Scientia

Pharmaceutica

Article
Effect of Vernonia amygdalina Del. Leaf Ethanolic
Extract on Intoxicated Male Wistar Rats Liver
Maria Immaculata Iwo 1,2, *, Sergia Louisa Sjahlim 1 and Siti Farah Rahmawati 1
1 Pharmacology Clinical Pharmacy Research Group, School of Pharmacy, Institut Teknologi Bandung,
Jl. Ganesha 10, Bandung 40132, Indonesia; [email protected] (S.L.S.); [email protected] (S.F.R.)
2 Biotechnology Biomedical Research Center, Institut Teknologi Bandung, Jl. Ganesha 10,
Bandung 40132, Indonesia
* Correspondence: [email protected]; Tel.: +62-22-856-220-2445

Academic Editor: Gernot Eller

Received: 29 December 2016; Accepted: 16 March 2017; Published: 23 March 2017

Abstract: Vernonia amygdalina has been shown to have antioxidant activity, and is also expected to
have hepatoprotective activity. This study was conducted to study the effect of V. amygdalina ethanol
extracts on intoxicated rat livers. Fresh leaves were extracted in ethanol, and the hepatoprotective
activity was tested on male Wistar rats induced with a combination of isoniazid (INH) and rifampicin.
Parameters observed were the activity of the enzyme alanine transferase (ALT), serum albumin levels,
liver index, and histopathological of the rat liver. The results showed that 50 and 100 mg/kg rat
body weight of V. amygdalina ethanol extracts could prevent liver intoxication, starting on day 14.
Based on serum albumin concentrations and ALT activity, the high dose extract (100 mg/kg) was
more potent as a hepatoprotective agent compared to the extract at a low dose (50 mg/kg). The group
of rats treated with a high dose extract showed normal liver index compared to the positive control.
Through histology examination, the liver of rats treated with a high dose extract (100 mg/kg) showed
minimal liver cell structure damage, and showed similar patterns to the normal rat. Based on these
results, it can be concluded that V. amygdalina ethanol extracts can be used to protect the liver in
a combination of INH and rifampicin as antituberculosis treatment.

Keywords: intoxicated rat liver; Rifampicin; INH; Vernonia amygdalina

1. Introduction
The metabolism of drugs and other exogenous compounds mainly takes place in the liver [1].
In addition, most drugs are administered orally and are absorbed from the gastrointestinal tract to the
tissues through the liver. Thus, the liver is liable to damage from such drugs and their metabolites [2].
Three of the main drugs responsible for liver damage in patients include tuberculosis (TB) medication,
anticancer drugs, and antibiotics.
Tuberculosis remains a major health problem in South-East Asia and is one of the highest causes of
morbidity and mortality. It is also the second highest cause of adult deaths after cardiovascular disease,
and is the deadliest pathogen out of all the communicable diseases; there are about half a million
cases of smear-positive TB in Indonesia. Due to this high rate of incidence of TB, many patients
prescribed anti-TB drugs also suffered damage to the liver. According to research by Nurazminah
conducted in a hospital in the cities of Jakarta and Cisarua, Indonesia, the percentage of respondents
who experienced liver damage caused by anti-TB drugs was 52.2% [3].
In tuberculosis patients, chemotherapy combination drugs are known to cause toxicity of the liver
(hepatotoxiticy). Previous studies have shown a 10% elevation of serum hepatocellular enzymes, and a
withdrawal of 1–2% of patients who took a standard combination chemotherapy of isoniazid (INH)
and rifampicin was seen because of severe hepatotoxicity leading to fulminant hepatitis [4]. Rifampicin

Sci. Pharm. 2017, 85, 16; doi:10.3390/scipharm85020016 www.mdpi.com/journal/scipharm

