotrienes, which are responsible for
the breakdown of articular cartilage, pannus formation, and
synovial hypertrophy [50, 51]. Clinical management of arthritis is primarily aimed at reducing the intensity of pain,
joint swelling, and then preventing or significantly reducing
bone erosion and the various joint damages observed [52]. In
this study, aqueous and ethanolic extracts of D. thollonii
significantly reduced joint swelling up to the fifth hour of
single administration, and the effect remains significant until
the fifth day in continuous treatment. Similarly, the histological analysis of the joints has shown that the extracts of
this plant, as well as diclofenac, induce a protective effect on
the alteration of the synovial membrane, on bone erosion
and considerably reduce cartilage lesions.
The caudal injection-induced polyarthritis of CFA in rats
is a very good experimental model for preclinical evaluation
of new antiarthritic agents; this model has many similarities
with human rheumatoid diseases [53–55]. In addition, CFA
injection induces hyperalgesia and allodynia by altering
sensitivity to high-threshold nociceptor transduction [56].
In the present study, D. thollonii extracts showed an antiarthritic potential on all evaluated inflammatory parameters.
These extracts significantly reduced the diameter of the joint
in arthritic animals, and they significantly reduced the
susceptibility of arthritic animals to mechanical pain, and
they significantly improved the arthritic score. On these
important parameters, the inhibitory effect of the extracts
was significantly greater than that of diclofenac. In many
disease states such as rheumatoid arthritis, decreased body
weight is an important predictor of health [57]. The results of
this study show that the extracts improved the body mass of
the treated animals when compared with the untreated
animals. During the development of rheumatoid arthritis,
Evidence-Based Complementary and Alternative Medicine
13
B
A
C
(a)
(b)
(c)
(d)
(e)
Figure 8: Histopathological analysis of ankle joints stained with H&E. (a) Healthy control showing normal structure with small joint space;
(b) arthritic control showing very large joint space (A), destruction of cartilage (B), bone erosion (C), and cellular infiltration (D); (c)
diclofenac 5 mg/kg treated, (d) aqueous extract (AE) 250 mg/kg treated, and (e) ethanolic extract (EE) 250 mg/kg treated showing a decrease
in joint space, a reduction of cells infiltration and reduction of cartilage destruction.
several enzymes are highlighted and represent good indices
according to their importance (case of alkaline phosphatase
and transaminases). The increase in serum levels of these
enzymes would be implicated in periarticular osteopenia, in
bone erosion and also indicate severe liver injury resulting in
the production of biologically active substances in the inflammatory process [58–61]. In the present study, arthritic
rat serum values ALP, AST, and ALT significantly increases,
whereas in animals submited to different treatments
(aqueous and ethanolic extracts of the leaves of D. thollonii
or diclofenac) increased levels of these enzymes have been
significantly reduced.
In the pathogenicity of rheumatoid arthritis, ROS, which
are considered to be enhancers of inflammatory proliferation
of the synovial membrane, play a key role in that their
increased production increases the destruction of cartilage
and even bones, activates or removes NF-κB transcription
factor, induces the production of many cytokines, and activates enzymes such as COX and 5-lipoxygenase or even
inducible nitrogen monoxide [62–65]. The NF-κB transcription factor is present in the cytosol of many cells where it
is bound with the factor I-kB, especially those expressing
cytokines, growth factors, chemokines, and adhesion molecules. Activation of the cell causes the phosphorylation of I-kB
which is degraded followed by the release of the factor NF-κB,
which is introduced into the nucleus of the cell and causes the
transcription of many proinflammatory mediators including
iNOS, COX-2, and TNF-α and IL-1β, IL-6, and IL-8 [66];
thus, bone erosion and cartilage destruction observed in
rheumatoid arthritis are due to overproduction of cytokines
14
and inflammatory mediators. The aqueous and ethanolic
extracts of the leaves of D. thollonii showed excellent antioxidant power, and in this study, the inhibition of the production of extracellular ROS in whole blood and in various
phagocytic cells was significative (neutrophils and macrophages). This activity is thought to be due to the inhibition of
pro inflammatory cytokine production (TNF alpha), the
inhibition of denaturation of protein and the inhibition of the
activity of proinflammatory enzymes such as COX and 5lipoxygenase.
