Caralluma dalzielii as a Potential Therapy for Alzheimer’s Disease: Insights from a Transgenic Drosophila melanogaster Model

Caralluma dalzielii as a Potential Therapy for Alzheimer’s Disease: Insights from a Transgenic Drosophila melanogaster Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Caralluma dalzielii as a Potential Therapy for Alzheimer’s Disease: Insights from a Transgenic Drosophila melanogaster Model Chinenye Jane Ugwah-Oguejiofor, Evelyn Hassan, Dhaakirah Yakubu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6128472/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigated the neuroprotective potential of the methanol extract of Caralluma dalzielii (CDME) against Alzheimer’s disease (AD) using transgenic Drosophila melanogaster as a model organism. Fresh plant materials were collected, authenticated, shade-dried, and subjected to maceration extraction with 70% methanol. Phytochemical screening and High-Performance Liquid Chromatography (HPLC) analysis identified secondary metabolites in the extract. Transgenic AD model flies expressing human Aβ-42 peptide were generated and exposed to dietary supplementation with CDME at 1 mg, 10 mg, and 20 mg/10 g diet for 14 days as the case may be. Behavioral assays, including negative geotaxis, grooming, and aversive phototaxis suppression (APS), were conducted to assess locomotion, anxiety-like behavior, and memory performance. Neurobiochemical assays evaluated glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), acetylcholinesterase (AChE), and triacylglycerol (TAG) levels in fly heads. Results showed significant improvements in survival rate, locomotor activity, and learning performance at both CDME doses compared to the control (p<0.05). CDME administration led to dose-dependent increases in GSH and SOD levels, reductions in MDA and AChE activity, and enhanced TAG levels. Molecular docking analysis revealed strong binding affinities of CDME-derived compounds to glycogen synthase kinase 3 (GSK3) and β-secretase (BACE 1), key enzymes in AD pathogenesis, with several compounds outperforming the reference drug donepezil. These findings suggest that CDME possesses neuroprotective properties, potentially mitigating AD symptoms through antioxidative and cholinergic mechanisms. Further studies are recommended to isolate active compounds and explore their therapeutic potential. This study highlights the promise of Caralluma dalzielii as a natural candidate for AD management. Caralluma dalzielii Neuroprotection Alzheimer’s disease Drosophila melanogaster Phytochemicals Molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Introduction Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, memory loss, and synaptic dysfunction and represents one of the most pressing global health challenges of the 21st century (Botella Lucena and Heneka, 2024). It is the most common cause of dementia worldwide, affecting millions of individuals and placing a significant burden on healthcare systems. Pathologically, AD is associated with the accumulation of amyloid-beta (Aβ) plaques, tau protein hyperphosphorylation, oxidative stress, and neuroinflammation (Ekundayo et al., 2024). Despite extensive research, there is currently no cure for AD, and available treatments only provide symptomatic relief without addressing the underlying disease mechanisms. With over 150 million projected cases worldwide by 2050, the urgency to identify effective therapeutic interventions has never been greater (Alves et al., 2025). Current treatments, such as acetylcholinesterase inhibitors and NMDA receptor antagonists, offer only symptomatic relief and are often associated with significant side effects (Chin et al., 2022). This therapeutic gap has spurred interest in natural products, particularly medicinal plants with ethnopharmacological relevance, as potential sources of novel neuroprotective agents. Caralluma dalzielii N. E. Brown (Asclepiadaceae), a shrub indigenous to sub-Saharan Africa, has long been used in traditional medicine to treat neurological conditions, including epilepsy and pain (Shehu et al., 2019; Ugwah-Oguejiofor et al., 2024). Recent preclinical studies have validated its neurobehavioral properties, demonstrating anxiolytic, anti-hyperactivity, and memory-enhancing effects in murine models (Ugwah-Oguejiofor et al., 2024). These effects are hypothesized to arise from GABAergic and glutamatergic modulation, coupled with antioxidant activity—a critical feature given the role of oxidative stress in AD pathogenesis. Gas chromatography-mass spectrometry (GC-MS) analyses of C. dalzielii extracts have identified bioactive compounds such as 3,5-Dimethylpyrazole and Acetophenone, which may underlie its neuropharmacological potential (Ugwah-Oguejiofor et al., 2024). However, its efficacy against AD-specific pathologies remains unexplored. The transgenic Drosophila melanogaster model has emerged as a powerful tool for studying AD mechanisms and screening therapeutic candidates. Drosophila shares approximately 75% of its genes with humans, allowing researchers to study human disease mechanisms effectively within a simpler organism (Kasanin et al., 2022). By expressing human amyloid-beta (Aβ42) and tau proteins, these flies recapitulate key AD hallmarks, including amyloid aggregation, synaptic dysfunction, and memory deficits within days—a temporal advantage over vertebrate models (Bongiorni et al., 2024). The transgenic Drosophila model provides a cost-effective, genetically tractable system with a short lifespan, enabling rapid screening of potential therapeutics (Tsintzas and Niccoli 2024). Additionally, natural products can be easily administered through feeding, and behavioral assays, such as memory and locomotor tests, provide measurable outcomes. This study seeks to bridge traditional medicine and modern neurogenetics by investigating C. dalzielii ’s therapeutic potential in a transgenic Drosophila AD model. We hypothesize that its methanol extract will mitigate Aβ42-induced neurodegeneration through dual mechanisms: (1) enhancing cholinergic signaling via acetylcholinesterase inhibition, and (2) reducing oxidative stress through free radical scavenging. By correlating behavioral outcomes (memory, locomotion) with molecular markers (amyloid load, tau phosphorylation), this work aims to validate ethnopharmacological claims while identifying bioactive fractions for future drug development. Materials and methods Plant collection and extraction Fresh C. dalzielii (CD) batches were collected from Sokoto in August 2024. Identification and authentication were done in the Laboratory of Pharmacognosy and Ethnopharmacy, UDUS where the voucher specimen (Pcg/UDUS/Asdy/001) was stored at the herbarium unit. The CD was shade-dried at room temperature for a month, after which it was pounded with mortar and pestle. Three hundred and nineteen grams (319 g) of the pounded plant material was extracted using the maceration method with 70% Methanol for 72 hrs. The filtrate was concentrated over a rotary evaporator at 45 o C while the remaining moisture was evaporated completely over a water bath set at 45 o C to yield dry extract (CCDE). Phytochemical screening of the extract Phytochemical screening of the extract was performed in order to determine the presence of secondary metabolites using standard phytochemical methods as described by Evans (2002). HPLC analysis The HPLC analysis was carried out using an Agilent 1100 system, which features dual binary pumps, a column oven, and a UV/Vis detector. The method employed a C-12 normal phase column (Phenomenex, Gemini 5 µm) with dimensions of 200 mm in length and 4.8 mm in internal diameter. The mobile phase consisted of acetic acid-acidified deionized water (pH 2.8) as solvent A and acetonitrile as solvent B, with a flow rate set at 0.8 mL/min. To maintain optimal conditions, the column was equilibrated with 5% solvent B for 20 minutes after each sample injection. The temperature was kept at 38°C, and a sample volume of 20 µL was injected. Detection of phytochemicals occurred at a wavelength of 280 nm. The identification and quantification of these compounds were performed by comparing their retention times and peak areas to those of pure standard compounds, utilizing an external standard approach to develop a calibration curve. The gradient elution procedure was structured as follows: 0–5 minutes: Increase from 5–9% solvent B 5–15 minutes: Hold at 9% solvent B 15–22 minutes: Increase from 9–11% solvent B 22–38 minutes: Increase from 11–18% solvent B 38–43 minutes: Increase from 18–23% solvent B 43–44 minutes: Rapid increase to 90% solvent B 44–45 minutes: Decrease from 90–80% solvent B 45–55 minutes: Continue at this rate This systematic methodology facilitated the effective separation and analysis of the phytochemical components present in the samples (Kaisoon et al., 2011). Fruit fly (Drosophila melanogaster) culturing Wild type fruit fly (Oregon strain) stock culture was obtained from Drosophila Laboratory, at CAMRET, UDUS. The flies were maintained and reared on normal diet made up of cornmeal medium containing 1%w/v brewer’s yeast and corn meal at room temperature under 12 h dark/light cycle conditions in the Drosophila Research Laboratory. Generation of Transgenic flies for the study Transgenic fly lines that express wild-type human Aβ-42 peptide (UAS-Aβ42) and a pan-neuronal elav-Gal4 driver were obtained from CAMRET. The AD model flies were obtained by crossing Gal4 virgin females to Aβ-42 males (Wei-Wei et al., 2017). In the F1 progeny, AD flies were UAS-Aβ42::ElavGAL4, and they could be distinguished from healthy siblings based on their phenotypic characteristics (Bongiorni et al., 2024). These F1 progeny flies (AD flies) were used for the study. Behavioral Assay Flies were raised on a standard Drosophila cornmeal agar diet at 25 o C and 42% relative humidity. The 1–2-day-old AD flies were grouped into five each fly containing 20 flies. Group I was the control group, Group II received donezepil 0.05 mmol while Groups III and IV received two doses of CDME, 1mg/10g diet and 10 mg/10g diet for 14 days. Negative geotaxis To evaluate the locomotor activity of each experimental group, 20 male flies were selected using a pooter and placed in individual empty 20 mL measuring cylinder. The flies were adjusted to room temperature for 10 min for adaptation (Bonilla-Ramirez et al., 2011). To measure the climbing ability, the flies were gently tapped down to the bottom of the test vial. A mark was made at 8 cm from the bottom of each cylinder. The cylinder was gently tapped, causing all flies to fall to the bottom, and the number of flies that crossed the 8 cm mark within 6 seconds was counted. This procedure was performed three times to determine the average pass rate per group with 2 min resting time. The data collected was then analyzed based on the average of the three replicates (n = 60) (Yankuzo et al., 2024). Grooming To assess anxiety-like behaviors, a grooming test was conducted, with slight modifications to the procedure described by Kaur et al. (2015). Each experimental group consisted