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Evaluation of anti-inflammatory and gastric anti-ulcer activity of Phyllanthus niruri L. (Euphorbiaceae) leaves in experimental rats

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Background: The medicinal plants signify a massive basin of potential phytoconstituents that could be valuable as a substitute to allopathic drugs or considered as an analogue in drug development. Phyllanthus niruri L. (Euphorbiaceae) is generally used in traditional medicine to treat ulcer and inflammation. In this project we investigated the methanolic extract of leaves of Phyllanthus niruri for anti-inflammatory and anti-ulcer activity. Methods: The anti-inflammatory activity of methanol extract of Phyllanthus niruri leaves was evaluated at the doses of 100, 200 and 400 mg/kg, p.o. while using ibuprofen (20 mg/kg, p.o) as the standard drug. The animals used were Swiss albino rats. Inflammation was induced by injecting 0.1 ml carrageenan (1% w/v) into the left hind paw. Paw tissues from the different groups were examined for inflammatory cell infiltration. On the other hand, antiulcer activity of methanolic extract of P. niruri leaves at the doses of 100, 200 and 400 mg/kg, p.o. were examined against ethanol-acid induced gastric mucosal injury in the Swiss albino rats - keeping omeprazole (20 mg/kg, p.o.) as reference. The rats were dissected and the stomachs were macroscopically examined to identify hemorrhagic lesions in the glandular mucosa. Results: P. niruri significantly (p < 0.01) decreased carrageenan-induced paw edema; it exhibited a reduction of 46.80%, 55.32% and 69.14% at doses of 100, 200 and 400 mg/kg, respectively. These findings were further supported by the histological study. The methanolic extract also disclosed good protective effect against ethanol-acid induced gastric mucosal injury in the rats. Administration of the extract's doses (100, 200 and 400 mg/kg) demonstrated a significant (p < 0.01) reduction in the ethanol- acid induced gastric erosion in all the experimental groups when compared to the control. The methanolic extract at the higher dose (400 mg/kg) resulted in better inhibition of ethanol-acid induced gastric ulcer as compare to omeprazole (20 mg/kg). Histological studies of the gastric wall revealed that toxic control rats revealed mucosal degeneration, ulceration and migration of numerous inflammatory cells throughout the section. On the other hand, MEPN treatment groups showed significant regeneration of mucosal layer and significantly prevented the formation of hemorrhage and edema. Conclusions: The investigation suggests that methanolic extract of P. niruri leaf possess anti-inflammatory activity and promotes ulcer protection as ascertained by regeneration of mucosal layer and substantial prevention of the formation of hemorrhage and edema.
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R E S E A R C H A R T I C L E Open Access
Evaluation of anti-inflammatory and gastric
anti-ulcer activity of Phyllanthus niruri L.
(Euphorbiaceae) leaves in experimental rats
Ronia Mostofa
1
, Shanta Ahmed
1
, Mst. Marium Begum
2
, Md. Sohanur Rahman
3
, Taslima Begum
1
,
Siraj Uddin Ahmed
1
, Riazul Haque Tuhin
1
, Munny Das
4
, Amir Hossain
1
, Manju Sharma
5
and Rayhana Begum
1*
Abstract
Background: The medicinal plants signify a massive basin of potential phytoconstituents that could be valuable as a
substitute to allopathic drugs or considered as an analogue in drug development. Phyllanthus niruri L. (Euphorbiaceae)
is generally used in traditional medicine to treat ulcer and inflammation. In this project we investigated the methanolic
extract of leaves of Phyllanthus niruri for anti-inflammatory and anti-ulcer activity.
Methods: The anti-inflammatory activity of methanol extract of Phyllanthus niruri leaves was evaluated at the doses of
100, 200 and 400 mg/kg, p.o. while using ibuprofen (20 mg/kg, p.o) as the standard drug. The animals used were Swiss
albino rats. Inflammation was induced by injecting 0.1 ml carrageenan (1% w/v) into the left hind paw. Paw tissues from
the different groups were examined for inflammatory cell infiltration. On the other hand, antiulcer activity of methanolic
extract of P. niruri leaves at the doses of 100, 200 and 400 mg/kg, p.o. were examined against ethanol-acid induced
gastric mucosal injury in the Swiss albino rats - keeping omeprazole(20mg/kg,p.o.)asreference.Theratsweredissected
and the stomachs were macroscopically examined to identify hemorrhagic lesions in the glandular mucosa.
