The PHILIPPINE JOURNAL OF

Veterinary Medicine
Volume 55 Special Issue December 2018

Published by the College of Veterinary Medicine

University of the Philippines Los Baños

ISSN 0031-7705
The 2ndVeterinary Medicine
International Conference
Surabaya, Indonesia
4-5 July 2018
 The Philippine Journal of Veterinary Medicine
Volume 55 Special Issue December 2018

The Philippine Journal of Veterinary Medicine is a peer-reviewed international journal of

basic and applied research in veterinary medicine and science. It is published semi-annually, for
the periods January-June and July-December each year, by the College of Veterinary Medicine,
University of the Philippines Los Baños. All articles are subjected to double-blind review.
Authors of articles appearing in the journal are solely responsible for opinions expressed therein.
All rights reserved. No article of the journal may be reproduced in any form and by any means
without a written permission from the publisher or the Editor-in-Chief.

EDITORIAL BOARD:

Jezie A. Acorda, DVM, MAgr, PhD

Editor-in-Chief

Dennis V. Umali, DVM, PhD

Associate Editor

Joseph F. dela Cruz, DVM, MS,

PhD Remil L. Galay, DVM, PhD
Technical Editors

Jesalyn L. Constante, DVM, MS

Business Manager

SUPPORT STAFF:
Ms. Jocelyn E. Arcinas
Mr. Fernando P. Micosa

The annual subscription price is US$100.00 (net) for foreign subscribers (inclusive of mailing cost) and
Philippine PhP1,500.00 plus mailing cost for local subscribers. Prices for current single issue and back issues
are available on request. Subscriptions are accepted on a prepaid basis only and are entered on a calendar year
basis. Issues are sent by air delivery to foreign subscribers.

All communications should be addressed to:

The Editor-in-Chief
Philippine Journal of Veterinary Medicine
College of Veterinary Medicine
University of the Philippines Los Baños
Laguna, Philippines 4031
Telefax Nos. +63-49-536-2727, +63-49-536-2730
Email: pjvm1964@gmail.com, vetmed_uplb@yahoo.com.ph

This journal is Abstracted/Indexed by: SCOPUS, Biological Abstracts, Focus on: Veterinary Science &
Medicine, Zoological Records, CAB Abstracts, Index Veterinarius, Veterinary Bulletin, Parasitology Database,
Helminthological Abstracts, Protozoological Abstracts, Review of Medical and Veterinary Entomology, EBSCO,
ASEAN Citation Index, Prescopus Russia, i-journals (www.ijournals.my), i-focus (www.ifocus.my), i-future
(www.ifocus.my), Philippine E-Journals (https://ejournals.ph) and UPLB Journals Online (http://journals.
uplb.edu.ph/index.php/PJVM).

© 2018 College of Veterinary Medicine, University of the Philippines Los Baños

 The Philippine Journal of Veterinary Medicine
Volume 55 Special Issue December 2018

CONTENTS

Original Articles

Medicine
Viability of Rabbit Adipocyte Stem Cells Cultured Under Different Oxygen
Concentrations In Vitro. ........................................................................................................... 1
E Safitri, P Srianto, TV Widiyatno, W Sandhika and RH Prasetyo

Microbiology
Antigenic Site of Glycoprotein Encoding Gene in Rabies Virus Isolate from
Indonesia. .................................................................................................................................. 9
J Rahmahani, S Suwarno and FA Rantam

Characterization of Newcastle Disease Virus Lentogenic Strain Infected

Native Chickens from Surabaya, Indonesia. ........................................................................ 17
FA Rantam, R Ernawati, AP Rahardjo, IL Rahmawati, D Kartika,
NS Widjaja and J Rahmahani

Nutrition
Effect of Concentrate to Forage Ratio on Milk Urea Nitrogen, Milk Production
and Reproductive Performance of Dairy Cows. .................................................................. 25
S Utama, S Mulyati, W Wurlina and I Mustofa

Pathology
Toxicity, Stability and Renal Histopathology of Alkaloid of Jarong (Achyranthes
aspera Linn.) (Caryophyllales: Amaranthaceae) Leaf on Mice. ......................................... 35
DK Meles, W Wurlina, I Mustofa, S Zakaria, A Basori, M Hariadi, E Safitri,
DKSC Putri and N Suwasanti

