Protective Effect of Phyllanthus Amarus by Modulation of Endogenous Biomarkers and DNA Damage in Acetic Acid induced Ulcerative Colitis: Role of Phyllanthin and Hypophyllanthin

ProtectiveeffectofPhyllanthusamarusbymodulationof
endogenousbiomarkersandDNAdamageinaceticacid
inducedulcerativecolitis:Roleofphyllanthinandhypophyllanthin

Research Article
Protective effect of Phyllanthus amarus by modulation of
endogenous biomarkers and DNA damage in acetic acid
induced ulcerative colitis: Role of phyllanthin and
hypophyllanthin
Amit D. Kandhare, Pinaki Ghosh, Arvindkumar E. Ghule, Girish N. Zambare,
Subhash L. Bodhankar*
Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane, Pune 411038, Maharashtra,
India
a r t i c l e i n f o
Article history:
Received 6 March 2012
Accepted 8 January 2013
Available online 23 January 2013
Keywords:
Apoptosis
Inflammatory bowel disease
Oxidative stress
Phyllanthus amarus
Tumor necrosis factor-a
a b s t r a c t
Aim: To evaluate the protective effect of phyllanthin and hypophyllanthin of standardized
aqueous extract of Phyllanthus amarus in acetic acid induced colitis model of inflammatory
bowel disease in Wistar rats.
Methods: Rats were rendered colitic by a colonic instillation of 2 ml (4%) acetic acid solution.
Rats were pretreated orally for 7 days before induction of colitis with P. amarus (50, 100 and
200 mg/kg) or vehicle (1 ml of distilled water). Colonic inflammation was evaluated by
disease activity index, gross morphologic damage, histological injury and different bio-
chemical markers of colonic inflammation such as superoxide dismutase (SOD), reduced
glutathione (GSH), malondialdehyde (MDA), myeloperoxidase (MPO), nitric oxide (NO) and
tumor necrosis factor-a (TNF-a) were performed in colonic contents from colitic rats. DNA
damage, a marker of apoptosis was also assessed in colonic contents.
Results: The results show that P. amarus (100 and 200 mg/kg) exerted a preventive anti-
inflammatory, antioxidant and anti-apoptotic effect in this model of rat colitis, as evi-
denced by a significant increase in SOD and GSH content as well as reduction of MPO activity,
by a decrease of MDA and reduction in NO as well as TNF-a production which is unregulated
as a consequence of the inflammatory status. It also significantly decreased the extent of DNA
fragmentation.
Conclusion: In conclusion, it is strongly suggested that the potent anti-inflammatory and
anti-apoptotic effects of P. amarus in rats with acetic acid induced colitis are mediated via
the neutrophil infiltration inhibition, inhibition of pro-inflammatory mediator production
and reducing DNA damage due to the presence of phyllanthin and hypophyllanthin phy-
toconstituents which offers a promising means for the treatment of diseases characterized
by inflammation of the gastrointestinal tract.
Copyright ª 2013, Indraprastha Medical Corporation Ltd. All rights reserved.
Abbreviations: GSH, glutathione; IBD, inflammatory bowel disease; MDA, malondialdehyde; MPO, myeloperoxidase; NO, nitric oxide;
PA, Phyllanthus amarus; SOD, superoxide dismutase.
* Corresponding author. Tel.: þ91 20 25437237, þ91 20 25437229; fax: þ91 20 25439383.
E-mail address: sbodh@yahoo.com (S.L. Bodhankar).
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/apme
a p o l l o m e d i c i n e 1 0 ( 2 0 1 3 ) 8 7 e9 7
0976-0016/$ e see front matter Copyright ª 2013, Indraprastha Medical Corporation Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.apme.2013.01.006

1. Introduction
Inflammatory bowel disease (IBD) is a chronic, relapsing in-
flammatory disease that is characterized by gastrointestinal
tract inflammation that leads to gastrointestinal mucosal
breakdown and ulcerations.1,2
Ulcerative colitis and Crohn’s
disease are the two primary disease processes that are asso-
ciated with IBD. In spite of rapid strides in research, the eti-
ology of IBD remains unknown. It has been demonstrated that
IBD is a combination of one or more factors firstly, dysregu-
lation of immune system (caused by genetic or environmental
factors),3,4
secondly, abnormal gastrointestinal tract luminal
factors, primarily dietary factors and the microorganisms
constituting the gastrointestinal tract flora, and thirdly, de-
fects in the GI mucosal barrier that allow luminal factors to
penetrate into the mucosa.5e7
Acetic acid induced colitis exhibit increased oxidation and
lipid peroxidation during initiation of colitis.8
It also asso-
ciated with alterations in the mucosal antioxidant defenses in
ulcerative colitis.
It is well accepted that reactive oxygen species play an
important role in the pathogenesis of IBD. Increased ROS
production resulting from respiratory burst of infiltrating
phagocytic cells which causes decreased antioxidant capacity
is a major pathogenic mechanism in IBD.9
Tumor necrosis
factor-a, interleukin-1 and interleukin-8 are the cytokines that
are secreted from macrophages and induces the production of
other inflammatory mediators such as ROS, and it also acti-
vates oxidative stress-responsive genes which amplify and
prolong inflammation during IBD.10
Synthetic chemical moieties which have been proposed for
their free radical scavenger activity such as 5-aminosalicylic
acid (5-ASA), sulfasulfapyridine (SASP) and glucocorticoids
helps in down regulation of the immune and inflammatory
responses of IBD. But their adverse reactions during prolonged
treatment and the high relapse rate limit their use.11,12
There
is paucity of ameliorative medication to treat IBD.
