neuroprotective effect of hypericum thymopsis against

American Journal of Pharmacy and Pharmacology 2016; 3(3): 20-28

http://www.aascit.org/journal/ajpp

ISSN: 2375-3900

Keywords Aluminum,

Alzheimer’s Disease,

Hypericum Thymopsis Extract

(HTE) Neuroprotection,

Mice

Received: January 25, 2016

Accepted: February 9, 2016

Published: May 19, 2016

Neuroprotective Effect of Hypericum thymopsis Against Chronic Exposure to Aluminum Chloride and Alzheimer's Disease

Salima Douichene, Kheira Hammadi*, Noureddine Djebli

Department of Biology, Laboratory of Pharmacognosy Api Phytotherapy, Faculty of Life and

Natural Sciences, University, Mostaganem, Algeria

Email address [email protected] (K. Hammadi) *Corresponding author

Citation Salima Douichene, Kheira Hammadi, Noureddine Djebli. Neuroprotective Effect of Hypericum

thymopsis Against Chronic Exposure to Aluminum Chloride and Alzheimer's Disease. American

Journal of Pharmacy and Pharmacology. Vol. 3, No. 3, 2016, pp. 20-28.

Abstract The effect of Aluminum chloride was investigated to describe the associated behavioral

and brain modifications. We don’t know enough about the biological chemistry of

chronic and sub- acute exposure to Aluminium to be able to predict its impact on human

health. Although the hypothesis of a link between Aluminium and Alzheimer’s disease

(AD) has been supported by several epidemiological studies. Extract of Hypericum

thymopsis (HTE), a well known medicinal plant, is used for the treatment of depression,

has been explored in the present study for its protective role against Aluminum

neurotoxicity. This data suggests that HTE may be a candidate for application in

neurodegenerative diseases such as Alzheimer’s disease. Results of this study

demonstrate that Aluminiumneurotoxicity play an important role in the development of

anxiety disorders, depression and memory deficit in mice, these alterations of behavioral

activities can be a cause of development of Alzheimer’s disease. HTE possesses

significant antioxidant activity and renders neuroprotection which was more pronounced

at the dose of 200mg/ kg against Al induced neurotoxicity. Chronic administration of

Hypericum thymopsis significantly improved retention in both tasks, attenuated

oxidative damage, Aluminum concentration in Aluminum treated mice (p 0,05).

Hypericum have neuropotective effects against Aluminum-induced cognitive

dysfunction and oxidative damage.

1. Introduction

Aluminum (Al) is one of the most abundant metals in the earth’s crust.

It’s a light weight metal widely used by man, this element appears mainly in food

products, driking water derived from both natural sources and treatment methods [1].

The human toxicological effects include encephalopathy [2], bone disease [3] and

anemia [4]. Aluminum is recognized as a risk factor for neurological disorder’s, such as

Parkinson’s disease, amyotrophic lateral sclerosis and Alzheimer’s disease (AD) [5].

Today, there is no cure for this neurodegenerative disease and there for it is of great

interest for researchers to find new drugs that can inhibit the acetylcholin esterase and N-

Methyl-D- Aspartate (NMDA)- receptor antagonist.

These drugs improve the function of still intact neurons, but do not inhibit the ongoing

degenerative process leading the neuronal cell death [6].

21 Salima Douichene et al.: Neuroprotective Effect of Hypericum thymopsis Against Chronic Exposure to Aluminum

Chloride and Alzheimer's Disease

Eighty-nine Hypericum species occur in turkey, 43 of which

are endemic as recorded in the flora of Turkey.

Hypericin was first identified in hypericum species and

servers as a chemotaxonomic marker of the genera [7].

Antioxidant activity of ethanol and water extracts of the

flowers of hypericum was investigated. It was observed that

antioxidative activities of ethanol extracts of Hypericum

thymopsis are comparable with vitamin E [8].

