Assessment Of Neuroprotective Potential of Freshwater Clam Lamellidens Corrianus Extract: An Invitro Exploration Against Alzheimer’s Disease

Alzheimer’s Disease

Alzheimer’s disease is defined as premature ageing of the brain. Usually beginning in mid-adult life and progressing rapidly to extreme loss of mental powers. Similar to that seen in very old age. The clinical features of Alzheimer’s disease include

5. An amnestic type of memory impairment
5. Deterioration of language
5. Motor and sensory abnormalities and gait disturbance

Alzheimer's disease is the most prevalent form of dementia and is a degenerative, incurable, and ultimately terminal condition. The exact cause of Alzheimer's remains unclear, but three potential risk factors are believed to play a role in the development of the disease.

5. vascular factor
5. genetic factor
5. behavioral factor

Alzheimer’s disease is a progressive neurodegenerative condition that typically begins gradually and worsens over time. The earliest symptom is often difficulty remembering recent events, or short-term memory loss. As the disease progresses, individuals may experience language difficulties, disorientation (including frequent disorientation in familiar places), mood swings, lack of motivation, and difficulty with self-care. As the condition advances, people may withdraw from family and social interactions. Eventually, essential body functions are impaired, leading to death.

The exact cause of Alzheimer's disease remains unclear. It is believed that around 70% of the risk is genetic, with multiple genes likely contributing to its development. Additional risk factors include a history of head injuries, depression, and hypertension. A definitive diagnosis requires examination of brain tissue. Engaging in regular mental and physical exercise, as well as maintaining a healthy weight, may help reduce the risk of developing Alzheimer's.

Stages Of Alzheimers

Effects Of Ageing on Memory

5. Occasionally misplacing items.
5. Forgetting that memory lapses occurred
5. Minor short-term memory loss

Early-stage Alzheimer’s (2 to 4)

5. Absent mindedness
5. Forgetting appointments
5. Subtle changes noticeable by close family or friends
5. Some confusion in unfamiliar situations

Middle stage Alzheimer’s(2to12)

5. Greater difficulty recalling recently learned information
5. increased confusion in various situations
5. Speech impairment

Late late-stage Alzheimer’s (up to a year)

5. More aggressive or passive behavior
5. Some loss of self-awareness and debilitating cognitive deficit

Risk Factors

5. Age

Age is the most significant risk factor for Alzheimer's disease, with the number of cases doubling every five years after the age of 65. According to the U.S. Alzheimer's Association, 1 in 8 individuals aged 65 and older are affected by Alzheimer's. Although less common, Alzheimer's can also impact younger individuals.

5. Gender

More women than men develop Alzheimer's disease, but this is likely due to the fact that women generally live longer than men, increasing their risk of developing the condition as age is a key factor.

5. Race and Ethnicity

Africans and Americans are at greater risk for developing Alzheimer’s disease than whites. This may be due in part to their higher prevalence of medical conditions such as high blood pressure and diabetes, which are associated with increased risk for Alzheimer’s.

5. Family History

People with a family history of Alzheimer's are at higher-than-average risk for the disease

Etiology

5. Accumulation of beta amyloid, an insoluble protein, which form sticky patches surrounded by debris of dying neurons, significant loss of neuron and volume in brain region devoted to memory and higher mental functioning.
5. Neurofibrillary tangles (twisted nerve cell fibers that are the damaged remains of microtubules-support structures that permit nutrients to flow through neurons)
5. An excessive accumulation of metal ions, such as zinc and copper, in the brain

Other possible factors being researched are:

5. Deficiencies of vitamin B_6, Vitamin B₁₂ and folate
5. Degenerative disorders of CNS
5. Parkinson disease
5. Vascular disease: hypertension, circulatory disturbance
5. Intracranial space occupying lesion
5. Metabolic disorders: Hepatic failure, Renal failure
5. Endocrine disorders, Myxedema, Addison’s disease
5. Infection, AIDS, Meningitis, Encephalitis
5. Intoxication, Alcohol, Heavy metals, Chronic barbiturate poisoning
5. Anoxia, anemia, post-anesthesia, chronic respiratory failure
5. Vitamin E deficiency, especially deficiency of thymine and nicotine
5. Miscellaneous- Epilepsy, Electric injury

