HISTOPATHOLOGICAL CHANGES IN LIVER AND KIDNEY OF

WISTAR RATS THAT GIVEN ETHANOL EXTRACT OF ALLIUM

CHINENSE AND PHALERIA MACROCARPA WITH
ADMINISTRATION OF ISONIAZID-RIFAMPICIN COMBINATION
Velen Etania1, Nurhayati Siregar2, Priskah Ade Putri Lubis3, Ali Napiah Nasution4
Faculty of Medicine, Universitas Prima Indonesia, Medan, Indonesia
Email : velenetania999@gmail.com

ABSTRACT
Allium chinense often used as herbs and spices in batak cuisine specialities meanwhile
Phaleria macrocarpa used as traditional medicine. Which is both of them are widely spread
all over Noth Sumatra, and contains high antioxidant. The purpose of this research is to find
out the histopathological description of liver and kidney that induced by INH-RIF, and that
were being given ethanol extract of Allium chinense and Phaleria macrocarpa. This research
is an experimental research. In this experiment, thirty rats are divide into ten groups and each
group are given a different treatment, this experiment was carried out for 10 days. The result
shows that INH-RIF cause harm to the liver and kidneys. Given that EEBB and EEBMD with
dosage 3,4 g/ 200 g BB can act as hepatoprotective and nephroprotective from the toxicity of
isoniazid and rifampicin and the result of histopathology shows minimal damage to almost
resemble to normal. Administration ethanol extract of Allium chinense and Phaleria
macrocarpa together with anti-tuberculosis drugs shows the protective effect of the liver and
kidneys againist the INH-RIF drugs.

