INTRODUCTION:

NEURODEGENERATIVE DISORDERS:

Any clinical illness affecting neurons is included in the category of disorders with neurodegenerative symptoms. A wide range of neurological illnesses that impair particular aspects of neuronal function are collectively referred to as "neurodegenerative disorders."

These illnesses can arise for a variety of causes and can worsen over time if left untreated. The majority of media coverage has gone to a select few of these various neurodegenerative disorders, including Alzheimer's disease (A.D.), Parkinson's disease (P.D.), amyotrophic lateral sclerosis (ALS), and Huntington's disease (H.D.). One common risk factor for neurodegenerative diseases, especially A.D. or P.D., was getting older.¹

ALZHEIMER’S DISEASE:

Alzheimer's disease is one kind of dementia, or loss of brain function, that results in issues with thinking, memory, and behaviour. Degenerative diseases include Alzheimer's as well. Alzheimer's disease is believed to be brought on by a confluence of environmental and genetic factors, while the precise etiology of the illness is yet unknown. Only when a person exhibits certain symptoms and does not exhibit signs of other forms of dementia can an Alzheimer's disease diagnosis be made.²

The illness emerges as difficulties with language, memory, perception, emotional behaviour, personality, and cognitive abilities, among other aspects of mental function. The disease's first symptoms are limited to forgetfulness. The symptoms of Alzheimer's disease worsen and become more noticeable as the condition advances. Additionally, the symptoms start to interfere more with the person's daily activities, making self-care increasingly challenging.² While there isn’t a cure for Alzheimer’s there are treatments that can help control the disease’s development, behavioural issues, sleep disruptions, and disorientation in the patient. A further objective of AD treatment is to alter the patient’s living situation and assist in educating and supporting family members and caregivers.³ The two main pathophysiological features of AD are intracellular neurofibrillary tangles and the formation of amyloid beta plaques.⁴ Neurofibrillary tangles, which cause loss of synapses and neurons, are intracellular gigantic paired helical filaments of hyperphosphorylated tau proteins.⁵ The main brain regions affected by Alzheimer's disease in humans are the hippocampus and the cerebral cortex association areas.⁶

Physicians divide symptoms of Alzheimer's disease into two categories: "cognitive" and "behavioural and psychiatric" before beginning treatment.⁷ Currently, galantamine, rivastigmine, and donepezil are the three cholinesterase inhibitors that are often administered. In addition to cholinesterase inhibitors, memantine has an approved use in the management of Alzheimer's disease. Glutamate activity in the brain is modulated by memantine. The complicated pathophysiology of Alzheimer's disease means that, despite great advancements in contemporary medicine, there is still no proven cure. Not only may medicinal herbs function on a wide range of targets through different mechanisms, but they are also harmless.⁷ Certain active ingredients in the phylloclade of the plant Epiphyllum oxypetalum (DC) have been revealed to contain glycosides, saponins, steroids, phenols, proteins, resins, tannins, and terpenoids.⁸

The many pharmacological properties of leaves of Epiphyllum oxypetalum have been demonstrated by science. Additionally, studies have demonstrated the anti-inflammatory, anti-helminthic, anti-cancer, anti-bacterial, and anti-Urolithiatic properties that may be responsible for potential antioxidant and anti-Alzheimer's effects.⁹

METHODOLOGY:

Collection and authentication of plant material:

The leaves of Epiphyllum oxypetalum Leaves were collected from the local vendor in Hassan, Karnataka. They were authenticated by Dr V. Rama Rao (Research officer Dep. of Botany), Central Ayurveda Research Institute #12, Uttarahalli Manavarthe Kaval, Kanakapura Main Road, Thalaghattapura post, Bangalore - 560109.

Preparation of plant extract:

The leaves of Epiphyllum oxypetalum Leaves were thoroughly cleaned with fresh water. The leaves were dried properly under shade for 40 days. Dried leaves were coarsely powdered and utilized for Methanolic extract. The Methanolic extraction was carried out by the Soxhlet extractor technique. Coarsely powdered leaves were Soxhlet with methanol for two cycles in 6 hours (Temperature-30^o). The extract was filtered through the Whatman filter paper. The extract was weighed and stored in air-tight containers for further Phytochemical and pharmacological studies. The percentage of extract yield was calculated by using the formula.