Histochemical Expression of Transforming Growth Factor Beta and Tumor

Necrosis Factor Alpha in Rabbits Infected with Sarcoptes scabiei. ............................... 43
SM Rizki, LT Suwanti and NDR Lastuti

Pharmacology
Effect of Alkaloid of Achyrantes aspera Linn. (Caryophyllales: Amaranthaceae) on
Increasing Caspase 9, Caspase 3 and Apoptosis in Mice with Breast Cancer. ................. 51
W Wurlina, DK Meles, I Mustofa, E Safitri, S Zakaria, A Basori, DKSC Putri
and N Suwasanti

Theriogenology
Effect of Aluminum Silicate on the Spermatozoa, Plasma Membrane and
Seminiferous Tubules of Mice Exposed to Fusarium graminearum
(Sordariomycetes: Hypocreales: Nectriaceae) ..................................................................... 59
Samik, S Mulyati, T Hernawati and E Safitri
 Research Notes

Microbiology
Isolation and Identiftcation of Lactic Acid Bacteria from the Digestive Tract of
Kampung Chicken (Gallus gallus domesticus). ...................................................................67
B Yulianto, WP Lokapirnasari

In Vitro pH Tolerance, Bile Salt Resistance and Antimicrobial Activity of

Lactobacillus plantarum Isolated from Crossbred Cattle. ................................................. 73
WP Lokapirnasari, AM Sahidu, L Maslachah, K Soepranianondo,
AB Yulianto, D Afikasari, TB Pribadi and I Hariyati

Nutrition
Amino Acid Sequence of Signal Transducers and Activators Transcription
Proteins From Broilers. ........................................................................................................ 79
A Ma’ruf, NMR Widjaja, N Hidajati and R Damayanti

Parasitology
Antigenic Protein Proftle of Anisakis spp. Larvae Isolated from Mackerel
Tuna Fish (Euthynnus sp.).................................................................................................... 85
ZN Wastomi, NDR Lastuti, R Ernawati, LT Suwanti, S Koesdarto,
M Mufasirin and HM Raharjo

Morphological Detection of the Intestinal Parasite Blastocystis sp. in Fresh

and Cultured Feces of Pet Sugar Glider (Petaurus breviceps) in Surabaya,
Indonesia. .............................................................................................................................. 91
F Natalia, LT Suwanti, E Suprihati, Kusnoto, S Koesdarto and P Srianto

Pathology
Comparative Histopathologic Changes in Rabbit (Oryctolagus cuniculus) Skin in
Relation to Degree of Infestation with Sarcoptes scabiei. ............................................ 97
A Azhimah, NDR Lastuti, A Arimbi, D Legowo, P Hastutiek and LR Yustinasari

Pharmacology
Effect of Sapogenin from Sambiloto (Andrographis paniculata) (Lamiales:
Acanthaceae) on Creatinine and BUN Levels and on Gentamicin-Induced
Nephrotoxicity in Rats. ....................................................................................................... 103
S Zakaria, W Wurlina, DK Meles, I Mustofa, M Hariadi, S Susilowati, E Safitri,
A Basori, DKSC Putri and N Suwasanti

Public Health
Identiftcation of Shiga Toxin-Producing Escherichia coli in Raw Milk Samples
from Dairy Cows in Surabaya, Indonesia. ....................................................................... 109
MH Effendi, N Harijani, SM Yanestria and P Hastutiek

Tetracycline Resistance Gene in Streptococcus agalactiae Isolated from Bovine

Subclinical Mastitis in Surabaya, Indonesia. ................................................................. 115
MH Effendi, A Oktavianto and P Hastutiek

Theriogenology
Bacterial Isolates from the Cervical Mucus of Dairy Cattle at Follicular and
Luteal Phases. ..................................................................................................................... 121
K Sudrajad, SP Madyawati, W Tyasningsih, R Rimayanti, P Srianto and
OS Widodo
 Human Chorionic Gonadotropin (hCG) from Urine of Pregnant Women for In Vitro
Maturation of Madura Cattle Oocytes. ...............................................................................127
HA Hermadi, RTS Adikara, M Hariadi and E Safitri

