全 文 ： 362 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 2013 年 7 月 第 11 卷 第 4 期

Chinese Journal of Natural Medicines 2013, 11(4): 03620370
doi: 10.3724/SP.J.1009.2013.00362
Chinese
Journal of
Natural
Medicines







Dose-response effects of Elephantopus scaber methanolic extract
on N-nitrosodiethylamine-induced hepatotoxicity in rats
A. Linza1, P. J. Wills2, P. N. Ansil1, S. P. Prabha1, A. Nitha1, B. Latha1,
K. O. Sheeba1, M. S. Latha1*
1 Biochemistry and Pharmacognosy Research Laboratory, School of Biosciences, Mahatma Gandhi University, P.D. Hills. P.O.,
Kottayam-686560, Kerala, India;
2 MIMS Research Foundation, Malabar Institute of Medical Sciences, Calicut-673007, Kerala, India

Available online 20 July 2013
[ABSTRACT] AIM:A decoction of Elephantopus scaber (Asteraceae) root is used to treat liver disorders in Indian and Chinese
traditional medicine. The study was designed to examine the dose response effects of E. scaber methanolic extract on rats exposed to
N-nitrosodiethylamine (NDEA) induced hepatotoxicity (0.02% NDEA in water five days per week, per oral) in preventive and curative
models. METHODS: In preventive groups, NDEA was administered for six weeks. Daily doses of E. scaber methanolic extract (200
and 100 mg·kg−1) started one week before the onset of NDEA intoxication and continued for six weeks. In curative animals, NDEA was
administered for six weeks followed by treatment with the methanolic n-hexane extract of E. scaber (200 and 100 mg·kg−1) for ten days.
RESULTS: E. scaber extract treatment significantly (P ≤ 0.05) reduced the levels of AST, ALT, and MDA in both experimental
groups. The extract also enhanced the antioxidant enzyme and protein levels in rats intoxicated with NDEA. Treatment with the extract
dose dependently protected the liver from NDEA-induced hepatotoxicity with normal hepatocytes and uniform sinusoids, but in some
areas showed degenerating hepatic cells in both treatment groups. CONCLUSION: E. scaber methanolic extract dose dependently
prevented and reversed the hepatotoxicity induced by NDEA in both experimental models.
[KEY WORDS] Elephantopus scaber; Asteraceae; N-nitrosodiethylamine; Hepatotoxicity; Root extract
[CLC Number] R965 [Document code] A [Article ID] 1672-3651(2013)04-0362-09

1 Introduction
N-Nitrosodiethylamine is a dialkyl nitrosoamine consi-
dered to be a potent hepatocarcinogen-producing hepato-
cellular carcinoma (HCC) after repeated administration in
experimental animals [1-4]. The presence of nitroso com-
pounds, like N-nitrosodiethylamine, N-nitrosodimethylamine,
N-nitrosopyrrolidine and N-nitrosopiperidine, has been
widely reported in various foodstuffs, such as milk products,
meat products, soft drinks, and alcoholic beverages [5-7].
Moreover, their reported presence in tobacco smoke accounts
for one of the biggest causes for individual exposure to these
nitrosamines [8]. N-Nitrosodiethylamine metabolism results in

[Received on] 20-Dec.-2011
[Research funding] This project was supported by the Kerala State
Council for Science, Technology and Environment (KSCSTE), Ker-
ala, India
[*Corresponding author] M. S. Latha: Dr., Tel.: 91-481-2731035;
Fax: 91-481-2731009; E-mail: mslathasbs@yahoo.com
These authors have no conflict of interest to declare.
the formation of reactive oxygen species (ROS) leading to
oxidative stress and cellular injury, which may be one of the
key factors in the etiology of cancer. Development of HCC is
known to be triggered by factors that lead to chronic hepatic
injury and deregulation of the normal process of wound
healing, which promotes the persistent stimulation of profi-
brotic and proangiogenic processes that lead to significant
structural changes in the liver, and functional changes in he-
patic physiology [9-11]. Several plant drugs have been evalu-
ated for their potential as liver protectants against
N-nitrosodiethylamine-induced hepatocarcinogenesis in ex-
perimental models [2-4, 12-14].
Elephantopus scaber L. (Asteraceae) is a small herb,
commonly known as elephant’s foot. In Ayurvedic medicine,
a mixture of E. scaber with other herbs is used to treat Vatika
granthi (minor neoplasms). Also, the leaves of E. scaber,
Ficus glomerata, and Tectona grandis are used together with
a honey-mixed fine paste of selected herbs to treat Pittaja
arbuda (major neoplasms) [15]. It is reported that the root
juice of the plant (two teaspoons three times per day) has
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2013 年 7 月 第 11 卷 第 4 期 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 363