Percentage of extract yield (Weight in gm of extract obtained)/(weight in gm of plant material taken) × 100

ACUTE TOXICITY STUDY:

OECD guideline No. 425 was used in order to assess acute oral toxicity. Before the drugs were given, Male swiss albino mice were obtained and fasted throughout the whole night. The dosage of the 100% ethanolic extract from the leaves of the Epiphyllum oxypetalum (DC.) plant was determined by weighing the mice. The extract was administered orally after being dissolved in distilled water. Three swiss male mice were given a dosage of 2000mg/kg in accordance with OECD norms. The animals' consciousness, mood, CNS activity, muscular tone, reflexes, and autonomic profile were all monitored for four, eight, twelve, and twenty-four hours. Additionally, the animals were monitored for 15 days to look for any abnormalities.¹⁰

EXPERIMENTAL ANIMALS:

Swiss Albino Mice of Male sex animal weighing 30-35g used in the study. The animals were collected from Sri Venkateshwara Enterprises, Bangalore and housed in polypropylene cages in groups of six rats per cage and kept under controlled environmental conditions at 23± 2°C, relative humidity 50±1% with 12 h light/dark cycle. Care of animals were taken according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The animals were fed with standard feed and water ad libitum and acclimatized for 15 days before the study. The experiments on animals were conducted in accordance with the Experimental Protocols approved by Institutional Animal Ethics Committee (IAEC).

EXPERIMENTAL DESIGN:¹¹

1. Normal control: Fed with normal diet and water ad libitum.

2. Disease control: Normal Saline will be Administered for 15 days + Scopolamine butylbromide (1mg/kg b.w. I.P) from day 10 to day 15.

3. Standard group: Donepezil tablet (2.5mg/kg b. w. P.O) for 15 days + Scopolamine butylbromide (1mg/kg b.w. I.P) from day 10 to day15.

4. Treatment group I (Low Dose): Drug Epiphyllum oxypetalum extract low dose (250mg/kg b.w. P.O) for 15 days + Scopolamine butylbromide (1mg/kg b.w. I.P) from day 10 to day 15.

5. Treatment group II (High Dose): Drug Epiphyllum oxypetalum extract High dose (500mg/kg b.w. P.O) for 15 days + Scopolamine butylbromide (1mg/kg b.w. I.P) from day 10 to day 15.

ANTI-ALZHEIMER’S STUDY:

· ELEVATED PLUS MAZE (EPM):

The elevated plus-maze will be used, with two arms that are open (165cm) and two arms that are closed (16×5×12 cm). The maze will be raised to a 25-centimeter height. Rats will be positioned one at a time at the end of an open arm, facing away from the central platform, and the amount of time it takes for them to go from one open arm to the other (transfer latency, or TL) will be noted. The animal will be gently pushed into one of the two enclosed arms and the time limit will be set to 90 seconds if it does not enter one of the arms within that time. After ten more seconds of maze exploration, the rat will be able to return to its home cage. Holding onto this,¹²

IR = (L1-LO) LO.

Where, LO is the TL on 15^th day

L1 is the initial TL on 1^st day trail (i.e 14^th day) in seconds.

· MORRIS WATER MAZE:

The MWM is a round, white pool with walls that rise to a height of 20cm and an interior diameter of 110cm. Thirteen centimeters of regular tap water were poured into it. The animals did not exhibit any negative effects from the modest amount of milk powder added to the room temperature (±22°C) water to make it opaque. The two imaginary diagonal lines that ran across the middle of the pool split the entire area into four equal-sized quadrants. A circular, detachable escape platform with a diameter of 10 cm is placed in the centre of one of the four quadrants, or one centimetre below the water's surface (the target quadrant).

A dimly lighted rectangle chamber with white lighting at its far end housed the pool. Over the course of the trial, the room's walls were fitted with a range of spatial clues that stayed in place.

Throughout the experiment, the platform remained in the same location. On the tenth day of the treatment period, the test was started, and the mice were given 120 seconds to swim without a platform so they could become used to it.

Over the course of the following four days (days 11, 12, 13, and 14), every animal was given four 120-second learning trials with a 60-second intertrial gap. The rat was positioned in the water diagonally opposite the platform's quadrant, facing the pool wall for every learning experiment. The escape latency time for each trial was determined by timing how long it took the animal to find the submerged platform.

The animals were instructed to find the platform and given 60 seconds to rest there if they couldn't find it in 120 seconds. In this instance, the escape latency time was 120 seconds. These meetings served as the acquisition tests or trials for the concealed platform and were recorded.

