EFFECT OF ETHANOL LEAF EXTRACT OF PAPAYA

(Carica papaya Linn.) ON THE BLOOD PROFILE OF

MICE (Mus musculus Linn.) INFECTED BY
Trypanosoma evansi Steel.
Muhammad Rizki1*, Anni Nurliani1 and Heri Budi Santoso1
1

Department of Biology, Faculty of Mathematics and Natural Sciences,

Lambung Mangkurat University. Jl. A. Yani Km 35.8 South Kalimantan,
Indonesia. Tel. +62-856-51181479, *email: rizkiriver@gmail.com
ABSTRACT
Surra is a disease caused by Trypanosoma evansi S. in cattle. This disease can
cause death in cattle. The resistance to some trypanocidal drugs such as
isometamidium and diminazen azeturat has been reported. This study was
designed to determine the effect of ethanol extract of papaya leaf to blood
profile mice infected T. evansi. The design of this study using RAL with twenty
five male mice were divided into five groups with five repetitions. Control
group P1, P2 group only infected with T. evansi while P3, P4 and P5 is a group
with infected T. evansi with the addition of ethanol extract of papaya each as
much as 75 mg/kg bw, 150 mg/kg bw and 300 mg/kg bw. Infection was
performed by intraperitoneal injection as much 3x107 / 0,2 mL T. evansi.
Parameters observed that the quantity and quality of blood. Papaya extract
was made by maceration method. Data were analyzed using Analysis of Varian
test and duncan test. The results showed that ethanol extract of papaya
leaves can increase the value of erythrocyte, Packed Ccorpuscular Volume,
hemoglobin and platelet were decreased due to infection of T. evansi. Whereas
to the values of leukocytes, eosinophils, lymphocytes, monocytes, Mean
Corpuscular Volume, Mean Corpuscular Hemoglobin dan Mean Corpuscular
Hemoglobin Concentration is decrease. All treatment does not affect the value
of basophils. The conclusion from this study is papaya leaf extract dose of 300
mg / kg was able to restore blood profile in mice approached normal values.
Keywords :T. evansi, blood profile, Carica papaya L., mice and surra.
INTRODUCTION
Surra or trypanosomiasis is a disease which is often suffered by livestock,
especially cattle and buffalo. This disease is caused by a blood parasite
Trypanosoma evansi L. According to Harahap et al. (2013) losses incurred in
cattle due to surra is a form of weight loss, low reproduction even death.
Besides, infections reported to cause immunosuppression or reduced immune
response of the host. As reported by Luckins (1998) economic losses due to
surra in Asian is estimated to reach US $ 1.3 billion every year. Trypanocidal is
a drug which has activity against the parasite Trypanosoma. However, in some
Trypanosoma there has been a resistance to several kinds trypanocidal (Astuti
et al., 2006).
Based on the report Harahap et al. (2013) and Astuti et al. (2006) that the
plants have antiplasmodium activity it can be used antitrypanosoma as such
sernai (Wedelia biflora) and Mindi (Melia azedarach). This is because
Trypanosoma and plasmodium attacks erythrocyte, causing anemia in the
host. Papaya (Carica papaya L.) Is a plant which has a function as

