Assessment of the Antiurolithiatic and Antioxidant Properties of Ficus pseudopalma Blanco Leaves (Moraceae)

Abstract

The present study investigates the antiurolithiatic and antioxidant potential of Ficus pseudopalma Blanco. The crude dichloromethane (DCM) leaf extract was fractioned by silica gel column chromatography. Ethyl acetate fraction showed an IC50 of 0.2586 mg/mL against ·OH radical and 5.289 mg/mL against H202. The crude DCM extract was assessed for antiurolithiatic property on ethylene‐glycol induced male Sprague‐Dawley rats. It was evaluated in preventive and therapeutic regimen. Forty‐ two rats were used with 6 rats per group. Preventive groups (Group 3 & 4) received induction with simultaneous treatment of extract from Day 1‐28. Therapeutic groups (Group 5‐7) received induction on Day 1, co‐administration of extract started on Day 15. The experimental groups were as follows: 1 – Vehicle, 2 – Induction control, 3 – 1000 mg/kg, 4 – 500 mg/kg, 5 – 1000 mg/kg, 6 – 500 mg/kg, and 7 – 10 mg/kg lupeol. The induction of ethylene glycol resulted to significant increase in Malondialdehyde (MDA) levels, serum creatinine, urine oxalate and kidney calcium content (all p values are <0.05), consistent with the histopathologic scoring of the kidney tissues wherein induction control group had the most number of crystal deposits. However, treatment of F. pseudopalma crude DCM extract in both preventive and therapeutic design as well as lupeol significantly decreased all the parameters (all p values are <0.05). Therapeutic dose of 1000 mg/kg F. pseudopalma crude DCM extract was comparable to the effect of lupeol in significantly decreasing serum creatinine (p=0.998) and urine oxalate levels (p=0.158). The present study established the antioxidant and antiurolithiatic potential of the plant.

Full text

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RESEARCHPAPER
22
Assessment of the Antiurolithiatic and Antioxidant Properties of Ficus
pseudopalma Blanco Leaves (Moraceae)
Christine Joy H. Acosta*1, Allan Patrick H. Macabeo2, Librado A. Santiago2
*1Graduate School, University of Santo Tomas, Manila, Philippines
christinejoy.acosta@yahoo.com1
2Research Center for Natural and Applied Sciences, University of Santo Tomas, Manila, Philippines
allanpatrick_m@yahoo.com2
librado_santiago@yahoo.com2
Abstract
The present study investigates the antiurolithiatic and antioxidant potential of FicuspseudopalmaBlanco.Thecrude
dichloromethane(DCM)leafextractwasfractionedbysilicagelcolumnchromatography.EthylacetatefractionshowedanIC50
of0.2586 mg/mLagainst·OHradical and5.289mg/mL against H202.ThecrudeDCM extractwasassessed forantiurolithiatic
propertyonethylene‐glycolinducedmaleSprague‐Dawleyrats.Itwasevaluatedinpreventiveandtherapeuticregimen.Forty‐
tworatswereusedwith6ratspergroup.Preventivegroups(Group3&4)receivedinductionwithsimultaneoustreatmentof
extractfromDay1‐28.Therapeuticgroups(Group5‐7)receivedinductionon Day 1, co‐administration of extract startedon
Day15.Theexperimentalgroupswereasfollows:1 –Vehicle,2–Inductioncontrol,3–1000mg/kg,4– 500mg/kg,5–1000
mg/kg,6–500mg/kg,and7–10mg/kglupeol.Theinductionof ethylene glycol resulted to significant increase in
Malondialdehyde(MDA)levels,serumcreatinine,urineoxalateandkidneycalciumcontent(allpvaluesare<0.05),consistent
with the histopathologic scoring of the kidney tissues wherein induction control group had the most number of crystal
deposits.However,treatmentofF.pseudopalmacrudeDCMextractinbothpreventiveandtherapeuticdesignaswellaslupeol
significantlydecreasedalltheparameters(allpvaluesare<0.05).Therapeuticdoseof1000mg/kgF.pseudopalmacrudeDCM
extractwascomparableto theeffectof lupeol insignificantlydecreasing serum creatinine(p=0.998)andurine oxalate levels
(p=0.158).Thepresentstudyestablishedtheantioxidantandantiurolithiaticpotentialoftheplant.
Keywords
Ficuspseudopalma,antioxidant,antiurolithiatic,lipidperoxidation
INTRODUCTION
Urolithiasis (urinary calculi or stones) refers to
calcifications that form in the urinary system, primarily
in the kidney or ureter, and may also form in or migrate
into the lower urinary system which includes the
bladder or urethra (Bernier, 2005). In 2010, the
prevalence of the disease in the Philippines is at 4-20%,
with higher incidence in male as compared to female
with a ration of 2:1 (Lopez, 2010). Also, despite
considerable advancements in the treatment, recurrence
rate is high at 75% (Sandhya et al., 2010).
