Brain Derived Neurotrophin Factor (BDNF) Level in Aged Sprague
Dawley Rats Brain after the Treatment of Centella asiatica Leaf
Extracts
Indah Fitriani
*1
, Nathaniel Aditya
1
, Adisti Dwijayanti
2,3
, Desak Gede Budi Krisnamurti
2,3
, Erni
Hernawati Purwaningsih
2,3
, and Rani Wardani Hakim
2,3†
1
Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia
2
Undergraduate Student, Faculty of Medicine, Universitas Indonesia
3
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
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: 10.5220/0008357800830087
In Proceedings of BROMO Conference (BROMO 2018), pages 83-87
ISBN: 978-989-758-347-6
Copyright
c
2018 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
83
decline results from impaired neuronal plasticity
(Tapia A et al., 2008)
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
o
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
84
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
Brain Derived Neurotrophin Factor (BDNF) Level in Aged Sprague Dawley Rats Brain after the Treatment of Centella asiatica Leaf Extracts
85
CA extract 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.
REFERENCES
Cunha. (2010). A simple role for BDNF in learning and
memory? Frontiers in Molecular Neuroscience.
doi:10.3389/neuro.02.001.2010
Erickson, K. I., Miller, D. L., & Roecklein, K. A. (2012).
The Aging Hippocampus: Interactions between
Exercise, Depression, and BDNF.
The Neuroscientist, 18, 8297.
Gohil, K., Patel, J., & Gajjar, A. (2010). Pharmacological
review on Centella asiatica: A potential herbal cure-all.
Indian Journal of Pharmaceutical Sciences, 72, 546.
Khotimah, H., Riawan, W., & Kalsum, U. (2009). Efek
Neuroprotektif Ekstrak Pegagan (Centella asiatica)
terhadap BDNF, TNFaR, NFkB, dan Apoptosis pada
Kultur Sel Syaraf yang Diinduksi LPS.
Khotimah, H., Riawan, W., & Kalsum, U. (2012).
Neurostimulant and Neuroprotective Effect of Centella
asiatica : In Vitro and In Vivo Studies. International
Symposium of Austronesian Humanities
and Custom Medicine.
Khotimah, H., Sumitro, S. B., Ali, M., & Widodo, M. A.
(2015). Standardized Centella Asiatica Increased
Brain- Derived Neurotrophic Factor
and Decreased Apoptosis of Dopaminergic Neuron in
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
86
Rotenone-Induced Zebrafish. GSTF Journal of
sychology (JPsych), 2, 4.
Lokanathan, Y., Omar, N., Ahmad Puzi, N. N., Saim, A.,
& Hj Idrus, R. (2016). Recent Updates in
Neuroprotective and Neuroregenerative Potential of
Centella asiatica. The Malaysian Journal of Medical
Sciences: MJMS, 23, 414.
Orhan, I. E. (2012). Centella asiatica (L.) Urban: From
Traditional Medicine to Modern Medicine with
Neuroprotective Potential. Evidence-Based
Complementary and Alternative Medicine, 2012, 18.
Patterson, S. L., Grover, L. M., Schwartzkroin, P. A., &
Bothwell, M. (1992). Neurotrophin expression in rat
hippocampal slices: a stimulus paradigm inducing LTP
in CA1 evokes increases in BDNF and NT-3 mRNAs.
Neuron, 9, 10811088.
Rajakumari, S. (2010). Enhancement of memory in rats
with Centella asiatica (Vol. 21).
Sakr, H. F., Abbas, A. M., & El Samanoudy, A. Z. (2015).
Effect of vitamin E on cerebral cortical oxidative stress
and brain-derived neurotrophic factor gene expression
induced by hypoxia and exercise in rats. Journal of
Physiology and Pharmacology: An Official Journal of
the Polish Physiological Society, 66, 191202.
Singh, B., & Rastogi, R. P. (1969). A reinvestigation of
the triterpenes of Centella asiatica. Phytochemistry, 8,
917921.
Tanaka, J.-I., Horiike, Y., Matsuzaki, M., Miyazaki, T.,
Ellis-Davies, G. C. R., & Kasai, H. (2008). Protein
synthesis and neurotrophin-dependent structural
plasticity of single dendritic spines. Science (New
York, N.Y.), 319, 16831687.
Tapia-Arancibia, L., Aliaga, E., Silhol, M., & Arancibia,
S. (2008). New insights into brain BDNF function in
normal aging and Alzheimer disease. Brain Research
Reviews, 59, 201220.
United Nations: Department of Economic and Social
Affairs, Population Division. (2017). The end of high
fertility is near. (No. Population Facts No. 2017/3).
United Nations.
World Health Organization, & National Institute of Aging.
(2011). Global Health and Aging. Geneva.
Brain Derived Neurotrophin Factor (BDNF) Level in Aged Sprague Dawley Rats Brain after the Treatment of Centella asiatica Leaf Extracts
87