全 文 ： 38 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 2013年 1月 第 11卷 第 1期

Chinese Journal of Natural Medicines 2013, 11(1): 00380042
doi: 10.3724/SP.J.1009.2013.00038
Chinese
Journal of
Natural
Medicines







Cardioprotective effect of Urtica parviflora leaf extract against
doxorubicin-induced cardiotoxicity in rats
N. R. Barman1, Prasanna K. Kar1, Prakash K. Hazam1, Himadri S. Pal1, Arvind Kumar1,
Sanjib Bhattacharya2, Pallab K. Haldar3*
1Himalayan Pharmacy Institute, Majhitar, Rangpo East Sikkim 737136, India；
2Bengal School of Technology, Sugandha, Hooghly, West Bengal 712102, India；
3Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India
Available online 20 Jan. 2013
[ABSTRACT] AIM: The present study evaluated the cardioprotective property of the hydroethanol extract of Urtica parviflora leaf
material (EEUP) against doxorubicin-induced cardiotoxicity in rats. METHODS: Cardiotoxicity was produced by doxorubicin ad-
ministration (15 mg·kg-1 i.p. for 21 days). The rats received EEUP at 200 and 400 mg·kg-1 b.w. (i.p.) daily for 21 days. After 24 h,
serum cardiac biomarkers, i.e. creatine phosphokinase (CPK) and lactate dehydrogenase (LDH); serum lipid profiles, like high density
lipoprotein (HDL), low density lipoprotein (LDL) and triglyceride (TG); serum biochemical parameters, viz. aspartate aminotransferase
(AST), alanine transaminase (ALT) and alkaline phosphatase (ALP); myocardial antioxidant parameters, viz. malondialdehyde (MDA),
superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) were measured. RESULTS: EEUP treatment significantly
(P < 0.01) and dose dependently protected the myocardium by decreasing the elevated level of MDA; elevating the diminished levels
of GSH, SOD, CAT and HDL, with a concomitant decrease in the elevated levels of HDL, LDL, and TG. EEUP also significantly (P <
0.01) reduced the increased activities of AST, ALT, ALP, CPK and LDH. The results revealed that EEUP demonstrated dose dependent
cardioprotective efficacy by restoration of the serum biomarkers profile and antioxidant property. CONCLUSION: From the present
study, U. parviflora leaf extract showed promising cardioprotective effect against doxorubicin-induced cardiotoxicity in Wistar rats.
[KEY WORDS] Doxorubicin; Cardioprotective; Urtica parviflora; Antioxidant; Leaf material; Serum biomarkers
[CLC Number] R965 [Document code] A [Article ID] 1672-3651(2013)01-0038-05

1 Introduction
Doxorubicin (adriamycin) is an anthracycline compound
and highly efficacious anticancer drug that is widely used for
treatment of various neoplastic diseases[1]. However, its clin-
ical use very often becomes a limiting factor in anticancer
therapy due to its high irreversible cardiotoxicity. Congestive
heart failure, cardiomyopathy and electrocardiographic
changes were demonstrated after cumulative doxorubicin
administration[2]. Doxorubicin-induced cardiotoxicity has
been shown to be mediated through several different mecha-
nisms, including membrane lipid peroxidation[3], free radical
formation[4], mitochondrial damage[5], decreased activity of
Na+–K+ adenosine triphosphate[6], and increases in serum

[Received on] 06-Jan.-2012
[*Corresponding author] Pallab K. Haldar: Associate Prof., E-mail:
pallab_haldar@rediffmail.com
These authors have no conflict of interest to declare.
total cholesterol, triglyceride and low density lipoproteins[7].
Urtica parviflora Roxb. (Urticaeae), commonly known
as Sishnu in Nepalese, Nettle in English, and Paharah-bichuti
in Bengali is a monoecious, perennial herb consisting of long
stoloniferous rhizomes found in forests and amongst taller
herbaceous vegetation, at 1 700−2 800 meters, partly shady,
moist places of evergreen forests, along the streams and
roadsides of Nepal, Bhutan, Western China and in Northern
India. In India, it is mainly found in the Uttarakhand, Kash-
mir, Tamil Nadu, Assam and Sikkim states[8-9]. The leaves of
the plant have stinging hairs, causing irritation and rashes to
the skin. Its leaves are used as a culinary vegetable (after
treatment with boiling water) in Darjeeling and the Sikkim
Himalayan region of India, and the plant is commercially
cultivated and sold in these regions to be used as vegetable.
Young cooked leaves are very palatable and nutritious food,
rich in vitamins and minerals, especially of α-tocopherol and
vitamin C[9]. The plant has been traditionally used for several
medicinal purposes. Its seed oil is edible, as well as medicinal
N. R. Barman, et al. /Chinese Journal of Natural Medicines 2013, 11(1): 3842
2013年 1月 第 11卷 第 1期 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 39