Herbal drugs provide a ray of hope. Polyphenols and fla-
vonoids have been shown to alleviate chronic inflammation in
experimental model of IBD.13
The major bioactive constituents of P. amarus are lignans
viz., phyllanthin, a diarylbutane and hypophyllanthin an
aryltetrahydronaphthalene (Fig. 1). Other constituents of the
plant include hydrolyzable tannins viz. phyllanthusiin D,
amariin, amarulone and amaric acid; alkaloids viz. ent-
norsecurinins, sobubbialine, epibubbialine. P. amarus has
been evaluated for an array of diseases like diabetes, hepatitis,
viral infection, bacterial infection, cancer, oxidative stress,
diarrhea and ulcer.14e22
The contraceptive effect of the herb
has also been reported by Rao and Alice.23
However the effects
of P. amarus plant in IBD are unexplored.
Hence, the objective of present investigation was to eval-
uate the protective effect of P. amarus in acetic acid induced
colitis model of IBD in laboratory animals.
2. Material and methods
2.1. Animals
Healthy adult male swiss albino mice (20e30 g) and male Wistar
rats (230e250 g) were obtained from the National Toxicological
Centre, Pune (India). The animals were housed in solid bottom
polypropylenecagesandmaintainedat24
CÆ1
C,withrelative
humidity of 45e55% and 12:12 h dark/light cycle. The animals
were acclimatized for a period of two weeks. The animals had
free access to standard pellet chow (Pranav Agro Industries Ltd.,
Sangli) throughout the experimental period, with the exception
of overnight fasting before induction of the ulcer. The animals
were provided with filtered water. The research protocols no.
CPCSEA/06/2011 was approved by the Institutional Animal
Ethics Committee (IAEC) of Poona College of Pharmacy, Pune.
2.2. Drugs and chemicals
P. amarus standardized extract (2.5% phyllanthin and hypo-
phyllanthin) was purchased from Natural Remedies Pvt. Ltd.,
Bangalore (Batch No. PA/10001). Prednisolone was obtained as
a gift sample from Symed Pharmaceutical Pvt. Ltd., Hyder-
abad. Acetic acid, anesthetic ether, ethanol, formalin, 1,1’,3,3’-
Tetraethoxypropane, crystalline beef liver catalase, reduced
glutathione, 5,50
-dithiobis (2-nitrobenzoic acid), bovine serum
albumin, thiobarbituric acid, Tris buffer, sucrose, trichloro-
acetic acid, citric acid monohydrate, sodium nitrate, copper
sulfate, sodium potassium tartrate, ethylenediamine tetra
acetic acid disodium salt, Folin’s phenol reagent were pur-
chased from S.D. Fine Chemicals, Mumbai, India. Sulphani-
lamide, naphthylamine diamine HCl, phosphoric acid were
obtained from Loba Chemi Pvt. Ltd., Mumbai, India. TNF-a
ELISA kit was obtained from Thermo Scientific, USA.
2.3. Plant material
The leaves of P. amarus were shade dried, powdered, and
extract 3 times with 100% methanol under reflux condition at
room temperature. These 3 extract were combined as filtered.
The solvent was evaporated under reduced pressure in a ro-
tary evaporator at 40
C. To this thick paste colloidal silicon
dioxide was added and dried in vacuum tube dryer. The final
product P. amarus extract was evaluated for phyllanthin and
hypophyllanthin content by HPLC.
2.4. High performance liquid chromatography analysis
of P. amarus extract
The P. amarus extract was subjected to high performance liq-
uid chromatography with column of RP C18, 5 m, 250 Â 4.6 mm
Fig. 1 e Chemical structures of phyllanthin and
hypophyllanthin.

and flow rate of 1.5 ml/min. The mobile phase for isolation
and detection was acetonitrile:buffer (40:60). Buffer consists
of 0.136 g of potassium hydrogen phosphate and 0.5 ml of
o-phosphoric acid. The detection wavelength was 230 nm.
2.5. Standard stock, calibration standards and quality
control sample preparation
The standard stock solution of phyllanthin and hypo-
phyllanthin (0.2 mg/ml) was prepared by dissolving requisite
amount in methanol. The quality control sample was pre-
pared as 5 mg/ml of P. amarus extract in methanol.
2.6. Acute toxicity testing
Acute oral toxicity in swiss albino mice was performed ac-
cording to OECD guidelines using AOT 425 software. Weighed
quantity of P. amarus extract was dissolved in distilled water
and administered orally. The animals were observed for 2
weeks following administration. Body weight, food con-
sumption, fluid intake and psychoemotor activities were
recorded daily.24
2.7. Dosages of P. amarus extract and standard drugs
used
The freshly prepared aqueous solution of P. amarus in the three
different dosages (50 mg/kg, 100 mg/kg and 200 mg/kg)25,26
was
administered to animals orally for 7 days. On 8th day, the
disease was induced by acetic acid.6
The drug treatment was
continued even after administration of acetic acid. Standard
drug used for comparison was prednisolone. Prednisolone was
not administered as pretreatment. It was administered on the
day of acetic acid administration. Prednisolone was given in
a dose of 2 mg/kg/day orally in rats as suspension containing
0.5% of sodium carboxymethyl cellulose.
