Anti-Oxidative Potential of Acalypha indica L. Root Extract on Brain-
Derived Neurotrophic Factor Levels in Old Sprague-Dawley Rats
Callen Zulkifli
1
, Reganedgary Jonlean
1
, Adisti Dwijayanti
2,3
, Desak Gede Budi Krisnamurti
2,3
, Erni
Hernawati Purwaningsih
2,3
, and Rani Wardani Hakim
2,3
1
Undergraduate Student, Faculty of Medicine, Universitas Indonesia, Jakarta 10430;
2
Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jakarta 10430;
3
Drug Development Research Cluster, Indonesian Medical Education and Research Institute (IMERI), Jakarta
Keywords: Acalypha indica L., BDNF, aging, oxidative stress, anti-aging, anti-oxidative
Abstract: In 2020, life expectancy in Indonesia is estimated to be greater than 70 years. It implies an increasing
number of elderly and becomes a major problem in the health sector. Aging is a process that involves
oxidative stress; however, it can be anticipated by the presence of brain-derived neurotrophic factor
(BDNF). The aim of this study is to investigate the effect of Acalypha indica L. (AI) as a notable medicinal
plant to BDNF level changes as a marker of anti-aging. This experimental study was conducted in 28 days
using male Sprague-Dawley rats. There were four groups identified: negative control, positive control
(vitamin E 6 IU), treatment (AI 250 mg/kg BW), and young group (8-12 weeks of age rats). Data are
collected from terminating all the rats and the brain tissues are further checked in the biochemistry
laboratory. The test used for statistical analysis is one-way ANOVA. Compared with the negative control
group, levels of BDNF in brain tissues identified in the treatment group were increased, even though
statistically insignificant (p-value = 0,6545). Nevertheless, the BDNF level in the negative control group is
still higher than shown in the positive and young group. These results show that AI provides anti-oxidative
properties; it also implies that AI can be used to impede the aging process. More studies are still needed to
know the molecular mechanisms of action of AI that are particularly involved in increasing BDNF levels of
brain tissues.
1 INTRODUCTION
World Health Organization reported that life
expectancy has been increasing since 2000. This
reflects on numbers of elderly which are getting
higher. Studies show mortality due to degenerative
disease occurred approximately 2- to 3-fold (World
Health Organization, 2018)
.
Aging is a multifactorial
process; many factors are involved such as genetics,
nutrition, physical activity, exposure to pollutant,
radiation, and microorganism (Nigam, Knight et al.,
2012). Hence, reactive oxygen species (ROS) are
produced and accumulated; later contribute to
cellular and DNA damage if the body fails to
neutralize them.
Physiologically, there are endogenous substances
produced naturally inside human bodies, such as
Brain-derived Neurotrophic Factor (BDNF) that
makes neuronal cell able to survive and grow. As a
consequence of the aging process, BDNF expression
in brain tissues also reduce. It manifests in
decreasing neuronal plasticity and cognitive function
(Perovic, Tesic et al., 2012).
For this reason, many researchers are striving to
find and develop recent medicinal plants that are
estimated beneficial to prevent age-related diseases
(Ekor, 2014). Acalypha indica L., a notable plant
whose numerous therapeutic purposes; one of them
is its anti-oxidative effect. Role of Acalypha indica
L. root extract to BDNF level has been performed on
a study which used young rats exposed to a hypoxic
environment as subjects (Ibrahim, Rahadian et al.,
2012). This research was conducted to determine the
effect of Acalypha indica L. on BDNF expression
identified in old Sprague-Dawley rats.
Zulkifli, C., Jonlean, R., Dwijayanti, A., Krisnamurti, D., Purwaningsih, E. and Hakim, R.
Anti-Oxidative Potential of Acalypha indica L. Root Extract on Brain-Derived Neurotrophic Factor Levels in Old Sprague-Dawley Rats.
DOI: 10.5220/0009842000002406
In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1
ISBN: 978-989-758-347-6
Copyright
c
2022 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
1
2 MATERIALS AND METHODS
2.1 Study Design and Sample
This experimental study was conducted on the
neuronal cell cultures from brain tissues of male
Sprague-Dawley rats aged 20 - 24 months with
initial weights ranging from 183 to 308 g (young
rats), and 333 to 490 g (old rats). It was conducted at
the laboratory of National Institute of Health
Research and Development (NIHRD), Ministry of
Health (Indonesia) in August 2017 May 2018.
Based on the Federer formula, the number of
samples was 24, divided into four groups (3 control
groups and 1 treatment group). Ethical authorization
was obtained from the Committee of the Medical
Research Ethics of the Faculty of Medicine,
Universitas Indonesia.
2.2 Extraction of Plant
The roots of Acalypha indica L. were collected and
washed. The materials were dried in the room under
sunlight exposure. Then, the dried roots were ground
to get the smaller ones. Roots were extracted
completely with 200 mL of ethanol (56-60°C). This
procedure was done in 24 48 hours duration and
repeated until all extracts were dissolved within the
ethanol. The extract was later evaporated with a
rotary evaporator to separate the solute from the
solvent. With gravimetric analysis, the water content
is also measured to determine the solute percentage.
2.3 Treatment with Acalypha indica
L.extract
One treatment group was given the ethanol extract
of Acalypha indica L. with a dose of 250 mg/kg
BW. The negative control group was given rat chow
and water ad libitum; the positive control group was
given vitamin E with a dose of 6 IU; the young
group consisted of rats aged 8 - 12 weeks which
given the same with the negative control group got.
2.4 Measurement of BDNF Levels
Samples were taken from neuronal culture medium.
They were centrifuged, and the supernatant
produced was stored in temperature -20°C.
