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Carbon tetrachloride induced nephrotoxicity in

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 Journal of Ethnopharmacology 122 (2009) 91–99

Contents lists available at ScienceDirect

Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Carbon tetrachloride-induced nephrotoxicity in rats: Protective role of

Digera muricata
Muhammad R. Khan ∗ , Wajiha Rizvi, Gul N. Khan, Rahmat A. Khan, Saima Shaheen
Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 4400, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Digera muricata is used in renal disorders in folk medicine. Generation of reactive radicals has been
Received 8 April 2008 implicated in carbon tetrachloride-induced nephrotoxicity, which are involved in lipid peroxidation,
Received in revised form 5 November 2008 accumulation of dysfunctional proteins, leading to injuries in kidneys. The present study was aimed to
Accepted 4 December 2008
evaluate the efficacy of Digera muricata on the kidney function in CCl4 -induced injuries. CCl4 treatment
Available online 11 December 2008
(5 ml/kg body wt., i.p. CCl4 :olive oil; 1:9) significantly increased the level of urine creatinine, protein,
nitrite, urobilinogen, red blood cells (RBCs), leucocytes count, and levels of blood urea nitrogen (BUN).
Keywords:
Level of proteins and DNA fragmentation %, argyrophilic nucleolar organizer regions (AgNORs) count
Carbon tetrachloride
Digera muricata
in renal tissues was also significantly increased. Activity of antioxidant enzymes; catalase, peroxidase,
Histopathology superoxide dismutase and reduced glutathione (GSH) were decreased while thiobarbituric acid reac-
Kidney function tive substances (TBARSs) were increased with CCl4 treatment. DNA ladder assay was intimately related
Antioxidant with the DNA fragmentation assay. Telomerase activity was determined in the CCl4 -treated renal tis-
Anticancer sue homogenate. Treatment with n-hexane (HDMP) and methanolic (MDMP) extracts of Digera muricata
AgNORs (200 and 250 mg/kg body wt., oral, respectively) effectively attenuated the alterations in the biochemical
Telomerase markers, telomerase activity was inhibited and confirms the restoration of normalcy and accredits the
protective role of Digera muricata against CCl4 -induced nephrotoxicity.
© 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction injuries (Satyanarayana et al., 2001; Ogeturk et al., 2005; Manna et

Carbon tetrachloride (CCl4 ) is a potent environmental hep-
atotoxin (Abraham et al., 1999). It has been established that
trichloromethyl (CCl3 ) radical and Cl are formed as a result of the
metabolic conversion of CCl4 by cytochrome P-450 (Adewole et al.,
2007). Increased oxidative stress generally describes a condition in
which cellular antioxidant defenses are inadequate to completely
inactivate the reactive oxygen species (ROS) and reactive nitro-
gen species (RNS) generated because of excessive production of
ROS/RNS. A major consequence of oxidative stress is damage to
nucleic acid bases, lipids and proteins, which can severely com-
promise cell health and viability or induce a variety of cellular
responses through generation of secondary reactive species, ulti-
mately leading to cell death by necrosis or apoptosis. Oxidative
stress also contributes to cancer risk by numerous mechanisms that
are independent of genotoxicity (Dalle-Donne et al., 2006).
Free radicals induce lipid peroxidation and is believed to be one
of the major causes of cell membrane damage leading to a num-
ber of pathological situations by causing acute and chronic renal
∗ Corresponding author. Tel.: +92 51 9064 3086; fax: +92 51 920 5753.
E-mail address: mrkhanqau@yahoo.com (M.R. Khan).
al., 2006; Adewole et al., 2007). In addition, reports on various doc-
umented case studies established that CCl4 produces renal diseases
in humans (Perez et al., 1987; Manna et al., 2006).
CCl4 cause extensive DNA strand breaks and without prompt
repair may cause cell death and compensatory cell regeneration
(Moustacchi, 2000; Jia et al., 2002). Recent studies suggested that
CCl4 causes toxic effects to argyrophilic nucleolar organizer regions
(AgNORs) that would lead to increase in AgNORs count, size, mor-
phology or spreading in the nucleus (Bocking et al., 2001; Khanna
et al., 2001). Nucleoar organizer regions (NORs) are composed of
chromosomal sites endowed with ribosomal DNA (rDNA) and com-
plexes with a set of non-histone proteins characterized by a high
affinity for silver (Trere et al., 1996). Increased number of nucleoli
(NORs) with abnormal shapes and sizes, including small dots might
have been utilized as an indicator of genotoxicity to complement
other histological procedures. AgNORs quantification has been used
to characterize neoplasias and hyperplasias, since these are markers
of ribosomal gene activity and hence, markers of nucleolar activity
(Kanematsu et al., 1997).
Telomeres are repetitive (TTAGGG) non-coding DNA regions
at the ends of chromosomes, which are progressively shortened
(Grace et al., 2000) because of incomplete replication of the 3 -
ends (Segawa et al., 2003). In vertebrates, telomerase is the main

