Chinese

Journal of
Natural
Chinese Journal of Natural Medicines 2014, 12(7): 04880494 Medicines

Hepatoprotective activity of Gentiana veitchiorum Hemsl.

against carbon tetrachloride -induced hepatotoxicity in mice
ZHANG Zhi-Feng1 , LIU Yuan1 , LU Lu-Yang1 , LUO Pei2*
1
Ethnic Pharmaceutical Institute of Southwest University for Nationalities, Chengdu 610041, China;
2
State Key Laboratory for Quality Research of Chinese Medicines, Macau University of Science and Technology, Avenida Wai
Long, Taipa, Macau, China
Available online 20 July 2014

[ABSTRACT]
AIM: To study the hepatoprotective effect of methanol extract of Gentiana veitchiorum (MGV) against CCl4 -induced oxidative
stress and liver injury in mice.
METHO D: T he acute hepatic model was developed by injection of 20% CCl 4 in mice. ICR mice were divided into six groups, in-
cluding control, CCl4 , CCl4 + silymarin, and CCl4 + M GV (100, 200, and 400 mg kg1 ) groups. Hepatic enzymes including AST, ALT
and ALP levels in serum, and antioxidant enzymes, including SOD, CAT and GPX activity in liver tissue, were determined. Histo-
pathological examination and Western blot analysis were performed.
RESULTS: Oral administration of MGV at 200 and 400 mg kg1 for 15 days dose-dependently inhibited the serum elevations of
AST, ALT, and ALP, and recovered the reduct ion of SOD, CAT, and GPX in liver tissue. Hematoxylin and eosin staining examina-
tion performed in liver tissues suggested that MGV treatment ameliorated histopathological changes in CCl 4 -induced mice. West-
ern blotting analysis implied that MGV increased HO-1 expression and recovered T NF- alternation.
CO NCLUSIO N: G. veitchiorum can protect the liver against CCl4 -induced damage in mice, and this hepatoprotective effect was
due at least in part to its ability through scavenging CCl 4 -associated free radical activities. The study provided in vivo evidence that
G. veitchiorum can be used as a safe, cheap, and effective agent to reduce acute liver damage, supporting its folk medicine use.

[KEY WO RDS] Gentiana veitchiorum ; Hepatoprotective effect; Antioxidant enzymes; Western blot
[CLC Number] R965 [Document code] A [Article ID] 2095-6975(2014)07-0488-07

Liver dysfunction is caused mostly due to exposure to toxic

Introduction
The liver is a vital organ of the human body . It performs
the normal metabolic homeostasis of the body, as well as
biotransformation, detoxification, and excretion of many en-
dogenous and exogenous compounds, including pharmaceu-
tical and environmental chemicals. Hepatic damage is usually
associated with the disorder of its detoxifying functions [1-2].
[Re ceive d on] 29-Jan.-2013
[Research funding] This project was supported by the National Key
Technology R&D Program in the 12th Five year Plan of China (No.
2012BAI27B07), and the Macau Science and Technology Develop-
ment Fund (No. 073/2011/A3).
[*Corresponding author] LUO Pei: Assistant Prof., Tel:
86-853-63589159, E-mail: Pluo@must.edu.mo
T hese authors have no conflict of interest to declare.
Copyright 2014, China Pharmaceutical University.
Published by Elsevier B.V. All rights reserved
chemicals, and certain drugs and environmental pollutants,
and it has been increasing for the last few decades [3] . Efforts
have been made to search for effective hepatoprotective
agents. However, no effective hepatoprotective therapies are
available until now. Plant-based drugs play a major role in the
treatment of hepatic disorders. In the absence of reliable
hepatoprotective drugs in modern medicine, a number of me-
dicinal plants and their formulations are used to cure hepatic
disorders in traditional medicine [4].
Gentiana veitchiorum Hemsl., known as bangjianwen-
bao in Tibetan, belongs to the family Gentianaceae. It is used
in traditional Tibetan medicine for the treatment of liver jaun-
dice with damp-heat pathogen, as well as for headache and
chronic pharyngitis [5] . People also crushed this plant for tea
or soup cooking, alone, or sometimes with other traditional
plants. Phytochemical research has revealed that G. veitchi-
orum contains organic acids, gentiopicroside, isoorientin
3'-methyl ether, isovitexin, isoorientin, and many other con-