Results: P. niruri significantly (p< 0.01) decreased carrageenan-induced paw edema; it exhibited a reduction of 46.80%,
55.32% and 69.14% at doses of 100, 200 and 400 mg/kg, respectively. These findings were further supported by the
histological study. The methanolic extract also disclosed good protective effect against ethanol-acid induced gastric
mucosal injury in the rats. Administration of the extracts doses (100, 200 and 400 mg/kg) demonstrated a significant
(p< 0.01) reduction in the ethanol- acid induced gastric erosioninalltheexperimentalgroupswhencomparedtothe
control. The methanolic extract at the higher dose (400 mg/kg) resulted in better inhibition of ethanol-acid induced
gastric ulcer as compare to omeprazole (20 mg/kg). Histological studies of the gastric wall revealed that toxic control rats
revealed mucosal degeneration, ulceration and migration of numerous inflammatory cells throughout the section. On the
other hand, MEPN treatment groups showed significant regeneration of mucosal layer and significantly prevented the
formation of hemorrhage and edema.
Conclusions: The investigation suggests that methanolic extract of P. niruri leaf possess anti-inflammatory activity and
promotes ulcer protection as ascertained by regeneration of mucosal layer and substantial prevention of the formation of
hemorrhage and edema.
Keywords: Phyllanthus niruri, Phytochemical, Anti-inflammatory, Anti-Ulcer
* Correspondence: rayhana_kushum78@yahoo.com
1
Department of Pharmacy, Primeasia University, Dhaka 1213, Bangladesh
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Mostofa et al. BMC Complementary and Alternative Medicine (2017) 17:267
DOI 10.1186/s12906-017-1771-7
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
Currently, various steroidal and non-steroidal anti-
inflammatory drugs (NSAID) are being used to treat
inflammatory diseases. Gastrointestinal bleeding and
ulceration are the most recurrent and formidable prob-
lems linked with NSAID [1]. Because of these side
effects, researchers are in dire need to develop safer
compounds. The gastric mucosal lesions caused by
ethanol, were reported as by prying with the gastric de-
fensive mechanisms [2]. While there are many products
used against gastric ulcers, most of these drugs generate
several adverse reactions [3]. To study the effects of
drugs on the acute phase of inflammation, models were
designed to induce inflammation in rat paws by injecting
pro-inflammatory agents such as carrageenan, dextran,
formaldehyde etc. [4]. Carrageenan-induced paw edema
animal model is usually used to assess the contribution
of natural products in weathering the biochemical
changes associated with acute inflammation. While the
carrageenan model is typically associated with activation
of the cyclooxygenase pathway and is delicate to gluco-
corticoids and prostaglandin synthesis antagonists, the
early phase of the carrageenan reaction is due to the
release of serotonin and histamine [5].
Due to the mounting concentration in the alternative
therapies in current years, herbal products have become
popular [6, 7]. P. niruri L. (Euphorbiaceae), leaves
extract is one such herbal drug currently undertaken in
this study primarily to explore its anti-inflammatory and
anti-ulcerogenic potential in animal model. P. niruri can
be found in the tropical regions of Asia and America.
The common names of the plant are stonebreaker or
seed-under-leaf. P. niruri is a chief plant in the Ayurvedic
tradition to treat stomach, genitourinary system, liver,
kidney and spleen conditions. The medicinal use of the
plant in disorders includes dysentery, influenza, vaginitis,
tumors, diabetes, jaundice, dyspepsia etc. The various
extracts of the plant also proved to act as antiviral and
antibacterial agent [810]. Indigenous women have also
used the plant for menstruation and uterus problems [11].
Many active phytochemicals such as flavonoids, alkaloids,
terpenoids, lignin, polyphenols, tannins, coumarins and
saponins have been recognized from various parts of P.
niruri. Extracts of this herb have been proven to have
therapeutic effects in many preclinical studies. Phyllanthus
niruri has been reported to be an effective anti-
inflammatory [12], analgesic [13], gastroprotective [14],
anti-diabetic [15], hepatoproctive [1618], anti-malarial
[19, 14] and antispasmodic [20]. In Bangladesh, P. niruri
grows all over the country. According to a previous study,
the aerial part of this plant has been reported for its anti-
inflammatory activity [12]. Besides, it has been stated that
the leaves of P. niruri contain profound amount of flavo-
noids and polyphenolics [21] which possess significant
activity against inflammation and ulcer [22, 23]. However,
there were no reports on the anti-inflammatory and anti-
ulcer effect of P. niruri regarding Bangladeshi species,
which encouraged us to evaluate the anti-inflammatory
and antiulcer activity of P. niruri in rats. Because of the
potentials of P. niruri as a medicinal plant in Bangladesh,
interest in this plant is justifiable to seek anti-
inflammatory and antiulcer activities. In addition the effect
of P. niruri leave extract on inflammation and gastric ulcer
was also assessed histologically.
Methods
Plant material
The fresh leaves of Phyllanthus niruri L. (Euphorbiaceae)
were collected in the months of January-February 2015
from Banani, Dhaka, Bangladesh. The plant was authenti-
cated from the Bangladesh National Herbarium, where a
voucher specimen was deposited (voucher no.- 41,684).