of 15 flies, and each group was tested in triplicate. Throughout a 2-min observation period, the grooming behaviors of individual flies were carefully monitored and recorded by observers unaware of the treatment assignments. The experiment was performed twice for each group. Statistical analysis was used to compare the average grooming time among the groups. Memory assessment test To assess memory, an aversive phototaxis suppression (APS) assay was employed, with modifications to the protocol described by Poetini et al. (2022). The assay leverages the flies' natural inclination to move towards a light source. Initial testing in a T-maze was performed to confirm the flies' responsiveness to light, a critical component of the assay. Ten flies were placed in the dark side of the T-maze, and the first five to cross to the lit side within 10 s were selected for the following steps. For the training phase (PC0), filter paper dampened with chloroquine was placed on the lit side of the T-maze. After allowing the flies to acclimate in the dark, they were permitted to cross to the lit side, associating the light with the aversive bitter taste of chloroquine. This training was repeated five times to establish baseline results. Three hours later (PC3), short-term memory was evaluated by repeating the procedure, but without the chloroquine filter paper. The flies were then observed to see if they entered the lit side or remained in the dark. Choosing to remain in the dark was recorded as a "pass," indicating that they remembered the association of light with the bitter taste, while entering the lit side was recorded as a "fail." Fifteen flies per group, across three replicates, were tested in this manner, and the percentage of flies that passed was recorded by observers blinded to the treatment groups. Biochemical assays The 1–2-day-old AD flies were grouped into five, each containing 20 flies. Group 1 was the control group, Group II received donezepil 0.05mmol while Groups III to V received doses of CDME, 1mg/10g diet, 10 mg/10g diet and 20 mg/10g, respectively, for 14 days. Fly head extraction Following cold anesthesia, fly heads from each experimental group were carefully isolated (20 heads per group; three replicates per group) and placed into 1.5 mL Eppendorf tubes containing 0.1 M phosphate-buffered saline (PBS). Subsequently, the tissue samples were homogenized using a handheld homogenizer (Hangzhou Miou Instrument Co., Ltd., Hangzhou, China) to ensure thorough disruption of cellular structures. The resulting homogenate was then centrifuged at 2500 g for 10 minutes at 4°C using a high-speed refrigerated microcentrifuge. After centrifugation, the supernatant was gently aspirated and transferred into sterile 1.5 mL tubes for storage at − 20°C pending further biochemical analyses. Glutathione (GSH) content determination GSH levels were determined using a spectrophotometric approach reported earlier (Patterson and Lazarow, 1955). Accordingly, GSH levels were measured by reacting samples with alloxan, phosphate buffer, sodium hydroxide, and metaphosphoric acid, with absorbance read at 340 nm to quantify GSH content, expressed as mg/dL. Superoxide Dismutase (SOD) Activity SOD activity was evaluated using a modified version of the pyrogallol auto-oxidation assay, originally developed by Marklund and Marklund (1974). This method involves measuring the inhibition of pyrogallol oxidation in a reaction mixture containing Tris-HCl buffer and sample extracts. The reaction is initiated by adding pyrogallol, after which changes in absorbance are monitored over 3 min at 420 nm. SOD activity is quantified as the amount of protein required to inhibit 50% of pyrogallol auto-oxidation under these conditions. Malondialdehyde (MDA) Levels MDA, a key indicator of lipid peroxidation, was quantified using an adapted version of the thiobarbituric acid reactive substances (TBARS) assay, originally developed by Ohkawa et al. (1979). This method involves mixing trichloroacetic acid with the sample, followed by the addition of thiobarbituric acid. The mixture is then heated in a water bath to facilitate the reaction between MDA and thiobarbituric acid. After cooling and centrifugation, the resulting TBARS, with a characteristic red-pink color, were measured spectrophotometrically at 532 nm. The results were expressed as nmol/L of TBARS. Acetylcholinesterase (AChE) Activity AchE levels were quantified using a competitive ELISA kit according to the manufacturer’s protocol (PARS BIOCHEM™ Nanjing, China). The absorbance values were measured at 450 nm and compared to a standard curve to determine AChE concentrations in the samples. Triacylglycerol Quantification Triacylglycerol levels were measured by adding a triglyceride working reagent to the samples, a triglyceride standard, and distilled water. The absorbance was measured at 500 nm, and the results were expressed as mg/dL of triglyceride. In silico studies Protein Preparation The crystal structures of glycogen synthase kinase 3 (GSK3) and human β-Secretase (BACE 1), identified by PDB IDs 1Q5K and 4D8C respectively, were prepared following established protocols (Ugwah-Oguejiofor et al., 2025). The protein structures were retrieved from the Protein Data Bank and processed using the Glide software within the Schrodinger Suite (2020–3), specifically utilizing the Protein Preparation Wizard. This process involved the addition of hydrogen atoms, assignment of bond orders, formation of disulfide bonds, and reconstruction of any missing side chains and loops through the Prime module. Solvent molecules located more than 3.0 Å from heteroatoms were removed to streamline the structure, which was then minimized and optimized using the OPLS3e force field. Generation of Receptor Grid The receptor grid was created to define the spatial coordinates and dimensions of the enzyme's active site, essential for ligand docking. This was accomplished using the receptor grid generation tool in Schrodinger Maestro version 12.5, with the active site based on co-crystallized ligands serving as a reference for constructing the scoring grid (Apeh et al., 2023). Preparation of Ligands A total of twenty phytochemical compounds obtained from High-Performance Liquid Chromatography (HPLC) of CDME, along with the standard ligand donepezil, were prepared using the LigPrep tool in Maestro 12.5, Schrodinger Suite (2020–3). The preparation yielded three-dimensional structures at minimal energy with accurate chiral configurations. Each ligand's ionization state was adjusted to reflect a physiological pH of 7.2 ± 0.2. Stereoisomers were generated by stabilizing specific chiral centers while allowing variability in others (Ugwah-Oguejiofor et al., 2023). Protein-Ligand Docking Molecular docking was performed using the Schrodinger Suite (2020–3) through the Glide-Ligand Docking tool incorporated in Maestro 12.5. The prepared ligands and receptor grid file were integrated into Maestro's workspace for docking into the protein's binding site utilizing Glide's standard precision mode. During this process, adjustments were made to the van der Waals radius scaling factor for ligand atoms to 0.80, a partial charge threshold was set at 0.15, and flexible ligand sampling techniques were employed to enhance docking accuracy (Johnson et al., 2022). Data analysis The data were recorded in mean ± SD. One-way Analysis of variance (ANOVA) was used where necessary and Tukey post hoc was used to compare significance across groups. A value of P ≤ 0.05 was considered statistically significant. GraphPad Prism v8.0 (GraphPad Software Inc., San Diego, California) was used for the analyses. Results Percentage Yield The percentage yield of the methanol extract of CDME was 10.7%. Phytochemical analysis The phytochemical analysis showed the presence of carbohydrates, saponin, flavonoids and other bioactive compounds although anthroquinones were not detected (Table 1 ). Table 1 Phytochemical Screening of CDME S/N PHYTOCHEMICAL RESULT 1 Carbohydrates · Molich’s test + · Fehling’s test (Red. Sugars) + 2 Saponins · Froth’s test + 3 Phenols · Ferric chloride’s test 4 Flavonoids · Ferric chloride’s test + · Alkaline Test + · Shinoda’s Test + 5 Tannins · Ferric chloride’s test + · Lead acetate test + 6 Alkaloids · Mayer’s test + · Hager’s Test + · Wagner’s Test + · Dragendroff’s Test + 7 Anthraquinones · Bontrager’s Test - 8 Diterpenoids · Copper Acetate’s Test + 9 Triterpenoids/Steroids · Salkowki’s Test + · Libermann-Burchard’s Test + 10 Cardiac glycosides · Killer-killiani’s Test + 11 Proteins · Xanthoproteic Test + + present; - not detected HPLC for CDME Twenty-one chromatographic peaks were identified in the HPLC analysis of the extract (Fig. 1 ). The identified compounds, along with their peak areas, heights, and concentrations, are presented in Table 2 . Table 2 Compounds of CDME identified through HPLC Compounds Retention time (min) Peak area Peak height Conc (ug/ml) Kaempferol 0.14 4083.6304 389.518 4.7443 Steroid 3.473 4093.1305 320.77 2.8339 catechin 4.613 6355.7564 494.097 7.888 Cyanogenic glycoside 9.48 4859.8114 380.588 3.4056 Narigenin 10.866 4680.9656 366.718 6.0167 Dihydrocytisine 13.126 6488.9442 507.931 8.3477 Dihydrocytisine 14.886 5010.2373 392.627 6.4454 Quercertin 16.956 3363.6766 263539 0.2195 Aphyllidine 19.44 4580.4334 359.146 3.1075 Ammodendrine 22.883 8235.6251 642.946 4.0347 Tannin 25.713 7935.2493 618.966 4.3193 Flavonones 28.606 2956.965 231.988 5.072 cardiac glycoside 30.28 5551.2211 435394 6.8916 Spartein 32.73 4434.1058 347.282 5.5031 Flavone 34.633 4104.5175 321.457 5.0956 Ribalinidine 36896 7474.5404 583.338 9.2765 Phytate 38.526 4454.0796 349.461 5.5279 Phytate 39.326 8756.4876 683.81 10.8675 Oxalate 40.326 6201.0704 486.517 2.3389 Epihedrine 40.953 3567.976 280.808 4.4295 Sapogenin 41.816 5879.1171 494.615 7.2987 Effect of CDME on survival rate and behavioural assessment in AD flies After completing the 14-day treatment, there was a significant ( p < 0.01; < 0.001) improvement in fly survival when CDME was added to their diet at concentrations of 1 and 10 mg/10g diet, compared to the control group (Fig. 2 ). The behavioural assessment was carried out using negative geotaxis, grooming and learning and memory assessment. The locomotor behaviour was assessed using the negative geotaxis assay. CDME at 10 mg/ 10 g diet significantly (p < 0.05) increased the average climbing rate compared to the control (Fig. 3 A). Similar to the reference drug, at the treated doses, CDME caused reduced grooming frequencies (p < 0.01) compared to the control (Fig. 3 B). The APS was utilized to evaluate both learning and short-term memory. Learning performance was assessed using the PC0 parameter, while short-term memory was evaluated through the PC3 parameter. The results showed that CDME administered at two different doses significantly enhanced learning capabilities (Fig. 3 C; p < 0.05). However, no significant improvement in short-term memory was observed (Fig. 3 D). Effect of CDME on neurobiochemical analysis in AD flies Treatment of the AD flies with CDME caused a significant (p < 0.001) elevation