antiplasmodium (Rehene 2010). Under these conditions it is necessary to

know more about the effect of ethanol extract of papaya on Trypanosoma
evansi. Parameters measured include the quantity and quality of blood. The
quantity of blood measured value erythrocytes, leukocytes, platelets,
hemoglobin, Packed Corpuscular Volume, differential leukocyte and
erythrocyte indices, while the quality is a blood condition that observed using
blood smear.
MATERIALS AND METHODS
Plant materials, preparation of plant extracts and phytochemical test
Papaya were taken from the garden located at Landasan Ulin Banjarbaru.
Making the ethanol extract of papaya is done by using maceration method.
Papaya leaves as much 40 grams put in a closed erlenmeyer, then poured
ethanol 96%. The solvent used is replaced every 24 x 3 hours. phytochemical
test based by Harborne (1987). The extract given to the mice for 3 days after
the mice are known to experience parasitaemia. Parasitemia examination in
mice performed using blood smear.
Experimental design
The design of this study used was completely randomized design with twenty
five male mice were divided into five groups with five repetitions. Control
group P1, P2 group only infected with T. evansi while P3, P4 and P5 is a group
with infected T. evansi with the addition of ethanol extract of papaya each as
much 75 mg/kg bw, 150 mg/kg bw and 300 mg/kg bw.
Samples T. evansi
T. evansi isolates came from the collection of BVet Banjarbaru taken from
buffalo. Number of Trypanosoma used as much 3x10 7 T. evansi / 0,2 mL.
Infection was performed by intraperitoneal
Blood sampling
Mice were first anesthetized with ether before blood is taken through the
heart. Blood is drawn using 3 mL syringe. Blood inserted into the tube which
has been filled with a solution of EDTA.
Hematology Test
Calculation of erythrocytes, leukocytes and platelets using a Neubauer
chamber. Packed Corpuscular Volume using microhematocrit method (Pflanzer
2007). erythrocyte indices calculation using the formula described by Meyer
and Harvey (2004). Differential leukocyte count using Shiling method (Mitruka
and Rawnsley, 1997). Whereas examination of hemoglobin levels based by
Hesser (1960).
Data Analysis
The data were analyzed using Analysis of Varian test the level of 5% and
Duncan test to find out the real difference between the results of the
treatment.
RESULT
Papaya Leaf Extract Ethanol
The extraction process produces papaya leaf extract as much 6.3 grams.
rendemen obtained is 15.75%. Phytochemical test results shown in Table 1.

Table 1. Result of Phytochemical test of papaya leaf extract

N
o
1

Coumpoun
Reagent
d
Alkaloid
Wagner
Meyer
Dragendorf
Flavonoid
HCl + Mg
Saponin
Aquades
FeCl3
Tanin
(CHCO)2O + H2SO4
Steroid

2
3
4
5

Result
+
+
+
+
+
+
+

Table 2. Results of the analysis of the number of erythrocytes, leukocytes, PCV, Hb

and erythrocyte indices.

Treatm
ent

Erythrocyt
es

PCV

Hb

Leukocyt
es

MCV

MCH

MCHC

(1 x
10 /mm3)

(%)

(g/dl)

(1x103/m
m3)

(fL)

(pg)

(g/dL)

P0
P1
P2
P3
P4

10,56
47,80
11,00
14,23
45,39
10,40
23,07
0,54a
2,28a
1,00a
0,51a
3,86a
0,67a
2,56a
1,81
18,60
3,50
40,77
102,71
19,36
19,39
0,12b
3,21b
0,07b
0,60b
17,21b
0,97b
4,23b
2,12
23,60
3,80
36,97
111,62
17,98
16,20
0,15b
2,07c
0,12bc
0,60c
13,27b
1,65b
1,52b
3,55
26,20
4,28
34,53
74,90
12,18
16,38
0,47c
2,28c
0,23c
0,29c
13,96c
1,67c
0,92b
5,03
34,40
6,04
31,53
68,45
12,05
17,66
0,29d
2,61d
0,23d
1,51d
4,63c
0,97ac
1,77b
Description:
The number followed by the same letter are not significantly different, otherwise if
numbers followed by different letters are significant differences among treatments
(sig> 0.05).
Table 3. Results of the analysis of the number of Platelet and Differential leukocyte

Treatm
ent
P0
P1
P2
P3
P4

Platelets

Neutrophi
ls

Lymphoc
ytes

Eosinoph
ils

Monocyt
es

Basophil
s

(1 x
103/mm3)

(%)

(%)

(%)

(%)

(%)

35,60
2,88ad
19,80
3,11b
28,00
1,22c
34,00
1,58a
38,20
3,11d

61,20
2,58a
66,60
2,70b
61,80
1,78a
58,00
2,64c
55,80
1,78c

172,80
9,47a
114
9,69b
117,8
6,46b
119,4
8,9b
150
12,21c

1,80
0,83a
10,80
1,92b
8,80
1,30c
6,80
0,84d
4,60
1,14e

1,20
0,84a
2,40
0,55b
1,40
0,89a
0,80
0,84a
1,20
0,45a

0,20
0,45a
0,40
0,55a
0,20
0,45a
0,40
0,55a
0,20
0,45a

Description:
The number followed by the same letter are not significantly different, otherwise if
numbers followed by different letters are significant differences among treatments
(sig> 0.05)

Figure
1.
Blood

Description :
1. Normal Erythrocytes
2. Erythrocyte lysis
3. Trypanosoma evansi
4. Platelets
5. Lymphocytes
6. Macrocytic
7. Microcytic

picture mice in each treatment at 1000x magnification with the Giemsa. The
arrow ( ) in normal erythrocytes showed characteristic rounded shape, while the
arrow ( ) on the cell membrane of erythrocyte lysis showed visible wrinkle.

