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Ann Colorectal Res. 2015 June; 3(2): e29511. DOI: 10.17795/colorectalresearch29511
Published online 2015 June 1. Research Article
Antioxidant and Anti-Inflammatory Effects of Gel and Aqueous Extract of
Melilotus officinalis L. in Induced Ulcerative Colitis: A Rattus norvegicus Model
Ali Reza Safarpour 1; Fatemeh Kaviyani 2; Masood Sepehrimanesh 1,*; Nasrollah Ahmadi 3;
Omid Koohi Hosseinabadi 4; Nader Tanideh 2; Najmeh Showraki 5
1Gastroenterohepatology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran
2Transgenic Technology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran
3Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, IR Iran
4Laboratory Animals Center, Shiraz University of Medical Sciences, Shiraz, IR Iran
5Department of Oral Medicine, School of Dentistry, Shiraz University of Medical Sciences, Shiraz, IR Iran
*Corresponding author: Masood Sepehrimanesh, Gastroenterohepatology Research Center, Research Tower, Nemazee Teaching Hospital, Shiraz University of Medical Sciences, P. O.
Box: 7193-51311, Shiraz, IR Iran. Tel: +98-7136281442, E-mail: sepehrimaneshmasood@gmail.com
Received: May 3, 2015; Revised: June 9, 2015; Accepted: June 12, 2015
Background: Ulcerative colitis (UC) is a chronic condition of intestine illness accompanied by some unknown etiology with different
immune, genetic and environmental factors.
Objectives: The current study aimed to evaluate the antioxidant and anti-inflammatory effects of the Melilotus officinalis L. in the acetic
acid induced UC in rats.
Materials and Methods: The plant aqueous extraction and high performance liquid chromatography and Ferric Reducing Antioxidant
Power (FRAP) assay were performed on aqueous extract to identify its compounds and antioxidant activities. Also, 70 adult male rats were
and UC was induced using 3% acetic acid solution. They received different daily doses of M. officinalis L. in two forms (orally, 500 and 1000
mg/kg) and gel extract (10% and 20%). On the 7th day, the colon tissues were examined regarding the macroscopic and histopathology
lesions plus oxidative stress and compared to the positive and negative control groups.
Results: HPLC analysis revealed that five grams of the flower powder contained 9.7 mg gallic acid, 99 mg catechin, 21.9 mg caffeic acid,
0.86 mg chlorogenic acid, 1.13 mg quercetin, 548.9 mg cinnamic acid, 289 mg coumarin and 126 mg p-coumaric acid. The FRAP value of
the extract was 2.91 ± 0.14 μM/g. There were signicant dierences between the group of rats which received the gel or aqueous extract of
the flower compared to the negative control group using normal saline and the base gel and they had no significant differences with the
positive control group using the Asacol, regarding the pathologic, malondialdehyde, and weight improvements.
Conclusions: It can be concluded that the M. officinalis L. extract can be used as an effective medicine to treat UC in animal model and also
in human subjects.
Keywords: Colitis, Ulcerative; Inflammation; Extract; Ulcerative Colitis; Asacol
Copyright © 2015, Colorectal Research Center and Health Policy Research Center of Shiraz University of Medical Sciences. This is an open-access article distributed
under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits
copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.
1. Background
Inflammatory bowel diseases (IBD), including Crohn’s
disease (CD) and ulcerative colitis (UC) are chronic in-
flammatory disease of the gastrointestinal tract. In fact,
UC is a relapsing non-transmural inflammatory disease
restricted to the colon and is one of the idiopathic dis-
eases of IBD (1). The incidence of UC remained stable
over the past 30 years, but, the disease become more
prevalent due to the early age of onset and low mortal-
ity, albeit with significant morbidity (2-4). The etiolo-
gies of the IBD are unknown and therefore there are no
causal treatments. In addition, patients with UC have
substantial impairments in health-related quality of
life (QoL) due to their symptoms, particularly during
periods of disease activity (5). On the other hand, the
current treatment is not completely successful and it is
frequently connected with adverse effects (6). It is sug-
gested that the plant and its products may provide an
option for the alternative or supplementary treatment
of the patients with IBD (7).
There are several ways to induce UC in the animal
model; they include using trinitrobenzene sulfonic acid
(TNBS) (8), dextran sulfate sodium (9), and acetic acid
(10, 11). Induction of UC using acetic acid had no mortal-
ity and the general health of the rats was similar to that
of the controls (12). Therefore, this method is widely sug-
gested as a useful model for the studies on the patho-
physiology and treatment of UC and it was performed
in the current study.