of GSH levels in the flies with a 10 mg/10 g diet dose higher than all the treated doses (Fig. 4 A). There was a dose-dependent increase in the SOD levels at all doses with the highest dose, 20 mg/10 g diet, slightly higher but comparable to the standard drug (Fig. 4 B). Figure 4 C shows the level of MDA activity in the treated AD flies with 10 mg/10 g diet significantly lower (p < 0.001) than the control. The level was also lower than that of the standard drug. In Fig. 4 D, it was observed that there was a dose-dependent decrease in the AChE activity in all the treated groups. However, the activity of the standard drug was much lower than that in all CDME groups. TAG levels in the 1 mg and 10 mg/10 g diet were significantly higher (p < 0.001) than in the control. They were also found to be higher in concentration than both the standard drug and the highest dose level groups (Fig. 4 E). Molecular docking The molecular docking analysis of compounds from CDME against GSK3 and BACE 1 showed varying compounds with docking scores ranging from − 7.856 to -3.040 kCal/mol and − 6.742 to -2.541 kCal/mol respectively (Table 3 ). The docking results against 1Q5K showed that the first nine compounds had higher binding affinities than the reference compound, while in 4D8C, the first ten compounds outperformed the reference compound. The 2D and 3D views of the top five compounds and the standard compound against 1Q5K and 4D8C are presented in Figs. 5 – 16 . Their bond interactions are presented in Tables 4 and 5 respectively. Table 3 Docking scores of compounds against target proteins 1Q5K and 4D8C Compounds PubChem CID Docking scores (kCal/mol) Compounds PubChem CID Docking scores (kCal/mol) 1Q5K 4D8C Catechin 9064 -7.856 Catechin 9064 -6.742 Flavone 10680 -7.690 Aphyllidine 12306738 -6.682 Narigenin 439246 -7.658 Ephedrine 9294 -6.358 Flavonones 14259001 -7.589 Quercetin 5280343 -6.286 Ribalinidine 336322 -7.527 Narigenin 439246 -6.048 Quercetin 5280863 -7.304 Ammodendrine 442625 -5.920 Dihydrocytisine 91747228 -6.917 Kaempferol 5280863 -5.887 Ephedrine 9294 -6.901 Spartein 644020 -5.851 Ammodendrine 442625 -6.613 Flavonones 14259001 -5.719 Aphyllidine 12306738 -6.263 Steroid 131751786 -5.436 Kaempferol 5280863 -6.165 Dihydrocytisine 91747228 -5.354 Sapogenin 198016 -5.679 Flavone 10680 -4.610 Cyanogenic glycoside 131751786 -5.598 Ribalinidine 336322 -4.462 Phytate 890 -5.448 Phytate 890 -3.270 Spartein 644020 -5.070 Oxalate 71081 -2.767 cardiac glycoside 439501 -3.740 Cyanogenic glycoside 131751786 -2.541 Steroid 139082353 -3.517 Tannin 16165470 - Oxalate 71081 -3.040 Sapogenin 198016 - Tannin 16165470 - Cardiac glycoside 439501 - Donepezil hydrochloride 5741 -6.344 Donepezil hydrochloride 5741 -5.334 Table 4 Interactions of the top five compounds of Caralluma dalzielii and standard compound against the target protein of glycogen synthase kinase 3 PubChem CID Name of compound Interactions H-bond Other bonds 9064 Catechin ASP 133, VAL 135, ASP 200 ILE 62, VAL 70, GLY 63, GLY 65, LYS 85, ALA 83, CYS 199, LYS 183, ASN 186, LEU 188, VAL 110, LEU 132, TYR 134 10680 Flavone VAL 135 VAL 70, ILE 62, ARG 141, THR 138, GLU 137, PRO 136, TYR 134, ASP 133, LEU 132, CYS 199, VAL 110, LEU 188, ALA 83, LYS 85, ASP 200, GLU 97 439246 Narigenin VAL 135, LYS 85, ASP 200 LYS 183, GLN 185, ASN 186, ILE 62, LEU 188, THR 138, TYR 134, ASP 133, LEU 132, VAL 110, VAL 70, ALA 83, CYS 199 14259001 Flavonones ASN 186, VAL 135 GLY 63, ILE 62, ARG 141, THR 138, GLY 137, PRO 136, TYR 134, ASP 133, LEU 132, VAL 110, ALA 83, LYS 85, VAL 70, CYS 199, ASP 200, LEU 188, GLN 185, LYS 183 336322 Ribalinidine VAL 135, ASN 186 THR 138, TYR 134, ASP 133, LEU 132, ALA 83, LYS 85, VAL 110, ILE 62, GLY 63, GLY 65, CYS 199, ASP 200, VAL 70, GLY 183, GLN 185, LEU 188 5741 Donepezil hydrochloride LYS 85 ASN 186, LEU 188, LEU 132, TYR 134, VAL 135, PRO 136, VAL 70, GLU 137, THR 138, GLN 72, ARG 141, GLU 97, VAL 110, ALA 83, PHE 201, GLY 65, ASP 200, CYS 199, ILE 62, VAL 61, LYS 60 Table 5 Interactions of the top five compounds of Caralluma dalzielii and standard compound against the target protein of human β-Secretase PubChem CID Name of compound Interactions H-bond Other bonds 9064 Catechin LYS 107, GLN 73, GLY 11, ASP 32 TRP 115, ILE 118, GLY 74, THR 72, TYR 71, LEU 30, GLY 34, SER 35, GLY 74, PHE 108, ILE 110, THR 221, GLY 219, ASP 217 12306738 Aphyllidine GLN 73 LYS 107, PHE 108, TRP 115, LEU 30, ILE 118, TYR 71, THR 72, GLY 74, ARG 224, THR 221, THR 220, GLY 219 9294 Ephedrine ASP 217, THR 220 PHE 108, LEU 30, ASP 32, GLY 34, SER 35, TRP 115, ILE 118, ILE 215, GLY 219, ASP 217, GLY 219, VAL 321, TYR 71, THR 72, GLN 73, GLY 74 5280343 Quercertin THR 220, TYR 187, ARG 128, ILE 126 LEU 30, ASP 32, GLY 34, SER 35, SER 36, ILE 118, GLY 219, ASP 217, ILE 215, ARG 224, VAL 321, THR 72, TYR 71 PRO 70, TYR 187, ALA 127 439246 Narigenin ASP 217, PHE 108 GLN 73, THR 72, TYR 71, VAL 321, ARG 224, ILE 215, GLY 219, THR 220, GLY 34, ASP 32, LEU 30, ILE 118, TRP 115, ILE 110, PHE 109, LYS 107 5741 Donepezil hydrochloride - ILE 110, TRP 115, PHE 108, GLN 73, THR 72, TYR 187, PRO 70, VAL 69, ARG 128, LEU 30, ASP 32, THR 220, GLY 219, GLY 34, SER 35, ASP 217, ILE 215, VAL 321, THR 318 Discussion The present study aimed to explore the therapeutic potential of CDME in a transgenic Drosophila melanogaster model of AD. This investigation not only corroborates the ethnopharmacological use of C. dalzielii for treating neurological disorders but also extends our understanding of its mechanisms of action in the context of AD. Our results provide strong evidence that CDME can mitigate key AD pathologies, including oxidative stress, memory deficits, and neurodegeneration, highlighting its potential as a natural therapeutic agent for AD. The transgenic Drosophila melanogaster model employed in this study has proven to be a reliable platform for studying AD, as it recapitulates critical features of human disease, including Aβ aggregation and synaptic dysfunction (Kasanin et al., 2022). In this model, the UAS/GAL4 system drives the expression of human Aβ42 and human tau, fused to a secretion signal to facilitate extracellular localization (Tsintzas and Niccoli, 2024). Nervous system-specific overexpression of these proteins induces progressive structural and behavioral phenotypes, including locomotor dysfunction, age-dependent neurodegeneration, and reduced lifespan, mirroring key pathological features of AD (Bongiorni et al., 2024; Kasanin et al., 2022). This model offers several advantages, such as rapid behavioral assessments and the ability to screen potential therapies in a genetically tractable system. In this context, CDME demonstrated significant improvements in both the survival rate and behavioral performance of AD flies, especially at the 10 mg/10 g dose. The increase in fly survival and improvements in locomotor function, as measured by the negative geotaxis assay, suggest that CDME may help preserve neuronal integrity, potentially by mitigating the neurotoxic effects of Aβ aggregates. In terms of memory performance, the CDME treatment showed promising results in the learning phase (PC0) of the APS assay, although no significant effect on short-term memory (PC3) was observed. This finding suggests that CDME may facilitate learning but does not fully rescue memory retention, possibly due to the complex nature of memory processes, which involve various molecular pathways that may not be entirely modulated by CDME in this model. These results are consistent with previous studies that have highlighted the role of natural compounds in enhancing cognitive function in AD models (da Rosa et al., 2022), though further research is needed to better understand the time-dependent effects of CDME on memory consolidation and retention. Biochemical analysis further supports the neuroprotective role of CDME. Dysregulation of GSH levels is increasingly recognized as a significant factor in the pathogenesis of AD. Studies, including both in vitro and in vivo AD models, have shown that AD pathology is associated with reductions in GSH levels (Mandal et al., 2015; Hashim et al., 2024). Furthermore, postmortem examinations of AD brains have consistently revealed decreased GSH concentrations compared to controls (Chen et al., 2022; Foret et al., 2024). The significant elevation of glutathione (GSH) levels in treated AD flies suggests that CDME has antioxidant properties, which is a key mechanism given the role of oxidative stress in AD pathogenesis (Ekundayo et al., 2024). Moreover, the dose-dependent increase in SOD activity provides additional evidence that CDME can mitigate oxidative damage, which is known to contribute to neurodegeneration in AD. SOD is a crucial antioxidant enzyme that catalyzes the dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen. This is a critical step in reducing oxidative stress, as superoxide is a highly reactive free radical produced during normal cellular metabolism and exacerbated by various pathological conditions, including AD (Olufunmilayo et al., 2023). An increase in SOD activity following treatment with CDME would suggest a protective effect against oxidative stress. Again, in AD, the extracellular accumulation of Aβ is associated with the generation of hydrogen peroxide, lipid peroxides, and degradation products, including MDA (Xia et al., 2017). Studies have indicated that increasing SOD expression can mitigate lipid peroxidation, as demonstrated in a rat model of ischemic brain damage (Rao et al., 2021). The relationship between oxidative stress and lipid peroxidation is well-documented in models of transient brain injury. MDA levels are commonly measured to assess the extent of lipid peroxidation, as it is a primary end product of this process. In contrast, MDA levels, an indicator of lipid peroxidation, were significantly reduced in the CDME-treated groups, further supporting the extract's ability to alleviate oxidative stress. These findings are in line with the known antioxidant properties of various phytochemicals identified in C. dalzielii , including flavonoids and saponins, which have been linked to the modulation of oxidative stress pathways in the brain (Ugwah-Oguejiofor et al., 2024). AD in transgenic drosophila model caused a reduction in TAG level (Heier and Kühnlein, 2018), a lipid metabolism alteration, but on treatment with donepezil and CDME the levels were improved. This suggests that these interventions may help restore lipid metabolism, potentially mitigating neurodegenerative processes associated with altered lipid profiles in AD. Additionally, the reduction in acetylcholinesterase (AChE) activity, a hallmark of AD-associated cholinergic dysfunction, observed in the CDME-treated flies points to its potential role in enhancing cholinergic signaling. This effect, although less pronounced compared to the standard drug donepezil, suggests that CDME may offer a dual mechanism of action in AD therapy, both by enhancing cholinergic transmission and reducing oxidative damage. This finding is particularly noteworthy given the limited efficacy and side effects associated with current pharmacological