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Journal of Medicinal Plants Research Vol. 5(25), pp. 6055-6060, 9 November, 2011
Available online at http://www.academicjournals.org/JMPR
ISSN 1996-0875 ©2011 Academic Journals
DOI: 10.5897/JMPR11.987
Full Length Research Paper
Potassium bromate (KBrO3) induced nephrotoxicity:
Protective effects of n-hexane extract of Sonchus asper
Rahmat Ali Khan1*, Muhammad Rashid Khan2, Sumaira Sahreen2, Nasir Ali Shah2,
Jasia Bokhari2, Maria Shabbir2, Umbreen Rashid2 and Shumila Jan2
1Department of Biotechnology, University of Science and Technology KPK, Pakistan.
2Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad, Pakistan.
Accepted 25 August, 2011
Potassium bromate (KBrO3) is an oxidizing agent used in industries for the formation of hair solution,
cosmetics and as a by product during ozonation of water, causes infections in kidney and has been
classified as 2B group toxic chemical a probable human carcinogen. In the present study, Sonchus
asper non polar n-hexane extract (SAHE) is used against KBrO3 induced nephrotoxicity in rats. During
this study 32 male albino rats were randomly divided into 4 groups and experiment was proceeded for 6
weeks. Results revealed that induction of KBrO3 in rats significantly reduced activities of antioxidant
enzymes (p<0.01) while enhanced NORs/cell and telomerase enzyme activity and lipid peroxidation
which were markedly improved by co-treatment of SAHE. KBrO3 also considerably distorted (p<0.01)
DNA fragmentation in kidney comparatively to control. These changes are noticeably (p<0.01)
reimbursed with treatment of SAHE. These data proved that SAHE extract has ability of DNA
fragmentation repairing, reduces rgyrophilic nucleolar organiser regions (AgNORS), restoring enzyme
activity and telomerase enzymes, which might be due to the presence of plant bioactive compounds.
Key words: Sonchus asper; DNA fragmentation, argyrophilic nucleolar organiser regions (AgNORs),
telomerase enzyme inhibition, antioxidant enzymes.
INTRODUCTION
Medicinal plants play important role in improving human
health. These are composed of some bioactive
phytochemical substances, which regulate various
physiological and molecular action in living organisms
(Hill, 1952). Today many natural products extracted from
medicinal plants are being tested for the presence of new
drugs with new modes of pharmacological action. Special
features of higher plants are their capacity to produce a
large number of secondary metabolites (Castello et al.,
2002). Recent studies are involved in the identification
and isolation of new therapeutic compounds of medicinal
importance from higher plants for specific diseases (Khan
et al., 2009; Khan et al., 2010a, b; Sahreen et al., 2010).
Some bioactive compounds derived from plants include
tannins, alkaloids, cardiac glycosides, flavonoids, sterols,
triterpenes and anthraquinones which play main role in
*Corresponding author. E-mail: rahmatgul_81@yahoo.com. Tel:
+92 928 633425.
nutrition, physiology and control of various diseases
(Sahreen et al., 2011; Khan et al., 2011a, b) and in many
biological activities including; spasmolytic activity of
smooth muscles and as antioxidant in protecting the body
against oxidative stress (Tona et al., 2000). Sonchus
asper (L.) Hill. have spiny leaves and yellow flowers. The
leaves are simple, bluish-green in color, lanceolate, with
wavy and lobed margins.
The stem and leaves emit a milky sap on cutting. Their
flowers grow in clusters. This plant is native to Pakistan,
but also found as a common weed in North America in
open fields and road sides. According to binomial
nomenclature S. asper belongs to Kingdom Plantae,
Order Asterales, Family Asteraceae, Tribe Cichorieae,
Genus Sonchus, and Species asper. S. asper locally
named as Mahtari used in the treatment of liver injuries
and cardiac dysfunction (Khan et al., 2011) and kidney
inflammation (Khan et al., 2010b).
Therefore, the study is arranged to investigate
protective effects of SAHE against KBrO3 induced
oxidative depression of antioxidant enzymes, DNA
6056 J. Med. Plants Res.
fragmentations, enhanced AgNORs counts and
telomerase enzyme.