A number of traditional plants were reported in the
literature, but there was little scientific effort to validate
their effects. One of them is the genus of Melilotus, fam-
ily of Fabaceae. Data regarding traditional phytotherapy
Safarpour A et al.
Ann Colorectal Res. 2015;3(2):e295112
show that the Melilotus species (sweet clover) were used
to reduce spasm, in the liver diseases and as a diuretic
(13). Melilotus officinalis L. also named yellow sweet clover
is a plant not only used as food and forage but also as a
medicine. The preventive effect of the whole plant ex-
tract on the experimental atherosclerosis in rabbits was
reported (14). However, the number of reports on the M.
officinalis L. application in animal and human diseases is
scarce and there are no reports on the efficacy of M. offici-
nalis L. in UC treatment.
2. Objectives
The current study aimed to determine the healing ef-
fects of M. officinalis L. as dietary and gel form and com-
pare them with that of Asacol on the tissue histopatho-
logical changes and malondialdehyde (MDA) level in
male rats with experimentally acetic acid induced UC.
3. Materials and Methods
3.1. Ethical Statement
This study was approved by the animal care and use
committee of Shiraz university of medical sciences, Shi-
raz, Iran. All efforts were made to prevent any unneces-
sary and harmful animal handling.
3.2. Plant Materials and Aqueous Extraction
Melilotusofficinalis L. was collected from the Productive
Farm of Baradan Medicinal Plant Co., Abadeh, Iran (GPS
coordinates: 31.160833, 52.650556, with Altitude of 2500
m) during the spring season (May, 2013). All plants were
in the flowering stage of developing and the taxonomic
identification of each plant was confirmed by the Fars
agriculture Jihad educational complex, Fars, Iran and the
local herbarium number 14684. The plant also matched
with the digital herbarium of Botanical Garden and Bo-
tanical Museum Berlin-Dahlem, Freie university, Berlin
(http://ww2.bgbm.org/herbarium/ (Barcode: B -W 14163
-01 0 / ImageId: 385175).
3.3. Aqueous Extraction
The provided plants were dried for five days at room
temperature and then powdered by grinder. Finally, 100
g of plant powder was used for aqueous extraction based
on previous reported procedures (15).
3.4. High Performance Liquid Chromatography
(HPLC)
Phenolic acid constituents of M. officinalis L. were
identified by high performance liquid chromatogra-
phy (HPLC) according to the method reported by Kwok
et al. (16), with some modifications. Briefly, 5 g of plant
powder was dissolved in 50 mL of HPLC grad water and
after 24 hours, the solution was ltered across 0.22 μm
filter. HPLC analyses were performed using an Agilent
1200 HPLC system (Milford, MA, USA) coupled to a pho-
todiode array detector. The samples were separated on
a reversed-phase C18 column (150 mm × 4.6 mm). The
mobile phase consisted of water (solvent A) and formic
acid (solvent B). At time = 0, the solvent A and B were in
1:9 (v/v) ratio. The gradient mobile phase was gradually
changed to 89:11 (v/v) A to B ratio in 20 minutes. Mobile
phase rate was 1 mL/minute and the column tempera-
ture was adjusted at 30°C. The eluent was monitored
by a UV detector and the detection wavelength was set
at 280 and 360 nm. Galic acid, chlorogenic acid, caffeic
acid, quercetin, cumarin, p-cumaric acid, and carvacrol
were used as internal standards.
3.5. Ferric Reducing Antioxidant Power Assay
FRAP assay was performed on aqueous extract accord-
ing to the previous methods (17, 18) with some modifica-
tions. A 20 μL sample of extract with dierent concen-
trations, 31.25, 62.5, 125, 250, 500 mM and 1 M was mixed
with 180 μL of FRAP reagent in the pale wells. Blank
samples also were prepared; then, both real and blank
samples were incubated in water bath for 10 minutes
at 37°C and the absorbance of the samples was deter-
mined against blank ones at 593 nm. Series of stock so-
lution at 12.5, 25, 50, 100 and 200 μg/mL were prepared
(r2 = 0.999) using aqueous solution of FeSO4.7H2O as the
standard curve. The values obtained were expressed as
nanomole (μM) of ferrous equivalent Fe (II) per gram of
freeze dried sample.
3.6. Animal Housing, UC Induction and Treatment
Seventy 250 - 280 g male Sprague Dawley rats were pur-
chased from the Laboratory Animals Center, Shiraz Uni-
versity of Medical Sciences. Animals were randomly allo-
cated into seven equal independent groups and treated
according to the procedure shown in Figure 1.
