composed of high concentrations of polyunsaturated
fatty acids (PUFAs). In a study done by Dei, Takeda
et al. (2002), an increase of MDA levels with age
had been demonstrated in the cytoplasm of neurons
and astrocytes. To fulfill a high demand of energy,
brain also consumes a large amount of oxygen.
However, compared to other organ, it relatively
lacks antioxidant defenses, such as a lower activity
in glutathione peroxidase and catalase, making it
more vulnerable to oxidative stress. (Kedar, 2003).
Therefore, protecting the brain from excessive
oxidative damage might ameliorate the balance
between pro-oxidants and antioxidants, hence
promoting a healthier aging process.
One preventive effort to ensure this healthy
aging is reflected in phytotherapy, known as herbal
medicine, which utilizes therapeutic potential of a
certain plant (Ho, So et al., 2010). Centella asiatica
(CA), a medicinal tropical plant from the family
Apiaceae used commonly in Southeast Asia, had
shown to have neuroprotective and cognitive-
enhancement effect which could play an important
role in aging (Dev, 2009, Mukherjee, Kumar et al.,
2007, Tiwari, Singh et al., 2008, Veerendra Kumar
and Gupta, 2003). However, there were only a
limited number of researches examining the
antioxidative properties possessed by this plant,
especially its role in brain aging and lipid
peroxidation. The animal subjects which were used
was also limited to a single breed of rat; not to
mention the lack of comparison with a proven
exogenous antioxidant.
In the present study, we compared the brain
MDA levels between CA-treated aged Sprague-
Dawley rats and their younger counterparts. The
antioxidative properties of CA on aged rats were
also compared to a well-known antioxidant agent,
vitamin E. We hypothesized that aged rats which
were treated with CA extract would have a lower
level of brain MDA compared to those untreated,
thus raising the potential of CA as an antioxidant
which could promote a healthier aging process.
2 METHODS
2.1 Study Design and Subjects
The subject used in this experiment, the male
Sprague-Dawley rats, is a distinct outbred albino rat
used commonly in nutritional and medical research
settings. These rats were obtained from the National
Institute of Health Research and Development,
Ministry of Health Republic of Indonesia. Sprague-
Dawley rat has an elongated head structure and a tail
longer than its body. These rats are first bred by R.
W. Dawley from the Sprague-Dawley Animal
Company in Wisconsin, United States in 1925. Their
docile characteristics make them easy to handle.
The rats were divided into two groups according
to their age; young rats (8-12 weeks old) and aged
rats group (20-24 months old). The aged rats were
further divided into three final groups according to
the treatment given; negative control (water as
placebo), positive control (vitamin E), and treatment
(Centella asiatica ethanolic leaves extract) group. In
total, there were 4 experimental groups.
To differentiate individual rats in every group, a
color-coding system was used; each rat possessed a
distinct mark on a certain part of its body. The rats
have initial weights ranging from 183 to 308 g for
the young rats, and 333 to 490 g for the aged, all in
healthy state. Using Federer’s formula, a minimum
of 24 subjects was needed to achieve the optimal
sample size. However, to anticipate the possibility of
subject exclusion due to death or other unforeseen
causes, a total of 27 rats were used.
2.2 Extract Preparation
Centella asiatica (CA) leaves were dried under the
sunlight until the water content fully evaporated and
grinded to small fractions. The active substances of
these grinded particles were then extracted by
soaking them to a solvent, ethanol, for 24 to 48
hours repeatedly. To obtain and separate the active
substances from its solvent, a rotary evaporator was
utilized. Subsequently, the percentage of active
substances contained in the viscous solution
produced from this process was measured using
gravimetric analysis.
2.3 Treatments
Prior to the 28-day treatment, all rats underwent a
one-week acclimatization at the experiment room,
adapting to a 24
o
C temperature and a light-dark
cycle of 12:12 with lights on at 9.00 PM.
Throughout the study, all groups were fed daily with
10 g of standard pelleted chow (protein 18.5-20.5 %;
fat ± 4%; fiber ± 6%; calcium ± 0.9%; phosphor ±
0.7%) and provided with water ad libitum.
After the aged rats were randomly distributed
into the three groups, the following treatment was
started at day-1 and ended at day-28 accordingly;
water as placebo (negative control), CA leaves
ethanolic extract with 300 mg/kg bodyweight dosage
(treatment), and 6 IU of vitamin E (positive control).
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