Brain Derived Neurotrophin Factor (BDNF) Level in Aged Sprague

Dawley Rats Brain after the Treatment of Centella Asiatica Leaf

Extracts

Indah Fitriani

, Nathaniel Aditya

, Adisti Dwijayanti

2,3

, Desak Gede Budi Krisnamurti

2,3

, Erni

Hernawati Purwaningsih

2,3

, and Rani Wardani Hakim

2,3

Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia

Undergraduate Student, Faculty of Medicine, Universitas Indonesia

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

Keywords: Centella asiatica, BDNF, Sprague-Dawley Rats, Aging

Abstract: Functional decrease in learning and memory is one of the characteristics of the aging process. It has been

known that a lower concentration of Brain Derived Neurotrophin Factor (BDNF) found on the brain, plays

a role in the phenomenon. This study was designed to determine whether a herbal plant, Centella asiatica

(CA), would increase the BDNF level on the aging brain tissue. 27 Male Sprague-Dawley rats aged 20-24

months and 4 weeks which were used in the study were divided into: negative control (were given

aquadest), positive control (supplementation of Vitamin E), young rats as a comparison (4 weeks old), and

treatment groups, which were given ethanol extract of CA leaves administered orally (300 mg/kg BW/week)

for 28 days. At the end of the treatment, the rats were terminated and the brain BDNF levels were assessed.

The data were analyzed by using a one-way ANOVA. The results showed a mean concentration of BDNF

for negative control, positive control group, young and treatment groups were 44.09±3.854, 43.09±11.99,

65.88±13.46, and 30.2±12.33 mmol/ml, respectively (p<0.05 vs control group). The treatment group

showed a higher BDNF level compared to all the groups. Interestingly, the BDNF level showed in the

positive control group were found to be lower than the treatment group. This result showed that the

supplementation of CA was effective in increasing BDNF brain level, thus raising the potential of having a

neuroprotective effect. These results implied the need of further research to find out the mechanism of

neuroprotective function exerted by CA.

1 INTRODUCTION

The global population is currently going through a

phenomenon called epidemiological transition. This

transition showed a pattern of population shifting

from high mortality and fertility pattern of

population to low mortality and fertility.The life

expectancy of population aged 65 years old and

above will continue to increase. In 2020, for the first

time in history, the population aged 60 years or more

would be higher than the children population aged

below 5 years. This shows that in the future, our

population would be constructed with more elderly

than before. (United Nations, 2017; WHO, 2011)

However, living longer does not mean living

healthier. Almost a quarter (23%) of the global

burden on morbidity and mortality of disease are

from the age group of people aged 60 years and

more. Elders are also more prone to many

communicable and non-communicable diseases.

Therefore, with the predicament of the increase of

this particular group, the global healthcare system

should answer accordingly. Beside of the increasing

age-span, ageing well should also be the goal of

future global healthcare system.(WHO, 2011)

One of the most aging-influenced aspect of

human life is cognitive health. Cognitive health

concerns about the brain function in ensuring the

independence of one individual, including the ability

of learning, intuition, judgment, language, and

remembering. A decline in cognitive function could

mean a loss of independence in older individuals,

causing burden to self and others. This decline

results from impaired neuronal plasticity (Tapia A et

al., 2008)

Fitriani, I., Aditya, N., Dwijayanti, A., Budi Krisnamurti, D., Purwaningsih, E. and Hakim, R.

Brain Derived Neurotrophin Factor (BDNF) Level in Aged Sprague Dawley Rats Brain after the Treatment of Centella asiatica Leaf Extracts.

DOI: 10.5220/0009842400002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

The pathogenesis of such neurodegenerative

condition still has not been established definitely and

involves multiple factors that influences several

systems, however it was known to be related to the

declining level of BDNF responsible for the loss of

the neuron function and structure.(Erickson et al.,

2012) BDNF has been known to have an important

roles in proliferation, differentiation, target

innervation, and survival of neurons of the central

and peripheral neuron system.(Tapia-Arancibia et al.,

2008). Lower levels of BDNF were also associated

with poorer memory. (Cunha, 2010)

Centella asiatica (CA), a small annual herb from

the family Apiaceae and native to Indonesia, India,

and many part of Asia has always been used widely

as a traditional medicines such as Aryuvedic

medicine, Chinese medicine, and many Southeast

Asian countries traditional medicine. (Lokanathan et

al., 2016). The CA, also known as pegagan in

Indonesia or gotu kola in India, have a significant

number of reviews on their medicinal uses along

with their supportive evidences (Gohil, Patel, &

Gajjar, 2010; Lokanathan et al., 2016; Orhan, 2012;

Rajakumari, 2010). Indicating the strong potential of

the plant in medicinal sector.