Moreover, aqueous extracts prepared from the flowering

aerial parts of the hypericum species are being used in the

treatment of physiological diseases such as neurolgia,

anxiety, neurosis and depression. The assess a spectrum of

learning and memory functions, a battery of tests is needed.

These [9, 10]. tests are selected to assess behavior and

memory. The present studies are undertaken to assess the

neurotoxicity of Aluminum and the neuroprotective effect

of Hypericum thymopsis after chronic exposition of albinos

mice.

2. Materials and Methods

Twenty four healthy adult mice weighting from 32+-4 g

were obtained from Pasteur institute Algeria, they were

maintained at 25+-5°C with a 12 h light/ dark cycle, and have

been given a commercial pellet diet 18 g /day/ mouse

(ONAB) and fresh drinking water ad libitum.

The mice were randomly divided into four groups: each

group containing six mice: control group neurotoxicity and

Alzheimer model, intoxicated/ Alzheimer treated groups and

the control treated groups.

AlCl3 dissolved in distilled water administrated orally

(10mg /kg) for the intoxicated /Alzheimer’s model groups,

and intoxicated/Alzheimer’s treated groups with a D-

Galactose IP (120mg/kg) for the Alzheimer’s model given

for 90 days; in parallel of Hypericum thymopsis

administration (200mg/kg orally) respectively for the

intoxicated treated group and Alzheimer’s disease animal.

The control treated group received the same dose of

Hypericum thymopsis (200mg/kg) administrated orally.

Controls were treated with distilled water. Body weight was

recorded daily but no significant differences were observed

between the groups.

3. Behavioral Tests

Functional behavioral assessment is required as part of

testing the nervous status, these guidelines apply to animals

in special tests: locomotor activity, holeboared (curiosity)

[11]., black and white test box (anxiety) [12]. and forced

swimming (Persolt test) [13].

4. Memory Tests

The assessment of animal memory using different types of

mazes has been used in neuroscience [14].

Several models have been proposed recently mainly trying

to evaluate accuracy of choice between the alternatives

presented in the same day of the session, instead of looking

for the accumulated learning through successive days of

training.

Moris water maze:

The Morris water maze is widely used to study spatial

memory and learning [15]. The maze consists of circular pool

(1, 2 m in diameter and 0, 47 m high) made of white plastic

(9, 10). The pool was filled to a depth of 20 cm with water

(24°C-25°C) that was made opaque by the addition of no

toxic white paint. An escape platform (10cm in diameter),

made of white plastic with a grooved surface for a better grip,

was submerged 0, 5 cm under the water level.

The animal has to swim until it finds the hidden platform.

The animal generally uses cues outside the maze to develop a

spatial map of the environment and guide its performance.

The pool was divided into four equal quadrants labeled N

(north), E (east), S (south), W (west).

Their order of use was randomized daily. The time the

mouse needed to find the hidden platform (latency) so that it

can stop swimming was recorded. A trial was started by

placing the mouse into the pool close to the rim, facing the

wall of the tank into one of the four quadrants. The mice

were given four days of training with four 60 seconds

training trials per day. During a spatial reference memory

(SRM) training, the platform was always placed in the same

spatial location of the pool (NE quadrant) throughout the

training period in both paradigms. During spatial working

memory (SWM) training, the escape platform was placed

from the edge of the pool in one of the four possible locations

(designated N, S, E and W).

5. Histological Studies

(Preparation of Brain Tissue)

At the end of exposure, animals were food deprived for

24h

Mice from both studies were sacrificed with an overdose

of Chloral.

After the step-through avoidance test, the brains of mice

were removed right after sacrifices. Removed brains were

then quickly impressed in 10% neutral buffered

formaldehyde for 24hours. Serial coronal paraffin section

were cut at 4µm were obtained with a Leica microtome

thickness for hematoxylin &eosin (H&E) 1% for 40 seconds.