488
 ZHANG Zhi-Feng, et al. / Chin J Nat Med, 2014, 12(7): 488494
stituents. Among thes e compounds, gentiopicroside is one of
the most important secondary metabolites in gentian [6] . In
our previous research, quantitative detection of the genti-
opicroside of G. veitchiorum was performed by Liu [7]. Gen-
tiopicroside also offers remarkable hepatoprotection against
damage induced by d-GalN/LP S related with its an-
ti-apoptotic activities [8]. However, no scientific data in vivo
were reported to support the hepatoprotective effect of G.
veitchiorum. Based on the divers ified ethnopharmacological
applications in liver diseases of this medicine in Tibet, the
present study was conducted to validate the hep atoprotec-
tive activity of G. veitchiorum using the widely used hepa-
totoxin (CCl 4)-induced liver damage model in mice. Enzy-
matic alterations, histopathological changes, HO-1, and
TNF- expressions were evaluated, respectively.
Material and Methods
Plant materials
The sample of Gentiana veitchiorum (Fig. 1) was col-
lected from Kangding County, Sichuan Province, China. The
identity of the plant material was authenticated by Prof.
ZHANG Hao (West China School of Pharmacy, Sichuan
University, Chengdu, China). Voucher specimens (No.
LGV0074) were deposited at the Herbarium Center of West
China School of Pharmacy, Sichuan University.
Fig. 1 Photographs of G. veitchiorum (A: environment of G.
veitchiorum; B: dry sample of G. veitchiorum)
Preparation of the methanol extract of Gentiana veitchiorum
Hemsl. (MGV)
The fresh herb of G. veitchiorum was dried in the shade
for three days, in an oven overnight, and then cut into small
pieces for extraction. The dry herb (500 g) was pulverized
into coarse powder and then refluxed with methanol (1 500
mL) at 70 C three times, 2 h for each reflux. The methanol
extract solutions were pooled and evaporated under reduced
pressure and the concentrate was dried to powder, the G. ve-
itchiorum methanol extract (M GV) was then stored at 20 C
for further use. The yield of M GV was 14.18% W/W. The
constituents of M GV were standardized using the HPLC/
DAD fingerprint (Fig. 2). Experiments were performed on an
Agilent HPLC 1200 system (Agilent Corp., CA, USA) con-
sisting of a vacuum degasser, autosampler, and DAD detector.
For chromatographic fingerprint analysis, a Zorbax SBC
489
column (5 m, 150 mm 4.6 mm i.d.) with a guard column
(5 m, 7.5 mm 4.6 mm i.d.) was used. The mobile phase
consisted of methanol (M ) and water (V/V, W) using a gradi-
ent program of 15%20% M in 05 min, 20%28% M in
530 min, 28%34% M in 3033 min, 34%40% M in 3345
min, 40%15% M in 4550 min. The flow rate was 1.0
mL min1 and column temperature was maintained at 35 C.
The detection wavelength was set at 270 nm for acquiring
chromatograms. The content of gentiopicroside in M GV was
11.2%. This chemically consistent extract was suspended in
0.5% CM C solution and stored at 4 C for use in the follow-
ing animal experiments, in which the dosage was expressed as
the dry weight of the extract.
Fig. 2 HPLC chromatogram of (A) gentiopicroside standard
and (B) HPLC fingerprint of MGV. 1, gentiopicroside
Animals
ICR mice weighing (22 2) g of either sex (half male
and half female) were obtained from the Animal House,
Pharmacy Discipline, Sichuan University (Chengdu, P.R.
