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NEUROPROTECTIVE EFFECT OF GOTUKOLA (Centella asiatica (L.) URBAN) IN

CEREBRAL ISCHEMIC RAT MODEL

Article · November 2010

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 Proceedings of the Third International Conference on Mathematics and Natural Sciences
(ICMNS 2010)

NEUROPROTECTIVE EFFECT OF GOTUKOLA (Centella

asiatica (L.) URBAN) IN CEREBRAL ISCHEMIC RAT
MODEL
Hegar Pramastyaa ,Kusnandar Anggadiredja a*, Irda Fidrianyb

a Pharmacology and Clinical Pharmacy Research Group, School of Pharmacy

Bandung Institute of Technology, Bandung, West Java, Indonesia
bPharmaceutical Biology Research Group, School of Pharmacy Bandung Institute of

Technology, Bandung, West Java, Indonesia

*Corresponding author, e-mail: kusnandar@fa.itb.ac.id

Abstract. Cerebral ischemia, which causes neuronal damage, is one of the

contributing factors leading to stroke. Inhibition of neuronal damage, therefore, will
be important in reducing the risk of stroke. Traditionally, Centella asiatic a (L.)
Urban, also known as gotukola, has been known to maintai n memory integrity at
the central nervous system level. The objective of this research is to evaluate the
neuroprotective properties of gotukola (Centella asiatic a (L.) Urban) extract. The
evaluations of inhibitory effect of neural damage of gotukola extract were done in
rats made stroke by single common carotid artery occlusion (CCAO). The rats were
randomly assigned to the following groups: control (sham operated), CCAO, CCAO
+ 100 mg/kg extract, and CCAO + 320 mg/kg extract. Examinations were done
before the surgery and continued every day until 8 days after the operation.
Evaluation parameters included: exploratory distance and number of rearing in
open field apparatus; the width of the opening of the ptotic eye; the number of head
dip on the platform; hanging, reestablishment, and neurological scores; and the
percentage of neuronal damage. The results revealed that the extract improved the
number of rearing as well as exploratory distance in open field; hanging,
reestablishment, and neurological scores; and the width of the opening of the ptotic
eye, compared to those observed in CCAO group. The percentage of neuronal
damage found in group treated with CCAO + 100 mg/kg extract, CCAO + 320
mg/kg extract and CCAO was 16.33 ± 2.96%, 22.75 ± 2.93%, and 35.50 ± 4.09%
respectively. Taken together, the results showed that gotukola ( Centella asiatic a (L.)
Urban) extract inhibited the neuronal damage induced by cerebral ischemia, and
further indicated the potential use of the extract in preventing neuronal damage in
stroke.

Keywords: CCAO,Centella asiatic a, cerebral ischemia, neuroportective, rat.

1 Introduction
Stroke is one of the degenerative diseases with high mortality rate, and is
considered the third rank of the cause of clinical death in the world. Approxima tely
85% of stroke incidents are caused by cerebral ischemia (Jiang, 2006). As life
expectancy increases, so does the prevalence of stroke. In Indonesia, in 2008 the
life expectancy increased to 70.46 from 70.16 year in the previous year. The

77
 HEGAR PRAMASTYA , KUSNANDAR A NGGADIREDJA , IRDA FIDRIANY

prevalence of stroke in 2008 reached a figure of 500,000 per year, with mortality
rate of 25% of the total patients (Misbach & Ali, 2001).

Prognosis of stroke patient is even worse than cancer (Dyker & Lees, 1998). Almost
50% of stroke patients whose duration occur until one year and more, culminate in
death or disabilities (Dyker & Lees, 1998). Any end result is triggered by the brain
tissues damage that diminishes the brain function especially for coordination.
Medication of stroke is expected to involve the drug that can protect the brain from
suffering after stroke attack, including the use antiplatelet, thrombolityc, or
anticoagulant as for expediting the blood flow to the brain. Neuroprotectant is
expected to minimize the brain damage after the stroke onset. Therefore, after
stroke recovery will be more promising.