To investigate the effect of aqueous and ethanolic
extracts of the leaves of D. thollonii on another aspect of
the cellular immune response, the assay on T-cell proliferation was used. The results show that these D. thollonii
extracts significantly inhibit T-cell proliferation, with a
very significant result for the extracts compared with
prednisolone used as a positive control. The results
suggest that the compounds present in D. thollonii extracts are capable of significantly modulating, at different
stages, the immune response. The significant decrease in
the diameter of the joints observed macroscopically and
histopathologically, followed by a decreased insensitivity
to pain, clearly reveals the anti-inflammatory, antihyperalgic, and antiarthritic potential of D. thollonii extracts. It is possible that the effect of our different extracts
is associated with an inhibition of the phosphorylation of
the transcription factor NF-κB. This is justified by the fact
that compounds such as betulinic acid, ellagic acid,
β-sitosterol, and ajuronic acid present in the extract of D.
thollonii [30] have shown their ability to prevent degradation of the inhibitory IκB-α protein, inducing inhibition
of NF-κB nuclear transcription factor activation, subsequent reduction of COX-2 expression, iNOS, inflammatory cell quantity, the levels of TNF-α, IL-6, IL-8,
and increase the production of IL-10 [67–72]. Given that,
during the development of the inflammatory reaction, the
stimulation of the production of TNF-α, IL-1β, NO, and
PGE2 would be linked to the activation of the NF-κB/IκBα axis [73]; thus, the suppression of this axis has a significant therapeutic effect [72]. In addition, D. thollonii
extracts showed in vitro inhibitory effects on TNF-α
production, intracellular ROS production, leukocyte
migration, and cell-proliferative HeLa cell line [24].
Evidence-Based Complementary and Alternative Medicine
the isolation of new anti-inflammatory and/or antiarthritic
products.
Data Availability
All data supporting our findings are adequately contained
within the manuscript.
Ethical Approval
The experimental procedures have been approved by the
local Ethics Committee and are in accordance with the
guidelines for the study of pain in awake animals, published
by the NIH (publication no. 85-23, “Principles of Animal
Protection,” Laboratory, Study of Pain, Ministry of Scientific
Research and Technology, which adopted the European
Union Guidelines on Animal Care and Experimentation
(EWC 86/609).
For the donation of human blood samples, all processes
of collecting blood are accepted by the Independent Ethics
Committee, ICCBS, University of Karachi, N°: ICCBS/IEC008-BC-2015/Protocol/1.0. The blood donors were provided
informed approval for the use of their blood for the purposes
of this study.
Conflicts of Interest
MM (PhD) is a Senior Lecturer in the Department of Animal
Biology, Faculty of Science, University of Dschang, Cameroon. AAD (PhD) is a Senior Lecturer in the Department of
Animal Biology, Faculty of Science, University of Yaounde 1,
Cameroon. DNSF, TEG, YNW, MMMV, and ACF are PhD
students in the Department of Animal Biology, Faculty of
Science, University of Dschang, Cameroon. AG is an Associate Professor in the Department of Animal Biology,
Faculty of Science, University of Dschang, Cameroon. The
authors declare that they have no conflicts of interest.
Authors’ Contributions
DNSF, MM, and AG designed the work. DNSF, MM, TEG,
AAD, MMMV, and ACF conducted the work and collected
and analysed the data. MM, NYW, AG, and AAD drafted the
manuscript and revised it critically. All authors agree to be
accountable for all aspects of the work.
10. Conclusions
At the end of this study, the conclusion that we can deduce is
that D. thollonii is a plant containing several groups of
chemical compounds with anti-inflammatory and antiarthritic potential. These properties have been evaluated by in
vitro and in vivo studies. In vitro studies have shown that D.
thollonii has a very strong anti-inflammatory property: inhibition of protein denaturation, inhibition of 5-LOX, inhibition of COX and ROS and in vivo studies that have
shown antiarthritis activity of the plant on a zymosan-induced monoarthritis model and a model of CFA-induced
polyarthritis in the rat. These results confirm the utilization
of this plant in the traditional treatment of chronic inflammatory diseases and consider it a potential candidate for
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