Sci. Pharm. 2017, 85, 16 2 of 7

is a strong inducer of Cytochromes P450 (CYP450) in the liver, and its combination with INH has
been related to an increased risk of hepatotoxicity. Rifampicin induces INH metabolism, therefore
increasing hydrazine production (especially in slow acetylators), which can escalate the toxicity [5].
Liver damage can be prevented by using hepatoprotective agents—compounds that mitigate liver
damage caused by hepatotoxic agents. Hepatoprotective agents are used in patients suffering from
various liver diseases. Several medicinal plants have been known to have hepatoprotective activities,
including Eclipta alba (Asteraceae), Glycyrrhizaglabra (Leguminosae), Boerhaaviadiffusa (Nyctaginaceae),
Phyllanthusamarus (Euphorbiaceae), and Silybummarianum (Compositeae). Vernonia amygdalina is
a shrub that grows throughout tropical Africa and South-East Asia [6]. The plant is generally known
as bitter leaf due to the bitterness of its leaves. Based on previous research [7–10], V. amygdalina
has been shown to have antioxidant activities by scavenging free radicals, and is also expected to
have hepatoprotective activities. This study was conducted to evaluate the hepatoprotective effect of
V. amygdalina ethanolic extracts in an anti-TB drug-induced animal model.

2. Materials and Methods

2.1. Plant Material

Vernonia amygdalina plants were collected from Salaman Magelang, Central Java, and identified at
the Indonesian Institute of Sciences, Research Center for Biology. The identity of the plants collected
for this study was determined by the Research Center for Biology, Indonesian Institute of Sciences
No 1806/IPH.1.01/If.07/VIII/2016 as Vernonia amygdalina Delile, a plant of the Compositae family.

2.2. Extraction of V. amygdalina

Fresh leaves were air-dried and processed into crude drug. Then, the dried powder was extracted
in ethanol 96% (v/v) using the Soxhlet method. The obtained extract was then concentrated using
a rotary evaporator and was then characterized.

2.3. Extract Characterization and Phytochemical Screening

The characteristics of extract were determined according to methods described by Harborne
through the following quality parameters: water content, water extractable matter, ethanol extractable
matter, extract density, and phytochemical screening [11].

2.4. Animals
Male Wistar rats weighing about 200–250 g and ages 10–12 weeks old were obtained from PT Bio
Farma, Bandung. The animals were distributed into stainless steel cages (five rats per cage). They were
fed a standard diet and water ad libitum. The animals were maintained under standard laboratory
conditions: free air circulation, room temperature of about 22–25 ◦ C, and humidity of 55–60%. All rats
were acclimatized for 5 days before the experiment. Handling of the animals were guaranteed since
the principal investigator has an Animal Welfare Assurance by the office of laboratory animal welfare,
NIH, number A5925-01.

2.5. Evaluation of Hepatoprotective Activity

A combination of INH and rifampicin were used as inducers of liver damage [5,12], and silymarin
was used as the reference hepatoprotective drug. The drug combination was administered orally 1 h
before extract administration. The animals were divided into four groups, each group consisting of
five animals. Group 1 served as the control group, which was given INH at a dose of 27 mg/kg body
weight (bw) and rifampicin at a dose of 54 mg/kg bw. Groups 2 and group 3 were treated with the
inductor combination and V. amygdalina ethanol extract at a dose of 50 and 100 mg/kg bw, respectively,
for 35 days. In Group 4, the animals received the inductor combination and silymarin at a dose of
54 mg/kg bw. The parameters observed were alanine transferase (ALT) activity, serum albumin levels,
Sci. Pharm. 2017, 85, 16 3 of 7

and the liver index. Rats liver histopathological examination was also performed. The albumin level
and ALT were measured using analytical kits obtained from Sclavo Diagnostics International, Sovicille
Siena, Italy. The liver index was calculated as follows:

liver weight
Liver Index = × 100
body weight

After 35 days, the rats were killed through humane procedures using CO2 gas, and the livers
were then collected for histological assessment.

2.6. Histological Assessment

Livers from rats of different groups were fixated in 10% neutral formalin solution, dehydrated
in graded alcohol, and embedded in paraffin. Fine sections were obtained, mounted on glass slides,
and counter-stained with hematoxylin–eosin for light microscopic analysis.

2.7. Statistical Analysis

The results are presented as mean ± standard deviation (SD). Analyses were performed using
a Student’s t-test. p-values less than 0.05 were considered statistically significant [13].

3. Results and Discussion

The ethanolic extract of the plant showed the characteristics presented in Table 1.

Table 1. Characteristics of the extract.

Quality Parameters Results

Water extractable matter (%) 7.7
Ethanol extractable matter (%) 15.9
Density for 1% extract (g/mL) 0.7
Water content (%) 8

The extractable matter content can be used to determine the amount of active constituents that
were extracted in the solvent used, as well as the solubility of the active constituents of extract in
different solvents. Based on the results, the ethanol extract of V. amygdalina had poor solubility
in water but high solubility in ethanol. Furthermore, from the phytochemical screening (Table 2),
it can be observed that the V. amygdalina ethanol extract contained alkaloids, flavonoids, saponins,
and steroid/triterpenoids.