6. Statistical Analysis

The experimental results were reported as means with

SEM. Statistical analysis was performed using SPSS

Software. Analysis of variance (ANOVA) and an LSD test

were used to compare the experimental groups with the

controls. One-way ANOVA P value using the post hoc

Fischer’s LSD test. P-value<0.05 was considered significant.

American Journal of Pharmacy and Pharmacology 2016; 3(3): 20-28 22

7. Neurological Parameters

7.1. Behavioral Test

Test of locomotor activity:

The results obtained after chronic poisoning with

aluminum chloride followed by treatment with Hypericum

thymopsis shows hyperactivity in mice intoxicated and

Alzheimer’s and Alzheimer's mice treated compared to the

control model. Moreover, this activity is gradually decreased

during the experimental period in all groups.

Highly significant difference (P < 0.01)

Figure 1. locomotor activity in mice intoxicated by aluminum (10 mg/kg), Alzheimer model mice poisoned by AlCl3 (10 mg/kg) with D-galactose (120 mg/kg)

intraperitoneally and mice Alzheimer model treated Hypericum thymopsis (200 mg/kg) compared to control mice for 12 weeks.

7.2. Test of Curiosity

The results obtained during test curiosity showed that mice treated with the extract of Hypericum thymopsis are curious

during the 1st phase of experimentation, the result is closer to that of the control group, this curiosity is decreased gradually

during the following phases.

By cons mice poisoned by AlCl3, and Alzheimer mouse model poisoned by AlCl3 orally and D-galactose intraperitoneally

and less exploratory than the other groups.

Figure 2. Test of curiosity in mice intoxicated by aluminum (10 mg/kg), Alzheimer model mice poisoned by AlCl3 (10 mg/kg) with D- galactose (120 mg/kg)

intraperitoneally and mouse model of Alzheimer's disease treated with Hypericum thymopsis (200 mg/kg) compared to control mice for 12 weeks.

7.3. Test of Anxiety

The results obtained during the test of anxiety show that the residence time in the dark compartment in the experimental

groups is very important for the duration of experimentation.

We note that the mouse model Alzheimer spend much more time in the dark during the third and fourth phases compared to

other groups.



Figure 3. Test anxiety in mice intoxicated by aluminum (10 mg/kg), Alzheimer model mice poisoned by AlCl3 (10 mg/kg) with D- galactose (120 mg/kg) by IP

and Alzheimer model mice treated with Hypericum thymopsis (200 mg/kg) compared to control mice for 12 weeks.

7.4. Test Cross

In this test it was observed that the intoxicated and Alzheimer model group, the treated and control mice spend more time in

the hallway arm protected throughout the experiment.

Figure 4. Test elevated plus maze in mice intoxicated by aluminum (10 mg/kg), Alzheimer model mice poisoned by AlCl3 (10 mg/kg) with D- galactose (120

mg/kg) IP and Alzheimer model mice treated with Hypericum thymopsis (200 mg/kg) compared to control mice for 12 weeks way.

7.5. The Forced Swimming Test

During the forced swim test was observed that the time recorded in the control mice treated immobility is much larger than

mice poisoned by AlCl3 orally at a dose of 10mg/kg and intoxicated by AlCl3 at the same dose and D- galactose

intraperitoneally at a dose of 120 mg/kg have recorded a short time.

Figure 5. Test Persolt in mice intoxicated by aluminum (10 mg/kg), Alzheimer model mice poisoned by AlCl3 (10 mg/kg) with D- galactose (120 mg/kg)

intraperitoneally and mouse model of Alzheimer's disease treated with Hypericum thymopsis (200 mg/kg) compared to control mice for 12 weeks.


7.6. Memory Test

8-arm radial maze

A. Spatial working memory:

In the spatial working memory test shows that the number

of repeated passages in mice and treated Alzheimer model

and witness higher unlike intoxicated during the first four

days of learning group.

