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3rd International Conferences and Workshops on Basic and Applied Sciences 2010

ISBN: 978-979-19096-1-7

The Effect of Alkaloid Fraction


Jarong (Achyrantes aspera linn) Leaf
on Viability and Mitotic Myeloma Cell Mice
I.D.P. Anom Adnyana*, W. Meles** D.K. Meles**, and Y.E. Puspitasari***
*Faculty of Veterinary
University of Brawijaya, Malang, Indonesia
e-mail: dewanom_adnyana@yahoo.com
Faculty of Veterinary
Airlangga University, Surabaya, Indonesia
**

***Faculty of Fisheries and Marine Sciences


University of Brawijaya, Malang, Indonesia

Abstract
Keywords: Achyrantes aspera linn, alkaloid,
myeloma cell, mitotic, metaphase, viability cell

Extract of Achyranthes aspera Linn leaves


wellknown consist saponin, achyrantin, ramnose
glucose, galactose, glicosida triterpenoid, alkaloid
and flavonoid. The aim of the research was to study
the effect of alkaloid fraction of jarong (Achyrantes
aspera linn) leaf as anticancer through cultured cell
of myeloma cell of mice (in vitro) and mitotic
process of cromosom divided. Percentage of the
perceived cell viability of the living myeloma cell is
compared with the dead cell using the negative and
positive control. This research divided into five
group: P0 is control negative,P1 liquid test 1 ppm,
P2 liquid test 10 ppm, P3 liqiud test 100 ppm, P4
liquid test 1000 ppm and P5 positive control of 100
ppm colchisin. The result showed that the cultured
cell in the concentration of 100 ppm and 1000 ppm
have lower viability compared with the
concentration of 1 ppm, 10 ppm and positive
control. In the concentration 1 ppm, the alkaloid
fraction of jarong (Achyrantes aspera linn) killed
more than 50 percentage of myeloma. Myeloma
cell devide in to 3 groups, the first group are given
the concentration of
alkaloid fraction of
Achyranthes aspera Linn, the second groups are
given RPMI 1640 media and DMSO in 3 dillution,
and the third groups are given the concentration of
colchicine were 100 ppm. Determination of
cromosom devided activity by coloration
cromosom methode and analyse by light
microscope in 100 X and 400X. The effect of
alkaloid fraction on the mitotic process of myeloma
cell inhibits cromosom mitotic at metaphase. In
conclusion, the effect of alkaloid fraction of
Achyranthes aspera Linn leaves in concentration
100 ppm and 1.000 ppm on the mitotic process of
myeloma cell inhibits cromosom mitotic at
metaphase stage.

Introduction

Cancer is a chronic disease became the second


largest cause of death in developed countries in the
world, after coronary artery. World Health
Organization (WHO) report that the number of
cancer cases expected to be at least doubled in most
countries over the next 25 years (Siswandono,
1983).
Cancer treatment can be done in various ways such
as
surgery,
radiation,
chemotherapy,
immunotherapy, and endochrynotherapy. Recently
the treatment able to cure one-third of cancer
patients through surgery or radiation therapy unless
before metastases (Gao et al., 2000).
Chemotherapy or anticancer drugs should have a
selective toxicity. Currently, the anticancer drugs
damage cells and cause toxicity due to inhibits the
growth of normal cells such as proliferation rapidly
such as bone marrow, epithelial germinatitum,
gastrointestinal tract and mucosal tissue
lymphocytes. The anticancer drugs kill cancer cells
through a mechanism. It call necrosis, but it cause
inflammatory necrosis and the other involving a
group of cells. Ideally, anticancer drugs kill cancer
cells without harming normal cells but until now
there is no anticancer drug that fulfill these
requirements (Coundry, 1995).
Development of anticancer drugs was done by
empirical screening, rational design, discovery of
new drug compounds and genetic therapy. Plants is
the one of drugs source for chemotherapy. One of
the plants used as anticancer drugs in the
B023

I.D.P. Anom Adnyana, The Effect of Alkaloid Fraction Jarong (Achyrantes aspera linn) Leaf on Viability and
Mitotic Myeloma Cell Mice

community is (Achyrantes aspera Linn) or known


by the name jarong, jarong lalaki or remek getih.
Implementation of the High Throught Screening
(HTS) program to search anticancer drugs from
plants in Indonesia conducted on 22 types of plants
including the plants. Jarong (Achyrantes aspera
Linn) used as an anticancer drug in the breast and
uterus (Sutawijaya, 2001; Nala, 2002).

