INTRODUCTION
Alzheimer’s is characterized by selective neuronal cell death, the presence
of extra cellular amyloid deposits in the core of neuritic plaques and the formation
of intraneuronal neurofibrillary tangles in the brain of affected individuals.
Alzheimer’s disease is a progressive neurodegenerative disease clinically
characterized by dementia and neurobehavioral deterioration (Farbood
et al., 2009). Neurochemically, these deficits are associated with
dramatic losses of cortically projecting cholinergic neurons and by a reduction
in the presynaptic markers of the cholinergic system, particularly in the areas
of the brain related to memory and learning (Iqbal and Grundke-Iqbal,
2008).
The major component of the amyloid plaque core is the pathologically deposited
amyloid-beta-peptide (Aβ), which is derived from Amyloid Protein Precursor
(APP). Alterations in APP processing to secrete higher levels of the longer
Aβ 42 peptide and a clinical correlation between elevated levels of amyloid
β-peptide in the brain and cognitive decline has recently been reported
(Ghosh et al., 2008). The pathological features
of Alzheimer’s disease include deposition of senile plaques in different
brain zones formed by aggregates of fibrillar Aβ peptide (Aβ), a neurotoxic
metabolic product (Alinovi et al., 2006; Sadik
et al., 2001).
Alzheimer’s Disease (AD) affects up to 15% of people over age 65 years
and nearly half of people aged 85 years. Selective neuronal and synaptic loss
in the hippocampus and cerebral cortex correlates with clinical symptoms and
therefore, a clearer understanding of the mechanisms that cause neuronal death
and dysfunction should lead to a greater understanding of the underlying pathophysiology
of the disease and unveil potential therapeutic opportunities (LaFerla
and Oddo, 2005; Hebert et al., 2003). Prevalence
rates for more than 20 million people worldwide including 4.5 million Americans.
This number is expected to double every 20 years as world populations continue
to age. AD is now considered to be the third major cause of death in developed
countries, after cardiovascular disease and cancer (Ma and
Gang, 2008).
The total worldwide cost of dementia care is estimated to be US$315.4 billion
annually and 77% of these costs occurred in the world’s more developed
regions. The course of AD often takes a decade or more to progress, bringing
with it a severe load to patients, families and society (Sato
et al., 2002).
The course of AD often takes a decade or more to progress, bringing with it
a severe load to patients, families and society. There is currently no known
cure for AD. However, several drugs for treatment of AD symptoms have been approved
by the US Food and Drug Administration (FDA) in recent years. Stem cell technology
has emerged in the face of extraordinary advances for prevention, diagnosis
and treatment of human diseases such as heart disease, diabetes, cancer and
diseases of the nervous system, such as Parkinson’s disease and Alzheimer’s
disease (AD). It offers great promise for tissue regeneration, cell replacement
and gene therapy but clinical applications remain limited (Saeed
and Mesaik, 2005).
Current AD therapies are merely palliative and only temporarily slow cognitive
decline and treatments that address the underlying pathologic mechanisms of
AD are still lacking. In this review, we focus on the current aspects of AD
ranging from the key risk factors for AD, the underlying pathogenic events and
the novel medications including disease-modifying properties (Khairallah
and Kassem, 2011). Researches done by FDA and other associations have claimed
that herbs contain natural composition of various compounds that affects our
internal system (brain, heart and mind). In conventional medicine, those formulations
are synthesized by chemical associations and are given in different doses to
the person. We should also keep in mind that around 30% of the ingredients that
are added in conventional medicine are extracted from natural resources like
plants and herbs (Winston, 2005).
The leads of Central Nervous System (CNS) active medicinal plants which have
emerged besides Rauwolfia serpentina, Mucuna pruriens for Parkinson’s
disease, Ocimum sanctum as an anti stress agent, Withania somnifera
as anxiolytic, Centella asiatica and Bacopa monnieri for learning
and memory disorders. Bacopa monnieri, Benincasa hispida (Roy
et al., 2008), Areca catechu (Jaiswal
et al., 2011) and Ginkgo biloba for Alzheimer’s disease.
The study related to Alzheimer’s Disease (AD) is focused towards the traditionally
used rejuvenating and neurotonic agents. The recent trends in the pharmacological
studies are based on the biochemical and molecular mechanism which leads to
the development of CNS active principles from the herbal drugs (Anekonda
and Reddy, 2005; Vinutha et al., 2007; Hussian
and Manyam, 1997; Moustafa et al., 2007).