Table 2. Phytochemical screening.

Compound Results
Alkaloids +
Flavonoids +
Saponins +
Tannins −
Quinones −
Steroid/Triterpenoids +
+ = contained; − = not contained.

The serum ALT activities of the animals in each group are presented in Table 3. It can be observed
that after seven days of INH and rifampicin administration, the ALT activity of all groups was elevated,
indicating that all of the animals’ livers were damaged. The significant increase in ALT activity on day
Sci. Pharm. 2017, 85, 16 4 of 7

7 shows that the combination of INH and rifampicin damage the liver. Throughout the experiment,
the ALT activity of the control (+) group remained significantly higher (p < 0.01) compared to those of
the treated groups. This indicates that V. amygdalina ethanol extract at the doses used can prevent liver
damage. The results also show that the two doses of V. amygdalina extract used seemed to have same
effect in lowering ALT activity starting after 7 days. Of the two doses, it seems that the higher dose
was more potent compared to the lower dose. This is seen from the ALT activity of the group treated
with V. amygdalina extract at a dose of 100 mg/kg bw, in which after only 14 days the value was no
longer significantly different than that at day 0. Meanwhile, the ALT activity of the group treated with
50 mg/kg bw of the extract needed 35 days to return to normal conditions.

Table 3. Effect of ethanolic extract of Vernonia amygdalina (VA) on serum alanine transferase
(ALT) activity.

Dose ALT (U/L)

Group
(mg/kg bw)
D0 D7 D14 D21 D28 D35
Control (+) 0 43.47 ± 16.24 61.63 ± 17.16 x 80.00 ± 9.09 z 79.57 ± 8.46 z 82.14 ± 6.64 z 84.00 ± 5.29 z
50 39.17 ± 10.04 58.14 ± 9.58 z 56.75 ± 10.63 c,z 51.37 ± 3.58 c,z 50.13 ± 5.79 c,y 49.50 ± 4.81 c,x
VA extract
100 44.93 ± 15.01 63.01 ± 3.01 z 55.75 ± 4.65 c,x 54.00 ± 5.47 c 49.00 ± 4.24 c 47.37 ± 4.89 c
Reference
54 42.56 ± 12.22 54.56 ± 7.88 y 49.42 ± 3.10 c 50.42 ± 6.39 c 48.86 ± 4.74 c 46.57 ± 5.80 c
(silymarin)
Values are expressed as mean ± standard deviation (SD). n = 5; Data was analyzed by students t-test, where a = p < 0.1,
b = p < 0.05, c = p < 0.01 compared to induced control (+) group; x = p < 0.1, y = p < 0.05, z = p < 0.01 compared to D0.

In addition to the ALT activity, to determine the effect of the extract on liver function, the albumin
concentrations were also analyzed. Albumin is synthesized in the liver; therefore, a decrease in serum
albumin concentration could indicate liver damage. The albumin levels of each group are presented
in Table 4.

Table 4. Effect of ethanolic extract of V. amygdalina on serum albumin concentration.

Dose Albumin Concentration (g/dL)

Group
(mg/kg bw)
D0 D7 D14 D21 D28 D35
Control (+) 0 3.57 ± 0.35 3.16 ± 0.16 y 2.91 ± 0.35 z 3.00 ± 0.32 z 2.86 ± 0.4 z 2.71 ± 0.31 y
50 3.37 ± 0.47 2.99 ± 0.19 a,y 3.45 ± 0.39 b 3.60 ± 0.38 c 3.69 ± 0.40 c 3.67 ± 0.56 c
VA Extract
100 3.67 ± 0.49 3.24 ± 0.29 x 3.46 ± 0.40 b 3.70 ± 0.43 c 3.76 ± 0.47 c 3.79 ± 0.47 c
Reference
54 3.43 ± 0.35 3.11 ± 0.23 x 3.46 ± 0.40 b 3.79 ± 0.29 c 3.83 ± 0.80 c 3.87 ± 0.46 cx
(silymarin)
Values are expressed as mean ± SD. n = 5; Data was analyzed by students t-test, where a = p < 0.1, b = p < 0.05,
c = p < 0.01 compared to induced control (+) group; x = p < 0.1, y = p < 0.05, z = p < 0.01 compared to D0.