Drugs and chemicals
Ibuprofen and omeprazole were obtained from the pharma-
ceutical industry ESKAYEF BANGLADESH LIMITED.
Carrageenan was obtained from Sigma Aldrich Chemicals,
Germany. All other chemicals were obtained from Merck
(Darmstadt, Germany) and were of analytical grade.
Extraction procedure
Fresh leaves of P. niruri were cleaned and dried in an
oven at 45 °C. Dried sample was pulverized to a coarse
powder using a grinder. About 200 g of coarse powders
were soaked in 95% methanol in a conical flask (600 ml),
plugged with cotton and then covered with aluminum
foil for seven days with occasional stirs. After seven days
the preparation was filtered and the filtrate was collected
for the preparation of extract. The filtrate was reduced
by rotary evaporator and kept in normal air for few days
to facilitate evaporation of the remaining solvent. The
residue was then weighed (26 g) and stored in a sealed
container.
Phytochemical analysis
Phytochemistry is the branch of chemistry, deals with
the chemical nature of the plant or plant products
(chemistry of natural products). Plants contain many
chemical constituents which are therapeutically active or
inactive like carbohydrates, triterpenoids, alkaloids,
glycosides, tannins, flavonoids, essential oils and other
similar secondary metabolites. Qualitative phytochemical
analyses were done using the standard procedures [24].
Test for carbohydrates
Molischs test
To 2 ml of extract, 2-3 drops of alpha naphthalene solu-
tion in alcohol was added and shaken for 2 min. 1 ml of
Mostofa et al. BMC Complementary and Alternative Medicine (2017) 17:267 Page 2 of 10
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concentrated sulphuric acid was added slowly from the
sides of the test tube. A deep violet colour at the junction
of two layers indicated the presence of carbohydrates.
Test for saponin
Foam test
The methanol extract (50 mg) was diluted with distilled
water and made up to 20 ml. The suspension was
shaken in a graduated cylinder for 15 min. Appearance
of persistent foam indicated the presence of saponins.
Test for alkaloids
Dragendorffs test
The methanol extract (6 g.) of the plant was dissolved in
10 ml of distilled water then 2 M hydrochloric acid was
added until acidify, Dragendorff s reagent (2 ml) was
added and an orange red precipitate indicated the pres-
ence of alkaloids.
Test for glycosides
Borntragers test
For the detection of glycosides, 50 mg of methanol extract
was hydrolysed with concentrated hydrochloric acid for
2 h on water bath, filtered and the hydrolysate (4 ml) of
filtered hydrolysate was taken in a test tube; 6 ml of
chloroform was added and shaken. Chloroform layer was
separated and 10% ammonia solution was added to it pink
colour indicated the presence of glycosides.
Test for sterols/terpenes
Hosss reaction
In this test, the methanol extract (20 mg) was taken in
chloroform (2 ml) and concentrated sulphuric acid was
poured from side of the test tube. The colour of the ring
at the junction of the two layers was noted. A violet green
colour indicated the presence of cholesterol, sitosterol. A
red colour ring showed the presence of sterol/terpenes.
Test for flavonoids
Shimoda test
To dry methanol extract (30 mg), ethanol (2 ml) was
added and dropped small piece of Magnesium ribbon.
The drop wise addition of conc. HCl leads to the devel-
opment of colour ranging from orange to red was
confirmatory for flavonoids.
Test for phenolics and tannins
Ferric chloride test
The extract (20 mg) was added in 2 ml of 1% ferric
chloride solution, a purple or red colour indicated the
presence of phenols.
One to 2 ml of methanol extract, a few drops of 5%
aqueous ferric chloride solution was added. A bluish
black colour was produced which disappears on addition
of few ml of dilute sulphuric acid followed by the forma-
tion of a yellowish-brown precipitate indicated the pres-
ence of tannins.
Test for anthraquinone
Borntrager test
3 ml of extract, 3 ml Benzene and 5 ml 10% ammonia
solution were added and thereafter shaken properly.
Appearance of a pink, red or violet colour in the ammo-
niacal (lower) phase was taken as the presence of free
anthraquinones.
Test for coumarin glycosides
NaOH test
A small amount of methanol extract was placed in test
tube and covered the test tube with a filter paper moist-
ened with dilute sodium hydroxide solution. The
covered test tube was placed on water bath for several
minutes. Removed the paper and exposed it to ultraviolet
(UV) light, the paper showed green fluorescence.
