The 3 rd Annual International Conference Syiah Kuala Unive rsity (AIC Unsyiah) 2013 [614821]

Proceedings of
The 3 rd Annual International Conference Syiah Kuala Unive rsity (AIC Unsyiah) 2013
In conjunction with
The 2 nd International Conference on Multidisciplinary Rese arch (ICMR) 2013
October 2-4, 2013, Banda Aceh, Indonesia

319
Asses humoral immunity of wistar strains rats
(Rattus norvegicus L.) which given ethanol extract-
buas buas (Premna pubescens Blume) leaves
Martina Restuati and Restianna Eliawaty Simanjuntak
Department of Biology, State University of Medan, Me dan, 20221, Indonesia.
Coorresponding Author: [anonimizat]

Abstract. The aim of the research was to to assess the effect of ethanol extract Premna pubescens leaves
on humoral immunity and the body weight of rats ( Rattus norvegicus L.). The white rats use 24 tails and
devided into 4 groups with each group replicates 6 times. There are A0 as control which give with
aquadest, A1 give with ethanol extract of Premna pubescens leaves, A2 give with ethanol extract of
Premna pubescens leaves and Sheep Red Blood Cells (SRBC), and A3 give with SRBC only. White Wistar
rats aged 3 months with body weight 100-300 grams. Aquadest and ethanol extracts of Premna pubescens
given orally every day and SRBC given by injections on the 8th and 15th day . The rats blood taken b y
decapitation on 31th day. Measurements antibody titer of the rat as humoral immunity used
hemagglutination method using a V microplate 96. Th e results were analyzed statistically with ANOVA. The
result showed that the antibody titer at A2 increas ed significantly , where t (65,78) > t table (4,94 ). This
suggests that the ethanol extract of Premna pubescens leaves influence the humoral imunnity of Wistar
strains rats using SRBC, The result of body weight statistically, t count 0.23 < t table 3.10. It means that ethanol
extract of Premna pubescens leaves did not influence the body weigh.
Keywords : Haemagglutination, Premna pubescens Blume, SRBC, antibody titers

Introduction
The efforts to improve body immunity system against sp ecific antigen is needed to be
concern. Medicinal plants are source of important the rapeutic aids for alleviating human
ailments. It is also good for self medication because i t could not occur any side effects. The
utilization of medicinal plants is less expensive d an easy to be found compared with
synthetic medicinal. Thus natural products have be en a major source of drugs for centuries.
In tune with this effort, the objective set for the p resent investigation is to identify
the leaves of Premna in order to determine the nature of the principle com ponent
responsible for its medicinal property. Premna pubescens Blume is the one of genus of
Premna that can be found in Indonesia but not familiar to be used as medicinal plant. It
has reported that Premna pubescens has a biological activity. The leaves are anti-
inflammatory, antibacterial, antitumour (Patel,2007) . Similarly, Thiruvenkatasubramaniam
(2010) defined that the extracts of P. corymbosa on blood glucose levels and serum lipid
profile possesses a hypoglycemic effect. There was s ignificant reduction in total cholesterol,
LDL cholesterol, VLDL cholesterol and improvement in HD L cholesterol in diabetic rats.
These biological activity is along with the chemical constituent in Premna pubescens .
This plant has an apigenin from flavonoid. Apigenin i s a nontoxic dietary /uniFB02avonoid that has
been shown to possess anti-tumor propertie and therefor e poses special interest for the
development of a novel chemopreventive and/or chemothe rapeutic agent for cancer. Fang
(2005) demonstrated that apigenin inhibits expression of vascular endothelial growth factor
(VEGF) in human ovarian cancer cells. VEGF plays an important role in tumor angiogenesis
and growth. It was found that apigenin inhibited VE GF expression at the transcriptional level
through expression of hypoxia-inducible facto 1 (HIF-1 ).. These /uniFB01ndings reveal a novel role
of apigenin in inhibiting HIF-1 and VEGF expression that is important for tumor angiogenesis
and growth.
Gupta (2001) also defined that apigenin is capable o f selectively inhibiting cell
growth and inducing apoptosis in cancer cells withou t affecting normal cell. Apigenin has

Proceedings of
The 3 rd Annual International Conference Syiah Kuala Unive rsity (AIC Unsyiah) 2013
In conjunction with
The 2 nd International Conference on Multidisciplinary Rese arch (ICMR) 2013
October 2-4, 2013, Banda Aceh, Indonesia

320
been shown to suppress angiogenesis in melanoma and ca rcinoma of the breast, skin, and
colon (Liu, 2005). Regarding apoptosis, apigenin de creased in the protein expression of Bcl-
2 protein and this anti-apoptotic factor might be also participated in the apoptotic process
(Zheng, 2005)
Based on these recent studies, it need to investigat e the utilization of Premna
pubescens Blume as immunomodulator by measurements antibody t iter of the rat ( Rattus
norvegicus L.) as humoral immunity.