Gotukola (Centella asiatic a (L.) Urban) has been known traditionally as herb that
affects the central nervous system especially the memory. Thus, in the ayurvedic
medical system, gotukola is known as me dhyarasayana (drug that works on the
nervous system). There has been a research on neuroprotective effect of gotukola in
pup’s hippocampus along with the memory activity enhancement (Madhyastha et
al., 2007). Administration of gotukola water extract at 20 mL/kg during the
postanatal day of 7-60 enhanced the rats’ memory and learning ability, also protect
the hippocampus from the prenatal stress. (Madhyasta et al., 2007). Gotukola
extract has been proved to enhance the spurt of dendrite in the hippocampus CA3
area (as the cognitive area) (Rao et al., 2006). It has been also known that C.
asiatic a showed stimulating effect on wounded tissue recovery (Suguna et. al,
1996 dalam Zheng & Qin, 2007). Wound healing activity is related to the damaged
cells reparation and cellular growth stimulation. Research using human SH -SY5Y
cell line in vitro showed that ethanolic extract of C. asiatic a, at 100µg/mL,
significantly enhanced the neurite elongation with the presence of nerve growth
factor (NGF) (Soumyanath et al., 2005). These results suggested that C. asiatic a
extract can protect the brain from neuronal damage after cerebral ischemia.

The objective of the present study was to evaluate the neuroprotective activity of C.
asiatic a extract on cerebral ischemic rats, and to identify the optimal dose that
gives the brain protection, in paricular. The evaluation was done in rats made
stroke by occlusion of one of the common carotid artery. Parameters that were
observed include the behavior of rats and histological presentation of the brain.

2 Materials and Methods

2.1 Materials and Apparatus
Ethanol 95%, ethanol 70%, povidone iodine 10%, Sodium chloride 0.9%,
Thiopenthone-Na (Pentothal®) reconstitution injection, amoxicillin reconstitution
injection, 3-0 silk and 4-0 nylon threads, tragacanth, formalin (formaldehyde
38%), monosodium phosphate and disodium phosphate, xylol, paraffin,
hematoxylin-eosine stain, Percolator, rotary evaporator, open field apparatus of 90
cm x 90 cm x 35 cm (L:W:H) in dimention, 50 cm height platform, hanging track,
microtome.

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 Evaluation of Neuroprotective Effect of Centella asiatica

2.2 Plant Materials

Gotukola (Centella asiatic a (L.) Urban) leaves harvested after about 4 months of
growing, taken from Medicinal and Aromatic Plants Research Bureau (BALITRO)
Manoko, Lembang. The plant was identified in Hebarium Bandungense, School of
Life Sciences and Technology ITB.

2.3 Experimental Animals

Inbred male Wistar rats of 75-90 days old weighting 180-270 g were used. The rats
were taken from animal husbandry of School of Pharmacy, ITB. Animals were fed
with standard animal food pellet and water ad libitum.

2.4 Methods

2.4.1 Leaves extraction

Gotukola leaves were extracted by maceration with ethanol 95% for 24 hours. The
maceration was replicated three times. The extract was then concentrated by
vacuum rotary evaporator at 50°C and 30 rpm.

2.4.2 Experimental group

There were four experimental groups in this study: the sham, CCAO (given
tragacanth 0.5%, the vehicle), CCAO + gotukola extract 100 mg/kg (CCAO + GE
100), CCAO + gotukola extract 320 mg/kg (CCAO + GE 320).

2.4.3 Cerebral ischemia induction

Unilateral common carotid artery occlusion (CCAO) was the method used for
inducing the cerebral ischemia, as performed in a previous study (Chen et al,
1986). The rats were anesthetized with thiopentone sodium (50 mg/kg, i.p.).
Surgery began with midline incision of the ventral neck. After being isolated, the
left common carotid artery was squeezed for two minutes. It was the permanently
occluded by ligation with two 4-0 nylons threads above and below the squeezed
point. Afterwards, the incision was sutured with 3-0 silk thread (USP). For
preventing any post-operation infection, povidone iodine was applied topically
along the incision, and 45 mg/kg amoxicillin was administered i.p.