0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2008.12.006
92 M.R. Khan et al. / Journal of Ethnopharmacology 122 (2009) 91–99
mechanism presently known that can stabilize the loss of telom-
eres. High telomerase activity is observed in malignant tumors,
neoplasias and hyperplasias, whereas low or no telomerase activ-
ity is observed in normal somatic cells. These findings suggest that
telomerase activation is a crucial step in cell immortalization and
oncogenesis (Segawa et al., 2003). Telomerase seems to be a mean
whereby cancer cells bypass normal cellular senescence therefore,
plays a critical role in carcinogenesis. Approximately 60% of renal
cell carcinomas have revealed telomerase activity that is not related
to stage or grade (Grace et al., 2000).
A number of studies also showed that various herbal extracts
and plant derived pure chemicals could protect organs against
CCl4 -induced oxidative stress by altering the level of antioxidant
enzymes (Ko et al., 1995; Rajesh and Latha, 2004). Endogenous
antioxidants in medicinal herbs may play an important role in
antioxidative defense against oxidative damage, possibly protect-
ing the biological functions of cells (Tirkey et al., 2005; Adewole et
al., 2007). Knowing the extent of nephrotoxicity and the grade are
helpful to assess the curing potential of a particular plant extract.
Digera muricata is used ethnopharmacologically in renal disorders
(Anjaria et al., 2002) aperient, refrigerant (Hocking, 1962) however,
its medicinal values have not been evaluated so far. Renal functions
may be evaluated at various levels ranging from the excretion of
creatinine, blood urea nitrogen (BUN) to the physical examination
of urine including the presence of red blood cells, leucocytes, pH
and specific gravity. Histopathology, AgNORs count, DNA damages
in the kidney tissue may allow for more accurate assessment of
kidney injuries.
In this study aside from the determination of renal function bio-
chemical assays of the urine and its physical properties, serum eval-
uation, and tissues damage, i.e., protein contents, DNA fragmenta-
tion and ladder assay, antioxidant enzymes level and AgNORs count
were conducted to assess the renal toxicity induced with chronic
dose of CCl4 and its protection with plant extract of Digera muricata.
2. Material and methods
2.1. Plant collection
Digera muricata (L.) Mart. locally also named as “Tandla” or
“Lulur” at maturity were collected, shade dried and used as plant
material. Plants were identified and submitted vide accession #
125127 in the Herbarium of Pakistan at Quaid-i-Azam University
Islamabad, Pakistan.
2.2. Preparation of plant extract
Air dried aerial parts of Digera muricata (1.50 kg) were ground
and extracted with methanol and n-hexane of 5.0 l successively,
with occasional shaking and filtered. The residue was re-extracted
twice with the respective solvent and dried under vacuum.
Methanolic extract of Digera muricata plant (MDMP) yielded 19 g
green viscous material, while 7 g yellowish green extract was
obtained with n-hexane solvent of Digera muricata plant (HDMP).
2.3. Animals and treatment
Forty healthy male albino rats, weighing 250–260, were pro-
vided by the Animal House of National Institute of Health (NIH)
Islamabad and were maintained at the Primate Facility maintained
at Quaid-i-Azam University, Islamabad. Food and fresh water was
available to the rats. Among them 10 were chosen randomly as con-
trol, and were administered with DMSO 5.0 ml (oral) after 72 h by
olive oil 5.0 ml (i.p.) once a week for 4 months. Rest of the rats were
divided into three groups; CCl4 group received only CCl4 (5 ml/kg of
rat body weight) mixed with olive oil in the ratio of 1:9, respectively
once a week for 4 months. However, the other two groups; HDMP
group and MDMP group were treated with the respective extract
(HDMP, 200 mg and MDMP, 250 mg/kg body weight of rats) after
72 h of CCl4 treatment for 4 months.
2.4. Assessment of renal functions
Before sacrifice rats were kept individually in metabolic cages
for 24 h to collect urine for estimation of renal function. Urine and
serum samples were assayed for BUN and creatinine by using stan-
dard diagnostic kits (Pioneer Diagnostic kit, New York, U.S.A for BUN
and Spinreact Diagnostic kit, Santa Coloma, Spain for creatinine).
2.5. Assessment of urobilinogen and protein contents in urine
Urine samples were assayed with the standard diagnostic kits;
urobilinogen (MediScreen Urine Strips, Orgenics, France) and urine
protein concentration (Rat Urinary Protein Assay Kit, Chondrex Inc.,
USA).
2.6. Nitrite assay
Nitrite assay was conducted by using Griess reagent. Urine sam-
ples were deproteinized by equal volumes of 0.3 M NaOH and 5%
ZnSO4 and centrifuged at 6400 × g for 20 min and supernatant was
collected. 1.0 ml of Griess reagent was added into the cuvette and
blank the spectrophotometer at 540 nm. Then 20 ␮l urine super-
natant was added in cuvette containing Griess reagent. Nitrite con-
centration was calculated using a standard curve for sodium nitrite.
2.7. Physical analysis of urine
Urine samples were assayed for red blood cells (RBCs) count,
white blood cells (WBCs) count and specific gravity by using stan-
dard diagnostic kits (MediScreen Urine Strips, Orgenics, France).
However, pH was determined by using pH meter.
2.8. Preparation of kidney homogenate
A midline abdominal incision was performed and both the kid-
neys were isolated, the left kidney was dried in liquid nitrogen and
stored at −70 ◦ C till further analysis, whereas, the right kidney was
stored in 10% formalin for the histological studies.
2.9. Estimation of protein and assay of antioxidant enzymes in
kidney homogenates
About 100 mg of kidney tissue was homogenized in 10 vol-
ume of 100 mM KH2 PO4 buffer containing 1 mM EDTA, pH 7.4
and centrifuged at 12,000 × g for 30 min at 4 ◦ C. The supernatant
was collected and used for the following experiments as described
below. Protein concentration of the supernatant of kidney tissue
was determined by the method of Lowry et al. (1951) using crys-
talline BSA as standard.
2.10. SOD assay
The activity of superoxide dismutase (SOD) was assayed by
using the protocol of Stewart and Bewely (1980) to measure its
ability to inhibit the photochemical reduction of nitroblue tetra-
zolium (NBT). The reaction solution (3 ml) contained; 50 ␮M NBT,
1.3 ␮M riboflavin, 13 mM methionine, 75 nM EDTA, 50 mM phos-
phate buffer (pH 7.8), 20–50 ␮l of enzyme extract. The test tubes
containing the reaction solution were irradiated under a light (15 W
fluorescent lamps) at 78 ␮mol m−2 s−1 for 15 min. The absorbance
of solution was noted at 650 nm. One unit of SOD activity was
 M.R. Khan et al. / Journal of Ethnopharmacology 122 (2009) 91–99 93