been consumed to overcome liver troubles[16-17]. The metha-
nolic extract from the leaves and roots is reported as an effec-
tive hypoglycemic agent against streptozotocin-induced dia-
betic rats [18]. Furthermore, the methanolic extract exerted
high inhibitory effect on the nitric oxide (NO) production in
lipopolysaccharide (LPS)-stimulated RAW264.7 macro-
phages [19]. Teng-Khia-U (E. scaber, E. mollis, and Pseu-
doelephantopus spicatus) is a Taiwan traditional medicine
formulated for treating nephritis, edema, dampness, chest
pain, fever/cough or pneumonia, and scabies/arthralgia [20].
Studies have showed that Teng-Khia-U possesses
anti-inflammatory and hepatoprotective activity. The aqueous
extract from the whole plant at a dose of 300 mg·kg−1 sig-
nificantly inhibited the development of pad swelling in an
acute experimental arthritis model, while a higher dose of the
extract at 500 mg·kg−1 was required to inhibit the develop-
ment of chronic joint swelling in rats [21]. The hepatoprotec-
tive effect of the E. scaber extract was studied in rats on
D-galactosamine, acetaminophen, and carbon tetrachloride
induced hepatic damage [22-23]. Acute toxicity studies revealed
the non-toxic nature of the crude extract of E. scaber [18]. Fur-
ther, the median lethal dose (LD50) of E. scaber extract was
212 3.24 mg·kg−1, with a 95% confidence limit of 1 (742.86
mg·kg−1)~2 (503.62 mg·kg−1), which means that E. scaber
showed relatively low acute toxicity and high safety [24].
Guaianolide glucosides that include four sesquiterpene lactones
are reported in E. scaber [25]. Two novel germacranolide ses-
quiterpene lactones, namely 17, 19-dihydrodeoxyelephantopin
and iso-17, 19-dihydrodeoxyelephantopin with marked
anti-tumor activity have also been isolated from E. scaber [20].
A published report establishes the presence of lupeol (18%),
stigmasterol (8.25%) and 11, 13-dihydrodeoxyelephantopin
(0.03%) in the methanolic extract of E. scaber [26]. The present
investigation was aimed at studying the dose response effect of
E. scaber methanolic extract in preventive and curative treat-
ments on NDEA-induced hepatotoxicity and oxidative stress in
albino rats.
2 Materials and Methods
2.1 Animals
Female Wistar rats weighing between 130–160 g were
used in this study. The rats had free access to food and water.
Animal studies were conducted according to Institute Animal
Ethics Committee (IAEC) regulations approved by the
Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA) (Reg. No. B 2442009/1)
and conducted humanely. The rats received standard pellet
diet and water ad libitum. The animals were maintained at a
controlled temperature of 26–28 ºC with a 12 h light: 12 h
dark cycle.
2.2 Chemicals
N-Nitrosodiethylamine (NDEA) and silymarin were
purchased from Sigma Chemical Co., St. Louis, MO., USA.
Assay kits for aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) were purchased from Agappe, India.
Hematoxylin and eosin were procured from Nice Chemicals,
Kochi, India. Methanol and Tween-80 was obtained from
Merck, Mumbai, India. All the other chemicals used were
also of highest purity grade.
2.3 Plant extraction
Plant materials were collected from their natural habitat
in Kerala, India and authenticated by the second author. A
voucher specimen (SBSBRL.03) is maintained in the
Biochemistry Research laboratory, School of Biosciences, M
G University. Roots of the plant were separated, cleaned,
chopped, shade-dried and powdered. Ten g of the dried
powder was Soxhlet-extracted with methanol (400 mL). The
Soxhlet extraction was continued until a drop of the solvent
from the siphon tube when evaporated does not leave a
residue. Then the extract was collected and the solvent
evaporated under vacuum in a rotary evaporator. This step
was repeated with a new set of dried powder and solvent until
the required quantity was achieved. The yield of the extract
was 16.6%. Extract was suspended in 5% Tween 80 to
respective dosages and stored at –20 ºC.
2.4 N-Nitrosodiethylamine-induced hepatotoxicity
2.4.1 Preventive effect of the extract
Thirty rats were divided into five groups, group I was
normal control; group II was the NDEA control; group III
was the NDEA and standard drug, silymarin (70 mg·kg−1)
treated rats; group IV was NDEA and E. scaber methanolic
extract (200 mg·kg−1) treated rats; and group V was the
NDEA and E. scaber methanolic extract (100 mg·kg−1)
treated rats. Hepatotoxicity induced by N-nitrosodiethylamine
(NDEA) administration was studied for a period of six weeks.
All of the groups, except Group I, received 0.02% NDEA in
water five days per week (per oral) for six weeks [27]. Oral
treatments with E. scaber methanolic extract at 200 mg·kg−1
and 100 mg·kg−1 doses were started for group IV and V
animals, respectively. Simultaneously, the standard drug
silymarin (70 mg·kg−1) treatment was conducted in group III
animals. Treatment with E. scaber extract and silymarin was
started one week before the onset of NDEA administration
and continued up to six weeks. The rats were sacrificed 48 h
after the last dose of NDEA administration (Fig. 1a).
2.4.2 Curative effect of the extract
Thirty rats were divided into five groups, group I was
normal control, group II was NDEA control, group III was
NDEA and standard drug silymarin (70 mg·kg−1) treated rats,
group IV was NDEA and E. scaber methanolic extract
(200 mg·kg−1) treated rats, and group V was NDEA and E.
scaber methanolic extract (100 mg·kg−1) treated rats. Groups
II–V animals received 0.02% NDEA in water five days per
week (per oral) for six weeks. After six weeks of exposure to
NDEA, group IV and V rats received E. scaber methanolic
extract orally (200 and 100 mg·kg−1, respectively, suspended
in 5% Tween 80) daily for 10 days. Also, group III rats were
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364 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 2013 年 7 月 第 11 卷 第 4 期