Anti-inflammatory activity
Experimental animal
Female Swiss albino rats weighing 120-150 g were used
in the experiment. Animals were housed in polypropyl-
ene cages in groups of six per cage and were kept in a
room maintained at 25 ± 2 °C with a 12 h light-dark
cycle, and were allowed to acclimatize for one week
before the experiment commenced. They were given free
access to standard laboratory animal feed and water ad
libitum. They were fasted over night before the experi-
mental procedures began and all surgeries were
performed under isoflurane (5% in 100% oxygen)
anesthesia. The procedures were conducted with efforts
to minimize preventable harm to the rats. Animal care
and research protocols were centered on values and
guidelines sanctioned by the Guide for the Care and Use
of Laboratory Animals (NIH publication No: 85-23,
revised in 1985). The prior approval for conducting the
experiments on rats was obtained from the Departmental
Ethics Committee of Dhaka University.
Induction of inflammation in experimental animals
The methanolic extract of P. niruri (MEPN) was evalu-
ated for anti-inflammatory activity as recommended by
Winter et al. [25], injecting the edematogenic agent,
explicitly carrageenan on adult albino rat [26].
There were six groups containing six rats each.
Ibuprofen (20 mg/kg) was used as the reference standard.
The extract was administered orally in the form of a sus-
pension with 2 to 3 drops of tween 80 at doses of 100, 200
and 400 mg/kg respectively in the treatment group. The
normal healthy group received distilled water only. All the
test samples were administered orally (0.5 ml) 30 min
Mostofa et al. BMC Complementary and Alternative Medicine (2017) 17:267 Page 3 of 10
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prior to injection of carrageenan (0.1 ml of 1% w/vsolu-
tion in distilled water) in the sub planted region of right
paw of each rat. However, the control group received no
carrageenan injection. The swelling of the paws were mea-
sured by slide calipers in one hour intervals. The observa-
tions were tabulated. The percentage of inhibition of paw
edema was calculated at the end of the 6th hour.
The experimental animals were divided into the fol-
lowing groups and received the subsequent treatments
accordingly:
The increase in paw thickness and percentage of in-
hibition in control/treatment were calculated using the
following formula:
Increase in paw thickness in control or treatment
group ¼PC or PT ¼Pt Po
Percentage of inhibition in paw thickness in the
treatment group ¼PC PT 100=PC
Where, Pt = paw thickness at time t, Po = initial paw
thickness, PC = Increase in thickness of paw of the con-
trol group and PT = Increase in thickness of paw of the
treatment group [27].
Induction of ulcer
The animals were barred from access to any nutrients
for a day and were only allowed access to drinking water
for two hours before the experiment commenced. Dur-
ing the fasting period, the rats were placed individually
in separate cages to prevent coprophagy. Thirty minutes
after pre-treatment with standard (omeprazole at the
dose of 20 mg/kg, p.o.) and test samples (MEPN at the
doses of 100, 200 and 400 mg/kg p.o.), gastric ulcers
were induced with ethanol-acid in these groups of rats
(25 ml per kg of 0.3 M HCl in 60% ethanol) [28]. These
rats were sacrificed 90 min after induction and their
stomachs were immediately excised. Each stomach was
opened along the larger curvature, washed with distilled
water. The gastric mucosa was examined for ulcers by
magnifying lens and scoring of ulcer was made as
follows [29].
Mean ulcer score for each animal was expressed as
ulcer index. The percentage of ulcer protection was
determined as follows:-
%protection ¼control mean ulcer indextest mean ulcer index
control mean ulcer index 100
The experimental animals were divided into six
groups, each consisting of six rats and received fol-
lowing treatment:
Histological investigation
At the end of the studies animals were sacrificed
while they were under isoflurane (5% in 100% oxygen)
anaesthesia. For histological examination, paw tissues
were taken 6 h after edema was induced by carra-
geenan. The tissue slices were immediately fixed in
freshly prepared 10% neutral buffered formalin for a
minimum of 24 h. On the other hand, specimens of
the gastric walls from each rat were kept in 10%
buffered formalin for 24 h for histopathological
Groups Treatment
Group I 0.5 ml/day distilled water,
p.o.; (Normal control, NC)
Group II 0.5 ml/day distilled water,
p.o. + 0.1 ml of carrageenan;
(Carrageenan control, CC)
Group III 20 mg/kg/day ibuprofen,
p.o. + 0.1 ml of carrageenan;
(IP-20)
Group IV 100 mg/kg/day methanolic
extract of P. niruri, p.o. + 0.1 ml
of carrageenan; (MEPN-100)
Group V 200 mg/kg/day methanolic
extract of P. niruri, p.o. + 0.1 ml
of carrageenan; (MEPN-200)
Group VI 400 mg/kg/day methanolic
extract of P. niruri, p.o. + 0.1 ml
of carrageenan; (MEPN-400)
Scoring of Ulcer
Normal
stomach
Red
coloration
Spot
ulcer
Hemorrhagic
streak
Ulcers Perforation
0 0.5 1 1.5 2 3
Groups Treatment
Group 1 5 ml/kg/day distilled water, p.o.;
(Normal control)
Group 2 5 ml/kg/day distilled water, p.o. + 25 ml
per kg of 0.3 M HCl in 60% ethanol;
(Ethanol control)
Group 3 20 mg/kg/day omeprazole, p.o. + 25 ml
per kg of 0.3 M HCl in 60% ethanol;
Group 4 100 mg/kg/day methanolic extract of
P. niruri, p.o. + 25 ml per kg of 0.3 M
HCl in 60% ethanol;
Group 5 200 mg/kg/day methanolic extract of
P. niruri, p.o. + 25 ml per kg of 0.3 M
HCl in 60% ethanol;
Group 6 400 mg/kg/day methanolic extract of
P. niruri, p.o. + 25 ml per kg of 0.3 M
HCl in 60% ethanol;
Mostofa et al. BMC Complementary and Alternative Medicine (2017) 17:267 Page 4 of 10
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examination following the assessment of ulcer score
for anti-ulcer activity. Then the tissue specimens were
processed for paraffin embedding tissue sections. The
samples were sectioned with a microtome, stained
with hematoxyline and Eosin (H and E) and mounted
on Canada balsam. All sections were examined under
light microscope. Photographs of the lesions were
taken with an Olympus photo microscope for
observation and documentation of histopathological
changes such as oedema, inflammation, infiltration
and erosion.
