Objective: Siddha system is an ancient traditional system of medicine treats many chronic ailments and neurological disorders. Muthu parpam is one of the herbo marine Siddha drugs which have the indication for neurocognitive dysfunction. The main objective of this current study was to evaluate the acetylcholine esterase (AChE) inhibition of Muthu parpam.
Methods: AChE activity was evaluated using a modified 96-well microplate assay based on Ellman’s method. Physostigmine (5, 10, 20, and 40 μg/ml) was used as the positive control.
Results: The result of this study clearly indicates that the test drug Muthu Parpam was effective in inhibiting AChE enzyme at the specified concentration dose dependently. Maximum percentage inhibition of about 71.68% was observed at 500 μg/ml when compared to that of the Physostigmine, a known AChE inhibitor with the maximum inhibition 84.87% at the concentration of 40 μg/ml.
Conclusion: Hence, this preliminary screening has proven the efficacy of Muthu parpam through AChE inhibition potential in the management of Alzheimer disease

L, SAKTHIMANIPRIYA

MA, SINEKHA

N, SABARI GIRIJA

P, SHANMUGAPRIYA

S, GUPTAJ

English

Innovare Academic Sciences: E-Journals

Vol 13, Issue 8, 2020Online - 2455-3891 Print - 0974-2441IN VITRO SCREENING FOR ACETYLCHOLINESTERASE ENZYME INHIBITION POTENTIAL OF MUTHU PARPAM – THERAPEUTIC LEAD FOR ALZHEIMER’S DISEASESABARI GIRIJA N1*, GUPTAJ S2, SINEKHA MA3, SAKTHIMANIPRIYA L1, SHANMUGAPRIYA P11Department of Nanju Maruthuvam, National Institute of Siddha, Chennai, Tamil Nadu, India. 2Sri Sairam Siddha Medical College and Research Centre, Chennai, Tamil Nadu, India. 3Department of Noinaadal, National Institute of Siddha, Chennai, Tamil Nadu, India. Email: drgirijakrish@gmail.comReceived: 27 Apil 2020, Revised and Accepted: 08 June 2020ABSTRACTObjective: Siddha system is an ancient traditional system of medicine treats many chronic ailments and neurological disorders. Muthu parpam is one of the herbo marine Siddha drugs which have the indication for neurocognitive dysfunction. The main objective of this current study was to evaluate the acetylcholine esterase (AChE) inhibition of Muthu parpam.Methods: AChE activity was evaluated using a modified 96-well microplate assay based on Ellman’s method. Physostigmine (5, 10, 20, and 40 µg/ml) was used as the positive control.Results: The result of this study clearly indicates that the test drug Muthu Parpam was effective in inhibiting AChE enzyme at the specified concentration dose dependently. Maximum percentage inhibition of about 71.68% was observed at 500 µg/ml when compared to that of the Physostigmine, a known AChE inhibitor with the maximum inhibition 84.87% at the concentration of 40 µg/ml.Conclusion: Hence, this preliminary screening has proven the efficacy of Muthu parpam through AChE inhibition potential in the management of Alzheimer disease.Keywords: Acetylcholine esterase, Muthu parpam, Alzheimer disease, Acetylcholine esterase inhibition, Cognitive function, Siddha.INTRODUCTIONAlzheimer’s disease (AD) is characterized that occur during the formation of amyloid aggregates within the core of neuritic plaque, and therefore, the formation of intraneuronal neurofibrillary tangles within the brain of afflicted individuals [1]. In the human brain, there are two major forms of cholinesterases, namely, acetylcholine esterase (AChE) and butyrylcholinesterase [2]. The foremost remarkable biochemical change in AD patients may be reduced of ACh levels within the hippocampus and cortex of the brain [3]. ACh is the principal neurotransmitter which functions all told autonomic ganglia and is the only neurochemical that triggers motor division of the somatic systema nervosum. The deterioration of cholinergic neurons within the brain, and therefore, the loss of neurotransmission is the key causes of the decline in cognitive function in patients with AD [4]. In normal cholinergic activity consisting the sequence of release, binding and enzymatic deactivation of ACh by AChE, while abnormal cholinergic activity is characterized by a deficit or short-fall in cholinergic transmissions at synapses and has been attributed to reduced production of ACh or its excess deactivation/hydrolysis by AChE. Therefore, inhibition of AChE, the enzyme liable for the hydrolysis of ACh at the cholinergic synapse, is currently the foremost established approach to treating AD [5]. Impairment in signal transduction across synapses is the main etiology for cognitive impairment [6,7] and thus regulating the activities of AChE has become a crucial research focus [8].Synthetic acetyl-cholinesterase inhibitors (AChEIs) include donepezil, rivastigmine, galantamine, and tacrine are commercially available drugs which influence the dynamics of ACh by inhibiting the activity of AChE. These actions taken place on cholinergic receptors of synaptic cells. Due to the side effects of synthetic AChEIs