treatments targeting the cholinergic system (Chin et al., 2022). The molecular docking analysis of compounds from CDME against GSK3 and BACE1 revealed significant insights into their potential as therapeutic agents for AD. The docking scores for these compounds ranged from − 7.856 to -3.040 kCal/mol for GSK3 and − 6.742 to -2.541 kCal/mol for BACE1, indicating a wide range of binding affinities. The top compounds, such as catechin, flavone, naringenin, flavanones, and ribalinidine, showed higher binding affinities against GSK3 compared to the reference compound donepezil hydrochloride. Similarly, against BACE1, compounds like catechin, aphyllidine, ephedrine, quercetin, and naringenin demonstrated strong binding affinities, often surpassing that of donepezil hydrochloride. These findings suggest that these compounds could be effective inhibitors of GSK3 and BACE1, enzymes implicated in AD pathology (Shri et al., 2023). The interaction analysis revealed that these compounds formed multiple hydrogen bonds and other interactions with key residues in both GSK3 and BACE1. For instance, catechin interacted with residues like ASP 133, VAL 135, and ASP 200 in GSK3, while forming bonds with LYS 107, GLN 73, and ASP 32 in BACE1. Hydrogen bonds were the primary contributors to the energetic interactions between the compounds from CDME and their protein targets, underscoring their pivotal role in molecular recognition (Ugwah-Oguejiofor et al., 2025). These bonds, often described as crucial for protein-ligand interactions, were complemented by other significant interactions such as pi-cation, salt bridges, and pi-pi stacking. These hydrophobic interactions are essential for stabilizing the ligand-protein complexes, highlighting the multifaceted nature of molecular binding (Johnson et al., 2022). The identification of compounds with strong binding affinities for both GSK3 and BACE1 is significant for AD treatment. GSK3 is involved in tau phosphorylation, a hallmark of Alzheimer's pathology, while BACE1 plays a critical role in amyloid-beta production. Compounds that can inhibit both enzymes may offer a more comprehensive therapeutic approach by targeting multiple aspects of the disease. Catechins the best-performing compound have been extensively studied. Extensive research in both human and animal models has documented catechins' multifaceted biological activities (Ide et al., 2018; Sheng et al., 2023). These polyphenolic compounds demonstrate anti-inflammatory properties through modulation of key inflammatory signaling pathways like NF-κB (Ravindranath and Ravindranath, 2011) and subsequent downregulation of cytokines including TNF-α and IL-6 (Coșarcă et al., 2019). Their antioxidant mechanisms involve dual action - neutralizing free radicals through electron donation (Bawono et al., 2023) and binding transition metals to prevent Fenton reactions (Cai et al., 2018). At the molecular level, catechins exhibit neuroprotective potential by interfering with pathological processes in neurodegenerative diseases. Experimental evidence suggests they inhibit abnormal tau protein hyperphosphorylation through PP2A activation while disrupting amyloid-beta fibril formation via direct molecular interactions (Basurto-Islas et al., 2025). The compounds also regulate apoptotic pathways by balancing Bcl-2 family proteins and caspases (Özduran et al., 2023). Current investigations focus on optimizing catechin bioavailability and synergies with other bioactive compounds to maximize therapeutic efficacy. Flavones (Wang et al., 2025), naringenin (Zhu et al., 2024), quercetin (Elreedy et al., 2023) and kaempferol (Dong et al., 2023) have been reported as potent against AD while others such as aphyllidine and ribalinidine are yet to be determined. Further studies are needed to validate some of these findings through experimental assays and to assess their pharmacokinetic properties. Additionally, molecular dynamics simulations could provide insights into the stability and dynamics of these protein-ligand interactions, further supporting their potential as therapeutic agents. Conclusion Our study demonstrates that CDME holds considerable promise as a potential therapy for AD. The extract’s ability to reduce oxidative stress, enhance learning, and improve locomotor function in the transgenic Drosophila melanogaster model of AD suggests that it may serve as a valuable source for developing novel therapeutic strategies for AD. However, future studies are needed to isolate and identify the specific bioactive compounds responsible for these effects, as well as to explore the long-term therapeutic potential of CDME in more complex vertebrate models. Additionally, clinical trials will be crucial to evaluate the safety and efficacy of CDME in human populations affected by AD. Declarations Acknowledgements The authors thank the technical staff of CAMRET, Usmanu Danfodiyo University for their technical support and Nigerian Tetfund for the financial support. Authors Contributions Conceptualization: Chinenye J. Ugwah-Oguejiofor, Funding acquisition: Chinenye J. Ugwah-Oguejiofor, Mustapha U. Imam, Aliyu H. Ahmed, Zainab Almustapha, Formal analysis, and Investigation: Evelyn Hassan and Dhaakirah Yakubu, Supervision: Chinenye J. Ugwah-Oguejiofor and Mustapha U. Imam, Writing: Chinenye J. Ugwah-Oguejiofor, Evelyn Hassan and Dhaakirah Yakubu, Review and editing: Mustapha U. Imam and Chinenye J. Ugwah-Oguejiofor. Funding The study was funded from the grant from the Institutional Based Research (IBR) funds TETF/DR&D/CE/UNI/SOKOTO/IBR/2024/VOL.II Financial Interests The authors have no relevant financial or non-financial interests to disclose. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6128472","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":424620268,"identity":"14cef7e3-22b9-4d65-a81f-96c91cccad72","order_by":0,"name":"Chinenye Jane Ugwah-Oguejiofor","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABL0lEQVRIie2RMUvEMBTHXzk4l3KuKdW7r9ASqIsfJuGGW3ougnTQMyLEReja+xYFP4ApB+0S90xyXZw6RLzhRtM7EQ4jXQX7g5B/CD/eywtAT88fxGHfsWCQAIwBiDmIr9WpSADcqRxiFMq6lMHDS7GG5IamzupuLZLXWV7FWGsJ45Eiw3pjaezxYhqArOiSFfeBkJfzXDaRlynAniJH+MT2ljhCDi9pLgruv3Myz1UcDVwNNDdVfGRR0uZs2yrPRkEFJ7NAxfjDKLe/KlkcgcOvaQ57hRgl8F0FJpChp21KgxGRAmdi9xYSLuXblZdJZELNfcvEwjQOtU4Wp2lW1WZiZDKqpk9al+dtKL2tRWHQ/t0KAImDC7SbzcD9qUz22wLgmFmacCxVenp6ev4dn4eCfSe+2NVfAAAAAElFTkSuQmCC","orcid":"","institution":"Usmanu Danfodiyo University","correspondingAuthor":true,"prefix":"","firstName":"Chinenye","middleName":"Jane","lastName":"Ugwah-Oguejiofor","suffix":""},{"id":424620271,"identity":"d07b09af-41f8-4632-a8dd-af3658af3dfc","order_by":1,"name":"Evelyn Hassan","email":"","orcid":"","institution":"Usmanu Danfodiyo University","correspondingAuthor":false,"prefix":"","firstName":"Evelyn","middleName":"","lastName":"Hassan","suffix":""},{"id":424620274,"identity":"79c26795-5968-445c-b81c-8c487b1bc3e8","order_by":2,"name":"Dhaakirah Yakubu","email":"","orcid":"","institution":"Usmanu Danfodiyo University","correspondingAuthor":false,"prefix":"","firstName":"Dhaakirah","middleName":"","lastName":"Yakubu","suffix":""},{"id":424620275,"identity":"fb86ade4-23b1-44e9-a290-0492b3ddc12c","order_by":3,"name":"Aliyu Hamid Ahmed","email":"","orcid":"","institution":"Usmanu Danfodiyo University","correspondingAuthor":false,"prefix":"","firstName":"Aliyu","middleName":"Hamid","lastName":"Ahmed","suffix":""},{"id":424620279,"identity":"ca585f3c-d1f8-4589-a1eb-f9c762907b7f","order_by":4,"name":"Zainab Almustapha","email":"","orcid":"","institution":"Usmanu Danfodiyo University","correspondingAuthor":false,"prefix":"","firstName":"Zainab","middleName":"","lastName":"Almustapha","suffix":""},{"id":424620282,"identity":"ccf69002-a18d-43f2-8735-6f15e2e320a0","order_by":5,"name":"Mustapha Umar Imam","email":"","orcid":"","institution":"Usmanu Danfodiyo University Sokoto","correspondingAuthor":false,"prefix":"","firstName":"Mustapha","middleName":"Umar","lastName":"Imam","suffix":""}],"badges":[],"createdAt":"2025-02-28 12:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6128472/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6128472/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78230641,"identity":"65b552c8-bf92-4a82-a6c5-2a958c5dd6dd","added_by":"auto","created_at":"2025-03-11 07:25:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":266474,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC spectrum imprint profile of methanol extract of \u003cem\u003eCaralluma dalzielii\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/ef096e032785dffca84e89ca.png"},{"id":78228131,"identity":"5888f919-a6b1-4aa9-9f6d-fafec2bd84a2","added_by":"auto","created_at":"2025-03-11 07:09:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37338,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of methanol extract of\u003cem\u003e Caralluma dalzielii\u003c/em\u003e on survival rate\u003c/p\u003e\n\u003cp\u003eData presented as mean±SD. **p\u0026lt;0.01; ***p\u0026lt;0.001; CDME= methanol extract of\u003cem\u003e Caralluma dalzielii\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/07968050949084544180a3d8.png"},{"id":78229029,"identity":"b504060d-b6dd-400a-981a-e9da1129b6b8","added_by":"auto","created_at":"2025-03-11 07:17:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90244,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of CDME on behavioural assessment in Alzheimer’s disease flies\u003c/p\u003e\n\u003cp\u003eA= negative geotaxis, B= grooming; C=learning assessment; D= short-term memory assessment\u003c/p\u003e\n\u003cp\u003eData presented as mean ± SD. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/c7c65d5a4c22ef66e86e0acf.png"},{"id":78228133,"identity":"66625449-c6f4-4a3e-9563-f64774f9f57a","added_by":"auto","created_at":"2025-03-11 07:09:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":134983,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of CDME on biochemical parameters in Alzheimer’s disease flies\u003c/p\u003e\n\u003cp\u003eA= Glutathione levels; B= Superoxide dismutase; C=Malondialdehyde; D=Acetylcholinesterase; E= Triacylglycerol. Data presented as mean ± SD. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/b68c6c9c0c8dad60ab2aa57d.png"},{"id":78229028,"identity":"d7d61af3-edb1-4696-9e0e-4a9b9f617bbd","added_by":"auto","created_at":"2025-03-11 07:17:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":345994,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of glycogen synthase kinase 3 with catechin, 2D left 3D right.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/ad6c955b4068a8911dd4a7d0.png"},{"id":78228137,"identity":"4bc03c95-1b68-45a8-970c-bc835d251785","added_by":"auto","created_at":"2025-03-11 07:09:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":332059,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of glycogen synthase kinase 3 with flavone, 2D left 3D right.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/7c4e14a36f351486056be1d1.png"},{"id":78228178,"identity":"ee50fc89-6f6c-4475-b190-b55e4dcc0d75","added_by":"auto","created_at":"2025-03-11 07:09:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":327286,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of glycogen synthase kinase 3 with naringenin, 2D left 3D right.