Steamed Watermelon (Citrullus lanatus Thunb.) Juice Improves

Spatial Memory in Dementia Rat Model

Maitrianti, Hadi Sunaryo and Dedi Suryadi

Faculty of Pharmacy and Science, Universitas Muhammadiyah Prof. DR. HAMKA, Jakarta, Indonesia

Keywords: Watermelon, Lycopene, Dementia, Spatial Memory

Abstract: Watermelon (Citrullus lanatus Thunb.) is one of the fruits that containing lycopene. Lycopene is able to

reduce oxidative damage that causes dementia. This research was aimed to determine the activity of steamed

watermelon juice in improving the spatial memory in dementia rats model induced by Electroconvulsive

Shock (ECS). The animal test was divided into 6 groups, namely normal control group, negative control

group, positive control group (vitamin E, 20.7 mg/kg BW), and 3 treatment groups that received steamed

watermelon juice with different dose, i.e 0.92 g/kg BW,1.85 g/kg BW, and 3.7 g/kg BW. All groups were

induced with ECS, except for the normal control group. The spatial memory was measured with the Radial

Arm Maze (RAM) test and the observed results were analyzed with ANOVA and a post-hoc Tukey’s HSD test.

The ANOVA test results showed signicant differences between all treatment groups. Meanwhile, the post-

hoc Tukey’s HSD test at 95% condence level resulted in a signicant difference between the the treatment

groups with the doses of 0.92 g/kg BW and 1.85 g/kg BW and the negative control group. However, this

test indicated an insignicant difference between the positive control group and the normal control group.

This nding conrms that the administration of steamed watermelon juice at doses of 0.92 g/kg BW and

1.85 g/kg BW improves the spatial memory in a rat mode of dementia comparable to the positive control.

The treatment groups receiving these two doses showed corresponding results, meaning that both doses of

steamed watermelon juice have an equivalent spatial memory enhancement activity.

1 INTRODUCTION

Dementia is a syndrome of decreased intellectual

function that is severe enough to interfere with social

and economic activities in daily life, and is, usually

followed by behavioral changes not attributable

to delirium or major psychiatric disorders. The

prevalence of dementia increases rapidly with life

expectancy. Even though there has been no national

research data regarding the prevalence of dementia

the growing number of elderly population predictably

increases the incidence of dementia in Indonesia.

The Stroke Registry Indonesia 2013 support this

assumption. It reports that stroke become more

prevalent at a younger age, and 60.59% of stroke

patients experience impaired cognition after returning

home from hospital treatment. This registry also

explains that the stroke risk factors in the country

are fairly high (Perhimpunan Dokter Spesialis Saraf

Indonesia, 2015).

Oxidative stress is signicantly involved in the

pathology of dementia even at the early stage of

the disease (Mao, 2013). The brain and nerve cells

are susceptible to damages by free radicals because

the membrane lipids contain many side chains of

polyunsaturated fatty acids. Oxidative stress also

depends on other factors, namely the quantity of brain

oxygen consumption, iron and other metals transition

that responsible for the formation of Reactive

Oxygen Species (ROS), and antioxidants in the brain.

Antioxidants have been widely studied in relation to

neurogenerative diseases (Rao et al., 2003. Research in

Parkinson’s patients and vascular dementia indicates

low serum lycopene levels (Foy, et al., 1999), which,

according to a stroke prevention study in Austria,

are associated with a high risk of microangiopathy

(Schmidt et al., 1997). In elderly patients, a high level

of lycopene is positively connected to their functional

capacity, as indicated by their ability to perform self-

care (Snowdon et al., 1996). Given the importance of

Maiﬁtrianti, ., Sunaryo, H. and Suryadi, D.

Steamed Watermelon (Citrullus lanatus Thunb.) Juice Improves Spatial Memory in Dementia Rat Model.

DOI: 10.5220/0008240000960101

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 96-101

ISBN: 978-989-758-349-0

attention to neurogenerative diseases, studies related

to the effectiveness of lycopene in the management of

dementia are necessary.

Lycopene is fat-soluble antioxidant synthesized by

many plants and microorganisms. It is responsible for

giving the red color in various vegetables and fruits.