Keywords : Allium chinense, Phaleria macrocarpa, Isoniazid, Rifampicin

INTRODUCTION and anti-proliferation activity and is
World Health Organization (WHO) reported to be potentially anti-tumor
contrive strategy to end tuberculosis in chemotherapy (Wang et al., 2016). The
2030 (WHO, 2019). Indonesia government results of Parapaga, Durry and Lintong,
also targeting to be free from tuberculosis 2018 shows that antioxidant activity are
in 2050 (Depkes RI, 2018). Compliance in capable to inhibit and prevent oxidative
taking anti-tuberculosis drugs is plays an damage. Antioxidants can also help
important role in the success of the suppress the production of free radicals
treatment of tuberculosis (Sitorus, and protect cells from oxidative stress
Fatmawati and Rahmaniah, 2017). (Oktaria, 2019).
The most common side effects that are The purpose of this research is to
taking first-line anti-tuberculosis are discover the effect Phaleria macrocarpa
hepatotoxicity and followed by extract and Allium chinense extract on the
nephrotoxicity (Pratiwi, 2018). side effects arising from the combination
Histologically, the combination of of anti-tuberculosis drugs based on the
isoniazid and rifampicin causes mild to regimens list on the liver and kidneys in
moderate kidney damage (Muzika et al., order to be applied as herbal medicine in
2016). among the community.
The Crown of Gods (Phaleria
RESEARCH METHODS
macrocarpa) is traditional medicinal
The research use an experimental
ingredients that are often used to treat
design with the post-test only control
various diseases and contains antioxidant,
group design pattern. This research was
antimicrobial, and anti-inflammatory
conducted in the Pharmaceutical
activities (Hendra et al., 2011). Phaleria
Laboratory, the University of North
macrocarpa can protect the liver of rat that
Sumatra for 3 months starting from
induce by paracetamol based on the Lestari
October-December 2019.
research, 2019. Ramadhan research, 2019
30 male white rats Wistar strains
also states that this fruit have an effect as a
(Rattus Novergicus) samples, weighing
nephroprotector to rat that induced by
150-200 grams. Handling rats according to
paracetamol.
the experimental animal code of ethics
Batak onions (Allium chinense) are
(Ethical clearance from the Universitas
spices that use in typical Batak dishes and
Prima Indonesia, Faculty of Medicine-
have intense antioxidant activity (Sinaga,
Animal Research Ethics Commision).
2016). A. chinense have anti-inflammatory
 Tools used: Laboratory glassware,
blender, incubator, oven, rotary
evaporator, water bath, animal scales,
animal house, injection equipment,
surgical equipment, microtome, glass
object, glass cover, microscope. The
materials are Phaleria macrocarpa, Allium
chinense, aquadest, isoniazid, rifampicin,
CMC-Na 0.5%, buffer formalin 10%,
ethanol 96%, Hematoxylin-eosin dye. The
phytochemical analysis using Dragendorff,
Mayer, Bouchardat reagent , HCl, Mg
powder, amyl alcohol, Pb(CH3COO)2,
H2SO4, chloroform, isopropanol, Molish
reagent, FeCl3, Liebermann-Burchard
reagent.
Making simplisia
Wash the Allium chinense and Phaleria
macrocarpa with flowing water. Slice
thinly the bulbs and cut the fruit into small
pieces after that dry and blend it until
delicate into the powder then put into a
tightly sealed container.
Making ethanol extract
Macerate 500 mg simplisia with ethanol
96%. Evaporate the product by rotary
evaporator at a temperature of ≤ 70oC. Re-
evaporate with water bath until the product
of the extract becomes viscous. (Lestari,
2019; Ramadhan, 2019).
Phytochemical analysis
Phytochemical analysis using
Harborne (2006) methods: alkaloids test: 1
ml HCl and 9 ml distilled water dissolve
0,5 gram simplisia. Add 3 of reaction tubes
containing 0.5 ml filtrate with 2-drop
solution of Mayer, Bouchardat and
Dragendorff reagent. The result is positive
when there is a deposition on 2 or 3
attempts. Saponins and flavonoids test: 10
gram simplisia dissolve in 10 ml of hot
water and shaken firmly, the saponins are
positive if the foam formed as high as 1-10
cm. Add 0.1 grams of Mg powder, 1 ml of
HCl and 2 ml of amyl alcohol to the
filtrate, if there is a red, yellow or orange
color on the amyl alcohol layer then the
flavonoids are positive. Glycosides test:
Dissolve 3 g simplisia with ethanol 96%
and added 10 ml of H2SO4. Add 20 ml
filtrate with 25 ml Pb(CH3COO)2 and then
filter it 3 times, after that mix 20 ml
mixture of chloroform and isopropanol in
each filter process. Add 2 ml aquadest, 5
drops of Molish reagent and 2 ml of
H2SO4 to 0,1 ml filtrate. The glycosides
are positive when forming a purple ring on
the boundary of two liquid. Tannins test: 1
g simplisia boil in 100 ml Aquadest. Add 2
drops of reagent FeCl3 to 2 ml filtrate,
stated positive if there are change to blue
or dark green. Steroids- triterpenoids test:
macerate 1 g simplisia with 20 ml n-
hexane. Add a few drops of the
Liebermann-Burchad reagent. The Steroids
is considered positive when the solutions
change to blue or turquoise color,
otherwise the triterpenoids are positive
when the solution change to red, pink or RESULT AND DISCUSSION
violet. Phytochemical analysis

Grouping of Research Treatment A phytochemical analysis is one way to

The samples are divided into 10 determine the secondary metabolite

groups, so each group numbered 3 rats. content of plant sample.

The treatment of each group is as follows: Table 1 Qualitative Phytochemical

Group control (Group 1) without treatment Analysis Results

and aquadest only. Group 2, 3 and 4 No. Content EEBB EEBMD

1 Alkaloids (+) (+)
Sequential-induced isoniazid 300 mg/ 70
2 Flavonoids (+) (+)
kg BB, rifampicin 450 mg/ 70 kg BB and
3 Saponins (+) (+)
combination of INH-RIF. Give ethanol
4 Tannins (-) (+)
extract of Phaleria macrocarpa with
5 Glycosides (+) (-)
dosage 3,4 g/ 200 g BB that induced by
Steroids/
isoniazid, rifampicin and combination into 6 (-) (-)
Triterpenoids
group 5, 6 and 7. Add ethanol extract of Description : (+) : contain classes of compounds; (-) :
Allium chinense 3,4 g/ 200 g BB that does not contain classes of compounds.