2.4.4 Behavioral and motor examination

Behavioral and motor examinations included open field, platform, hanging and re -
establishment tests, measurement of the width of ptotic eye, and examination for
neurological scores. Open field examination was done by recording the
spontaneous movement of rats in open field apparatus for seven mi nutes (Wahl et
al., 1992). Hanging score (ranged from 1 to 4) was based on the number of limbs
the rats used to hang on the hanging track. Reestablishment score (1 to 4) was
determined by the ability of rats to hold on to the hanging track after being forced
to hang with the front limbs (scored between 1 and 4). The degree of ptotic eye was
measured as the space width between the lower and upper eyelids. Neurological
scoring was done according to a previous procedure (Rogers et el., 1997).

79
 HEGAR PRAMASTYA , KUSNANDAR A NGGADIREDJA , IRDA FIDRIANY

2.4.5 Brain tissue examination

Brains were isolated on the eighth day after the surgery. Rats were sacrificed by
cervical dislocation. The brains were soaked in the 10% formalin in buffer solution
for at least two days. The brains were then sliced at 6 mm from the frontal pole
with 2-3 µm of thickness. The slices were stained with Hematoxylin-eosin. The
measurement of the brain infarction was done by counting the dead neurons per
100 counted neurons and expressed in percent (Ito et al., 1999).

2.4.6 Statistical analysis

In statistical analysis one-way ANOVA was used followed by LSD for post hoc
analysis. Differences were considered significant at P<0.05.

3 Results
3.1 Exploratory distance
Results of exploratory distance observations are depicted in Figure 1. There was
not any significant difference of exploratory distance among experimental groups
(P< 0.05). Sham group showed the highest of exploratory distance followed by
CCAO group + GE 320, CCAO group, and CCAO + GE 100.

In first day after surgery, there were significant differences among the sham and
other groups. The exploratory distance of the CCAO, CCAO + GE 100, CCAO + GE
320, and sham group was 325.67 ± 59.47, 227.48 ± 56.45, 509.90 ± 174.4, and
1282.85 ± 480.54 cm, respectively. The significant difference between the sham
group and others indicated the motor impairment after CCAO surgery.

Observation on the second day after surgery revealed significant difference between
CCAO group and sham group (P< 0.05). Meanwhile, there was no significant
difference between CCAO + GE 100 and sham group, and the lack of significant
difference was not observed between CCAO + GE 320 and sham group. Exploratory
distance of CCAO group and sham group was 201.73 ± 73.09 cm and 821.42 ±
239.2 cm, respectively. The CCAO + GE 100 and CCAO + GE 320 groups showed
improvement on day 3 after the surgery.

80
 Evaluation of Neuroprotective Effect of Centella asiatica

3000.00

2500.00

Exploratory Distance (cm)

2000.00

1500.00

1000.00

*
500.00
* *
* *
* *
0.00
Pre-op I II III IV V VI VII VIII

Hari
Day

Figure 1 Effects of gotukola extract on exploratory distance as tested in open field

test. Open field tests were done for 7 minutes. ♦ Sham (rats underwent surgery
without occlusion of common carotid artery). ■ CCAO (CCAO operation with only
vehicle administration).▲ CCAO + 100 mg/kg extract. ● CCAO + 320 mg/kg extract.
Data represents means ± SEM of 6-7 rats. *P<0.05 (one-way ANOVA followed by LSD).

On the sixth day after surgery the CCAO + GE 100 and CCAO + GE 320 groups
showed significant difference compared to sham group. The exploratory distance of
CCAO + GE 100, CCAO + GE 320, and sham group was 252. 57± 82.89 cm, 548.60
± 252.37 cm, and 1196.50 ± 234.26 cm, respectively. Significant di fference were
also observed on the day seven after surgery between sham and CCAO + GE 320
groups. Exploratory distances throughout the observation period were so
fluctuated, and tended to decrease. Several reasons might underlie this
observation, including those related to mood and anxiety and pain after operation.

3.2 Rearing
Results of rearing observations are depicted in Figure 2. Rearing was observed
concomitantly with exploratory distance in open field apparatus. Rearing number
of all groups seemed to be decrease during the days following the surgery. There
was significant difference (P<0.05) on day 2 after surgery between CCAO (1.00 ±
0.68) and sham (9.00 ± 3.55) groups. On day 6, there were differences among
CCAO (4.20 ± 1.28), CCAO + GE 100 (4.00 ± 2.28), CCAO + GE 320 (6.67 ± 1.45)
and sham (13.60 ± 1.81) groups. On the day 8, there were no differences among
the groups, albeit increased rearing in CCAO + GE 320 group. Again, the number
of rearing was fluctuated during the observation period, and it might be related to
mood and anxiety.