Materials and Methods

Animals
Male and female Wistar rats ( Rattus norvegicus ) weighing 100-300 g. were collected from
Chemistry Laboratory in State University of Medan. T hey were housed under standard
laboratory conditions of light, temperature and relat ive humidity. The animals were given
standard rat pellets and tap water ad libitum . The rats were randomly divided into four
experimental groups: A 0 (control), A 1 (Etanol extract of Premna pubescens ), A 2 (Etanol
extract of Premna pubescens + SRBCs), A 3 (SRBCs).

Antigen
The antigen that used in thus research was sheep re d blood cells (SRBCs) Fresh blood was
collected from sheep sacrified in the local slaughte r house, in Veterinary Laboratory, Medan.

The design of research
The white rats use 24 tails and devided into 4 grou ps with each group replicates 6 times.
There are A 0 as control which give with aquadest, A 1 give with ethanol extract of Premna
pubescens leaves, A 2 give with ethanol extract of Premna pubescens leaves and Sheep Red
Blood Cells (SRBC), and A 3 give with SRBC only. Aquadest and ethanol extract s of Premna
pubescens given orally every day and SRBC given by injections on the 8th and 15th day .
The rats blood taken by decapitation on 31 th day.

Materials and Methods
Preparation of premna pubescens leaf aqueous extract
3450 Premna pubescens fresh leaves were air-dried at room temperature. 1 250 gr of
the air-dried leaves of the plant was milled into fin e powder. The powdered leaf was
macerated in etanol and extracted twice, on each occa sion with 12.5 1itre of etanol at room
temperature for 5 days. The combined aqueous extract s olubles were concentrated to
dryness in a rotary evaporator. The resulting aqueous extract was freeze-dried, powdery
crude aqueous leaf extract of Premna pubescens . Aliquot portions of the crude plant extract
residue were weighed for use on each day of experimen ts.

Determining dose of premna pubescens
Doses of the test formulation were determined based on Vadivu (2009), 250 mg/kg
p.o. It was administered orally to Wistar rats for 31 days. The etanol extract mixed CMC 1
%.

Determining antibody levels by the haemagglutinatio n technique
a) Preparation serum
Blood was collected by decapitation process on day 30 th . Then the blood centrifuged and
serum was obtained.
b) Hemaglutination reaction

Proceedings of
The 3 rd Annual International Conference Syiah Kuala Unive rsity (AIC Unsyiah) 2013
In conjunction with
The 2 nd International Conference on Multidisciplinary Rese arch (ICMR) 2013
October 2-4, 2013, Banda Aceh, Indonesia

321
The antibody titer was determined by a two-fold seri al dilution of one volume (100 /uni00B5l) of
serum and one volume (50 /uni00B5l) of physiological salin e (reagent). Quantitation of serum
antibodies were carried out by antibody titre plate technique containing respective antigens.
50/uni00B5l of physiological saline was added into all wells of microtitre plate, and then 100/uni00B5l of
antiserum added into the first well of microtitre pla te, the antiserum was serially diluted in
the well of the first row till the 11 th well of the microtitre plate leaving the 12 th well as
positive control. Then 25/uni00B5l of 1% test antigen (SRBC s) in saline was added to all the wells
of the microtitre plate. The plate was were mixed tho roughly for the effective mixing of
reagents and incubated for an hour at 37° C for about 1 day until the control tube showed a
negative pattern (a small button formation). The hi ghest serum dilution showing visible
hemagglutination was taken as the antibody titer .
Statistical analysis
The data was analysed using one-way analysis of vari ance (ANOVA), P<0.05 was
considered significant.