Earlier, only tomato and its products were considered

as the potential sources of lycopene, but nowadays

there are scientically proven facts that watermelon

also contains an appreciable amount of cis-congured

lycopene. Therefore, people gradually shift their

preference towards watermelon and its allied products

to deal with their health concerns. Nevertheless,

the quantity of lycopene varies depending upon the

variety and growing conditions (Fish and Davis,

2003). Overall, in the cells, lycopene is present in the

form of crystalline with a range of 2.30-7.20 mg/100 g

fresh weight basis (Huh et al., 2008; Artes-Henandez

et al., 2010). In general, the human body only absorbs

10-30% of fresh lycopene in fresh lycopene in fruits

or vegetables. Lycopene is more effectively absorbed

when consumed from processed foods, such as sauce

and juice. The increased absorption of lycopene in

tomato products is due to the presence of cis-isomers

of lycopene. Lycopene naturally appears as ‘trans’,

which is thermodynamically stable, soluble in non-

polar solvents, and identiable at a wavelength of

446-506 nm. Lighting and heating can potentially

change the isomer form ‘trans’ to ‘cis’ (Rao & Rao,

2007). Maulida & Zulkarnaen (2010) conclude that

the optimum temperature for watermelon to release

lycopene from its cell structure is 70

This research aimed to determine the activity of

steamed watermelon juice in improving the spatial

memory in a rat model of Electroconvulsive Shock

(ECS)-induced dementia and to determine the

effective dose to increas spatial memory. Steamed

watermelon juice was expected to be able to

improve spatial memory and function as a natural

treatment, which would be potentially developed

into phytopharmaceutical preparations, as well

as a reference for further research to support the

development of science.

2. MATERIALS AND METHODS

2.1 Materials

The research apparatus included an electroconvulsive

meter Model EC 02 from Orchid Scientic and

Innovative India Pvt. Ltd. (B 59, MIDC, Ambad,

Nashik-422010 India) and a radial arm maze designed

with eight equidistantly spaced arm (53.5 cm x 12.5

cm) extending from a 24 cm diameter central platform

elevated 11.5 of the oor.

The fresh watermelons (Citrullus lanatus

Thunb.) were obtained from Cipanas, West Java and

determined at LIPI Biological Research Center, Bogor

from the Herbarium Bogoriense collection, Vitamin

E (tocopherol) was acquired from PT. Cortico Mulia

Sejahtera. The test animals were white male rats

(Rattus norvegicus) strain Sprague Dawley aged 2-3

months and weighed ± 200 gram.

2.2 Methods

2.2.1 Identication of Watermelon

The watermelons were taxonomically identied

from the Herbarium Bogoriense collection at LIPI

Biological Research Center, Bogor.

2.2.2 Preparation of Steamed Watermelon

Juice

Around 100 grams of watermelons were steamed for 5

minutes at a temperature of 70

C and the temperature

was measured with a thermometer (Maulida &

Zulkarnaen, 2010). The steamed watermelon was

drained and the juice was extracted using a juicer

machine.

2.2.3 Phytochemical Screening

The steamed watermelon juice was tested for the

presence of alkaloids, avonoids, steroids, tannins,

triterpenoids, and saponins.

2.2.4 Treatment of Test Animal

This research is approved by the Ethics Committee of

Health Research, Faculty of Medicine, University of

Indonesia No. 278/UN2. F1/ETHICS/2017.

A total of 30 white male Sparague Dawley rats

(Rattus norvegicus) aged 2-3 months old and weighed

with ± 200 g was divided into six (6) groups, each

consisting of ve (5) rats. These six groups were:

Group 1 as normal control, Group 2 as negative

control, Group 3 as positive control, Group 4 as a

treatment group receiving 0.92 g/kg BW of steamed

watermelon juice, Group 5 as a treatment group

receiving 1.85 g/kg BW of steamed watermelon juice,

and Group 6 as a treatment group receiving 3.7 g/kg

BW of steamed watermelon juice.