Statistical analysis
The values are represented as mean ± S.E.M, and
statistical significance between treated and control
groups was analyzed using One way ANOVA, followed
by DunnettstestwhereP< 0.05 was considered
statistically significant.
Results
Preliminary phytochemical analysis
The traditional use of the species was scientifically vali-
dated through the identification of the phytochemicals
responsible for their use in indigenous systems of health
care. The result of qualitative chemical analysis of the
methanolic extract of P. niruri is tabulated in Table 1.
Carrageenan induced acute inflammation
Ibuprofen was used as the reference drug during the
anti-inflammatory evaluation of the methanolic extract
of the leaves of P. niruri in carrageenan induced acute
inflammation model. Animals that were treated with
ibuprofen (20 mg/kg, p.o.) and methanolic extract (100,
200 and 400 mg/kg p.o.) exhibited significant reduction
in paw thickness from 1st to the 6th hour (Table 2).
After 6 h of carrageenan treatment, swelling and redness
were observed in carrageenan control group, while
swelling and redness were significantly reduced in the
groups which were given MEPN. Fig. 1 showed the im-
ages of the inflamed paw of the groups viz., carrageenan
control, standard and treatment groups at 6 h after car-
rageenan injection. The results obtained at the end of
the study disclosed that the extract exhibited significant
(p< 0.01) anti-inflammatory activity in inflamed rat
paws, when compared with carrageenan control. At the
end of the study, ibuprofen (20 mg/kg) treated group
showed 64.89% of inhibition. Oral administration of
methanolic extract at the doses of 100, 200 and 400 mg/kg
reduced paw edema by 46.80, 55.32 and 69.14%, respect-
ively when compared with control group at the 6
th
hour
after carrageenan injection.
Histopathology of paw tissue
Fig. 2a showed a section from the rat paw received only
distilled water without carrageenan injection. The tissue
architecture was preserved, showing dermal collagen and
minimal number of leukocytes. Fig. 2b demonstrated the
carrageenan induced section, elicit migration of numerous
inflammatory cells throughout the section was observed.
On the other hand, ibuprofen (20 mg/kg) treated
group revealed appearance of only few inflammatory
cells (Fig. 2c). However, groups treated with methanolic
extract of P. niruri at doses of 100 and 200 mg/kg
displayed moderate to minimal number of inflammatory
cell infiltration (Fig. 2d, e, respectively). The inflammatory
cellsinfiltration was almost completely reduced by the
treatment with methanolic extract of P. niruri at the dose
of 400 mg/kg when compared with CC group (Fig. 2f).
Effect of P. niruri on ethanol-induced gastric ulcer
In ethanol control animal, oral administration of ethanol
produced characteristic lesions in the glandular portion
of rats stomach which appeared as elongated bands of
thick, black & dark red lesions. MEPN showed signifi-
cant protection index of 69.59, 74.32 and 80.40% with
the dose of 100, 200 and 400 mg/kg/day, p.o. respect-
ively in comparison to ethanol control. Whereas
omeprazole (standard drug) reduced ulcer by 75.00%
(Results are tabulated in Table 3).
Gross evaluations of gastric lesions
Ethanol controlled rats exhibited severe mucosal injury
whereas, the rats that were treated with P. niruri leaves
extract before ethanolic induction had significantly
reduced areas of gastric ulceration revealing flattening of
gastric mucosal folds compared to rats treated with only
distilled water. There were no significant differences
between doses of 200 and 400 mg/kg methanolic extract
in terms of area of ulceration. It was also observed that
protection of gastric mucosa was more prominent in rats
treated with 400 mg/kg methanolic extract (Fig. 3).