Results and Discussion
Antibody titer
Antibody titer in this research was found by calcul ated total antibody titer.
Measurement antibody titer of the rat as humoral immu nity used hemagglutination method
using a V microplate 96
Table 4.1. The average humoral immunity of Rattus norvegicus by antibody titre after
treating.
No. Treatment Antibody Titer
1. A 0 1
2. A 1 1,67
3. A2 7,17
4. A3 6,67

Antibody titre by HA Test and weight of Rattus norvegicus for each treatment had a
various result after giving ethanol extract of Premna pubescens . The highest level was A 2
with 7,17 of the average antibody titre. The data w as tested by ANOVA one way test.
Table 4.2 The analysis of ANOVA one way, the effect of ethanol extract of Premna
pubescens treatment toward humoral immunity at Rattus norvegicus.
Variance Df JK KT F count F table
0,05 0,01
Treatment 3 189,13 37,82 65,78 3,10 4,94
Error 20 11,5 0,575 – – –
Total 23 200,63 – – – –

Based on the statistic by using ANOVA one way, it wa s found that F count = 65,78>
Ftable (0,01) = 4,94. This result indicated that there wa s an effect of ethanol extract of
Premna pubescens toward humoral immunity at Rattus norvegicus .

Proceedings of
The 3 rd Annual International Conference Syiah Kuala Unive rsity (AIC Unsyiah) 2013
In conjunction with
The 2 nd International Conference on Multidisciplinary Rese arch (ICMR) 2013
October 2-4, 2013, Banda Aceh, Indonesia

322

Body weight
Tabel 4.3. The average of Rattus norvegicus weight after administered the treatment.
No. Experiment Weight
1. A 0 12,33
2. A 1 8,5
3. A2 13,17
4. A 3 18,5

To investigate the difference of the average for ea ch group, data was tested statistically
with ANOVA one way (Table 4.5).

Table 4.4. Analysis of Variance (Anova) of the effe ct ethanol extract of Premna pubescens
toward Rattus norvegicus body weight.
Variance Df JK KT F count F table
0,05 0,01
Treatment 3 305,46 61,09 0,23 3,10 4,94
Error 20 5221,18 261,06 – – –
Total 23 5526,62 – – – –

Based on statistic test by using Anova one way, it w as found that F count = 0,23 >
Ftable (0,01) = 4,94. This result indicated that H 0 accepted and H a rejected. It means that
the effect of ethanol extract of Premna pubescens has not an effect toward body weight of
Rattus norvegicus .
The effect of ethanol extract of Premna pubescens tow ard humoral immunity of Rattus
norvegicus
Positive reaction show a positive effect if SRBCs depos ition dispersed covering wells of
microtitre plate. Deposition proceed from a webbing be tween antibody with SRBCs until
cover much of the wells of microtitre plate. Whereas the negative reaction show SRBCs
deposition collected among the wells of microtitre plat e because there has not antibody and
antigent is excessive. Regarding weight effect of an tigent, so it will collected among the
wells of microtitre plate construct an deposition like a pink button (Achyat, dkk. 2008).
Regarding the result of this research, the average of antibody titer for each
treatments had a different value. The group that ha d a highest value was A 2. This caused
the imunization with SRBCs for twice. This result wa s along with A 3 that antibody titer had a
high value. It caused A 3 already experienced with SRBCs. Group of A 0 and A 1 also formed an
imunity humoral although these group never had experi enced with SRBCs.
Serum of A 0 had an hemaglutination reaction but in low value ab out 0-1. Meanwhile
A1 had a higher value between 1-3. This caused by ser um contain anti antigent of SRBCs
although it never experience with SRBCs. Similar wit h the previous research, Achyat, et al
(2008) reported that the antigent is natural antibo dy. In A 3 was found that the antibody
titer was high. It caused by A 3 had an imunization with SRBCs for twice. The anti body titer
of A 3 was higher than A 0 and A 1 because the group had produced antibody in large scal e to
dissolved the antigent when imunization.

The effect of ethanol extract of Premna pubescens tow ard the alteration of Rattus
norvegicus body weight
The average of Rattus norvegicus body weight A 1 was lower than A 0. The average of Rattus
norvegicus body weight A 0 had lower than A 2 and A 3. This caused by the difference of disire

Proceedings of
The 3 rd Annual International Conference Syiah Kuala Unive rsity (AIC Unsyiah) 2013
In conjunction with
The 2 nd International Conference on Multidisciplinary Rese arch (ICMR) 2013
October 2-4, 2013, Banda Aceh, Indonesia

323
for each day. Based on statistic test by using Anova on e way, it was found that ethanol
extract of Premna pubescens has not an effect toward body weight of Rattus norvegicus .

Conclusions
1. Ethanol extract of Premna pubescens has a significant effect in increasing humoral
imunity by using antibody titer at Rattus norvegicus .
2. Giving ethanol extract of Premna pubescens toward body weight of Rattus
norvegicus has not a significant effect.

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