Steamed Watermelon (Citrullus lanatus Thunb.) Juice Improves Spatial Memory in Dementia Rat Model

The animals were acclimatized for at least ve (5)

days to the laboratory conditions before the behavioral

experiments. They were placed in a natural light/dark

cycle (12 h each) and given free access to standard

food and water. From the second to the fourth day

of the acclimatization, they were trained in the

maze environment. After all group of rats fasted for

12 hours, they were subjected to preliminary radial

arm maze test for 12 days. This test was called the

Radial Arm Maze Test I (RAM I). The rats were

placed on the central platform and the food (pellets)

were hidden in a container at the end of the arm. The

rat performance represented the ratio of the number

of reward eaten pellets to the total number of arms

entered in 10 minutes. There were a total of eight (8)

arms in the maze.

After the RAM I was comleted, Groups 2, 3, 4, 5,

and 6 received ECS with a magnitude of 112 mA/150

V for 1 sec/day. Afterward, the experimental animals

were rested for 10 minutes. Then, they were placed

in the maze again and re-tested to prove that the ECS

induced a dementia model. The ECS was performoed

for eight (8) days.

The positive control group (Group 3) received

Vitamin E at a dose of 20.7 mg/kg BW, Groups 4,

5, and 6 were given steamed watermelon juice at

different doses, namely 0.92 g/kg BW, 1.85 g/kg

BW and 3.7 g/kg BW, respectively. The steamed

watermelon juice and vitamin E were administered

orally once a day every morning for 14 days.

The next stage was a post-test, i.e.,a radial arm

maze test for 12 consecutive days, referred to as RAM

II. Before the post-test the rats fasted for 12 hours.The

performance of the test rats in the RAM assay was

calculated with the formula (1) (Sari & Ar Rochman,

2015):

Performance (%)

x100%=

(Number= of eaten reward (food))/

(Number of arms entered by the rats)

(1)

2.2.5 Data Analysis

The mean percentage of the performance of the

experimental groups in RAM II was tested for normality

using the Shapiro-Wilk test and homogeneity using

the Levene’s test. Afterward, the data were analyzed

with one way ANOVA at 95 % signicance level (ɑ

= 0.05). If the results showed a statistically signicant

difference, then the data analysis was continued with

the Tukey test (Priyatno, 2012).

3 RESULTS AND DISCUSSION

The phytochemical screening showed that steamed

watermelon juice was positive for alkaloids,

terpenoids, steroids and saponins (Table 1).

Before the induction of dementia with ECS, all

groups of experimental animals were subjected to the

radial arm maze test I; henceforth referred to as RAM

I, this test identied the basic memory of each rat and

the whole six groups, and afrmed that all of them

had the same condition before the treatment. The

results of RAM I are described in Table 2. The entire

groups showed remarkable spatial memory every day

during the 12 days observation. Spatial learning and

memory help animals nd locations that provide,

among other things, food and safety, and are thereby

crucial for survival (Dogru et al., 2003).

The performance of each test group in selecting the

arms and feeding in RAM I showed an increase every

day. Even though Group 1 had lowest average score

Table 1: The phytochemical screening results of steamed

watermelon juice.

Compounds Results

Alkaloids +

Flavonoids -

Tannins -

Steroids and Triterpenoids +

Saponins +

Table 2: The observation and assessment results of the

performance (%) of the experimental rat groups in RAM I.

Day The Performance of the Groups (in %)

1 2 3 4 5 6

1 23.3 5 3.3 20 0 10.6

2 0 26.6 23.3 32.5 9.8 5.3

3 68 23.1 57.6 58.6 43.3 76

4 20 53.8 28.3 37.5 34.4 39.2

5 25 73.8 74.2 68 61.1 100

6 20 64.2 35 63.6 73 37.5

7 7.3 46.2 74.2 60 55 57.5

8 20 56.6 40 46 65.8 57.1

9 20 56.6 40 46 65.8 53.1

10 79.6 33.3 89.2 36.6 100 68

11 100 97.1 100 42 87.1 76.6

12 100 80 96 40 90 80

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

of performance among all the test groups (38.3%), it

scored 100% on Day 11 and 12. Meanwhile, Group 5

had the highest average (57.1%), followed by Group 3

and 6 (55%), Group 2 (51.4%), and Group 4 (45.8%).