One of the pathway in urolithiasis is presented by
hyperoxaluria, one of the major risk for calcium oxalate
stone formation, it leads to activaton of Renin
Angiotensin System which will increase the levels of
Angiotensin II, thereby activating Nicotinamide adenine
dinucleotide phosphate oxidase, which is an important
source of receptor-mediated reactive oxygen species
(ROS) generation (Pareta et al., 2011). Renal cellular
exposure to oxalate leads to the production of ROS and
development of oxidative stress. Free radical mediated
oxidation promotes lipid peroxidation which greatly
contributes to cell membrane destruction and damage.
Inflammation of renal cells subsequently facilitates the
retention of calcium oxalate crystals and growth of
stones in renal tubules (Selvam, 2002). Hence,
addressing this problem is of great and immediate
concern.
Ficus pseudopalma Blanco (Family Moraceae) is an
endemic plant species in the Philippines. It is commonly
known as Philippine Fig, “Niog-niogan” and “Lubi-lubi”
in vernacular. A claim reported by Stuart in 2011
believed that the decoction of the leaves is used for the
treatment of kidney stones and diabetes. This is
corroborated by a study of Ragasa et al. in 2009 where
one of the constituents isolated from the leaves of the
plant is lupeol, a pentacyclic triterpene which has been
reported to exhibit antiurolithiatic property (Vidya et al.,
2002). Evidences in previous studies showed that lupeol
demonstrates antiurolithiatic property by inhibiting lipid
peroxidation (Sudhahar et al., 2008).
Presently, there are no published scientific evidences as
yet to prove this claim in Ficus pseudopalma Blanco.
Hence, this research aimed to assess the antiurolithiatic
and antioxidant properties of Ficus pseudopalma
Blanco leaves and looked into the possible correlation
of the two properties.
MATERIAL AND METHODS
Chemicals and reagents
Analytical grade dichloromethane (DCM), petroleum
ether, ethyl acetate (EtoAc) and acetone used for the
extraction and fractionation of FP leaves were
purchased from Bellman Corp. Lupeol ( ≥ 94%) was
purchased from Sigma Chemicals Co. Ascorbic acid,
used as the standard for free radical scavenging activity

Christine Joy H. Acosta et al,Current Research in Biological and Pharmaceutical Sciences, 2 (2), March-April 2013, 22-30
23
was purchased from University of Santo Tomas –
Office of Laboratory Equipments and Supplies (OLES).
Standard malondialdehyde (MDA) and Standard oxalate
reagents were purchased from BioAssay systems and
Trinity Biotech, respectively.
Plant material preparation
Fresh leaves samples were collected in Bgy. San Ramon,
Buhi, Camarines Sur. It was identified and
authenticated by Noe Gapas of National Museum,
Philippines. Leaf samples were air dried at room
temperature and grinded using Wiley mill grinder.
Approximately 4 kilograms of dried ground leaves were
subjected to exhaustive percolation using
dichloromethane (DCM) as extracting solvent.
Percolate was collected every 48 hours then
concentrated under reduced pressure using rotary
evaporator at a temperature below 40°C. The
concentrated extract was preserved at 2-8°C for further
use.
Fractionation
The concentrated leaf extract was fractioned by silica
gel column chromatography. Elution was carried out
using the following solvents: petroleum ether,
petroleum ether-ethyl acetate (50:50), ethyl acetate,
ethyl acetate-acetone (50:50) and acetone. One (1) gram
of sample extract was loaded on top of the silica gel and
6 mL of the eluents were added according to increasing
polarity (gradient elution). Addition of next solvent was
done after the complete elution of the former solvent
was achieved. Collection of eluates was done by
volume of 2 mL in test tube.
Screening for Triterpenes
Liebermann Burchard Test for Triterpenes
The different fractions gathered were screened for the
presence of triterpenes by Liebermann-Burchard test.
Ten mg of the fractions was dissolved in 1 mL of
chloroform; 1 mL of acetic anhydride was added
following the addition of 2 mL of conc. H2SO4. The
positive result of the test is the appearance of colors
ranging from blue to green, red, pink, purple or violet
(Guevarra, 2005). Comparable results were obtained
from triplicate determinations.
Thin Layer Chromatography (TLC)
TLC was performed on a silica gel plate (5 × 20 cm,
Kieselgel 60F, 0.25 mm, Merck). An aliquot of the
different fractions was spotted on the silica gel plate
with a solvent system of toluene : methanol (9:1 v/v).
The developed chromatogram was observed after
exposure to Iodine crystals. Comparable results were
obtained from triplicate determinations.
