INTRODUCTION:

Neurotransmitters are the body’s chemical messengers. They transmit signals from one nerve cell across a space to another nerve, muscle or gland cell. Acetylcholine is a neurotransmitter that has a wide variety of functions in the brain and other organ systems of the body. It is a neurotransmitter that acts as a chemical message that is released by neurons and allows them to communicate with other specialized cells such as myocytes and cells of glandular tissues. Acetylcholine plays an important role in muscle movement, sensation of pain, learning and cognitive function, the regulation of the endocrine system and rapid eye movement (REM) sleep cycles. Acetylcholinesterase (AChE), is an enzyme that catalyses the breakdown of acetylcholine to acetate and choline.^1,2

In recent years, interest in medicinal plants therapy has increased, leading to a greater focus on the medicinal use of the plants in treating disease and improving overall health withoutany significant side effects. Herbal medicines and natural products therapy are the oldest remedies known to themankind³

Alzheimer's disease is a brain disorder and it is a progressive diseases that gets worse over time. Alzheimer's disease causes the brain to shrink and ultimately make the brain cells to eventually die. Alzheimer’s disease is a neurodegenerative disorder that affects brain by slowly destroying memory and thinking skills and eventually, the ability to perform the simplest tasks.

About 6.5 million people in the United States living in age 65 and older live with Alzheimer's disease. About 55 million people worldwide with suffering with dementia, 60% to 70% are estimated to have Alzheimer's disease

In AD patients, significant deficits in cholinergic transmission have been associated with cognitive decline. A careful evaluation of AD medication includes anticholinergic medications to reduce the severity and characteristics of cognive deficits. Until date, FDA for treatment of cognitive impairement, namely rivastigmine, neostigmine, donepezil a cholinesterase inhibitor has approved as medication while these drugs shows unwanted side effects such as gastrointestinal tract irritation, hepatotoxcity.^4,5So now days there are growing interest of finding AchE inhibitors from natural sources.

Drugs acting on CNS may have general stimulatory or inhibitory action with anticonvulsant and psychopharmacological activities. Memory impairment is a major global health problem. Current therapies are showing resistance and exhibits various adverse effects. Therefore there is an emergency need for possible alternative treatments for AD and memory deficit. Most of the medicinal plants are prescribed in ayurvedic to enhance the memory.^6,7

Several medicinal plants have been used for decades in different cultures to improve memory such as Valeriana officinalis, Punica granatum, Salvia officinalis, Myristica fragrans Bacopa monnieri linn, Centella asiatica linn and Evolvulus alsinoides linn.

These herbal plants shows various potential activities like free radical scavenging activities, Anti-amyloidogenic, anticholinesterse, Hypolipidemic, antioxidant and anti-inflammatory activities. Terpenoids, Flavonoids phytochemical constituents present in plants used as acetylcholinesterase inhibitor in AD.^8,9

Hence in current study the extracts of Oxystelma esculentum were screened for invitro acetylcholinesterase inhibitory activity.

MATERIALS AND METHODS:

The leaves of Oxystelma esculentum were collected from Tamil Nadu (forest of kalakatu) Tirunelveli District, India. Taxonomic identification was made from botanical survey of medicinal plants, Siddha Unit, Government of India, Palayamkottai authenticated by V.Chelladurai Botanists. Authenticated register No:XCH 40379.

1.5kg of dried powdered aerial parts of Oxystelma esculentum was extracted sequentially by hot continuous percolation method by soxhlet apparatus using hexane, chloroform, ethyl acetate and ethanol as solvent. The collected extracts were concentrated by using rotatory vacuum evaporator and kept in desiccator.

Preliminary phytochemical analysis of extracts:

All the extracts were subjected to preliminary phytochemical screening for the detection of various plant constituents. The nature of the constituents present in the plant helps to determine the biological and pharmacological activity. The hexane, chloroform, ethyl acetate and ethanolic extracts of Oxystelmaesculentum. Was subjected to preliminary phytochemical analysis to identify the various active constituents present.¹⁰

In vitro acetylcholinesterase inhibitory assay:

All the four extracts were subjected to in-vitro anti-alzheimer’s activity by acetylcholinesterase enzyme inhibition assay.