After the 12-day observation in RAM I, the animal

model of dementia was prepared. Except for the

normal control group, all of the experimental groups

were given ECS with an intensity of 150 V/112mA for

1 sec/day for eight (8) days. Each rat was electrocuted

at the top of the ear and jammed until the electricity

caused muscle spasms and stiffness. After rested for

10 minutes, the rats were tested in the maze to prove

that the group had already developed dementia. The

observation results are summarized in Table 3.

The results of the RAM assays after the ECS

showed a decrease in the percentage of feeding and

arm entries. When compared with normal controls

(no ECS), all of the treatment groups showed low

performances. Groups 4 even failed seven times

(0%) and it was the lowest performance of all test

groups. These results are the effect of ECS-induced

memory degradation. They indicate poor working

and reference memory capabilities, as well as low

learning abilities in rats. During the maze test, the rats

behaved as if in confusion and remained silent in the

maze. They moved slowly and did not recognize the

food placed at the end of the arm. The ECS decreased

their memory because it involved the formation

of free radicals that attacked the walls and nerve

cells of their brains. These cells are composed of

unsaturated fatty acids. Therefore, ECS would lead to

peroxidation of fatty acids and neurotoxicity, causing

neurotransmitter death and, subsequently a decline in

memory (Luo et al., 2011).

Groups 4, 5, and 6 received steamed watermelon

juice at different doses namely 0.92 g/kg BW, 1.85 g/

kg BW, and 3.7 g/kg BW respectively, for 14 days.

The steamed watermelon juice was freshly made each

day. The watermelon was prepared in small pieces,

weighed using an analytical scale according to the

needs of the three (3) treatment groups steamed at

70°C for 5 minutes and rested until the temperature

decreased. Afterward, the steamed watermelon was

drained and ready for juice extraction. The positive

control received Vitamin E at a dose of 20.7 mg/kg

BW dissolved in Na CMC. After these treatments, the

rats were subjected to the second performance test, i.e.,

RAM II to identify any increases in spatial memory.

The observation results are presented in Table 4.

Table 4 shows an increase in spatial memory

during the 12 day observation in RAM II. The spatial

memory of the treatment groups began to increase

on Day 4 and reached its peak on the last day of the

observation. Group 4 showed the highest performance

with an average of 74.6%. The spatial memories of

Groups 3, 5, and 6 increased by 65%, 68.1% and

42% respectively. The normal control (Group 1) had

a different performance with negative control (Group

2). Furthermore, the performance of the negative

control in RAM II was signicantly different from its

performance in RAM I. This group showed the lowest

performance demonstrating the poor working and

reference memory capabilities and the low learning

ability of rats.

Group 4 and 5 each receiving steamed watermelon

juice at the doses of 0.92 g/kg BW and 1.85 g/kg BW,

respectively, scored 100% three times and ranked as

the highest performance groups. This result suggest

that these doses are effective to increase the spatial

Table 3: The performance (%) of the experimental rat

groups in the RAM test after the induction of ECS.

Day The Performance of the Groups (in%)

1 2 3 4 5 6

1 50.3 24 0 0 59.3 20

2 54 40 20 0 30 55

3 60.3 37.3 0 0 46.3 53.3

4 81.5 52 0 0 20 18.3

5 94 14 16.6 10 6.6 40

6 85.6 20 6.6 0 26.6 0

7 87 80 12 0 50 10

8 86.6 55 35 0 40.6 20

Table 4: The Performance (%) of the Experimental Rat

Groups in RAM II.

Day The Performance of the Groups (%)

1 2 3 4 5 6

1 39.3 13.3 22.1 18.7 0 0

2 40 0 29.1 50 37.5 0

3 51.6 13.3 50 62.5 33.5 27

4 94.4 21 87.5 100 83.3 46.6

5 66.6 10 45.8 55.3 100 66.6

6 50 20 26.1 80 72.2 56.2

7 66.6 20 66.6 87.5 91.6 75

8 66.6 13.2 61.1 83.3 44.4 66.6

9 50 20 91.6 100 66.6 66.6

10 66.6 20 100 100 88.8 33.3

11 66.6 20 100 83.3 100 33.3

12 66.6 0 100 75 100 33.3

Steamed Watermelon (Citrullus lanatus Thunb.) Juice Improves Spatial Memory in Dementia Rat Model

memory of rats with ECS induced dementia. The

memory improvement may caused by an increase

in the synaptic strength between the neurons in rat’s

hippocampus or referred to as neuronal plasticity,

especially in the gyrus dentatus and CA1 hippocampus

that are responsible for spatial information (Poirer et

al., 2008).