Effect of Bovine Seminal Protein on the Quality of Frozen Spermatozoa from

Goats. ..................................................................................................................................... 133
S Susilowati, IN Triana, TW Suprayogi, A Arimbi and W Wurlina

Editorial Policies. ............................................................................................................................. 139

Guidelines for Authors......................................................................................................................141

1
Sciences 16(1): 40-52.
 51
ORIGINAL ARTICLE

EFFECT OF ALKALOID OF Achyrantes aspera Linn. (CARYOPHYLLALES:

AMARANTHACEAE) ON INCREASING CASPASE 9, CASPASE 3 AND
APOPTOSIS IN MICE WITH BREAST CANCER

Wurlina Wurlina*1, Dewa Ketut Meles1, Imam Mustofa1, Erma Safttri1, Sunarni
Zakaria2, Achmad Basori 2, Desak Ketut Sekar Cempaka Putri3
and Niluh Suwasanti 3

1
Department of Veterinary Reproduction; 2Department of Pharmacology; 3Faculty of Medicine,
Universitas Airlangga, Surabaya, East Java, Indonesia

ABSTRACT

It has been proven that Achyranthes aspera Linn. alkaloid can improve
apoptosome (cytochrome C and APAF-1) formation in the mitochondria of cancer
cells. However, it is yet to be seen whether chromosome fragmentation by caspase 9
and caspase 3 can induce apoptosis in breast cancer cells in mice. Fifty two-month
old adult female mice were used in this study and were equally divided into ftve
groups. Healthy mice (C-) were given orally with 0.5 ml of carboxy methyl cellulose
(CMC) solvent, while mice with breast cancer (C+) were given 0.5 ml of 15 mg/kg
body weight methotrexate. To test for the effect of alkaloid on breast cancer cells, T0
group, mice with breast cancer, was only administered with 0.5 ml of CMC solvent.
T1, T2 and T3 each received 0.5 ml of A. aspera alkaloid, at a dose of 75, 100 and 125
mg/kg body weight. respectively. All groups received the same treatment daily for
eight weeks. Rate of apoptosis in mice breast cancer cells and expression of caspase 9
and caspase 3 were measured. This study suggests that Achyranthes aspera alkaloid
can increase number of apoptotic cells and enzymes caspase 9 and caspase 3.

Key words: Achyranthes aspera, alkaloid, apoptotic cell, breast cancer, caspase 3,
caspase 9