Fig. 1 Schematic representation of experiments. a. Preventive group. Rats were administered with NDEA for six weeks and ongoing
treatment with E. scaber extract at 200 and 100 mg·kg−1 and silymarin. b. Curative group. Animals administered with NDEA for six
weeks and then treated with E. scaber extract at 200 and 100 mg·kg−1 and silymarin for 10 days. denote NDEA administration. Dotted
regions denote treatment with E. scaber extracts and Silymarin

treated with standard drug, silymarin (70 mg·kg−1) daily for
ten days. Animals were sacrificed 48 h after the last dose of
the extract administration (Fig. 1b).
2.5 Biochemical assays
Sera from different groups were separated by centrifuga-
tion at 2 000 r·min−1 at 4 ºC for 15 min. Dissected livers were
excised, and washed thoroughly in ice-cold saline to remove
the blood. Ten percent of homogenate was prepared in 0.1
mol·L−1 Tris HCl buffer (pH 7.4). The homogenate was centri-
fuged at 3 000 r·min−1 for 20 min at 4 ºC and the supernatant
was used for the estimation of reduced glutathione (GSH),
glutathione-S-transferase (GST), glutathione peroxidase (GPx),
catalase, lipid peroxidation (Thiobarbituric Acid Reactive Sub-
stances–TBARS), and total protein.
2.5.1 Liver function tests
AST (EC 2.6.1.1) [28] and ALT (EC 2.6.1.2) [29] levels
were estimated at 340 nm by semi-auto analyzer.
2.5.2 Estimation of reduced glutathione (GSH)
Reduced glutathione (GSH) was estimated by its reac-
tion with dithio-bis-2-nitrobenzoic acid (DTNB) which gives
a yellow colored complex with an absorption maximum at
412 nm [30].
2.5.3 Estimation of glutathione-S-transferase (GST)
GST (EC 2.5.1.18) activity was determined from the rate
of increase in conjugate formation between reduced gluta-
thione and CDNB [31].
2.5.4 Estimation of glutathione peroxidase (GPx)
GPx (EC 1.11.1.9) activity was determined by measuring
the decrease in GSH content after incubating the sample in
the presence of H2O2 and NaN3 [32].
2.5.5 Estimation of catalase
Tissue catalase (EC 1.11.1.6) activity was determined
from the rate of decomposition of H2O2 [33].
2.5.6 Estimation of malondialdehyde (MDA) formation
Lipid peroxidation in liver was measured by the forma-
tion of malondialdehyde (MDA) and measured by the thio-
barbituric acid reactive substance (TBARS) method [34].
2.5.7 Estimation of protein
Protein content in the tissue was determined [35] using
bovine serum albumin (BSA) as the standard.
2.6 Histopathological studies
Dissected livers were cut into small pieces and fixed in
10% neutral buffered formalin for histopathological analysis.
The liver (5–6 mm thick pieces) fixed in buffered formalin
for 12 h was processed for paraffin embedding. Five µm
thick sections of the paraffin embedded liver were stained
with hematoxylin and eosin and examined for histopa-
thological changes under the microscope (Motic AE 21,
Germany). The microphotographs were taken using a Moti-
cam 1000 camera at an original magnification of 100 ×.
2.7 Statistical analysis
Results were expressed as x ± s, and all statistical
comparisons were made by means of one way ANOVA test
followed by Tukey post hoc analysis. P-values less than or
equal to 0.05 were considered significant.
3 Results
3.1 Body weight
The body weight of the NDEA-treated animals de-
clined significantly (P ≤ 0.05) by the end of the 6th week
of exposure when compared with the normal rats. Ongo-
ing treatment with E. scaber extract prevented the de-
cline of animal body weight remarkably in rats exposed
to NDEA for six weeks (Fig. 2a). In post-treatment
animals, E. scaber extract improved the body weight
during a period of ten days after intoxication with