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/82598fe3a3447c13d8dc0dec.png"},{"id":78230642,"identity":"972fb3da-9533-4905-8142-4a9d29ea8e74","added_by":"auto","created_at":"2025-03-11 07:25:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":367551,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of glycogen synthase kinase 3 with flavonone, 2D left 3D right\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/2ab68dd93da9d0d3bdd92523.png"},{"id":78229030,"identity":"eae31211-2439-476d-8066-81b0588b8caa","added_by":"auto","created_at":"2025-03-11 07:17:44","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":368617,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of glycogen synthase kinase 3 with ribalinidine, 2D left 3D right\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/c72823f837056cbecb277044.png"},{"id":78231050,"identity":"2374f8aa-6dfb-464e-819a-c82056be3820","added_by":"auto","created_at":"2025-03-11 07:33:45","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":416407,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of glycogen synthase kinase 3 with donepezil hydrochloride, 2D left 3D right\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/0729dd5b559ebba6c4de6af9.png"},{"id":78228142,"identity":"bdb69744-cdfc-4723-ab13-79337930d61e","added_by":"auto","created_at":"2025-03-11 07:09:44","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":270397,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of human β-Secretase with catechin, 2D left 3D right\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/77bc33610554d9023f817cf3.png"},{"id":78229031,"identity":"8e16fd26-00d0-4798-a8c0-5455d0438ec6","added_by":"auto","created_at":"2025-03-11 07:17:45","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":285413,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of human β-Secretase with aphyllidine, 2D left 3D right\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/a621478eb2962303059a6327.png"},{"id":78228152,"identity":"1ea73666-c358-46e4-912f-75ce3fd46807","added_by":"auto","created_at":"2025-03-11 07:09:45","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":284515,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of human β-Secretase with ephedrine, 2D left 3D right\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/7f0ef23a8abf6aeb551e2bb7.png"},{"id":78230643,"identity":"f26fd49b-ca81-4d35-b693-105e395222a1","added_by":"auto","created_at":"2025-03-11 07:25:45","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":360859,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of human β-Secretase with quercertin, 2D left 3D right\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/45f1a0819e8f3a9fe42a8f36.png"},{"id":78229037,"identity":"d1c8cdcd-48fd-4f40-af71-640b585652c7","added_by":"auto","created_at":"2025-03-11 07:17:45","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":335021,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of human β-Secretase with naringenin, 2D left 3D right\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/159880aae6bb1ef8693aaf0c.png"},{"id":78230647,"identity":"34a2cd0d-624c-4dcf-ad45-f8a008032618","added_by":"auto","created_at":"2025-03-11 07:25:45","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":411253,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular interaction of amino-acid residues of human β-Secretase with donepezil hydrochloride, 2D left 3D right\u003c/p\u003e","description":"","filename":"16.png","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/71b61bc1e5d92cc3059e04c8.png"},{"id":83368288,"identity":"9316eedc-9eab-4c22-894a-76ceaa0108e0","added_by":"auto","created_at":"2025-05-23 22:31:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5621032,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6128472/v1/63f42ef3-d66d-43e1-bcae-78b8285ae6ce.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Caralluma dalzielii as a Potential Therapy for Alzheimer’s Disease: Insights from a Transgenic Drosophila melanogaster Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, memory loss, and synaptic dysfunction and represents one of the most pressing global health challenges of the 21st century (Botella Lucena and Heneka, 2024). It is the most common cause of dementia worldwide, affecting millions of individuals and placing a significant burden on healthcare systems. Pathologically, AD is associated with the accumulation of amyloid-beta (Aβ) plaques, tau protein hyperphosphorylation, oxidative stress, and neuroinflammation (Ekundayo et al., 2024). Despite extensive research, there is currently no cure for AD, and available treatments only provide symptomatic relief without addressing the underlying disease mechanisms. With over 150\u0026nbsp;million projected cases worldwide by 2050, the urgency to identify effective therapeutic interventions has never been greater (Alves et al., 2025). Current treatments, such as acetylcholinesterase inhibitors and NMDA receptor antagonists, offer only symptomatic relief and are often associated with significant side effects (Chin et al., 2022). This therapeutic gap has spurred interest in natural products, particularly medicinal plants with ethnopharmacological relevance, as potential sources of novel neuroprotective agents.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCaralluma dalzielii\u003c/em\u003e N. E. Brown (Asclepiadaceae), a shrub indigenous to sub-Saharan Africa, has long been used in traditional medicine to treat neurological conditions, including epilepsy and pain (Shehu et al., 2019; Ugwah-Oguejiofor et al., 2024). Recent preclinical studies have validated its neurobehavioral properties, demonstrating anxiolytic, anti-hyperactivity, and memory-enhancing effects in murine models (Ugwah-Oguejiofor et al., 2024). These effects are hypothesized to arise from GABAergic and glutamatergic modulation, coupled with antioxidant activity\u0026mdash;a critical feature given the role of oxidative stress in AD pathogenesis. Gas chromatography-mass spectrometry (GC-MS) analyses of \u003cem\u003eC. dalzielii\u003c/em\u003e extracts have identified bioactive compounds such as 3,5-Dimethylpyrazole and Acetophenone, which may underlie its neuropharmacological potential (Ugwah-Oguejiofor et al., 2024). However, its efficacy against AD-specific pathologies remains unexplored.\u003c/p\u003e \u003cp\u003eThe transgenic Drosophila melanogaster model has emerged as a powerful tool for studying AD mechanisms and screening therapeutic candidates. Drosophila shares approximately 75% of its genes with humans, allowing researchers to study human disease mechanisms effectively within a simpler organism (Kasanin et al., 2022). By expressing human amyloid-beta (Aβ42) and tau proteins, these flies recapitulate key AD hallmarks, including amyloid aggregation, synaptic dysfunction, and memory deficits within days\u0026mdash;a temporal advantage over vertebrate models (Bongiorni et al., 2024). The transgenic Drosophila model provides a cost-effective, genetically tractable system with a short lifespan, enabling rapid screening of potential therapeutics (Tsintzas and Niccoli 2024). Additionally, natural products can be easily administered through feeding, and behavioral assays, such as memory and locomotor tests, provide measurable outcomes.\u003c/p\u003e \u003cp\u003eThis study seeks to bridge traditional medicine and modern neurogenetics by investigating \u003cem\u003eC. dalzielii\u003c/em\u003e\u0026rsquo;s therapeutic potential in a transgenic Drosophila AD model. We hypothesize that its methanol extract will mitigate Aβ42-induced neurodegeneration through dual mechanisms: (1) enhancing cholinergic signaling via acetylcholinesterase inhibition, and (2) reducing oxidative stress through free radical scavenging. By correlating behavioral outcomes (memory, locomotion) with molecular markers (amyloid load, tau phosphorylation), this work aims to validate ethnopharmacological claims while identifying bioactive fractions for future drug development.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant collection and extraction\u003c/h2\u003e \u003cp\u003eFresh C. dalzielii (CD) batches were collected from Sokoto in August 2024. Identification and authentication were done in the Laboratory of Pharmacognosy and Ethnopharmacy, UDUS where the voucher specimen (Pcg/UDUS/Asdy/001) was stored at the herbarium unit. The CD was shade-dried at room temperature for a month, after which it was pounded with mortar and pestle. Three hundred and nineteen grams (319 g) of the pounded plant material was extracted using the maceration method with 70% Methanol for 72 hrs. The filtrate was concentrated over a rotary evaporator at 45\u003csup\u003eo\u003c/sup\u003eC while the remaining moisture was evaporated completely over a water bath set at 45\u003csup\u003eo\u003c/sup\u003eC to yield dry extract (CCDE).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhytochemical screening of the extract\u003c/h3\u003e\n\u003cp\u003ePhytochemical screening of the extract was performed in order to determine the presence of secondary metabolites using standard phytochemical methods as described by Evans (2002).\u003c/p\u003e\n\u003ch3\u003eHPLC analysis\u003c/h3\u003e\n\u003cp\u003eThe HPLC analysis was carried out using an Agilent 1100 system, which features dual binary pumps, a column oven, and a UV/Vis detector. The method employed a C-12 normal phase column (Phenomenex, Gemini 5 \u0026micro;m) with dimensions of 200 mm in length and 4.8 mm in internal diameter. The mobile phase consisted of acetic acid-acidified deionized water (pH 2.8) as solvent A and acetonitrile as solvent B, with a flow rate set at 0.8 mL/min. To maintain optimal conditions, the column was equilibrated with 5% solvent B for 20 minutes after each sample injection. The temperature was kept at 38\u0026deg;C, and a sample volume of 20 \u0026micro;L was injected. Detection of phytochemicals occurred at a wavelength of 280 nm. The identification and quantification of these compounds were performed by comparing their retention times and peak areas to those of pure standard compounds, utilizing an external standard approach to develop a calibration curve.\u003c/p\u003e \u003cp\u003eThe gradient elution procedure was structured as follows:\u003c/p\u003e \u003cp\u003e0\u0026ndash;5 minutes: Increase from 5\u0026ndash;9% solvent B\u003c/p\u003e \u003cp\u003e5\u0026ndash;15 minutes: Hold at 9% solvent B\u003c/p\u003e \u003cp\u003e15\u0026ndash;22 minutes: Increase from 9\u0026ndash;11% solvent B\u003c/p\u003e \u003cp\u003e22\u0026ndash;38 minutes: Increase from 11\u0026ndash;18% solvent B\u003c/p\u003e \u003cp\u003e38\u0026ndash;43 minutes: Increase from 18\u0026ndash;23% solvent B\u003c/p\u003e \u003cp\u003e43\u0026ndash;44 minutes: Rapid increase to 90% solvent B\u003c/p\u003e \u003cp\u003e44\u0026ndash;45 minutes: Decrease from 90\u0026ndash;80% solvent B\u003c/p\u003e \u003cp\u003e45\u0026ndash;55 minutes: Continue at this rate\u003c/p\u003e \u003cp\u003eThis systematic methodology facilitated the effective separation and analysis of the phytochemical components present in the samples (Kaisoon et al., 2011).