Neurodegenerative diseases such as Alzheimer’s disease are presently the primary contributor to disability and the second most prevalent cause of death globally. Over the past three decades, there has been a notable increase in both fatalities and disabilities originating from neurological conditions, particularly in low- and middle-income nations.^1,2Alzheimer’s disease (AD) is a neurodegenerative illness marked by the deterioration and eventual death of neurons in various brain areas, leading to memory loss, cognitive decline, language difficulties, and problems with daily functioning. It affects 75% of individuals with dementia.³

Despite extensive efforts , no effective disease modifying therapy available for Alzheimer's disease.Over 20 compounds have undergone large phase 3, double-blind, randomized controlled trials with patients at various stages of AD, yet none has proven effective in slowing cognitive decline or improving overall functioning. These trial failures underscore the need for new approaches to clinical trial design for AD⁴. Existing treatments only partially alleviate symptoms and cognitive issues, making the development of an effective therapeutic agent for AD an urgent and critical challenge. Current medical research focuses on factors believed to contribute to AD, such as tau proteins and amyloid-beta deposits. Thus, the search for new, more effective drugs is very important and plants remains the paramount source of drugs. Dracaena reflexa (DR)is a tropical plant that is traditionally believed to have high medicinal value. Ancient medicinal practitioners have long held Dracaena reflexa for poisoning, dysentery, diarrhoea, and dysmenorrhea⁵. Tetracosane a phytoconstituent of Dracaena reflexa, which is identified from the GC-MS analysis also having potent inhibitory action on beta-amyloid aggregation. In vitro and in silico studies revealed that Dracaena reflexa inhibit acetylcholinesterase and butylcholinesterase enzyme that are known for hydrolysing acetylcholine in the synaptic cleft in the brain⁶.Moreover, Dracaena reflexa possess antioxidant and anti-inflammatory properties, suggesting potential benefits for neuroinflammatory conditions tied to Alzheimer’s disease ^7,8. So, This study is designed to evaluate the effect of hydroalcoholic extract of Dracaena reflexa in the management of Alzheimer’s disease using Aluminium chloride induced AD model in mice.

MATERIALS AND METHODS

Plant Collection and Authentication

Dracaena reflexa leaves were collected from Medical College Campus, Thiruvananthapuram on December 2023 and was authenticated at Department of Botany, Karyavattom Campus, University of Kerala.

Preparation of Plant Extract

The shade-dried powdered leaf was initially defatted with petroleum ether at 60–70°C. After drying, the remaining marc was extracted using the Soxhlet extraction method with an ethanol and water mixture in an 80:20 ratio, respectively, for up to 36 cycles. The hydroalcoholic extract was then concentrated under reduced pressure using a Rotary evaporator.

Preliminary Phytochemical Screening

The defatted hydroalcoholic extract of Dracaena reflexa leaves was subjected to quantitative test to identify various phytochemicals such as alkaloids, flavonoids, saponins, steroids and tannins.

Approval by IAEC

The study protocol was approved by the Institutional Animal Ethics Committee of Govt. Medical College, Thiruvananthapuram dated 30/01/2024 as per the approval No;01/01/IAEC/2024/GMCT.

Animal Selection, Maintenance, and Care

28 healthy adult male Swiss albino mice weighing 25-40g were used for the study. Animals were procured from Sree Chitra Tirunal Institute for Medical Sciences and Technology , Poojapura. Animals were housed in polypropylene cages with stainless steel lids with provisions for keeping food and drinking water ad libitum as per Committee for Control and Supervision of Experiments on Animals (CCSEA).

Experimental Design

Mice were divided into four groups of seven animals each (n =7).Alzheimer’s model was induced in mice by administering 150mg/kg of AlCl₃ orally for 28 days. Hydroethanolic extract of Dracaena reflexa leaves was administered at doses of 200mg/kg(LDDR) and 400 mg/kg (HDDR) orally. The control group received 0.1% of CMC alone. The disease group received only 150mg/kg of AlCl₃ orally. The treatment groups received plant extract suspended in 0.1% CMC along with 150mg/kg of AlCl₃ orally. The drug treatment is considered as a preventive regimen and given concurrently along with the disease induction.

Evaluation of Cognition

Novel Object Recognition Test^9-13

The novel object recognition test is a fast and efficient way to assess learning and memory in mice. It is non-invasive and spontaneous. This test is based on the fact that when animals are exposed simultaneously to familiar and novel objects, they approach frequently and spend more time exploring the novel object than the familiar one in order to satisfy their innate curiosity/exploratory instinct. The test assessment has three sessions: habituation, training, and test. Different sets were administered with corresponding drugs. The experiment started with habituation on the first day followed by training on day 2 and testing on the 3rd day. The result on day 3 was taken as response before the treatment and the procedure is again repeated on day 14 and day 28 of the treatment period.