Supernatant separated
from
the sediment and
then the cells grown in a medium using a mixture
of 10% FBS in the culture bottles. Antibiotics such
as canamycin, streptomycin and penicillin added to
media to prevent contamination. In addition,
FBS was added HEPES and NaHCO3 then kept it
in an incubator culture 95% O2 and C025% at 37
C for 24 hours.

Jarong (Achyrantes aspera Linn) contain various


substances such as saponins, alkaloids, betaine,
akirantin, ramnosa and glucose. Recently, alkaloid
fraction of leaves jarong (Achyrantes aspera Linn)
is still unknown so it is unknown chemical formula
alkaloidnya type. Alkaloid fraction of leaves jarong
(Achyrantes aspera Linn) is known to inhibit cell
division cycle at the metaphase stage (Adyana,
2006). The aim of the research was to study the
effect of alkaloid fraction of jarong (Achyrantes
aspera linn) leaf as anticancer through cultured cell
of myeloma cell of mice (in vitro) and mitotic
process of cromosom divided.

From the results obtained cell number as many as 9


x 10 5 cells/ml. This amount is considered
adequate, so that each microwell plate can be
charged a flat 24 holes 0,2 ml test solution and 0.8
ml of myeloma cell cultured subsequently stored
in 95% O2 incubator and C025% at 37C for
24 hours.
Cell culture harvested with eroded wall pitting each
treatment and the examined under a microscope.
Each treatment is inserted into the vial and 1 ml
was taken, added trhypan blue dye 0.4% 1 ml. Each
treatment is inserted into the vial and 1 ml was
taken, added with the tryphan blue dya 0.4% 1 ml.
Using
the
counting technique
under
the
microscope the Thoma calculated percentage cell vi
ability cell is the number of live cells divided by the
number
of total cells (live
cells
and dead
cells) times 100% (Meyer and Harvey, 2003). The
distance between the staining with cell counting
done not less than 3 minutes and a maximum for 10
minutes this is to avoid false positive results
(Freshney, 1987).

2 Methodology
2.1 Plants Material
This study uses the alkaloid fraction of jarong
(Achyrantes aspera Linn) leaves. A. aspera Linn
leaves were collected from Surabaya, East Java.
Myeloma cell mice get by thawing from myeloma
cell of mice type P3UI.

Calculated the number of living cells (not stained)


and the number of dead cells (stained) are visible in
the area of hemocytometer count. Hemocytometer
count has 9 squares on each side. There are two
rules to calculate the cell, if the cells that touch the
left and top of each count area was incorporated
areas count the cells that touch on the right and
bottom lines count areas were not included in the
count (Meyer and Harvey, 2003). Media containing
myeloma cells prior triptan blue staining diluted
with 0.4%.Before entering the cell to be counted,
hemocytometer closed with a cover glass and filling
the cells first have to meet the space
provided. Calculations
performed
with
a
magnification of 100X.

2.2 Dose selection and mode of administration


Determination doses of alkaloid fraction from
jarong leaves based on preliminary study was
conducted by Wurlina (2000), Wurlina and
Sastrowardoyo (2003), Wurlina et al., (2003) and
Meles (2004). Doses of alkaloid fraction are
100 mg /kg bw (in vivo) so that on studies in
vitro the dose used is a concentration of 1 ppm,
10 ppm, 100 ppm and 1000 ppm.
Preparation of the test conducted by weighing
alkaloid fraction 50 mg and putting it into beaker
glass. Added 1 ml of sterile 10 % DMSO solution.
Then added to 10 ml of distilled water are
mixed until homogeneous and
then,
put
it
into sterile screw-tube in order to obtain 5000 ppm
concentration.

The design of this study using Completely


Randomized Design, the data was analyzed using
one-way analysis of variance (ANOVA) if there is a
real difference test followed by Least Significant
Difference (LSD) (Kusriningrum, 1989).

Negative controls were given only 0.1 ml of 10%


DMSO medium, added 9 ml distillated water,
homogeneous. 0.1 ml was taken and add it 9 ml of
distillated water, homogenenous
Positive control is obtained by add 50 ml Kolkhisin
in a beaker glass, 1 ml of 10 % DMSO, stir it until
dissolved.