NDEA for six weeks (Fig. 2b). The standard drug sily-
marin (70 mg·kg−1) also showed a remarkable gain in
body weight from NDEA-induced liver damage in both
treatments.
3.2 Liver function
AST and ALT activities in the serum of NDEA adminis-
tered rats were elevated in contrast to the normal rats. In the
preventive treatment, E. scaber remarkably prevented the rise
of AST and ALT levels, and in the curative treatment animals,
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2013 年 7 月 第 11 卷 第 4 期 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 365




Fig. 2 Graph showing the body weight pattern of rats adminis-
tered with NDEA and E. scaber extract in preventive (a) and
curative (b) treatments. The mean of each of the groups is repre-
sented, with error bar indicating the standard deviation. Group
I- Normal control, Group II-NDEA control, Group III- Silymarin
and NDEA treated, Group IV-E. scaber 200 mg·kg−1 and NDEA
treated, and Group V-E. scaber 100 mg·kg−1 and NDEA treated
animals. Significant difference (0.05) observed between normal
control and NDEA control and between NDEA control and
treated groups during final body weight of animals

it reversed the toxic effect of NDEA as evidenced by the
normal levels of AST and ALT displayed. The results were
comparable to standard drug silymarin.
3.2.1 Estimation of aspartate aminotransferase (AST)
Administration of E. scaber methanolic extract (200
mg·kg−1) exerted its protection by 81% and 94% in the pre-
ventive and curative groups, respectively. E. scaber methano-
lic extract (100 mg·kg−1) treatment exerted its protection by
61% and 86% in preventive (Fig. 3a) and curative (Fig. 4a)
groups, respectively. In the preventive and curative treat-
ments, silymarin (70 mg·kg−1) exerted its protection by 62%
and 70%, respectively when compared to the normal control.
3.2.2 Estimation of alanine aminotransferase (ALT)
E. scaber methanolic extract (200 mg·kg−1) treatment ex-
erted its protection by 85% and 90% in preventive and cura-
tive groups, respectively compared to the normal control.
Administration of E. scaber methanolic extract (100 mg·kg−1)
exerted its protection by 62% and 78% in the preventive
(Fig. 3b) and curative (Fig. 4b) groups, respectively. The
standard drug silymarin (70 mg·kg−1) exerted its protection by
65% and 82% in the preventive and curative groups, respec-
tively when compared to the normal control.
3.3 Estimation of reduced glutathione
Reduced glutathione (GSH) levels were lowered signifi-
cantly (P ≤ 0.05) in rats exposed to NDEA compared to nor-
mal control. Percent protection induced by E. scaber metha-
nolic extract at a dose of 200, 100 mg·kg−1 and silymarin (70
mg·kg−1) treatment caused an increase in the levels of GSH by
58, 36, and 50%, respectively in the preventive groups (Table 1).
In the curative groups, the percent protection yielded was 71, 51,
and 57%, respectively for doses of 200, 100 mg·kg−1 and sily-
marin (70 mg·kg−1) when compared to the normal control
(Table 2).
3.4 Estimation of GST
Rats administered NDEA alone were found to have signifi-
cantly (P ≤ 0.05) lowered levels of GST. In the preventive
groups, treatment with 200 and 100 mg·kg−1 methanolic extract
exhibited a significant increase i.e., 54% and 39%, respectively
in the GST levels compared to untreated rats (Table 1). Sily-
marin treatment also prevented the lowering of GST level by
46%. In the curative groups, treatment with 200 and 100
mg·kg−1 exerted a significant increase of 82 and 63%, respec-
tively in GST levels (Table 2). Silymarin exhibited a 70%
increase in GST levels when compared to normal control.

