SYNOPSIS OF THE PROPOSED RESEARCH
Title:
Isolation and characterization of
anticholinesterase agents and antioxidants from the polyphenols of Loranthus globosus.
Introduction:
Alzheimer’s
disease (AD) is a progressively developing neurodegenerative disorder of the
brain in the elderly people with the symptoms of memory loss, cognition and behavioral
abnormalities.
The
pathological hallmarks of AD are profound loss of cholinergic neurons, senile
plaques consisting of A-Beta protein, and neurofibrillary tangles of
microtubule –associated protein tau [1-3]. Although there has been
tremendous progress in understanding the etiology and pathogenesis of AD, but
still the effective drugs remain limited.Cholinergic dysfunction and oxidative
stress have been implicated as the major contributing factors in the
pathogenesis of AD[4]. Therefore,
inhibition of cholinesterase and oxidation, offer the two promising strategies
in treatment of AD [5]. Currently, only three cholinesterase
inhibitors (ChEI) such as donepezeil, galantamine and rivastigmine have been
approved by the Food and Drug Administration (FDA) to treat AD[5]
.These drugs have ameliorated the symptoms and improved the functioning of
patients with AD , however ,none of these drugs can completely restrict or
reverse the disease progression associated with AD. Therefore, it is an urgent
need for agents that are pharmacologically safe, cost effective and immediately
available with minimal side effects.
The potential beneficial roles of natural antioxidants have been emphasized in various diseases including AD. Plant extracts such as Ginkgo bilboa and curucumin have been reported to reduce oxidative stress associated with AD [6-7]. Polyphenol are the most abundant antioxidants in the plant kingdom, and it is claimed that,they have neuroprotective effect[8]. Loranthus globosus (Roxb), belonging to the family loranthaece, is a parasite and has been used by the local people to treat diseases of the nervous system including AD. A previous study from our laboratoruy revealed that the ethylacetate fraction of L. globosus is in rich phenolics and has strong cholinesterase inhibitory and andtioxidant activities. A strong correnlation has also been observed between phenolic content and anticholinesterase and antioxidant activities, indicating that the polyphenols might be responsible for the activities. Therefore, the present work has been designed to evaluate the cholinesterase inhibitory activity and antioxidant property of the polyphenolic extracts from L.globosus and to isolate the active compound using modern chromatographic techniques.
Objectives of the Work:
The potential beneficial roles of natural antioxidants have been emphasized in various diseases including AD. Plant extracts such as Ginkgo bilboa and curucumin have been reported to reduce oxidative stress associated with AD [6-7]. Polyphenol are the most abundant antioxidants in the plant kingdom, and it is claimed that,they have neuroprotective effect[8]. Loranthus globosus (Roxb), belonging to the family loranthaece, is a parasite and has been used by the local people to treat diseases of the nervous system including AD. A previous study from our laboratoruy revealed that the ethylacetate fraction of L. globosus is in rich phenolics and has strong cholinesterase inhibitory and andtioxidant activities. A strong correnlation has also been observed between phenolic content and anticholinesterase and antioxidant activities, indicating that the polyphenols might be responsible for the activities. Therefore, the present work has been designed to evaluate the cholinesterase inhibitory activity and antioxidant property of the polyphenolic extracts from L.globosus and to isolate the active compound using modern chromatographic techniques.
Objectives of the Work:
The
major objective of this study is to develop drug from our indigenous medicinal
plants for the treatment of Alzheimer’s disease. This study involves attempts to evaluate the acetylcholinesterase
& butylcholinesterase inhibitory and antioxidant activities of the
polyphenolic extract of Loranthus globosus in vitro
and to isolate the active compound by using modern chromatographic technique.
Methodology:
A) Preparation of Polyphenolic extract from crude methanol extract: Dried plant powders will be exhaustively extracted with methanol. After the methanol solution was concentrated in vacuo, it will be suspended in water and fractionated with ethyl acetate after defatting with petroleum ether. The resulting extract will be subjected to dia-ion resin column, for the extraction of phenolics; which will be reffered to as polyphenolic extract.
A) Preparation of Polyphenolic extract from crude methanol extract: Dried plant powders will be exhaustively extracted with methanol. After the methanol solution was concentrated in vacuo, it will be suspended in water and fractionated with ethyl acetate after defatting with petroleum ether. The resulting extract will be subjected to dia-ion resin column, for the extraction of phenolics; which will be reffered to as polyphenolic extract.
B) Assessment
of the polyphenolic extract for acetylcholnesterase inhibitory activity in vitro.
I.
Preparation of acetylchinesterase enzyme: For the enzyme source, the rat brains will
be homogenized in a homogenizer with 5 volumes of a homogenization buffer [10
Mm Tris-HCL
(pH 7.2), 1 M NaCl, 50 Mm MgCl2 and 1 % Triton X-100], and centrifuged at 10,000 g for 30 min. The resulting supernatant will be used as an enzyme source. All the extraction steps will be carried out at 40C.
(pH 7.2), 1 M NaCl, 50 Mm MgCl2 and 1 % Triton X-100], and centrifuged at 10,000 g for 30 min. The resulting supernatant will be used as an enzyme source. All the extraction steps will be carried out at 40C.
II.
Assay of anti-acetylcholinesterase activity: The
inhibitory avtivity of the polyphenolic extract against acetylcholinesterase
(AChE) will be evaluated according to the modified Ellman’s coupled enzyme assay.
III. Preparation of butylcholinesterase enzyme:
For the butylcholinesterase enzyme (BChE) source, human blood from anonymous healthy volunteer will be collected in EDTA treated (1 mg/ml) glass tubes. These tubes will be centrifuged at 2000 g for 10 minutes to eliminate the red blood cells. The resulting plasma (supernatant) will be recuperated, diluted (1/200) with 50 Mm phosphate buffer (pH 7.4) and will be used as an enzyme source.