2.8. Induction of colitis
Colonic inflammation was induced in fasted rats following the
method of Kandhare et al.6
The study comprised six different
groups of six animals in each groups as follows:
Group 1: normal animals: received 1 ml of distilled water for
11 days.
Group 2: control animals: received 2 ml of 4% acetic acid so-
lution (once, intrarectally) and 1 ml of distilled water for 11
days.
Group 3: drug treated animals: received 7 days pretreatment
with P. amarus (50 mg/kg p.o.) and 2 ml of 4% acetic acid so-
lution, intrarectally on 8th day. Drug treatment was continued
till 11th day.
till 11th day.
till 11th day.
Group 6: prednisolone treated animals: received prednisolone
(2 mg/kg p.o., for 3 days) and acetic acid (2 ml of 4% solution,
once, intrarectally). Prednisolone and acetic acid treatment
was started on the same day.6,27
On the 11th day the blood was withdrawn by retro-orbital
puncture and then animals were sacrificed by cervical dis-
location and colons were collected and the spleen from each
animal was also weighed. Portions of colonic specimens were
stored in 10% formalin for histopathological studies.
2.9. Evaluation of the disease
The disease induced in experimental animals was evaluated
based on its macroscopic characteristics. Evaluation pattern
for macroscopic characteristics of isolated colon was used, as
reported by Morris et al.28
2.9.1. Determination of ulcer area and ulcer index
The evaluation of ulcer area and ulcer index was performed
according to Dengiz and Gursan.29
For determination of ulcer
area, each colon was incised and washed with normal saline
and was scanned using CCD scanner at a magnification of
2400 dpi. The images were processed using image J software
and Adobe Photoshop to determine ulcer area.
2.9.2. Determination of hematological parameters
Hematological parameters were determined using an auto-
mated hematological analyzer (Sysmex KX-21) with specific
software for rat blood samples.30
The parameters analyzed
were white blood cell (WBC) number, red blood cell (RBC)
number, hemoglobin (Hb) concentration, hematocrit (HCT)
and platelet count (PLT).
2.9.3. Biochemical assays
Five hundred milligrams tissue from the colon was excised,
washed, chopped and homogenized at 3000 rpm in chilled Tris
buffer (10 mM, pH 7.4) and supernatant of homogenate was
employed to estimate various biochemical parameters.
2.9.3.1. Determination of colonic superoxide dismutase (SOD),
glutathione (GSH), malondialdehyde (MDA) and myeloperox-
idase (MPO) contents. The mucosal pathological alteration
occurs due to the overproduction of ROS. Colonic SOD, GSH,
MDA and MPO assay were determined as previously reported
method elsewhere.31e33
2.9.3.2. Determination of colonic nitrite/nitrate level. Colonic
NO level was estimated as nitrite and nitrate by the acidic
Griess reaction after reduction of nitrate to nitrite by vana-
dium trichloride according to the previously reported method
elsewhere.34
The Griess reaction relies on a simple colori-
metric reaction between nitrite, sulfonamide and N-(1-
naphthyl) ethylenediamine to produce a pink Azo-product
with maximum absorbance at 543 nm. The concentrations
were determined using a standard curve of sodium nitrate and
the results were expressed as mg/mg of wet tissue.
2.9.3.3. Determination of colonic TNF-a levels. The quantifi-
cations of TNF-a were performed with the help and
a p o l l o m e d i c i n e 1 0 ( 2 0 1 3 ) 8 7 e9 7 89

instructions provided by Thermo Scientific, USA manufac-
tured Rat TNF-a immunoassay kit. The assay employs the
sandwich enzyme immunoassay technique. The values were
expressed as pg/ml.6
2.9.3.4. Determination of DNA fragmentation as an index of
apoptosis. DNA isolation from colon tissue was performed
according to standard phenol chloroform cetyl trimethyl
ammonium bromide (CTAB) method mentioned by Tiwari
et al.35
10 ml of the DNA isolated from the colonic homogenate
was added to 3 ml of loading buffer (20 ml of glycerol 50%,
25 mg of bromophenol blue and 3 drops of 1N NaOH) and
subjected to 2D gel electrophoresis in 2% agarose gel. The gel
was examined in gel documentation instrument (Alpha
Innotech) and gel image was captured.
2.9.4. Evaluation based on microscopic (histologic) characters
Freshly excised colon of one animal from each group was
washed with saline and preserved in 10% formaldehyde so-
lution for histopathological studies. It was processed for 12 h
using isopropyl alcohol, xylene and paraffin embedded for
light microscopic study (Nikon E200). Paraffin embedded tis-
sue sections cut at 5 mm thickness were prepared and stained
after deparaffinization using hematoxylin and eosin stain (H
E) to verify morphological assessment of colon damage. Pho-
tomicrographs were captured at a magnification of 40Â.35
2.10. Statistical analysis
All the results were expressed as mean Æ S.E.M. Data analysis
was performed using GraphPad Prism 5.0 software (GraphPad,
SanDiego,CA).Statisticalcomparisonsweremadebetweendrug
treated groups and colitis control animals. Data of biochemical
parameters were analyzed using one way analysis of variance;
Dunnett’s multiple range test was applied for post hoc analysis.