Clinical Manifestation

5. Personality changes-Lack of interest in day-to-day activities, easy mental fatigability, self- centered, decreased self-care
5. Memory impairment-Recent memory is prominently affected.
5. Cognitive impairment-Disorientation, poor judgment
5. Affective impairment, including mood swings, irritability, and depression
5. Behavioral impairment, including stereotyped behaviors, changes in sexual drives and activities, and neurotic or psychotic behaviors
5. Neurological impairment-Aphasia, apraxia, seizures, headache.
5. Catastrophic reaction, characterized by agitation and attempts to compensate for cognitive deficits by using strategies to avoid revealing failures in intellectual performance, such as changing the subject.
5. Sundowner syndrome, characterized by symptoms such as drowsiness, ataxia, and other related signs.

Treatment Of Alzheimer’s Disease

Most drugs currently used to treat Alzheimer's disease, as well as those under investigation, focus on slowing the progression of the condition. However, there are no cures available to date, and the improvements offered by some of these drugs may be so modest that patients and their families may not notice significant benefits. The U.S. Food and Drug Administration (FDA) has approved two main classes of drugs to treat the cognitive symptoms of Alzheimer's disease:

• Cholinesterase inhibitors (commonly used for mild-to-moderate Alzheimer's; donepezil is also approved for treating severe dementia)
• N-methyl-D-aspartate (NMDA) receptor antagonists (used for moderate-to-severe Alzheimer's)

All FDA-approved medications for Alzheimer's disease are expensive. While these drugs are generally considered safe, they can cause a range of side effects, including indigestion, nausea, vomiting, diarrhea, loss of appetite, muscle cramps, and fatigue. These pharmacological treatments primarily address the symptoms of Alzheimer's, such as memory loss and cognitive decline. Drugs like tacrine, donepezil, rivastigmine, and galantamine work by inhibiting acetylcholinesterase, an enzyme that breaks down acetylcholine. By preventing this breakdown, these drugs help increase the levels of acetylcholine in the synapses, thereby improving communication between neurons.

Cholinesterase Inhibitors

Although there are four approved cholinesterase inhibitors, Donepezil, Rivastigmine and Galantamine are indicated for mild to moderate Alzheimer’s. Memantine is indicated for moderate to severe Alzheimer’s.

There are two types of cholinesterase, acetylcholinesterase and butyryl cholinesterase (BuChE). Acetyl cholinesterase is found primarily in the blood and neural synapses. Butyryl cholinesterase is found primarily in the liver. The primary distinction between the two lies in their substrates. Acetylcholinesterase breaks down acetylcholine (ACh) at a faster rate, while butyrylcholinesterase hydrolyzes butyrylcholine (BuCh) more efficiently. Acetylcholinesterase (AChE) has emerged as the most promising therapeutic target for symptomatic relief in Alzheimer’s disease (AD), as a cholinergic deficit is a consistent and early characteristic of the condition. Inhibition of acetyl cholinesterase was considered to be achievable as a therapeutic target because of proven efficacy of inhibition of peripheral acetyl cholinesterase as a treatment for myasthenia gravis (MG) proving that the approach was feasible. However, selective inhibition of the central nervous system (CNS) acetyl cholinesterase initially proved to be daunting. Before tacrine, physostigmine, the classic acetyl cholinesterase inhibitor (AChEI) was investigated as a treatment for Alzheimer’s. Physostigmine was subsequently abandoned because of poor tolerability. Four drugs are currently available for Alzheimer’s treatment: galantamine, rivastigmine, donepezil, and memantine. The first three are acetyl cholinesterase inhibitors and memantine is not.