High Performance Liquid Chromatography (HPLC)
The fractions which confirmed the presence of
triterpenes were further screened for identification of
lupeol by HPLC. One mg of sample was diluted with 5
mL methanol. The test solution was injected to a C18
column of an Agilent Series II-HPLC machine. The
mobile phase consisted of 50:50 methanol and 0.5
percent phosphoric acid in water, respectively. The
gradient elution program was run at a flow rate of 0.9
mL/min; total run time was 30 minutes. Absorbance
was detected at 280 nm. Lupeol was generated at the
following concentrations: 5 ppm, 25 ppm, 50 ppm and
100 ppm. A standard curve of the different
concentrations of lupeol was prepared and the
concentration of lupeol in the sample was computed by
linear regression formula.
Free radical scavenging activity
Hydroxyl radical (·OH) scavenging activity
The fraction that yielded the highest concentration of
lupeol was tested for free radical scavenging activity.
The hydroxyl radical scavenging ability was determined
following the method described by Samak et al. (2009)
using Fenton reaction. Fenton reaction mixture
containing 3 mM deoxyribose, 0.1 mM ferric chloride,
0.1 mM EDTA, and 0.1 mM ascorbic acid and 2 mM
H2O2 in 20 mM phosphate buffer pH 7.4 was added to
various concentrations of sample (63 μg/mL - 1000
μg/mL in 95% ethanol). The reaction mixture was
incubated for 30 minutes at 37°C and was added to 0.5
mL of 5% trichloroacetic acid (TCA) and 0.5 mL of 1%
thiobarbituric acid (TBA) to yield a final volume 3 mL.
The reaction mixture was kept in boiling water bath for
30 minutes and cooled. The absorbance was measured
at 532 nm against an appropriate blank solution. All
tests were performed three times. Ascorbic acid was
used as a positive control. Percent inhibition in
hydroxyl radical was calculated by the following
expression: Percentage of inhibition = [(Ao – A1) / Ao] x
100, where Ao is the absorbance of the control and A1 is
the absorbance of the sample.
Hydrogen peroxide (H2O2) scavenging activity
The hydrogen peroxide scavenging ability was
determined according to the method described by
Ebrahimzade et al. (2010). Different concentrations of
sample (1 mg/ml – 10 mg/ml in 95% ethanol) and
standard ascorbic acid in distilled water was added to
0.6 mL solution of 40 Mm H2O2 in phosphate buffer pH
7.4. After 10 min, absorbance of H2O2 was recorded at
230 nm against blank solution without H2O2. Percent
inhibition in H2O2 was calculated by the following
expression: Percentage of inhibition = [(Ao – A1) / Ao] x
100, where Ao is the absorbance of the control and A1 is
the absorbance of the sample.
Animal study
Test animal
Experimental protocol was approved by the University
of Santo Tomas Institutional Animal Care and Use
Committee (IACUC). All experimental study was
performed at the University of Santo Tomas – Tomas
Aquinas Research Center Animal House. The institution
also served as the housing facility for the test animals
and site of experimentation. All test animals were
procured from Food and Drugs Administration
laboratory. Test animals were properly acclimatized for

Christine Joy H. Acosta et al,Current Research in Biological and Pharmaceutical Sciences, 2 (2), March-April 2013, 22-30
24
7 days prior to experimental protocol and given regular
rat chow diet and water supply daily.
OECD Main test
The Organization for Economic Cooperation and
Development (OECD) Main test for acute toxicity is
performed using five Sprague-Dawley rats which were
observed for 14 days. The first rat was given a 175
mg/kg initial dose of F. pseudopalma crude DCM
extract orally. The rat was observed for 48 days. The
dose administered to the other four rats was determined
from the observation of the first rat, if the rat survives
the next dose is increased and if it the rat dies the next
dose is decreased.
Antiurolithiatic study
Forty two rats were used and six rats were randomized
and assigned per experimental group. Induction of
urolithiasis was done by daily oral administration of
0.75% ethylene glycol and 1% ammonium chloride
from Day 1-5, followed daily oral administration of
0.75% ethylene glycol alone from Day 6-28. All
experimental groups received urolithiatic induction for
28 days except for the vehicle control. Administration
of F. pseudopalma crude DCM extract was done via
oral gavage daily using 5% tween 80 solution as vehicle.
The effect of F. pseudopalma crude DCM extract was
evaluated in two treatment design: preventive and
therapeutic regimen. Preventive groups received stone
induction with simultaneous treatment with extract from
Day 1-28. Therapeutic groups received stone induction
on Day 1, co-administration of extract started on Day
15. Experimental protocol was completed in 28 days.
The experimental groups for the antiurolithiatic study
were as follows: Group 1 – Vehicle control, Group 2 –
Induction control, Group 3 – 1000 mg/kg dose of F.
pseudopalma crude DCM extract (Preventive), Group
4 – 500 mg/kg dose of F. pseudopalma crude DCM
extract (Preventive), Group 5 – 1000 mg/kg dose of F.
pseudopalma crude DCM extract (Therapeutic), Group
6 – 500 mg/kg dose of F. pseudopalma crude DCM
extract and lastly Group 7 – 10 mg/kg dose of lupeol
(Therapeutic- Positive control).