81
 HEGAR PRAMASTYA , KUSNANDAR A NGGADIREDJA , IRDA FIDRIANY

25.00

20.00

Number of rearing
15.00

10.00

5.00

0.00
Pre-op I II III IV V VI VII VIII

Hari
Day

Figure 2 Effects of gotukola extract on number of rearing. Open field tests were done
for 7 minutes in open field apparatus. ♦ Sham (rats underwent surgery without
occlusion of common carotid artery). ■ CCAO (CCAO operation with only vehicle
administration). ▲ CCAO + 100 mg/kg extract. ● CCAO + 320 mg/kg extract. Data
represents means ± SEM of 6-7 rats. *P<0.05 (one-w ay ANOVA followed by LSD).

4.50

4.00

3.50 **
3.00 *
Hanging score

2.50
* *
*
2.00 *

1.50

1.00

0.50

0.00
Pre-op I II III IV V VI VII VIII

Day
Hari

Figure 3 Effects of gotukola extract on hanging score. ♦ Sham (rats underwent

surgery without occlusion of common carotid artery). ■ CCAO (CCAO operation with
only vehicle administration).▲ CCAO + 100 mg/kg extract. ● CCAO + 320 mg/kg
extract. Data represents means ± SEM of 6-7 rats. *P<0.05 (one-w ay ANOVA followed
by LSD).

82
 Evaluation of Neuroprotective Effect of Centella asiatica

3.3 Hanging score

The results are described in Figure 3. The score were decreased in grups
undergoing CCAO surgery. The score in CCAO + GE 100 (2.29 ± 0.18) and CCAO +
GE 320 (2.67 ± 0.33) was significantly different ( P < 0.05) with that of the sham
group. Of day 5, there was significant difference between CCAO (3.00 ± 0.26) and
CCAO + GE 100 (3.00 ± 0.22) compared to sham (3.67 ± 0.21). Hanging score of the
extract-treated groups increased gradually throughout the 8-day observation
period, anda there was no difference between CCAO + GE 100 and CCAO + GE
320. The result showed motor function improvements of ischemic rats after the
administration of gotukola extract.

3.4 Reestablishment score

The data is presented in Figure 4. Reestablishment score of all CCAO-treated
groups was decreased. Reestablishment score of CCAO group was not more than
2.5. On the first day after the surgery, reestablishment score of CCAO (2.33 ± 0.21)
and CCAO + GE 100 (2.29 ± 0.18) was significantly different ( P < 0.05) compared to
the sham (3.17 ± 0.31) group. Reestablishment score of CCAO + GE 100 and CCAO
+ GE 320 group were gradually increased towards day 8 after the surgery. The
score of CCAO + 100 (3.00 ± 0.3) and CCAO + 320 (3.17 ±0.4) were significantly
higher (P < 0.05)than that of the CCAO.
4.00

3.50
**
Reestablishment score

3.00
**
2.50

2.00 ***
1.50

1.00

0.50

0.00
Pre-op I II III IV V VI VII VIII

Hari
Day

Figure 4 Effects of gotukola extract on reestablishment score. ♦ Sham (rats

underwent surgery without occlusion of common carotid artery). ■ CCAO (CCAO
operation with only vehicle administration). ▲ CCAO + 100 mg/kg extract. ● CCAO +
320 mg/kg extract. Data represents means ± SEM of 6-7 rats. *P<0.05 vs. sham,
**P<0.05 vs. CCAO, ***P<0.05 vs. CCAO + gotukola extract 320 mg/kg (one-w ay
ANOVA followed by LSD). (one-w ay ANOVA followed by LSD).

3.5 Number of head-dips

Numbers of head dips were on a platform, placed 50 cm above the floor. It is
supposed to be a strange place for expe rimental animals. Almost all of the groups
showed decline in the number of head dip when tested after the surgery. From the

83
 HEGAR PRAMASTYA , KUSNANDAR A NGGADIREDJA , IRDA FIDRIANY

observation, there was no significant difference between the groups with regard to
this parameter. Yet, number of head dips in CCAO + GE 100 and CCAO + 320 were
usually higher than CCAO group.