The platform was taken out of the water on day 15, or 24 hours after the last learning trial, and they participated in a probing trial session to gauge their memory recall.

Every mouse was dropped into the water diagonally across from the target quadrant, and it was given sixty seconds to swim around and locate the quadrant where the platform had been before. The amount of time the animal spent in the target quadrant was noted.¹³

BIOCHEMICAL TEST:

· Preparation of Brain Sample:

After assessing the learning and memory paradigms in Scopolamine induced amnesia, rats from each group will be euthanized by using a carbon dioxidechamber; brains will be removed quickly weighed (400mg) and homogenized in cold phosphate buffer (0.05M, pH 7.4) after being kept in cooled 0.9% saline in a small container. The frontal cortex, hippocampus, and septum will be quickly dissected out on a petri dish chilled on crushed ice. The samples of mice brain were centrifuged at 4000rpm at 4⁰C degrees centigrade for 20min after centrifugation homogenates will be collected in different test tubes to analyze Acetylcholinesterase, Catalase, SOD. The supernatant is used for enzymatic assays.¹⁵

· Estimation of Acetylcholinesterase activity:

Ache is a marker for cholinergic neurons in the forebrain. The Ellman method is used for assessing the activity of Ache. Ache activity was assayed spectrophotometrically by measuring the rate of hydrolysis of acetylcholine (ATCI) in phosphate buffer (pH 8). The reaction was started by adding 200µl of homogenate to 2.8ml reaction mixture containing 250µl 10mM DTNB, 100µl 30mM ATCI and 2.45ml of 0.1M phosphate buffer. Change in absorbance will be measured for 2min at 30sec intervals at 412nm. The result will be reported in µMol of acetylcholine iodide hydrolysed/min of protein.¹⁴

ANTIOXIDANT ENZYME ANALYSIS:

· Determination of Catalase (CAT):

In a cuvette containing 1.95ml of phosphate buffer (pH 7.0), a volume of 0.05 ml of supernatant was added and 1ml of H2O2 was added for the reaction to start. The absorbance was measured spectrophotometrically at 240 nm by UVSpectrophotometry. The percentage of inhibition of free radicals by CAT was calculated by using the equation Where A0 is the absorbance of the control without extract and A1 is the absorbance of the sample extract.¹⁶

· Determination of Superoxide dismutase (SOD):

SOD Activity was determined by measuring pyrogallol autoxidation. Briefly, the reaction was started by adding 0.5 ml of pyrogallol (2mM) to a reaction mixture containing 500µl of homogenate, 0.5ml of distilled water and 2ml of 0.1M Tris-HCL buffer (pH 8.2). The change in absorbance was monitored for 3min at 420nm and the activity was expressed as enzyme units required to inhibit 50% pyrogallol autooxidation.¹⁷

HISTOPATHOLOGICAL EXAMINATION:

The rats were sacrificed, their brains were isolated with cold PBS solution followed by fixation with 10% formalin. The samples were then sent to pathological lab for investigation in the cross sections of hippocampus.

· STATISTICAL ANALYSIS:

The results are expressed as Mean±SEM from n=6 rats in each group. Data was analysed using statistical software Graph Pad Prism version 10. The significance of difference among the groups were assessed using One Way Analysis of Variance (ANOVA) followed by Turkey multiple comparisons test, where all the groups are compared with disease control group. Value p<0.05 was considered as statistically significant.

RESULT:

Behavioural Models for Anti-Alzheimer’s Study:

1. Elevated Plus Maze

Inflexion ratio in Elevated plus maze:

Effect of MEEO on Inflexion ratio in Elevated plus maze:

Values are expressed as Mean ±SEM (n=6), ^#P<0.05 Significant compared with Normal control, High dose (**P≤0.005) moderate significant, Standard (***P<0.0005) very Significant, low dose(*P<0.05) significant compared with Disease control, using One way ANOVA followed by Tukey's multiple comparisons test.

2. Morris Water Maze:

Escape latency time on day 11-14.

Time spent in target quadrant

Effect of MEEO on time spent in target quadrant in MWM in mice

Values are expressed as Mean±SEM (n=6), ^***P<0.001 compared with disease control, **P<0.01 compared with disease control and ^#p<0.05 compared with normal control. Data was analyzed using one- way ANOVA followed by the Tukey's multiple comparisons test.