for sciatica, rheumatism, and several skin ailments; a hair
wash from the leaf extract is believed to avoid baldness. The
leaves are used in dysentery, joint pain and liver disorders[9].
The fresh roots are employed for the treatment of fractures of
the bone and dislocations of the joints[8]. Leaves and inflo-
rescences are prescribed as a general tonic, and as a cleaning
agent after parturition[10]. Its leaves and stems produce an
inflammatory rash, accompanied by a considerable burning
and itching sensation that is attributed to the presence of his-
tamine and 5-hydroxytryptamine[11]. Previous research stud-
ies have reported the hepatoprotective[12], hypoglycemic[13],
and in vitro antioxidant potential of U. parviflora[14]. The
present study attempted the evaluation of the cardioprotective
effect of U. parviflora leaf extract in a doxorubicin-induced
cardiotoxicity model in Wistar rats.
2 Materials and Methods
Plant material The mature leaves of Urtica parviflora
Roxb. (Urticaceae) were collected during the month of Au-
gust 2009 from Majhitar, East Sikkim, India. The plant spe-
cies was identified and authenticated from the Botanical
Survey of India, Sikkim Circle, Gangtok, Sikkim, India, and
a voucher specimen (No. HPI/121) was retained in the Phar-
macology Department of Himalayan Pharmacy Institute,
Majhitar, Sikkim, India for future reference.
Drugs and chemicals Doxorubicin was obtained from
Get Well Pharmaceuticals, New Delhi, India. Superoxide
dismutase, catalase references were obtained from Sig-
ma-Aldrich, Germany and reduced glutathione was from
Loba Chemie, Mumbai, India. Enzyme analyzing reagent kits
were obtained from Span Diagnostics Ltd., Surat, India and
Transasia Bio-medicals Ltd., Solan, India. All of the other
chemicals used for the biochemical estimation were of ana-
lytical grade obtained commercially.
Preparation of extract Air-dried fresh leaves (218 g)
were powdered in a mechanical grinder, and the powdered
plant material was extracted with 50% ethanol by maceration
at room temperature 24−26 °C for 96 h. Then the solvent was
completely removed under reduced pressure to yield the dry
extract (EEUP, yield: 6.50%) which was stored in a vacuum
desiccator for future use. Preliminary phytochemical studies
on EEUP indicated the presence of flavonoids, steroids, gly-
cosides and tannins[15].
Animals Adult male Wistar albino rats (100−150 g) were
used for the present study. The rats were grouped and housed
in polyacrylic cages (38 cm × 23 cm × 10 cm), with not more
than four animals per cage and maintained under standard
laboratory conditions (temperature (25 ± 2) °C and dark/light
cycle 14/10 h) for seven days prior to commencement of the
experiment. They were allowed free access to standard dry
pellet diet (Hindustan Lever, Kolkata, India) and water ad
libitum. All experimental procedures described were re-
viewed and approved by the Institutional Animal Ethics
Committee, Himalayan Pharmacy Institute (IAEC Reg. No.
HPI/09/60/0071).
Acute toxicity EEUP was administered orally to
male Swiss albino mice to evaluate the acute toxicity as per
the reported method[16].
Experimental design The rats were divided into 4
groups (n = 6). Group I served as normal control and received
normal saline 5 mL·kg-1 body weight i.p. Except for group I,
doxorubicin was administered (5 mg kg-1 body weight i.p.)
to all other groups of animals in three equal injections on
the 7th, 14th and 21st days for a total cumulative dose of 15
mg kg-1 body weight. Group II served as the doxorubicin
control. Groups III and IV received EEUP at doses of 200
and 400 mg kg-1 body weight i.p. daily for 21 consecutive
days.
Enzyme assays Twenty-four hours after the last treat-
ment, blood samples were collected by cardiac puncture from
all of the groups of rats. Serum samples were separated for
the estimation of cardiac biomarkers, viz. creatine phos-
phokinase (CPK)[17] and lactate dehydrogenase (LDH)[18];
lipid profile like cholesterol[19], triglycerides[20], high density
lipoprotein (HDL), low density lipoprotein (LDL)[21]; serum
enzymes like aspartate amino transferase (AST)[22], alanine
amino transferase (ALT)[23], and alkaline phosphatase
(ALP)[24]. After collection of blood, all animals were sacri-
ficed by cervical dislocation, and heart vessel was isolated
and homogenized in buffered isotonic saline for estimation of
myocardial antioxidant parameters, including malondialde-
hyde (MDA)[25], superoxide dismutase (SOD)[26], catalase
(CAT)[27], and reduced glutathione (GSH)[28].
Histopathological studies The hearts form the experi-
mental rats were fixed in 10% buffered formalin and were
processed for microtome sectioning. Sections of about 5 µm
thickness were stained with haematoxylin and eosin for his-
topathological studies.
Statistical analysis All results are expressed as the
mean ± standard error of the mean (SEM). The results were
analyzed for statistical significance by one-way analysis of
variance (ANOVA) followed by Dunnett’s post hoc test using
GraphPad InStat version 3.05 (GraphPad Software, USA).
3 Results
Acute toxicity: The EEUP did not show any toxic ef-
fects or deaths up to the dose of 4 000 mg·kg-1, b.w., p.o.
Serum lipid levels: Animals treated with doxorubicin
produced a significant (P < 0.01) increase in cholesterol,
triglycerides and LDL levels, and a slight increase in the
HDL levels as compared to the normal control group. How-
ever, treatment with EEUP significantly (P < 0.01) decreased
the cholesterol, triglycerides, HDL and LDL levels as com-
pared to the doxorubicin control group (Table 1).
Serum enzyme biomarkers: Doxorubicin-treated ani-
mals showed a significant (P < 0.01) increase in CPK, LDH,
AST, ALT, and ALP levels as compared to the normal con-
trol group. After treatment with EEUP, the levels of CPK,
N. R. Barman, et al. /Chinese Journal of Natural Medicines 2013, 11(1): 3842
40 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 2013年 1月 第 11卷 第 1期

