SHORT COMMUNICATION Anti Lithiasis Activity of Avocado (Persea americana Mill) Leaves Extract in White Male Rats

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HAYATI Journal of Biosciences March 2012
Vol. 19 No. 1, p 49-52
EISSN: 2086-4094
Available online at:
http://journal.ipb.ac.id/index.php/hayati
DOI: 10.4308/hjb.19.1.49
SHORT COMMUNICATION
Anti Lithiasis Activity of Avocado (Persea americana Mill)
Leaves Extract in White Male Rats
IETJE WIENTARSIH∗∗
∗∗
∗, RINI MADYASTUTI, BAYU FEBRAM PRASETYO, ANGGARA ALDOBRATA
Department of Clinic, Reproduction, and Pathology, Faculty of Veterinary Medicine,
Bogor Agricultural University, Darmaga Campus, Bogor 16680, Indonesia
Received February 17, 2011/Accepted February 24, 2012
In Indonesia, avocado leaves have been used as traditional medicines for diureticum to cure urolithiasis. This
research was to determine anti lithiasis activity of avocado leaves (Persea americana Mill) extract on white male
rats nefrolithiasis model induced by ethylene glycol. Ethanol extraction method was used to get extract of
avogadro leaves. Twenty adult male white rats were divided into 4 different induction treatments i.e. aquadest,
ethylene glycol 0.75% and ammonium chloride 2%, and extract of avocado leaves with different levels of 100 and
300 mg/kg bw respectively. Their body weight was measured daily to determine their growth ratio. And at the end
of the trial, the kidney was analyzed its calcium level and inhibitory activity to formation of calcium oxalate
crystals. The results showed that the amount of calcium level in the kidney of rats treated with extract of
avogadro leaves was significantly decreased than that of rats treated with ethylene glycol 0.75% and ammonium
chloride 2% (P < 0.05). The extract avocado leaves as a herbal remedy can be recommended as a phytotherapeutic
agent especially for preventive action for urolithiasis diseases.
Key words: Persea americana Mill, anti lithiasis, kidney stone, ethylene glycol
___________________________________________________________________________
_________________
∗∗
∗∗
∗Corresponding author. Phone/Fax: +62-251-8623940,
E-mail: wien_tje@yahoo.com
INTRODUCTION
Nephrolithiasis is the third prevalent disorder in the
urinary system. Approximately, 80% of these nefrolith are
composed of calcium oxalate (CaOx). Nephrolith is a solid
particle like gravels that exists in every part of urinary
system. It is formed by excessive salt in the blood-stream
and then it crystallizes in the kidney. The size and shape
of the gravel is vary, from the very tiny particle that flowing
undetectably through the blood stream till the gravel’s
size. However, even the tiniest gravel or stone is able to
cause the great pain when it moves out of the kidney.
Minor bleeding can be seen when the gravel causes the
wound inside urinary system. In most cases kidney stone
are source of discomfort and inconvenience, progressive
lost of renal function can occur after repeated episodes of
stone disease (Park & Pearle 2007).
Chemical medicines are used only to prevent growing
stones to be bigger and helping excretion of kidney stones
(nephrolith) spontaneously. Diuretics are commonly used
in the treatment of hypertension and other disorders
associated with edema and heart disease.
Apparently avocado leaves are one natural ingredient
that can be used as a traditional medicine. The leaves
have been used as a diuretic, anti-inflammation, anti-
hypertension, hypoglycaemia, diarrhea, sore throat, and
bleeding (Brai et al. 2007). However, people only notice
the good effect of avocado leaves is based on empirical
evidence. Therefore, this study determined the effect of
avocado leaves extract on lithiasis.
MATERIALS AND METHODS
Persea americana Mill. leaves were collected from
Research Institute for Herbs and Spicy Bogor Indonesia.
Persea americana powder was macerated with ethanol
70% for 48 h and the extract was concentrated to get a
semisolid extract using a rotary evaporator. Temperature
of the evaporator was maintained below 60 oC. The P.
americana (avocado) leave extract was evaluated for
presence of various phytoconstituents by performing
different qualitative chemical tests.
Twenty male Sprague Dawley (200-250 g) were used
in this study. Experimental Animals were acclimated for
seven days for adaptation. Animals were fed ad libitum.
Animals were divided randomly into 4 groups (n = 5) and
were treated as follow: group A were untreated and served
as normal control, group B, C, and D were induced with
0.75% ethylene glycol and 2% ammonium chloride in
drinking water ad libitum for ten days. Group C and D
were fed orally with ethanol extract P. americana solution
with different levels (100 and 300 mg/kg bw).