Mechanism Of Action of Cholinesterase Inhibitor in AD

1. Naturally Derivative

1.1. Huperzine A

Huperzine A (HupA) is a Lycopodium alkaloid extracted from the Chinese medicinal herb Huperzia. It is a highly selective, reversible, and potent acetylcholinesterase inhibitor. The synthetic racemic mixture of huperzineA exhibits weaker acetylcholinesterase inhibitory effects compared to the natural form. Many of the initial derivates demonstrated lower potency than the natural huperzineA. HuperzineA has also been hybridized with tacrine and donepezil. While the donepezil hybrid demonstrated reduced effectiveness, the HupA-tacrine hybrids, known as huprines Y and X, have shown greater efficacy in amplifying acetylcholinesterase inhibition in vitro compared to tacrine. Additionally, huprines Y and Z exhibited stronger inhibitory activity than either of the parent compounds. Huperzine has a higher oral bioavailability compared to tacrine and donepezil. HupA is also shown to be more potent than tacrine, rivastigmine, and galantamine in terms of inhibition activities, and it had the least amount of activity against butrylcholinesterase .

1.2. Huperzine B

Natural Huperzine B (HupB) is a Lycopodium alkaloid isolated from the Chinese medicinal herb Huperziaserrata which has been demonstrated as an effective and reversible inhibitor of acetyl cholinesterase.

1.3. Nelumbo Nucifera

Nelumbonucifera, commonly known as the lotus, is an aquatic plant with a variety of medicinal properties. The mechanism of action is felt to be acetyl cholinesterase inhibition. One new compound and four known compounds were isolated from the n-butanol fraction of the N. nucifera. The new compound is a beta-cyclogeranioldiglycoside, nuciferoside, and the four known compounds are cycloartenol , p-hydroxybenzoic acid, vanilloloside, and 5′-O-methyladenosine. Compounds 5 and 1–3 demonstrated good and noncompetitive acetyl cholinesterase inhibition and compounds 1, 2, and 5 showed exhibited butryl cholinesterase inhibition. Compounds 1– 3 and 5 have possible cholinesterase inhibitory effects with the potential to be used for Alzheimer’s treatment.

1.4. Himatanthuslancifolius

Himatanthuslancifolius is a shrub that contains several indole alkaloids with a number of medicinal properties such as antimicrobial effects, gastro protection, and the ability to affect the vascular and nonvascular smooth muscle responsiveness. It shows acetyl cholinesterase inhibiting properties

1.5. Galangin

A flavonol isolated from RhizomaAlpiniaeOfficinarum called galangin demonstrated the highest inhibitory effects on acetyl cholinesterase activity. This suggests that galangin could be developed as a potential treatment for Alzheimer’s because of the dual mechanism of action of cholinesterase inhibition and free radical scavenging properties.

