Antioxidant and Hepatoprotective Activity of Garlic Chives (Allium tuberosum)
Ethanolic Extract in Doxorubicin-induced Renal and Liver injured Wistar Rats
Ika Rahmawati Sutejo
Departement of Biochemistry
Faculty of Medicine University of Jember
Jember, Indonesia
e-mail: ikarahmawati.fk@unej.ac.id
Abstract—The plant phenolic compounds such as flavonoids
and isoflavons have an important role in the treatment of many
diseases and some of them induce a potent antioxidant and
hepatoprotective effect. The objective of this study is to
investigate the antioxidant and hepatoprotective activity of
flavanoids compound from ethanolic extract of Allium
tuberosum (EAT) against doxorubicin-induced hepatotoxicity
in male Wistar rats. Preliminary phytochemical tests of EAT
were done by Thin Layer Chromatography. Antioxidant and
hepatoprotective activity of EAT was evaluated by treating
groups of rats with 500 &1000 mg/kg body weight per oral of
EAT for 14 days and at the same time (day-1 and day-8)
challenging with 4.67 mg/kg body weight of Doxorubicin. On
day-15 the hepatoprotective and antioxidant effects of EAT
were evaluated by measuring liver function and MDA serum
spectrophotometrically using standard procedures. Thin Layer
Chromatography indicate the presence of the flavanoids on
EAT. Results showed that intraperitoneal injection of
Doxorubicin caused a significant (p<0.001) elevation in the
serum levels of SGOT and SGPT. However, elevations in the
measured biochemical parameters were significantly (p<0.05
and p<0.01) attenuated in rats treated with EAT, in dose
related fashion. Oral administration of EAT also decreases
levels serum MDA (p<0.01). It can be concluded that ethanolic
extract of Allium tuberosum has a significant hepatoprotective
and antioxidant activity. In addition, Allium tuberosum may be
useful for adjuvant chemotherapy doxorubicin.
Keywords; Allium tuberosum, antioxidant, doxorubicin,
hepatoprotective
I. INTRODUCTION
Cancer is the one of the major causes of death in the
world. In 2012, there were 14 million new cases and 8.2
million deaths caused by cancer.[1] In Indonesia, the
prevalence of cancer in 2013 at 1.4% and the WHO
estimates that the number of new cases of cancer will
continue to increase by 70% in the next two decades.[2] The
high prevalence and mortality of cancer encourage the
development of an effective therapy with minimal side
effects.
Currently, chemotherapy is the primary modality of
cancer therapy, and the most frequently used is doxorubicin.
Erfan Efendi
Departement of Biochemistry
Faculty of Medicine University of Jember
Jember, Indonesia
e-mail:erfanefendi.fk@unej.ac.id
This drug is effective in treating various types of cancer such
as breast, lung, prostate, cervical, bone, etc.[3] but
Doxorubicin has many side effects including cardiotoxic,
hepatotoxic, nephrotoxic, and immunosuppression.[4], [5]
Doxorubicin also increased SGOT, SGPT, and γ-GT serum.
[6]
Adjuvant chemotherapy is a cancer treatment strategies
by combining a compound with chemotherapeutic agents to
increase treatment efficacy while decreasing the toxicity of
chemotherapy itself. [7] One of the potential plants in this
case is garlic chives (Allium tuberosum). Allium tuberosum
is known to have flavonoids, especially allicin that is proven
to has hepatoprotector effect, but the mechanism is not
certain.[8]
II. MATERIAL AND METHODS
A. Plants
Garlic chives (Allium tuberosum) were collected from
area around Purworejo, Ngantang, Malang, Indonesia, in
March 2016. The plant was authenticated by a taxonomist at
Departement of Botany, Faculty of Mathematics, University
of Jember, Indonesia. Garlic chives specimens stored in the
herbarium of Faculty of Mathematics, University of Jember.
B. Animals
Wistar rats (weighing 100-150 g) were housed at a
constant temperature (25±2°C) with a constant relative
humidity (60±10%) on the an automatically controlled 12:12
h light-dark cycle (light on at 7:00 a.m.). Rats were fed with
standard rat chow and water as libitum. The rats were
acclimatized and quarantined for at least one week prior to
the experiment.
C. Research Design
Thirty seven male Sprague Dawley rats ere divided into
five groups consisting of 6-7 rats each as follows: 1) normal
controls, rats were given 0.9% NaCl intraperitoneally, 2)
negative control, Doxorubicin rats were given 4.67 mg/kgW
in 0.5% CMC-Na po, 3) extracts controls, rats were given
EAT 1000 mg/kgW in a solvent 0.5% CMC-Na po, 4) first
group treatment, Doxorubicin-induced rats 4.67 mg/kgW ip
and at the same time got EAT 500 mg/kgW, 5) Second group
treatment, Doxorubicin-induced Rat 4.67 mg/kg i.p. and at
the same time got EAT 1000 mg/kgW. Induction
Doxorubicin (Ebewe, obtained from P.T. Ferron Par
Pharmaceutical Cikarang, Indonesia) performed on days 1
and 8. The treatment of EAT were administered for 14 days.
D. Extraction Method
Garlic chives which have been dried in the oven smoothed
by using a blender. A total of 599 grams of chives powder
macerated using ethanol 70% with a ratio of 1: 3. The
macerate was filtered using filter paper to obtain the filtrate
which then evaporated to obtain as much as 125.56 grams of Figure 1. TLC chromatogram in UV (254 nm) for the
concentrated extract. samples of EAT, arrows indicate flavanoids
component.