Through the course of the experiments blood samples
were extracted by tail clipping method and urine
samples were collected using improvised metabolic
cage on day 0, 14 and 28. On the 28th day, all rats were
euthanized via cervical dislocation and kidney samples
were collected afterwards. Serum samples were
determined for creatinine levels, Urine samples were
analyzed for the presence of calcium oxalate crystals by
chemical and microscopic analysis, oxalate content was
also quantified. Kidney samples were used for calcium
determination, histopathologic scoring and
Thiobarbituric Acid Reactive Substances (TBARS)
assay.
Serum creatinine determination
Blood samples were collected by tail clipping method
then centrifuged at 5000 rpm for 5 minutes and serum
was collected. Automated creatinine determination was
performed at United Diagnostics laboratory, Dapitan St.,
Sampaloc, Manila.
Urine analysis
Urine samples were collected using improvised
aluminum metabolic cage. Oxalate content was
measured on urine samples collected on day 0, 14 and
28 following the procedure of Trinity Biotech Oxalate
kit. The 24-hour collective urine was used for the assay.
On 14th day of treatment, 3-hour morning urine sample
was collected and evaluated for the presence of calcium
oxalate crystal by chemical and microscopic analysis.
The fresh, concentrated morning urine sample is
recommended for microscopic analysis (Chandhoke et
al., 1999). Chemical test was performed by adding three
drops of concentrated HCl and a pinch amount MnO2 to
1 mL of urine sediments. The presence of oxalate in the
urine reacts with HCl and MnO2, releasing CO2, which
is evident by formation of effervescence (Aligada,
1993).
Kidney calcium content determination
Left Kidney was harvested and dried at 80°C in an oven,
dried kidney was boiled in 10 ml of 1 N HCl for 30
minutes and homogenized. Homogenate was
centrifuged at 2000 rpm for 10 minutes and the
supernatant was separated (Divakar et al., 2010).
Automated calcium determination was performed at
United Diagnostics laboratory, Dapitan St., Sampaloc,
Manila.
Histopathologic evaluation
Right kidney was harvested, cleaned off extraneous
tissue and fixed in 10 percent neutral buffered formalin.
Samples were processed in series of graded alcohol and
xylene, embedded in paraffin wax, sectioned at 5µm
and stained with Haematoxylin and Eosin. Preparation
of kidney tissue samples were done by Hi Precision
diagnostics. An anatomical pathologist graded the
severity of the amount of crystal deposits present in the
tissue according to the parameters in the study of
Chandhoke et al. (1999), where crystal deposits were
graded using the mean value as: “-” no crystal
deposition; “+” less than 5 crystals per high power field
(x40); “++” 6-10 crystals per high power field (x40);
and “+++” more than 10 crystals per high power field
(x40). The pathologist was blinded by the identity of the
samples.
Thiobarbituric Acid Reactive Substances Assay
Extent of lipid peroxidation was estimated by the
amounts of thiobarbituric acid (TBA) reactive product
Malondialdehyde (MDA).Two milliliters of TBA-TCA-
HCl reagent, which was prepared by mixing in a ratio of
1:1:1 0.37% thiobarbituric acid, 0.25M HCl, and 15%
trichloroacetic acid (TCA), was added to 0.1 milliliters
of the left kidney tissue homogenate. Subsequently the
mixture was placed in a water bath for 15 minutes,
cooled to room temperature and was centrifuged at
1000 rpm for ten minutes. Finally the absorbance was
read against a reference blank at 535nm (Niehaus&
Samuelsson, 1968; Kalpana et al., 2007; Sudheer et al.,

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e
0
of ethyl aceta
t
as compared
t
5
percent con
f
fraction and
a
/
mL and 0.15
5
f
ethyl acetate
f
s compared
t
5
percent con
f
fraction and
a
/
mL and 3.35
P
arameter Log
a
venging Activ
a
rameter Log
i
S
cavenging Ac
t
013, 22-30
i
on) of ascorb
i
f
erent concent
r
e
rmined using
m
edian in
h
btained from
p
n
d IC
50
value
s
m
eter logistic
e
1A and 1B).
t
e fraction wa
s
t
o 0.1601 mg/
m
f
idence interv
a
scorbic acid i
5
3 to 0.1651
m
f
raction was f
o
t
o 3.59 mg/
m
f
idence interv
a
scorbic acid i
0 to 3.856
m
istic Regressi
o
ity Determina
t
i
stic Regressi
o
t
ivity Determi
n
i
c acid
r
ations
mean
h
ibitory
p
lotted
s
were
(4PL)
s
found
m
L for
al, the
s from
m
g/mL,
o
und to
m
L for
al, the
s from
m
g/mL,
o
n for
t
ion
o
n for
n
ation

Christine Joy H. Acosta et al,Current Research in Biological and Pharmaceutical Sciences, 2 (2), March-April 2013, 22-30
26
Acute oral Toxicity
Zero mortality was observed for 14 days in all the five
rats up to 2000 mg/kg dose of F. pseudopalma crude
DCM extract. Gross necropsy of organs was regarded as
unremarkable and histopathologic evaluation of liver
and kidneys revealed normal tissues and necrosis was
not observed. Thus, F. pseudopalma crude DCM extract
is non toxic up to 2000 mg/kg.