Ficus hispida Linn. belongs to the family Moraceae is a widely distributed
and most commonly found in the interior and coastal regions distributed throughout
India, Srilanka, Myanmar and Southern region of the Republic china. A mixture
of honey and the juice of these fruit is a good antihemorrhagic (Peraza-Sanchez
et al., 2002) but the barks and leaves are of particular interest
from a medicinal point of view as an antidiarrhoeal (Mandal
and Kumar, 2002), antidiabetic (Gosh et al.,
2004) and as cardio protective (Shanmugarajan et
al., 2007) among others.
The current study was undertaken to evaluate the ameliorating effect of ethanolic leaf extract of Ficus hispida Linn (EEFH) , till now no pharmacological evaluation has been done on FH especially in leaf for its anti-alzhemerian activity. This prompted us to pursue the activity and was examined for their efficacy.
MATERIALS AND METHODS
Plant material: The fresh Ficus hispida Linn (FH) leaves were
collected from (Perannakavur, Changlepet, Tamil Nadu, India) western Ghats of
South India during June 2008. The plant was identified and authenticated by
Dr. G.V.S. Murthy, Joint Director, Botanical Survey of India, Southern Circle,
Coimbatore (BSI/SC/5/23/08-09/Tech-738). The specimen voucher was deposited
in the Department of Pharmacology and Toxicology, C.L. Baid Metha College of
Pharmacy, Chennai, Tamil Nadu, India.
Preparation of the ethanolic extract of FH: The fresh leaves of FH were
collected and washed with running water and shade dried at room temperature.
One kilogram of the dried leaf was made in to coarse powder. The powder was
passed through a 60 No. mesh sieve. The grounded power was extracted with ethanol
in water bath at room temperature. The solvent was then removed by filtration
and fresh solvent was added to the plant material (Ravishankara
et al., 2002). The extract process was twice repeated. The combined
filtrates were then evaporated under reduced pressure to give a dark green viscous
mass. The extract was stored at 0-4°C. The percentage yield was 20.2%w/w.
Phytochemical screening: The freshly prepared ethanol leaf extract of
FH (EEFH) was qualitatively tested for the presence of chemical constituents.
Phytochemical screening of the extract was performed using the following reagents
and chemicals: Alkaloids with Mayer’s, Hager’s and Dragendorffs reagent;
Flavonoids with the use of sodium acetate, ferric chloride, amyl alcohol; Phenolic
compounds and tannins with lead acetate and gelatin; carbohydrate with Molish’s,
Fehling’s and Benedict’s reagent; proteins and amino acids with Millon’s,
biuret and xanthoprotein test. Saponins was tested using hemolysis method; Gum
was tested using Molish’s reagent and Ruthenium red; Coumarin by 10% sodium
hydroxide and quinones by Concentrated Sulphuric acid. These were identified
by characteristic color changes using standard procedures (Trease
and Evans, 1989).
These were identified by characteristic color changes using standard procedures.
The screening results were as follows: Alkaloids +ve; carbohydrates +ve; proteins and amino acids +ve; saponins +ve, steroids -ve; sterols +ve; phenols +ve; flavonoids +ve; gums and mucilage +ve; glycosides +ve; saponins +ve; terpenes +ve and tannins -ve. Where +ve and –ve indicates the presence and absence of compounds.
Animals: Young adult Swiss albino mice of either sex, weighing (18-20
g) were obtained from animal house of C.L. Baid Metha College of Pharmacy, Chennai,
Tamil Nadu, India. Animals were kept in raised mesh bottom cages to prevent
coprophagy. The animals were maintained in colony cages at 25±2°C,
relative humidity 50-55% maintained under 12:12 h light and dark cycle. The
animals were fed with Standard animal feed (Hindustan Lever Ltd., Bangalore,
India) and water ad libitum, animals were acclimatized to the laboratory
conditions prior to experimentation. All the experiments were conducted between
10-00 and 17.00 h and were in accordance with the ethical guidelines of the
International association for Study of Pain (Zimmermann,
1983). All experiments were carried out according to the guidelines for
care and use of experimental animals and approved by Committee for the Purpose
of Control and Supervision of Experiments on Animals (CPCSEA). the study was
approved by the Institutional Animal Ethical Committee. C.L. Baid Metha College
of Pharmacy, Chennai, Tamil Nadu, India.