MATERIALS AND METHODS
Extraction and experimental design
Crude methanolic extract of S. asper was obtained as previously
described by Khan et al. (2010b) and was further fractionated with
n-hexane to get non polar fraction, stored at 4°C for in vivo
investigation in rats. To check the activities of antioxidant enzymes,
lipid peroxidation, DNA damages, AgNORs count and telomerase
enzyme activity 32 male albino rats were purchased from National
Institute of Health (NIH), Islamabad, Pakistan, acclimatized for 7
days, then randomly divided into 4 groups. Group 1 was remain
untreated, group II was given 20 mg/kg bw KBrO3 in aqueous
saline, group III and group IV was given 100 and 200 mg/kg SAHE
respectively for 6 weeks. Study protocol was approved by ethical
committee of Quaid-i-Azam, University, Islamabad, Pakistan. After
completion of experiments kidney was treated with liquid nitrogen
for further analysis.
Effect of SAHE on antioxidant status
For determination of antioxidant status, 10% solution of tissues was
made in phosphate buffer (100 mmol) and EDTA (1 mmol),
centrifuged at 12,000 × g for 30 min at 4°C to c ollect the
supernatant. Activities of antioxidant enzymes; c atalase assay
(CAT) (Chance and Maehly, 1955), superoxide dismutase (SOD)
(Kakkar et al., 1984), glutathione-S-transferase (GST) (Habig et al.,
1974), glutathione reduct ase (GSR), (Carlberg and Mannervik,
1975), glutathione peroxidase (GSH-Px) (Mohandas et al., 1984)
and contents of reduced glutathione (GSH) were estimated with
protocol of Jollow et al. (1974) while lipid peroxidation (TBARS) was
estimated with Iqbal et al. (1996).
DNA fragmentation% assay
DNA fragment ation % assay was conducted using the procedure of
Wu et al. (2005) with some modifications. The tissue (50 mg) was
homogenized in 10 volumes of a TE solution pH 8.0 (5 mmol Tris-
Hcl, 20 mmol EDTA) and 0.2% triton X-100. 1.0 ml aliquot of each
sample was centrifuged at 27,000 × g for 20 min to separate the
intact chromatin (pellet, B) from the fragmented DNA (supernatant,
T). The pellet and supernatant fractions were assayed for DNA
content using a freshly prepared Diphenylamine (DPA) solution for
reaction. Optical density was read at 620 nm with (SmartSpecTM
Plus Spectrophotometer catalog # 170-2525) spectrophotometer.
The results were expressed as amount of % fragmented DNA by
the following formula:
% Fragmented DNA = T×100/T+B
AgNORs count
Silver staining technique was used according to Trere et al. (1996).
The AgNORs technique was performed on dried slides as follows;
unstained fixed slides were dewaxed by dipping for 3 min in xylene.
After complete removal of wax the slides were hydrated in
descending order of ethanol concentration (90, 70 and 50%) and
washed in distilled water for 10 min and dried in an oven. After
drying slides wer e treated with one drop of colloidal solution (2%
gelatin and 1% formic acid) and two drops of 50% AgNO3
solution onto the slide and incubated at 35°C for about 8 to 12 min.
The progressive staining was followed under microscope to get
golden colored nuclei and brown/black NORs. Then, the slide was
washed in distilled water, treated for 1 min with 1% sodium
thiosulphate at room temperature to stop the reaction, and washed
in tap water. The cells were examined under light microscope at
100 × magnification and number of AgNORs was counted per cell.
DNA ladder assay
DNA was isolated by using the methods of W u et al. (2005) to
estimate DNA damages. 5 µg of r at DNA was separately loaded in
1.5% agarose gel containing 1.0 µg/ml ethidium bromide including
DNA standards (0.5 µg per well). Electrophoresis was performed for
45 min at 100 Volt. Aft er electrophoresis gel was studied under gel
doc system and was photographed through digital camera.
RT-PCR analysis (TRAP assay)
Telomerase activity was determined by the protocol of Wen et al.
(1998) with some modifications.100 mg kidney was washed in ice-
cold wash buffer (10 mM Hepes-KOH pH 7.5, 1.5 mM MgCl2, 10
mM KCl, 1 mM dithiothreitol, 20 µl RNAs inhibitors), and
homogenised in 200 µl ice cold lysis buffer. The homogenate was
incubated on ice for 30 min and then centrifuged at 10,000 xg for 30
min at 4°C.
PCR reaction mixture (total 48 µl) consisted of 36.6 µl DEPC
treated water, 2 µl (6 µg pr otein) extract, 5 µl 10xTRAP reaction
solution, 2 µl (50 µM) each dNTP, 0.4 µl (2 U) Taq DNA
polymerase, and 2 µl (0.1 µg) of TS primer sequence (5'-
AATCCGTCGAGCAGAGTT-3'). The PCR r eaction mixture was
incubated at 25°C in water bath f or 30 min for extension of TS
primer. CX primer sequence (5'-
CCCTTACCCTTACCCTTACCCTAA-3') 2 µl (0.1 µg)) was added.