China). A minimum of ten animals was used in each group.
The animals were allowed to stay in the animal room main-
tained at (25 1) C with a 12 h light/dark cycle for 3 days
before any experiment to acclimate with the environment and
fed with a rodent standard diet with free access water ad libi-
tum. All experimental protocols were in accordance with the
Principles of Laboratory Animal Care and Use in Research
stipulated by the M inistry of Health of China and approved by
the local Animal Ethics Committees of the Faculty of M edi-
cine, Sichuan University.
Drugs and chemicals
Assay kits for ALT (Ref No.0930-101), AST (Ref No.
 ZHANG Zhi-Feng, et al. / Chin J Nat Med, 2014, 12(7): 488494
0920-101) and ALP (Ref No. 0900-151) were purchased from
Stanbio Laboratory (Boerne, TX, USA). SOD assay kit was
purchased from Dojindo Laboratories (S311, Kumamoto,
Japan). CCl4 (Product No. 319961-500M L) and silymarin
(Product No. S0292 -50G) were purchased from Sigma
Chemical Co. (St. Louis, M O, USA). A reference standard
of gentiopicros ide (purity > 98%) was purchased from the
National Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China). All other chemicals
were of analytical grade and obtained from Chengdu Che m-
icals Co. (Sichuan, China).
CCl4-induced acute liver injury model in mice and treatment
with MGV
The liver injury model w as established according to the
method of Yadav and D ixit [9] with some modifications.
Animals were randomly divided into six groups with ten
mice in each group. All animals except the first group were
intraperitoneally (i.p.) injected with 20% CCl4 in corn oil
(10 mL kg1 body weight) every 72 h, twice, until the liver
injury was confirmed in each animal by significantly el e-
vated serum levels of A ST, ALT, and ALP. Then, the
treatments were started. Animals of the first group served as
normal control and were given only the same volume of
corn oil. Animals of the second group served as negative
control and were orally given vehicle (0.5% CM C solution,
10 mL kg1 body weight). Animals of the third group served
as positive control and received silymarin at a dose of 25
mg mL kg1 by oral administration for 15 days. The fourth,
fifth, and sixth groups were given M GV by oral administra-
tion in the doses of 100, 200, and 400 mg mL kg 1 body
weight, respectively.
After 15 days of the test drug treatment, blood samples
were collected from the retro-orbital sinus. The serum sam-
ples were separated by centrifugation at 2 000 g for 5
minutes at 4 C and used to determine the levels of ALT,
AST, and A LP. Then, the animals were anesthetized with
diethyl ether and sacrificed by decapitation. The liver was
perfused with 1.15% ice-cold KCl to remove all red blood
cells completely. Then it was carefully dissected, cleaned of
extraneous tissue and minced in 10% ( W/V) ice-cold 0.1
mol L1 phosphate buffer (pH 7.4). Liver tissue from the left
liver lobe was cut into two pieces, one w as used for hist o-
pathological examination and the other was for the prepara-
tion of liver tissue homogenate.
Measurements of AST, ALT and ALP levels in serum
Hepatic enzymes, such as AST, ALT, and ALP, are usual-
ly used as the biochemical markers of hepatic damage. The
separated serum samples were employed for the measurements
of ALT, AST [10], and ALP [11] using assay kits. The measure-