Open Field Test ^14-16

Open field test consists of exposing an animal to an open arena, a new environment without any aversive or appetitive stimuli where the animal is left to explore freely for a fixed amount of time.

Day 1 Training: At any nook of the box the mice were placed and their behaviour was monitored for 5 min. After that, they were removed from the open field and returned to home cage.

 Day 2 Testing: After 24 hrs, the mice were again set within the open field, the duration outlaid in the central area was recorded.

Day 7 test: The same procedure on day 2 is repeated. The result on day 7 was taken as the response before the treatment and the procedure was again repeated on day 14 and day 28 of the treatment period.

Radial Arm Maze Test^{17 -18}

The Radial arm maze task is a spatial discrimination task that has been extensively used to investigate specific aspects of spatial working and reference memory. The radial arm maze consists of baited and unbaited arms. Food is the reward used to make the correct choice of entry into the arms. If the mice choose the baited arm, then it is recorded as a correct response, and the mice are considered to have retained memory.

Radial arm maze test consists of different sessions like food deprivation, handling and shaping, training, and testing sessions.  Day 0 (Food deprivation): Food deprivation was carried out by restricting each animal with 2 grams of food pellets per day in the animal department.  Day 1-3:Habituation The animals were habituated individually to the environment by placing it on the central platform and allowed to explore the RAM for 15 min/Day. Reinforcers or bait were placed in each arm. On the last day of habituation (day 3), the number of reinforcers was reduced to four arms, and the sessions ended when all eight arms had been visited.  Day 4-11:Training The animals were trained individually in one session per day for 8 days. 1 piece of feed was positioned at the culmination of arms 1,3,5 and 7 and all the other four arms remain closed. The bait was placed in effectively conceal the food from view, while the animal was permitted to freely navigate the maze.

Day 12:Test session On the day of the test, the working memory error (WME), reference memory error (RME), and reference-working memory error (RWME) were analysed

Shuttle Box Avoidance Test^19,20

Passive and active avoidance tests are behavioral tasks widely used to assess the different forms of learning and memory. Avoidance happens when a person or animal learns to predict an unpleasant event through conditioning and takes action to prevent it. Active avoidance requires performing a specific behaviour in order to escape or avoid the aversive stimulus. The procedure consists of habituation followed by a learning phase of five consecutive days. Retention trials are done on days seven and fourteen, following the learning phase.

Day 0: Habituation The mice were brought to the lab and made familiar with the apparatus by keeping them in the chamber one by one. Later they were returned to the home cage.  Day 1-5 : Learning sessions  The learning session consisted of 1 trial/day for five consecutive days. During the trial, mice exposed to conditioned stimuli (CS) after 30 seconds which is light and buzzer sound for 8 sec, followed by unconditioned stimuli (US) which is a foot shock of 0.4mA for 10 seconds. The mice kept in one compartment (right chamber) of the shuttle box and were exposed to buzzer stimuli followed by the foot shock through the grid floor. As the learning session progressed, the animal learned to escape to the unelectrified compartment (left chamber) as soon as the CS is delivered. This was recorded as an avoidance response. The stimuli were terminated when the mice escaped to the safe compartment or when 12 seconds after CS had elapsed, whichever occurred first. This learning session was continued till the end of five days. Day 7 and 14: Retention test Retention test was performed on days seven and fourteen soon after the end of the learning sessions. The retention trial consisted of the same experimental procedure repeated without giving a foot shock. Here the mice were individually placed in the right compartment and the CS of light and the buzzer sound were delivered. Jumping of the animal to the left safe compartment upon receiving the CS was recorded as an avoidance response. The learning and memory capacity of the animal was assessed based on the number of avoidance responses exhibited by the animal during 10 trial sessions on the test days.

Sacrifice of the Animal²¹

After the completion of the behavioral test, the animals were sacrificed by cervical dislocation. the skull was carefully cut open and the brain was exposed from its dorsal side. The whole brain was quickly excised and transferred to a petridish containing ice cold normal saline. The isolated brain was adopted for further  works

Biochemical investigation of Brain homogenate

Brain Homogenate Preparation

The brain tissue was first washed with ice-cold isotonic saline. It was then homogenized in a 0.1M Phosphate Buffer (pH-7.4) at a ratio of 10 parts buffer to 1 part tissue by weight. After homogenization, the mixture was centrifuged at 10,000 rpm for 15 minutes. The resulting supernatant was separated and used in aliquots for further biochemical analysis.