\u003c/p\u003e\n\u003ch3\u003eFruit fly (Drosophila melanogaster) culturing\u003c/h3\u003e\n\u003cp\u003eWild type fruit fly (Oregon strain) stock culture was obtained from Drosophila Laboratory, at CAMRET, UDUS. The flies were maintained and reared on normal diet made up of cornmeal medium containing 1%w/v brewer\u0026rsquo;s yeast and corn meal at room temperature under 12 h dark/light cycle conditions in the Drosophila Research Laboratory.\u003c/p\u003e\n\u003ch3\u003eGeneration of Transgenic flies for the study\u003c/h3\u003e\n\u003cp\u003eTransgenic fly lines that express wild-type human Aβ-42 peptide (UAS-Aβ42) and a pan-neuronal elav-Gal4 driver were obtained from CAMRET. The AD model flies were obtained by crossing Gal4 virgin females to Aβ-42 males (Wei-Wei et al., 2017). In the F1 progeny, AD flies were UAS-Aβ42::ElavGAL4, and they could be distinguished from healthy siblings based on their phenotypic characteristics (Bongiorni et al., 2024). These F1 progeny flies (AD flies) were used for the study.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral Assay\u003c/h2\u003e \u003cp\u003eFlies were raised on a standard Drosophila cornmeal agar diet at 25\u003csup\u003eo\u003c/sup\u003eC and 42% relative humidity. The 1\u0026ndash;2-day-old AD flies were grouped into five each fly containing 20 flies. Group I was the control group, Group II received donezepil 0.05 mmol while Groups III and IV received two doses of CDME, 1mg/10g diet and 10 mg/10g diet for 14 days.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNegative geotaxis\u003c/h3\u003e\n\u003cp\u003eTo evaluate the locomotor activity of each experimental group, 20 male flies were selected using a pooter and placed in individual empty 20 mL measuring cylinder. The flies were adjusted to room temperature for 10 min for adaptation (Bonilla-Ramirez et al., 2011). To measure the climbing ability, the flies were gently tapped down to the bottom of the test vial. A mark was made at 8 cm from the bottom of each cylinder. The cylinder was gently tapped, causing all flies to fall to the bottom, and the number of flies that crossed the 8 cm mark within 6 seconds was counted. This procedure was performed three times to determine the average pass rate per group with 2 min resting time. The data collected was then analyzed based on the average of the three replicates (n\u0026thinsp;=\u0026thinsp;60) (Yankuzo et al., 2024).\u003c/p\u003e\n\u003ch3\u003eGrooming\u003c/h3\u003e\n\u003cp\u003eTo assess anxiety-like behaviors, a grooming test was conducted, with slight modifications to the procedure described by Kaur et al. (2015). Each experimental group consisted of 15 flies, and each group was tested in triplicate. Throughout a 2-min observation period, the grooming behaviors of individual flies were carefully monitored and recorded by observers unaware of the treatment assignments. The experiment was performed twice for each group. Statistical analysis was used to compare the average grooming time among the groups.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMemory assessment test\u003c/h2\u003e \u003cp\u003eTo assess memory, an aversive phototaxis suppression (APS) assay was employed, with modifications to the protocol described by Poetini et al. (2022). The assay leverages the flies' natural inclination to move towards a light source. Initial testing in a T-maze was performed to confirm the flies' responsiveness to light, a critical component of the assay. Ten flies were placed in the dark side of the T-maze, and the first five to cross to the lit side within 10 s were selected for the following steps. For the training phase (PC0), filter paper dampened with chloroquine was placed on the lit side of the T-maze. After allowing the flies to acclimate in the dark, they were permitted to cross to the lit side, associating the light with the aversive bitter taste of chloroquine. This training was repeated five times to establish baseline results. Three hours later (PC3), short-term memory was evaluated by repeating the procedure, but without the chloroquine filter paper. The flies were then observed to see if they entered the lit side or remained in the dark. Choosing to remain in the dark was recorded as a \"pass,\" indicating that they remembered the association of light with the bitter taste, while entering the lit side was recorded as a \"fail.\" Fifteen flies per group, across three replicates, were tested in this manner, and the percentage of flies that passed was recorded by observers blinded to the treatment groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical assays\u003c/h2\u003e \u003cp\u003eThe 1\u0026ndash;2-day-old AD flies were grouped into five, each containing 20 flies. Group 1 was the control group, Group II received donezepil 0.05mmol while Groups III to V received doses of CDME, 1mg/10g diet, 10 mg/10g diet and 20 mg/10g, respectively, for 14 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFly head extraction\u003c/h2\u003e \u003cp\u003eFollowing cold anesthesia, fly heads from each experimental group were carefully isolated (20 heads per group; three replicates per group) and placed into 1.5 mL Eppendorf tubes containing 0.1 M phosphate-buffered saline (PBS). Subsequently, the tissue samples were homogenized using a handheld homogenizer (Hangzhou Miou Instrument Co., Ltd., Hangzhou, China) to ensure thorough disruption of cellular structures. The resulting homogenate was then centrifuged at 2500 g for 10 minutes at 4\u0026deg;C using a high-speed refrigerated microcentrifuge. After centrifugation, the supernatant was gently aspirated and transferred into sterile 1.5 mL tubes for storage at \u0026minus;\u0026thinsp;20\u0026deg;C pending further biochemical analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eGlutathione (GSH) content determination\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eGSH levels were determined using a spectrophotometric approach reported earlier (Patterson and Lazarow, 1955). Accordingly, GSH levels were measured by reacting samples with alloxan, phosphate buffer, sodium hydroxide, and metaphosphoric acid, with absorbance read at 340 nm to quantify GSH content, expressed as mg/dL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSuperoxide Dismutase (SOD) Activity\u003c/h2\u003e \u003cp\u003eSOD activity was evaluated using a modified version of the pyrogallol auto-oxidation assay, originally developed by Marklund and Marklund (1974). This method involves measuring the inhibition of pyrogallol oxidation in a reaction mixture containing Tris-HCl buffer and sample extracts. The reaction is initiated by adding pyrogallol, after which changes in absorbance are monitored over 3 min at 420 nm. SOD activity is quantified as the amount of protein required to inhibit 50% of pyrogallol auto-oxidation under these conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMalondialdehyde (MDA) Levels\u003c/h2\u003e \u003cp\u003eMDA, a key indicator of lipid peroxidation, was quantified using an adapted version of the thiobarbituric acid reactive substances (TBARS) assay, originally developed by Ohkawa et al. (1979). This method involves mixing trichloroacetic acid with the sample, followed by the addition of thiobarbituric acid. The mixture is then heated in a water bath to facilitate the reaction between MDA and thiobarbituric acid. After cooling and centrifugation, the resulting TBARS, with a characteristic red-pink color, were measured spectrophotometrically at 532 nm. The results were expressed as nmol/L of TBARS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAcetylcholinesterase (AChE) Activity\u003c/h2\u003e \u003cp\u003eAchE levels were quantified using a competitive ELISA kit according to the manufacturer\u0026rsquo;s protocol (PARS BIOCHEM\u0026trade; Nanjing, China). The absorbance values were measured at 450 nm and compared to a standard curve to determine AChE concentrations in the samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTriacylglycerol Quantification\u003c/h2\u003e \u003cp\u003eTriacylglycerol levels were measured by adding a triglyceride working reagent to the samples, a triglyceride standard, and distilled water. The absorbance was measured at 500 nm, and the results were expressed as mg/dL of triglyceride.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIn silico studies\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003eProtein Preparation\u003c/h2\u003e \u003cp\u003eThe crystal structures of glycogen synthase kinase 3 (GSK3) and human β-Secretase (BACE 1), identified by PDB IDs 1Q5K and 4D8C respectively, were prepared following established protocols (Ugwah-Oguejiofor et al., 2025). The protein structures were retrieved from the Protein Data Bank and processed using the Glide software within the Schrodinger Suite (2020\u0026ndash;3), specifically utilizing the Protein Preparation Wizard. This process involved the addition of hydrogen atoms, assignment of bond orders, formation of disulfide bonds, and reconstruction of any missing side chains and loops through the Prime module. Solvent molecules located more than 3.0 \u0026Aring; from heteroatoms were removed to streamline the structure, which was then minimized and optimized using the OPLS3e force field.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of Receptor Grid\u003c/h2\u003e \u003cp\u003eThe receptor grid was created to define the spatial coordinates and dimensions of the enzyme's active site, essential for ligand docking. This was accomplished using the receptor grid generation tool in Schrodinger Maestro version 12.5, with the active site based on co-crystallized ligands serving as a reference for constructing the scoring grid (Apeh et al., 2023).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Ligands\u003c/h2\u003e \u003cp\u003eA total of twenty phytochemical compounds obtained from High-Performance Liquid Chromatography (HPLC) of CDME, along with the standard ligand donepezil, were prepared using the LigPrep tool in Maestro 12.5, Schrodinger Suite (2020\u0026ndash;3). The preparation yielded three-dimensional structures at minimal energy with accurate chiral configurations. Each ligand's ionization state was adjusted to reflect a physiological pH of 7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2. Stereoisomers were generated by stabilizing specific chiral centers while allowing variability in others (Ugwah-Oguejiofor et al., 2023).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eProtein-Ligand Docking\u003c/h2\u003e \u003cp\u003eMolecular docking was performed using the Schrodinger Suite (2020\u0026ndash;3) through the Glide-Ligand Docking tool incorporated in Maestro 12.5. The prepared ligands and receptor grid file were integrated into Maestro's workspace for docking into the protein's binding site utilizing Glide's standard precision mode. During this process, adjustments were made to the van der Waals radius scaling factor for ligand atoms to 0.80, a partial charge threshold was set at 0.15, and flexible ligand sampling techniques were employed to enhance docking accuracy (Johnson et al., 2022).