A value of P 0.05 was considered to be statistically significant.
3. Result
3.1. Chromatographic investigation of P. amarus
standardized extract
Plants of genus P. amarus are a key source of moieties such as
phyllanthin and hypophyllanthin. The representative HPLC
finger-print of P. amarus extract was provided by the Natural
Remedies Pvt. Ltd., Bangalore (Batch No. PA/10001) and it is
shown in Fig. 2. The HPLC analysis of P. amarus extract reveal
the presence of phyllanthin and hypophyllanthin as evident
from HPLC studies for the plant extracts and standard phyl-
lanthin and hypophyllanthin sample. The retention time for
phyllanthin and hypophyllanthin on the HPLC column was
25.243 min and 26.832 min in a total run time of 50 min
(Fig. 2B). The percent area for phyllanthin and hypo-
phyllanthin were 68.14 and 31.95 respectively.
3.2. Acute toxicity testing
Acute toxicity studies of the aqueous extract of P. amarus
showed no signs and symptoms such as restlessness,
respiratory distress, diarrhea, convulsions and coma and it
was found safe up to 5000 mg/kg.
3.3. Acetic acid induced colitis
Intracolonic administration of acetic acid (4%) resulted in
colonic inflammation, which was evidenced after 48 h with
severe necrosis of the mucosa, regeneration and inflamma-
tory reaction. P. amarus treated group showed mild lesions,
regeneration and inflammatory reaction. The prednisolone
treated group showed suppressed inflammatory reaction.
3.3.1. Effect of P. amarus on wet weight of colon
As depicted in Table 1, wet weight of colon from acetic acid
administered rat was considerably higher (2.69 Æ 0.12 g) as
compared with normal rats (1.16 Æ 0.13 g), which serves as
marker of edema. There were significant reductions in wet
weight of colon in rats pretreated with P. amarus (100 mg/kg
and 200 mg/kg p.o.) (1.99 Æ 0.07 g and 1.55 Æ 0.09 g, P 0.01 and
P 0.001 respectively), as well as in rats treated with a pred-
nisolone (2 mg/kg p.o.) (1.39 Æ 0.11 g, P 0.001).
3.3.2. Effect of P. amarus on colon weight to length ratio
The ratio of colon weight/length ratio was found to be sig-
nificantly higher (0.21 Æ 0.011) in acetic acid control group as
compared to normal group (0.08 Æ 0.008). Pretreatment with
P. amarus (100 mg/kg and 200 mg/kg p.o.) for 7 days
decreased the colon weight to length ratio (0.15 Æ 0.008 and
0.12 Æ 0.011 respectively) and the decrease was found to be
significant (P 0.01 and P 0.001 respectively) as compared
to acetic acid control group in dose dependant manner. Rats
treated with prednisolone (2 mg/kg p.o.) also showed the
significant decreased the colon weight to length ratio
(0.10 Æ 0.009, P 0.001) as compared to acetic acid control
group [Table 1]
3.3.3. Effect of P. amarus on spleen weight
The rat with acetic acid induced colitis exhibited splenic
enlargement (2.32 Æ 0.12 g) as compared with normal
(1.19 Æ 0.98 g). Pretreatment with P. amarus (100 mg/kg and
200 mg/kg p.o.) for 7 days inhibiting spleen enlargement
(1.83 Æ 0.14 and 1.57 Æ 0.13 g respectively) and was found to be
significant (P 0.05 and P 0.001 respectively) as compared to
acetic acid control group. Treatment with prednisolone (2 mg/
kg p.o.) significantly attenuated (P 0.001) this increased
weight of spleen (1.43 Æ 0.09 g) as compared to acetic acid
control group [Table 1]
3.3.4. Effect of P. amarus on macroscopic scores
Colonic damage score represents macroscopic evidence of
extensive colonic mucosal injury along the 1e3 cm segment
at the site of instillation after 24 h of induction of colitis. The
mucosa appeared ulcerated, edematous and haemorrhagic
compared to normal control group. Pretreatment with
P. amarus (100 mg/kg and 200 mg/kg p.o.) for 7 days reduced
the severity of gross lesion score when compared with
acetic acid control rats. Prednisolone (2 mg/kg p.o.) treated
rats also showed the significant reduction (P 0.001) in
macroscopic score as compared to acetic acid control rats
[Table 1].