Acute toxicity study: This was performed for the extracts to ascertain safe dose by the acute oral toxic class method by the Organization of Economic Cooperation and Development (OECD). A single administration of starting dose of 2000 mg kg-1 body weight p.o. of the EEFH was administered to three female mice and the mice were observed for three days to evaluate considerable changes in body weight and other signs of toxicity. There was no considerable change in body weight before and after treatment and no sign of toxicity were observed. When the experiment was repeated again with same dose level, 2000 mg kg-1 b.wt. p.o. of plant extract for 7 more days and observed for fourteen days no change was observed from the experiments.
Induction of amnesia
Intracerebroventricular injection of Aβ peptide: The administration
of a Aβ 25-35 was performed by identifying the bregma pointing the skull,
each mouse was injected at bregma with a 50 μL Hamilton micro syringe fitted
with a 26-gauge needle that was inserted to a depth of 2.4 mm in brief, the
needle was inserted unilaterally 1 mm to the right of the middle point equidistant
from each eye, at an equal distance between the eyes and ears and perpendicular
to the plane of the skull. Mice exhibited normal behavior with in 1 min after
injection. The injection volume was 10 μL; the memory impairment due to
the injection was examined on the second day of i.c.v. injection by step down
inhibitory avoidance (Laursen and Belknap, 1986).
Experimental design: Animals were divided into four groups of each ten
animals:
| Group I: |
Animals injected by 1% w/v CMC |
| Group II: |
Animal injected with β-Amyloid (25-35) 10 μL by intracerebroventricular
injection (i.c.v.) |
| Group III: |
Animals injected by Aβ peptide and treated with 200 mg kg-1
(p.o.) EEFH |
| Group IV: |
Animals injected by Aβ peptide and treated with 400 mg kg-1
(p.o.) EEFH |
Amnesia is induced by intracerebroventricular injection (i.c.v.) of Aβ peptide, i.c.v. Injection for the II, III and IV groups were performed on the 21st day of the pre treated and continued for 7 days. Control animals are injected with only 1% w/v CMC.
In vivo pharmacological examination (behavioral studies)
Step down inhibitory avoidance: The apparatus consist of 50x25x25 cm acrylic
box, whose floor consisted of parallel 1.0 cm apart. A 7.0 cm wide, 2.5 cm high,
25.0 cm long platform occupied the centre floor. In the training session, immediately
after stepping down placing their four paws on the grid the animals received
a 0.4 MA, 2.0 sec scrambled foot shock. In test session no foot shock was given
and step-down latency was used as a measure of retention (to a ceiling of 300
sec) one-trial step-down inhibitory avoidance in rats and mice involves the
activation of two separate memory types, a Short-Term Memory (STM) system and
a Long-Term Memory (LTM) system. Therefore, retention tests were carried out
90 min after training to evaluate STM and 7 days after training to evaluate
long-term memory. The same mice were used for both tests, as testing for STM
has been found not to affect LTM retention scores in previous studies (Dhingra
et al., 2006).
Open field habituation: In order to control for possible effects on
locomotor activity, animals were explored twice, with a 24 h interval, to a
40x50x60 cm open field whose brown linoleum floor was divided into 12 equal
squares by white lines. In both sessions, the animals were placed in the rear
left square and left to explore it freely for 5 min during which time the number
of lines crossing, head dippings and rearings were counted (Saitoh
et al., 2006; Yamada et al., 2005).
Water maze task: The experimental apparatus consisted of a circular
water tank (diameter-100 cm; height-35 cm), containing water at 28°C to
a depth of 15 cm and rendered opaque by adding titanium dioxide. A platform
(diameter 4.5 cm; height 14.5 cm) was submerged 1 cm below the water surface
and placed at the midpoint of one quadrant. After several trials, the test was
conducted after injection of β amyloid peptide. In each training trial,
the time required to escape on to the platform was recorded (Pietropaolo
et al., 2008).
Y-maze task: Y-maze task is used to measure the spatial working through
the spontaneous alternation of behaviour. The maze is made of black painted
wood. Each arm is 40 cm long, 13 cm high, 3 cm wide at the bottom, 10 cm wide
at the top and converges at an equal angle. Each mouse is placed at the end
of one arm and allowed to move freely through the maze during an 8 min session.