UC was induced according to the previously reported
protocols (10, 11). Briefly, all animals were fasted over-
night and their bowels were cleaned before induction
of colitis. A polyurethane cannula (2 mm diameter) was
applied for the rectal entrance of acetic acid and the
tip was inserted up to 8 cm proximal to the anus verge.
Two milliliters of 3% acetic acid was administered tran-
srectally into the colon by a cannula during 30 seconds
to induce UC under ketamine and xylazine anesthesia.
The main substance of gel was carboxymethyl cellulose
(CMC) and the gel was produced by mixing 2% sodium
CMC in 5% glycerol and it was continuously stirred with
a mixer in order to prepare a gel-forming agent. In the
next step, M. officinalis L. extracts (10%, and 20%) were
added to deionized water. The mixtures were gradually
added to the Na-CMC with glycerol and finally the pre-
pared gel was homogenized for 30 minutes and all for-
mulations were collected in an aluminum tube in the
refrigerator (11).
Safarpour A et al.
3
Ann Colorectal Res. 2015;3(2):e29511
70 male SD rat
10 male SD rat
10 male SD rat
M. oficinalis
Gel 10%
Enema
2 ml per day
M. oficinalis
Gel 20%
Enema
2 ml per day
M. oficinalis
Aqueous extract
(500 mg/kg)
Oral
1 ml per day
M. oficinalis
Aqueous extract
(1000 mg/kg)
Oral
1 ml per day
10 male SD rat 10 male SD rat 10 male SD rat
10 male SD rat 10 male SD rat
Grouping and UC induction
Group I
Group IV Group VGroup VI Group VII
Group II Group III
Normal saline
Oral
1 ml per day
Asacol
Enema
2 ml per day
Gel base
Enema
2 ml per day
• Daily weigh measurement
• Histopathological evaluations
• MDA assay
Colon tissue sampling After 7 days
Figure 1. Experimental Setup and Rat Groupings
3.7. Tissue Sampling, Macroscopic and Microscop-
ic Evaluations
After seven days, rats were euthanized under deep
ether anesthesia. Then, the distal 8 cm of the colon was
removed and opened by longitudinal incision. The mu-
cosal surface was washed with saline buffer and the mu-
cosal injury was macroscopically assessed using Morris
et al. (19) grading scale. Colon tissue was processed and
stained according to the previously reported procedures
(10, 20-25). Briefly, colon tissues were free of surrounding
fat and fixed in 10% buffered formalin solution for histo-
pathological examination. Formalin-fixed tissues were
processed routinely and embedded in paraffin. Blocks
were cut at 5 μm and stained with hematoxylin-eosin. All
sections were studied and photographed using a light
microscope. The degree of inflammation of the colon was
graded as previously described (26).
3.8. Malondialdehyde Measurement
Colon tissue MDA assessment was performed by mea-
suring thiobarbituric acid reactive substances (TBARS)
in PBS tissue homogenate (27); since MDA is one of the
end-products of lipid peroxidation (LPO), and the extent
of LPO is most frequently measured by estimating MDA
levels (28).
3.9. Statistical Analysis
The results were presented as mean. Differences among
the groups were determined using a one-way ANOVA and
Duncan post-hoc test. All the statistical analyses were per-
formed by SPSS software (version 18, Chicago, IL, USA). P
value less than 0.05 was considered significant.
4. Results
The current study aqueous extraction efficiency was
Safarpour A et al.
Ann Colorectal Res. 2015;3(2):e295114
39%. Chromatogram of M. officinalis L. in 280 nm is shown
in Figure 2. As demonstrated, the comarin had the high-
est concentration in the M. officinalis L. phenolic acid con-
stituents; and comarin levels in aqueous extract of M. offi-
cinalis L. are presented in Table 1. Catechin and cinnamic
acid were the highest compounds in the phenolic acid
and comarin constituents of M. officinalis L. Antioxidant
activity of aqueous extract of M. officinalis L. was mea-
sured based on the FRAP assay.
The current study analysis demonstrated that the FRAP
value for this extract was 2.91 ± 0.14 μM/g. Percentage of
weight changes in different groups during the experi-
mental period is shown in Figure 3. The most weight
change was observed in the normal saline control group
(13.67%), while, the lowest weight changes were observed
in the Asacol control, 20% extract enema, and 1000 mg
oral extract with 1.44%, 1.62%, and 2.31%, respectively.