The primary active components of CA are

saponins (also called triterpenoid). (Singh & Rastogi,

1969) Saponins include asiaticoside, a trisaccharide

with aglycone asiatic acid, madecosside, and

madasiatic acid. These components are responsible

for some of the CA medicinal effects such as wound

healing and vascular effects. (Gohil et al., 2010)

CNS, cognitive, and antioxidant actions of CA

has been studied in many research and maybe due to

its brahmoside and brahminoside components, but

are yet to be confirmed by clinical studies. (Gohil et

al., 2010; Khotimah, Sumitro, Ali, & Widodo, 2015).

Khotimah et al also found that methanolic extract of

CA could increase the BDNF level in neuronal tissue

of Rattus noevegicus strain Wistar that were exposed

to lipopolysaccharide. (Khotimah, Riawan, &

Kalsum, 2009)

However, there has been a gap in the current

research, in which study of the neuroprotective

effects of the CA has never been demonstrated on a

subject with aging. Using 20-24 months old Sprague

Dawley rats, we hypothesized that treatment with

CA extract could effectively induce a

neuroprotective effect on old age rats, hence this

study aims to expand the knowledge in CA

specifically its effectiveness to the level of BDNF

found on the brain tissue of aged rats.

2 METHODS

2.1 Study Design & Subjects

The Sprague-Dawley (SD) young (2-3-month-old)

and aged (20-24-month-old) male rats, were obtained

from Research and Development Department of the

Ministry of Health. In total, there were 27 rats that

were used in this study. SD is a strain of albino rat

which is used in many researches because its

calmness and ease of handling.

Before the experiment, the rats were acclimatized

for 1 week with lighting of 12 hours (light on from

06:00 p.m. to 06:00 a.m.), constant room temperature

of 24

C, were given standard food and ad libitum

drink. The rats were divided into 4 groups: the aged

rats with no treatment as negative control, the aged

rats with treatment of CA extract (300mg/kgBW),

the aged rats with Vitamin E treatment of 6 IU and

the young rats. The Vitamin E treatment were used

as a positive control, whereas the young rats were

used to provide comparation between aged and

young rats. The animals were kept for 28 days under

the same environment where the rats underwent

acclimatization. The treatment were given once

every 7 days.

2.1.1 Daily Nutrients

The rats were fed with a type of pellets made from a

mixture of cornmeal, rice bran powder, fishmeal,

soybean, coconut, meat and bone meal, oat, ground

nut, canola, skimmed milk, and fish pellet with brand

names of SPA-Z and FF999. This standard pellets

contained 18.5%-20.5% protein, 4% fat, 6% fiber,

8% ash, 0,9% calcium, 0,7% phosphor and has

metabolized energy of 3100-3200 kcal/kg. The rats

were given water, ad libitum.

2.2 Extraction of CA

The CA leaves were dried on a drying racks or

sundried until all the water content evaporated. After

being dried, the CA leaves were then grinded until

the leaves become powdery. Then, the grinded CA

leaves were extracted/macerated with an ethanol

solvent, until all the active constituents were

dissoluted into the solvent. This extraction process

were performed for 24-48 hours and then proceeded

with a separation of the active component from the

solvent. This was achieved by evaporation using

rotary evaporator. At last, the gravimetry analysis

were performed to analyze the water content so that

the solute percentage can be determined.

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

2.3 BDNF Measurement by ELISA

After 28 days of treatment, the rats were sacrificed

by an intraperitoneal injection of ketamine and

xylacine. Consequently, the rats were dissected and

the tissue were obtained. The brain tissues were then

isolated and contained in an alcohol solution in

separate tubes. The tubes then were stored in a

refrigerated storage.

BDNF ELISA kit were used to evaluate the

BDNF level in the brain tissue. The ELISA kit that

were used were from ELabScience

. This ELISA kit

utilizes a Sandwich-ELISA method. The ELISA kit

has micro plates precoated with antibodies specific

to the rat BDNF. The samples were then added to the

micro plate wells and combined with the antibody.

Next, added to the well were biotinylated antibodies

specific for rat BDNF and Avidin-Horseradish

Peroxidase (HRP) conjugates. The microplates were

then incubated. After incubation, the free

components were washed away. The next step was to

add a substrate reagent to each wells, those wells that

contained rat BDNF, biotinylated detection antibody,

and Avidin-HRP conjugate would appear blue. The

termination of the enzyme-substrate reaction was

achieved by adding Stop Solution, changing the

samples appearance yellow in color. A

spectrophotometry were then used to measure the

optical density (OD) of the samples at the 450nm ± 2

nm wavelength. The OD value would be

proportional to the BDNF level. A comparation of

the samples OD to the standard curve would show

the level of BDNF in the samples. Here, we

measured every each of the subjects brain tissue

BDNF level in a grouped manner.

2.4 Statistical Analysis

All the grouped data from the ELISA tests were then

analyzed with an ordinary one-way analysis of

variance (ANOVA) using the Graphpad Software,

Inc statistical analyzer, Prism 7 for Windows. The

results obtained from this analysis were presented as

mean data ± SD. P values of less than 0.05 were

considered indicating statistical significance.