which include gastrointestinal disturbances, moderate effectiveness, high cost and short half-life of AChEIs, there always a need for exploring the character for newer potent and long-lasting AChE inhibitors with minimal side effects [9-11]. There always a need for exploring the character for newer potent and long-lasting AChE inhibitors with minimal side effects. An outsized number of plant species from different parts of the globe are examined for cholinesterase inhibitory activity [8,12].Traditional medicine is defined as the sum total of the knowledge, skill, and practices based on the theories, beliefs, and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health as well as in the prevention, diagnosis, improvement, or treatment of physical and mental illness [13]. Siddha system is one of the traditional systems of medicine mainly practiced in Southern India. This is a standard system of drugs mainly practiced within the southern a part of India. As per Siddha literature, Muthu Parpam is the herbo - marine Siddha formulation which has the indication for Marutchi (i.e., confusion and perplexity of mind). Muthu (Pearl) is one in all the nine gems possess the expectorant, styptic, tonic, and spasmodic action [14,15]. Hence, this study was an attempt to find out the ACh esterase (AChE) inhibition potential of Muthu Parpam.METHODSThe purification process of Muthu [14]Soak the pearl (35 g) in sour curd and should be kept under the sunlight for 3 days. Daily morning sour curd should be poured on the pearl. To stay dry, it should be kept under the daylight for 2 days without adding the curd. Repeat this process 2 times and then a pearl should be washed and dry it.Ingredients [14]•	 Purified Muthu (Pinctada margaritifera) – 1 palam (35 g)•	 Notchi (Vitex negundo) – 2 palam (70 g)•	 Nilappanai kizhangu (Curculigo orchioides) – 2 palam (70 g).© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2020.v13i8.38062Research ArticleMethod of preparationThirty-five grams of purified Muthu (pearl) should be soaked in 70 g of notchi juice for 2 days and then grind it with the notchi juice for 2 days. Then, it should be made into villai (pellets) and dry it in the sunlight. The pellets should be placed in an earthen vessel which should be closed with another vessel and the margin of vessels sealed with seven layered clay cloths and the set up should be dried in sunlight for 1 day. After that, it should be placed in a deep pit and pudam process (calcination process) should be done with 40 cow dung cakes. Then, this same procedure should be repeated with the juice of nilappanai kizhangu and then make it as a fine powder. The final product, the parpam should be stored in the airtight glass container.Indication and adjuvants [16]•	 Nirvidam (Asparagus racemosa) [15] – Nithiraiyil undaagum marutchi (Confusion and perplexity of mind during the sleep)•	 Water – Maalaikan noi (Night blindness)•	 Milk – Pithathaal undaagum veppam (Heat due to pitham)•	 Nilathulasi juice – valippu (epilepsy)•	 Nilavilaa juice – pitha diseases•	 Devadhaaru decoction – Vatha diseases•	 Butter – moolam (Piles)•	 Vilvam leaf juice – Adhisaaram (diarrhoea)•	 Brown sugar – vaanthi (Vomit).Test drug for analysisThe test drug Muthu Parpam prepared with the above manner was procured from the GMP certified pharmaceutical (Earth India Naturals) company and given for the analysis.In vitro AChE enzyme inhibition assay – methodology [17]AChE activity was measured employing a modified 96-well microplate assay supported Ellman’s method. Enzyme hydrolyses the substrate acetylthiocholine leading to the merchandise thiocholine which reacts with Ellman’s reagent (DTNB) to provide 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which might be detected at 412 nm. 50 mM Tris–HCl pH 8.0 was used as a buffer throughout the experiment. AChE enzyme stock solution (518 U/ml) was stored at ‒80°C and therefore, the enzyme-dilution was done in 0.1% BSA within the buffer. DTNB was dissolved within the buffer containing 0.1 M NaCl and 0.02 M MgCl2. ATCI was dissolved in deionized water. Within the 96-well plates, 100 µl of three mM DTNB, 20 µl of 0.26 U/ml of AChE, and 40 µl of buffer (50 mM tris pH 8.0), to which 20 µl of test drug in various concentrations (25, 50, 100, 250, and 500 µg/ml) dissolved in a buffer containing no more than 10% methanol was added to the wells. After mixing, the plate was incubated for 15 min (25°C). The enzymatic reaction was initiated by the addition of 20 µl of 15 mM acetylthiocholine iodide and therefore the hydrolysis of acetylthiocholine was monitored by reading the absorbance every 5 min for 20 min at 412 nm. Physostigmine (5, 10, 20, and 40 µg/ml) was used because of the positive control. All the reactions were performed in triplicate.RESULTS AND DISCUSSIONAChE activity has been shown to be increased within and around amyloid plaques to develop the assembly of amyloid beta-peptides