LDH, AST, ALT and ALP were significantly (P < 0.01)
restored towards normal in a dose dependant manner (Ta-
bles 2 and 3).

Table 1 Effect of EEUP on cholesterol, triglycerides, HDL
and LDL level in doxorubicin-treated rats (mean ± SEM, n = 6)
Treatments Cholesterol /(mg·dL-1)
Triglycerides
/(mg·dL-1)
HDL
/(mg·dL-1)
LDL
/(mg·dL-1)
Normal
control 112.60 ± 1.13 176.46 ± 0.96 23.54 ± 0.29 50.06 ± 0.73
Dox
(5 mg·kg-1) 199.02 ± 1.09
a 195.91 ± 0.84a 35.21 ± 0.61a 85.69 ± 1.29a
EEUP
(200 g·kg-1)
+ Dox
136.46 ± 0.83b 175.13 ± 0.67b 28.36 ± 0.44b 61.75 ± 0.88b
EEUP
(400 g·kg-1)
+ Dox
137.625 ± 1.081b 177.99 ± 1.11b 25.11 ± 1.31b 71.63 ± 0.72b
P < 0.01 vs anormal control and bdoxorubicin control. Dox: Doxoru-
bicin.

Table 2 Effect of EEUP on CPK and LDH in doxorubi-
cin-treated rats (mean ± SEM, n = 6)
Treatments CPK/(IU·L-1) LDH /(IU·L-1)
Normal control 137.13 ± 1.99 134.33 ± 1.64
Dox (5 mg·kg-1) 631.70 ± 9.39a 221.78 ± 1.29a
EEUP (200 mg·kg-1) + Dox 361.72 ± 2.80b 178.41 ± 1.21b
EEUP (400 mg·kg-1) + Dox 206.87 ± 2.28b 166.73 ± 1.04b
P < 0.01 vs anormal control and bdoxorubicin control. Dox: Doxoru-
bicin.