Philipp. J. Vet. Med., 55(SI): 51-58, 2018

INTRODUCTION

Non-contagious diseases are the primary
cause of death among Indonesians. Cancer
ranked sixth in causing mortality in Indonesia,
only a rank behind cardiovascular disease.
Consquently, the right chemotherapeutic and
anticarcinogenic medications are needed, the
ideal remedy of which are target-specific drugs,
without causing damage to the normal cells. It
has, however, remained a challenge to develop
these drugs, especially when resistance is
involved.
*FOR CORRESPONDENCE:
(email: wurlina_made@yahoo.co.id)
The indigenous herb Achyranthes aspera
is one of the anticancer herbal medicines
empirically used for its alkaloid, which
has been shown to induce antimitosis and
antitelomerase in mice myeloma cell type
P3UI, stopping at metaphase stage. It has the
ability to bind tubulin, a protein that arranges
microtubules by inhibiting or blocking protein
polymerization into microtubules, thus
damaging the microtubules and inducing
apoptosis or cell death (Wurlina, 2005;
Wurlina, 2006; Wurlina et al., 2008; Meles et
al., 2017a).
One way by which apoptosis occurs is
52 WURLINA et al.
by mitochondrial damage. Mitochondria is
responsible for oxidation reaction and cell
reduction in producing energy in the form
of adenosine triphosphate (ATP). Likewise,
it plays a role in cell metabolism, and fat
and protein synthesis. Molecular approach
shows that mitochondrial damage will lead
to increased cytochrome C synthesis, which
stimulates apoptotic protease activating factor
1 (APAF-1) formation, and, along with dATP,
will form apoptosome, causing the PT pore to
open. These changes induce the caspase 9, the
caspase initiator, and activate caspase 3, the
caspase executor, causing excessive DNAase,
which leads to chromosome fragmentation
and ultimately causes cell death (Lantuejoul
et al., 2004; Wurlina et al., 2010a; Adnyana et
al., 2012).
Based on empirical principle, theoritical
and research results, alkaloid from
Achyranthes aspera containing caspase 9 and
caspase 3 can induce apoptome formation,
causing chromosome fragmentation and
eventual cell death of breast cancer cells in
mice induced by benzo(a)pyrene. This study
assumes that increase in apoptosis in mice
breast cancer cells are associated with the
increase in caspase 9 and caspase 3 induced
by the alkaloid A. aspera.
MATERIALS AND METHODS
Alkaloid processing
Achyranthes aspera powdered leaves
were weighed to the desired amount, soaked
in n-hexane solvent and filtered with Buchner
filter using a vacuum pump. The pulps were
macerated (to dissolve fat and chlorophyll)
with the same solvent until the solution was
clear. Samples were macerated with 10%
tartaric acid methanol, filtered and sediments
were remacerated. NH 4OH was added to
filtrates and polar salt. Chloroform was mixed,
and the filtrate was evaporated until a thick
mass had formed. Alkaloid concentration was
measured by HPLC, examined by thin-layer
chromatography and processed with column
cromatography to purify alkaloid (Meles et al.,
2017a).
A. aspera alkaloid
Dosage determination of A. aspera
alkaloid as an anticancer drug was based on
prior research conducted by Wurlina (2005).
The dose rate of 60 mg/kg body weight in vitro
was deemed an appropriate amount, such that
in vivo research correlated with the volume of
mice body fluids, which consist of extracellular
and intracellular fluid, making up 60% of
body weight. If all given drug dosages were
distributed to extracellular and intracellular
fluid, the effective dose in vivo would be 100/60
× 60 mg/kgbw. According to Wolf and Wagner
(1977) as cited in Wurlina (2005), minimum
and maximum dose are based on drug dose
logarithm: log 10, log 30, log 100, log 300, log
1,000 and so on. Effective dose for 100 mg/kg
bodyw weight is between dose of 30 and 300, so
the minimum dose would be equal to log 30/log
100 × 100 mg/kg body weight = 75 mg/kgbw;
effective dose would be equal to log for 100/
log 100 × 100 mg/kg body weight = 100 mg/
kg body weight. Meanwhile, maximum dose
would be equal to log 300/log 100 × 100 mg/
kgbw = 125 mg/kg body weight. As a result,
drug dosage for this study was set at 75 mg/kg
body weight as the minimum dose, 100 mg/kg
body weight as the effective dose and 125 mg/
kg body weight as the maximum dose.
Inducing breast cancer in mice
Fifty two-month old adult female mice with
breast cancer were injected subcutaneously,
around the nipple, with 10 mg/kg body weight
of benzo(a)pyrene at 3-day interval for eight
weeks. Meanwhile, 10 approximately two-
month old healthy female adult were used
as negative control. Based on Wurlina et al.
(2010a), breast cancer in mice proliferated
between 6th to 8th week. Its occurence was
proven by macroscopic and microscopic tests,
as evident from swollen mammary gland and
mass of cancer cells seen during biopsy. Cancer
cells are marked by more than one nuclear
cell, defined by active growth and division.
Alkaloid treatment
Fifty two-month old female with breast
cancer were divided into five groups, consisting
of 10 mice per group; another 10 healthy female
mice were used as negative control. Negative
 EFFECT OF ALKALOID A. aspera ON MICE WITH BREAST CANCER 53