1.6. Cardanol Derivatives

It is a new acetyl cholinesterase from non isoprenoid phenolic lipids (NIPLs) of Anacardium occidentale. Cardols, cardanols, anacardic acids, and methylcardols are the primary nonisopreniod phenolic lipids components of cashew nut-shell liquid (CNSL) and have been used to generate potential bioactive compounds. It shows accetylcholinesterase inhibiting properties, so also used in the treatment of Alzheimer’s disease. Oceans are probably the Earth’s most valuable natural resource, providing food mainly in the form of fish and shellfish. Marine invertebrates, which constitute one of the major groups of marine organisms, are a source of a wide range of medicinal benefits, in addition to the large numbers of marine natural products (MNPs) that have been discovered. Seafood from edible marine invertebrates, such as mollusks, has been linked with various medicinal benefits for human health. In some cultures, shelled gastropods and bivalves are considered as a healthy food, and they also contributed in a range of traditional natural remedies. Marine organisms live in complex habitats and are exposed to extreme conditions, which leads them to produce a wide variety of specific and potent active substances that cannot be found elsewhere. Among the 34 fundamental phyla of life, 17 are found on land, while 32 are found in the sea (with some overlaps). Marine organisms represent a largely unexplored domain, offering the highest potential for identifying compounds with novel biological activities and higher potency. Recent research has focused on isolating novel chemical structures and compounds from MNPs, demonstrating the immense value of the marine world. The field of marine research spans across more than 80 nations, with over 2,700 scientists concentrating on diversity, distribution, and the discovery of potential drugs from marine sources. The marine system produces an inexhaustible and rich source of potential natural products, which have a wide range of nutraceutical, cosmeceutical, and unique pharmaceutical activities. More than 30,000 compounds have been identified from the marine environment, each with unique structures and associated pharmaceutical activity. Extensive bioprospecting of marine fauna, flora, and microbes has had a significant impact on the biomedical industry, producing small molecules with anti-infective, anticancer, anti-inflammatory, analgesic, immunomodulatory, antiviral, neuroprotective, antifouling, and a variety of other biological activities. This evidence proves that the marine reservoir holds an infinite number of pharmacologically promising and exciting drug candidates for human health. Freshwater bivalves (Mollusca: Order Unionoida) are classified into six families and approximately 165 genera worldwide. The global rate of extinction for these bivalves remains poorly understood. In North America, the freshwater fauna north of Mexico includes 297 taxa from two families. Among these, 19 taxa are presumed extinct, 44 species are listed or proposed as federally endangered, and another 69 species may be endangered. Many of these endangered species are considered functionally extinct, meaning that while individuals of the species survive, they do not reproduce. The decline of North American unionoid bivalves can be traced to the impoundment and inundation of riffle habitats in major rivers, such as the Ohio, Tennessee, Cumberland, and Mobile Bay Basin. The construction of dams led to the local extinction of the bivalves' host fish, which are essential for their life cycle. The combined effects of host fish loss, increased siltation, and various forms of industrial and domestic pollution have driven a rapid decline in unionoid bivalve populations across North America. In Europe, while some local unionoid populations have been extirpated, no unionoid species have gone extinct. However, freshwater communities in countries like China, which face soil erosion, industrial pollution, and the construction of numerous dams on rivers, are likely experiencing local extirpation or even the extinction of their endemic freshwater bivalve species. Similarly, nations in South America, such as those along the Rio Paraná, face similar challenges to their freshwater bivalve populations. Furthermore, three taxa from Israel are now officially recognized as extinct.

Profile Of Lamellidens Corrianus

Fig.2 Lamellidens corrianus

Scientific classification

Scientific Name: Lamellidenscorrianus

Kingdom: Animalia

Phylum:   Mollusca

Class:       Bivalvia

Sub class: Autobranchia

Order:      Unionida

Family:    Unionidae

Genus:     Lamellidens

Species:   Lamellidenscorrianus

Lamellidenscorrianus is a freshwater bivalve species that inhabits large lowland rivers, typically found in substrates made up of sand, silt, and mud. It is a food source for many aquatic animals and has been part of human diet in India.

Origin and distribution

Lamellidenscorrianus is a freshwater bivalve species native to South Asia, specifically found in India, Bangladesh, and Myanmar. It primarily inhabits large lowland rivers and their associated floodplains, preferring slow-flowing or standing waters with sandy, silty, or muddy substrates. This species is commonly distributed in regions where the water bodies support a rich macrozoobenthic community.