The reaction mixture (total 50 µl) was subjected to PCR cycles (25)
at 94°C for 30 s, 55°C for 30 s, and 72°C for 90s (then 10 min for
the final step). After amplification 5 µl of loading dye (0.25%
bromophenol blue, 0.25% xylenocyanol and 50% glycerol) was
mixed to each PCR product and 25 µl of each sample were loaded
onto a 12.5% non-denaturing polyacrylamide gel. After complete
running of gel it was fixed in fixing solution (0.5% acetic acid,10%
ethanol) and stained with 0.2% AgNO3 for 10 min, followed by 15
min incubation in developing s olution (0.1% formaldehyde and 3%
NaOH) and then photographed.
Statistical analysis
To determine the treatment effects one way analysis of variance
was carried by computer software SPSS 13.0. Level of significance
among the various treatments was determined by LSD at 0.05%
level of probability.
RESULTS
Effect of SAHE on antioxidant status
Protective effects of SAHE against KBrO3-induced toxicity
are shown in Table 1. SAHE significantly increased
(p<0.01) the activities of antioxidant enzymes; CAT,
SOD, GST, GSH-px and GSR as well as GSH contents
while reduced contents of TBARS in kidney as was
altered by induction of KBrO3.
Khan et al. 6057
Table 1. Effect of SAHE on antioxidant status in rat.
Treatment CAT
(U/min)
SOD
(U/min)
GST (nM
/min/mg protein)
GSH-Px (nM
/min/mg protein)
GSR (nM
/min/mg protein)
GSH
(M/g tissue)
TBARS (nM
/min/mg protein)
Control 12.233±0.250++ 45.33±5.65++ 223.3±10.3 ++ 173.8±15.3++ 59.61±2.5++ 1.7±0.57++ 5.5±0.075++
20 mg/kg KBrO3 5.535±0.185** 25.35±2.08 ** 134.5±24.7** 113.8±26.1** 33.99±0.9** 0.76±0.59** 11± 0.68**
100 mg/kg SHE+ KBrO3 9.893±0.0992++ 32.87±2.68**+ 169.3±9.92**++ 151.2±32.8++ 46.13±1.3**++ 1.3±0.06++ 8.0±0.49++
200 mg/kg SHE+ KBrO3
11.251±0.171++ 42.65±3.88++ 205.17±17.1++ 167.50±7.78++ 58.46±3.6++ 1.7±0.09++ 6.0±0.27++
Mean ± SE (n = 8 number). ** indicate significance fr om the control group at p<0.01 probability level. ++ indicate significance from the KBrO3 group at p<0.01 probability level.
AgNORS (NORS/cell)
0
1
2
3
4
5
6
7
8
9
1 2 3 4
Trea tment group
NORS/cell
Figure 1. Effects of SAHE on AgNORs c ounts.
Effects of SAHE on kidney AgNORs in rat
Preventive efficacy of SAHE against KBrO3
administration in rat on AgNORS count as shown
in Figure 1.
Administration of KBrO3 significantly increased
(p<0.01) AgNORS count than control. Post-
treatment with SAHE erased the KBrO3 toxication
and significantly (p<0.01) reversed the level of
AgNORs towards the control rats.
Effects of SAHE on kidney % DNA
fragmentation in rat
Preventive efficacies of SAHE against KBrO3
administration in rat on DNA fragmentation are
6058 J. Med. Plants Res.
% DNA Fragmentation
0
5
10
15
20
25
30
35
40
1 2 3 4
Trea tment groups
%Fragmentation
Figure 2. Effects of SAHE on % DNA fragmentation.
150
0
b
p
5
0
b
p
Figure 3. Agarose gel showing DNA damage by KBrO3 and preventive effect of SAHE, Lanes
(from left) DNA marker (M), Control (1 to 4), KBrO3 (5 to 8), 100, 200 mg/kg b.w., SAHE (9, 10).
shown in Figure 2. Administration of KBrO3 significantly
increased (p<0.01) % DNA damages than control. Post-
treatment of SAHE erased the KBrO3 toxication and
significantly (p<0.01) reversed DNA damages towards
the control rats.