control (C-) received oral 0.5 ml of carboxy positive reaction denoted by chocolate color.
methyl cellulose (CMC) solvent. Positive Apoptotic or necrotic cell death data and
control (C+) was given 0.5 ml of 15 mg/kg body caspase 9 and caspase 3 expression were then
weight methotrexate (standard anticancer analyzed with analysis of variance (ANOVA)
drug). To test for the effect of the alkaloid on using SPSS. Significant differences were
breast cancer cells, T0 group, mice with breast further tested with Duncan’s Multiple Range
cancer, was only given 0.5 ml of 0.5% CMC Test.
solvent. T1 to T3 each received 0.5 ml of A.
aspera alkaloid, at 75, 100 and 125 mg/kg body
weight, respectively. Same treatments were RESULTS AND DISCUSSION
given for eight weeks. Immunohistochemistry
Breast cancer cells post-alkaloid
(IHC) was used to determine the rate of
treatment
apoptosis in breast cancer cells and expression
Table 1 shows that a higher dose of A.
of caspase 9 and caspase 3.
aspera alkaloid induced a higher rate of
apoptosis in mice breast cancer cells than
Calculation of necrotic and apoptotic
lower dose. There was a significant difference
cells
(P<0.05) among negative control (C-), positive
Treated mice breast cancer cells control (C+) and alkaloid treatment groups
were dissected and prepared for (T1-T3) in terms of cancer cell death that led
immunohistochemistry by staining with to necrosis. This shows the effect of A. aspera
ethidium bromide and acridine orange. alkaloid and methotrexate treatment on breast
Specifically, 100 ppm acridine orange solution cancer cell death. However, no significant
was mixed in a vial with equal amount of difference (P>0.05) was seen between positive
ethidium bromide in PBS solution. Twenty- control and treatment groups. It means
five µl cell suspension was mixed with 1 µl that the alkaloid at a dose of 75 mg/kg body
mixed dyes and observations were made weight to 125 mg/kg body weight produced
immediately. Ten µl cell suspension was the same effect as that of 15 mg/kg body
placed on a glass object with glass cover and weight methotrexate. Meanwhile, there was
observed with a minimum of 300 cells under a no significant difference among treatments
fluoresence microscope at 400× magnification. T1, T2 and T3, suggesting no significant effect
Apoptotic cells appeared orange with on increasing alkaloid dosage in inducing
a bright spot, which denotes chromatin apoptosis on mice breast cancer cells.
condesation and DNA fragmentation; necrotic Cancer cells positive for apoptosis show
cells were orange but without the bright spot; significant difference (P<0.05) among negative
and normal cells were green (Fig. 1). The control, positive control and all treatment
percentage of apoptopic cells was calculated groups. This shows significant apoptosis
according to the following formula: in breast cancer cells given with A. aspera
apoptotic cells alkaloid or methotrexate, when compared with
% apoptotic cells = x 100% negative control. Meanwhile, there was no
∑������������������ ��� � � � + ∑���������������� ���� � � � + ∑�� ����� ���� � � � significant difference (P>0.05) between positive
control and treatments given alkaloid dose of
75 and 125 mg/kg body weight, suggesting no
Immunohistochemical examination substantial difference in apoptosis in breast
Processed breast cancer cells were cancer cells, regardless of increasing alkaloid
subjected to a labeled strepavidin biotin II dose when compared to methrotrexate dose.
and neutral buffer formalin fixation with 3-4µ In the positive control group, cell death
incised paraffin block. Antibody monoclonal by necrosis (7.38+0.58%) and apoptosis
caspase 9 and caspase 3 kit was used. (0.44+0.06%) occurred. This was expected
Observations were made under a microscope since normal cells that grow and develop
at 400× magnificaiton. Caspase 9 and caspase will normally die either from physiological or
3 expression were measured by quantifying
54 WURLINA et al.

Fig. 1. Staining of living (green), apoptotic (orange with yellow spot) and necrotic (brown) in mice
with breast cancer.
C-: healthy female mice given only 0.5 ml of 0.5 % carboxy methyl cellulose (CMC) solvent; C+:
mice with breast cancer given 0.5 ml of 15 mg/kg body weight methotrexate; T0: mice with
breast cancer given 0.5 ml of 0.5% CMC; T1: mice with breast cancer given 0.5 ml of 75 mg/
kg body weight A. aspera alkaloid; T2: mice with breast cancer given 0.5 ml of 100 mg/kg
body weight A. aspera alkaloid; and T3: mice with breast cancer were given 0.5 ml of 125 mg/
kg body weight A. aspera alkaloid.