Table 1 (TBARSs) formation by following the protocol of Esterbauer and

Reaction solutions for TRAP assay.
Cheesman (1990).
Lysis buffer TRAP reaction solution

10 mM Tris–HCl (pH 7.5) 20 mM Tris–HCl (pH 8.3)

2.15. DNA fragmentation % assay
1 mM MgCl2 1.5 mM MgCl2
1 mM EGTA 63 mM KCl DNA fragmentation % assay was conducted using the proce-
0.1 mM AEBSF [4-(2-Aminoethyl)- 0.005% Tween-20 dure of Wu et al. (2005) with some modifications. The kidney
benzenesulphonyl-fluoride-hydrochlorine] tissue (50 mg) was homogenized in 10 volumes of a TE solu-
5 mM mercaptoethanol 1 mM EGTA
tion pH 8.0 (5 mM Tris–Hcl, 20 mM EDTA) and 0.2% triton X-100.
0.5% CHAPS (3-Cholamidopropyl- 0.1 mg/ml bovine serum albumin
dimethylammonio-1-propane-sulphonate) 1.0-ml aliquot of each sample was centrifuged at 27,000 × g for
10% glycerol RNAs inhibitors (20 ␮l) 20 min to separate the intact chromatin (pellet, B) from the frag-
RNAs inhibitors (20 ␮l) mented DNA (supernatant, T). The pellet and supernatant fractions
were assayed for DNA content using a freshly prepared dipheny-
defined as the amount of enzyme, which caused 50% inhibition of lamine (DPA) solution for reaction. Optical density was read at
photochemical reduction of NBT. 620 nm at (SmartSpecTM Plus Spectrophotometer catalog # 170-
2525) spectrophotometer. The results were expressed as amount
of % fragmented DNA by the following formula:
2.11. CAT assay
T × 100
% fragmented DNA =
The enzyme catalase (CAT) catalyzes the conversion of H2 O2 T +B
into water. The CAT activity was measured using the method of
Chance and Maehly (1955) with some modification. The CAT reac- 2.16. DNA ladder assay
tion solution 3 ml contained; 50 mM phosphate buffer (pH 7.0),
5.9 mM H2 O2 , and 0.1 ml of enzyme extract. The reaction was ini- DNA ladder was carried out according to the procedure of Wu et
tiated by adding the enzyme extract. Changes in absorbance of the al. (2005). 5 ␮g of DNA of rats separately was loaded in 1.5% agarose
reaction solution at 240 nm were noted after every 30 s. One unit gel containing 1.0 ␮g/ml ethidium bromide including DNA stan-
CAT activity was defined as an absorbance change of 0.01 unit/min. dards (0.5 ␮g per well). Electrophoresis was performed for 45 min
at 100 V. After electrophoresis gel was studied under gel doc system
2.12. POD assay and was photographed.