Estimation of Acetylcholine Esterase Activity^22,23

An aliquot of about 0.4ml of brain homogenate was added to a cuvette containing 2.6 ml phosphate buffer (0.1 M pH 8) and 100µl of 0.01M DTNB. This mixture was thoroughly mixed with bubbling air and absorbance is measured at 412 nm in the spectrometer. Absorbance is recorded as the basal reading when it attains a stable value. 20 µl of Acetylthiocholine (substate) was added and the ?A was recorded. Changes in the absorbance per minute were then determined.

Estimation of Malondialdehyde (MDA) Levels^24-27

MDA is commonly used as a marker for evaluating lipid peroxidation and is typically assessed using the Ohkawa method. To the tissue homogenate samples (less than 0.2 mL), add .2 mL of 8.1% SDS, 1.5 mL 20?etic acid solution (pH adjusted to 3.5 with NaOH), and 1.5 ml 0.8% aqueous TBA solution. Raise the total bulk to 4.0 mL with distilled water, then heat the mixture at 95°C for 60 minutes. After cooling with tap water, add 1.0 mL of distilled H2O & 5 of ml15:1 (v/v) mixture of n-butanol and pyridine, and shake vigorously. Following centrifugation at 4000 rpm for 10 minutes, the organic layer was collected and its absorbance at 532 nm was recorded against a blank. Tetra methoxy propane (TMP) served as an external standard, and the lipid-peroxide levels presented as nanomoles (nmol) of malondialdehyde (MDA).

Estimation of Catalase (CAT) Levels ²⁸

Catalase activity is assessed using UV spectroscopy. In the UV spectrum, hydrogen peroxide (H?O?) exhibits increasing absorption as the wavelength decreases. Catalase levels in the brain homogenate were measured as follows: 0.2 ml of tissue homogenate was mixed with 1.2 ml phos- buffer (0.05 M, pH 7.0), and the reaction was initiated by adding 1.0 ml of hydrogen peroxide (0.03 M). The absorbance decrease at 240 nm was recorded over 3 minutes, with an enzyme blank run simultaneously using 1.0 ml of distilled water instead of hydrogen peroxide. Catalase activity was calculated as micromoles of H₂O₂ degraded / min/milligram of protein.

Estimation of Glutathione Level ^29-32

The GSH level in the hippocampal and cortical regions of the brain was ascertained by Ellman method. The deproteination of brain homogenate was performed by mixing it with an equal volume of 10% TCA and centrifugated at 2000 rpm for 10 min. The supernatant was separated and used for GSH estimation. The supernatant was joined with 2 ml of phosphate buffer, 0.5 ml DNTB, and 0.4 ml of water. The mixture was shaken thoroughly and vortexed. The absorbance was taken at 412 nm within 15 min against the blank.

Estimation of Serotonin Level ^33-34

The serotonin level was determined by Schulmpf method, The serotonin combined with o-phthaldiadehyde results in the development of fluorophore and is measured spectrofluorimetrically.The brain was isolated and the brain tissue was weighed and homogenized in 5ml HCI -Butanol for 1 min. The sample was centrifuged for 10 mins at 2000 rpm. The supernatant was collected and mixed with a centrifuge tube containing 2.5 ml of heptane and 0.31 ml of 0.1 M HCI. After 10 min of shaking and centrifuging under the same condition the two phases separated and the aqueous phase (0.2ml) was used for serotonin estimation.To the Aliquot(0.2ml) of aqueous extract, 0.25ml of OPT reagent was added. The mixture was heated for 10 min at 1000 c for the development of fluorophore. Then the sample was allowed to equilibrium with ambient temperature and absorbance was measured at 360-470 nm in the spectrofluorometer. A concentrated HCI of about 0.25 ml without OPT was taken as tissue blank. Serotonin (500µg/ml)was used as an Internal standard and was prepared by dissolving in distilled water and HCI-Butanol mixture in a ratio of 1:2.

Histopathological Evaluation of Brain ^35-36

Histopathological analysis was conducted on the brains of 2-3 mice randomly chosen from each group. The brains were promptly fixed in 10% phosphate-buffered formaldehyde, embedded in paraffin, and 5 µm longitudinal sections were made. These sections were stained with hematoxylin and eosin (H&E) and Congo Red stain examined under a microscope at 200 x magnification.

Statistical analysis

Results are expressed as Mean ± SEM

Results

Percentage yield

% yield value of hydroethanolic extract of Dracaena reflexa leaves found to be 19%

Preliminary phytochemical screening

Investigation revealed the presence of Carbohydrate, Alkaloids, Phenols, Flavonoid, Tannins, Saponins and Terpenoids

Evaluation of Cognition by Novel Object Test

On day 0 all groups spent almost similar time near the novel object as shown in the  Fig.no.1. All groups of animals except the disease group had spent more time with novel object on day 14 and day 28. The disease group showed comparatively lesser exploratory behaviour when compared to control group. Thus, the increased exploration time near novel object in drug treated groups shows the potential of the drug to prevent the cognitive impairment by AlCl_3.