10.00
Number of head dip on platform

9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
*
0.00
Pre-op I II III IV V VI VII VIII

Hari
Day

Figure 5 Effects of gotukola extract on the number head-dip. ♦ Sham (rats

underwent surgery without occlusion of common carotid artery). ■ CCAO (CCAO
operation with only vehicle administration).▲ CCAO + 100 mg/kg extract. ● CCAO +
320 mg/kg extract. Data represents means ± SEM of 6-7 rats. *P<0.05 (one-w ay
ANOVA followed by LSD).

7.00

6.00
Width of ptotic eye (mm)

5.00 ** ** **
**
** **
4.00 **
** **
**
3.00

2.00

1.00

0.00
Pre-op I II III IV V VI VII VIII

Day
Hari

Figure 6 Effect of gotukola extract on the width of the ptotic eye. ♦ Sham (rats
underwent surgery without occlusion of common carotid artery). ■ CCAO (CCAO
operation with only vehicle administration).▲ CCAO + 100 mg/kg extract. ● CCAO +
320 mg/kg extract. Data represents means ± SEM of 6-7 rats. *P<0.05 vs. sham,
**P<0.05 vs. CCAO (one-way ANOVA followed by LSD).

84
 Evaluation of Neuroprotective Effect of Centella asiatica

3.6 Width of ptotic eye opening

The data is presented in Figure 6. All CCAO-treated groups showed ptotic eye,
though the size of ptotic eye among the groups were not uniform. The CCAO group
showed the most squinted eye. Measurement on day 6 until day eight after the
surgery showed marked difference (P < 0.05) between the CCAO and CCAO + GE
100 and CCAO + GE 320 group.

3.7 Neurological score

Results on the observation of neurological score are presented in Figure 7. The
score of three groups undergoing CCAO increased significantly on day 1 after the
surgery. Observation on the following days after the surgery showed the decline in
the neurological score especially in CCAO + GE 100 and CCAO + GE 320 groups.
From day 6 until day 8 after the surgery, there were significant differences ( P <
0.05) between groups CCAO + GE 100 and CCAO + GE 320 compared and that of
CCAO.
3.00

2.50

2.00
Neurological score

1.50

1.00
**
**
0.50 ** **

** **
0.00
Pre-op I II III IV V VI VII VIII
-0.50
Day
Hari

Figure 7 Effects of gotukola extract on neurological score. ♦ Sham (rats underwent

surgery without occlusion of common carotid artery). ■ CCAO (CCAO operation with
only vehicle administration).▲ CCAO + 100 mg/kg extract. ● CCAO + 320 mg/kg
extract. Data represents means ± SEM of 6-7 rats. *P<0.05 vs. sham, **P<0.05 vs.
CCAO (one-w ay ANOVA followed by LSD).

3.8 Brain tissue examination

Results of brain tissue examination are presented in Figure 8. It was shown that
the three CCAO-treated groups had extensive brain damage. CCAO, CCAO + GE
100, and CCAO + GE 320 groups had markedly brain damage compared to sham
group (P < 0.05). However, CCAO + GE 100 and CCAO + GE 320 groups had less
brain damage compared to CCAO group (P < 0.05). The percentage of dead cells
was 1.5 ± 0.5%, 35.5 ± 4.09%, 16.33 ± 2.96%, and 22.75 ± 2.93% in sham, CCAO,
CCAO + GE 100, and CCAO + GE 320 group, respectively.

85
 HEGAR PRAMASTYA , KUSNANDAR A NGGADIREDJA , IRDA FIDRIANY

* **
* **

**

Figure 8 Effects of gotukola extract on the percentage of damaged neurons. Brains

were isolated on day 8 after the surgery. Neuronal damage was calculated as
percentage of dead neurons out of the 100 counted neurons. The histology w as done
in brain tissue slides of 2-3 μm of thick, taken from the area 6 mm from the frontal
pole of cerebrum. The slides were stained with Haematoxyllin -eosin. Data represents
means ± SEM of 6-7 rats. *P<0.05 vs. sham, **P<0.05 vs. CCAO (one-w ay ANOVA
followed by LSD).