Table 1 Preliminary phytochemical analysis of P. niruri leaves extract
Phytoconstituents Methanolic extract of P. niruri
Carbohydrates +
Saponins +
Alkaloids +
Glycosides
Terpenoids +
Steroids +
Flavonoids +
Phenolics and Tannins +
Anthraquinone
Coumarins glycosides +
+ = Present, = Absent
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Histological evaluation of gastric lesions
Fig. 4a shows a section from the subject that received
only distilled water without induction of ulcer. The
section of gastric mucosal layer showed normal tissue
architecture and absence of gastric tissue degeneration.
Whereas the ethanol control group demonstrated mucosal
degeneration, ulceration and migration of numerous
inflammatory cells throughout the section (Fig. 4b). How-
ever, administration of omeprazole (20 mg/kg) showed no
significant change in histopathology and in turn revealed
Table 2 Anti-inflammatory effect of Phyllanthus niruri methanolic extract on carrageenan induced changes in paw thickness in
experimental rats
Groups NC CC IP-20 MEPN-100 MEPN-200 MEPN-400
Dose mg/kg ——————— ————— 20 mg/kg ibuprofen 100 mg/kg extract 200 mg/kg extract 400 mg/kg extract
Initial paw thickness (cm) 0.49 ± 0.07 0.42 ± 0.06 0.45 ± 0.09
b*
0.44 ± 0.05
b*
0.44 ± 0.08
b*
0.43 ± 0.06
b*
Paw thickness (cm) after 1 h 0.49 ± 0.05 0.74 ± 0.08
a*
0.52 ± 0.07
b*
0.6 ± 0.07
b,c
0.58 ± 0.05
b,c
0.51 ± 0.07
b*
Paw thickness (cm) after 2 h 0.49 ± 0.08 0.83 ± 0.07
a**
0.57 ± 0.05
b**
0.67 ± 0.04
b,c
0.63 ± 0.05
b*
0.57 ± 0.06
b**
Paw thickness (cm) after 3 h 0.49 ± 0.05 0.91 ± 0.09
a**
0.62 ± 0.10
b**
0.7 ± 0.06
b,c
0.66 ± 0.06
b*
0.6 ± 0.08
b**
Paw thickness (cm) after 4 h 0.49 ± 0.06 0.99 ± 0.11
a**
0.58 ± 0.10
b**
0.68 ± 0.09
b*
0.62 ± 0.07
b**
0.56 ± 0.09
b**
Paw thickness (cm) after 5 h 0.49 ± 0.08 0.96 ± 0.09
a**
0.49 ± 0.07
b**
0.65 ± 0.10
b*
0.59 ± 0.07
b**
0.48 ± 0.06
b**
Paw thickness (cm) after 6 h 0.48 ± 0.06 0.94 ± 0.07
a**
0.33 ± 0.09
b**
0.50 ± 0.06
b**
0.42 ± 0.07
b**
0.29 ± 0.07
b**
Each value is Mean ± S.E.M (n=6). (*) indicates statistically significant difference from respective group using one way analysis of variance, followed by Dunnetts
multiple comparison test (*p< 0.05 and
**
p< 0.01).
c
indicates statistically no significant difference from respective group using one way analysis of variance,
followed by Dunnetts multiple comparison test (p> 0.05).
a
when compared with normal control,
b
when compared with carrageenan control; NC- Normal control,
CC- Carrageenan control, IP-20 ibuprofen 20 mg/kg/day, MEPN-100 -methanolic extract of P. niruri 100 mg/kg/day, MEPN-200- methanolic extract of P. niruri
200 mg/kg/day, MEPN-400 - methanolic extract of P. niruri 400 mg/kg/day.
Fig. 1 Effect of methanolic extract of P. niruri on carrageenan induced paw edema after 6 h. a= Normal control: no erythema and inflammation
were observed. bCarrageenan control: severe erythema and swelling were observed. c20 mg/kg ibuprofen: mild amount of erythema and
swelling were observed. d100 mg/kg methanolic extract of P. niruri: moderate amount of erythema and swelling were observed e200 mg/kg
methanolic extract of P. niruri: moderate amount of erythema and swelling were observed. f400 mg/kg methanolic extract of P. niruri: mild
amount of erythema and swelling were observed. The small arrow indicates the absence of swelling and erythema whereas the large arrow
indicates severe swelling and erythema in the rats paw
Mostofa et al. BMC Complementary and Alternative Medicine (2017) 17:267 Page 6 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
regeneration of structure and prevention of hemorrhage
and edema (Fig. 4c). MEPN at the dose of 100 mg/kg ex-
hibited moderate regeneration (Fig. 4d). On the other
hand, MEPN at the doses of 200 and 400 mg/kg displayed
significant regeneration of mucosal layer and expressively
prevented the development of hemorrhage and edema
(Fig. 4e, f respectively).