Evaluation of Cognition by Open Field Habituation test

On day 0 all groups spend almost similar time in the central area of the open field apparatus. As shown in Fig.no.2.All groups of animals except the disease group had spent more time in the central area of the open field apparatus on day 14 and day 28. The disease group showed comparatively lesser exploratory behaviour when compared to the control group. The group which received high dose and low dose of hydroalcoholic extract of Dracaena reflexa showed a significant increase in time spent in the central area.

Evaluation of Cognition by Shuttle Box Avoidance Test

On day 0, all groups show almost similar no of avoidance responses in the shuttle box. On day 14, the disease group showed a statistically significant decrease in avoidance response compared to the avoidance responses of the control group on the same day as shown in Fig.no.3.The group that received high dose and low dose of hydroalcoholic extract of Dracaena reflexa showed a significant increase in  no of avoidance response compared to the disease group on the same day

On day 28, the avoidance responses of the control group were found to be increased However, the disease group had a decrease in avoidance responses when compared to the control group. Similarly, low and high doses hydroalcoholic extract of Dracaena reflexa treated groups showed a statistically significant  increase in no of avoidance responses compared to the disease group.

Evaluation of Cognition by Radial arm maze test

From Fig.no.4a, On 14^th day, working memory error of mice in the disease group is significantly increased compared with that of control group whereas the working memory error of the animals in the low dose and high dose group is significantly decreased compared with that of disease group.

Similarly, by 28^th day, working memory error of mice in the disease group is significantly increased  compared with that of control group whereas the working memory error of the animals in the low dose and high dose group is significantly decreased compared with that of disease group indicating retaining of memory.

From Fig.no.4b by 14^th day, reference memory error of the animals in the low dose and high dose group is significantly decreased compared with that of disease group.

Similarly, by 28^th day, reference memory error of mice in the disease group is significantly increased compared with that of control group whereas the reference memory error of the animals in the low dose and high dose group is significantly decreased compared with that of disease group indicating retaining of memory.

Biochemical Estimation of Brain Homogenate

Evaluation of brain Acetylcholinesterase in Albino mice

The Aluminum chloride treatment showed an increased level of AChE that was significantly higher compared to the control . However the level is lowered for hydroalcoholic extract of Dracaena reflexa low dose and high dose treated groups compared to the control group.

From Fig.no.5, the brain AChE level was substantially elevated in the AlCl_3-treated group compared to the control group  indicating AlCl₃ induced learning and memory impairment. The high dose and low dose treatments considerably decreased AChE levels compared to the AlCl₃ treated group, which was statistically significant.

Estimation of Serotonin

From Fig.no.6, the brain serotonin level was significantly reduced in AlCl₃ treated group compared to the control group  and indicating AlCl₃ induced learning and memory impairment. The high dose and low dose treatment considerably increased serotonin levels compared to the AlCl₃ treated group, which was statistically significant.

Estimation of Catalase Activity

From Fig.no.7, the brain catalase level was significantly reduced in AlCl₃ treated group compared to the control group and indicating AlCl₃ induced learning and memory impairment. The high dose and low dose treatment considerably increased catalase levels compared to the AlCl₃ treated group, which was statistically significant.

Estimation of Glutathione level

From Fig.no.8, the brain glutathione level was significantly reduced in AlCl₃ treated group compared to the control group and indicating AlCl₃ induced learning and memory impairment. The high dose and low dose treatment considerably increased glutathione levels compared to the AlCl₃ treated group, which was statistically significant.

Estimation of Malondialdehyde level.

Fig.no.9, the brain MDA  level was significantly increased in AlCl₃ treated group compared to the control group and indicating AlCl₃ induced learning and memory impairment. The high dose and low dose treatment considerably decreased MDA levels compared to the AlCl₃ treated group, which was statistically significant.

Histopathological Analysis of Brain

Histopathological Examination of Mice Brain using H and E Staining

1. Control
2. AlCl₃ 150mg/kg
3. AlCl₃ 150mg/kg + 200mg/kg of hydroalcoholic extract of Dracaena reflexa
4. AlCl₃ 150mg/kg +400mg/kg of hydroalcoholic extract of Dracaena reflexa

Fig 5.10 (A,B,C,D) represents the brain sections of Swiss albino mice after different types of treatment for 4 weeks. Compared to the normal group, there is presence of amyloid plaques in disease group. At the same time, these plaques decreased in low dose and high dose treated groups, thus indicating the capacity of hydroalcoholic extract of Dracaena reflexa to inhibit the formation of amyloid plaque in the brain of albino mice.

Histopathological Examination of Mice Brain using Congo Red  Staining