The POD activity was assayed by using the protocol of Chance
and Maehly (1955) using the guaiacol oxidation method. The POD
reaction solution 3 ml contained; 50 mM phosphate buffer (pH
5.0), 20 mM guaiacol, 40 mM H2 O2 , and 0.1 ml of enzyme extract.
Changes in absorbance of the reaction solution at 470 nm were
noted after every 20 s. One unit POD activity was defined as an
absorbance change of 0.01 unit/min.
2.13. GSH assay
Reduced glutathione (GSH) level was measured by the method
of Ellman (1959). The homogenate (720 ␮l) was double diluted and
5% TCA was added to it to precipitate the protein content of the
homogenate. After centrifugation (10,000 × g for 5 min) the super-
natant was taken. DTNB (Ellman’s reagent) was added to it and the
absorbance was measured at 412 nm. A standard graph was drawn
using different concentrations of a standard GSH solution and GSH
contents were calculated.
2.14. Estimation of lipid peroxidation products
Degree of lipid peroxidation in kidney tissue homogenates was
determined in terms of thiobarbituric acid reactive substances
2.17. TRAP assay
For the quantitative assessment of telomerase activity ‘Telom-
eric Repeat Amplification Protocol’ was used according to Wen et al.
(1998) with some modifications. Liver tissue (100 mg) were washed
once in ice-cold wash buffer (10 mM Hepes–KOH pH 7.5, 1.5 mM
MgCl2 , 10 mM KCl, 1 mM dithiothreitol, 20 ␮l RNAs inhibitors),
and homogenized in 200 ␮l ice-cold lysis buffer (Table 1). The
homogenate was incubated on ice for 30 min and then centrifuged
at 10,000 × g for 30 min at 4 ◦ C. PCR reaction mixture (total 48 ␮l)
consisted of 36.6 ␮l DEPC-treated water, 2 ␮l (6 ␮g protein) extract,
5 ␮l 10× TRAP reaction solution (Table 1), 2 ␮l (50 ␮M) each dNTP,
0.4 ␮l (2 U) Taq DNA polymerase, and 2 ␮l (0.1 ␮g) of TS primer
sequence (5 -AATCCGTCGAGCAGAGTT-3 ). The PCR reaction mix-
ture was incubated at 25 ◦ C in water bath for 30 min for extension of
TS primer. CX primer sequence (5 -CCCTTACCCTTACCCTTACCCTAA-
3 ) 2 ␮l (0.1 ␮g) was added. The reaction mixture (total 50 ␮l) was
subjected to PCR cycles (25) at 94 ◦ C for 30 s, 55 ◦ C for 30 s, and
72 ◦ C for 90 s (then 10 min for the final step). 5 ␮l of a buffer
containing 0.25% bromophenol blue, 0.25% xylenocyanol and 50%
glycerol was added to each PCR product. 25 ␮l of each sample were
loaded onto a 12.5% non-denaturing polyacrylamide gel in 0.5× TBE
buffer. Electrophoresis was carried out at 140 V in 0.5× TBE buffer

Table 2
Effect of MDMP and HDMP on creatinine, BUN and urobilinogen in urine and serum.

Variables Control CCl4 CCl4 + MDMP CCl4 + HDMP

Urine
Creatinine (␮mol/l) 251.4 ± 6.3 1465.7 ± 52.2** 432.8 ± 15.1** , ‡ 395.9 ± 25.3**,‡
Urobilinogen (mg/dl) 1.2 ± 0.5 11.6 ± 0.7** 1.8 ± 0.5* , ‡ 4.0 ± 0.7** , ‡

Serum
Creatinine (␮mol/l) 99.9 ± 6.3 171.9 ± 9.9** 119.7 ± 8.6‡ 116.8 ± 5.8‡
BUN (mg/dl) 13.9 ± 0.8 19.4 ± 0.8** 15.2 ± 0.8‡ 14.4 ± 0.7‡

Results are expressed as mean ± S.E. (n = 10).

*
Indicate significance at P < 0.05 probability from control group.
**
Indicate significance at P < 0.01 probability from control group.
‡
Indicate significance at P < 0.01 probability from CCl4 group.
94 M.R. Khan et al. / Journal of Ethnopharmacology 122 (2009) 91–99

Table 3
Effect of MDMP and HDMP on protein and nitrite concentration in urine.

Variables Control CCl4 CCl4 + MDMP CCl4 + HDMP

Protein (mg/dl) 25.0 ± 3.1 267.1 ± 9.5 **

118.1 ± 13.1 **,‡
61.3 ± 9.0* , ‡
Nitrite (␮mol/ml) 49.5 ± 2.2 70.7 ± 1.6** 56.3 ± 1.2** , ‡ 58.9 ± 0.4** , ‡

Results are expressed as mean ± S.E. (n = 10).

*
Indicate significance at P < 0.05 probability from control group.
**
Indicate significance at P < 0.01 probability from control group.
‡
Indicate significance at P < 0.01 probability from CCl4 group.

at room temperature until the bromophenol blue just ran off the
gel.
The gel was transferred to a fixing solution containing 0.5% acetic
acid and 10% ethanol with gentle shaking for 15 min. After addition
of 0.2% AgNO3 , the gel was stained for 10 min followed by washing
twice in distilled water and then incubated in developing solu-
tion (0.1% formaldehyde and 3% NaOH) for about 10 min. The gel
was handled after staining, maintained wet and photographed with
Digital Camera.
2.18. Histopathological examination
3. Results
Urinalysis for certain physical properties, solutes, cells, casts,
crystals, or particulate matter were analyzed after CCl4 -induced
toxicity and the repairing potential of Digera muricata extract in rat.
3.1. Assessment of renal function
MDMP and HDMP treatment significantly (Table 2) ameliorated
the effects of CCl4 with the decrease in level of urine and serum
creatinine, urine level of urobilinogen and serum level of BUN.

