The Effect of Centella asiatica on

Brain Malondialdehyde Levels of Aged Rats

Nathaniel Aditya

, Indah Fitriani

, Desak Gede Budi Krisnamurti

2,3

, Siti Farida

2,3

, Erni Hernawati

Purwaningsih

2,3

, and Rani Wardani Hakim

2,3

Undergraduate Student, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Jakarta Pusat, Indonesia

Department of Medical Pharmacy, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Jakarta Pusat,

Indonesia

Drug Development Research Cluster, Indonesian Medical Education and Research Institute (IMERI), Jl. Salemba Raya

No. 6, Jakarta Pusat, Indonesia

Keywords: Aging, Antioxidant, Oxidative Stress, Lipid Peroxidation, Malondialdehyde, MDA, Centella asiatica

Abstract: Background: In 2050, the number of elderly with 65 years of age or more is estimated to reach 1.5 billion. To

better anticipate this problem, a shift of paradigm, from chronological to biological aging, is needed. Aging

is a multifactorial process closely related to oxidative stress, a phenomenon in which the rate can be indicated

through its secondary metabolite level, malondialdehyde (MDA). Objective: This study examines the effect

of a well-known traditional medicinal plant used for its anti-inflammatory properties, Centella asiatica (CA),

to brain MDA levels in aged Sprague-Dawley rats. Methods: The aged male rats were divided into three

groups: negative control, positive control (vitamin E 6 IU), treatment (CA leaves ethanolic extract 300 mg/kg),

with one additional group of untreated young rats. Throughout 28 days, each rat was given the corresponding

treatment. The brains then were collected to be studied using the Lipid Peroxidation (MDA) Assay Kit. One-

way ANOVA is the choice of the statistical analysis method. Results: We found that the level of MDA in the

brain tissues of the treatment group rats had a lower value compared to that of the control group, although

statistically insignificant (P = 0.5683). Unquestionably, the MDA concentration in the vitamin E-treated rats

is the lowest of all. Conclusion: These results implied that CA may exhibit an antioxidative effect on aged

rats which could hinder an aging process, if not prevent it.

1 INTRODUCTION

Today, the world is in a state of demographical

shift. In 2050, the number of elderly with 65 years of

age or more is estimated to be 1.5 billion; four times

the number of that in 2010 (WHO, 2011). Being the

fastest among all age groups, this increasing rate of

older population will be felt mostly in developing

countries such as Indonesia (Jones, 2010). As the

elderly number grows, its financial weight on national

health service sector will also continue to rise because

older people are more susceptible to external and

internal stress; a result of declining physiological

function (Cesari, Prince et al., 2016, WHO, 2015).

Besides, one should also consider accompanying

diseases, e.g. depressive disorders and anxiety, as the

main cause of quality of life deterioration. To better

anticipate this imminent problem, a shift of paradigm,

from chronological to biological aging, is urgently

needed (Cesari, Prince et al., 2016). One way to

address this challenge is by changing the focus of

therapy; from just lengthening lifespans into

increasing health span (Ho, So et al., 2010, WHO,

2015).

Aging is a multifactorial process closely related to

oxidative stress. The theory of free radical aging

stated that aging process is caused by an imbalance of

an oxidative and antioxidative process (Finkel and

Holbrook, 2000, López-Otín, Blasco et al., 2013).

One biological consequences of the cellular oxidative

stress is lipid peroxidation, a phenomenon in which

the rate can be indicated through its secondary

metabolite, malondialdehyde (MDA) (Lieberman,

Marks et al., 2013). One of the most important organs

which is vulnerable to the harmful effects of lipid

peroxidation is brain, for it is composed of high

Aditya, N., Fitriani, I., Krisnamurti, D., Farida, S., Purwaningsih, E. and Hakim, R.

The Effect of Centella asiatica on Brain Malondialdehyde Levels of Aged Rats.

DOI: 10.5220/0009844700002406

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

ISBN: 978-989-758-347-6

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

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).