ments were conducted on a semi-automatic biochemistry ana-
lyzer (MD-150 PLUS, Sanhe M edical Equipment, China) ac-
cording to the manufacturers instructions.
Assessment of antioxidant enzymes activity in liver
A sample (300 mg) of fresh liver tissue was weighed and
490
homogenized in 4 C 0.9% sodium chloride (10% W/V) using
a Teflon homogenizer. The homogenate was centrifuged at 4
000 g for 20 min to remove cell debris, unbroken cells, nuclei,
erythrocytes, and mitochondria. The resultant supernatant was
subjected to the measurement of SOD, CAT, and GPX. SOD
activity was determined by the method described by M isra
and Fridovich [12]. CAT activity was determined by the
method described by Beers and Sizer [13] based on the de-
composition rate of hydrogen peroxide (H 2O2). GPX activity
was determined by measuring the decrease of GSH content
after incubating the sample in the presence of H 2O2 and NaN 3
[14]
. Protein content in the tissue was determined according to
Lowrys method [15].
Histopathological examination
Liver tissue for histopathological analysis was fixed in
10% buffered formalin solution (pH 7.0) for at least 24 h,
subsequently dehydrated and embedded in wax. The embed-
ded tissue was cut into 3 m thick sections in a M R-3 rotary
microtome (Becthai Bangkok Equipment & Chemical Co.,
Thailand). The sections were stained with haematoxylin-eosin
(HE). The histopathological characters, including fatty
changes, necrosis, ballooning degeneration, and cell infiltra-
tion, were observed and recorded under an Olympus BX41
micro-imaging system at original magnification of 10 10.
Western blotting analysis of TNF- and HO-1
In brief, frozen liver tissues were homogenized in
ice-cold 1x RIPA lysis buffer (Cell Signaling Technologies,
USA) for 3 min at 4 C. The homogenate was immediately
centrifuged at 12 000 r min1 (M icrofuge 22R centrifuge,
Beckman Conlter, USA) for 15 min at 4 C, the supernatant
was gently collected and stored at 80 C until use. The con-
tent of total protein in the supernatants was determined by
using a Bio-Rad kit (Bio-Rad Laboratories, Hercules, CA,
USA). Equal protein extract (70 g) were separated on a 12%
sodium dodecyl sulfate (SDS) polyacrylamide gel and trans-
ferred to PVDF membrane. Blots were then washed with H 2O,
blocked with 5% skimmed milk powder in TBST [10 mmol L1
Tris-HCl (pH 7.6), 150 mmol 1
L NaCl, 0.05% Tween-20] for 1 h
and immunoblotted using against TNF-, HO-1 and b-actin pro-
teins (1 : 1 000 for overnight at 4 C) antibody (Cell Signaling
Technology, or Santa Cruz Biotechnology, USA). Then the
membrane was washed and primary antibodies were detected
with goat anti-rabbit and/or mouse-IgG conjugated to horseradish
peroxidase detected by an enhanced chemiluminescence proce-
dure (Amersham, Buckinghamshire, England). Band intensities
were quantified using a densitometer analysis system (Quantity
One software, Bio-Red).
Statistical analysis
All data were expressed as x s. Results were statisti-
cally analyzed by one-way ANOVA followed by post hoc
analysis with Turkeys multiple comparison tests using SPSS
software (Students version 11.5). P < 0.05 was considered
statistically significant while P < 0.01 was considered ex-
tremely significant.
 ZHANG Zhi-Feng, et al. / Chin J Nat Med, 2014, 12(7): 488494