But during the fifth day, we see that the number of

repeated corridors is much greater in the control group

compared to other groups.

Figure 6A. Test of spatial working memory in mice intoxicated by aluminum (10 mg/kg), Alzheimer model mice poisoned by AlCl3 (10 mg/kg) with D-

galactose (120 mg/kg) IP and Alzheimer model mice treated with Hypericum thymopsis (200 mg/kg) compared to control ice for 12 weeks way.

B. Non-spatial memory preferably condition:

The results obtained in testing non-spatial working memory preferably packaged show that the residence time in the arms

illuminated in four groups approached ente them this time is increased in controls and Alzheimer treated with Hypericum

thymopsis during the second and third tests unlike intoxicated intoxicated group and Alzheimer model.

On the fifth day, we note that the residence time in the baited arm was higher in the group Alzheimer 's and Alzheimer's

compared to controls (non-significant results) Treaty by against the residence time in the intoxicated group is reduced

compared to control.

Figure 6B. Test of non-spatial memory preferably packaged in mice intoxicated by aluminum (10 mg/kg), Alzheimer model mice poisoned by AlCl3 (10 mg/kg)

with D-galactose (120 mg/kg) intraperitoneally and Alzheimer model mice treated with Hypericum thymopsis (200 mg/kg) compared to control mice for 12

weeks.

C. Regardless of position:

When testing regardless of position it was observed that the treated group and model Alzheimer choose baited the control

arm, this choice is decreased in the intoxicated during the first two phase’s group. On Alzheimer mouse model registered in the

baited arm number is reduced during the third and fourth day of learning.

On the fifth day, it is clear that the control mice compared to choose the baited and poisoned mouse model Alzheimer arms

more frequently.



Figure 6C. Test position of distinction in mice intoxicated by aluminum (10 mg/kg), Alzheimer model mice poisoned by AlCl3 (10 mg/kg) with D- galactose

(120 mg/kg) by IP and Alzheimer model mice treated with Hypericum thymopsis (200 mg/kg) compared to control mice for 12 weeks.

Morris water maze:

A. Spatial working memory (MST):

The results of the test of spatial working memory show that poisoned mouse model Alzheimer take much longer to detect

the visible platform as compared to controls and intoxicated group treated during the first and second day of the experiment.

On the other hand, it was noticed that the mouse model Alzheimer take a long period to detect the platform relative to the

control and treated.

On the fourth day, the mouse Alzheimer model takes a long time to detect the visible platform compared with witnesses who

latency reduced progressively testing.

Figure 7A. Test Pool Morris ' spatial working memory (MST) "in mice intoxicated by aluminum (10 mg/kg), Alzheimer model mice poisoned by AlCl3 (10 mg/kg)

with D- galactose (120 mg/kg) intraperitoneally and Alzheimer model mice treated with hypericum thymopsis (200 mg/kg) compared to control mice for 12 weeks.

B. Spatial reference memory (MSR):

During the spatial reference memory (MSR) the journey to the invisible platform is significantly higher in Alzheimer model

group from the second trial until the end of the experiment.

However, we note that the journey to the invisible platform is significantly reduced in intoxicated compared to controls.

Significant difference (P < 0.05)

Figure 7B. Test Pool Morris ' spatial reference memory (MSR) "in mice intoxicated by aluminum (10 mg/kg), Alzheimer model mice poisoned by AlCl3 (10 mg/kg)

with D- galactose (120 mg/kg) intraperitoneally and Alzheimer model mice treated with Hypericum thymopsis (200 mg/kg) compared to control mice for 12 weeks.


8. Results of Histological Studies

H&E staining shows that there are typical

neuropathological changes in the hippocampus of

intoxicated/Alzheimer’s model. In the control groups the

neurons were full and arranged tightly, the nuclei were light

stained. By comparison in the model group mice the

cytoplasm of neuron were shrunken, the nuclei were side

moved and dark stained, neurofibrillary degeneration and

neurons loss were observed in hippocampus. Curcuma

administration shows moderated neuropathological changes.