4 Discussion
Gotukola is well known in several regions of Indonesia, Sundanese call it antanan
besar while the people of Jawa call it kerok betok. Most of compounds isolated
from gotukola belong to pentayclic triterpenic acid, their respective glycoside,
belonging to ursane- or oleanane-type including asiaticoside, asiatic acid,
centelloside, madecassic acid, madecassoside, brahmoside, brahmic acid,
brahminoside, thankuniside, isothankuniside, madaisatic acid, centic acid, cenellic
acid, betulinic acid, and indocentic acid. There were also several asiaticosides
found in the butanol fraction including asiaticoside C, D, E, and F. Terpenoid
group has several important therapeutic effects. In addition, the substances are
also used as marker compound for quality control standard. There were also
several essential oil found in gotukola, mostly caryophyllene, farnesol, and elemene
(Zheng & Qin, 2007).

Gotukola is known as both vegetable and medicinal herb. As medicinal herb,

people believe that this plant can be used for ameliorating hypertension, treatment
of dermatitis, healing the wound, purification of the blood, and as memory
enhancer (Satake et al., 2007). Gotukola, also noted in Ayurvedic system of
medicine, has been used for a long time in India. In this system, gotukola included

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 Evaluation of Neuroprotective Effect of Centella asiatica

in medhya ras ayana group of medicine as also Acorus cal aums, jatamansi, and
Bacopa monnieri (Sharma, 1992 in Rao et al., 2006). Medhya ras ayana have been
claimed to improve mental ability (Sharma, 1992 in Rao et al., 2006).

Gotukola also proved to give stimulation effect on brain. Several effects on central
nervous system influenced by gotukola including nerve tonic stimulation, sedation
effect, tranquillizer, memory and intelligence enhancement. Extract of gotukola not
only can enhance the spatial ability of memory but also increase the dendritic
arborization in CA-3 area of hippocampus of the rats (Rao et al. 2005). Water
extract of gotukola in dose of 4-6 mL/kg has been found to be effective in
stimulating the elongation of dendrite for about 100µm from the cell body after 4-6
weeks of observations (Rao et al., 2005).

Results of the present study showed that common carotid artery occlusion
significantly decreased brain function to certain degree. This procedure inhibits
blood supply to several areas of the brain such as motor and sensory cortices,
hippocampus, amygdala, globus palidus, and midbrain (Barr, 1979). However,
several behavioral parameters might be confounded by post -operative condition of
the rats, especially pain. Hence, motor function decrease was also seen in the
sham group, though it was not significant compared to CCAO -treated groups.
Observation on behavioral parameters showed that gotukola-treated groups
demonstrated better brain function compared to that undergoing CCAO without
extract (CCAO group).

Histological examination revealed that CCAO group had most extensive brain
damage than other groups. Groups treated with gotukola extract showed less brain
damage, with the group given 100 mg/kg demonstrated the least brain damage.
This seems to be slightly diffe rent with behavioral results in which the groups
treated with 320 mg/kg gotukola extract showed the most promising improvement.
This inconsistency between behavioral and histological results might be due the
choice of brain are for histological examination. It might be that the area observed
was not the one responsible for the behavioral parameters evaluated. Furthermore,
behavioral observations sometimes do not give best representation of brain damage
due to certain degree of bias and confounding (Wahl et al, 1992). Altogether,
however, results from both behavioral observation and histological examination
provide evidence that gotukola extract had the ability to protect the neuron from
damage due to cerebral ischemia.

The ability of gotukola to protect the neuron cannot be separated from the potential
activity of asiaticoside, as the major content of gotukola extract, and another
terpenoid such as madecasoside, and asiatic acid. Murdoch and Hall (1990) have
summarized the causal mechanisms that lead and aggravate neural damage after
brain ischemia, as follow: 1) Failure in ion regulation; influx of Ca 2+, Na+ and Cl -;
excessive efflux of K+ caused by pump protein impaired and drastic decline of ATP
as energy source, 2) Accumulation of Ca 2+ leading to the cascade of cellular
reaction, like activation of phospholipase enzyme, that could worsen the membrane
integrity; This phenomenon will also lead NMDA receptor excessive stimulation that
causes neurotoxicity, 3) Excessive lactate production through glycolysis
metabolism pathways as compensatory energy catabolism during anaerobic
condition, and 4) Free radicals generation.