Antiurolithiatic Study
Serum creatinine
Mean creatinine levels were compared on day 0, 14 and
28. Statistical analysis showed that on day 14 there was
a significant increase in the mean creatinine levels of
the induction control (p=0.016), groups treated with F.
pseudopalma crude DCM extract at 1000 mg/kg
(p<0.001), 500 mg/kg (p=0.001) and lupeol (p<0.001)
in therapeutic regimen. Conversely, creatinine levels in
groups treated with F. pseudopalma crude DCM extract
at 1000 mg/kg (p=0.001) and 500 mg/kg (p=0.035) in
preventive regimen were significantly decreased while
vehicle treated control did not have significant change
in creatinine levels (p=0.146); these data are shown in
Figure 2A.
After 28 days of treatment, groups treated with F.
pseudopalma crude DCM extract at 1000 mg/kg
(p=0.007) and 500 mg/kg (p<0.001) in preventive
regimen together with therapeutic regimen at 1000
mg/kg (p=0.003), 500 mg/kg (p=0.001) as well as
lupeol (p<0.001) all had significant decrease in the
mean creatinine levels, while the induction control
progressively had significant increase (p=0.005) in the
mean creatinine levels, vehicle control did not
significantly differ (p=0.228) in the mean creatinine
levels as shown in Figure 2B. Post hoc analysis showed
that therapeutic regimen at 1000 mg/kg (p=0.998) were
comparable to the effect of lupeol; also preventive dose
at 1000 mg/kg (p=0.072) and 500 mg/kg (p=0.122)
were able to maintain creatinine levels comparable to
the vehicle treated control.
Figure 2A: Mean serum creatinine levels compared on
day 0 and 14. A= Vehicle control; B= Induction control;
C= FP DCM extract 1000 mg/kg (Preventive); D= FP
DCM extract 500 mg/kg (Preventive); E= FP DCM
extract 1000 mg/kg (Therapeutic); F= FP DCM extract
500 mg/kg (Therapeutic); G= Lupeol 10 mg/kg
(Therapeutic).
Figure 2B: Mean serum creatinine levels compared on
day 0 and 14. A= Vehicle control; B= Induction control;
C= FP DCM extract 1000 mg/kg (Preventive); D= FP
DCM extract 500 mg/kg (Preventive); E= FP DCM
extract 1000 mg/kg (Therapeutic); F= FP DCM extract
500 mg/kg (Therapeutic); G= Lupeol 10 mg/kg
(Therapeutic).
Urine analysis
Analysis of urine samples on Day 14 showed that all
experimental groups administered with ethylene glycol
except the vehicle treated group exhibited effervescence
and revealed the presence of envelope-like calcium
oxalate crystals in chemical and microscopic analysis as
shown in Figure 3 and 4, respectively. This indicates
that all the groups treated with ethylene glycol
induction demonstrated calcium oxalate crystal
formation.
Figure 3: Representative images of spot urine chemical
test on day 14. A= Vehicle control; B= Induction control;
0
20
40
60
80
ABCDEFG
MeanserumcreatinineLevels(mg/dL)
Experimentalgroups
SerumCreatinineLevels(Day0&
14)
Day0
Day14
0
10
20
30
40
50
60
70
80
90
ABCDE FG
MeanserumcreatinineLevels(mg/dL)
Experimentalgroups
SerumCreatinineLevels(Day14&
28)
Day14
Day28

C
D
e
5
(
F
o
c
m
(
(
(
B
U
M
a
w
t
h
p
(
l
u
r
c
u
a
t
h
c
c
5
<
p
c
m
w
F
c
c
t
h
u
c
C
= FP DCM
e
D
CM extract
e
xtract 1000
m
5
00 mg/kg
(
Therapeutic).
F
igure 4: Repr
e
o
xalate crystal
c
ontrol; B= In
d
m
g/kg (Preve
n
Preventive);
Therapeutic);
Therapeutic);
B
lack arrows p
U
rine oxalate
c
M
ean urine ox
a
a
nd 28. Statist
i
w
as a significa
n
h
e inductio
n
p
seudopalma
preventive),
1
u
peol (all p
v
r
egimen at 10
0
c
ontrol (p=0.0
9
u
rine oxalate c
o
a
fter 28 days
o
h
at there is a
c
ontent of th
e
c
rude DCM ex
t
5
00 mg/kg (t
h
<
0.001) whil
p
rogressively
c
reatinine lev
e
m
g/kg (p=0.7
4
w
ere able to
m
F
igure 5B. Fu
r
c
o-administrati
c
rude DCM
h
erapeutic (p
=
u
rine oxalate
c
ontrol. While
Christine Joy H.
e
xtract 1000
m
500 mg/kg (
P
m
g/kg (Therap
e
(
Therapeutic);
e
sentative mic
r
under HPO (
X
d
uction control
n
tive); D= FP
E= FP DC
M
F= FP D
C
G= Lupeol
oint to calciu
m
c
ontent determ
a
late content
w
i
cal analysis s
h
n
t increase in
t
n
control,
g
crude DCM
1
000 and 500
v
alues are <0.