3.3.5. Effect of P. amarus on ulcer area and ulcer index
Rectal administration of 4% acetic acid produced ulcers of
colon in acetic acid control and all drug treated animals. The
mean ulcer area (39.67 Æ 1.35 mm2
) and ulcer index
(66.04 Æ 4.66) of acetic acid control group showed high ul-
cerogenic effect of acetic acid. Pretreatment with P. amarus
(100 mg/kg and 200 mg/kg p.o.) for 7 days significantly
decreased the ulcer area (31.83 Æ 1.81 and 25.67 Æ 1.11 mm2
respectively) as well as ulcer index (41.76 Æ 2.79 and
29.15 Æ 2.13) of colon (P 0.01 and P 0.001 respectively) as
compared to acetic acid control group in a dose dependant
manner. These increased ulcer area (9.00 Æ 0.96 mm2
) as well as
ulcer index (15.27 Æ 2.75) was significantly decreased (P 0.001)
in the prednisolone (2 mg/kg p.o.) treated rats [Table 1].
Fig. 2 e (A) HPLC finger-print of reference standard. (B) HPLC finger-print of Phyllanthus amarus aqueous extract.

3.3.6. Effect of P. amarus on hematology
The acetic acid treated rats showed a significant decrease in the
hematological parameters WBC, RBC count, Hb and platelet
compared to normal rats [Table 2]. These reductions were sig-
nificantly attenuated in the P. amarus pretreated group
(P 0.001). No effect was observed in the rest of the hemato-
logical parameters analyzed. The decreased levels of hemato-
logical parameters including WBC, RBC, Hb and platelet were
significantly increased (P 0.001) by the treatment of prednis-
olone (2 mg/kg p.o.) as compared to acetic acid control group.
3.3.7. Effect of P. amarus on colonic SOD concentrations
Induction of colitis produced a significant decrease in colonic
SOD content (2.88 Æ 0.69 U/mg of protein) as compared with
the normal group (13.93 Æ 1.17 U/mg of protein). Pretreatment
with P. amarus (100 mg/kg and 200 mg/kg p.o.) for 7 days sig-
nificantly increased SOD content (8.13 Æ 0.56 and
10.83 Æ 0.71 U/mg of protein) as compared with acetic acid
control group (P 0.01 and P 0.001 respectively). Predniso-
lone also protected against SOD depletion induced by acetic
acid (12.25 Æ 0.96 U/mg of protein, P 0.001) [Table 3]
3.3.8. Effect of P. amarus on colonic GSH concentrations
As depicted in Table 3, induction of colitis produced a signifi-
cant decrease in colonic GSH content (12.96 Æ 1.41 mg/mg
protein) compared with the normal group (28.47 Æ 1.87 mg/mg
protein). P. amarus (100 mg/kg and 200 mg/kg p.o.) pretreat-
ment for 7 days significantly increased GSH content as com-
pared with acetic acid group (20.49 Æ 0.97 mg/mg protein and
22.46 Æ 1.41 mg/mg protein, P 0.01 and P 0.001 respectively).
Prednisolone also significantly protect (P 0.001) against GSH
depletion induced by acetic acid (24.80 Æ 1.48 mg/mg protein).
3.3.9. Effect of P. amarus on colonic LPO concentration
Levels of LPO in the colons of acetic acid control group were
substantially higher (76.78 Æ 2.96 nmol/mg of protein) than in
normal group (21.40 Æ 2.83 nmol/mg of protein) (Table 3). In
rats pretreated with P. amarus (100 mg/kg and 200 mg/kg p.o.)
for 7 days, the levels of colonic LPO were reduced significantly
(51.23 Æ 4.15 and 42.23 Æ 3.57 nmol/mg of protein, P 0.001
respectively) as compared to acetic acid control group. Rats
treated with prednisolone (2 mg/kg) also had significantly
reduced (P 0.001) colonic LPO levels (29.51 Æ 2.72 nmol/mg of
protein) as compared to acetic acid control group [Table 3].
3.3.10. Effect of P. amarus on colonic MPO concentration
Colonic MPO concentration in acetic acid control group was
higher (20.12 Æ 0.81 U/mg) in comparison to the normal group
(5.54 Æ 0.55 U/mg). Pretreatment with P. amarus (50 mg/kg,
100 mg/kg and 200 mg/kg p.o.) for 7 days produced significant
decrease in MPO concentration (15.78 Æ 1.07, 13.10 Æ 0.91 and
11.13 Æ 0.72 U/mg, respectively) as compared to acetic acid
control group (P 0.01 and P 0.001, respectively). Predniso-
lone (2 mg/kg) also provided protection against the elevation
in MPO concentration induced by acetic acid treatment
(7.32 Æ 0.65 U/mg, P 0.001) [Table 3].
3.3.11. Effect of P. amarus on colonic nitrite/nitrate level
Acetic acid induced colitis resulted in increased colonic ni-
trite/nitrate level (63.40 Æ 3.38 mg/mg) in comparison with
normal group (26.86 Æ 2.97 mg/mg). Pretreatment with
P. amarus (100 mg/kg and 200 mg/kg p.o.) for 7 days produced
a significant reduction in colonic nitrite/nitrate level com-
pared to acetic acid induced colitis group (51.76 Æ 2.11 and
42.33 Æ 3.04 mg/mg, P 0.05 and P 0.001 respectively). Rats
Table 1 e Effect of Phyllanthus amarus on colon weight to length ratio, spleen weight, macroscopic score, ulcer area and
ulcer index of rat in acetic acid induced colitis.