Table 1. Mean percentage of different cells of mice given increasing doses of Achyranthes aspera.
Treatment Living cells (%) Necrotic cells (%) Apoptotic cells (%)
C- 92.2+2.59a 7.4+0.58b 0.4+0.06c
C+ 14.2+7.15d 56.4+4.33a 29.4+3.76a
T0 32.5+3.48b 56.3+6.66a 11.2+4.36b
T1 23.4+3.99c 50.2+6.29a 26.4+4.69a
T2 10.6+3.71d 53.1+3.62a 36.3+3.37a
T3 8.3+4.42de 58.7+5.47a 33.0+3.61a
Means with different superscripts within the same column differ significantly (P<0.05).

pathological changes. According to Yalon et and disturbances in mitosis or meiosis.

al. (2004), almost a thousand cells that grow
and develop in the body, either young or old,
will eventually meet cell death by apoptosis or
necrosis, which can be pathological, because
of exposure to chemicals and ultraviolet
rays, or physiological, due to certain bodily
processes that affect the immune responses
Cell death by necrosis begins with swelling
of the cells. If left untreated, the organelles
will swell, including the nucleus, leading
to malfunctioning organelles, including the
mitochondria, golgi apparatus, endoplasmic
reticulum and lysosome – a condition called
cell degeneration. At this stage, cell damage
 EFFECT OF ALKALOID A. aspera ON MICE WITH BREAST CANCER
is irreversible. This condition will cascade
to necrosis, such that the nucleus becomes
pyknotic, followed by karyorrhexis and
karyolysis. The cells that swell undergo
ischemia, marked by decrease in the tissues’
blood supply, causing ATP supply in the cells
to decrease and lead to Na-K-ATPase being
interrupted. In essence, an imbalance in
intracellular and extracellular fluids occurs,
which cause the cells to swell. This can lead
to cell degeneration and necrosis because of
impaired ATP supply, therefore, interfering
with ATPase synthesis, especially the Na-K-
ATPase synthesis, which leads to Na-K pump
malfunction.
Consequently, Na+ ions accumulate
inside the cells, while K+ ions build outside,
causing the cells to swell. If this persists,
ATPase formation will be interrupted and the
organelles, specifically the mitochondria, golgi
body, lysosome, ribosome, nucleus (and its
components) will swell. When swelling reaches
its peak, the cell leaks, damages the cell
membrane, and ultimately leads to lysis and
necrosis, marked by pyknosis, karyorrhexis
and karyolysis (Yalon et al., 2004; Wurlina et
al., 2010).
Necrosis induced by alkaloids from A.
aspera is due to the activation of the lysosome.
Lysosome produces lysozyme, an enzyme that
digests the cell membrane, which eventually
disrupts the entire cell, spills its contents and
leads to lysis and necrosis (Yalon et al., 2004).
Cell death through apoptosis happens
without prior occurrence of cell swelling
or inflammation. Rather, it is preceded by
shrinkage then disruption of the cell, while the
nucleus and the chromosome form apoptotic
bodies, which sustain the lysis. This process
ends with phagocytosis (Lanteujoul et al.,
2004; Meles et al., 2017b).
Pathological apoptosis occurs through
the activation of protein kinase C (PKC) and
cystein protein kinase-2 (CPK2). PKC and
CPK2 activate the protein P53 inside the
nucleus, then affect the transcription process of
protein P21, which causes the inhibition of the
cyclin-dependent kinase enzyme (CDK) form,
including CDK1, CDK2, CDK4 and CDK6.
CDK binds the protein cycline to initiate cell
cleavage. Inhibition of the CDK causes the
55
protein cycline to be inactive. This causes the
cell cycle involved in DNA synthesis in the S
phase, RNA synthesis in G1 and G2 phase,
and mitosis in M phase to become dormant,
so that the cell is unable to undergo cleavage,
leading to condensation of chromosome then
apoptosis (Andrew et al., 2002; Yalon et al.,
2004; Huang et al., 2005).
Pathological apoptosis is caused by protein
P53 activity, through the activation of protein
Bax, which stimulates the mitochondria to
produce cytochrome C excessively, causing
apoptosis. Cytochrome C activates the
apoptotic protease activating factor (APAF-1),
activating caspase 9 to interact with caspase 8
to form the caspase executor, caspase 3. Then
caspase 3 activates DNAase to digest the DNA,
fragmenting the DNA and causing cell death
(Yalon et al., 2004; Zhou et al., 2004; Meles et
al., 2017b).
Caspase 9 and caspase 3 expression
The results in Table 2 show that A.
aspera alkaloid increased the amount of
caspase 9. Significant difference (P<0.05)
between C+ and T0 indicates that treatment
with methotrexate for eight weeks in mice
with breast cancer significantly increases the
amount of caspase 9. When C- is compared
with T1, T2 and T3, alkaloid treatment shows
significant difference (P<0.05), suggesting
that alkaloid at a dose from 75 to 125 mg/kg
body weight for eight weeks can substantially
increase the amount of caspase 9. Also, there
was a significant difference (P<0.05) between
T1 and T2, which indicates that increase
in alkaloid dose from 75 to 100 mg/kg body
weight can increase caspase 9. However, this
was not observed between T2 and T3. Overall,
maximum dose to increase caspase 9 is 100
mg/kg body weight.
Further, results show that A. aspera
alkaloid can also increase the amount of
caspase 3. The amount of caspase 3 between
C- and C+ is significantly different (P<0.05).
Likewise, there was a significant difference
between caspase 3 in C+ and C- (P<0.05). This
shows that exposure to methotrexate for 8
weeks can contribute to significant increase
in the amount of caspase 3. The same pattern
was observed between C- and treatment
56 WURLINA et al.