0
0
0 mg/kg (p=
0
9
5) did not h
a
o
ntent as sho
w
o
f treatment,
s
significant de
c
e
groups trea
t
t
ract at 500 m
g
h
erapeutic), an
e the indu
c
had significa
n
e
ls. Still, pre
v
8) and vehicl
e
m
aintain urine
r
thermore, po
s
on of 500 mg/
k
extract in
p
=
0.125) regi
m
content com
p
1000 mg/kg
Acosta
et al,Curr
e
m
g/kg (Preven
t
P
reventive);
E
e
utic); F= FP
D
G= Lupeol
r
oscopic imag
e
X
40) on day 1
4
; C= FP DCM
DCM extrac
t
M
extract
1
C
M extract
10 mg/kg (
T
m
oxalate cryst
a
ination
w
as compared
h
owed that on
t
he urine oxal
a
g
roups treate
d
extract at
mg/kg (ther
a
0
01). Howeve
r
0
.065) and v
e
a
ve significan
t
w
n in Figure 5
A
s
tatistical ana
l
c
rease in the
u
t
ed with F.
p
g
/kg (preventi
v
d lupeol (all
p
c
tion contro
l
n
t increase i
n
v
entive regi
m
e
treated cont
r
oxalate conte
n
s
t hoc analysi
k
g dose of F.
p
p
reventive (p
=
m
en was able
p
arable to ve
h
dose of F.
p
e
nt Research in Bio
l
t
ive); D= FP
E
= FP DCM
D
CM extract
10 mg/kg
e
s of calcium
4
. A= Vehicle
extract 1000
t
500 mg/kg
1
000 mg/kg
500 mg/kg
T
herapeutic).
a
ls
on day 0, 14
day 14 there
a
te content of
d
with F.
500 mg/
k
g
a
peutic), and
r
, preventive
e
hicle treated
t
increase in
A
. Moreover,
l
ysis showed
u
rine oxalate
p
seudopalma
v
e), 1000 and
p
values are
l
(p<0.001)
n
the mean
m
en at 1000
r
ol (p=0.073)
n
t, shown in
s found that
p
seudopalma
=
0.079) and
to decrease
h
icle treated
p
seudopalma
l
ogical and Pharm
a
crude
D
creatin
i
treated
(p=0.1
5
decrea
s
Figure
and 1
4
FP DC
extract
1000
m
mg/kg
(Thera
p
Figure
and 2
8
FP DC
extract
1000
m
mg/kg
(Thera
p
0.000
0.500
1.000
1.500
2.000
Urineoxalatecontent(mmol/L)
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
Urineoxalatecontent(mmol/L)
a
ceutical Sciences, 2
D
CM extract
i
ne levels (
p
control. T
h
5
8) is comp
a
s
ing urine oxa
l
5A: Mean o
x
4
. A= Vehicle
M extract 100
0
500 mg/kg
(
m
g/kg (Thera
p
(Therapeut
i
p
eutic).
5B: Mean ox
. A= Vehicle
M extract 100
0
500 mg/kg
(
m
g/
k
g (Thera
p
(Therapeut
i
p
eutic).
AB
Urine
o
AB
Urine
O
(2), March-April 2
(preventive)
w
p
=0.925) co
m
h
erapeutic do
s
a
rable to the
l
ate content.
x
alate content
control; B= I
n
0
mg/kg (Prev
e
(
Preventive);
E
p
eutic); F= F
P
i
c); G= L
u
alate content
c
control; B= I
n
0
mg/kg (Prev
e
(
Preventive);
E
p
eutic); F= F
P
i
c); G= L
u
CD
Experimentalgr
o
o
xalatecontent(
CD
Experimentalgro
u
O
xalateContent(D
a
013, 22-30
w
as able to m
a
m
parable to
v
s
e of 1000
effect of lu
p
compared on
n
duction cont
r
e
ntive); D= F
P
E
= FP DCM
e
P
DCM extra
c
u
peol 10
c
ompared on
d
n
duction cont
r
e
ntive); D= F
P
E
= FP DCM
e
P
DCM extra
c
u
peol 10
EF
o
ups
Day0&14)
EF
u
ps
a
y14&28)
a
intain
v
ehicle
mg/kg

p
eol in
day 0
r
ol; C=
P
DCM
e
xtract
c
t 500
mg/kg
d
ay 14
r
ol; C=
P
DCM
e
xtract
c
t 500
mg/kg
G
Day
0
Day
14
G
Day0
Day14

Christine Joy H. Acosta et al,Current Research in Biological and Pharmaceutical Sciences, 2 (2), March-April 2013, 22-30
28
Kidney calcium content determination
Mean kidney calcium content was compared among the
groups. One way ANOVA established that the kidney
calcium content of ethylene-glycol induction control
was significantly elevated (p<0.001) as compared to the
vehicle control group. On the other hand, the kidney
calcium content of groups treated with F. pseudopalma
crude DCM extract in both preventive and therapeutic
regimen at 500 and 1000 mg/kg dose as well as the
group treated with lupeol were significantly reduced (all
p values are <0.001) than ethylene glycol induction
control. However, Tukey’s HSD post hoc analysis
showed the effect of F. pseudopalma crude DCM
extract in lowering kidney calcium content is not
comparable (all p values are <0.001) to that of lupeol
and negative control. These findings were further
supported by Figure 6, a graph showing the mean
kidney calcium content of the different treatment groups.
