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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 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.

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.

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.

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).

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.

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.

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

References:

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7. Mishra M, Palanivelu k. The effect of curucumin (curucumin) on alzheimers disease: an overview. Ann Indian Acad Neurol 2008, 11:13-19
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13. Chattipakorn S, Pongpanparadorn A, Pratchayasakul W, Pongchaidacha A, Ingkaninan K, Chattipakorn N. Tabernaemontana divaricata extract inhibits neuronal acetylcholin-esterase activity in rats. J Ethnopharmacol. 2007; 110:61-68.