Mean comparison of macroscopic and microscopic
changes as quantitative values plus MDA level in differ-
ent groups are presented in Table 2. As observed, normal
saline control group had the highest and Asacol control,
20% extract enema, and 1000 mg oral extract showed the
lowest pathological changes in both macroscopic and
microscopic evaluations. Also, changes of MDA levels in
response to different treatments indicated the antioxi-
dant activity of M. officinalis L. extract. The lowest MDA
level (highest antioxidant level) was detected in the gel
20% in enema rout of administration, which was compa-
rable to that of Asacol treatment (Table 2).
mAU
800
700
600
500
400
300
200
100
0
0510 15 20 25 30 min
10.864
12.124
16.640
17.048
17.919
15.450
17.49
3
Figure 2. Chromatogram of Aqueous Melilotus officinalis Extract Provided
by High Performance Liquid Chromatography
Table 1. Characterization of Melilotus officinalis Compounds by
High Performance Liquid Chromatography
Compounds Level (mg/L)
Phenolic acid compounds
Gallic acid 9.75
Catechin 99.06
Caffeic acid 21.99
Chloregenic acid 0.87
Quercetin 1.13
Comarin compounds
Cinnamic acid 548.91
Comarin 289.00
p-comaric acid 126.00
Rutin -
Carvacerol -
Figure 3. Rate of Weight Change and its Trend in Different Groups Dur-
ing the Study Period
16
14
12
8
6
4
2
0
Percentage of weight change
Control (Normal saline)
Control (Base gel)
Control (Asacoi)
OE 500
OE 1000
EE 10%
EE 20%
Group
a
13.67 b
12.16
d
5.69 d
4.81
c
2.31
c
1.44
c
1.62
Differences among the groups were determined using a one-way ANOVA
and Duncan post-hoc test. Significant difference between the groups is
indicated by different superscript letters (P < 0.05).
Table 2. Comparison Between Macroscopic and Microscopic Scores Plus Malondialdehyde Levels in Different Experimental Groupsa, b
Groups Macroscopic Score Microscopic Score b MDA Level (μM)
Control groups
Normal saline 19.0 10.7 1.80
Base gel 18.1 9.9 1.75
Asacol 1.9 1.2 0.93
Melilotus officinalis
500 mg/kg oral extract 8.2 4.7 1.13
1000 mg/kg oral extract 2.1 1.0 1.00
10% gel enema 7.5 4.2 1.13
20% gel enema 1.8 0.8 0.93
a Differences among the groups were determined using a one-way ANOVA and Duncan post-hoc test.
b Significance: P < 0.05.
Safarpour A et al.
5
Ann Colorectal Res. 2015;3(2):e29511
Comparison of histopathological lesions in the colon
tissue of healthy rats and seven different experimental
groups are shown in Figure 4. As indicated, acetic acid in-
duced UC in the colon, demonstrated by lack of epithelial
cells plus infiltration of inflammatory cells in the sub-
mucosal layers. However, by administration of oral and
enema of M. officinalis L., the healing process of the colon
tissue was improved and using 1000 mg/kg oral aqueous
extract or gel 20% enema can completely cure the colon
tissue, comparable to Asacol treatment.
Figure 4. Histopathological Evaluations of the Colon Tissues
A, healthy rat colon with complete crypt, submucosal layer and muscular layer plus healthy epithelium; B, normal saline control group, lack of epithelial
cells plus inflammatory cells infiltration; C, base gel control group, damage of epithelium and inflammation of subepithelial layers; D,500 mg/kg oral
aqueous extract of Melilotus officinalis, formation of epithelial cells but existence of inflammatory cells in the submucosal layers; E, enema of Melilotus
officinalis L. 10% gel, formation of epithelial cells but existence of inflammator y cells in the submucosal layers; F, enema of M. officinalis L. 20% gel, approxi-
mately complete formation of epithelial cells plus crypts and muscularis mucosa formation, mild inflammation of the submucosal layers; G, 1000 mg/kg
oral aqueous extract of Melilotus officinalis L., complete formation of epithelial cells, crypts and muscularis mucosa, very mild inflammation of the sub-
mucosal layers; H, Asacol control group, complete formation of epithelial cells, crypts and muscularis mucosa without any inflammation (H& E staining).
Safarpour A et al.
Ann Colorectal Res. 2015;3(2):e295116
5. Discussion
The current study compared the healing effects of M. of-
ficinalis L. extract in the 500 and 1000 mg/kg orally and
10% and 20% gel forms against acetic acid induced UC by
measuring tissue histopathology and MDA level in rats.