Figure 6: Mean kidney calcium content among the
experimental groups. A= Vehicle control; B= Induction
control; C= Preventive 1000 mg/kg (Day 1-28); D=
Preventive 500 mg/kg (Day 1-28); E= Therapeutic 1000
mg/kg (Day 15-28); F= Therapeutic 500 mg/kg (Day
15-28); G= Lupeol (Day 15-28)
Histopathologic evaluation
Histopathological score on the severity of crystal
depositions found in the kidney tissues revealed that
induction control had the most number of crystal
deposits observed and vehicle treated group was shown
to have no crystal deposits while groups treated with F.
pseudopalma crude DCM extract in both preventive and
therapeutic design as well as lupeol were able to
decrease the count of crystal deposits as illustrated in
Figure 7. Statistical analysis confirms that treatment
with F. pseudopalma crude DCM extract and lupeol
were able to significantly reduce the number of crystal
deposits compared to the induction control (p=0.002).
However, the reduction in crystal deposits exhibited by
the groups treated with lupeol (p<0.001) and F.
pseudopalma crude DCM extract (p=0.010) were not
comparable to the negative control. These findings are
supported in Figure 8.
Figure 7: Representative microscopic images of kidney
crystal deposits under HPO (X40). A= Vehicle control;
B= Induction control; C= FP DCM extract 1000 mg/kg
(Preventive); D= FP DCM extract 500 mg/kg
(Preventive); E= FP DCM extract 1000 mg/kg
(Therapeutic); F= FP DCM extract 500 mg/kg
(Therapeutic); G= Lupeol 10 mg/kg (Therapeutic).
Black arrows point to crystal deposits
Figure 8: Comparison of the severity of crystal deposits
among the experimental groups. A= Vehicle control; B=
Induction control; C= Preventive 1000 mg/kg (Day 1-
28); D= Preventive 500 mg/kg (Day 1-28); E=
Therapeutic 1000 mg/kg (Day 15-28); F= Therapeutic
500 mg/kg (Day 15-28); G= Lupeol (Day 15-28)
0
1
2
3
4
5
6
ABCDEF G
Meankidneycalciumcontent(mg/dL)
Experimentalgroups
Kidneycalciumcontent
A
B
C
D
E
F
G

Christine Joy H. Acosta et al,Current Research in Biological and Pharmaceutical Sciences, 2 (2), March-April 2013, 22-30
29
TBARS Assay
Mean malondialdehyde (MDA) levels were compared
among the groups (Appendix X). One way ANOVA
(Appendix XI, Table XI) found that the MDA levels in
ethylene-glycol induction control was significantly
higher (p<0.001) as compared to the vehicle control
group. Conversely, levels of MDA in groups treated
with F. pseudopalma crude DCM extract in both
preventive and therapeutic regimen at 500 and 1000
mg/kg dose as well as the group treated with lupeol
were significantly lower (all p values are <0.001) than
ethylene glycol induction control. However, Tukey’s
HSD post hoc analysis showed that the effect of F.
pseudopalma crude DCM extract in lowering MDA
levels is not comparable (all p values are <0.001) to that
of lupeol and negative control. These findings were
further supported by Figure 9, a graph showing the
mean MDA levels of the different treatment groups.
Figure 9: Mean MDA levels among the experimental
groups. A= Vehicle control; B= Induction control; C=
FP DCM extract 1000 mg/kg (Preventive); D= FP DCM
extract 500 mg/kg (Preventive); E= FP DCM extract
1000 mg/kg (Therapeutic); F= FP DCM extract 500
mg/kg (Therapeutic); G= Lupeol 10 mg/kg
(Therapeutic).
DISCUSSION
This study aimed to assess the antioxidant and
antiurolithiatic properties of Ficus pseudopalma Blanco
since there are no published evidences as yet to prove
these claims. However, the leaves of the plants are
beneficial in the treatment of kidney stones according to
folkloric claims. This is further corroborated by the
identification of lupeol as present in the plant which
possesses antioxidant and antiurolithiatic properties
(Vidya et al., 2002). Phytochemical screening
substantiated the presence of lupeol in the ethyl acetate
fraction from the F. pseudopalma crude DCM extract as
demonstrated in Liebermann Burchard test, TLC and
HPLC.