Interestingly, unlike the rest of the treatment

groups, Group 6 that recieved steamed watermelon

juice at a dose of 3.7 g/kg BW showed a decreased

performance, even lower than normal control by 42%.

This group failed two times (i.e., 0% performance)

on the rst and second day of observation. Its second

lowest constant rate (33.3%) ocurred on Day 10, or one

day after the increased performance of the negative

control group, until the end of the observation. The

poor working memory may be behind this result.

This memory is responsible only for the information

received from one session of RAM test. It is erased

shortly before the next session of RAM test, and

therefore the rats try to habituate themselves to the

maze environment again and form a new working

memory, while making mistake (i.e., incorrect arm

entries). According to Crusio & Schwegler (2005), the

working memory is closely related to spatial memory.

The RAM test also observed reference memory that,

for example, encoded and retained information about

the food at the end of some arms. This memory is

always useful in any maze test because it has already

faded befor 24 hours. In this case, the rats must form

new memory again from scratch allowing for a 24-

hou maze test (Rao et al., 2003).

The average percentage of the group performance

in RAM II was subjected to homogeneity and

normality tests. The normality test yielded p-value

0.142 (p>0.05), while the homogeneity test resulted

in p-value 0.1 (p>0.05). The data analysis was

continued with a one-way ANOVA to determine

any statistically signicant difference between the

treatment groups in completing the maze test after the

induction of ECS. The ANOVA test produced p-value

0.000 (p<0.05), representing a signicant difference

between the groups in RAM II.

The subsequent Tukey test proved that in the

positive control group (Group 3), as well as the

treatment groups receiving 0.92 g/kg BW (Group 4)

and 1.85 g/kg BW (Group 5) of steamed watermelon

juice, the spatial memory in the rat model of dementia

improved. These three groups had a similar spatial

memory improvement to the normal control group.

The two treatment groups i.e., Group 4 and 5 showed

comparable results as well, meaning, that the groups

receiving 0.92 g/kg BW (Group 4) and 1.85 g/kg BW

(Group 5) experience an equivalent spatial memory

enhancement activity. Meanwhile, receiving steamed

watermelon juice at a dose of 3.7 g/kg BW, Group

6 exhibited signicantly different activities with

the normal and positive control groups, but it was

comparable to the negative control. In other words, the

treatment in Group 6 was not effective in increasing

the spatial memory of the experimental animals. The

effective doses of steamed watermelon juice in this

study were 0.92 g/kg BW and 1.85 g/kg BW. The

average percentage of spatial memory improvement

in these doses exceeded the positive control group

(Vitamin E, 20.7 mg/kg BW).

Theoretical oxidative damage is preventable if free

radicals and antioxidants in the body are in balance.

In this condition antioxidants will not exhibit any

effects (Asri, 2014). The antioxidative properties of

lycopene contained in steamed watermelon juice are

allegedly one of the factors that prevent ECS-induced

oxidative damage. Lycopene is a highly unsaturated

straigh-chain hydrocarbon with a total of 13 double

bonds, eleven (11) of which are conjugated. This

unique nature makes lycopene molecule a very potent

antioxidant. An in vitro study afrms that lycopene

is twice as potent as β-carotene and its singlet

oxygen quenching ability is ten times greater than ɑ

tocopherol (Rao et al., 2003).

4 CONCLUSIONS

This study concludes that the administration of

steamed watermelon juice at the doses of 0.92 g/

kg BW and 1.85 g/kg BW can improve the spatial

memory in a rat model of dementia, which is

comparable to the positive control. Treatment groups

that recieved these doses had corresponding spatial

memory enhancement activities.

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Steamed Watermelon (Citrullus lanatus Thunb.) Juice Improves Spatial Memory in Dementia Rat Model

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