Description and biology

Lamellidenscorrianus is a medium-sized freshwater bivalve belonging to the family Unionidae. The shell is typically oval to elongated, with a moderately convex shape. The outer surface of the shell is usually brown or grayish, often with darker markings, while the inner surface can appear white or pale. This species can grow up to 7 cm in length, though individuals can vary in size depending on environmental conditions. The shell is often smooth, but some may exhibit fine ridges or growth lines. The hinge of the shell has a relatively simple structure, with a small ligament holding the two valves together. Lamellidenscorrianus has an asymmetrical, moderately inflated, and elongated shell with a smooth or slightly sculptured exterior. The left valve tends to be more convex than the right, which is typical of many bivalves. The anterior margin of the shell is slightly curved, while the posterior margin is more rounded. The beaks (hinges) are positioned towards the posterior end of the shell, and they are not very prominent, blending well with the general shape of the bivalve. The ligament that connects the two shells is elastic, and this feature allows the valves to open slightly and filter food when conditions are right. Lamellidenscorrianus primarily inhabits large lowland rivers and floodplains with slow-moving or standing water.Lamellidenscorrianus thrives in clean, fresh water with low to moderate flow. It is commonly found in regions of rivers that have moderate water velocities, which facilitate its filter-feeding behavior. The species requires well-oxygenated water, often found in areas where the water quality is relatively high. Temperature-wise, it generally prefers tropical to subtropical water conditions, with temperatures typically ranging from 20°C to 30°C. The species is found in regions of rivers where water clarity is high and where sediment load is moderate, ensuring it can feed effectively by filtering out organic particles. The growth rate of Lamellidenscorrianus can vary depending on environmental conditions such as water quality, food availability, temperature, and habitat stability. This fresh water bivalve generally spawns in the warmer months when water temperatures are optimal. The males release sperm into the water, which fertilizes the eggs inside the female. After fertilization, the eggs develop into larval stages called glochidia. The glochidia larvae are parasitic and must attach to a suitable host fish to complete their development. The larvae latch onto the gills or fins of host fish, where they undergo further growth and development. This parasitic phase is a crucial part of the species' life cycle. Once the glochidia complete their development and undergo metamorphosis, they detach from their host and fall to the riverbed where they begin their life as juvenile clams. This post-larval phase is when they begin to burrow into the substrate, growing into adult bivalves.

Biochemical composition

The biochemical analysis of Lamellidenscorrianus flesh showed the presence of proteins, lipids, carbohydrates, and minerals.

Pharmacological activities

Lamellidenscorrianus extract possess the following activities.

1. Antioxidant
2. Anti-inflammatory
3. Antibacterial
4. Antiviral
5. Anticancer
6. Immunomodulatory Effects
7. Antidiabetic
8. Hepatoprotective
1. Analgesic
10. Neuroprotective

MATERIALS AND METHODS

Collection Of Lamellidens Corrianus

The Lamellidans corrianus specimens were harvested from their natural habitat Olipramkadavu,Malappuram District (Kerala, India). They were rinsed with a strong water jet and meticulously cleaned to remove any adhering algae and debris. The species was verified by Dr.

C.D. Sebastian, Professor and Head of the Department of Zoology at the University of Calicut, Kerala.

Extraction

A total of 300 g of mussel tissue was blended and subjected to two rounds of extraction with 600 ml of ethyl acetate using mechanical stirring overnight. The resulting mixture was then centrifuged at 8,000 rpm for 20 minutes at 4°C. The residual material was further extracted using the same procedure with 600 ml of methanol and a 7:3 water-ethanol mixture. The three resulting supernatants were evaporated to dryness under reduced pressure at temperatures between 35°C and 55°C using a rotary evaporator, yielding 1.8%, 6.4%, and 3.2% for ethyl acetate, methanol, and water-ethanol, respectively. The extracts were then stored in sealed glass vials at -25°C until needed.

The EELC was subjected to qualitative chemical tests for the detection of various constituents like carbohydrates,protiens,glycosides and amino acids , fixed oils and fats , gums and mucilage , alkaloids , phytosterols and flavonoid , tannins and phenolic compounds , saponins , triterpenoids etc.

1. Detection of alkaloids.

The extract was dissolved in dil.Hcl and filtered.The filtered extract was subjected to detection of alkaloids.

Mayer’s Test

Filtrates were treated with Mayer's reagent (Potassium Mercuric Iodide). The formation of a yellow coloredprecipitate indicates the presence of alkaloids.

Dragendroff's Test:

Fitrates were treated with Dragendroffs reagent (solution of Potassium Bismuth Todide). The formation of red precipitate indicates the presence of alkaloids.