Effect of SAHE on kidney DNA damages (DNA ladder
assay)
KBrO3 forming DNA-free radical adduct, induces DNA
damages in the kidney tissues of rats. DNA ladder assay
showed that DNA damage was present in control as well
as DMSO treated group. However, KBrO3 group showed
severe DNA damages. Post-treatment of SAHE reduced
the DNA damages as indicated by DNA band of SAHE
comparatively to KBrO3 group (Figure 3).
Effect of SAHE on RT-PCR analysis
Telomerase enzyme play important role in oxidative
stress and cancer. The results for telomeric repeat
amplification protocol assay showed a single band in
control group (Lane 5 to 6) which revealed the absence
of telomerase enzyme activity while (Lanes 7 to 8) showed
Figure 4. Polyacrylamide gel shows the telomerase
enzyme activity in various groups of the study. From left
to right marker Lane (M), control group Lane (1), KBrO3
group Lane (2) and SAHE Lane (3).
TRAP amplification product in group treated with KBrO3.
Figure 4 (Lanes 1 to 4) shows that telomeric repeats
bands were not present, indicated the protective effects
of S. asper. These result suggested that SAHE possess
potent anticancer as well as anti telomerase activity might
be the presence of bioactive anticancer compounds in
the extract.
DISCUSSION
The data revealed that the treatment of KBrO3 causes
significant alteration in antioxidant enzymes and oxidative
DNA damage in kidneys of rats which are visualize on
agarose gel by staining with ethidium bromide. Treatment
with SAHE significantly improved the activities of CAT,
SOD, GSH-px, GST, GSR and reduced DNA damages.
Similar investigation was reported by Khan et al., (2009,
2010a, b) during study of protective effects of Digera
muricata against carbon tetrachloride induced
nephrotoxicity in rats. Khan and Sultana (2005) reported
that the induction of KBrO3 caused oxidative DNA
damages in rats, which support our investigations. These
results show that the SAHE contain bioactive compounds
which play important role in DNA repair. Silver stained
nucleolar organelles (NORs) per cell and chemical
toxicity are directly correlated each other. Various studies
reported that the quantity of protein AgNORs/cell is
directly related with cell proliferation. It has also been
reported from various investigation that number of
AgNORs counts per cell and the prognosis of malignant
tumour are directly related to one another. According to
Irazusta et al. (1998), the quantification of AgNORs
proteins per cell has been useful in diagnostic pathology
especially in the differentiation of benign from malignant
tumors and helpful in limitrophic lesions recognitation. In
present study, statistically significant difference indicated
Khan et al. 6059
the presence of invasive neoplasia (Wilkinson , 1990).
The results inferred from the current data revealed that
KBrO3 induce telomerase activity in rats. The highly
sensitive TRAP assay was used to detect the telomerase
activity. The rat treated with KBrO3 showed amplification
of telomeres which was completely devoid by
administration with SAHE. Similar results were obtained
by our laboratory group of researcher. Those free
radicals induced telomeric activity was reversed by post-
treatment with Sonchus asper (Khan et al., 2009) and in
other studies (Ramachandran et al., 2002). This
antitelomeric and anticancer effect of various fractions
showed that this might be possible due the presence of
bioactive natural telomerase inhibitors compounds, which
needs further isolation and purification.
REFERENCES
Castello MC, Phatak A, Chandra N, Sharon M (2002). Antimicrobial
activity of crude extracts from plant parts and corresponding calli of
Bixa orellana L. Indian J. Exp. Biol., 1378-1381.
Carlberg I, Mannervik EB (1975). Glutathione level in rat brain. J. Biol.
Chem., 250: 4475-4480.
Chance B, Maehly AC (1955). Assay of catalase and peroxidases. Met.
Enzymol., 11: 764-775.
Habig W H, Pabst MJ, Jakoby W B (1974). Glutathione-S-transferases:
the first enzymatic step in mercapturic acid formation. J. Biol. Chem.,
249: 7130-7139.
Hill AF (1952). Economic Botany. A textbook of useful plants and plant
products. 2nd edn. McGraw-Hill Book Company Inc, New York.
Irazusta SP, Vassallo J, Magna LA (1998). The value of PCNA and
AgNOR staining in endoscopic biopsies of gastric mucosa. Pathol.
Res. Prac., 194: 33-39.
Iqbal M, Sharma SD, Zadeh HR, Hasan N, Abdulla M, Athar M (1996).
Glutathione metabolizing enzymes and oxidative stress in ferric
nitrilotriacetate (Fe-NTA) mediated hepatic injury. Redox Report, 2:
385-391.
