Anti-Oxidative Potential of Acalypha indica L. Root Extract on

Brain-Derived Neurotrophic Factor Levels in Old Sprague-Dawley

Rats

Callen Zulkifli

, Reganedgary Jonlean

, Adisti Dwijayanti

2,3

, Desak Gede Budi Krisnamurti

2,3

Erni Hernawati Purwaningsih

2,3

, and Rani Wardani Hakim

2,3

Undergraduate Student, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Drug Development Research Cluster, Indonesian Medical Education and Research Institute (IMERI), Jakarta, Indonesia

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: 10.5220/0008357400580061

In Proceedings of BROMO Conference (BROMO 2018), pages 58-61

ISBN: 978-989-758-347-6

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.

Anti-Oxidative Potential of Acalypha indica L. Root Extract on Brain-Derived Neurotrophic Factor Levels in Old Sprague-Dawley Rats

Figure 1: BDNF Level Expression in Negative

Control, Vitamin E (Positive Control), Acalypha

indica L., and Young Group.

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 insignificant

with p-value 0.6545.

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

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

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 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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Anti-Oxidative Potential of Acalypha indica L. Root Extract on Brain-Derived Neurotrophic Factor Levels in Old Sprague-Dawley Rats