Wagner’s Test

The filtrates were treated with Wagner’s reagent (a solution of iodine in potassium iodide). The appearance of a brownish-red precipitate signifies the presence of alkaloids.

Hager’s Test:

The filtrates were exposed to Hager’s reagent (a saturated solution of picric acid). A yellow precipitate indicates the presence of alkaloids.

2. Detection of carbohydrates

The extract was dissolved in 5 ml of distilled water and filtered. The filtrate was then used for carbohydrate testing.

Molisch's Test

Two drops of alcoholic α-naphthol solution were added to the filtrate in a test tube. A violet ring at the junction indicates the presence of carbohydrates.

Benedict's Test

The filtrate was treated with Benedict's reagent and heated gently. The formation of an orange-red precipitate indicates the presence of reducing sugars.

Fehling's Test

The filtrate was hydrolyzed with dilute HCl, neutralized with an alkali, and heated with Fehling's A and B solutions. A red precipitate confirms the presence of reducing sugars

Barfoed's Test

A few drops of Barfoed's reagent were added to the test solution and heated in a boiling water bath for 1-2 minutes, followed by cooling. The appearance of a red color indicates the presence of reducing sugars.

3. Detection of Glycosides

Test for Anthraquinone Glycosides:

The extract was hydrolyzed with dilute HCl, then tested for glycosides.

Borntrager's Test

To the extract, add H?SO?, boil, and filter. To the cold filtrate, add equal volumes of benzene. The appearance of a pink to red color in the ammoniacal layer indicates the presence of anthraquinone glycosides.

Modified Borntrager's Test

The extract was treated with ferric chloride solution and heated in boiling water for 5 minutes. The mixture was cooled, extracted with equal volumes of benzene, and treated with ammonia solution. The formation of a rose-pink color in the ammoniacal layer confirms the presence of anthranol glycosides.

Test for Hydroxyl Anthraquinones

The test solution was treated with potassium hydroxide, resulting in the appearance of a red color.

Test for Phenolic Glycosides

The test solution was treated with 3 drops of a mixture of 1 ml 1% ferric chloride and 1 ml 1% potassium ferrocyanide. A green-blue color indicates the presence of phenolic glycosides.

Test for Coumarin Glycosides (Fluorescence Test)

To 1 ml of test solution, add 1 ml of 1 N NaOH. The development of a blue-green fluorescence indicates the presence of coumarin glycosides.

Test for Cardiac Glycosides

Legal's Test

The extract was treated with sodium nitroprusside in pyridine and sodium hydroxide. The formation of a pink to blood-red color indicates the presence of cardiac glycosides.

Keller-Killiani Test

A small portion of the extract was shaken with 1 ml of glacial acetic acid containing a trace of ferric chloride. One ml of concentrated H?SO? was added carefully along the sides of the test tube. A blue color in the acetic acid layer and a red color at the junction of the two liquids indicates the presence of glycosides.

Baljet Test

The test solution was treated with sodium picrate solution. The appearance of a yellow to orange color indicates the presence of glycosides.

Raymond's Test

The test solution was treated with hot methanolic alkali. The development of a violet color indicates the presence of glycosides.

1. Detection of Saponins

Froth Test

The extract was diluted with distilled water to 20 ml and shaken in a graduated cylinder for 15 minutes. The formation of a 1 cm layer of foam indicates the presence of saponins.

Foam Test

0.5 g of the extract was shaken with 2 ml of water. If the foam persists for 10 minutes, it indicates the presence of saponins.

2. Detection of Phytosterols

Salkowski's Test

The extract was treated with chloroform and filtered. The filtrate was then treated with a few drops of concentrated sulfuric acid, shaken, and allowed to stand. The appearance of a golden yellow color indicates the presence of triterpenes.

Liebermann-Burchard's Test

The extract was treated with chloroform and filtered. The filtrate was then treated with a few drops of acetic anhydride, boiled, and cooled. Concentrated sulfuric acid was added, and the formation of a brown ring at the junction indicates the presence of phytosterols.

