Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder associated with aging, characterized by progressive cognitive decline, cholinergic dysfunction, and memory impairment. Current statistics indicate that nearly 50 million people worldwide live with dementia, with this number expected to reach 152 million by 2050. Existing treatments primarily rely on acetylcholinesterase inhibitors and NMDA receptor antagonists, which offer limited efficacy.
Leucaena leucocephala (Lam.) de Wit, a fast-growing tropical leguminous tree native to Central America, is traditionally used as fodder in India but also possesses numerous therapeutic properties attributed to its diverse secondary metabolites. Previous studies have demonstrated its antifungal, anthelmintic, anticancer, antioxidant, and antidiabetic activities. This research aims to evaluate the potential anti-AD effects of the plant’s ethanolic leaf extract, focusing on phytochemical profiling, compound isolation, characterization, and assessment of nootropic activity.
Materials and Methods
Chemicals and Instruments
All chemicals and solvents were of analytical grade and used without further purification. A Soxhlet extractor facilitated leaf extraction. Thin-layer chromatography (TLC) and column chromatography were employed for separation and isolation, while IR, ^1H NMR, ^13C NMR, and mass spectrometry enabled compound characterization.
Plant Collection and Authentication
Fresh, healthy leaves of Leucaena leucocephala were collected from Moradabad, India, and authenticated with a voucher specimen (No. 3914-15-2) deposited at the NISCAIR Herbarium, New Delhi.
Extraction
The leaves were shade-dried at room temperature, pulverized, and subjected to Soxhlet extraction using ethanol. One hundred grams of powdered leaves were placed in a porous thimble, while 200 g of ethanol was added to the boiling flask. The extraction continued until the solvent became dark green. The ethanolic extract was then concentrated under reduced pressure, weighed, and stored at 5°C in amber bottles.
Phytochemical Screening
Standard qualitative tests were performed to detect major secondary metabolites, including tannins, alkaloids, flavonoids, terpenoids, steroids, carbohydrates, proteins, saponins, lipids, fixed oils, and phenolic compounds.
Isolation and Characterization
TLC was performed on silica gel 60 F254 plates, with visualization under iodine vapor. A solvent system of toluene:ethyl acetate:chloroform:glacial acetic acid (4:3:2:1) was used. The main compound (LLQ) was isolated by column chromatography using n-hexane-chloroform eluents. The isolated compound’s melting point and spectral properties were recorded using IR, ^1H NMR, ^13C NMR, and mass spectrometry.
Biological Evaluation
Experimental Animals
Male and female Wistar albino rats (140–200 g) were obtained from IFTM University’s animal facility. The animals were housed under standard conditions (24±1°C; 12/12 h light/dark cycle) with ad libitum access to food and water. Animal experiments were conducted in compliance with CPCSEA guidelines (approval No. 2021/837ac/PhD/02).
Acute Toxicity
Acute toxicity was evaluated following OECD guideline 423. Rats fasted overnight were given either ethanolic extract (2000 mg/kg) or normal saline. Animals were monitored continuously during the first hour, periodically for 24 h, and daily for 14 days for behavioral changes, signs of toxicity, or mortality.
Nootropic Activity
Novel Object Recognition Test (NORT): Rats were divided into six groups, including normal control, scopolamine (1 mg/kg) control, three extract-treated groups (100, 200, 400 mg/kg), and a donepezil-treated group (5 mg/kg). Treatments lasted 21 days. Animals underwent habituation, familiarization, and testing phases in a standard open-field apparatus with identical and novel objects. Exploration times were recorded, and the discrimination index (DI) was calculated.
Y Maze Test: The same grouping was used. Rats were placed at the center of the Y maze, and spontaneous alternation behavior was observed for 8 minutes. Arm entries were recorded, and percentage alternation (memory index) was calculated.
Results and Discussion
Extraction Yield and Physical Properties
The ethanolic extraction yielded 8.36% w/w of a dark brown, sticky extract.
Phytochemical Screening
Phytochemical tests confirmed the presence of carbohydrates, alkaloids, saponins, tannins, flavonoids, lipids, fixed oils, and phenolic compounds (see Table 1).
Table 1. Phytochemical Constituents in Ethanolic Extract
| Component | Test Used | Result |
| Carbohydrates | Fehling’s | + |
| Alkaloids | Dragendorff’s | + |
| Saponins | Foam Test | + |
| Steroids | Salkowski | – |
| Tannins | Ferric Chloride | + |
| Glycosides | Borntrager’s | – |
| Flavonoids | Shinoda | + |
| Fats/Oils | CuSO₄ | + |
| Phenols | Ferric Chloride | + |
TLC and Column Chromatography
TLC profiling revealed five spots, and compound LLQ was successfully isolated using column chromatography.
Characterization of Isolated Compound
The isolated compound (LLQ) was yellow with a melting point of 314–316°C. Spectral data matched quercetin:
- IR: characteristic peaks for C-C, C=C, C-H, C=O, C-O, and O-H.
- ^1H NMR and ^13C NMR: confirmed flavonoid structure.
- MS: [M+1]+ peak at m/z 303.02, consistent with quercetin.
Acute Toxicity
No mortality or behavioral changes were observed in rats up to 2000 mg/kg, indicating the extract’s safety.
Nootropic Activity
NORT Results: Scopolamine significantly reduced DI compared to controls (p<0.05). Extract doses of 200 and 400 mg/kg, and donepezil significantly restored DI in a dose-dependent manner.
Y Maze Results: Scopolamine-treated rats showed reduced alternation, which was significantly improved by extract (200, 400 mg/kg) and donepezil treatment, indicating reversal of memory deficits.
Conclusion
The ethanolic extract of Leucaena leucocephala leaves yielded notable amounts of bioactive phytochemicals. The isolated compound was identified as quercetin. The extract demonstrated significant nootropic activity in scopolamine-induced memory impairment models, supporting its potential as a neuroprotective agent.
Acknowledgement: The present research work is the part of PhD degree from Department of Experimental Biology. Universidad Autónoma Metropolitana-Iztapalapa San Rafael Atlixco No. 186, Col. Vicentina, Iztapalapa, 09340, Mexico. Authors want to say the thanks for the administration of University for providing the facilities.
Conflict of interest: None
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