Fig. 3 Effect of E. scaber methanolic extract on (a) aspartate aminotransferase (AST) levels and (b) alanine aminotransferase (ALT) in the
serum of preventive treatment groups. *P ≤ 0.05 vs normal control, †P ≤ 0.05 vs NDEA control

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366 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 2013 年 7 月 第 11 卷 第 4 期


Fig. 4 Effect of E. scaber methanolic extract on (a) aspartate aminotransferase (AST) and (b) alanine aminotransferase (ALT) in the se-
rum of curative treatment groups. *P ≤ 0.05 versus normal control. †P ≤ 0.05 versus NDEA control

3.5 Estimation of GPx
In NDEA-exposed animals, a significant (P ≤ 0.05)
lowering of GPx level compared to normal control was
observed. E. scaber treatment at 200 mg·kg−1 increased the
GPx level by 77% and 84% in the preventive and curative
groups, respectively. Administration of E. scaber metha-
nolic extract (100 mg·kg−1) yielded its protection level by
59% and 77% in the preventive and curative groups, re-
spectively. Silymarin (70 mg·kg−1) treated rats exerted its
protection by 67% and 77% in the preventive and curative
groups, respectively when compared to the normal control
(Tables 1 & 2).
3.6 Estimation of catalase
Catalase levels were lowered significantly (P ≤ 0.05) in
rats exposed to NDEA compared to the normal control. In the
pretreatment groups, E. scaber methanolic extract at 200 and
100 mg·kg−1 exhibited significant (P ≤ 0.05) protection by
69% and 39%, respectively in catalase levels (Table 1).
Silymarin-treated rats also prevented the lowering of catalase
by 72%. In post-treatment groups, E. scaber methanolic ex-
tract at 200 and 100 mg·kg−1 exhibited significant (P ≤ 0.05)
reversal by 79 and 55%, respectively in catalase levels (Table
2). Silymarin treated rats also reversed the decline of catalase
by 85% (Table 2).
Table 1 Effect of E. scaber methanolic extracts and Silymarin on different parameters in NDEA exposed rats (preventive trea-
tment groups) ( x ± s, n = 3)
Treatment groups GSH (mg/100 mg tissue)
GPx
(min/mg protein)
GST
(enzyme unit/mL)
Catalase
(millimoles/min/mg protein)
TBARS
(mmol/100 mg tissue)
Protein
(mg/mg tissue)
Normal control 1.26 ± 0.07 2.53 ± 0.69 0.73 ± 0.11 1.03 ± 0.19 1.33 ±0.24 46.54 ± 9.18
NDEA control 0.59 ± 0.08* 1.25 ± 0.39* 0.35 ± 0.05* 0.45 ± 0.14* 4.26 ±0.95* 26.83 ± 6.38*
Silymarin (70 mg·kg−1)
+ NDEA 0.87 ± 0.05
† 2.11 ± 0.70† 0.52 ± 0.06† 0.74 ± 0.10† 2.19 ±0.09† 41.03 ± 8.06†
E. scaber (200 mg·kg−1)
+ NDEA 0.97 ± 0.10
† 2.23 ± 0.58† 0.56 ± 0.06† 0.85 ± 0.10† 2.03 ±0.10† 42.23 ± 9.35†
E. scaber (100 mg·kg−1)
+ NDEA 0.83 ± 0.09
† 2.01 ± 0.63† 0.50 ± 0.07† 0.68 ± 0.08 2.25 ± 0.13† 40.03 ± 9.11†
* P ≤ 0.05 vs normal control, †P ≤ 0.05 vs NDEA control