Mice tend to explore the maze systematically, entering each arm in turn. The
ability to alternate requires that the mice know which arm they have already
visited. The series of arm entries, including possible returns into the same
arm, are recorded visually. Alternation is defined as the number of successive
entries into the three arms, on overlapping triplet sets. The percentage of
alternation is calculated as the ratio of actual alternations, defined as the
total number of arm entries minus two and multiplied by 100 (Hiramatsu
and Inoue, 2000).
Estimation of neuro transmitter and metabolic enzymes
Estimation of acetylcholinesterase enzyme: Twenty milligram of brain tissue
per mL of phosphate buffer (pH 8, 0.1 m) was homogenized in a Potter-Elvehjem
homogenizer. A 0.4 mL aliquot of brain homogenate was added to a cuvette containing
2.6 mL of 0.1 m phosphate buffer (pH 8). One hundred microliter of the DTNB
reagent was added to the photo cell. The absorbance was measured at 412 nm then
acetylthiocholine iodide was added. Changes in absorbance were recorded and
the change in absorbance per minute was calculated (Vinutha
et al., 2007). The enzyme activity is expressed as μmol/min/mg
tissue.
Estimation of monoamine oxidase (MAO) enzyme: The assay mixture contain
4 mm of serotonin as specific substrates for MAO-A, 250 μL solution of
the mitochondrial fraction and 100 mm sodium phosphate buffer (pH 7.4) up to
the final volume of 1 mL, the reaction was allowed to proceed at 37°C for
20 min and stopped by adding 1 m HCl (200 μL), the reaction product was
extracted with 5 mL of butyl acetate, the organic phase was measured at wavelength
of 280 nm in a spectrometer. Blank samples were prepared by adding 1 m HCl (200
μL) prior to the reaction and worked subsequently in the same manner (Muralidharan
et al., 2009).
Estimation of dopamine: On the day of experiment mice were sacrificed,
whole brain was dissected out and separated the subcortical region (including
the striatum). Weighed a specific quantity of tissue and was homogenized in
3 mL HCl Butanol in a cool environment. The sample was then centrifuged for
10 min at 2000 rpm. Supernatant phase was removed and added to an Eppendorf
reagent tube containing heptane and HCl. After 10 min, shake the tube and centrifuged
under same conditions to separate two phases. Upper organic phase was discarded
and the aqueous phase was used for dopamine assay (Schlumpf
et al., 1974).
Dopamine assay: To 0.02 mL of the HCl phase, 0.4 mL HCl and 0.01 mL
EDTA/Sodium Acetate buffer (pH 6.9) were added, followed by 0.01 mL iodine solution
for oxidation. The reaction was stopped after 2 min by the addition of sodium
thiosulphate in 5 M Sodium hydroxide. 10 M Acetic acid was added 1.5 min later.
The solution was then heated to 100°C for 6 min. When the sample again reaches
room temperature, excitation and emission spectra were read (330 to 375 nm)
in a spectrofluorimeter. Compared the tissue values (fluorescence of tissue
extract minus fluorescence of tissue blank) with an internal reagent standard
(fluorescence of internal reagent standard minus fluorescence of internal reagent
blank). Tissue blanks for the assay were prepared by adding the reagents of
the oxidation step in reversed order (sodium thiosulphate before iodine). Internal
reagent standards were obtained by adding 0.005 mL double distilled water and
0.1 mL HCl Butanol to dopamine standard.
Estimation of serotonin: Three milligram of mice brain is homogenized
in 0.1 mL of HCl-n butanol for 1 min in glass homogenizer. The sample was centrifuged
for 10 min at 2000 rpm. The supernatant phase was removed and added to Eppendorf
reagent tubes containing 0.2 mL of heptane and 0.025 mL of HCl 0.1 M. After
10 min of vigorous shaking the tube was centrifuged under the same conditions
as above in order to separate the two phases. The aqueous phase was taken and
o-phthaldialdehyde was added. The fluorophore was developed by heating to 100°C
for 10 min. After the sample reach the equilibrium with ambient temperature
the intensity readings at 360-470 nm was taken in microcuvette (Kepe
et al., 2006).
Estimation of glutamate: Weighed quantity of brain portion was homogenized
with perchloric acid and centrifuge for 10 min at 3000 rpm. Adjust supernatant
fluid to pH 9 with phosphate solution. Allow to stand 10 min, in an ice bath
and then filter through a small, fluted filter paper. Allow to warm to room
temperature, dilute and take 1.0 mL for the assay (Sivaraman
et al., 2010).