Sulfur Test

The test solution was treated with sulfur powder. If the sulfur sinks to the bottom, it indicates the presence of steroids and terpenoids.

3. Detection of Tannins and Phenols:

Ferric Chloride Test

The extract was treated with 3-4 drops of ferric chloride solution. The formation of a bluish-black color indicates the presence of phenols.

Gelatin Test

To the extract, 1% gelatin solution containing sodium chloride was added. The formation of a white precipitate indicates the presence of tannins.

4. Detection of Flavonoids

Alkaline Reagent Test

Extracts were treated with a few drops of sodium hydroxide solution. The formation of a yellowcolour, which fades to colourless upon the addition of dilute acid, indicates the presence of flavonoids.

Lead Acetate Test

A few drops of lead acetate solution were added to the extracts. The appearance of a yellow precipitate indicates the presence of flavonoids.

5. Detection of Proteins and Amino Acids

Ninhydrin Test

To the extract, a 0.25% w/v ninhydrin solution was added and heated for a few minutes. The formation of a blue colour indicates the presence of amino acids.

Xanthoproteic Test

The extract was treated with a few drops of concentrated nitric acid. The appearance of a yellow color indicates the presence of proteins.

Millon’s Test

The test solution was treated with Millon’s reagent. A white precipitate that turns red upon gentle heating indicates the presence of proteins.

6. Detection of Fixed Oils and Fats

Oily Spot Test

A drop of the extract was placed on filter paper, and the solvent was allowed to evaporate. An oily stain on the paper indicates the presence of fixed oils.

7. Detection of Vitamin C

Sodium Nitroprusside Test

When the test solution was treated with sodium nitroprusside solution, a blue color was produced.

Sodium Bicarbonate Test

The test solution was treated with sodium bicarbonate solution. A violet color was produced, indicating the presence of vitamin C.

Pharmacological Studies

Determination Of Anti-Alzheimer’s Activity by Invitro Method

Cholinesterase inhibitory activity was determined using Ellman’s technique,which was previously described in a study.The enzyme hydrolyses the substrate acetylthiocholine to create thiocholine,which interacts with Ellman’s reagent (DTNB) to yield 2-nitrobenzoate - mercaptothiocholine and 5-thio-2-nitrobenzoate which can be detected at 405 nm.In this assay , 25 µL of acetylthiocholine iodide (5 mM) , 125 µL of DTNB (3 mM), 50 µL of buffer B (50 mM tris-HCl , PH-8, 0.1 % BSA) , and test extract solution at different volume (20,40,80,160 and 320 µL) or a negative control (25 % DMSO in methanol) were mixed and incubated for 10 minutes at 37^oc . 25 µL of 0.05 U/MlAChEwas added to start the reaction. At 405 nm , the absorbance was measured.

Identification

The fresh water clams were identified as Lamellidens corrianus of Unionidae family by Dr. C.D. Sebastian, Professor and Head of the Department of Zoology at the University of Calicut, Kerala.

Preparation Of Extract

The flesh of fresh water clam Lamellidens corrianus was extracted with ethanol,methanol and ethyl acetate by maceration process.The extraction was carried out as per procedure in chapter 4.The percentage yield of extract of Lamellidens corrianus was shown in table 2.

Table No.2 Percentage yield of extract of Lamellidens corrianus.

Biochemical Analysis

Pharmacological Study

Enzyme Inhibition Assay of Lamellidens Corrianus Extract of Anti-Alzheimer’s Activity

The Enzyme inhibition assay was conducted to evaluate the potential anti-Alzheimer’s activity of lamellidens corrianus extract by measuring its impact on enzyme absorbance at various concentration. The absorbance value recorded at different concentration are as follows:

Table No.4 Absorbance of different concentration

The results indicate a concentration-dependent increase in absorbance, suggesting enzyme inhibition activity. A significant rise in absorbance at higher concentrations implies that the extract may effectively inhibit acetylcholinesterase (AChE), supporting its potential role in mitigating Alzheimer’s disease symptoms.