Table 2. Mean percentage of caspase 9 andcaspase 3 in mice with breast cancer given
increasing doses of Achyranthes aspera.
Treatment caspase 9 expression caspase 3 expression
(%) (%)
C- 87.2±5.95 a 93.3±7.30a
C+ 80.0±7.70 b 85.9±9.12b
T0 55.7±6.68d 60.5±6.96d
T1 62.2±6.14 c 68.8±2.93e
T2 74.4±3.47 b 80.1±8.23b
T3 77.8±6.49 b 84.8±10.71b
Means with different superscript letters differ significantly (P<0.05).

groups, signifying that alkaloid treatment
from 75 to 125 mg/kg body weight can also
increase caspase 3 expression compared to
solvent alone. Between T1 and T2, however,
no significant difference was seen (P<0.05),
implying that effective dose of alkaloid to
increase caspase 3 is 100 mg/kg body weight.
The immunohistochemical appearances
of breast cancer in mice with benzo(a)pyrene-
induced caspase 9 and caspase 3 enzyme

Fig. 2. Immunohistochemistry of benzo(a)pyrene-induced caspase 9 enzyme expression in mice

with breast cancer (denotes development of malignant cell).
C-: healthy female mice given only 0.5 ml of 0.5 % carboxy methyl cellulose (CMC) solvent; C+:
mice with breast cancer given 0.5 ml of 15 mg/kg body weight methotrexate; T0: mice with
breast cancer given 0.5 ml of 0.5% CMC; T1: mice with breast cancer given 0.5 ml of 75 mg/
kg body weight A. aspera alkaloid; T2: mice with breast cancer given 0.5 ml of 100 mg/kg
body weight A. aspera alkaloid; and T3: mice with breast cancer were given 0.5 ml of 125 mg/
kg body weight A. aspera alkaloid.
 EFFECT OF ALKALOID A. aspera ON MICE WITH BREAST CANCER 57

Fig. 3. Immunohistochemistry of benzo(a)pyrene-induced caspase 3 enzyme expression in mice

with breast cancer (denotes development of a malignant cell).
C-: healthy female mice given only 0.5 ml of 0.5 % carboxy methyl cellulose (CMC) solvent; C+:
mice with breast cancer given 0.5 ml of 15 mg/kg body weight methotrexate; T0: mice with
breast cancer given 0.5 ml of 0.5% CMC; T1: mice with breast cancer given 0.5 ml of 75 mg/
kg body weight A. aspera alkaloid; T2: mice with breast cancer given 0.5 ml of 100 mg/kg
body weight A. aspera alkaloid; and T3: mice with breast cancer were given 0.5 ml of 125 mg/
kg body weight A. aspera alkaloid.