After ten days experiment period, the animals were
anesthetized by ether and the kidney samples were taken
for analysis. The left kidney was dried in oven at 100 oC

for 24 h, then weighed and minced in a beaker glass
containing 7 ml of 0.5 N nitric acid. The mixture was heated
until the liquid became transparant. Calsium concentration
was determined by atomic absorption spectroscopy
method.
RESULTS
Phytochemical Assay. Persea Americana (Avocado)
leaves powder which was macerated with ethanol and then
evaporated produced 34% yield extract. The result of
phytochemical assay showed that extract of avocado
leaves contains alkaloid, tannin, saponin, tritherphenoid,
and flavonoid (Table 1).
Body and Kidney Weight, and Their Ratio. There was
body weight increasing until eighth day of experiment in
the control. On the other hand there was significant body
weight decreasing (P < 0.05) on the rats treated with extract
Avogadro leaves 100 and 300 mg/kg bw compared to that
of treated with 0.75% ethylene glycol and 2% ammonium
chloride (Figure 1 and Table 2).
Average body and kidney weight and its ratio of
control was 314.98, 1.12, and 0.71 g, respectively.
Meanwhile on the rats treated with 0.75% ethylene glycol
and 2% ammonium chloride, the body weight, kidney
weight and ratio was 278.8, 1.12, and 0.77 g, respectively.
Average of body weight of the rats treated with extract of
Avogadro leaves 100 and 300 mg/kg bw was 238.45 and
253.73 g, respectively. And the kidney weight was 0.86
and 0.97 g respectivey, and the ratio was 0.72 and 0.76
respectively.
There was a decreasing in ratio of body and kidney.
However, statistical analysis showed that there was no
significant effect (P > 0.05) on the creasing of body and
kidney weight ratio. Compared to that of treated with 0.75%
ethylene glycol and 2% ammonium chloride, the
differences of decreasing of body weight on rats treated
with extract of Avogadro leaves 100 mg/kg bw was up to
7 %, while on rats treated with extract of Avogadro leaves
300 mg/kg bw was 1%.
Calcium Concentration in the Rat Kidney. Statistical
analysis of calcium concentration in kidney renal showed
that there was significant different (P < 0.05) on treatments
extract of Avogadro leaves 100 and 300 mg/kg bw, compared
to positive control of 0.75% ethylene glycol and 2%
ammonium chloride (Table 3). The data showed that extract
of avocado leaves could decrease concentration of calcium
in the kidney.
DISCUSSION
Extraction process in general used 70% ethanol to
extract active compounds such as polyphenols,
flavonoids, tannin, and alkaloids. Ethanol’s polarity is less
than water’s polarity, therefore degrading cell walls and
seeds that have unpolar character will be efficient (Lapomik
et al. 2005).
Phytochemical assay of extract of avocado leaves was
conducted to observe the type of secondary metabolite
compounds of extract contents. The result showed that
the presence of flavonoid is much higher than other
Table 1. Phytochemical assay of Persea americana (avogadro)
leaf extract of ethanol extraction
Compound Result
Flavonoid
Alkaloid
Tannin
Saponin
Tritherphenoid
++++
++
+
+
+
+ = detected.
200
225
250
275
300
325
350
12345678910
Body weight (g)
Time (day)
Figure 1. Body weight of adult male white rats induced with four
different treatments in 10 days of experimental period.
= normal control, = positive control, =
treated dose 100 mg/kg bw, = treated dose 300 mg/
kg bw.
Table 2. Average of body and kidney weight and their ratio of adult male white rats induced with four different treatments
Weight A B C D
Body (g)
Kidney (g)
Ratio
314.98 + 7.97c
1.12 + 0.08
0.71
278.80 + 23.63b
1.07 + 0.24
0.77
238.45 + 18.47a
0.86 + 0.26
0.72
253.72 + 17.64a
0.97 + 0.26
0.76
Different superscript indicates that is significantly different (P < 0.05, n = 5). A = normal control, B = positive control, C = treated dose
100 mg/kg bw, D = treated dose 300 mg/kg bw.
Tabel 3. Renal calcium concentration
A B C D
Ca concentration (mg) 0.075 + 0.013b0.139 + 0.079a0.060 + 0.012b0.067 + 0.009b
Different superscript indicates that is significantly different (P < 0.05, n = 5). A = normal control, B = positive control, C = treated dose
100 mg/kg bw, D = treated dose 300 mg/kg bw.
50 WIENTARSIH ET AL. HAYATI J Biosci

compounds such as alkaloid, tannin, saponin, and
tritherpenoid (Table 1). According to Wientarsih et al.