Groups Colon weight Colon weight
to length ratio
Spleen weight
(g)
Macroscopic
score
Ulcer area
(mm2
)
Ulcer index
Normal 1.16 Æ 0.13 0.08 Æ 0.008 1.19 Æ 0.98 e e e
Acetic acid control 2.69 Æ 0.12###
0.21 Æ 0.011###
2.32 Æ 0.12###
9.16 Æ 0.40###
39.67 Æ 1.35###
60.03 Æ 1.88###
Prednisolone (2 mg/kg) 1.39 Æ 0.11*** 0.10 Æ 0.009*** 1.43 Æ 0.09*** 2.66 Æ 0.33*** 9.00 Æ 0.96*** 15.27 Æ 2.75***
PA (50 mg/kg) 2.31 Æ 0.15 0.18 Æ 0.015 2.04 Æ 0.13 7.83 Æ 0.47 35.50 Æ 1.87 51.45 Æ 2.43
PA (100 mg/kg) 1.99 Æ 0.07** 0.15 Æ 0.008** 1.83 Æ 0.14* 6.83 Æ 0.40** 31.83 Æ 1.81** 41.76 Æ 2.79**
PA (200 mg/kg) 1.55 Æ 0.09*** 0.12 Æ 0.011*** 1.57 Æ 0.13*** 4.83 Æ 0.60*** 25.67 Æ 1.11*** 29.15 Æ 2.13***
Data are expressed as mean Æ S.E.M. from five rats and analyzed by one way analysis of variance followed by Dunnett’s test. *P 0.05, **P 0.01,
***P 0.001 as compared to acetic acid control group and #
P 0.05, ##
P 0.01, ###
P 0.001 as compared to normal group (n ¼ 5).
Table 2 e Effect of Phyllanthus amarus on hematological parameters of rat in acetic acid induced colitis.
Parameter Normal Acetic acid control Prednisolone (2 mg/kg) PA
50 mg/kg 100 mg/kg 200 mg/kg
WBC (X103/mL) 21.78 Æ 1.20 6.93 Æ 0.6###
18.55 Æ 1.32*** 9.48 Æ 1.23 13.48 Æ 1.23*** 14.15 Æ 0.75***
RBC (X106/mL) 14.57 Æ 1.42 4.22 Æ 0.56###
10.86 Æ 0.89*** 5.38 Æ 0.61 8.35 Æ 0.92*** 9.29 Æ 0.78***
HGB (g/dL) 14.75 Æ 0.82 8.24 Æ 0.58###
13.81 Æ 0.30*** 10.13 Æ 0.78 11.41 Æ 0.72** 12.43 Æ 0.38***
PLT (X105/mL) 12.51 Æ 0.96 6.78 Æ 0.50###
11.22 Æ 0.62*** 8.39 Æ 0.51 8.67 Æ 0.15 10.66 Æ 0.36***
P 0.05, ##
P 0.01, ###

treated with prednisolone (2 mg/kg) also showed the signifi-
cant protection against elevated levels of colonic nitrite/ni-
trate (37.85 Æ 2.04 mg/mg, P 0.001) as compared to acetic acid
control group [Table 3].
3.3.12. Effect of P. amarus on colonic TNF-a level
There was significant increase in colonic TNF-a in acetic acid
control (245.8 Æ 8.41 pg/mg) group as compared to normal
animals (87.22 Æ 6.59 pg/mg). Rats pretreated with P. amarus
(100 mg/kg and 200 mg/kg p.o.) for 7 days showed significant
decrease (183.5 Æ 10.50 and 139.5 Æ 8.48 pg/mg, P 0.05 and
P 0.001 respectively) in this elevated levels of TNF-a as
compared to acetic acid control group. These elevated levels of
TNF-a (117.3 Æ 9.69 pg/mg) were significantly decreased
(P 0.001) by the treatment of prednisolone (2 mg/kg) as
compared to acetic acid control group [Fig. 3]
3.3.13. Effect of P. amarus on colonic DNA fragmentation
Higher degree of apoptosis was observed in rats treated with
acetic acid which was reflected by maximum fragmentation of
DNA. Pretreatment with P. amarus (100 mg/kg and 200 mg/kg
p.o.) for 7 days reduced the extent of DNA fragmentation.
Treatment with prednisolone (2 mg/kg) also showed reduction
in the extent of DNA fragmentation. There was no DNA
damage observed in normal group [Fig. 4].
3.3.14. Histological analyses
Microscopically, colon samples from normal group [Fig. 5A]
showed the normal histology of the rat colon. In the acetic
Fig. 3 e Effect of aqueous extract of Phyllanthus amarus on
colon TNF-a of rats in acetic acid induced colitis. Data are
expressed as mean ± S.E.M. from five rats and analyzed by
one way analysis of variance followed by Dunnett’s test.
*P 0.05, **P 0.01, ***P 0.001 as compared to acetic
acid control group and #
P 0.05, ##
P 0.01, ###
P 0.001
as compared to normal group (n [ 5).
Table 3 e Effect of Phyllanthus amarus on various antioxidant parameters of rat colon in acetic acid induced colitis.