into fibrils and to extend the cytotoxicity of those peptides. The results obtained from the four extracts of Muthu Parpam against AChE enzyme inhibition activity and also the percentage inhibition was evaluated and tabulated (Tables 1 and 2). Muthu parpam showed very potent inhibition (71.68 ± 2.38%) at the concentration of 500 µg/ml in comparison with physostigmine (84.87 ± 4.78%) at the concentration of 40 µg/ml (Graph 1).Physostigmine was first isolated from Calabar beans (Physostigma venenosum) in 1864 [18] and is an AChE inhibitor [19]. Although physostigmine can cross the blood brain barrier, this drug contains a narrow therapeutic index due to its short half-life and various side effects. The common side effects of physostigmine are hypersalivation, nausea, vomiting, and arrhythmia in arterial blood vessel patient and bradycardia [20]. Some studies have suggested that the AChE inhibitor, rivastigmine could also be fatal [21], while cholinesterase inhibitors Table 2: Percentage inhibition of AChE enzyme by standard drugThe concentration of physostigmine in µg/mlPercentage inhibition of AChE enzyme by std drug5 38.87±1.9910 44.48±3.5320 65.12±4.6440 84.87±4.78Each value represents the mean±SD. n=3Table 1: Percentage inhibition of AChE enzyme by test drug - MPThe concentration of MP in µg/mlPercentage inhibition of AChE enzyme by test drugMP 25 14.71±4.24MP 50 28.75±3.92MP 100 36±3.78MP 250 48.63±2.19MP 500 71.68±2.38Each value represents the mean±SD. n=3. MP: Muthu ParpamGraph 1: Percentage inhibition of standard - physostigmine on AChE enzyme inhibition assay57 Girija et al.Asian J Pharm Clin Res, Vol 13, Issue 8, 2020, 56-58remain to be a vital therapeutic tool against AD. AChE inhibitors in contemporary medicines should be undertaken for the treatment to prevent the side effects.This study revealed that the Muthu Parpam significantly inhibited AChE in a very dose-dependent manner. Notchi (V. negundo) juice employed in the preparation of Muthu Parpam has the pharmacological activity of anxiolytic and anti-amnesic [22,23]. Some pharmacological studies are reported that the Nilappanai kizhangu (C. orchioides) have the anticonvulsant activity and alleviate the cerebral ischemic injury in in vivo and in vitro experimental models [23,24]. Hence, adding these juices additionally enhance the cognitive function of the brain.CONCLUSIONAging is a natural and normal process; it is decaying the body continuously. Inappropriate aging may be preventable through principles suggested by ancient sages (Siddhars) which are called as Noianugavidhi (prevention of diseases). Promoting mental health state is that the need of the hour. Effectiveness of Muthu Parpam in the treatment of AD was proven through this study on the inhibition potential of acetylcholine choline esterase. It had been a preliminary screening for evaluating the efficacy. Hence, further clinical studies on Muthu Parpam should be necessary to position the footprints within the neurocognitive field.ACKNOWLEDGMENTThe author wishes to acknowledge the Noble research solutions, Perambur and Prof. Dr. R. Meenakumari, Director of National Institute of Siddha, Chennai, for their extended support in conducting this research work.AUTHORS’ CONTRIBUTIONThe authors, Dr. N. Sabari Girija, Dr. S. Guptaj, and Dr. M. A. Sinekha conceived of the presented idea. The corresponding author prepared the manuscript with the help of Dr. L. Sakthimanipriya. Experimental and manuscript work was supervised by Dr. P. Shanmugapriya. All the authors have equally contributed in preparing this manuscript.CONFLICTS OF INTERESTThe authors have declared that they have no conflicts of interest in this study.REFERENCES1. Badami O, Dongr SH, Suresh B. In vitro Antioxidant property of Solanum pseudocapsicum leaf extract. Indian J Pharmacol 2005;37:251-2.2. Giacobini E. Cholinergic function and Alzheimer’s disease. Int J Geriatr Psychiatry 2003;18 Suppl 1:S1-5.3. DeKosky ST, Harbaugh RE, Schmitt FA, Bakay RA, Chui HC, Knopman DS, et al. Cortical biopsy in Alzheimer’s disease: Diagnostic accuracy and neurochemical, neuropathological, and cognitive correlations. Intraventricular bethanecol study group. Ann Neurol 1992;32:625-32.4. Bartus RT, Dean RL, Beer B, Lippa AS. The cholinergic hypothesis of geriatric memory dysfunction. Science 1982;217:408-17.5. Sims NR, Bowen DM, Allen SJ, Smith CC, Neary D, Thomas DJ, et al. Presynaptic cholinergic degeneration in patients with dementia. J Neurochem 1983;40:503-9.6. Brenner GM. 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IN VITRO SCREENING FOR ACETYLCHOLINESTERASE ENZYME INHIBITION POTENTIAL OF MUTHU PARPAM – THERAPEUTIC LEAD FOR ALZHEIMER’S DISEASE

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IN VITRO SCREENING FOR ACETYLCHOLINESTERASE ENZYME INHIBITION POTENTIAL OF MUTHU PARPAM – THERAPEUTIC LEAD FOR ALZHEIMER’S DISEASE

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