induced by isoniazid, rifampicin and Allium chinense and Phaleria

combination into group 8, 9 and 10. The
macrocarpa have the same chemical
treatment is given orally bases once every compound in the form of alkaloids,
day for 10 days. flavonoids and saponins. Besides
Preparation of a Liver Surgery glycosides are found in A. chinense and
Preparation tannins in P. macrocarpa. Ethanol is the
At the end of the experiment, rats safest and more effective way to bind these
were anesthetized using chloroform. phytochemical compounds. (Gberikon,
Perform surgery to the rats in order to take Adeoti and Aondoaclaa., 2015).
the liver and kidneys then put it into a
Histopathology of the liver
sample bottle which contains 10% The histopathology study of liver was
formalin buffer solution and label it. The also performed which shows
preparation is made with a thickness of 4-6 hepatoprotection effect by looking at the
mm using microtome and dyed with staging of liver cell damage (hydropic
hematoxylin-eosin stain then seen under degeneration, inflammatory cells
the microscope to see histopathology infiltration and necrosis).
changes.
 Rats that are given ethanol extract of
Phaleria macrocarpa with isoniazid
induction only (group 5) have hydropic
degeneration and mild inflammatory cell
infiltration. Group 6 that given same
extract induced with rifampicin only,
Figure 1 Photomicrograph of the normal
pointing to the normal without significant
liver (HE, 10x).
damage. Group 7 (Fig 2) with visible mild
In the control rats, group 1 (Fig 1) is hydropic degeneration, inflammatory cell
seen normal hepatocyte cells arranged infiltration and had slightly necrosis.
radial with a centralist vein in the center.
Rats induced with isoniazid 300 mg/ 70 kg
BB (Group 2) are obtained by moderate
hydropic degeneration with severe
inflammatory cell infiltration and necrosis.
In group 3, rats induced with rifampicin
Figure 3 Photomicrograph of the liver rat
450 mg/ 70 kg BB are obtained moderate
Group 10 = Ethanol extract of Allium
hydropic degeneration, moderate
chinense + INH-RIF. (HE, 40x)
inflammatory cell infiltration and necrosis.
Description: hydropic degeneration (red
The combination of INH-RIF have severe
arrow), inflammatory cell infiltration (blue
hydropic degeneration with severe
arrow), necrosis (black arrow).
inflammation and found necrosis.
Rats given ethanol extract of Allium
chinense that induced by isoniazid (Group
8) and with induced by rifampicin (Group
9) are not found having liver cell damage
and lead to normal histology. In Figure 3,
there is mild inflammatory cell infiltration
Figure 2 Photomicrograph of the liver rat
without any damage.
Group 7 = Ethanol extract of Phaleria
It shows the Batak onion extract and
macrocarpa + INH-RIF. (HE, 40x)
the Crown of God fruit extract are capable
Description: hydropic degeneration (red
to protect the liver induced by isoniazid
arrow), Inflammatory cells infiltration
and rifampicin.
(blue arrow), necrosis (black arrow).
Histopathology of the kidney
The histopathology study of the
kidney is also performed which shows
nephroprotection effect by looking at the
staging of renal damage (glomerular
Figure 5 Photomicrograph of the renal rat
atrophy, widening lumen of tubules and
group 7 = Ethanol extract of Phaleria
necrosis).
macrocarpa + INH-RIF. (HE, 10x)
Description: glomerular atrophy with
necrosis (black arrow).

In Figure 5, the presence of mild

glomerular atrophy with necrosis and a
slight widening of lumen of the tubule.
Figure 4 Photomicrograph of the normal
Rats that are given Allium chinense
renal (HE, 10x).
extract with Isoniazid induction (Group 8)
In control rats (Fig 4), the glomerular were seen has a mild glomerular atrophy
and the kidney tubules look normal, the and by induction of rifampicin (Group 9)
nuclei are clearly visible and the bowman has a mild glomerular atrophy with slight
capsule looks apparent. Rats that induced necrosis.
by isoniazid (Group 2) had moderate
glomerular atrophy and necrosis. In group
3 that induced by rifampicin found
moderate glomerular atrophy with necrosis
and mild widening lumen of tubules.
Combination of INH-RIF (Group 4) had
severe glomerular atrophy with necrosis Figure 6 Photomicrograph of the renal rat

and a moderate dilatation of tubular. Group 10 = ethanol extract of Allium

Rats that are given ethanol extract of chinense + INH-RIF. Description:

Phaleria macrocarpa with induced glomerular atrophy (black arrow) and

isoniazid only (Group 5) are seen mild widening the lumen of the tubule (blue

glomerular atrophy with necrosis, whereas arrow).

induced by rifampicin (Group 6) is found

mild glomerular atrophy only.
 In the treatment of group 10 (Fig 6), An overview of histopathology
there is a mild glomerular atrophy with assessed in the liver in the form of
necrosis and a dilation of mild tubular hydropic degeneration characterized by
tubules. swelling of the cell with a small to large
The administration of Allium chinense vacuole contains water, inflammatory cell
extract and Phaleria macrocarpa extract infiltration and necrosis characterized by a
are capable to protect the kidneys from the change in cell morphology and The cell
induced isoniazid and rifampicin. nucleus becomes smaller, crushed until it
vanish. In the kidney organ, glomerular
Discussion
atrophy is characterized by decreased
This study is conduct to find out the
glomerular tissue size due to reduced cell
differences between the ethanol extract of
count. Dilation of tubular tubules is due to
Allium chinense and Phaleria macrocarpa
protein cast or protein deposits in the
in the white rat-induced isoniazid and
tubules till the lumen is widening.
rifampicin. Administration ethanol extract
Isoniazid and rifampicin are shown to
of Allium chinense and Phaleria
cause damage to liver and kidneys organs
macrocarpa at a dose 3.4 g/200 g BB can
compared with use of isoniazid or
neutralize liver and kidney damage caused
rifampicin only, from the liver
by isoniazid and rifampicin with
histopathology results found severe
histological indicators of the liver and
hydropic degeneration, inflammatory cell
kidneys in rats.
infiltration as well as necrosis and on
Based on the results of the study in the
kidney is start atrophy and glomerular
negative control group i.e. the mouse
necrosis accompanied by widening of the
without treatment only given aquadest
lumen of tubule. It is in sync with the
only has mild inflammation. The negative
contraindicated administration of isoniazid
control group should have no damage to
and rifampicin due liver disorders and
the cells of hepatocytes, it can be caused
chronic renal failure (Rian, 2010).
by an outside variable that cannot be
Interaction isoniazid with rifampicin is
controlled, such as the condition of rat
rifampicin increased hepatotoxic
psychology or the initial condition of the
metabolites of isoniazid drugs (Permala et
rat liver and kidneys before treatment .The
al., 2010).
environment changes also affect rats
10 day of giving the ethanol extract of
behavior patterns (Suharyadi, Sukohar and
Allium chinense and ethanol extract of
Muhartono, 2014).
Phaleria macrocarpa has no toxicity,
since there is no death and poisoning in
white rats during the research. Based on
the histopathology that has
researched, Ethanol extract of Allium
chinense and Phaleria macrocarpa are
able to protect the liver and kidneys due to
the induction of isoniazid and rifampicin
because they have high antioxidant.
The antioxidant content of flavonoids
such as CYP3A as activity inhibitors also
able to acts as free radical scavenger,
tannins can directly bind to free radicals
and toxic metabolites of drugs and
saponins serve as antibacterial and anti-
fungi (Hassanin, Abd El-Kawi
Hashem, 2013). Flavonoids compounds in
Allium chinense bulbs and Phaleria
macrocarpa fruit has influence
protecting the liver and kidneys.
CONCLUSION
The results of this study showed influence
after administration of ethanol extract of
Allium chinense and ethanol extract of
Phaleria macrocarpa at a dose of 3,4 g/
200 g BB reduced liver and kidney tissue
damage as evidenced by histopathology
evaluation. Co-administration of them
along with anti-tuberculosis drugs showed
hepatoprotection and nefroprotection in
some extent againist isoniazid
rifampicin drugs. Combination of isoniazid
and rifampicin are more toxic rather than
using only one of them.
ADVICE
a) For the next studies to administering
ethanol extract of Allium chinense and
been
Phaleria macrocarpa with different
dosages.
b) For the next studies to extending trial
time.
c) For the next studies to do quantitative
phytochemical analysis of ethanol
extract of Allium chinense and
Phaleria macrocarpa
ACKNOWLEDGMENTS
Author are thankful to the dr. Ali Napiah
Nasution, M.K.T as supervising lecturer
who has been helping guide and be a
counselor during the research.
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