Table 3 Effect of EEUP on AST, ALT and ALP in doxoru-
bicin-treated rats (mean ± SEM, n = 6)
Treatments AST /(IU·L-1)
ALT
/(IU·L-1)
ALP
/(IU·L-1)
Normal control 5.55 ± 0.36 14.63 ± 0.46 48.11 ± 0.65
Dox (5 mg·kg-1) 23.62 ± 0.87a 55.27 ± 0.71a 135.81 ± 2.70a
EEUP (200 mg·kg-1) + Dox 16.36 ± 0.69b 29.63 ± 0.61b 64.43 ± 1.57b
EEUP (400 mg·kg-1) + Dox 11.60 ± 0.39b 22.29 ± 0.57b 34.68 ± 1.18b
P < 0.01 vs anormal control and bdoxorubicin control. Dox: Doxoru-
bicin.

Table 4 Effect of EEUP on MDA, GSH, CAT and SOD in
doxorubicin-treated rats (mean ± SEM, n = 6)
Treatments
MDA
/(nmol/g of
wet tissue)
GSH
/(µg/g of
wet tissue)
CAT
/(Units/mg
of protein)
SOD
/(Units/mg
of protein)
Normal
control 13.73 ± 0.53 24.17 ± 0.43 36.95 ± 1.66 25.95 ± 0.81
Dox
(5 mg·kg-1) 44.96 ± 1.32a 13.22 ± 0.96a 6.93 ± 0.75a 9.93 ± 0.69a
EEUP
(200 mg·kg-1)+
Dox
34.55 ± 0.69b 17.86 ± 0.46b 17.28 ± 1.01b 15.96 ± 0.62b
EEUP
(400 mg·kg-1)
+ Dox
24.74 ± 1.30b 20.72 ± 0.39b 26.02 ± 1.36b 18.04 ± 0.78b
P < 0.01 vs anormal control and bdoxorubicin control. Dox: Doxoru-
bicin.

Antioxidant status: The MDA level was increased,
whereas the GSH, SOD and CAT activities were significantly
(P < 0.01) decreased in the doxorubicin-treated group as
compared with normal control animals. In the EEUP-treated
groups, there was significantly lower MDA and higher GSH,
CAT and SOD levels as compared to the doxorubicin con-
trol group (Table 4).
Cardiac histopathology: Figs. 1-4 illustrate the histo-


Fig. 1 Cardiac section of normal control rats


Fig. 2 Cardiac section of Dox control (5 mg·kg−1) rats


Fig. 3 Cardiac section of EEUP (200 mg·kg−1) treated rats


Fig. 4 Cardiac section of EEUP (400 mg·kg−1) treated rats
N. R. Barman, et al. /Chinese Journal of Natural Medicines 2013, 11(1): 3842
2013年 1月 第 11卷 第 1期 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 41