All treatments were given twice daily. As for the

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

young rats group, no additional treatment was given

(water as placebo).

2.4 Termination

In the last day of treatment (day-28), all rats were

sedated under ketamine and xylazine prior to

termination and brain collection procedure. The

brains were weighed, put in a sterile container, and

preserved in a -20

C container.

2.5 Outcomes

2.5.1 Tissue Homogenate

One hundred milligrams of tissue from each brain

was dissolved with 1 ml of 0.01 M phosphate-

buffered saline (PBS) with a pH of 7.4 before

homogenized. It was then centrifuged at 3500 rpm for

10 minutes. Then, the supernatant was obtained and

kept in a -20

C container.

2.5.2 MDA Calculation

Four hundred microliters mixture of water, MDA

standard, and supernatant were put into each of two

1.5 mL tubes. Into every tube, 200 µL of

trichloroacetic acid (TCA) 20% was added, then

vortexed and centrifuged at 5000 rpm for 10 minutes.

After the supernatants were transferred to 2 mL tubes,

400 µL of thiobarbituric acid (TBA) 0.67% was

added before the tubes were incubated for 10 minutes

in a water bath with a temperature of 96-100

Following the incubation, the tubes were left out in

the air until they reached room temperature before

their wave absorbance at 530 nm were measured

using a spectrophotometer. The final MDA

concentrations were calculated based on the MDA

wave absorbance standard curve.

2.6 Statistical Analysis

The data acquired were processed and analyzed

through GraphPad Prism ver. 7.00 statistical

software. The results are shown as mean ± SEM.

Shapiro-Wilk normality test was performed to see if

the data came from a Gaussian distribution. Ordinary

one-way ANOVA was the chosen parametric test,

followed by Tukey’s multiple comparisons test with

a single pooled variance as the follow-up. The

statistical significance was defined as a P value of

<0.05.

2.7 Ethical Consent

The study protocol and the usage of rats as

experimental subjects was approved by the Health

Research Ethical Committee, Faculty of Medicine,

Universitas Indonesia – Cipto Mangunkusumo

Hospital in December 2016 with the registration

number 1016/UN2.F1/ETIK/2016.

3 RESULTS & DISCUSSION

Of all 27 healthy male rats involved at the beginning

of the study, only 21 rats were alive at the time of

termination. In different periods, each of the 6 rats

appeared sick initially, and then died for unknown

reasons.

From our data, brain MDA concentration was

found to be lowest in those treated with vitamin E

(positive control) with a mean and SEM of 3.12 ±

0.39 nmol/L. In the negative control group consisting

of untreated aged rats, the MDA concentration

measured 3.78 ± 0.44 nmol/L, closely followed by the

young rats at 3.70 ± 0.21 nmol/L as the second

highest. With a mean difference of only 0.29 nmol/L

with the vitamin E-treated group, CA-treated aged

rats had brain MDA concentration of 3.41 ± 0.25

nmol/L (Figure 1).

Although the findings were not statistically

significant (P = 0.5683), the decrease of brain MDA

seen in CA-treated aged rats from that of untreated

aged rats correlates with a similar previous finding

(Kumar and Gupta, 2002). That study demonstrated a

significant decrease of brain MDA in male Wistar rats

treated with 200 and 300 mg/kg of CA whole-plant

aqueous extract. However, the research did not

provide information about the age of the rats used;

The Effect of Centella asiatica on Brain Malondialdehyde Levels of Aged Rats

Figure 1: Brain MDA concentration (nmol/L) in different

groups of aged male Sprague-Dawley rats and young

Sprague-Dawley rats. Data are shown in mean ± SEM (P =

0.5683).

albeit the weight range was stated to be 200-250 g.

This was almost half of the aged Sprague-Dawley rats

used in current study (333-490 g). This reduction of

MDA as a lipid peroxidation marker indicate that

there was also a decrease in the lipid peroxidation

process itself. This decrease may be due to the

electron and H

donating capacity of flavonoids

present in CA (Subathra, Shila et al., 2005).