Estimation of total protein: Total protein was estimated in brain homogenate
by using suitable reagents method (Lowry et al., 1951).
Estimation of antioxidant enzyme
Estimation of superoxide dismutase (SOD): The assay mixture contained 1
mL of pyrogallol-tris-DEPTA, 0.2 mL of suitably diluted tissue and 0.8 mL of
water. The rate of pyrogallol autoxidation is taken from the increase in absorbance
at 420 nm. The activity of SOD was expressed as units/minutes/mg protein. One
unit of the enzyme is defined as the amount of enzyme which inhibits the rate
of pyrogallol auto oxidation by 50%. The SOD is expressed as units/min/mg protein
(Byers et al., 2006).
Estimation of catalase (CAT): Homogenize the tissue with phosphate buffer
at 1 to 4°C and centrifuged. Stirred the sediment with cold phosphate buffer
and allowed to stand in the cold condition with occasional shaking. Repeat the
extraction once or twice, supernatants were combined and used for assay. H2O2-phosphate
buffer was taken in one cuvette, added 0.01- 0.04 mL sample and read against
a control cuvette containing enzyme solution without H2O2
phosphate buffer at 240 nm. It was noted for a decrease in the optical density
from 0.450 to 0.400. This value was used for the calculations (Pari
and Latha, 2004).
Estimation of glutathione peroxidase (GPx): The reaction mixture consisting
of 0.2 mL each of EDTA, sodium azide and 0.4 mL of phosphate buffer, 0.1 mL
of suitably diluted tissue was incubated at 37°C at different time intervals.
The reaction was arrested by the addition of 0.5 mL of TCA and the tubes were
centrifuged at 2000 rpm. To 0.5 mL of supernatant, 4 mL of Disodium hydrogen
phosphate and 0.5 mL DTNB were added and the color developed was read at 420
nm immediately (Resende et al., 2008). The activity
of GPx is expressed as μmoles of glutathione oxidized/minutes/mg protein.
Estimation of glutathione reductase (GR): The reaction mixture containing
1 mL of phosphate buffer, 0.5 mL of EDTA. 0.5 mL of oxidized glutathione and
0.2 mL of NADPH was made up to 3 mL with water. After the addition of suitably
diluted tissue, the change in optical density at 340 nm was monitored for 2
min at 30 sec intervals (Sapakal et al., 2008).
The activity of GR is expressed as nmoles of NADPH oxidized/minute/mg protein.
Estimation of lipid peroxidation: About 0.2 mL of tissue homogenate,
acetic acid, sodium dodecyl sulphate and thiobarbituric acid were added. The
volume of the mixture was made up to 4.0 mL with distilled water and then heated
at 95°C in a water bath for 60 min. After incubation the tubes were cooled
to room temperature and final volume was made to 5.0 mL in each tube, n-butanol:
pyridine mixture was added and the contents were vortexed thoroughly for 2 min.
After centrifugation at 3000 rpm for 10 min, the organic upper layer was taken
and its optical density read at 532 nm against an appropriate blank without
the sample (Alper et al., 1998). The levels of
lipid peroxides were expressed as nmoles of Malondialdehyde (MDA)/min/mg protein
in brain homogenate.
Histopathological examination: The mice from each group were anesthetized
by i.p. (intraperitoneal) injection of thiopentone (100 mg kg-1),The
brain was removed with out any injury after opening the skull. The collected
sample were washed with normal saline and fixed in 10% neutral formalin for
48 h for further histological observation. Paraffin section were taken at 5
μm thickness processed in alcohol-xylene series and was stained with Haematoxylin-eosin
dye. The section were examined microscopically for histopathological changes
especially in the hippocampus and in the cortex (Takanaga
et al., 2003).
Statistical analysis: All values are expressed as Mean±SEM. Data
were analyzed by non-parametric ANOVA followed by Dunnett’s multiple comparison
tests and other data were evaluated using Graph Pad PRISM software. A p-value<0.05
was considered significantly different.
RESULTS
Effect of EEFH on step down inhibitory avoidance: There was a significant difference between the control group (I) and the negative control group (II) in the step down latency in the STM and LTM. The EEFH treated groups show significant tendency to increase the retention of STM and LTM. The low dose and high dose shown significance (p<0.05) when compared with the negative control was shown in Fig. 1.