Daily oral administration of INH and rifampicin for seven consecutive days significantly (p < 0.01)
altered the rats’ liver function, which was characterized by a decrease in serum albumin concentration.
However, after 14 days, both extract groups showed no significant changes in albumin concentration
compared to the initial condition (D0). In fact, the albumin concentration of these groups increased
after the 14th day. These results suggest that both doses of the extract can prevent liver damage.
At end-point of the experiment (D35), the animals were sacrificed and their body weights
measured. The livers of the animals were then isolated and weighed to determine the liver index.
This parameter was measured to determine the presence of liver enlargement associated with
liver damage.
The results of the liver index calculation (Figure 1) showed that the liver index of the group of
rats given extract at a dose of 100 mg/kg bw and silymarin were significantly (p < 0.05 and p < 0.01,
respectively) lower compared to the control group. Additionally, the liver index of the 100 mg/kg bw
extract group was no different compared to the group given silymarin, suggesting that the extract at
a high dose (100 mg/kg bw) has comparable hepatoprotective activity to the reference drug.
 Sci.Sci. Pharm.
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1616 5 of58of 8

Sci. Pharm. 2017, 85, 16 5 of 7

extract
extract group
group wasnonodifferent
was differentcompared
compared to
to the
the group
group given
givensilymarin,
silymarin,suggesting
suggesting that thethe
that extract at at
extract
a high
a high dose
dose (100mg/kg
(100 mg/kgbw)bw)has
hascomparable
comparable hepatoprotective
hepatoprotective activity
activitytotothe
thereference drug.
reference drug.

** pp << 0.05,
0.05,##pp<<0.01
0.01
Compared to control
Compared to control
#
#

Figure 1. The liver index of the group of rats treated with V. amygdalina (VA) extract.
Figure 1. The liver index of the group of rats treated with V. amygdalina (VA) extract.
Figure 1. The liver index of the group of rats treated with V. amygdalina (VA) extract.
The pattern and severity of liver damage of each animal in every group was observed by
The pattern
histological
The and
andseverity
observation.
pattern ofofliver
liver damage
The microscopic
severity of each
cross-sections
damage animal
eachareanimal in every
presented
in every
in group
Figure 2. was
group wasobserved
observed
byby
histological observation.
histological observation.The
Themicroscopic
microscopiccross-sections
cross-sections are presented
presentedin
inFigure
Figure2.2.

(d)

(d)

Figure 2. Cont.
Sci. Pharm. 2017, 85, 16 6 of 7
Sci. Pharm. 2017, 85, 16 6 of 8

Figure 2. Histopathological sections of rat’s liver. All pictures are magnified 10×10 times. (a) Normal
Figure
control 2. Histopathological
group; (b) Histology ofsections of rat’s
the normal liver.
liver All pictures
structure are magnified
from literature [14]; 10×10 times.control
(c) Positive (a) Normal
group;
(d)control
Histologygroup; (b) intoxication
of the Histology of liver
the normal
structureliver structure
from from
literature literature
[12]; [14],treated
(e) Group (c) Positive control at
with extract
group;
a dose (d)mg/kg
of 50 Histology
bw of
andthe
(f)intoxication
100 mg/kgliver structure
bw; and (g) Thefrom literature
reference [12];VS
group. (e)marks
Groupthe treated with
central vein,
extract
yellow at a dose
marks showofnormal
50 mg/kg bw and (f)green
hepatocytes, 100 mg/kg
marksbw;showandKupffer
(g) Thecells,
reference
blackgroup.
marksVS marks
show the
sinusoid
central vein,
dilatation, blueyellow
marks marks show normal
show hydropic hepatocytes,
degeneration, andgreen marks
orange show
marks Kupffer
show cells,cells.
necrotic black marks
show sinusoid dilatation, blue marks show hydropic degeneration, and orange marks show necrotic
cells.
Histopathological examination of the liver sections from normal rats showed normal parenchymal
Histopathological
structure, normal sinusoidal examination of the
architecture, andliver sections from
no significant lesionsnormal
were rats showedIn normal
observed. addition,
parenchymal structure, normal sinusoidal architecture, and no significant
Kupffer cells—phagocytic cells that engulf pathogens, cell debris, and damaged blood cells—could lesions were observed. In be
addition, Kupffer cells—phagocytic cells that engulf pathogens, cell debris, and
found in the lining of the sinusoids. According to Sahai et al. [14] and Junqueira [15], profiles of normal damaged blood
cells—could
liver histologybe found
show in the lining
hepatocytes of the
with sinusoids. According
a polyhedral shape andtoaSahai
roundetnucleus
al. [14] and Junqueira
at the [15],
center [15,16].
profiles of normal liver histology show hepatocytes with a polyhedral shape and a round nucleus at
The positive control group showed changes in liver structure that includes inflammation, hydropic
the center [16,15]. The positive control group showed changes in liver structure that includes
degeneration, and necrosis. The parenchyma showed loss of the normal sinusoidal architecture due
inflammation, hydropic degeneration, and necrosis. The parenchyma showed loss of the normal
to numerous foci of lobular inflammation, hepatocyte dropout, and rosette formation. These results
sinusoidal architecture due to numerous foci of lobular inflammation, hepatocyte dropout, and
were in line with studies that have shown that the special characteristics of liver damage caused
rosette formation. These results were in line with studies that have shown that the special
by a combination of INH and rifampin are inflammation and cell necrosis [16,17]. According to
characteristics of liver damage caused by a combination of INH and rifampin are inflammation and
Mitchell et al., [16,17].
cell necrosis hydropic degeneration
According occursetdue
to Mitchell al., to a disturbance
hydropic of active
degeneration transport
occurs due tothat prevents the
a disturbance
cellular efflux of Na + ions resulting in an increased concentration of intracellular Na+ ions. This causes
of active transport that prevents the cellular efflux of Na ions resulting in an increased concentration
+