pathological assessments of the cardiac segments of all ex-
perimental rats. Doxorubicin administration caused disor-
ganization of normal cellular architecture of the heart. EEUP
treatment reduced such changes in a dose related manner
almost similar to that of the normal control.
4 Discussion
The present work was aimed to study the cardioprotec-
tive activity of the hydroethanol extract of U. parviflora leaf
(EEUP) in doxorubicin-induced cardiotoxicity in rats. The
results of this study revealed that EEUP at the doses of 200
and 400 mg·kg-1 body weight dependently and significantly
ameliorated the cardiotoxicity by restoring serum and myo-
cardial biochemical parameters towards the normal values.
The existing experimental evidence suggests that dox-
orubicin-induced myocardial oxidative stress is due to the
generation of free radicals in the heart tissues[29]. In biologi-
cal systems, doxorubicin is enzymatically reduced to the
doxorubicin semiquinone radical. This semiquinone radical
directly transfers its electron to molecular oxygen, generating
free radicals, namely, superoxide and hydrogen peroxide[30].
This free radical generation plays an important role in the
cardiotoxicity of doxorubicin. Cardiac tissue is especially
susceptible to free radical injury because of the lower activi-
ties of the free radical detoxifying mechanisms, such as SOD,
CAT and GSH. Further, doxorubicin also has a high affinity
for the phospholipid component of the mitochondrial mem-
brane in cardiac myocytes, leading to selective accumulation
of doxorubicin in the heart tissue[31]. The doxorubi-
cin-induced mitochondrial injury is critical to the heart be-
cause it would presumably have extreme adverse effects on
the contractile functioning of the cardiac myocytes by pro-
ducing alterations in the energy metabolism[32].
EEUP treatment was able to reduce the doxorubi-
cin-induced cardiotoxic manifestations in multiple ways.
Increase in the level of plasma triglycerides, total cholesterol
and low density lipoproteins in the doxorubicin-treated group
indicate doxorubicin may be interfering with metabolism or
the biosynthesis of lipids. Treatment with EEUP showed a
reduction in serum lipid profile levels in a dose related fash-
ion. The lipid lowering effect of EEUP may be due to inhibi-
tion of hepatic cholesterol biosynthesis, increased fecal bile
acid secretion and stimulation of receptor mediated catabo-
lism of LDL cholesterol, and an increase in the uptake of
LDL from blood by the liver[33].
A deficiency of oxygen supply or glucose may damage
the myocardial cells, and the cell membrane becomes per-
meable or ruptures, resulting in leakage of enzymes from
cardiac tissues to the blood. It has been reported that doxoru-
bicin-induced free radical generation triggers membrane de-
gradation and disruption of cardiac myocytes, which can lead
to elevations of LDH and CPK in the serum[34-35]. In the pre-
sent study, an increase in the activities of LDH, CPK, AST,
ALP and ALT was observed in doxorubicin-treated rats.
Treatment with EEUP decreased the enzyme activities in
serum and restored the same in the heart. This could be due to
a protective or membrane-stabilizing effect of EEUP on the
myocardium, reducing the cardiac damage, and thereby re-
stricting the leakage of these enzymes. The present results are
in good agreement with those reported by previous work-
ers[36-37].
Cardioprotective activity of EEUP was further supported
by EEUP-induced activation of myocardial non-enzymatic
and enzymatic endogenous antioxidant defense mechanisms.
Cellular GSH depletion is closely related to the lipid peroxi-
dation and disturbance of Ca2+ influx induced by toxic agents.
Overproduction of oxidative free radicals enhances lipid pe-
roxidation. Here it was evident by an increase in MDA,
which is the end product of lipid peroxidation. Intraperitoneal
administration of EEUP in doxorubicin-treated rats reduced
the MDA level and elevated the GSH content to near-normal
levels, which prevented degradation of cellular macromole-
cules and thus cell disruption, probably by decreasing the
Ca2+ influx[38]. The present study has shown that doxorubicin
induced lipid peroxidation and decreased the levels of protec-
tive antioxidant enzymes in the heart tissues. Treatment with
EEUP significantly reduced the lipid peroxidation and in-
creased the activities of SOD and CAT which detoxify su-
peroxide and hydrogen peroxide radicals, respectively[39].
These results indicated the protective effect of EEUP on
doxorubicin-induced cardiotoxicity by boosting the endoge-
nous non-enzymatic and enzymatic antioxidant systems,
which entailed scavenging of oxidative free radicals.
Histopathological examination of different cardiac sec-
tions revealed that doxorubicin caused anomalous histologi-
cal changes in the cardiac tissue. However, EEUP treatment
prevented the changes and maintained the histological struc-
ture almost similar to that of normal control.
Preliminary phytochemical studies showed the presence
of flavonoids, tannins, glycosides and steroids in EEUP. Fla-
vonoids and tannins are well known polyphenolic natural
antioxidants the presence of which may be responsible for the
antioxidant role of EEUP and protection of myocardial tis-
sues from doxorubicin-induced oxidative injury.
In the present investigation, administration of EEUP to
doxorubicin-intoxicated rats demonstrated prominent reduc-
tion in serum biomarker enzymes, normalization of serum
lipid profiles compared to doxorubicin control rats in a
dose-dependent manner. Also, EEUP treatment resulted in
significant modulation of lipid peroxidation, endogenous
non-enzymatic (GSH) and enzymatic (SOD and CAT) anti-
oxidant and detoxification systems. Therefore, it can be con-
cluded that the hydroethanol extract of the leaves of the edi-
ble plant Urtica parviflora is remarkably effective against
doxorubicin-induced cardiotoxicity in Wistar rats. plausibly
by virtue of its lipid lowering property and augmenting en-
dogenous antioxidant mechanisms.
N. R. Barman, et al. /Chinese Journal of Natural Medicines 2013, 11(1): 3842
42 Chin J Nat Med Jan. 2013 Vol. 11 No. 1 2013年 1月 第 11卷 第 1期

Acknowledgements
The authors are thankful to the authorities of the Hima-
layan Pharmacy Institute, Sikkim and Jadavpur University,
Kolkata for the necessary facilities.
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