Effect of EEFH on water maze: There is an increase in escape latency in negative control group when compared with the control group (p<0.01) of the two groups of amnesia induced animals, both showed decreased time to escape on to the escape platform. The group treated with 200 mg kg-1 EEFH group 400 mg kg-1 treated showed the significance of (p<0.01 and p<0.01), respectively as shown in Fig. 2.
Effect of EEFH on exploratory behavior: The amnesia induced group negative control indicated decrease in the exploratory behavior i.e., head dippings, rearings, line crossings by the (p<0.05, p<0.05 and p<0.05), respectively in comparison with the control group I. The results presented by the treated groups shows significance (p<0.05, p<0.05 and p<0.01) increase in the head dippings and line crossings in respect of 200 mg kg-1 when compare with the negative control group. The 400 mg kg-1 treated group shown significance of (p<0.01, p<0.01 and p<0.01) head dippings, rearings and line crossings, respectively as shown in Fig. 3.
Effect of EEFH on Y-maze: The amnesia induced group indicated decrease in the alternation of behaviour by the (p<0.01) in comparison with the control group I. The results presented by the treatment groups shows significance by (p<0.01 and p<0.01) increase in the alternation of behaviour in respect of 200 mg kg-1 of EEFH and 400 mg kg-1 of EEFH when compared with negative control group as shown in Fig. 4.
|
| Fig. 1: |
Effect of EEFH on step down inhibitory avoidance (*p<0.05) |
|
| Fig. 2: |
Effect of EEFH on water maze (**p<0.01) |
|
| Fig. 3: |
Effect of EEFH on exploratory behavior (*p<0.05, **p<0.01) |
Effect of EEFH on AchE (activities of acetylcholine esterase) activity: Injection of Aβ 25-35 significantly (p<0.01) increased the AchE activity when compared with control group. In the treated group there was a significance (p<0.01) reduction in enzyme levels on both 200 and 400 mg kg-1 of EEFH treated mice as shown in Fig. 5.
|
| Fig. 4: |
Effect of EEFH on Y-maze (**p<0.01) |
|
| Fig. 5: |
Effect of EEFH on AchE activity (**p<0.01) |
Effect of EEFH on MAO (monoamine oxide) -A: The enzyme activity in Aβ 25-35 induced group had significantly increased (p<0.01) when compared with control group I. The activity of MAO-A in 200 mg kg-1 of EEFH treated animals showed a significant reduction (p<0.01). The activity of MAO-A in 400 mg kg-1 of EEFH treated animals significant reduction (p<0.01) in comparison negative control group as shown in Fig. 6.
Effect of EEFH on dopamine: The enzyme activity of Aβ 25-35 induced group had decreased (p<0.01), respectively when compared with control group I. The dopamine activity in 200 and 400 mg kg-1 of EEFH treated groups showed a significance increase in (p<0.01 and p<0.01) on comparison with the negative control as shown in Fig. 7.
Effect of EEFH on serotonin: The enzyme activity in Aβ 25-35 induced group had decreased (p<0.01), respectively.
|
| Fig. 6: |
Effect of EEFH on MAO-A (**p<0.01) |
|
| Fig. 7: |
Effect of EEFH on dopamine (**p<0.01) |
When compared with control group. The serotonin activity in 200 and 400 mg kg-1 of EEFH treated groups showed a significance increase in (p<0.01 and p<0.01) on comparison with the negative control as shown in Fig. 8.
Effect of EEFH on glutamate level: There was an increase in the glutamate level in Aβ 25-35 induced group (p<0.01), respectively. The glutamate activity in 200 and 400 mg kg-1 EEFH treated groups showed a significance increase decreased in (p<0.01 and p<0.01) on comparison with the negative control as shown in Fig. 9.
Effect of EEFH on protein level: In the present study the protein level of Aβ 25-35 induced group show significant (p<0.05) increase when compared with control group. A significant decrease (p<0.01) in the level of protein was observed for 200 and 400 mg kg-1 of EEFH treated group as the results shown in Fig. 10.