· Biochemical Studies For Anti-Alzheimer’S Study: 3. Acetylcholinesterase Enzyme Levels

Effect of MEEO on acetylcholinesterase level in mice:

Values are expressed as Mean±SEM (n=6), (^####P<0.0001) Highly significant compared with Normal control, High dose (****P≤0.0001), Standard (**P<0.0005), low dose (***P<0.05) is Significant compared with Disease control, using One way ANOVA followed by Tukey's multiple comparisons test.

· Antioxidant Analysis:

1. Catalase activity:

Effect of MEEO on Catalase activity level in mice:

Values are expressed as Mean±SEM (n=6), (^###P<0.0001) Highly significant compared with Normal control, High dose (**P<0.005), Standard (**P<0.005), low dose (*P<0.05) is Significant compared with Disease control, using One way ANOVA followed by Tukey's multiple comparisons test.

2. SOD level:

Effect of MEEO on SOD level in mice:

Values are expressed as Mean ±SEM (n=6), (^###P<0.0001) Highly significant compared with Normal control, High dose (**P<0.005), Standard (**P<0.005), low dose (*P<0.05) is Significant compared with Disease control, using One way ANOVA followed by Tukey's multiple comparisons test.

· Histopathologic Studies:

A. Normal group

b. Disease group

C. Standard group

d. Low dose group

E. High Dose:

RESULT:

Histopathological sections if mice brain. (A) Normal Group given with water ad libitum, (B) Disease Group induced with scopolamine butylbromide (1mg/kg b.w. I.P), (C) Standard Group administered with Donepezil (2.5mg/kg b.w. P.O), (D) Low dose Group administered with MEEO at low dose of 250mg/kg b.w. P.O), (E) High dose Group administered with MEEO at high dose of 500mg/kg b.w. P.O).

DISCUSSION:

In the current study, the Anti-Alzheimer's effect of Epiphyllum oxypetalum leaves of Methanolic extract was evaluated using different behavioural models, i.e., Elevated plus maze and Morris water maze, one biochemical model to determine the Ache activity, Anti-oxidant estimations like SOD, CAT, and histopathological evaluation was carried out. Donepezil was used as a standard drug, and Scopolamine induced Alzheimer’s model in male mice.

The findings from the elevated plus maze model indicated the memory-enhancing activity of the MEEO. The Methanolic extract showed a significant protective effect in transfer latency during the retention period indicating the drug reaction to overcome memory conflicts produced by the scopolamine administration.

The findings from Morris water maze suggested to test spatial learning and memory of the scopolamine-induced group was increased compared to the control group, which means there is a lack of memory in the toxic group. It is widely used in behavioural neuroscience to study spatial learning memory. In this model, a reduction in escape latency (EL) indicated improvement of learning and memory.

One of the critical theories for AD is the cholinesterase hypothesis. Compared to a healthy person, an Alzheimer's patient has much greater levels of acetylcholinesterase. This study used Ellman's technique to measure the amount of acetylcholinesterase throughout the rat brain. Acetylcholinesterase undergoes catalyzation of choline and choline esters that functions as neurotransmitters. The results indicated that the acetylcholinesterase level was higher in the Scopolamine-induced group, confirming memory impairment compared to the control group.

As per the oxidative stress hypothesis, patients with Alzheimer's disease do face a deficit in antioxidant levels. In this experiment role, antioxidants such as super oxide dismutase (SOD) and Catalase (CAT) have been studied and determined their levels. Catalase enzyme catalyzes the decomposition of hydrogen peroxide to water and oxygen, Catalase levels were increased in treatment group. Superoxide dismutase undergoes catalyzation to form hydrogen peroxide and molecular oxygen, SOD levels increase in treatment group.

The hippocampus area of the mice brain was examined for histopathological analysis. The dentate gyrus is the part of the brain where adult neurogenesis takes place and it is also implicated in hippocampal neurogenesis. This cell death in the hippocampus dentate gyrus was significantly prevented by a pretreatment with MEEO.

CONCLUSION:

The conclusion shows present study indicates that Methanolic extract of Epiphyllum Oxypetalum Leaves may have showed the potential in becoming an alternative treatment in scopolamine induced AD model. The amount of acetylcholinesterase was found to be more in the negative control (Scopalamine2) and low dose (250mg/kg). Histopathological studies of the brain showed recovery in the cell arrangements when treated with the MEEO. The results of the present study contribute to the ability of Epiphyllum Oxypetalum Leaves extract in ameliorating the memory impairment effects of Scopolamine and can be used as a potential drug for neurodegenerative diseases like Alzheimer’s Disease. Additional studies are suggested to classify the active phytochemicals and illustrate the mode of action.