Results CCl4-induced liver injury model mice have been shown in

Effects of MGV on levels of ALT, AST and ALP in serum
Liver injury was successfully induced in mice by re-
peated administration of CCl4 , shown by the signif icant
elevation of serum level of AST (302.7 52.4) U L1, ALT
1
(321.2 63.7) U L , and ALP (122.7 19.8) IU L1 in
mice when compared w ith the control group (117.516.4)
U L1, (50.4 10.2) UL1, (21.3 2.4) IU
L1 , respectively,
P < 0.01). The effects of M GV at three different doses on
t h e s e r u m l ev e l s of A S T , A LT , an d A L P i n
Table 1. It was demonstrated that oral administration of
M GV at 200 and 400 mg kg1 significantly and dose-depen-
dently inhibited the elevation of serum levels of AST, ALT,
and ALP when compared with the negative control group (P
< 0.05). M GV at 400 mg kg1 showed similar potency of
hepatoprotective effect to the positive control silymarin.
However, the lower dose of M GV (100 mgkg1) failed to
demonstrate a significant hepatoprotective effect in terms of
suppressing the elevation of serum levels of ALT, AST, and
ALP level induced by CCl4 in mice.

Table 1 Effe cts of methanol extract of G. veitchiorum on liver markers levels in CCl4 -induced mice ( x s, n = 10)
Groups T reatment L 1 )
AST /(U L 1 )
ALT /(U L 1 )
ALP/(IU
Normal control Vehicle 117.5 16.4 50.4 10.2 21.3 2.4
^^ ^^
Negative control Vehicle 302.7 52.4 321.2 63.7 122.7 19.8 ^^
Silymarin
kg1 )
Positive control/(mg 156.3 22.9 ** 104.4 17.3 ** 48.6 7.5 **
25
kg1 )
MGV low dose/(mg 100 210.6 47.3 264.8 33.6 94.8 18.7
1 * *
MGV medium dose/(mg
kg ) 200 190.7 11.5 208.1 41.5 75.4 6.5 *
1 ** **
MGV high dose/(mg
kg ) 400 165.2 20.4 136.8 13.3 60.9 2.4 **
^
P < 0.05; ^^P < 0.01 vs normal control group; * P < 0.05; ** P < 0.01 vs negative control group

Effects of MGV on activities of SOD, CAT and GPX in liver congestion, cells infiltration, loss of cellular boundaries and
tissues ballooning degeneration, loss of architecture and fatty chang-
The effects of M GV at three different doses on liver an- es (Fig. 3B), obviously different from those observed in nor-
tioxidant enzymes (SOD, CAT, and GPX) in CCl4-induced mal control group. However, treatments with MGV at the
liver injury model are shown in Table 2. In CCl4-induced doses of 200 and 400 mg kg1, and of silymarin for 15 days
liver injury control group, compared with normal control
group, SOD dropped from (2.18 0.06) to (1.73 0.07) ( P <
0.05), CAT decreased from (49.6 2.5) to (20.8 3.4) (P <
0.01), and GPX reduced from (16.57 0.10) to (13.49 0.08)
(P < 0.05). Similar to the effect on liver enzymes, oral ad-
ministration of M GV at 200 and 400 mg kg1 significantly
and dose-dependently recovered the decreases of liver lev els
of SOD, CAT, and GPX when compared with the CCl4 con-
trol groups. M GV at 400 mg kg1 g even showed a potency of
hepatoprotective effect to recover the SOD, CAT, and GPX in
liver tissue back to normal levels, as did the positive control
silymarin. A low dose of M GV (100 mg kg1) only demon-
strated a trend of amelioration on these indicators without a
statistical difference.
Histopathological examination
The results of histopathological examinations once again
revealed that CCl4-induced liver injuries were attenuated by
the oral administration of M GV. As shown in Fig. 3, normal
liver showed typical hepatolobular architecture, consisting of
clear central vein with radiating cords of hepatocytes sep a-
rated by sinusoids; Hepatic cells were polygonal in shape,
with distinctive nuclei and uniform cytoplasm. Few binucle-
Fig. 3 Histopathological changes in liver section from mice
ated cells were also present, and the cytoplasm was regularly after CCl 4 intoxication and prevention by the treatment with
distributed (Fig. 3A). Hepatic injuries in mice induced by Silymarin or MGV (HE staining, Original magnificati on, 10;
CCl4 were demonstrated by marked vacuolization of hepato- A. Normal control; B. Negative control; C. Positive control; D.
cytes, necrosis around central vein, sinusoidal dilation and MGV Low dose ; E. MGV Medium dose ; F. MGV High dose )

491
 ZHANG Zhi-Feng, et al. / Chin J Nat Med, 2014, 12(7): 488494

Table 2 Effe cts of the methanol extract of G. veitchiorum on liver enzymatic antioxidant activities in CCl 4-induced acute he patic
injury in mice ( x s, n = 10)
SOD CAT GPX
Groups T reatment
/(U/mg protein) /(U/mg protein) /(mU/mg protein)
Normal Control Vehicle 2.18 0.06 49.6 2.5 16.57 0.10
Negative Control Vehicle 1.73 0.07 ^ 20.8 3.4 ^^ 13.49 0.08 ^
Positive Control kg1
Silymarin 25 mg 2.16 1.52 45.4 2.76 ** 16.22 0.13 **
MGV Low Dose kg1
100 mg 1.76 0.13 29.7 3.1 15.51 0.06 *
1 * *
MGV Medium Dose 200 mg
kg 1.94 0.09 38.3 2.9 15.59 0.03 *
MGV High Dose kg1
400 mg 2.06 0.05 ** 48.2 1.8 ** 16.18 0.07 **
^
P < 0.05; ^^P < 0.01 vs normal control group; * P < 0.05; ** P < 0.01 vs negative control group