Effect of EEFH on superoxide dismutase: SOD levels in the brain was
significantly reduced (p<0.01) in Aβ 25-35 induced group when compared
to control group.
|
| Fig. 8: |
Effect of EEFH on serotonin (**p<0.01 ) |
|
| Fig. 9: |
Effect of EEFH on glutamate level (**p<0.01 ) |
|
| Fig. 10: |
Effect of EEFH on protein Level (*p<0.05, **p<0.01) |
Treatment with EEFH at 200 and 400 mg kg-1 dose level showed significant
increase (p<0.01 and p<0.01), respectively when compared with negative
control group as the results were shown in Fig. 11.
Effect of EEFH on glutathione peroxidase: The GPx in the dementia induced
mice (group II) shown significant (p<0.01) reduction in the enzyme activity
when compared with control group.
|
| Fig. 11: |
Effect of EEFH on Super oxide dismutase (**p<0.01) |
|
| Fig. 12: |
Effect of EEFH on Glutathione Peroxidase (**p<0.01) |
The treatment with EEFH at 200 and 400 mg kg-1 shown the significance
(p<0.01 and p<0.01), respectively when compared with negative control
group as shown in Fig. 12.
Effect of EEFH on glutathione reductase: The glutathione reductase activity of Aβ 25-35 induced group shown significant (p<0.01) decrease in the activity when compared with control group. The two groups with 200 and 400 mg kg-1 doses of EEFH treated animals showed significance increase (p<0.01) when compared with negative control group. The results are shown in Fig. 13.
Effect of EEFH on catalase level: There was a decrease in the catalase
level in Aβ 25-35 induced group when compared with the control group. The
two groups with 200 and 400 mg kg-1 doses of EEFH treated animals
showed significant increase (p<0.01) when compared with negative control
group. The results are shown in Fig. 14.
Effect of EEFH on lipid peroxidation level: There was an increase in
the lipid peroxidation level in Aβ 25-35 induced group when compared with
the control group.
|
| Fig. 13: |
Effect of EEFH on glutathione reductase (**p<0.01) |
|
| Fig. 14: |
Effect of EEFH on catalase level (**p<0.01) |
The two groups with 200 and 400 mg kg-1 doses of EEFH treated animals
showed significant decrease (p<0.01) when compared with negative control
group. The results are shown in Fig. 15.
Effect of EEFH on hippocampai neuronal degeneration: There was a increase
in neuronal degeneration and decrease in the number of neuronal cells in hippocampal
region in Aβ 25-35 induced group (negative control) group when compared
with the control group .The group treated with 200 and 400 mg kg-1
EEFH showed the significant decrease in brain cell edema and improves the neuronal
configuration when compared with negative control group as shown in Fig.
16.
|
| Fig. 16(a-d): |
Effect of EEFH on hippocampai neuronal degeneration: (a) Control
group, (b) Aβ 25-35 Treated group, (c) EEFH 200 mg kg-1
Treated group and (d) EEFH 400 mg kg-1 Treated group |
DISCUSSION
The present study has revealed the neuroprotective effect of EEFH on Aβ
25-35 induced cognitive deficits in mice. Ficus hispida Linn. is a medicinal
plant with antioxidant properties. Intracerebroventricular injection of Aβ
25-35 induced impairment of memory assessed by passive avoidance and Morris
water tests. Aβ 25-35 has the potential to induced oxidative stress in
the brain cholinergic hypo function, elevation of AchE and MAO (Akiyama
et al., 2003). Moreover, it has been reported that it induces the
production of hydrogen peroxide and lipid peroxide in hippocampal neurons of
the mice brain. And the induction of 4-hydroxy-2-nonenal and 8-hydroxy-2-deoxyguanosine
(a marker of oxidative damage to DNA) immunoreactivities following infusion
of Aβ 1-42 in mice brain. In the present study, we have found a significant
decrease in the level of antioxidant enzymes and the elevated of AchE and MAO
in mice brain after a single injection with Aβ 25-35 (D’Almeida
et al., 1996). Furthermore in balance each antioxidant enzyme were
also observed and demonstrated that continuous i.c.v. infusion of Aβ 1-42
in mice resulted in a significant decrease in protein expression of SOD, GPx
and glutathione-s-transferase in mice brain. Glutathione (GSH) is the major
non-protein thiols antioxidant in mammalian cells and it is considered to be
the main intracellular redox buffer. GSH protects cellular protein-thiols against
irreversible loss, thus preserving protein function.