The acetylcholinesterase inhibitor activity of the test samples (hexane, chloroform, ethyl acetate and ethanol extracts) were assayed by the following adaptation of spectrophotometric method reported by elman et al.^11,12

The cuvette used as a blank to control for the nonenzymatic hydrolysis of acetylcholine contained a mixture of 500μl of 3mm DTNB solution (in 0.1m potassium phosphate ph 8), 100μl of 15mm achi (in water), 275μl of 0.1M potassium phosphate ph 8, and 100μl of each cork and corkback ethanol-water extract solutions at the different concentrations (25, 50,100,200,400 and 800µg/ml).In the reaction cuvette, 25 μl of buffer was replaced by ache solution 0.16u/ml. the resulting solutions were placed in a spectrophotometer. Thiocholine formed during the hydrolysis of acetylcholine reacts rapidly with DTNB and a yellow compound is formed. the reaction was monitored for 5min at 405nm and the absorbance registered every minute. velocities of reaction were calculated enzyme activity was calculated as a percentage of the velocities compared to that of the assay using buffer solution instead of inhibitor (cork or corkback ethanol-water extracts). Positive assays were performed in triplicate.

% of inhibition = [(od of control - od of test)/(od of control)]×100

The extract exhibiting minimum IC₅₀value in comparison with the standard drug was selected as an active extract.

RESULTS AND DISCUSSION:

As illustrated in Table 1, Hexane extract shows the presence of terpenoids, saponins and oils and fats. Chloroform extract shows the presence of alkaloids, carbohydrate, phytosterols, tannins, glycosides and oil and fats. Ethyl acetate extract shows the presence of alkaloids, carbohydrates, saponins, phytosterols, phenols, terpenoids, glycosides and flavonoids. ethanolic extract shows the presence of glycosides, flavonoids, phytosterol, alkaloids, terpenoids and phenolic compounds.

The above phytochemical investigation has revealed that the ethanolic extract on the aerial parts of Oxystelma esculentum possess rich phytochemical constituents compared to other extracts like glycosides, flavonoids, phytosterol, alkaloids, terpenoids and phenolic compounds. These active constituents can exert pharmacological activities like anti-microbial, anti-inflammatory and to treat stress related disorders and as diuretics.

Table 1: Phytochemical screening on aerial parts of Oxystelma esculentum R.BR

Compounds	Hexane extract	Chloroform extract	Ethyl acetate extract	Ethanol extract
Alkaloids	-VE	+VE	+VE	+VE
Reducing sugar	-VE	+VE	+VE	+VE
Saponins	+VE	+VE	-VE	-VE
Phytosterols	-VE	+VE	+VE	+VE
Tannins	-VE	+VE	-VE	+VE
Flavonoids	-VE	+VE	+VE	+VE
Protein and Amino acid	-VE	-VE	-VE	+VE
Terpenoids	+VE	+VE	+VE	+VE
Glycosides	-VE	+VE	+VE	+VE
Fixed oils and fats	+VE	+VE	-VE	-VE

Note: +veindicate the presence of phytoconstituent

-ve indicate the absence of phytoconstituent

Fig. 1: Graph showing acetylcholinesterase inhibition assay of different extracts of Oxystelma esculentum R.BR

As illustrated in the above table 2, all the extracts were subjected to acetylcholinesterase inhibition assay. It was observed that ethanol extract of Oxystelma esculentum has good acetylcholinesterase inhibition compared with other extracts with an IC₅₀value of 38.54. The Galantamine was used as a standard, and its IC₅₀values was found to be 14.22. The percentage inhibition of acetylcholinesterase for the extracts was described in Figure 1.

Enzyme inhibitors are the agents that interact with the enzyme to thwart it from working in the normal manner. They have great physiological and pharmacological significance. There are various types of inhibitors reported in the scientific literature are nonspecific, irreversible, and reversible (competitive and noncompetitive).

The primary therapeutic stratagem against Alzeimer disease (AD) till date engrosses the use of cholinesterase inhibitors (ChEIs) which increase residual cholinergic activity, improve cognition thinking ability, and reduce behavioral disturbances.

Based on the recent developments in the scientific field and emphasis of the scientific community on the usage of natural compounds for the treatment of various human diseases, the bioactive compound polyphenols such as quercetin, resveratrol, curcumin, gallocatechins, cinnamic acid, caffeine, and caffeic acid are the potential inhibitor of cholinesterase for the treatment of AD.