significantly attenuated the liver injuries, shown by the ab-

sence of focal or bridging necrosis or mild hepatitis. The his-
topathological changes induced by CCl4 were improved by
the treatment with MGV in a dose-dependent manner, and
the recovery was significant in the high dose group (400
mg kg1) which was comparable to that of silymarin (F igs.
3C-F). In short, histopathological examination demonstrated
that MGV remarkably ameliorated CCl4-induced liver injury,
which supported the results obtained from the investigations
on aminotransferases and liver oxidative stress levels with
each other.
Effects of MGV on expression of TNF- and HO-1 in liver
tissue
Previous results from biochemical assay and hist olog-
ical evaluation suggested that M GV exhibited a prote ctive
action on the liver function in mice. To elucidate the pos-
sible mechanis m of M GV in CCl4-induced hepatotoxicity
in mice, therefore the expression of TNF- and HO -1 in
the liver of mice treat ed by CCl4 was examined in the
abs ence or pres ence of M GV therapy. As s how n in F ig. 4,
CCl4 administrat ion s ignificant ly elevated the exp res -
sions of TNF - compared wit h the normal control group.
Treatment w ith M GV (400 mg kg 1 ) and posit ive cont rol
silymarin s ignificant ly inhibit ed the overexp res sion of
TNF- in liver t issue. Int erestingly, CCl 4 s light ly in-
creased the express ion of HO-1, w hile it w as further un-
regulat ed by M GV treat ment w ith a significant difference
compared w ith CCl4 group. H owever, s ilymarin treat-
ment did not s how obvious influence on t he express ion
Fig. 4 Western blot analysis of the effect of MGV on the
of OH-1.
expression of TNF- and HO-1 in CCl4 -induced hepatotoxi ci-
Discussion ty. The blot was reported sequentially with antibodies spe cial
for TNF-, HO -1 or actin. Normal, Normal control group;
H epat itis is a high incidence ailment around the CCl4 , Negative control group; CCl4 + Sily, Positive control
world, which is generally induced by toxic chemicals , group (Silymarin treatment at 25 mg kg 1); CCl4 + MGV,
virus es, alcohol, lip id peroxidat ive products, and various MGV treatment group (MGV treatment at 400 mg kg1 ).
drugs [ 16] . A lthough some synthetic medicines w ere rec- Ide ntical results were obtained in 3~5 separate mice live rs.
ommended for liver therapy in the last years, most of Representative immunoblot pictures and densitometric anal-
them are immunos upp ressive [17] . Therefore, studies on ysis of proteins. Normalization of Western blot was ensure d
novel therapeutic strategies from plant medicine are attract- by actin. Bars were Mean SEM of 3~5 experiments. * P <
ing more and more attention in recent years [18-20]. 0.05 vs normal control group and # P < 0.05 vs CCl 4 group