3.2. Effect of MDMP and HDMP protein and nitrite concentration

For microscopic evaluation kidneys were fixed in a fixative
in urine
(absolute alcohol 60%, formaldehyde 30%, glacial acetic acid 10%)
and embedded in paraffin, sectioned at 4 ␮m and subsequently
CCl4 treatment for 16 weeks significantly increased the protein
stained with hematoxylin/eosin. Sections were studied under light
and nitrite concentration in urine as compared with the control
microscope (DIALUX 20 EB) at 40 and 100 magnifications. Slides of
group. Administration of MDMP and HDMP significantly decreased
all the treated groups were studied and photographed.
the rise in protein and nitrite contents (Table 3).

2.19. AgNORs count

3.3. Effect of MDMP and HDMP physical properties of urine

AgNORs count was conducted according to the method of
Rosana et al. (2005). After the fixed slides dewaxed with xylene
for three 3 min were rehydrated by grading of alcohol (90–50%)
for 3 min followed by hydration in distilled water for 10 min the
slides were dried in oven. To visualize the AgNORs one drop of col-
loidal solution (2% gelatin and 1% formic acid) was added along
with two drops of 50% AgNO3 solution onto the hydrated slides and
incubated at 37 ◦ C for 10 min in the dark. The progressive staining
was followed under microscope to get golden colored nuclei and
brown/black NORs. Then, the slides were washed in distilled water
followed with 1% sodium thiosulfate treatment at room tempera-
ture to stop the reaction, and washed with distilled water. The cells
were examined under light microscope (DIALUX 20 EB) to note the
number of the nucleoli.
2.20. Statistical analysis
Parametric data, expressed as mean and standard error of
the mean (S.E.M.), were analyzed through repeated measure and
two-way ANOVA, followed by the post hoc Fisher least signifi-
cant difference (LSD) for comparison of various treatments using
the SPSS 13.0. Differences were considered statistically significant
when P < 0.05.
In urine RBCs count, WBCs count were significantly increased by
CCl4 administration as compared to control group (Table 4), how-
ever, it did not significantly affect the pH and specific gravity of
urine. Treatment with MDMP and HDMP produced a significant
decrease in RBCs and WBCs count in CCl4 -treated rats, while pH
was increased with the extract administration.
3.4. Effect of MDMP and HDMP on CCl4 -induced changes in the
antioxidant profile
Effects of the MDMP and HDMP on CAT, POD, and SOD activity
for all experimental groups are given in Table 5. Treatment of rats
with CCl4 significantly decreased the activities of CAT, POD and SOD
enzymes in kidney tissue homogenates compared to the control
group. This reduction was improved significantly by the treatment
with MDMP and HDMP and the enzyme activities were increased
as compared to the CCl4 -treated group.
3.5. Effect of MDMP and HDMP on lipid peroxidation
The results are shown in Table 6. TBARS contents were increased
while GSH level decreased significantly in CCl4 -induced toxicity
group against the control group. Treatment with MDMP and HDMP

Table 4
Effect of MDMP and HDMP physical properties of urine.

Variables Control CCl4 CCl4 + MDMP CCl4 + HDMP

RBC/␮l 0.0 ± 0.0 17.1 ± 5.9** 3.3 ± 1.5‡ 3.0 ± 1.2‡

WBC/␮l 8.7 ± 2.8 369.7 ± 13.5** 39.1 ± 6.3* , ‡ 45.0 ± 5.9** , ‡
pH 7.3 ± 0.2 7.6 ± 0.2 7.7 ± 0.2 7.9 ± 0.2*
Specific gravity 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0

Results are expressed as mean ± S.E. (n = 10).

*
Indicate significance at P < 0.05 probability from control group.
**
Indicate significance at P < 0.01 probability from control group.
‡
Indicate significance at P < 0.01 probability from CCl4 group.
 M.R. Khan et al. / Journal of Ethnopharmacology 122 (2009) 91–99 95

Table 5
Effect of MDMP and HDMP on CCl4 -induced changes in the antioxidant profile.

Variables Control CCl4 CCl4 + MDMP CCl4 + HDMP

** , ‡
CAT, units/min 3.6 ± 0.13 1.1 ± 0.09 **
4.5 ± 0.17 1.9 ± 0.22** , ‡
POD, units/min 2.4 ± 0.06 1.4 ± 0.05** 2.3 ± 0.11‡ 1.8 ± 0.42* , ‡
SOD, units/mg protein 10.2 ± 1.22 0.4 ± 0.50** 3.6 ± 0.30** , ‡ 6.7 ± 0.36** , ‡

Results are expressed as mean ± S.E. (n = 10).

*
Indicate significance at P < 0.05 probability from control group.
**
Indicate significance at P < 0.01 probability from control group.
‡
Indicate significance at P < 0.01 probability from CCl4 group.

Table 6
Effect of MDMP and HDMP on lipid peroxidation.

Variables Control CCl4 CCl4 + MDMP CCl4 + HDMP

TBARS, ␮g/mg protein 45.1 ± 1.4 102.9 ± 2.3** 70.6 ± 2.6** , ‡ 85.7 ± 2.0** , ‡
GSH, nmol/mg protein 35.7 ± 1.4 10.2 ± 0.6** 21.8 ± 1.0** , ‡ 24.5 ± 0.9** , ‡

Results are expressed as mean ± S.E. (n = 10).