Also, the phenolic acid compounds of the plant and an-
tioxidant activity of aqueous extract were determined
using HPLC and FRAP test. The analysis revealed that cat-
echin and cinnamic acid were the highest phenolic acid
and comarin constituents of this plant. Also findings of
the current study demonstrated that both dietary and gel
forms had significant obvious healing effects and these
effects were dose dependent. However, the antioxidant
activity of the 20% gel in enema rout was clearly higher
than those of the other concentrations and routs of ad-
ministration.
Although, the UC symptoms can sometimes reduce
their own, the disease usually requires treatment to go
into remission. There are several strategies to treat UC
including pharmacotherapy (aminosalicylates, glucocor-
ticoids, immunosuppressive) and surgical procedures,
but the current therapies are not completely successful
and frequently cause adverse effects (7). Anti-inflamma-
tory drugs, such as aminosalicylates, corticosteroids and
immunosuppressive agents, were frequently applied to
treat this disease. However, there were serious side effects
and the recrudescence rates of IBD were rather high (29,
30). Therefore, scientists noticed the use of medicinal
plants as alternative or complementary therapies for UC.
The current study authors previously reported the bene-
ficial effects of some medicinal plants in different animal
species. The plants included Pistacia atlantica (11), Berberis
vulgaris (31), strawberry (32) and Hypericum perforatum
(10) in rats and Teucrium polium (33) and Calendula offici-
nalis (34) in dogs. However, no reports were issued on the
healing effects and antioxidant properties of M. officinalis
L. extract and its mechanism on UC until now.
Authors found that the aqueous extract of M. officinalis
L. contained high level of catechin and cinnamic acid as
the phenolic acid and comarin compounds. Catechin is a
type of natural phenol and antioxidant found in medici-
nal plants Catechins is reported to have anti-inflammatory
effects on TNBS and acetic acid induced rat colitis (35, 36).
This may be through selective immunomodulatory effects
mediated, at least in part, by inhibition of nuclear factor-
κB (NF-κB) and activator protein-1 (37). It is clarified that cat-
echins can exert their antioxidant activities via decreasing
nitric oxide, MDA, and increasing superoxide dismutase.
They also ameliorate mucosal inflammation by inhibit-
ing the production of TNF-α, IFN-γ and NF-κBp65 (36). The
current study findings regarding beneficial anti-colitis
activities of M. officinalis L. are in line with the above men-
tioned reports. It can be concluded that catechin, with the
highest amount of phenolic acid compound in the plant
extract, plays antioxidant and anti-inflammatory activities
due to the reported mechanisms.
Finally, MDA is frequently used to measure lipid perox-
ide levels and, exhibiting good correlation with degree
of lipid peroxidation. In the present study, the MDA lev-
els decreased in all M. officinalis L. treated groups com-
pared with those of the control groups. This decrease
may in fact support the protective effect of M. officinalis
L. against lipid peroxidation in UC. Although, scientific
information concerning antioxidant properties of the M.
officinalis L. extract is still rather scarce (13), such effects
against lipid peroxidation were previously reported in
cell culture experiments (38).
In conclusion, the mucus layer contributes significantly
to gut barrier function and protection from pathogens
(39) and the current findings are interesting as a strategy
to select the medicinal plants to treat UC. The antioxidant
and anti-inflammatory effects of M. officinalis L. extract
seem to be clear, especially by identifying catechin and
cinnamic acid as the most phenolic acid and comarin
constituents, but its molecular anti-inflammatory mech-
anisms are not fully investigated yet. Therefore, perform-
ing further studies to identify this mechanism in cell
culture and also animal and human studies are highly
recommended.
Acknowledgements
The animals were kindly provided by Mahjoob Vahedi
at the Laboratory Animals Center, Shiraz University of
Medical Sciences, Shiraz, Iran. Authors wish to thank all
people who kindly helped with the technical and scien-
tific phases of this study.
Authors’ Contributions
Ali Reza Safarpour: Study concept and design, analysis
and interpretation of data, critical revision of the manu-
script for important intellectual content; Fatemeh Kavi-
yani: Gathering, analysis and interpretation of data, Draft-
ing the manuscript; Masood Sepehrimanesh: Gathering,
analysis and interpretation of data, drafting the manu-
script, critical revision of the manuscript for important
intellectual content, statistical analysis, administrative,
technical, and material support, study supervision; Nas-
rollah Ahmadi: Data gathering, study supervision; Omid
Koohi Hosseinabadi: Gathering, analysis and interpre-
tation of data, drafting the manuscript; Nader Tanideh:
Study concept and design, drafting the manuscript, criti-
cal revision of the manuscript for important intellectual
content, study supervision.
Funding/Support
The study was supported by Shiraz University of Medi-
cal Sciences, Shiraz, IR Iran.
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