Jollow DJ, Mitchell JR, Zampaglione N, Gillete JR (1974).
Bromobenzene induced liver necrosis. Protective role of glutathione
and evidence for 3,4-bromobenzene oxide as a hepatotoxic
metabolite. Pharmacology, 11: 151-169.
Kakkar P, Das B, Viswanathan PN ( 1984). A modified
spectrophotometric assay of superoxide dimutase. Indian J. Biochem.
Biophys., 21: 130-132.
Khan MR, Haroon J, Khan RA, Bokhari J, Rashid U (2011). Prevention
of KBrO3-induced cardiotoxicity by Sonchus asper in rat. J. Med.
Plants Res., 5(12): 2514-2520.
Khan MR, Rizvi W , Khan GN, Khan RA, Sheen S (2009). Carbon
tetrachloride-induced nephrotoxicity in rats: Protective role of Digera
muricata. J. Ethnopharmacol., 122: 91-99.
Khan N, Sultana S (2005). Chemomodulatory effect of Ficus racemosa
extract against chemically induc ed renal carcinogenesis and
oxidative damage response in Wister rats. Lif e Sci., 77: 1194-1210.
Khan RA, Khan MR, Sahreen S (2010a). Evaluation of Launea
procumbens use in renal disorders: a rat model. J. Ethnopharmacol.,
128: 452-461.
Khan RA, Khan MR, Sahreen S (2011a). Protective effect of Sonchus
asper extracts against experimentally-induced lung injuries in rats: A
novel study. Exp. Toxicol. Pathol., doi:10.1016/j.etp.2011.01.007.
Khan RA, Khan MR, Sahreen S, Bukhari J (2010b). Prevention of CCl4-
induced nephrotoxicity with Sonchus asper in rat. Food Chem.
Toxicol., 23: 1304-1321.
Khan RA, Khan MR, Sahreen S, Bukhari J (2010c). Antimicrobial and
Phytotoxic activity of various fractions of Sonchus asper. Afr. J.
Biotechnol., 47: 3877-3683.
Khan RA, Khan MR, Sahreen S, Jan S, Bokhari J, Rashid U (2011b).
Phytotoxic characterization of various fractions of Launaea
6060 J. Med. Plants Res.
procumbens. Afr. J. Biotechnol., 10: 5377-5380.
Mohandas J, Marshal JJ, Duggin GG, Horvath JS, Tiller DJ (1984).
Differential distribution of glutathione and glutathione-related
enzymes in rabbit kidney. Possible implications in analgesic
nephropathy. Biochem. Pharmacol., 33: 1801-1807.
Ramachandran C, Fonseca HB, Jhabvala P, Escalon EA, Melnick SJ
(2002). Curcumin inhibits telomerase activity through human
telomerase reverse transcritpase in MCF-7 breast cancer cell line.
Cancer Lett., 184: 1-6
Sahreen S, Khan MR, Khan RA (2010). Evaluation of antioxidant
activities of various solvent extracts of Carissa opaca fruits. Food
Chem., 122: 1205-1211.
Sahreen S, Khan MR, Khan RA (2011). Hepatoprotective effects of
methanol extract of Car issa opaca leaves on CCl4-induced damage in
rat. BMC Compl. Alter. Med., 11:48 doi: 10.1186/1472-6882-11-48.
Tona L, Kambu K, Ngimbi N, Mesia K, Penge O, Lusakibanza M,
Cimanga K, De BT, Apers S, Totte J, Pieters L, Vlietinck AJ (2000).
Antiamoebic and spasmolytic activities of extracts from some
antidiarrhoeal traditional preparations used in Kinshasa, Congo.
Phytomedicine, 7: 31-38.
Trere D, Zilbering A, Dittus D, Kim P, Ginsberg PC, Daskal I (1996).
AgNOR quantity in needle biopsy specimens of prostatic
adenocarcinomas: correlation with proliferation state, Gleason score,
clinical stage, and DNA c ontent. J. Clin. Pathol., 49: 209-213.
Wilkinson EJ (1990). Vulvar intraepithelial neoplasia and s quamous cell
carcinoma with Sofowara emphasis on new nomenclature. Prog.
Reprod. Urin. Tract Pathol., 2: 1-20.
Wu B, Ootani A, Iwakiri R, Sakata Y, Fujise T, Amemori S, Yokoyama
F, Tsunada S, Fujimoto K (2005). T cell deficiency leads to liver
carcinogenesis in Azoxymethane-treated rats. Exp. Biol. Med., 231:
91-98.