Examination of BDNF level was done by using
BDNF kit according to the standardized procedures
listed in its manual kit. The measurement was done
by counting optical density (OD) with
spectrophotometry at a wavelength of 450 nm ± 2
nm.
2.5 Statistical Analysis
Data were processed using GraphPad Prism 7
software and the results were provided as a mean ±
standard deviation (SD). After conducted normality
test by Shapiro-Wilk test, the statistical significance
of the difference between the BDNF level seen in
those four groups was tested by one-way analysis of
variance (ANOVA). The significance of a difference
was considered in p-value < 0.05.
3 RESULTS AND DISCUSSION
3.1 Results
The level of BDNF and protein content was
measured in each subject separately. BDNF level
was then divided by the concentration of protein to
get the representative BDNF level for further
identification.
As seen in Figure 1, there were differences in the
numbers of BDNF levels in each group. By
calculating the mean of BDNF levels in each group,
it has been found that treatment group Acalypha
indica L. root extract 250 mg/kg BW had the highest
BDNF content which reached 51.57 ± 9.713 pg/mg
protein, while the lowest was shown by positive
control group vitamin E 6 IU which was 43.09 ±
11.99 pg/mg protein. BDNF contained in the
negative control group was 44.09 ± 3.854 pg/mg
protein. This value was slightly higher than the value
of BDNF level in the young group, which was
43.18 ± 20.28 pg/mg protein. Despite the
differences, these data were statistically insignifican
with p-value 0.6545
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
2
Figure 1: BDNF Level Expression in Negative Control,
Vitamin E (Positive Control), Acalypha indica L., and
Young Group.
3.2 Discussion
This study showed that the root extract of Acalypha
indica L. could provide anti-oxidative potential
when given to aged subjects. This role was
facilitated through the increment of BDNF level of
brain tissues.
The high concentration of BDNF in the group
receiving Acalypha indica L. is synergic with many
preceding studies that have proven its positive
correlation. For example, when compared to
standard antioxidant L-ascorbic acid, Acalypha
indica L. provided moderate antioxidant activity
with different expressions depending on the material
used: root 53.27% and leaves 31.14%. This study
clearly indicated that the antioxidant expression of
Acalypha indica L. was higher in root extract
(Shanmugapriya, Ramanathan et al., 2011).
Bioactive constituents contained in the extracts, such
as phenolic and flavonoid components were known
to give radical scavenging activities by neutralizing
reactive oxygen species (ROS) which later prevent
neuronal damage and improve viability and
proliferation of neuronal cells (Vauzour, Vafeiadou
et al., 2008).
BDNF concentration was observed as the lowest
in the group with vitamin E supplementation, which
corresponded with a previous study on rats done by
Sakr, Abbas et al. (2015). BDNF gene expression
was known to be decreased in rats with sustained
hypoxia and chronic exercise when exposed to
vitamin E supplementation. Vitamin E given in this
research is 100 mg/kg BW of the doses and injected
intraperitoneally. This suggested that BDNF
expression of the cortical neuron was related to
oxidative stress induced by hypoxia and exercise.
However, this research was limited in young rats
aged 4 weeks.
According to one research which studied the
correlation between age and plasma BDNF level,
there were significant differences in BDNF levels
between younger and older subjects after undergo
horizontal bed rest for 14 days. In younger subjects,
BDNF levels were smaller (34.36 ± 15.24 pg/mL)
than older subjects (62.02 ± 18.31 pg/mL). These
results were affected by the resistance of the brain to
counteract acute stressors, which was declined as the
age increased. It contributed to a bigger increment in
BDNF level as a compensatory mechanism (Soavi,
Marušic et al., 2016). Therefore, this strongly
correlates with such differences of BDNF level
shown in both old and young groups observed in this
study.
As stated in a study on sixty frogs (Bufo
melanostictus Schneider) aimed to identify the
therapeutic effect provided by Acalypha indica L.,
there was a significant difference (p<0.05) of
neuroprotective effect among treated frogs when
compared to control group. In the experiment, the
extracts were made into four groups with various
amount of doses. It concluded that the
neuroprotective effect was observed highest at 200-
500 mg/kg BW of dose (Purwaningsih et al., 2010).
This range of dose was also synergic with a study
detecting another pharmacological activity of
Acalypha indica L. This study demonstrated that its
anti-inflammatory effect was observed best at dose
250 mg/kg BW which seen by maximum inhibition
that was comparable to phenylbutazone 100 mg/kg
BW as a standard drug (Saha, Ahmed et al., 2017).
Moreover, one in vitro study using hypoxic
primary cell culture of hippocampus obtained from
9-10 weeks of age rats stated the possible
mechanism underlying Acalypha indica L. role in
BDNF increment. It directly impedes protein
damage which contributes to the production of new
endogenous BDNF. Through experiment in which
the root extract of Acalypha indica L. with different
exposure of doses used, it was known that
proliferation of the neuronal cells was linear with
increasing doses applied (Ibrahim, Rahadian et al.,
2012).
Therefore, it might be a limitation of this
research that made the results insignificant. It could
be occurred due to relatively small doses of
Anti-Oxidative Potential of Acalypha indica L. Root Extract on Brain-Derived Neurotrophic Factor Levels in Old Sprague-Dawley Rats
3
Acalypha indica L. used or the course of the
experiment that was too short. In addition, there is a
possibility that the molecular mechanism of
increased BDNF level in both young and aged
subjects are not the same. Hence continued research
to gain more level of confidence in research results
is suggested.
We can conclude that Acalypha indica L. root
extract with the optimum doses can improve
neuronal cell survival by increasing BDNF levels in
aged subjects.
ACKNOWLEDGEMENTS
This research was funded by Hibah Publikasi
Terindeks Internasional Untuk Tugas Akhir
Mahasiswa UI (PITTA) 2018.
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