**
Indicate significance at P < 0.01 probability from control group.
‡
Indicate significance at P < 0.01 probability from CCl4 group.

erased the effects of CCl4 intoxication and MDA contents decreased
whereas GSH level was significantly increased in the kidney tissue
homogenate.
3.6. Effect of MDMP and HDMP on CCl4 -induced changes on renal
morphology
The histomorphological changes were graded and summa-
rized in Table 7. The sections of control group showed normal
glomeruli, afferent arterioles, and tubular cells. Marked histo-
logical changes were observed both in cortex and medulla in
kidneys of CCl4 -treated rats. Cortex was more severely affected
due to the CCl4 toxicity as compared to medulla. The renal sec-
tions showed marked tubular degeneration, tubular dilatation,
interstitial fibrosis, glomerular atrophy, glomerular hypertrophy,
glomerular degeneration and congestion in capillaries in CCl4 -
treated rats. Treatment with MDMP and HDMP markedly preserved
the normal morphology of the kidney and showed normal
glomeruli, tubular dilatation and prevented interstitial edema and
capillary congestion. However, histology of MDMP-treated group
was more closely related with the normal group (Fig. 1).
AgNORs staining sections and DNA ladder assay are presented in
Figs. 2 and 3, respectively. Treatment of rats with CCl4 increased
the percentage of DNA fragmentation and AgNORs count signifi-
cantly as compared to control group. By contrast, the kidneys of
rats showed significant lower percentage of DNA fragmentation and
AgNORs count in the MDMP- and HDMP-treated groups. A pecu-
liar type of continuous DNA fragmentation pattern was observed
in electrophoresis. DNA fragmentation percentage and DNA lad-
der assay were intimately correlated with each other. Treatment
of MDMP showed marked repairing potential against the HDMP
treatment.
3.8. Effect of MDMP and HDMP on telomerase activity
Telomerase activity was not detected in renal tissues of control
group whereas varying levels of telomerase activity was detected
in the form of bands that were multiple of 6 bp of the primer band.
Telomerase activity was inhibited with both MDMP and HDMP
treatment (Fig. 4).
4. Discussion

3.7. Effect of MDMP and HDMP on DNA fragmentation
percentage and AgNORs count
Percentage of DNA fragmentation and AgNORs count in kidney
tissues of various treatments are presented in Table 8 while the
Present study was conducted to evaluate the protective effect
of the methanol and n-hexane extract of Digera muricata against
CCl4 -induced renal disorders in rat. Results suggested that the both
extracts possess protective action against the renal dysfunction
induced by the potent toxin, CCl4 . Urinalysis may provide infor-

Table 7
Effect of MDMP and HDMP on CCl4 -induced changes on renal morphology.

Group Glomerular Glomerular Tubular Tubular Necrosis of Congestion in Interstitial edema

atrophy hypertrophy dilatation necrosis epithelium capillary loops

Control − − − − − − −
CCl4 +++ +++ +++ +++ +++ +++ +++
CCl4 + MDMP − − ± − − ± −
CCl4 + HDMP − ± ± ± − ± ±

−, normal; ±, mild; +++, severe disruption.

Table 8
Effect of MDMP and HDMP on DNA fragmentation percentage and AgNORs count.

Variables Control CCl4 CCl4 + MDMP CCl4 + HDMP

DNA fragmentation % 38.6 ± 3.0 61.8 ± 2.0** 54.6 ± 2.4** , ‡ 47.2 ± 2.3**,‡
AgNORs, count/cell 1.5 ± 0.2 9.1 ± 0.5** 4.0 ± 0.2**,‡ 5.0 ± 0.3** , ‡

Results are expressed as mean ± S.E. (n = 10).

**
Indicate significance at P < 0.01 probability from control group.
‡
Indicate significance at P < 0.01 probability from CCl4 group.
96 M.R. Khan et al. / Journal of Ethnopharmacology 122 (2009) 91–99

Fig. 1. (a) Hemotoxylin and eosin-stained sections of normal rat kidney. (b) Kidney section of CCl4 -treated rats showing glomerular degeneration, tubular brush border loss,
tubular dilatation, necrosis of epithelium and interstitial oedema. (c) Kidney section of CCl4 + methanol extract of Digera muricata (250 mg/kg)-treated rats showing almost
normal morphology. (d) Kidney section of CCl4 + n-hexane extract of Digera muricata (200 mg/kg)-treated rats showing almost normal morphology.