ACKNOWLEDGEMENT:

The authors of this article would like to express their gratitude to Dr. K. Ramesh, Director of the Karnataka college of Pharmacy, Bengaluru, for giving me access to use the college’s research facilities and for providing me with the materials required.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

REFERENCE:

1. R Howes MJ, J Houghton P. Ethnobotanical treatment strategies against Alzheimer's disease. Current Alzheimer Research. 2012; 9(1): 67-85.

2. Anns Morrrison, Constantine Lyketsos. A Review on the pathophysiology of Alzheimers disease and directions in treatment. Advanced Studies in Nursing, 3, 2005, 5.

3. Bischkopf J, Busse A, Angermeyer MC. Mild Cognitive impairment a review on prevalence, incidence, prevalence & mortality and outcome according to current approaches. Blackwell Munks. 2002; 106: 403-14.

4. Thanvi B., Robinson T. Sporadic cerebral amyloid angiopathy— an important cause of cerebral haemorrhage in older people. Age Ageing. 2006; 35: 565–571. 10.1093/ageing/afl108

5. Wang X., Michaelis M. L., Michaelis E. K. Functional genomics of brain aging and Alzheimers disease: focus on selective neuronal vulnerability. Curr. Genomics. 2010; 54.2174/138920210793360943.

6. NIH National Institute on Aging (NIA). NIA scientists and other experts review this content to ensure it is accurate and up to date. Content reviewed: May 16, 2017.

7. Bienias JL, Beckett LA, Bennett DA, Wilson RS, Evans DA. Design of the Chicago Health and Aging Project (CHAP). J Alzheimers Dis. 2003; 5: 349–355

8. Dandekar R, Fegade B and Bhaskar VH: GC-MS analysis of phytoconstituents in alcohol extract of Epiphyllum oxypetalum leaves, Journal of Pharmacognosy and Phytochemistry 2015; 148-54.

9. Mahmad A, Shaharun MS, Saad B and Dash GK: Epiphyllum oxypetalum Haw.: a lesser known medicinal plant. Indo American Journal of pharmaceutical Sciences 2017;

10. Narisawa T and Fukaura Y: N‐methylnitrosourea‐induced colon tumorigenesis by ursodeoxycholic acid in F344 rats. Japanese Journal of Cancer Research. 1998; 1009-13

11. Hanish Singh Jayasingh, C., Mohamed Mansor, M., Afiq, A., Yasothini, M., Punitha, C., and Pavithiraa, C. (2020). Neurocognitive effects of Prunus domestica fruit extract on scopolamine-induced amnesic mice. Journal of Applied Pharmaceutical Science. https://doi.org/10.7324/japs.2020.10118.

12. Khooshbu P, Ansari I. Evaluation of anti-Alzheimer activity of alcoholic extract of Costus pictus d. Don Leaves in Wistar albino rats. Asian J Pharm Clin Res. 2019; 13(2):36–43.

13. Shaikh RK. Kanase Evaluation of anti-alzheimer’s activity of alcoholic extract of hylocereus undatus fruits in wistar albino rats. ILPSR. 2022; 13(9): 1567-80.

14. Raviteja, S., Manandhar, S., Priya, K., Hari, G., & Ranganath Pai, K. S. (2021). Protective role of caffeic acid in cognitive dysfunction and oxidative stress induced by colchicine in rats. Indian Journal of Pharmaceutical Education, 55(2s), s457–s467. https://doi.org/10.5530/ijper.55.2s.117

15. Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol [Internet]. 1961; 7(2): 88–95. Available from: http://dx.doi.org/10.1016/0006-2952(61)90145-9

16. Ştefǎnuţ MN, Cǎta A, Pop R, Moşoarcǎ C, Zamfir AD. Anthocyanins HPLC-DAD and MS Characterization, Total Phenolics, and Antioxidant Activity of Some Berries Extracts. Anal Lett. 2011; 44(18).

17. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem [Internet]. 1974; 47(3): 469–74. Available from: http://dx.doi.org/10.1111/j.1432-1033.1974.tb03714

Received on 04.10.2024 Revised on 30.10.2024

Accepted on 12.11.2024 Published on 08.03.2025

Available online from March 12, 2025

Res.J. Pharmacology and Pharmacodynamics.2025;17(1):1-7.

DOI: 10.52711/2321-5836.2025.00001

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Dyuthi HY*, U. Rajashekhar