Discussion
Upon phytochemical screening the methanolic extract of
P. niruri disclosed the presence of alkaloids, phenols,
steroids, triterpinoids, flavonoids and coumarins. Many
studies have reported that certain terpenoids, steroids
and phenolic compounds (tannins, coumarins and
flavonoids) have protective effects due to their antioxi-
dant properties. [3032]. Lately, a number of natural
products of traditional medicines and ingredients of
healthy foods have been comprehensively explored and
subjected to clinical trials to establish as anti-inflammatory
agents [33]. Presence of major Phytoconstituents in the
methanolic extract of leaves of P. niruri makes it a potential
candidate for further investigation.
The edema induced by carrageenan was expressed in
two phases (first phase and second phase) [34]. In the
first phase: a rapid rise in edema was detected instantly
after sub-plantar injection of carrageenan. In the second
phase (at the end of 2nd hour), a significant increase in
edema was detected. The release of prostaglandins is
thought to be the main reason for the swelling in second
phase [35]. In this study, MEPN inhibited the carra-
geenan induced edema in a dose-dependent manner and
Fig. 2 Histological evaluation of anti-inflammatory effects of methanolic extract of P. niruri aNormal control,bCarrageenan control, c20 mg/kg
ibuprofen, d100 mg/kg methanolic extract of P. niruri,e200 mg/kg methanolic extract of P. niruri,f400 mg/kg methanolic extract of P. niruri.Eachgroup
was assessed at 400× magnification, scale bar: 20 μm; Iindicates numerous infiltrations of inflammatory cells in the specimen from the rats paw tissue
Table 3 Effect of P. niruri leaves extract on various parameters
in ethanol induced gastric ulcer
Groups Ulcer index % Protection
Normal control 1.56 ± 0.04 -
Ethanol control 14.8 ± 1.03
a**
-
Omeprazole 20 mg/kg 3.7 ± 0.06
b**
75.00
MEPN 100 mg/kg 4.5 ± 0.05
b**
69.59
MEPN 200 mg/kg 3.8 ± 0.03
b**
74.32
MEPN 400 mg/kg 2.9 ± 0.05
b**
80.40
MEPN- methanolic extract of P. niruri
Each value is Mean ± S.E.M (n=6). (*) indicates statistically significant alteration
from respective group using one way analysis of variance followed by Dunnetts
multiple comparison test (
**
p< 0.01).
a
when compared with normal control,
b
when compared with ethanol control
Mostofa et al. BMC Complementary and Alternative Medicine (2017) 17:267 Page 7 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
had a potential anti-inflammatory effect in the second
phase (2
nd
-6
th
hour). In the treatment groups, the devel-
opment of inflammation in the second phase was less.
MEPN might have demonstrated their anti-inflammatory
activity by inhibiting the synthesis and release of prosta-
glandins, proteases, and lysosomal enzymes.
In the present study, the histopathogical examin-
ation of the hind paw tissue showed that methanolic
extract of P. niruri suppressed the massive influx and
accumulation of inflammatory cells in the paw tissue
after carrageenan induction. The suppressive effects
were observed at all doses of the test drugs. However,
the present investigation concluded that methanolic
extract of P. niruri reduced the inflammatory cells
infiltration, in a dose-dependent manner and at the
higher dose the effect was similar to that of refer-
ence drug.
The anti-ulcer effect of the methanolic extract was eval-
uated using ethanol induced gastric ulcer model. Ethanol
induced gastric lesions formed due to interference in gas-
tric blood flow which contributes to the development of
the hemorrhage and necrotic aspects of tissue injury. Al-
cohol swiftly penetrates the gastric mucosa superficially
causing cell and plasma membrane damage leading to
augmented intracellular membrane permeability to
sodium and water. The mammoth buildup of calcium de-
scribes a chief step in the pathogenesis of gastric mucosal
injury. This sequence leads to the demise of cells and
erosion of epitheliums surface [36, 37].