492
 ZHANG Zhi-Feng, et al. / Chin J Nat Med, 2014, 12(7): 488494
In the Chinese Pharmacopoeia, several species from the
family Gentianaceae including G. manshurica Kitag., G.
scabara Bge., G. triflora Pall., and G. regescens Franch. are
used for treatment of jaundice with damp-heat pathogen. Also,
G. aldida Pall. and G. veitchiorum were usually used for
heat-clearing and detoxication in traditional Tibetan medicine
for many years [5] . The hepatoprotective effect of G. olivieri
Griseb. was studied using in vivo models in rats [21] . Previous
research suggested that gentiopicroside from Gentiana spe-
cies can prevent liver injuries induced by either CCl4 or
D-GALN [22] . It was documented that G. veitchiorum was
effective in suppressing inflammation and protecting the liver,
as shown by a marked reduction of hepatic fibrosis induced
by dimethylnitrosamine [23]. G. veitchiorum increased the
level of SOD and inhibited the formation of M DA, and then
showed a protective effect on NH 3-induced bronchitis [24] . In
the present study, the results demonstrated that the methanol
extract of G. veitchiorum has a significant hepatoprotective
effect on acute liver injury induced by CCl4. The groups
treated orally at the doses of 200 and 400 mg kg 1 showed a
significant decrease in the levels of SOD, CAT, and GPX,
which indicated the antioxidation effect. Regulation of the
balance of oxidation and antioxidation may be involved in the
mechanism of G. veitchiorum against oxidative-induced liver
injury. It also provides some scientific evidence for the t reat-
ment of liver injury in traditional folk medicine.
Carbon tetrachloride has been extensively used to i n-
vestigate chemical toxin-induced liver damage in animal
models. Fatty liver, cirrhosis, and necros is are the remarka-
ble pathological characteristics of CCl 4-induced hepatotoxi-
city [25] . Administration of CCl4 results in significant elev a-
tion of serum ALT, AST, and ALP, as these enzymes are
liberated into systematic circulation by liver cells. Generally,
ALT and AST activities reach a maximum in the first 24 h
and then gradually decrease, remaining at abnormal levels at
72 h after CCl4 treatment [26] . The study suggested that MGV
can reverse the elevation of transaminases and ALP, demon-
strating a marked protective effect to liver in this time course.
Oxidative stress may play an important role in many ret-
rogressive diseases, such as liver injury and chronic fatigue
syndrome. The acute toxic effects of CCl4 are well known to
be mediated through free radicals in vivo [11] . Thus, antioxi-
dant or inhibition of free radical generation could be i m-
portant in liver protection against CCl4-induced liver disease
[14]
. HO-1 plays an important anti-inflammatory role in a
number of inflammatory diseases, and also has anti-apoptotic
and anti-proliferative properties. Recently, many studies
demonstrated that the HO-1 mediated pathway can be acti-
vated in Kupffer cells after suppression of LPS-stimulated
TNF-. There is a growing appreciation that HO-1, in partic-
ular, is an important regulator and biomarker of the an-
ti-inflammatory effects of many hepatoprotective agents.
Interestingly, induction of HO-1 by Kupffer cells after chem-
ical inhibition of TNF- demonstrates that HO-1 is critical to
the suppression of hepatotoxic irritants stimulated TNF-
expression in response to the inflammatory action in hepatic
493
damage. The present studies demonstrated that G. veitchiorum
significantly reduced the oxidative stress by recovering the
decreased activities of SOD, CAT, and GPX in CCl4-induced
acute liver injury. This indicates the antioxidative and free radical
scavenging properties of G. veitchiorum. Histopathological re-
sults indicated the structural and functional integrity of the liver
cells, which further supported the hepatoprotective effect of G.
veitchiorum.
In conclusion, the study demonstrated that G. veitchi-
orum inhibited the elevations of AST, ALT, and ALP in se-
rum, and recovered the decrease of activities of SOD, CAT,
and GPX and ameliorated the histopathological changes in
liver by CCl4-induced mice. Importantly, these protective
effects of G. veitchiorum against hepatic damage in mice
were likely related with its potential to increase HO-1 expres-
sion and recover TNF- alternation. The present results sug-
gest that the hepatoprotective effect of G. veitchiorum was
due, at least partly, to its ability to reduce oxidative stress in
liver which is likely through scavenging CCl4-associated free
radicals and suppressing inflammatory response. The results
suggested that G. veitchiorum can be used as a safe and effec-
tive agent to reduce acute liver damage, thus providing scien-
tific evidence to its traditional medicinal value.
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