2.3 Ethical Consideration

The Healths Research Ethical Committee, Faculty of

Medicine, Universitas Indonesia – Cipto

Mangunkusumo Hospital approved the study

protocol and the usage of animal sibjects (December

2016). The ethic registration number is

1016/UN2.F1/ETIK/2016 and

402/UN2.F1/ETIK/IV/2018.

3 RESULTS & DISCUSSION

The results showed the BDNF level mean ± SD for

the negative control, the treatment groups, the Vit E

group, and the young group were 44.09±3.854,

65.88±13.46, 43.09±11.99, and 30.2±12.33

mmol/ml, respectively. In the rats treated with C.

asiatica (300mg/kgBW), the BDNF level were found

to be significantly increased (p<0.05 vs control

group). However, in the group treated with Vitamin

E, there was no significant change in the BDNF level

compared to the negative control. There were also no

significant difference of the BDNF level found

between the young and negative control group. The

highest concentration of BDNF was found on the CA

treatment group, meanwhile the lowest was found on

the Vitamin E group. (Figure 1)

O n e – w a y A N O V A d a t a

Figure 1: The BDNF levels (pg/mg protein) in 4 groups of

Sprague Dawley rats. The data are shown in mean ± SD (P

= 0.0014)

BDNF, a neurotrophin that functions in

maintaining the integrity of neuron system, have

long been thought to play a role in brain aging. Here

in this study we tried to demonstrate the effect of CA

on the brain BDNF level in an effort to reverse the

effect of declining BDNF level on the aging brain.

Our findings demonstrated that the ethanol extract of

CA succeeded in increasing the BDNF level in the

aged rat brain tissue compared to the negative

control. This results was consistent with previous

study that used LPS-induced rats (Khotimah et al.,

2012). Khotimah et al. also stated CA extract

Brain Derived Neurotrophin Factor (BDNF) Level in Aged Sprague Dawley Rats Brain after the Treatment of Centella asiatica Leaf Extracts

elevated BDNF level through induction on the

expression of BDNF in brain tissue. Moreover,

another study also showed the antioxidant and

antiinflammatory properties of CA play a role in the

neuroprotective effect of CA. (Gohil et al., 2010).

Studies about CA have showed its remarkable effects

on brain aging (Orhan, 2012). This effects have been

generally attributed to its tripertene saponosides

which covered many substances. However, the main

active component varies between studies. Some of

the tripertenes components that have been showed

responsible for CA neuroprotective effects are

asiaticoside, madecassoside, brahmoside, and

brahminoside. Unfortunately, the current studies

results are limited to answer this question. (Gohil et

al., 2010; Orhan, 2012)

In the results, it is apparent that the BDNF level

on the CA group was increased significantly but not

on the Vitamin E group. This contradicted our

former idea of the vitamin E activity as an

antioxidant was expected to enhance the BDNF

levels. However, study from Sakr et al. conformed

with this results (Sakr et al., 2015). According to

Sakr, this result might be due to the different

between CA and Vitamin E mechanism of action on

the BDNF. This previous research showed that CA

extract increased the expression of BDNF, while

Vitamin E treatment did not exert the same effect.

Hence suggestedly the absence of the BDNF level

increase in VIt E group. This could be the reason

why there was a difference shown by the 2 groups.

Still however, CA and Vitamin E both also achieved

their neuroprotective effect through antioxidant and

anti-inflammatory activities. (Khotimah et al., 2009;

Sakr et al., 2015)

Meanwhile, the young rats group had lower level

of BDNF than the control group (young:

44.09±3.854, control: 43.09±11.99 pg/mg protein)

although it was not significantly different. According

to the studies about the BDNF level during aging it

was shown that the BDNF expression could also be

induced by the process of neuron degeneration to

increase the growth of neuron, which happened

naturally in aged rats (Katoh-Semba et al., 1998).

Moreover, in accordance to this study by Katoh-

Semba et al., young and very young rats will have

lower BDNF. However, another research by Cunha

et al. showed that this phenomenon differs in rats

with neurodegenerative condition. In rats with

neurodegenerative condition, the BDNF level is

expected to be low beacuse of the defect on the

BDNF regulation, causing the compensation

mechanism to not occur. This would be manifested

in the clinical symptoms of the neurodegenerative

diseases (Cunha, 2010).

4 CONCLUSIONS

The results demonstrated that the BDNF levels were

increased with CA extract treatment. In addition, this

study also found evidence that Vitamin E treatment

attenuated the oxidative stress thus decreasing the

BDNF expression. Whereas, in young rats the BDNF

was lower than expected due to less oxidative stress

as the BDNF expression trigger. These results call

for further studies especially to determine the

molecular mechanism of how CA influences BDNF.

ACKNOWLEDGEMENTS

We would like to show our gratitude to Publikasi

Terindeks Internasional Untuk Tugas Akhir

Mahasiswa UI (PITTA) Grant by Universitas

Indonesia that give us opportunity to publish this

study.

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Brain Derived Neurotrophin Factor (BDNF) Level in Aged Sprague Dawley Rats Brain after the Treatment of Centella asiatica Leaf Extracts