The neurons recovered their characteristic shape, with

prolonged neurofibrillary tangles (Figure 8).

Figure 8. Microscopic study of cerebral cortex in mouse brain. Grossly (x 40). Histological sections of brain were stained with hematoxylin & eosin (H&E).

Exposed mice (A, B) to 10 mg/kg/day AlCl3 during 12 weeks. Control (C). Treated mice (D) with 200mg/kg of Hypericum

thymopsis during 12 weeks.

9. Discussion

The test results show that the locomotor activity and the

poisoned mouse Alzheimer model mice and mice treated

with Hypericum thymopsis are hyperactive compared to

control mice. These results are not consistent with the work

of Golub et al [16] and Kumar et al [17] have observed that

locomotor activity decreases due to the aluminum

intoxication.

The test results of curiosity, which measures the

exploratory behavior shown by mice reveal that poisoned

mice and Alzheimer mouse model are less exploratory than

the control and treated mice. This suggests that aluminum

toxicity induced damage to the nervous system that result

coincides with the work of Gourrier - Fréry [18]

The results obtained during the test anxiety indicate that

groups of intoxicated mice treated and control spend much

time in the dark. These results are not consistent with the

work and Rebai Djebli et al [19]; Costall et al [20]. Who

noticed that poisoned prefer the light compartment.

The arms are well protected anxiety in mice intoxicated by

aluminum and the results obtained when testing at elevated

cross maze show that poisoned mice and Alzheimer model

mice prefer to stay in the protected arm. Similarly, the control

mice and mice treated the same compartment prefer

unprotected. These results are consistent with the work of

Djebli Rebai [19] and who found the same result.

The forced swim test (Persolt) reveals that the immobility

time of control mice and mice treated Alzheimer model is

more important than the poisoned mice and Alzheimer mouse



model. These results are consistent with several studies Sahin

et al [21]. and Gardier Bouirin [22].

Test of 8-arm radial maze, in the event of spatial working

memory, it has been shown that the number of repeated

corridors is much higher in the control mice, Alzheimer

model and processed compared to intoxicated mice and our

results not confirm the number of errors is intoxicated higher

compared to controls.

These results translate into a deficit of spatial working

memory in procedural learning that is caused by an

accumulation of aluminum in the brain, this result is

consistent with the work of Santucci et al [23].

On the test regardless of position it was found that the

control mice and mice treated Alzheimer model choosing

baited arms unlike poisoned mouse.

As for the non-spatial memory test preference

conditioning, mice intoxicated is widely taken time to get to

the food than control mice and treated intoxicated, this result

is consistent with the work of Santucci et al [23].

In both versions of the test of the Morris water maze, spatial

working memory and reference it was noted that the poisoned

mice and Alzheimer mouse model takes longer to find

compared to mice treated and control platform. These results

are consistent with the work of Luo et al [24] noted that these

poisoned take much more time to find the platform compared

to controls. These poisoned mouse Alzheimer model shows a

deficit of spatial learning and the ability to store

10. Conclusion

In the past, aluminum was considered perfectly harmless

and no one had thought to study its methods of penetration

into the body and its behavior in the organs.

Today, aluminum toxicity is well established in laboratory

animals and in patients with renal failure, and an

epidemiological link has actually been demonstrated between

aluminum exposure and neurodegenerative diseases.

In our experimental study we attempted to evaluate the

antioxidant effect of Hypericum thymopsis on chronic

neurotoxicity of aluminum, for a period of 12 weeks.

The neurotoxic effect of aluminum has been proven by a

number of neurological disorders that result in a decrease in

body weight, disturbances stereotyped behavior, exploratory

activity (locomotor activity test holes) and anxiety and a

memory deficit and proved by the results of the test of the

Morris water maze learning

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