Result

The results of ANOVA test using SPSS 14.0


Windows showed count rates F = 161.264 at 95%
confidence level ( = 0.05). There are differences

Thawing
was
done
by
centifugated
myeloma cells in Rosewell Park Memorial Institute
medium(RPMI) at 1500 rpm for 5 min at 4 C.
B023

3rd International Conferences and Workshops on Basic and Applied Sciences 2010

between mean of the treatment groups and


inhibition of cell growth. From the results of LSD
showed that each test solution has a positive
difference with the control solution (kolkhisin 100
ppm). The greater of test concentration is the
smaller the percentage of cell viability.Therefore it
can be concluded that the test solution has a growth
inhibition activity against cancer cells (sitostatica)
which is one of the anticancer mechanism
(Swanson and Pezzuto, 1990). The results of the
study the effects of the alkaloid fraction of leaves
Jarong (Achyrantes aspera Linn) in 0.2 ml of each
treatment against the viability of myeloma cells in
vitro by using a hemocytometer assistance can be
seen
in
Table
1.
In this research showed that the
concentration that kills 50 percent of myeloma cells
is a dose of 1 ppm by the LC50 of 0.719 ppm.The
material under test with a LC50 1000 ppm can be
generally said to be cytotoxic (Mayer et al., 1992),
while based on the criteria of the National Cancer
Institute (NCI) extract is active as an anti-cancer
extracts with LC 50 20 ppm (Swanson and
Pezzuto, 1990). Table 2 showed that dose 1 ppm of
the alkaloid fraction of leaves jarong (Achyrantes
aspera Linn) has been able to kill cancer cells more
than 50 percent. According to Adyana (2006) of the
alkaloid fraction of leaves jarong (Achyrantes
aspera Linn) at doses of 100 ppm and 1000 ppm
give effect on cell cycle by inhibiting mitotic
myeloma cells at metaphase stage. Chabner et
al.(2001) suggests that the alkaloids derived from
plants Vinka specific work on the cell cycle by
inhibiting the process of mitosis. Alkaloid from the
plant also has the ability to bind to tubulin is a
protein that make up microtubules by inhibiting or
blocking polimerasi protein into microtubules.
Alkaloids from plants can cause interference with
the cell membrane so that the resulting components
of the membrane will change and the physiological
processes will interfere with membrane damage and
wrinkling occurs on these membranes (Gill et al.,
2001; Jujena et al., 2001). Myeloma cell death
above 50 percent contained at a dose of 1 ppm
alleged that the administration of the alkaloid
fraction of leaves jarong (Achyrantes aspera Linn)
at a dose of 1 ppm has resulted in the death of
myeloma cells through a process of degeneration,
necrosis and apapotosis.
4

ISBN: 978-979-19096-1-7

concentration of 100 ppm and 1000 ppm. At a


concentration of 1 ppm of the alkaloid fraction of
leaves Jarong (Achyrantes aspera Linn) can cause
the death
of myeloma cells more than
50 percent at 0.716 ppm.

References
[1] Adyana, I.D.P. 2006. Efek Anti Telomerase
Fraksi Alkaloid Terhadap Pembelahan dan
Mitosis Sel Mieloma Mencit. Fakultas
Kedokteran Hewan Universitas Airlangga.
[2] Chabner, B.A., D.P.Rian, L.Paz-Ares, R.G.
Carbonero, and P. Calabresi. 2001.
Antineoplastic Agents In Goodman &
Gilmans The Pharmacological Basis of
Therapeutics 10th. Edition. McGraw-Hill.
Medical Publishing Division. P.1417-1421.
[3] Coundry, E.V. 1995. Cancer Cell. W.B.
Sounder Company Philadelpia and London.
P.136-144.
[4] Gao,X.Y., D.W.Wang, and F.M. Li. 2000.
Determination of Acdysterone in Achyrantes
Bidentata and Its Activity Promoting
Proliferation of Osteoblast-Like Cell. Yao
Xue Xue Bao. Nov:35(11) : 868-870.
[5] Freshney, L.R. 1987. Culture of Animal Cell :
A Manual Basic of Technique . 2nd Edition.
Alan R. Liss Inc. New York. p. 227-292.
[6] Gao,X.Y., D.W.Wang, and F.M. Li. 2000.
Determination of Acdysterone in Achyrantes
Bidentata and Its Activity Promoting
Proliferation of Osteoblast-Like Cell. Yao
Xue Xue Bao. Nov:35(11) : 868-870.
[7] Gill, S.M.K., N. Balasioner, and P. Parte.
2001.
Intermitent
Treatment
With
Taxmoxiven on Reproduction in Male Rat.
Asian. J. Andri 3(2)-P 155-158.
[8] Jujena, P., S. M.K.Gill., S. Dsolisa., V.
Padwai., N. Balasimor., M. Aleem, and Zool.
2001. Anti Fertility Effect Estradiol in Adult
Female Rat. J. Endokrinol. Invest 249(8):
598-607.
[9] Meles , D.K. 2004. Efek Antimitosis Fraksi
Alkaloid Achyrantes aspera linn Pada
Pembelahan Sel Embrio. Disertasi, Pasca
Sarjana Universitas Airlangga.