osmosis and an influx

of intracellular of water
Na+ ions. This into theosmosis
causes cells, causing
and an the cells
influx ofto become
water into swollen
the cells,and havethe
causing ancells
enlarged
to
nucleus.
becomeGranular
swollen andin the
havenucleus are also
an enlarged evidentGranular
nucleus. [17]. in the nucleus are also evident [17].
AsAsforfor
the groups
the administeredV.
groupsadministered V.amygdalina
amygdalina extract
extract andandsilymarin,
silymarin,the theliver
livercell
cell damage
damage waswas
lighter than
lighter thanthe
the positive controlgroup.
positive control group. Liver
Liver tissue
tissue in theingroup
the group
given 50given
mg/kg 50bwmg/kg bw showed
of extract of extract
showed a microscopic
a microscopic appearance
appearance with an with an orderly
orderly arrangement.
arrangement. There
There was was
still still sinusoidal
sinusoidal dilation,
dilation, but
butneutrophils
neutrophils could be be
could observed
observedaccumulated
accumulated around the central
around vein, as
the central wellasaswell
vein, plentyas of Kupffer
plenty cells.
of Kupffer
Liver
cells. tissue
Liver of the
tissue group
of the given
group 100 100
given mg/kgmg/kgbw of bw extract showed
of extract lighter
showed liver liver
lighter damage than the
damage than50the
50 mg/kg
mg/kgbw bw group,
group,butbut
notnot
better than
better the group
than given
the group silymarin.
given The composition
silymarin. The composition of cellsof
in cells
the liver
in the
tissue of the high dose group was regularly arranged, and no visible
liver tissue of the high dose group was regularly arranged, and no visible sinusoidal dilation was sinusoidal dilation was
observed, although there was also a more regular arrangement of hepatocytes. After administration of
silymarin, the liver histology showed only slight damage and approached normal circumstances.
Sci. Pharm. 2017, 85, 16 7 of 7

4. Conclusions
A combination of INH and rifampicin given daily for seven consecutive days can cause significant
rat liver intoxication. Ethanolic extracts of V. amygdalina leaves at 46 and 92 mg/kg bw (corrected to its
water content)—equivalent to 3.58 and 7.17 g of dried leaves—were shown to be able to protect liver
intoxication caused by a combination of INH and rifampicin.

Acknowledgments: Thank to ITB Biotechnology Biomedical Research Center director for research facility.
Author Contributions: All the authors were involved in the design of the experiments; Segia Louisa Sjahlim
performed the experiments, analyzed the data and contributed reagents/materials/analysis tools and wrote the
paper; Maria Immaculata Iwo and Siti Farah Rahmawati involve in analyses the data and in the writing and
editing of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest

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