Parameter Normal Acetic acid control Prednisolone (2 mg/kg) PA
50 mg/kg 100 mg/kg 200 mg/kg
SOD (unit/mg protein) 13.93 Æ 1.17 2.88 Æ 0.69###
12.25 Æ 0.96*** 4.69 Æ 0.69 8.13 Æ 0.56** 10.83 Æ 0.71***
Reduced glutathione
(mg of GSH/mg protein)
28.47 Æ 1.87 12.96 Æ 1.41###
24.80 Æ 1.48*** 14.84 Æ 0.96 20.49 Æ 0.97** 22.46 Æ 1.41***
Lipid peroxidation
(nmol/mg protein)
21.40 Æ 2.83 76.78 Æ 2.96###
29.51 Æ 2.72*** 66.33 Æ 4.51 51.23 Æ 4.15*** 42.23 Æ 3.57***
MPO (U/mg) 5.54 Æ 0.55 20.12 Æ 0.81###
7.32 Æ 0.65*** 15.78 Æ 1.07** 13.10 Æ 0.91*** 11.13 Æ 0.72***
Colonic NO (mg/mg) 26.86 Æ 2.97 63.40 Æ 3.38###
37.85 Æ 2.04*** 60.80 Æ 2.93 51.76 Æ 2.11* 42.33 Æ 3.04***
P 0.05, ##
P 0.01, ###
Fig. 4 e Effect of aqueous extract of Phyllanthus amarus on
DNA fragmentation in acetic acid induced colitis. Lane 1:
normal; lane 2: acetic acid control; lane 3: prednisolone
(2 mg/kg); lane 4: Phyllanthus amarus (100 mg/kg); and lane
5: Phyllanthus amarus (200 mg/kg).

acid control group [Fig. 5B], colonic sections showed typical
inflammatory changes in colonic architecture such as trans-
mural necrosis, edema and diffuse inflammatory cell infil-
tration in the mucosa, desquamated areas and loss of the
epithelium. There were no inflammatory cells in the lamina
propria and the epithelium remained intact in prednisolone
(2 mg/kg) treated group [Fig. 5C]. Pretreatment with P. amarus
(200 mg/kg p.o.) showed significant attenuation in the severity
of colon injury, a higher integrity of mucosal architecture and
the epithelial loss [Fig. 5D] [Table 4].
4. Discussion
Amongst the various experimental models of inflammatory
boweldiseases, inductionofcolitisby acetic acid inrats isone of
the standardized and extensively used methods.6,30,36,37
It
mimics some characteristics of IBD, including transmural colon
inflammation, granuloma formation, scarring and fibrosis of
intestinal tissue, fecal impaction, stenosis, diarrhea and oxi-
dative stress.30
Increased production of inflammatory media-
tors, neutrophil infiltration and increased vasopermeability are
the major causative factors that are involved in the induction of
colitis by intrarectal instillation of acetic acid.38
IBD is charac-
terized by neutrophils and macrophages infiltration which in
turn cause increased levels of ROS in the inflamed tissue, as
well as a decreased antioxidant capacity.39
The present study
has shown that acetic acid induced ulcerative colitis was
associated with macroscopic, microscopic and biochemical
changes.
The wet weight of colon is hallmark of degree of inflam-
mation in colon.40
P. amarus significantly inhibited this
increasing wet weight of colon and colon weight to length
ratio by virtue of it healing property.
The spleen is the essential part of the immune system as
well as center of activity in the reticuloendothelial system. It is
responsible for destruction of redundant red blood cells. Pre-
vious investigations have revealed that splenic atrophy is
associated with complication in colitis in humans and rats.41,42
Our result is in coincident with previous investigations.
P. amarus significantly attenuated this increasing splenic
weight by virtue of its immune system modulatory property.
The gross morphological lesions characterized by ulcer and
necrotic area of various sizes,24
were healed depicting
Fig. 5 e Photomicrographs of sections of colons from rats stained with H E (403). Colon microscopic image of (A) normal rat
with intact epithelial (white arrow) and mucosal layer (black arrow); (B) acetic acid induced colitis rat with extensive damage
including edema in submucosa (red arrow) and cellular infiltration (blue arrow), hemorrhages, necrosis (yellow arrow); (C)
prednisolone (2 mg/kg p.o.) treated rat with infiltration and edema (red arrow); (D) Phyllanthus amarus (200 mg/kg p.o.) 7 days
pretreated rat with edema in submucosa (red arrow) and low degree of cellular infiltration. Images (403 magnification) are
typical and representative of each study group.

protection of microflora from the corrosive effect of acetic acid
by P. amarus. Ulcer area and ulcer index were quantitatively
determined and reflect the protective action of P. amarus.
Clinical manifestation of IBD includes exacerbated hemato-
logical imbalance leading to unexplained diarrhea and mal-
ena. This feature was reversed in P. amarus treated animals
showing its therapeutic potential. Hematology provides an
insight into the disease state of colitis. The various compo-
nents of blood were disproportionately altered.6,30
In the
vehicle treated group where as their ratios were unchanged in
P. amarus treated animals.
Overproduction of ROS contributes to pathobiological al-
terations in the mucosa.43e45
The principle reacting oxygen
metabolite altering the colonic milieu includes SOD, GSH and
MDA.32e34
Superoxide anions are transformed into secondary
antioxidant H2O2 by superoxide dismutase. P. amarus restored
the reduced level of SOD.