Fig. 2. (a) Silver-stained sections of normal rat kidney. (b) Kidney section of CCl4 -treated rats showing increased AgNORs count and nuclear membrane degeneration. (c) Kidney
section of CCl4 + methanol extract of Digera muricata (250 mg/kg)-treated rats showing lower number of AgNORs as compared to CCl4 with normal nuclear membrane. (d)
Kidney section of CCl4 + n-hexane extract of Digera muricata (200 mg/kg)-treated rats showing lower number of AgNORs as compared to CCl4 with normal nuclear membrane.
 M.R. Khan et al. / Journal of Ethnopharmacology 122 (2009) 91–99
Fig. 3. Agarose gel showing DNA damage by CCl4 and preventive effect of Dig-
era muricata extracts in different groups. Lanes (from left) high-molecular weight
marker (1), control (2 and 3), CCl4 (4 and 5), methanol (6 and 7) and n-hexane (8
and 9).
mation regarding the status of kidney and liver function and acid
base balance (Free and Free, 1972). Normally, there is little or no
urobilinogen excreted into the urine unless a pathological condi-
tion exists. Urobilinogen is the end product of conjugated bilirubin
after it has passed through the bile ducts and been metabolized
in the intestine. The presence of abnormally high levels of BUN
in serum, urobilinogen in urine and creatinine both in urine and
serum are possible indicators of hepatic and/or kidney injuries
induced through CCl4 treatment (Pels et al., 1989; Ozturk et al.,
2003; Ogeturk et al., 2005; Simerville et al., 2005). The serum cre-
atinine level does not rise until at least half of the kidney nephrons
are damaged or destroyed (Bhattacharya et al., 2005). It was also
reported that chronic renal injuries and BUN elevations developed
in Balb/c mice only after 12 weeks of CCl4 intoxication (Ogawa et
al., 1992).
Nitrites normally are not found in urine however, higher values
in urine were found with CCl4 intoxication in this study. Several
studies have reported that nitrite is produced in the liver of treated
rats with CCl4 (Muriel, 1998). Nitrites can turn into nitric oxide
(NO) in acidic pH. Peroxynitrite anions have been generated by the
reaction of nitric oxide and superoxide anion. These peroxynitrite
anions oxidize biomolecules, which finally leads to lipid peroxi-
dation and tubular damage (Muriel, 1998). Adewole et al. (2007)
found that nitrite excretion in urine was increased in CCl4 -treated
rats. Srinivasan et al. (2005) observed increased level of nitrite in
liver and kidneys during chronic administration of CCl4 .
Urinary specific gravity (USG) correlates with urine osmolality
and gives important insight into the hydration status. USG and urine
Fig. 4. Polyacrylamide gel showing telomerase activity in various groups. Lanes
(from left) high-molecular weight marker (1), control (2 and 3), CCl4 (4–8), methanol
(9 and 10) and n-hexane (11 and 12).
97
pH was not affected with CCl4 administration. Glomerular haema-
turia is typically associated with erythrocyte cases, dysmorphic red
blood cells and significant proteinuria (Simerville et al., 2005). The
glomerular capillary wall is permeable only to substances with a
low-molecular weight. Once filtered, low-molecular weight pro-
teins are reabsorbed and metabolized by the proximal tubule cells.
High level of proteinurea and haematuria in urine of this study
reflected the nephrotoxicity induced with CCl4 . Ogawa et al. (1992)
also reported chronic renal injuries after long administration with
CCl4 .
The present study revealed that oral administration of methanol
(MDMP) and n-hexane (HDMP) extracts significantly improved
creatinine and urobilinogen, and decreased the elevated levels of
proteinuria and haematuria, serum creatinine and BUN, however,
n-hexane extract did not change the BUN level. Earlier studies have
also shown that different plant extracts comprehensively amelio-
rated the renal injuries induced through CCl4 intoxication (Ogeturk
et al., 2005; Adewole et al., 2007).
Our study revealed that CCl4 treatment caused a significant
increase in protein content of kidney tissues. CCl4 administration
resulted in oxidative damage of proteins (Abraham et al., 1999) and
their accumulation (Srinivasan et al., 2005) due to poor degrada-
tion by proteasomal and lysosomal pathways, causing metabolic
dysfunction of kidneys (Dalle-Donne et al., 2006). Digera muri-
cata extract erased the intoxication and protein contents reverted
towards normalcy. More ameliorated effects were observed with
HDMP as compared to MDMP as long as the protein contents were
considered.
In vitro and in vivo studies indicated that CCl4 enhanced lipid
peroxidation, CCl3 radical formation from the metabolic conver-
sion of CCl4 by cytochrome P-450, and renal reduced/oxidized
glutathione ratio in kidney cortex as well as renal microsomes and
mitochondria (Adewole et al., 2007). As O2 tension rises, a greater
fraction of CCl3 radical present in the system reacts very rapidly
with O2 and many orders of magnitude more reactive free radical,
trichloromethoxy (CCl3 OO) radical has been generated from CCl3 .
This radical is more reactive and is capable of abstracting hydrogen
from polyunsaturated fatty acids (PUFAs) to initiate the process of
lipid peroxidation (Kamalakkannan et al., 2005). This phenomenon
resulted in the generation of ROS. With the establishment of oxida-
tive stress, the defense capacities against ROS become insufficient
(Halliwell and Gutteridge, 2000).
In the present study it has been observed that CCl4 induced a sig-
nificant decrease in the tissue level of CAT, POD, SOD and GSH. The
decreased activity of SOD in kidney in CCl4 -treated rat may be due
to the enhanced lipid peroxidation or inactivation of the antiox-
idative enzymes. This would cause an increased accumulation of
superoxide radicals, which would further stimulate lipid peroxida-