The position that GSH takes part in the redox-sensitive signaling cascade has
been receiving growing support. One of the most importance GSH-dependent detoxifying
processes involved is Glutathione Peroxidase (GPx) which plays a central role
in the removal of hydrogen and organic peroxides and leads to the formation
of oxidized Glutathione (GSSG). GSSG is reduced back to its thiols form (GSH)
by the ancillary enzyme Glutathione reductase (GRD), leading to the consumption
of NADPH which is mainly produced in the pentose phosphate pathway. GSH also
takes part in xenobiotic conjugation with the assistance of several glutathione
S-transferase isoenzymes. GSH conjugates or GSSG can be eliminated from the
cell by the family of ATP dependent transporter pumps. The inhibition GSH synthesis
leads to an increase in Aβ induced cell death and intracellular Aβ
accumulation (Polidori, 2003).
It’s been suggested that antioxidant might contribute to the prevention
of Alzheimer’s disease. Antioxidant such as beta carotene and vitamins
C, E and A may protect cells from the type of damage that leads to aging in
the brain and tissues (Saeed et al., 2005). Both
vitamin C and E are antioxidant which are likely reduce oxidative stress and
injury in the central nervous system, this may reduce the Aβ plaque deposition
in the neuronal cells (Cole et al., 2005).
Presently in present study, we found that treatment with EEFH ameliorated cognitive deficits in Aβ 25-35 injected mice, especially shows increase in step down inhibitory avoidance and in the exploratory behaviour. Step-down inhibitory avoidance task is a classic task of memory with a strong aversive component. EEFH exerted significant effects in short-term retention and increase in long-term retention of this task. We also examined the effect of EEFH on the open-filed test as it is presented in which allowed us to study the effect of the treatment on the general locomotor activity of the animals, results found that animals treated with EEFH had an increase in the number of head dippings, line crossings and rearings in the open filed. These results of our experiments have been consistent with the favorable effect on locomotor activity. In the water-maze test, consumption of EEFH decreases the escape latency almost to normal levels in the dose dependent manner. It is possible that neuroprotection plays a role in that favorable effect of EEFH on Aβ 25-35 induced cognitive deficits.
The AchE activity has been shown to be increased around Aβ plaques in
Alzheimer’s brain. The calcium influx followed by oxidative stress is involved
in the increase in activity of AchE induced by Aβ 25-35 peptide, decreasing
cell membrane order and ultimately leading to the exposure of more active enzyme
(Ban et al., 2006). The observation that Aβ
peptide increases AchE activity indicates that it can be possible to ameliorate
cholinergic function, by inhibiting Aβ induce increase in AchE activity.
The AchE activity in the brain was increased in mice treated with Aβ 25-35
when compared with the normal. In addition, the Aβ 25-35 induced increase
in AchE was attenuated by EEFH treatment.
MAO is an important enzyme in the metabolism of a wide range of monoamine neurotransmitters,
including noradrenalin, dopamine and 5-hydroxytryptamine. MAO exists in two
forms, A and B. MAO-A in the metabolism of the major neurotransmitter monoamines.
MAO-A inhibitors have been accepted to treat depression (Kaakkola
and Wurtman, 1992). MAO-A and MAO-B in the brain have been implicated in
the etiology of Alzheimer’s disease. Elevations in MAO-A in Alzheimer neurons
have been linked to increase in neurotoxic metabolites and neuronal loss. Free
radical generation by Aβ or other noxious stimuli could contribute to an
imbalance between the production of nitric oxide and oxygen radicals and participate
in oxidative stress. In conclusion, we suggest that markedly EEFH improves cognitive
deficits induced by Aβ 25-35 and this effect is mediated by the antioxidant
properties of EEFH.
CONCLUSION
In conclusion from the observation, the neuroprotective activity of the plant Ficus hispida Linn. on Alzheimer’s type of dementia is due to the inhibiting activity against AchE, MAO, glutamate, lipidperoxidation there by it elevates Dopamine, Serotonin and free radical scavenging activity. Further studies have to be anticipated on the various level of extracts and isolated principles of the plant for various studies with immunomodulatory changes with respect to the brain mitochondrial level DNA damage.
ACKNOWLEDGMENT
The authors are grateful to Dr. S. Venkataraman (Director of C.L. BAID METHA Foundation for Pharmaceutical Education and Research, Chennai) and for his technical and secretarial assistance. The authors have no conflict of interest to report.
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