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe data were recorded in mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. One-way Analysis of variance (ANOVA) was used where necessary and Tukey post hoc was used to compare significance across groups. A value of P\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant. GraphPad Prism v8.0 (GraphPad Software Inc., San Diego, California) was used for the analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003ePercentage Yield\u003c/h2\u003e\n \u003cp\u003eThe percentage yield of the methanol extract of CDME was 10.7%.\u003c/p\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003ePhytochemical analysis\u003c/h2\u003e\n \u003cp\u003eThe phytochemical analysis showed the presence of carbohydrates, saponin, flavonoids and other bioactive compounds although anthroquinones were not detected (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhytochemical Screening of CDME\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eS/N\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePHYTOCHEMICAL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRESULT\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCarbohydrates\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Molich\u0026rsquo;s test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Fehling\u0026rsquo;s test (Red. Sugars)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaponins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Froth\u0026rsquo;s test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhenols\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Ferric chloride\u0026rsquo;s test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavonoids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Ferric chloride\u0026rsquo;s test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Alkaline Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Shinoda\u0026rsquo;s Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTannins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Ferric chloride\u0026rsquo;s test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Lead acetate test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlkaloids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Mayer\u0026rsquo;s test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Hager\u0026rsquo;s Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Wagner\u0026rsquo;s Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Dragendroff\u0026rsquo;s Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnthraquinones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Bontrager\u0026rsquo;s Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDiterpenoids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Copper Acetate\u0026rsquo;s Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTriterpenoids/Steroids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Salkowki\u0026rsquo;s Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Libermann-Burchard\u0026rsquo;s Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCardiac glycosides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Killer-killiani\u0026rsquo;s Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProteins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026middot; \u0026nbsp; \u0026nbsp; \u0026nbsp; Xanthoproteic Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003cp\u003e+ present; - not detected\u003c/p\u003e\n \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\n \u003ch2\u003eHPLC for CDME\u003c/h2\u003e\n \u003cp\u003eTwenty-one chromatographic peaks were identified in the HPLC analysis of the extract (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The identified compounds, along with their peak areas, heights, and concentrations, are presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCompounds of CDME identified through HPLC\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompounds\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRetention time (min)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak area\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeak height\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConc (ug/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKaempferol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4083.6304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e389.518\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.7443\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSteroid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.473\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4093.1305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e320.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.8339\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecatechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6355.7564\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e494.097\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.888\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCyanogenic glycoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4859.8114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e380.588\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.4056\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNarigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.866\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4680.9656\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e366.718\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.0167\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDihydrocytisine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6488.9442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e507.931\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.3477\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDihydrocytisine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.886\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5010.2373\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e392.627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.4454\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQuercertin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3363.6766\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e263539\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.2195\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAphyllidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4580.4334\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e359.146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.1075\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmmodendrine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.883\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8235.6251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e642.946\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.0347\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTannin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.713\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7935.2493\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e618.966\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.3193\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavonones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.606\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2956.965\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e231.988\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.072\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecardiac glycoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5551.2211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e435394\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.8916\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpartein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4434.1058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e347.282\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.5031\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.633\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4104.5175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e321.457\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.0956\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRibalinidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36896\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7474.5404\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e583.338\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.2765\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhytate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.526\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4454.0796\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e349.461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.5279\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhytate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8756.4876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e683.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.8675\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOxalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6201.0704\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e486.517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.3389\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEpihedrine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.953\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3567.976\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e280.808\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.4295\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSapogenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41.816\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5879.1171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e494.615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.2987\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eEffect of CDME on survival rate and behavioural assessment in AD flies\u003c/h3\u003e\n\u003cp\u003eAfter completing the 14-day treatment, there was a significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u0026lt; 0.001) improvement in fly survival when CDME was added to their diet at concentrations of 1 and 10 mg/10g diet, compared to the control group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The behavioural assessment was carried out using negative geotaxis, grooming and learning and memory assessment. The locomotor behaviour was assessed using the negative geotaxis assay. CDME at 10 mg/ 10 g diet significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased the average climbing rate compared to the control (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similar to the reference drug, at the treated doses, CDME caused reduced grooming frequencies (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the control (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eThe