Hence based on the above evidence, we have carried out in vitro screening of extracts of Oxystelma esculentum against acetylcholinesterase target enzyme. It was observed that ethanol extract shows promising inhibition against the enzyme compared with other extracts. Furthermore ethanol extract was considered as an active extract and subjected for in vivo anti Alzheimer activity.

Table 2: Effect of extracts of Oxystelma esculentum on acetylcholinesterase inhibition

conc. (µg)	Standard (Galantamine)	Hhhexane (hex)	Chloroform (ch)	Eethyl acetate (ea)	Ethanol (e)
25	0.844868	0.158457	0.374019	0.261059	0.261059
50	1.395604	0.333459	0.261059	0.374019	0.414937
100	0.311203	0.274455	0.374019	0.374019	0.364303
200	0.158456	0.215940	0.374019	0.364303	0.364303
400	0.392732	0.274455	0.261059	0.059940	0.316913
800	0.364303	0.215940	0.261059	0.844868	0.571324
IC50 Value	14.22	726.07	171.41	72.01	38.54

CONCLUSION:

Based on the current investigation, it is quite clear that different polyphenols could be used for the treatment of Alzheimer disease. However, the mechanism of their action needs to be further studied. The present study suggests that ethanol extract of Oxystelma esculentum have the potential acetylcholinesterase inhibitory activity. The further research are to be carried out to investigate the active compounds responsible for the activity.

REFERENCES:

1. Dale P, George J, David Fitzpatrick, Lawrence C, Anthony L, James O, Mark Williams. Neuroscience. 2nd edition. Sinauer associates, Sunderland. 2001.

2. Siva Athitya LV, Muthu Vijai Bharath V, Jayshree P. Screening of Gracilaria corticata Extracts for Acetylcholinesterase Inhibitory Activity Research J. Pharm. and Tech. 2018; 11(9): 3848-3850.

3. Zheleva-Dimitrova D, Balabanova V. Antioxidant and acetylcholinesterase inhibitory potential of Arnica montana cultivated in Bulgaria. Turkish Journal of Biology. 2012; 36: 732–737.

4. Thatoi HN, Patra JK, Das SK. Free radical scavenging and antioxidant potential of mangrove plants: a review. Acta Physiologiae Plantarum.2014; 36(3)

5. Balkis A, Tran K, Lee YZ. Screening ﬂavonoids for inhibition of acetylcholinesterase identiﬁed baicalein as the most potent inhibitor.Journal of Agricultural Science. 2015; 7: 26–35.

6. Akram, Muhammad, and Allah Nawaz. Effects of medicinal plants on Alzheimer's disease and memory deficits. Neural Regeneration Research. 2017; 12(4): 660-670

7. Vasudev Pai CS, Chandrashekar KS, Venkatesh Kamath. Cognitive Enhancement and Neuroprotective Effects of Ancient Ayurvedic Medicinal Plant Celastrus Paniculatus: An Overview. Research J. Pharm. and Tech. August. 2016; 9(8): 1295-1298

8. Peet Thomas S, Victoria J, Tenzin C, Chennu Manisha, Justin A. Recent Plant Based Remedies for Alzheimer’s disease, Parkinsons disease and Cerebral Ischemic Stroke. Research J. Pharm. and Tech. 2019; 12(18): 3951-3959

9. Ki-Yeol Yoo, So-Young Park. Terpenoids as potential anti-Alzheimer's disease therapeutics. Molecules. 2012; 17(3):3524-38

10. Kokate CK. Practical Pharmacognosy. Preliminary Phytochemical Screening. Vallabh prakashan, New Delhi. 13th edition. 2009. 106-111

11. Ellman GL, Courtney KD, Andres V. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology. 1961; 7(2): 88–95.

12. Chitra V, Narayanan J. In vitro Screening for Anti-Cholinesterase and Anti-Oxidant Activity of Extract of Garcinia hanburyi. Research J. Pharm. and Tech. 2018;11(7): 2918 -2921

13. Islam BU, Tabrez S. Management of Alzheimer’s disease-An insight of the enzymatic and other novel potential targets. Int J Biol Macromol. 2017; 97: 700- 709.

14. Jiang Y, Gao H, Turdu G. Traditional Chinese medicinal herbs as potential AChE inhibitors for anti-Alzheimer’s disease: a review. Bioorg Chem. 2017; 75: 50- 61.

Received on 08.05.2022 Modified on 20.04.2023

Research J. Pharm. and Tech 2024; 17(2):467-470.

DOI: 10.52711/0974-360X.2024.00073