The results revealed that the ethanol administration in
the control group resulted in immense ulceration in com-
parison with the normal group. However, treatment with
omeprazole at the dose of 20 mg/kg and methanolic
extract of P. niruri at the doses of 100,200 and 400 mg/kg
prior to ethanol administration exhibited significant inhib-
ition. Among the test samples, the best result was
Fig. 3 Gross appearance of the gastric mucosa. aNormal control: no
mucosal damage was observed,bEthanol control: marked ulcers
along with hemorrhagic streaks and mucosal damage were observed,
c20 mg/kg omeprazole, mild injuries were observed in the gastric
mucosa as compared to the ethanol control group, d100 mg/kg
methanolic extract of P. Niruri:moderately reduced gastric mucosal
damage and ulcers were observed, e200 mg/kg ethanolic extract of
P. Niruri: significantly reduced gastric mucosal damage and ulcers were
observed, f400 mg/kg methanolic extract of P. Niruri: no damage to
the gastric mucosa was observed and gastric mucosa appeared flat as
compared to the ethanol control. Iindicates gastric mucosal damage
and hemorrhagic streaks
Fig. 4 Histological evaluation of anti-ulcer effect of methanolic extract of
P. niruri. aNormal control, bEthanol control, c20 mg/kg omeprazole,
d100 mg/kg methanolic extract of P. Niruri,e200 mg/kg methanolic
extract of P. Niruri,f400 mg/kg methanolic extract of P. Niruri.Eachgroup
was assessed at 400× magnification, scale bar: 20 μm; Iindicates normal
gastric mucosa, Jindicates degeneration of gastric mucosa and infiltration
of inflammatory cells frequently observed in the specimen from
the stomach, Kindicates almost complete regeneration of gastric mucosa
Mostofa et al. BMC Complementary and Alternative Medicine (2017) 17:267 Page 8 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
obtained with P. niruri at an optimum dose of 400 mg/kg
which was potentially effective as compared to the stand-
ard drug, omeprazole. Edema, cellular debris and damaged
mucosal epithelium were found in ulcerated stomach
membranes. Protections against these histopathological
changes by MEPN in pre-treated rats were observed,
similar to the result of omeprazole.
However, the findings observed in the current studies
support and extend previous results that reported the
anti-inflammatory and anti-ulcer activities of Phyl-
lanthus niruri aerial part and leave extract, respectively.
Furthermore, the present studies also revealed a better
inhibition of inflammation and gastric ulcer as compare
to the previously reported.
Conclusion
In our study the extract exhibited protection against
characteristic lesions produced by ethanol administra-
tion. This antiulcer effect of methanolic extract of P.
niruri may be due to both reductions in gastric acid
secretion and gastric cytoprotection. Further studies are
needed for their exact mechanism of action on gastric
acid secretion and gastric cytoprotection. However, the
present investigation concluded that the treatment of
extracts reduced the ethanol induced ulcer in a dose-
dependent manner and at the higher dose (400 mg/kg)
the effect was similar to that of reference drug.
In conclusion, MEPN exhibited anti-inflammatory and
antiulcerogenic activity. MEPN at the dose of 400 mg/kg
showed higher level of cytoprotection. The depletion in
inflammation may have occurred due to high flavonoid,
triterpenoids, steroids, saponins and tannin content. How-
ever, the mechanisms behind these events are still vague.
Therefore, further experiments should be undertaken to
identify which of the phytoconstituents and mechanisms
are involved in the actions illustrated by the results.
Abbreviations
P:Phyllanthus; MEPN: methanolic extract of leaves of Phyllanthus niruri;
NC: Normal control; CC: Carrageenan control; NSAID: non-steroidal anti-
inflammatory drugs
Acknowledgements
We are thankful to Department of Pharmacy, Primeasia University, Dhaka-1213,
Bangladesh.
Funding
Self-funded by research team.
Availability of data and materials
Data are all contained within the paper.
Authorscontributions
RB, MMB and MSR made substantial contributions to conception design and
conduction of research. RM, SA, TB, SUA and MD performed all of the
experiments in the laboratory. Data collection, analysis, graphical representation
and interpretation were done by RM and MSR. Article was written by RM and RB.
Critical revision of the article was done by RHT, AH, and MS. Critical statistical
analysis was done by RB. MSR made the necessary corrections in the write up.
Conception, experiment design, overall monitoring and final approval of the
article was done by RB. All Authors read and approved the final manuscripts.
Competing interests
All authors declare that they have no conflict of interests.
Consent for publication
Applicable.
Ethics approval and consent to participate
The use of experimental laboratory animals in this study was approved by
Biomedical Research Center, University of Dhaka, Bangladesh (DPT/BMRC/
2015-16/237).
PublishersNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Department of Pharmacy, Primeasia University, Dhaka 1213, Bangladesh.
2
Department of Pharmaceutical Technology, Faculty of Pharmacy, University
of Dhaka, Dhaka, Bangladesh.
3
Department of Biochemistry and Molecular
Biology, University of Rajshahi, Rajshahi 6205, Bangladesh.
4
Department of
Pharmaceutical Sciences, North South University, Dhaka 1229, Bangladesh.
5
Department of Pharmacology, Faculty of Pharmacy, Hamdard University,
New Delhi 110062, India.
Received: 15 July 2016 Accepted: 8 May 2017
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