(2000), avocado’s leaves contain saponin, alkaloida,
flavonoids, polyphenols, and quersetin. According to
Spigno and Faveri (2007), antioxidant activity was higher
when the extract contains more flavonoids. Flavonoids
are polyphenolic compounds which widespread in the
plant and have diverse pharmacological effects such as
anticancer, antioxidant, antiaging and antibacteria (Xu et
al. 2007). According to Singh (2005), derivate of flavonoid
can repair the damage of the kidney by capacity of
antioxidant activity and as radical scavenger. The effect
of avocado’s leaf extract in the both treatment significantly
decreased calcium level in the kidney compared to the
positive control (0.75% ethylene glycol and 2%
ammonium chloride treatment) because of much higher
flavonoid content in the extract. According to Fouada et
al. (2006), flavonoid property of extract of avocado’s leaves
(with ethanol extraction) can prevent attachment of CaOx
crystals and stop the further process in the formation of
kidney stones. Flavonoid can prevent adhesion of crystal
calcium oxalate by scavenging effects in prevent injury
by free radical and resulting more stable radical. In other
words, flavonoids stabilize the reactive oxygen species
by hydroxyl group which has high reactivity according to
the following equation : Flavonoid (OH) + R· → Flavonoid
(O·) + RH (Nijveldt et al. 2001).
Ethylen glycol (EG), as a nephrotoxic agent, is
commonly used as an experimental regiment to induce
animal models of calcium oxalate nephrolithiasis.
Therefore, EG was used in this experiment to make
hiperoxaluria condition.
Hyperoxaluria is a major risk factor of calcium oxalate
disease and leads to increasing of calcium oxalate
supersaturation and calcium oxalate stone formation.
Exposure to oxalate has been shown to be toxic to renal
epithelial cells, which results in lipid peroxidation mediated
by free radicals. Oxalate toxicity is mediated, in part, by
activation of lipid signaling pathways that produce
arachidonic acid, lysophospholipids, and ceramide. These
lipids, in turn, are able to disrupt mitochondrial function
by increasing production of reactive oxygen intermediates
(ROI), decreasing mitochondrial membrane potential, and
increasing mitochondrial permeability (Meimaridou et al.
2006).
Effect of the avocado’s leaf extract in different levels
in animals could decrease ratio of body and kidney weight.
Nephrolithiasis increase kidney weight due to
inflammatory reactions which has high mineral content in
the kidney. Cruzan et al. (2004) stated that white rats body
weight were decreased due to the high doses of ethylene
glycol poisoning as well as the increasing of kidney weight
and kidney weight ratio relative to body weight. Increasing
of kidney’s weight was due to inflammation process and
mineral content. In this research, the activity of flavonoid
as anti-inflammation made the ratio of kidney’s weight
decreased. The role of flavonoid in preventing formation
of crystal calcium oxalate due to the anti-inflammatory
action through inhibition of leukotriene synthesis,
prevention of histamine release (Kheir eldin et al. 2008)
and inhibition of neutrofil degranulation.
On the measurement of calcium level in the kidney
(Table 3), The calcium level of rat kidney in Avogadro leaf
extract treatment was lower than that of the positive control.
The extract of avocado’s leaves could decrease calcium
level in the kidney and therefore showed inhibition of
calcium oxalate nephrolith. According to Khan et al. (2000)
calcium oxalate crystal and high oxalate levels in the
nephrons can damage the epithelial cells and
consequently, the cell may produce some products as well
as free radical inducing heterogenous crystal nucleation
and cause aggregation of crystals.
Crystal formation and retention are critical events for
the formation of kidney stones. Oxalate and calcium
oxalate (CaOx) crystals are injurious to renal epithelium.
Membranes of injured cells promote crystal adherence
and retention.
Ratio of kidney and body weight depends on the size
and thickness of the kidney itself (Baker et al. 1979).
Kidney’s abnormality in the positive control treatment is
caused by inflammation and mineral deposit condition.
The ratio of positive control was 0.77 and the ratio of the
Avogadro leaf extract 100 mg/kg bw was 0.72, anti-
inflammatory and anti oxidant activity of avocado’s leaf
extract could decrease ratio of kidney and body weight
(Table 2).
In conclusion, extract of avocado’s leaves with ethanol
extraction is an efficient agent to inhibit formation of
calcium oxalate crystal in the rats’ kidney. The extract of
avocado’s leaves is potential natural antioxidant and anti
inflammation compounds that able to prevent formation
of calcium oxalate crystal by interfering process of
epithelial cell damage.
ACKNOWLEDGEMENT
This study was supported by Directorate of Higher
Education Competency Grant number 0154/023-04.2/XII/
2009 to Ietje Wientarsih.
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