DISCUSSION
The value of the group P1 erythrocytes decreased. This is according to
research conducted by Arifin (2001). Decrease of erythrocytes caused due to
infection of T. evansi. Damage to the erythrocytes can be caused tripanotoksin
released by T. evansi (Payne et al. 1991). PCV is a percentage value of
erythrocyte so that if the number of erythrocytes decreased, the value of PCV
also decreased (Widjajakusuma and Sikar 1986). The low value of hemoglobin
caused by infection of T. evansi. Value leukocytes of mice in group P1 has
increased. This is according to research conducted by Arifin (2001) that T.
evansi infection can cause leukocytosis.
The value of MCV, MCH, MCHC according to research conducted by Olayanju
et al. (2013) in mice infected with T. evansi. MCV value in P1 group increased.
Macrocytic anemia caused decreased erythrocyte so immature erythrocytes
should be released to sufficient erythrocytes in the body (Tsantes et al. 2004).
MCH will increase in macrocytic anemia caused by a deficiency of vitamin B12
and folic acid. Lack of vitamin B12 and folic acid can be caused by infection
with Trypanosoma (Sangwan et al. 1993). MCHC decrease in group P1 is the
effect of anemia. Platelet counts mice infected T. evansi experience
thrombocytopenia. This is consistent with research Kipper et al. (2011) in mice
infected with T. evansi.
Results In Value Neutrophils, monocytes and eosinophils appropriate by Arifin
(2001). the value of neutrophils in the group P1 decreased. According Happi et
al. (2012) a decrease of neutrophils due to the effects of anemia. Erythrocytes
have a role in protecting the neutrophil apoptosis. On the value of monocytes
and eosinophils increased in the group P1. This is because monocytes have
phagocytic function (Guyton and Hall 2008), while eosinophils have a function
as mediators from mast cells inactivated (Stockham and Scott 2002). Value of
lymphocytes in group P1 has increased. This is according to research
conducted by Paim et al. (2011) in mice infected with T. evansi. The value of

basophils in the group P1 is not different from other groups. This is because of
basophils does not play a role in fighting parasitic infections and the low
presence of basophils in the blood. Basophils usually play a role in allergic
reactions such as asthma and dermatitis (Obata et al. 2007).
Papaya has the potential to improve the process of erythropoiesis in mice
(Dharmarathna et al. 2013). Research Ikpeme et al. (2011) showed that the
ethanol extract of papaya leaves can increase the value of RBC, Hb and PCV in
mice. This is because papaya contains iron that can help in the formation of
red blood cells. Besides papaya has secondary metabolites are suspected to
have activity in inhibiting Trypanosoma. Flavonoids may inhibit cellular
respiration process protozoa. The ability of flavonoids to inhibit Trypanosoma
evidenced by Umar et al. (2010). Alkaloids will bind to heme and converted
into compounds hemozoin. hemozoin are toxic compounds that are harmful to
the parasite (Hans and Joachim Knlker 2014). Inhibiting the activity of
alkaloids in Trypanosoma evidenced by Merschjohann et al. (2000). Saponins
may also interact with the antigen in inhibiting Trypanosoma (Johnson et al.
1963). Tannins can destroy Trypanosoma membrane by a protein complex
bond and hydrophobic effect (Taylor 2000). Steroids have a role in inhibiting
the growth of the parasite by stimulating the production of Nitric oxiden (NO)
in macrophages (Balestieri et al. 2002).
CONCLUSION
Ethanol extract of papaya dose of 300 mg/kg bw dose is most influential in
improving the quality of the blood of mice from infection Trypanosoma evansi
ACKNOWLEDGMENTS
The authors are thankful to Department of Biology, Faculty of Mathematics
and Natural Sciences, Lambung Mangkurat University, Banjarbaru, Indonesian
and Bvet banjarbaru for providing financial support for the present work. The
authors are grateful to drh. Ichwan and drh. Nur Jannah for the laboratory
facilities provided.

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