Furthermore, beside its established role as an

oxidative stress indicator, MDA was also known for

causing yet another secondary oxidative stress to

proteins nearby. In one research which studied the

interaction between MDA and bovine serum albumin

(BSA), it was found that an oxidative process called

protein glycooxidation played the key role. The

research hypothesized that this process was one of the

main cause of molecular aging (Traverso, Menini et

al., 2004). Hence, by decreasing the MDA levels on

brain tissue, not only the lipid peroxidation of the

PUFAs will be reduced, but also the secondary

detrimental damage caused by MDA to proteins will

also be prevented.

In lipid peroxidation process of PUFAs, which can

be found at large amount in brain tissue, chemical

reactions induced by lipid peroxyl radical (LOO

)

appear to be responsible for aging and other age-

dependent diseases (Spiteller, 2007). Compared to

other organs in our bodies, brain also has a higher risk

for oxidative damage because (1) it requires

significant amounts of oxygen per weight

(approximately 20% of the total oxygen used in

humans) while (2) not highly equipped with

antioxidant protective mechanisms. In addition, key

ingredients behind the cause of cell membrane lipid

peroxidation, Fe and ascorbate, was found to be at

high concentration in brain tissue (Floyd, 1999). This

means that CA capability to lower lipid peroxidation

process in the aging brain could translate into a potent

antioxidant effect in an organ inherently faced with a

pro-oxidative state. Centella asiatica positive effects

on brain aging have been attributed to its two major

triterpene saponosides; asiatic and madecassic acides,

as well as their heterosides; asiaticoside and

madecassoside (Orhan, 2012).

Our data also demonstrate that vitamin E reduced

MDA brain concentration in aged rats. This finding

was relevant with a proven role of vitamin E as a

peroxyl radical scavenger which terminates chain

reactions and protecting long-chain PUFAs for

important cellular signaling events (Traber and

Atkinson, 2007). Nonetheless, one in vivo study

showed that supplemental vitamin E given to healthy

persons had no effect to the rate of lipid peroxidation

(Meagher, Barry et al., 2001). A difference on the

marker used on that observation (urinary isoprostane

called iPF

2α

-VI and urinary 4-hydroxynonenal) and

the fact that it measured whole-body lipid

peroxidation instead of a single organ might be the

cause of this contradicting finding, among many

others.

Unexpectedly, in this current study, one finding

raised questions; the resemblance between the brain

MDA levels of young rats to that found in the

untreated aged rats. Statistically, the comparison

between these two groups were proven to be the most

insignificant (P = 0.9987). This result was not

analogous with a previous study done by Subathra,

Shila et al. (2005) which displayed a significantly

lower level of MDA in various brain regions of young

rats when contrasted to untreated aged rats. Some

plausible explanation behind these disparities are the

difference in the strain of rat (Wistar vs. Sprague-

Dawley), the age range of young rats (3-4 months vs.

2-3 months) and aged rats (>24 months vs. 20-24

months), and the type of CA extract used (whole-

plant vs. leaves). A shorter duration of treatment in

present study (28 days) could also play a key role in

these different findings. Likewise, 6 rats which died

in the middle of the study, thus altering the previously

optimal number of subjects, might contribute to the

change in mean calculations.

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

4 CONCLUSION

From our results, we concluded that Centella asiatica

may exhibit an antioxidative effect on aged rats,

comparable to that of vitamin E, which was

demonstrated by its capacity to reduce

malondialdehyde levels in aged brain rats. Despite the

insignificance found, the study suggests a potential

future role of Centella asiatica in hindering aging

process, if not preventing it.

ACKNOWLEDGEMENTS

This research was funded and supported by the

PITTA (Publikasi Terindeks Internasional Untuk

Tugas Akhir Mahasiswa UI) grant provided by

DRPM (Direktorat Riset dan Pengabdian Kepada

Masyarakat) Universitas Indonesia.