One of the major factors in the pathogenesis of kidney
stone disease is hyperoxaluria or the excessive amount
of oxalate in the urine, which is known to promote
crystal formation (Pareta et al., 2011). A study by
Selvam in 2002, showed that hydroxyl radical were
generated in excess in oxalate-induced renal cell injury.
Thus, free radical scavenging activity of F.
pseudopalma was determined and found to have
scavenging activity at 0.2586 mg/mL and 5.289 mg/ml
against hydroxyl radical and hydrogen peroxide,
respectively.
The production of ROS develops into oxidative stress
causing degradation of lipids, demonstrated by elevated
TBARS levels (Khan, 2005). A study by Huang et al.
(2002) observed elevated TBARS (MDA levels) in rat
kidneys induced with ethylene glycol. The production
of MDA in tissue samples is indicative of renal cell
injury and inflammation which can be confirmed by
increased creatinine levels. Subsequently, loss of
membrane integrity facilitates crystal formation thus
giving way to crystal deposition. Ethylene glycol, as a
metabolic precursor of oxalate was used to induce the
state of oxidative stress as well as calcium oxalate
crystal formation in the rats.
In the present study, the group treated with ethylene
glycol induction alone had significantly elevated
(p<0.001) TBARS levels. The increased levels of
TBARS suggest that large amount of MDA had been
already produced from the process of free radical
induced lipid peroxidation by ethylene glycol induction.
In this context, ethylene glycol induction control rats
were found to have marked renal damage, consistent
with the elevated serum level of creatinine observed
after day 14 (p=0.016) and 28 (p=0.005).
Oxalate has been reported to cause renal tissue damage
by reacting with polyunsaturated fatty acids in cell
membranes (Divakar et al., 2010). Since it is stated that
hyperoxaluria is a promoter of crystal formation, the
changes in urinary oxalate levels are important
measures of the disease. Ethylene glycol induction
control showed significant increase (p<0.001) in urine
oxalate content observed after 14 and 28 days. The
formation of calcium oxalate crystals were evident after
14th day of ethylene glycol induction supported by urine
chemical and microscopic analysis findings.
Calcium oxalate crystals and high oxalate levels in renal
tissues can produce damages in the epithelial cells, and
consequently, the cells may generate some products, as
well as free radicals, inducing heterogeneous crystal
nucleation and causing aggregation of crystals (Prasad
et al., 2011). In this study, kidney calcium content was
measured and histopathologic examination was done to
evaluate crystal deposition. Results showed that
induction control had increased kidney calcium content
(p<0.001). In the same way, histopathologic evaluation
revealed severe crystal deposits (p=0.002).
Treatment with F. pseudopalma crude DCM extract in
both preventive and therapeutic regimen at 500 and
1000 mg/kg was able to significantly inhibit (all p
values are <0.001) lipid peroxidation as shown by
marked reduction in MDA levels. In this light,
‐1.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
ABCDE FG
meannmolMDA/gSample
Experimentalgroups
Malondialdehyde(MDA)levels
A
B
C
D
E
F
G

Christine Joy H. Acosta et al,Current Research in Biological and Pharmaceutical Sciences, 2 (2), March-April 2013, 22-30
30
impairment of renal functions of the ethylene glycol
induction control rats evidenced from the increased
serum creatinine levels was alleviated in the rats
administered with F. pseudopalma crude DCM extract,
which demonstrated significant decrease (all p values
are <0.05) in creatinine levels. Likewise, significant
decrease (all p values are <0.05) in urine oxalate levels
and kidney calcium content were exhibited by all the
groups treated with F. pseudopalma crude DCM extract.
Lastly, histopathologic evaluation showed reduction in
the amount of crystal deposits in F. pseudopalma
treated rats. Therapeutic dose of 1000 mg/kg F.
pseudopalma crude DCM extract was comparable to the
effect of lupeol at 10 mg/kg in significantly decreasing
serum creatinine (p=0.998) and urine oxalate levels
(p=0.158).
CONCLUSION
The results presented clearly establish the antioxidant
and antiurolithiatic potential of F. pseudopalma. The
ability of the plant to prevent crystal formation can be
attributed to lipid peroxidation inhibition as also shown
in studies of Khan et al. (2005) and Prasad et al. (2011)
ACKNOWLEDGEMENT
Acknowledgement is given to Department of Science
and Technology – Science and Education Institute,
University of Santo Tomas – Faculty of Pharmacy and
Research Center for Natural and Applied Sciences. The
researcher would like to thank Dr. Rowen C. Yolo, Dr.
Jovencio G. Apostol, Mr. Xandro Alexi Nieto and Dr.
Teresita Coloma for their great contribution in this
study.
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