Conclusions

[10] Meyer, D.J. and J.W. Harvey. 2003.


Veterinary
Laboratorium
Medicine.
Interpertation and Diagnosis. W.B. Sounders
Company. Philadelphia.

Jarong leaf extract alkaloid fraction (Achyrantes


aspera Linn) could inhibit the growth of myeloma
cell cultures.
Jarong leaf alkaloid concentrations(Achyrantes
aspera Linn) that
comes
closest to
the
control positive (kolkhisin
100
ppm) is
the

[11] Nala, N. 2002. Obat Tradisional Usada


Penyakit kanker. Upada Denpasar
B023

I.D.P. Anom Adnyana, The Effect of Alkaloid Fraction Jarong (Achyrantes aspera linn) Leaf on Viability and
Mitotic Myeloma Cell Mice

[12] Siswandono. 1983. Mekanisme Kerja Obatobat Anti Kanker. Buletin ISFI Jatim. Tahun
X. No. 1-2. Hal. 3.
[13] Sutawijaya. I.K. 2001. Berbagai Cara
Pengobatan
Menurut
Lontar
Usada.
Pengobatan Tradisional Bali. CV Indra Jaya.
Singaraja Bali.
[14] Swanson, S.M. and J.M. Pezzuto. 1990.
Bioscreening Tecnique for Cytotoxic Potential
and Ability to Inhibit
Macromolecule
Biosynthesis in : Thomson, E. B. Drug Bio
Screening : Drug Evaluation Tecnique in
Pharmacology. VCH publishers Inc. New
York. p. 273- 295.
[15] Wurlina, 2000. Efek Antifertilitas Infusa Daun
Achyranthes aspera linn Terhadap Siklus
Birahi Pada Mencit, Laboratorium. Ilmu
Kemajiran. Fakultas Kedokteran Hewan
Universitas Airlangga.
[16] Wurlina., W. Sastrowardoyo, dan
D.K.
Meles. 2003. Pengaruh Antimitosis Ekstrak
Etanol Achyranthes aspera linn Terhadap
Perkembangan Embrio (Cleavage) Mencit
(Mus musculinus). Lembaga Penelitian
Universitas Airlangga .
[17] Wurlina dan W. Sastrowardoyo. 2003. Efek
Alkaloid Daun Acyrantes aspera linn
Terhadap
Perkembangan
Sel
embrio
(Cleavage) Mencit (Mus musculinus).,
Lembaga Penelitain Universitas Airlangga.

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3rd International Conferences and Workshops on Basic and Applied Sciences 2010

Table

1:

Viability

Treatment

Myeloma

Cell

Replication
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4

P0

P1

P2

P3

P4

P5

Live
176
138
164
203
87
82
78
58
55
48
73
38
13
22
12
10
23
3
9
6
6
13
27
31

Cell
Dead
18
5
7
12
198
168
158
171
119
168
203
117
138
112
156
171
117
205
142
109
124
136
117
119

Table 2: LSD Myeloma Cell Test Results


Treatment

ISBN: 978-979-19096-1-7

Mean SD Optical Density


Live Cell

Dead Cell

P0

94.388 a 2,5964

5.612 c 2,5964

P1

30. 424 b 3,5838

69. 576 c 3,5838

P2

26. 199 b 3,9992

73.801b 3,9992

P5

13.189 c 7,7519

86.81 b 7,7519

P3

9.456 c 4,8238

90.547 a 4,8238

P4

7.262 c 6,4231

92.738 a 6,4231

B023

Total
194
143
171
215
285
250
236
229
174
216
276
155
151
134
168
181
140
208
151
115
130
149
144
150

% Viability
cell live

% Viability
cell death

90.7
96.50
95.90
94.4
30.5
32.8
33.1
25.3
31.6
22.2
26.4
24.5
8.7
16.4
7.1
5.5
16.4
1.4
6.0
5.2
4.6
8.7
18.8
20.7

9.3
3.50
4.1
5.6
69.5
67.2
66.9
74.7
68.4
77.8
73.6
75.5
91.3
83.6
92.9
94.5
83.6
98.6
94
94.8
95.4
91.3
81.2
79.3