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3rd International Conferences and Workshops on Basic and Applied
Sciences 2010 ISBN: 978-979-19096-1-7
B023
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: [email protected]
**Faculty of Veterinary Airlangga University, Surabaya,
Indonesia
***Faculty of Fisheries and Marine Sciences University of
Brawijaya, Malang, Indonesia
Abstract
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.
Keywords: Achyrantes aspera linn, alkaloid, myeloma cell,
mitotic, metaphase, viability cell
1 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
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I.D.P. Anom Adnyana, The Effect of Alkaloid Fraction Jarong
(Achyrantes aspera linn) Leaf on Viability and Mitotic Myeloma Cell
Mice
B023
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).
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.
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.
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.
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.
Thawing was done by centifugated myeloma cells in Rosewell Park
Memorial Institute medium(RPMI) at 1500 rpm for 5 min at 4 C.
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.
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 viability
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).
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.
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).
3 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
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3rd International Conferences and Workshops on Basic and Applied
Sciences 2010 ISBN: 978-979-19096-1-7
B023
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 Conclusions
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
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.
[10] Meyer, D.J. and J.W. Harvey. 2003. Veterinary Laboratorium
Medicine. Interpertation and Diagnosis. W.B. Sounders Company.
Philadelphia.
[11] Nala, N. 2002. Obat Tradisional Usada Penyakit kanker.
Upada Denpasar
-
I.D.P. Anom Adnyana, The Effect of Alkaloid Fraction Jarong
(Achyrantes aspera linn) Leaf on Viability and Mitotic Myeloma Cell
Mice
B023
[12] Siswandono. 1983. Mekanisme Kerja Obat-obat 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
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B023
Table 1: Viability Myeloma Cell
Table 2: LSD Myeloma Cell Test Results
Mean SD Optical Density Treatment
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
Cell Treatment Replication Live Dead Total
% Viability cell live
% Viability cell death
1 176 18 194 90.7 9.3 2 138 5 143 96.50 3.50 3 164 7 171 95.90
4.1
P 0
4 203 12 215 94.4 5.6 1 87 198 285 30.5 69.5 2 82 168 250 32.8
67.2 3 78 158 236 33.1 66.9
P1
4 58 171 229 25.3 74.7 1 55 119 174 31.6 68.4 2 48 168 216 22.2
77.8 3 73 203 276 26.4 73.6
P2
4 38 117 155 24.5 75.5 1 13 138 151 8.7 91.3 2 22 112 134 16.4
83.6 3 12 156 168 7.1 92.9
P3
4 10 171 181 5.5 94.5 1 23 117 140 16.4 83.6 2 3 205 208 1.4
98.6 3 9 142 151 6.0 94
P4
4 6 109 115 5.2 94.8 1 6 124 130 4.6 95.4 2 13 136 149 8.7 91.3
3 27 117 144 18.8 81.2
P5
4 31 119 150 20.7 79.3