E. Measurement of SGOT and SGPT Serum
After 8 weeks, the animals were killed and blood B. EAT Decreases SGOT and SGPT Serum Level
samples were obtained using cardiac puncture method. SGOT and SGPT serum was measured as an indicator of
Serum Glutamate Pyruvate Transaminase (SGPT) and Serum cellular injury of liver. Table 1 shows a significant increase
Glutamate Oxaloacetate Transaminase (SGOT) levels were (p <0.01) of SGOT and SGPT enzymes in doxorubicin-
measured using spectrophotometery method. induced group compared to control group. Levels both
F. Measurement of MDA Serum decreased significantly (p <0.01) in the group receiving
treatment EAT dose of 1000 mg/kgW and received
Data collection was performed by measuring levels of induction doxorubicin.
blood serum malondialdehida. At room temperature, 100 mL
of blood serum and 10 mL of BHT solution mixed in a glass C. EAT Decreases MDA Serum Level
tube. Successively added 700 mL of 1% orthophosphoric The repair mechanisms of liver serum is confirmed by
acid and 200 mL of 2-thiobarbituric acid (TBA) of 0.6%. measuring levels of MDA. MDA is used as an indicator of a
Tubes were incubated into a tub of hot water of 95 °C for 45 damage by lipid peroxidation. The increase of MDA indicate
minutes. After incubation, the tube is cooled in cold water. 1 oxidation processes or membrane damage caused by free
mL of n-butanol was added to the tube, then centrifuged at radicals. Figure 2 shows the doxorubicin significantly
2000 rpm for 10 minutes. The top layer is taken and increase the MDA serum levels (p<0.01). Meanwhile, EAT
measured by a spectrophotometer λ 535 nm. TBARS administration after Doxorubicin induction can lower MDA
(thiobarbituric Acid Reactive Substance) which is condensed serum levels (p<0.05 at a dose of 500 mg/kgW and p<0.01 at
bioproduct MDA with TBA calculated in μM using the a dose of 1000 mg/kgW).
extinction coefficient of 1, 56 x 105 M-1 cm-1.
G. Statistical Analysis Table 1. EAT effect on SGOT and SGPT serum level
Statistical significance was evaluated by one-way Groups SGOT (U/L) SGPT (U/L)
analysis of variance (ANOVA). Significance was Control 40.38 ± 2.43 20.52 ± 4.03
measured using Fisher’s least significant for the exact p 56.26 ± 4.04a 36.19 ± 2.60a
DOX
values and significant differences are noted in the
results. Differences with p<0.05 were considered EAT 1000 40.16 ± 4.47 20.74 ± 3.10
significant. DOX + EAT 500 45.01 ± 4.38 b
28.68 ± 1.84
b
DOX + EAT 1000 40.82 ± 2.63 25.90 ± 7.32b
III. RESULTS a
Significant difference (P<0.01) compared to the control group value. b
Significant difference (P<0.05) compared to the Dox group value. Data were
A. Presence of the Flavonoid on Thin Layer expressed as Mean±SD (n=6-7 rats)
Chromatography
Thin layer chromatography indicates the presence of the
flavonoid on EAT (figure 1). The static phase was using
silica gel 60F254, while the motion phase was using
buthanol, acetic acid, and water with a ratio of 8: 2: 10.

Figure 2. Effect of EAT on MDA serum level.*Significant
difference (P<0.01) compared to the control group
value. **Significant difference (P<0.05) compared
to the Dox group value. *** Significant difference
(P<0.01) compared to the Dox group value. Data
were expressed as Mean±SD (n=6-7 rats)
IV. DISCUSSION
Doxorubicin is one of the most effective
chemotherapeutic agent since 1970. [9] This drug is proven
to be effective in treating various types of cancer such as
breast cancer, lung, prostate, cervical, bone, etc. [3]
Doxorubicin is not selective in the mechanism of action. It
does not only affect cancer cells but also normal cells which
is actively dividing, this poses various problems. As a result
of the non-selective character, Doxorubicin also affect the
formation of cells that are actively dividing cells such as
bone marrow (the development of red blood cells),
lymphocyte activation, hair loss, and various organ toxicities.
[10]
In this study, the EAT found to be able restoring liver
function who had supressed due to the induction of
doxorubicin. SGOT and SGPT are specific enzymes as
indicators of hepatic injury. In the case of hepatitis due to
infection or other inflammatory reactions, SGPT levels
higher or the same height as SGOT.[11] But in this study
showed the opposite, where SGOT higher than SGPT. This is
possible because of toxic effects of doxorubicin on the heart
in addition to the liver. Increased significant levels of SGOT
without increased of SGPT levels are common in cardiac
injury.[12] Cardiotoxicity is also one of the most common
complication of doxorubicin, but this study did not measure
specific enzyme to assess the work of the liver.
MDA is used as an indicator of refurbishment by lipid
peroxidation. Doxorubicin toxicity effects are made possible
by the presence of oxidative stress. This study shows that the
induction of doxorubicin alone was shown to significantly
increase serum levels of MDA. After giving EAT, MDA
decreased indicating that lipid peroxsidase pathway plays a
role in the formation of lesion on the liver.
V. CONCLUSION
It can be concluded that doxorubicin causes
hepatotoxicity and EAT with a dose of 1000 mg/kgW after
induction of doxorubicin is able to restore hepatic function as
through antioxidant pathways.
ACKNOWLEDGMENT
The author would like to thank the Ministry of Research,
Technology and Higher Education of Indonesia also the
University of Jember, which has funded this research.
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