Glutathione is essential in scavenging of reactive oxygen
species and keeping the enzyme glutathione peroxidase in
a reduced state.46
Decreased glutathione levels, is indicative of
oxidant stress in experimental model of colitis.47
The effect of
P. amarus in inflammatory processes could be due to the
antioxidant properties of these compounds observed in this
study and also evidenced by the restoration of colonic gluta-
thione content.
The enhanced lipid peroxidation provides an index of
oxidative stress.6
Acetic acid has been known to elevate levels
of MDA to produce colitis. The present investigation indicated
inhibition of lipid peroxidation. The P. amarus was found to
decrease the elevated level of MDA in acetic acid induced
colitis.
The colonic MPO activity is an index of neutrophil infiltra-
tion and has been reported to be increased in acetic acid trea-
ted animals.48
During inflammation activated neutrophils
enter in inflamed mucosa and submucosa of the large intes-
tine, leading to overproduction of reactive oxygen and nitrogen
species, proteases and lipid mediators that result in intestinal
injury.44,48e50
P. amarus attenuated increase in MPO level and
there by neutrophil infiltration. This increase in MPO activity
was substantially reduced in rats treated with P. amarus.
Many studies have shown that nitric oxide (NO) takes part in
the pathogenesis of inflammatory bowel disease.51
Altered
regulation of NO has been implicated in many gastrointestinal
disease states. More specifically, NO production was shown to
be increased in ulcerative colitis.52,53
As an important inflam-
matory mediator, NOcould react with superoxide anion to form
more poisonous nitrite anion, which then disturbs the function
of inflammatory cells and further impairs the tissue.54e57
P. amarus attenuated this increase in level of nitric oxide.
It has been demonstrated TNF-a plays a vital role in
pathogenesis of IBD which caused migration of NF-KB into
nucleus of mucosal cells that responsible for overproduction
of other pro-inflammatory cytokines and further colonic
damage.58
Pretreatment with P. amarus reduced the elevated
level of TNF-a proving its anti-inflammatory potential.
Boirivant et al59
reported that IBD is associated with
reduced apoptosis of inflammatory cells. In IBD the elevated
level of oxidative stress is responsible for DNA damage and
the resultant injury.9
By virtue of its antioxidant potential, P.
amarus maintained cellular integrity by inhibiting free radical
generation along with lipid peroxidation and DNA damage.
It has been well documented that Phyllanthus is contain
phyllanthin and hypophyllanthin as a major active constitu-
ent.60
The HPLC finger-print of the present investigation also
revealed the same findings. Chirdchupunseree and Pramyo-
thin, 2010 and Chouhan and Singh, 2011 reported that the
phytoconstituent of Phyllantus via phyllanthin and hypo-
phyllanthin is mainly responsible for antioxidant and anti-
inflammatory potential of the plant. Result of the present
investigation agrees with the findings of Chirdchupunseree
and Pramyothin,61
2010 and Chouhan and Singh,62
2011.
In conclusion, the results of the present investigation
provide pharmacological credence of P. amarus in the patho-
genesis of IBD. The results of this study suggest that the
potent anti-inflammatory and anti-apoptotic effects of
P. amarus in rats with acetic acid induced colitis are mediated
via the neutrophil infiltration inhibition, inhibition of
pro-inflammatory mediator production and reducing DNA
damage due to the presence of phyllanthin and hypo-
phyllanthin phytoconstituents. These results strongly suggest
that phyllanthin and hypophyllanthin are two phytocon-
stituents which mainly responsible for the modulation of
pathophysiological activity of P. amarus which offers a prom-
ising means for the treatment of diseases characterized by
inflammation of the gastrointestinal tract.
Conflicts of interest
All authors have none to declare.
Acknowledgments
The authors would like acknowledge Dr. S. S. Kadam, Vice-
Chancellor and Dr. K. R. Mahadik, Principal, Poona College of
Pharmacy, Bharati Vidyapeeth Deemed University, Pune,
Table 4 e Effect of Phyllanthus amarus on pathological changes of rat colon in acetic acid induced IBD.
Group Ulceration Hyperemia Necrosis Edema Cellular infiltration Goblet cell hyperplasia
Normal 0 0 0 þ 0 0
Acetic acid control þþþþ þþþ þþþþ þþþþ þþþþ þþþ
Prednisolone (2 mg/kg) 0 þ þ þþ þ þ
PA (200 mg/kg) þ þþ þ þ þ þþ
0: No abnormality detected; þ: damage/active changes up to less than 25%; þþ: damage/active changes up to less than 50%; þþþ: damage/active
changes up to less 75%; þþþþ: damage/active changes up to more than 75%.

India, for providing necessary facilities to carry out the study.
We are also thankful to the All India Council of Technical and
Education (AICTE), India for financial support by awarding
GATE scholarship to one of the authors Mr. Kandhare Amit for
the research work.
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Protective Effect of Phyllanthus Amarus by Modulation of Endogenous Biomarkers and DNA Damage in Acetic Acid induced Ulcerative Colitis: Role of Phyllanthin and Hypophyllanthin

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Protective Effect of Phyllanthus Amarus by Modulation of Endogenous Biomarkers and DNA Damage in Acetic Acid induced Ulcerative Colitis: Role of Phyllanthin and Hypophyllanthin