87
 HEGAR PRAMASTYA , KUSNANDAR A NGGADIREDJA , IRDA FIDRIANY

Several studies about biological activity of gotukola extract or component of

gotukola extract have been done. Water extract of gotukola has been shown to
inhibit the iPLA2 and cPLA2 enzymes acitvity. Water extract of gotukola has been
demonstrated to inhibit the activity of iPLA 2 in the cerebellum of the rats at the
concetration between 125 μg/mL to 500 μmg/mL. At the concentration between
12.5 μg/mL until 500 μg/mL, water extract of gotukola could inhibit the activity of
cPLA2 (Barbosa et al, 2008). iPLA2 and cPLA2 are believed to be involved in cellular
damage following ischemic condition (Farooqui et al, 2006). Inhibition of those
enzymes can supress the production of eicosanoid substances like prostaglandin
and leukotrien which play important role in tissue inflammation as in stroke. Along
with that, suppression of phospholipase enzyme possibly can take a part in
keeping the integrity of the cells membrane. Hence, it can prevent massive inlfux of
ions like Na+ and Cl - or K + efflux followed by water intrusion, leading the edema
and cascade effect caused by depolarization of the cell (Murdoch and Hall, 1990).

Another studies on asiaticoside showed that the substance has the anxiolytic effect
in mice at the dose of 5 – 40 mg/kg body weight (Chen et al, 2006). Meanwhile,
antidepressant effect also can be detected in mice at the dose of 10 – 20 mg/kg
body weight (Liang et al, 2008). Results from experiment using elevated maze plus,
hole board, and light/dark box, showed that the dose of 10 mg/kg and 20 mg/kg
gave the highest anxiolytic effect (Chen et al, 2006). The highest antidepressant
effect was also observed after the dose of 10 mg/kg and 20 mg/kg (Liang et al,
2008). In this experiment, anxiolytic and antidepressant effects can be slightly seen
on the open field observation and number of head dip on the platform.

It seems that there is no relation between anxiolytic and antidepressant activities

and neuroprotective action, but several experiments have proved that anxiolytic
and antidepressant drugs showed potential neuroprotective effect. Imipramin, an
antidepressant, was reported to give neuroprotective effect at a concentration 3μm,
on the apoptotic neural stem cell induced by lipopolisaccharide (Peng et al, 2008).
In addition, diazepam at 10 mg/kg protected the CA 1 area of hippocampus for 35
days following cerebral ischemic induction (Schwartz – Bloom et al, 1998).

Antioxidant contained in gotukola extract might be expected to play a role in its

neuroprotective acitvity. Several compounds such as quercetin, catechin, rutin,
and narigin could be responsible for the antioxidant activity (Zheng and Qin, 2007).
As shown by Flora and Gupta (2007) at doses of 200 mg/kg and 500 mg/kg, water
extract of gotukola protected the Wistar rats from the oxidative reaction induced by
arsenic compounds.

5 Conclusion
Results of the current study have indicated neuroprotective effect of gotukola
extract on cerebral ischemia in induced rats. At 100 mg/kg the extract showed the
highest protection against neuronal damage following cerebral ischemia induced by
single common carotid artery occlusion. In the long run, the results further shed
light on the potential use of gotukola extract as an alternative for the management
of stroke.

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 Evaluation of Neuroprotective Effect of Centella asiatica

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H EGAR P RAMAS TY A
Pharmacology-Clinical Pharmacy Research Group
School of Pharmacy,
Institut Teknologi Bandung, Indonesia
E-mail: hegarpramastya@gmail.com

KUSNANDAR ANGGADI REDJA

Pharmacology-Clinical Pharmacy Research Group
School of Pharmacy
Institut Teknologi Bandung, Indonesia
E-mail: kusnandar@fa.itb.ac.id

I RDA FI DRIANY
Biological Pharmacy Research Group
School of Pharmacy
Institut Teknologi Bandung, Indonesia
E-mail: irda@fa.itb.ac.id

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