IV. Assay of anti-butylcholinesterase activity:
The assessment of butylcholinesterase enzyme (BChE) inhibition will be performed as described above, for acetylcholinesterase enzyme; except that the acetylcholine (ACh) iodide, will be replaced by S-butylrylthiocholine(BCh) iodide.
III. Preparation of butylcholinesterase enzyme:
For the butylcholinesterase enzyme (BChE) source, human blood from anonymous healthy volunteer will be collected in EDTA treated (1 mg/ml) glass tubes. These tubes will be centrifuged at 2000 g for 10 minutes to eliminate the red blood cells. The resulting plasma (supernatant) will be recuperated, diluted (1/200) with 50 Mm phosphate buffer (pH 7.4) and will be used as an enzyme source.
IV. Assay of anti-butylcholinesterase activity:
The assessment of butylcholinesterase enzyme (BChE) inhibition will be performed as described above, for acetylcholinesterase enzyme; except that the acetylcholine (ACh) iodide, will be replaced by S-butylrylthiocholine(BCh) iodide.
C) Assessment
of the polyphenolic extract for anti-oxidant activity in vitro.
I. DPPH
radical scavenging activity : The free
radical scavenging activity of the polyphenolic extract, based on the
scavenging activity of the stable 1,1-diphenyl -2-picrylhydrazine (DPPH) free
radical, will be determined by the method, described by Braca et al. (2001, J.
Nat. Prod.,64, 892-895).
II. Effect
of the polyphenolic extract on hydrogen peroxide-induced lipid peroxidation in
rat brain homogenate: The ability of the polyphenolic extract to
inhibit lipid peroxidation will be studied spectrophotometrically by incubating
mouse brain homogenates, treated with hydrogen peroxide (10μM) and different concentrations of the extract.
III.
Hydroxyl radical scavenging activity of the polyphenolic extract: Hydroxyl radical scavenging activity of the polyphenolic extract will
be determined spectrophotometrically, by
deoxy-D-ribose degradation method as described by Chung et al. (1997).
deoxy-D-ribose degradation method as described by Chung et al. (1997).
D)
Isolation and characterization of the active compounds from the polyphenolic
extract by using HPLC and spectroscopic methods of analysis: The active compounds in the polyphenolic extract will be initially
separated by conventional chromatographic techniques and finally, with HPLC. And
the purified compounds will be characterized by spectroscopic methods such as
IR,
1H-NMR, 13C-NMR, etc.
1H-NMR, 13C-NMR, etc.
Time
frame:
Work to be done
|
Approximate Time Needed
|
Collection of plants and preparation of
polyphenolic extract from the crude methanol extract
of Loranthus globosus.
In vitro assessments of the polyphenolic extract for acetylcholinerase & butylcholinesterase inhibitory and antioxidant activities. |
6 months
|
Fractionation of the polyphenolic extract by
column chromatography and isolation of the active compounds by HPLC or by
preparation thin layer chromatography; Characterization of the isolated
compounds by spectroscopic method of analysis.
|
6 months
|
Assessment of the isolated compounds for
acetylcholinerase & butylcholinesterase inhibitory and antioxidant
activities in vitro.
|
|
Total time = 1 year
|
|
Socio-economic contribution:
Alzheimer’s disease is AD is currently the fourth
leading cause of death after heart disease, cancer and stroke and the most
common cause of dementia among people age 60 and older. Twenty five million
people are estimated to suffer from AD world wide and 66 % of them live in the
third world countries. Only four drugs are approved for the treatment of this
disease, which offer limited use due to their side effects. Plant has already
proved to be a potential source of AD drugs, as two out of five drugs have been
developed from plants [9-13]. Bangladesh is a good repository of
medicinal plants and interestingly some plants are traditionally used by the
rural people to improve memory and cognition. If we succeed in developing an AD
drug from our Bangladeshi plants, treatment of this disease would be cheaper
and accessible to our poor people and such a discovery certainly contribute to
the progression of health sector and economy of our country.
Conclusion:
Although AD is an immense health problem
throughout the world including Bangladesh but the effective therapeutic options
for AD are limited and thus demand for new drugs. Plants serves as a
well-established source of rich diverse compounds with wide range of
pharmacological activities and two out of four drugs currently used to treat
AD, have been developed from plants. With the objective of developing new drugs
for the treatment of AD, the plant Loranthus
globosus has been selected for the evaluation of acetylcholinerase &
butylcholinesterase inhibitory and antioxidant activities in vitro, based on the followings facts. Firstly, the extract of L.globosus
is rich in polyphenols and possesses strong antioxidant activity in compared
with other medicinally important plants as found in a preliminary study under
the same experimental condition. Secondly,
it contains several bioactive compounds that have demonstrated anticancer, antioxidant
and inflammatory activities. Finally,
this plant is used by the rural people to treat various ailments and the
species of mistletoe are reputed to have neuroprotective, antiproliferative and
anti-inflammatory properties.
Evaluation of the protective effects of L.globosus polyphenolic extract on oxidative stress and acetylcholinerase & butylcholinesterase may render the development of drug in the treatment of AD.
Evaluation of the protective effects of L.globosus polyphenolic extract on oxidative stress and acetylcholinerase & butylcholinesterase may render the development of drug in the treatment of AD.
Supervisor:
Dr. Golam Sadik Professor Department of Pharmacy Rajshahi University
Mobile:
01721677207
E-mail: gsadik2@yahoo.com |
Applicant:
Md.Imran Nur Manik M. Pharm. Roll: 09065338 Reg. number : 2368
Department
of Pharmacy
Rajshahi
University
Mobile:01770967151 |
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