expression are shown in Figs. 2 and 3,
respectively. Development of malignant cells
can be observed in all groups.
Degeneration of both cancer and normal
cells generally occur through necrosis and
apoptosis. Cell death through apoptosis
involves the role and function of mitochondria,
specifically the excessive production of
cytochrome C, which spills out its contents due
to permeability changes on its membranes. In
addition, the mitochondrion is responsible for
ATP production derived from carbohydrates,
fats and proteins. In the body, carbohydrate
processing via oxidation breaks it down into
glucose, fat into fatty acids and protein into
amino acids. These products will be broken
down into acetyl CoA, which then enters the
mitochondrial matrix through the enzyme
from tricarboxylic acid, producing several
ATPs, H2O and CO2. ATP joins cytochrome
C synthesized by the mitochondria, which
then interact with APAF-1 to form the
apoptosome (Adnyana et al., 2012; Meles et
al., 2017b). Apoptosome activates procaspase
9, the enzyme initiator that induces caspase
9 (cysteine aspartate protease 9) formation.
Caspase 9 will then activate caspase 3, which
executes apoptosis as it increases the amount
of DNAse. This ultimately fragments the cell’s
chromosome, leading to cell death. This study
illustrates that Achyranthes aspera alkaloid
can increase the rate of apoptosis and caspase
9 and caspase 3 expression in mice with breast
cancer.
58 WURLINA et al.
REFERENCES
Adnyana DPA, Meles IDK and Meles W. 2012.
Alkaloid fraction of jarong (Achyranthes
aspera Linn) leaf induced apoptosis breast
cancer cell through p53 pathways. Advances
in Natural and Applied Sciences 6(2): 124-127.
Andrew W,Vicky D, Hill D, Keesey J and Manzow
S. 2002. Cell Death: Apoptotis and Necrosis
Proliferation. Boehriger Mannheim 2-11.
Huang DM, Guh JH, Huang YT, Chueh SC, Chiang
PC and Teng CM. 2005. Induction of mitotic
arrest and apoptosis in human prostate cancer
pc-3 cells by evodiamine. Journal of Urology
173(1): 256-261.
Lantuejoul S, Soria JC, Moro-Sibilot D, Morat L,
Veyrenc S, Lorimier P, Brichon PY, Sabatier
L, Brambilla C and Brambilla E. 2004.
Differential expression of telomerase reverse
transcriptase (hTERT) in lung tumors. British
Journal of Cancer 90(6): 1222-1229.
Meles DK, Wurlina and Adnyana DP. 2017.
Measurement of alkaloids Achyranthes aspera
Linn level using thin layer chromatography
method and high-performance liquid
chromatography. KnE Life Sciences 3(6): 378-
385.
Meles IDK, Meles W and Adnyana DPA. 2017.
The alkaloid fraction of Achyranthes aspera
Linn increase NK Cells in breast cancer cells.
Advances in Natural and Applied Sciences
11(9): 150-153.
Meles W. 2005. The influence of antimitotic of
Achyranthes aspera Linn extract in embryonal
cleavage in order to produce post coital
contraception. Publishing House of Ancient
China Medical Books.
Wurlina DK, Meles DK, Sastrowardoyo and Zakaria
S. 2010b. Activity of alkaloid Achyranthes
aspera Linn on apoptotic and necrotic
induction of breast cancer and influence on
the natural killer cells. Media Kedokteran
Hewan 26(1): 1-7.
Wurlina W, Sastrowardoyo W and Zakaria S.
2008. The effect of antitelomerase alkaloid
Achyranthes aspera Linn on the chromosome
division and apoptotic division on the mieoloma
culture cell. Research Report of Lemlit Unair.
Wurlina, Sastrowardoyo W, Zakaria S, Meles DK,
Putra MS and Suwasanti N. 2010a. The acivity
of alkaloid Achyranthes aspera L. causing
apoptosis on breast cancer cells. Veterinaria
Medika 3: 169-176.
Wurlina. 2006. The effect of antimitosis
Achyranthes aspera Linn in embryonic
cell division (cleavage) in rats. Journal of
Biological Researches 11(2): 161-166
Yalon M, Gal S, Segev Y, Selig S and Skorecki KL.
2004. Sister chromatid separation at human
telomeric region. Journal of Cell Science
117(10): 1961-1970.
Zhou J, Liu M, Aneja R, Chandra R and Joshi HC.
2004. Enhancement of paclitaxel-induced
microtubule stabilization, mitotic arrest, and
apoptosis by the microtubule-targeting agent
EM012. Biochemical Pharmacology 68(12):
2435-2441.