tion. Decrease in GSH activity during CCl4 toxicity might be due
to the decreased availability of GSH resulted during the enhanced
lipid peroxidation. Improvement of renal GSH levels in rats treated
with Digera muricata extracts in comparison to CCl4 administration
further demonstrated the antioxidative effect of the plant. Chronic
treatment of Digera muricata extracts also improved the levels of
three key antioxidant enzymes SOD, POD and CAT in CCl4 admin-
istered rats. CCl4 toxicity in rats induced a significant elevation in
renal TBARS that was improved with the extract of Digera muricata.
Carotenoids are present in the Digera muricata (Punia et al., 2004)
which may have different functional properties such as scaveng-
ing of active oxygen species, inhibition of the generation of free
radicals and chain breaking activity. Similar observations were also
reported with black tea extract on the level of TBARS in rats after
CCl4 exposure (Fadhel and Amran, 2002).
CCl4 administration mediated lipid peroxidation of lipid struc-
tures of renal tissues, resulting in subcellular damages as observed
in histopathological examination. In this study the kidneys of
98 M.R. Khan et al. / Journal of Ethnopharmacology 122 (2009) 91–99
CCl4 -treated rats have shown characteristic morphological findings
such as interstitial fibrosis, glomerular and tubular degenera-
tion, interstitial mononuclear cell infiltration. The vasoconstriction
induced by CCl4 produces an ischemic local environment, which
leads to a number of cellular damages such as deterioration in
membrane integrity. Marked histological changes are prominent
in the outer cortex and inner medullary region of the kidney. The
severe changes were not observed in the groups treated with Digera
muricata extracts suggesting the protective effects of Digera muri-
cata in attenuating CCl4 -induced morphological changes. Similar
histopathological changes were observed by Ogeturk et al. (2005) in
renal of rats treated with CCl4 and these histopathological changes
were disappeared in rats treated with CCl4 + caffeic acid phenyl
ester (CAPE).
Tubular epithelial cells alterations including vacuolization, atro-
phy and finally detachment of epithelial cells, indicated tubular
necrosis in kidneys of rats treated with CCl4 . Similar histopatho-
logical alterations were also recorded in other studies after long
exposure to CCl4 (Doi et al., 1991; Ozturk et al., 2003). It is believed
that with these histopathological changes the capacity of tubular
absorption may have been altered, thus bringing about functional
overload of nephrons with subsequent renal dysfunction (Adewole
et al., 2007).
It has been hypothesized that oxidative damage can occur in
DNA during the peroxidative breakdown of membrane polyunsat-
urated fatty acids. DNA damage affects homeostasis of various cells
leading to induced signal transductions associated with apoptosis
and cell proliferation (Khanna and Jackson, 2001; Jia et al., 2002;
Bonventre, 2003; Poirier, 2004). Administration of Digera muricata
extracts to CCl4 intoxicated rats protected the renal tissues and
markedly decreased the percentage of fragmented DNA that was
also revealed in DNA ladder assay.
This study indicated the protective role of Digera muricata at
several levels. It may contribute its protective effects by erasing the
damaging action of CCl4 at various metabolic cycles, proteasomal
protein degradation and the repair of DNA damage. The protective
potential may either involve antioxidant; signal transduction, gene
expression, and effective involvement in the metabolic pathways.
AgNORs count predicted the cellular activity and proliferation
level (Shiro et al., 1992; Trere et al., 1996). Quantitative evalua-
tion of AgNORs allowed us to determine the level of intoxication.
Increased number of nucleoli (NORs) with abnormal shapes and
sizes, including small dots, may be used to evaluate the cellular
damage. In present study significantly higher numbers of AgNORs in
the nuclei of glomerular and tubular cells in the renal tissues of rats
treated with CCl4 were recorded and were scattered through out the
nucleus. Higher number of AgNORs with CCl4 administration indi-
cated the presence of invasive neoplasia (Wilkinson et al., 1990).
Both types of extract of Digera muricata ameliorated the effects of
CCl4 thereby decreasing the AgNORs count. However, MDMP more
convincingly erased the effects of CCl4 intoxication.
Telomeres are specialized protein–DNA structures at chromo-
somal ends of highly conserved G-rich sequence TTAGGG in all
eukaryotic cells that are lost gradually during each cell division.
Telomerase is a key enzyme for synthesis of telomeric repeats.
In immortal cancer cells, telomerase found to be reactivated in
response to the onset of tumorigenesis (Wen et al., 1998). To date,
measurement of telomerase activity has been considered as a diag-
nostic marker for malignant tumors (Lin et al., 1997; Yoshida et
al., 1997) and in cell lines (Chakraborty et al., 2006). Telomerase
activity was determined in the present study indicating the onset
of tumorigenesis induced through CCl4 treatment in the renal tis-
sues. This activity was found absent with the treatment of MDMP
and HDMP suggesting the presence of telomerase inhibitors in both
the extracts. Similar results have been reported in various studies
(Ramachandran et al., 2002; Eitsukaa et al., 2004).
5. Conclusion
This study substantiated the scientific evidence in favour of its
pharmacological use in renal disorders in folk medicine. The plant
Digera muricata possesses the antioxidant as well as anticancer
compounds. In addition this study suggested to isolate and to eval-
uate the active compounds present in this plant.
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