Table 2 Effect of E. scaber methanolic extracts and Silymarin on different parameters in NDEA exposed rats (curative treat-
ment groups) ( x ± s, n = 3)
Treatment groups GSH (mg/100 mg tissue)
GPx
(min/mg protein)
GST
(enzyme unit/mL)
Catalase
(millimoles/min/mg protein)
TBARS
(mmol/100 mg tissue)
Protein
(mg/ mg tissue)
Normal control 1.27 ± 0.15 1.68 ± 0.41 1.07 ± 0.22 1.05 ± 0.21 1.20 ± 0.26 56.63 ± 4.99
NDEA control 0.71 ± 0.14* 0.94 ± 0.17* 0.67 ± 0.06* 0.62 ± 0.08* 3.15 ± 0.74* 41.5 ± 1.86*
Silymarin (70 mg·kg−1)
+ NDEA 1.03 ± 0.05
† 1.51 ± 0.42† 0.95 ± 0.14† 0.88 ± 0.12† 1.56 ± 0.27† 54.43 ± 4.99†
E. scaber (200 mg·kg−1)
+ NDEA 1.11 ± 0.12
† 1.57 ± 0.37† 1.00 ± 0.18† 0.96 ± 0.18† 1.42 ± 0.22† 55.50 ± 5.13†
E. scaber (100 mg·kg−1)
+ NDEA 1.00 ± 0.02
† 1.51 ± 0.38† 0.93 ± 0.13† 0.86 ± 0.13† 1.58 ± 0.26† 53.16 ± 4.47†
*P ≤ 0.05 vs normal control, †P ≤ 0.05 vs NDEA control
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2013 年 7 月 第 11 卷 第 4 期 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 367

3.7 Estimation of malondialdehyde
A significant (P ≤ 0.05) increase in tissue MDA level
was observed in the NDEA-treated rats. In the preventive
treatment, NDEA-induced elevation of tissue MDA concen-
trations, was lowered by 76% and 69% in rats treated with E.
scaber extract at a dose of 200 and 100 mg·kg−1, respectively
(Table 1). This effect was comparable to that of silymarin
(71%). In the curative treatment, E. scaber methanolic extract
at 200 and 100 mg·kg−1 exerted its protection by 88% and
80%, respectively (Table 2). Silymarin (70 mg·kg−1) treated
rats exerted its protection by 81%.
3.8 Estimation of protein
Protein levels were lowered significantly (P ≤ 0.05) in
rats exposed to NDEA. E. scaber methanolic extract (200
mg·kg−1) treatment caused an increase in the levels of protein
by 78% and 93% in the preventive and curative groups. E.
scaber methanolic extract (100 mg·kg−1 treatment caused an
increase in the level of protein by 67% and 77% in the pre-
ventive and curative groups, respectively. Silymarin (70
mg·kg−1) treated rats caused an increase in the level of protein
by 72% and 85% in the preventive and curative groups, re-
spectively compared to the normal control (Tables 1 & 2).
3.9 Histopathological studies
Histopathological analysis of normal rat liver showed
uniformly arranged cell plates with oval hepatocytes of the
same size. In both experimental groups, NDEA administered
rat liver showed irregularly formed cell plates with more
damage towards the central vein region. Further, scattered
masses of necrotic tissues were detected in most of the areas.
Enlarged nuclei were also spotted in NDEA treated rats. Rats
in preventive groups administered with E. scaber methanolic
extract at a dose of 200 and 100 mg·kg−1 showed normal
hepatocytes with uniform sinusoids, but in some areas
degenerating hepatic cells were detected (Fig. 5). In the
curative groups, E. scaber extract treatment completely
protected the liver from NDEA-induced hepatotoxicity as
evidenced by normal hepatocytes with uniform cell plates
(Fig. 6).