REFERENCES

Cesari, M. et al. 2016. Frailty: an emerging public

health priority. Journal of the American

Medical Directors Association 17(3) 188-192.

Dei, R. et al. 2002. Lipid peroxidation and advanced

glycation end products in the brain in normal

aging and in Alzheimer's disease. Acta

neuropathologica 104(2) 113-122.

Dev, R.D.O. 2009. Comparison on cognitive effects

of Centella asiatica in healthy middle age

female and male volunteers. Annals of Nutrition

and Metabolism 55 709.

Finkel, T. and Holbrook, N.J. 2000. Oxidants,

oxidative stress and the biology of ageing.

Nature 408(6809) 239-247.

Floyd, R.A. 1999. Antioxidants, oxidative stress, and

degenerative neurological disorders (44448).

Proceedings of the Society for Experimental

Biology and Medicine 222(3) 236-245.

Ho, Y.-S., So, K.-F. and Chang, R.C.-C. 2010. Anti-

aging herbal medicine—How and why can they

be used in aging-associated neurodegenerative

diseases? Ageing research reviews 9(3) 354-

362.

Jones, G.W. 2010. The 2010 – 2035 Indonesian

population projection. UNFPA Indonesia.

Kedar, N. 2003. Can we prevent Parkinson’s and

Alzheimer’s disease? Journal of postgraduate

medicine 49(3) 236.

Kumar, M.V. and Gupta, Y. 2002. Effect of different

extracts of Centella asiatica on cognition and

markers of oxidative stress in rats. Journal of

ethnopharmacology 79(2) 253-260.

Lieberman, M., Marks, A.D. and Peet, A. 2013.

Marks basic medical biochemistry: Wolters

Kluwer Health/Lippincott Williams & Wilkins.

López-Otín, C. et al. 2013. The hallmarks of aging.

Cell 153(6) 1194-1217.

Meagher, E.A. et al. 2001. Effects of vitamin E on

lipid peroxidation in healthy persons. Jama

285(9) 1178-1182.

Mukherjee, P.K., Kumar, V. and Houghton, P.J.

2007. Screening of Indian medicinal plants for

acetylcholinesterase inhibitory activity.

Phytotherapy research 21(12) 1142-1145.

Orhan, I.E. 2012. Centella asiatica (L.) Urban: from

traditional medicine to modern medicine with

neuroprotective potential. Evidence-based

complementary and alternative medicine 2012.

Spiteller, G. 2007. The important role of lipid

peroxidation processes in aging and age

dependent diseases. Molecular biotechnology

37(1) 5-12.

Subathra, M., Shila, S., Devi, M.A. and

Panneerselvam, C. 2005. Emerging role of

Centella asiatica in improving age-related

neurological antioxidant status. Experimental

gerontology 40(8) 707-715.

Tiwari, S. et al. 2008. Effect of Centella asiatica on

mild cognitive impairment (MCI) and other

common age-related clinical problems. Digest

Journal of Nanomaterials and Biostructures

3(4) 215-220.

Traber, M.G. and Atkinson, J. 2007. Vitamin E,

antioxidant and nothing more. Free Radic Biol

Med 43(1) 4-15.

Traverso, N. et al. 2004. Malondialdehyde, a

lipoperoxidation-derived aldehyde, can bring

about secondary oxidative damage to proteins.

The Journals of Gerontology Series A:

Biological Sciences and Medical Sciences 59(9)

B890-B895.

Veerendra Kumar, M. and Gupta, Y. 2003. Effect of

Centella asiatica on cognition and oxidative

stress in an intracerebroventricular

streptozotocin model of Alzheimer's disease in

rats. Clinical and Experimental Pharmacology

and Physiology 30(5‐6) 336-342.

WHO. 2011. Global health and aging. Geneva: World

Health Organization.

The Effect of Centella asiatica on Brain Malondialdehyde Levels of Aged Rats

WHO. 2015. World report on ageing and health.

World Health Organization.

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