Fig. 5 Histopathological features of liver in the preventive treatment group (A) Normal rat liver (B) NDEA control (C) NDEA + Silymarin
(70 mg·kg−1) treated rats (D) NDEA + E. scaber methanolic extract (200 mg·kg−1) (E) NDEA + E. scaber methanolic extract (100 mg·kg−1)
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368 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 2013 年 7 月 第 11 卷 第 4 期



Fig. 6 Histopathological features of liver in the curative treatment group. (A) Normal rat liver (B) NDEA control (C) NDEA + Silymarin (70
mg·kg−1) treated rats (D) NDEA + E. scaber methanolic extract (200 mg·kg−1) (E) NDEA + E. scaber methanolic extract (100 mg·kg−1)

The results were comparable with the standard hepatoprotec-
tive drug, silymarin at a dose of 70 mg·kg−1.
4 Discussion and Conclusion
Weight loss is one of the major symptoms of hepatotox-
icity [36]. Published reports indicate that repeated administra-
tion with NDEA reduces the body weight of animals, but
stimulates the body weight by the treatment with plant ex-
tracts [4, 37]. In the present investigation, a severe weight loss
was observed in rats exposed to NDEA in both experimental
groups. It is important to note that the treatment with E. sca-
ber methanolic extract enhanced the body weight in both
experimental groups, indicating its ability to protect the ani-
mals from NDEA-induced toxicity.
Glutathione (GSH) is a key player in reduction processes
in the cell. It also plays a role in the reduction of NTPs to
dNTPs, and in the detoxification of endogenous and exoge-
nous compounds, it serves as a cofactor for various enzymes,
stores and transports cysteine, and may be involved in cell
cycle regulation and thermotolerance [38-39], Lipid peroxida-
tion, initiated in the presence of NDEA, and resulting in the
production of malondialdehyde (MDA), directly produces
oxidative stress [40]. In a previous study, the E. scaber extract
showed medial antioxidant capacity with moderate electron
donating activity and reducing capacity [19]. In the present
study, the levels of GSH, GPx, GST, catalase, MDA, and
protein were improved to normal levels in rats treated with E.
scaber extract in both the preventive and curative groups
which reveals the antioxidant potential of the extract.
Amino transferases (AST and ALT) are the important
cytoplasmic enzymes present in the liver. During chemical
intoxication, the membrane integrity is lost due to necrosis,
and these enzymes move into the circulatory system followed
by elevated levels of aminotransferases in the serum [41]. In-
creased levels of serum AST and ALT was detected in
NDEA-treated rats, and was effectively down-regulated by
both treatments with the E. scaber methanolic extract indi-
cating the protective role of E. scaber in treating
A. Linza, et al. /Chinese Journal of Natural Medicines 2013, 11(4): 362370
2013 年 7 月 第 11 卷 第 4 期 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 369

NDEA-exposed hepatotoxicity. These biochemical restora-
tions may be due to the inhibitory effects of E. scaber extract
on the process of cellular damage in the liver. The phyto-
chemicals identified are more potent in inhibiting free radi-
cals, and this could be protective against the progression of
NDEA-induced hepatotoxicity. Thus protection yielded by
this extract may be due to the combined effects of these
compounds or fractions rather than any single component.
Histopathological observation also supported the protec-
tive role played by E. scaber in NDEA-treated rats, as was
evidenced by the presence of normal hepatocytes and small
emboli of degenerating hepatic cells in both experimental
groups. Thus the results presented in this study indicate that
the hepatotoxicity induced by N-nitrosodiethylamine was
effectively inhibited by the treatment with E. scaber extract
in a concentration dependant manner in both the preventive
and curative models. When compared to the pre-treatment
group, the post-treatment group showed better efficacy
against NDEA-induced liver injury. This might be due to
the self-recovery of the animals during ten days after the
toxin NDEA was administered.
The present study has unveiled the pharmacological ac-
tions of E. scaber against NDEA-induced hepatotoxicity for
the first time. This study has successfully drawn several con-
clusions that determine the hepatoprotective effect of E. sca-
ber. The present study reveals the root extract to have hepa-
toprotective properties against toxic chemicals that cause
chronic liver injury, which seems to validate the plant use in
traditional medicine. The result of the experiment indicates
that E. scaber methanolic extract can normalize the situation
by bringing the AST and ALT levels, as well as the antioxi-
dant levels, near to normal. That the E. scaber methanolic
extract at a dose of 200 mg·kg−1 experiences more protection
than the 100 mg·kg−1 dose confirms the dose response effect
of E. scaber methanolic extract. The present study also con-
cludes that the protective effect of E. scaber was comparable
to the protection yielded by the standard hepatoprotective
drug, silymarin. Treatment with E. scaber extract on patho-
logical conditions in chronic hepatic disorder showed re-
markable protection, and provides a new approach to study
the active principles responsible for chemoprevention in tu-
mor models.
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