APS was utilized to evaluate both learning and short-term memory. Learning performance was assessed using the PC0 parameter, while short-term memory was evaluated through the PC3 parameter. The results showed that CDME administered at two different doses significantly enhanced learning capabilities (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no significant improvement in short-term memory was observed (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of CDME on neurobiochemical analysis in AD flies\u003c/h2\u003e\n \u003cp\u003eTreatment of the AD flies with CDME caused a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) elevation of GSH levels in the flies with a 10 mg/10 g diet dose higher than all the treated doses (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). There was a dose-dependent increase in the SOD levels at all doses with the highest dose, 20 mg/10 g diet, slightly higher but comparable to the standard drug (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC shows the level of MDA activity in the treated AD flies with 10 mg/10 g diet significantly lower (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) than the control. The level was also lower than that of the standard drug. In Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, it was observed that there was a dose-dependent decrease in the AChE activity in all the treated groups. However, the activity of the standard drug was much lower than that in all CDME groups. TAG levels in the 1 mg and 10 mg/10 g diet were significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) than in the control. They were also found to be higher in concentration than both the standard drug and the highest dose level groups (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\n \u003ch2\u003eMolecular docking\u003c/h2\u003e\n \u003cp\u003eThe molecular docking analysis of compounds from CDME against GSK3 and BACE 1 showed varying compounds with docking scores ranging from \u0026minus;\u0026thinsp;7.856 to -3.040 kCal/mol and \u0026minus;\u0026thinsp;6.742 to -2.541 kCal/mol respectively (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The docking results against 1Q5K showed that the first nine compounds had higher binding affinities than the reference compound, while in 4D8C, the first ten compounds outperformed the reference compound. The 2D and 3D views of the top five compounds and the standard compound against 1Q5K and 4D8C are presented in Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e16\u003c/span\u003e. Their bond interactions are presented in Tables \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e respectively.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDocking scores of compounds against target proteins 1Q5K and 4D8C\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompounds\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePubChem CID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDocking scores (kCal/mol)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompounds\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePubChem CID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDocking scores (kCal/mol)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e1Q5K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e4D8C\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCatechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-7.856\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCatechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.742\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-7.690\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAphyllidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12306738\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.682\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNarigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e439246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-7.658\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEphedrine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.358\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavonones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14259001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-7.589\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQuercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5280343\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.286\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRibalinidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e336322\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-7.527\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNarigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e439246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.048\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQuercetin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5280863\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-7.304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmmodendrine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e442625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.920\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDihydrocytisine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91747228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.917\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKaempferol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5280863\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.887\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEphedrine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.901\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpartein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e644020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.851\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmmodendrine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e442625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavonones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14259001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.719\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAphyllidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12306738\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.263\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSteroid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131751786\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.436\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKaempferol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5280863\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDihydrocytisine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91747228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.354\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSapogenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e198016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-4.610\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCyanogenic glycoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131751786\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.598\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRibalinidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e336322\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-4.462\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhytate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e890\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhytate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e890\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.270\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpartein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e644020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.070\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOxalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.767\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecardiac glycoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e439501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.740\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCyanogenic glycoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131751786\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.541\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSteroid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139082353\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.517\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTannin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16165470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOxalate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSapogenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e198016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTannin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16165470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCardiac glycoside\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e439501\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDonepezil hydrochloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.344\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDonepezil hydrochloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.334\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eInteractions of the top five compounds of \u003cem\u003eCaralluma dalzielii\u003c/em\u003e and standard compound against the target protein of glycogen synthase kinase 3\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePubChem CID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eName of compound\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eInteractions\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-bond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOther bonds\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCatechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eASP 133, VAL 135, ASP 200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eILE 62, VAL 70, GLY 63, GLY 65, LYS 85, ALA 83, CYS 199, LYS 183, ASN 186, LEU 188, VAL 110, LEU 132, TYR 134\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAL 135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAL 70, ILE 62, ARG 141, THR 138, GLU 137, PRO 136, TYR 134, ASP 133, LEU 132, CYS 199, VAL 110, LEU 188, ALA 83, LYS 85, ASP 200, GLU 97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e439246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNarigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAL 135, LYS 85, ASP 200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLYS 183, GLN 185, ASN 186, ILE 62, LEU 188, THR 138, TYR 134, ASP 133, LEU 132, VAL 110, VAL 70, ALA 83, CYS 199\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14259001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavonones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eASN 186, VAL 135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGLY 63, ILE 62, ARG 141, THR 138, GLY 137, PRO 136, TYR 134, ASP 133, LEU 132, VAL 110, ALA 83, LYS 85, VAL 70, CYS 199, ASP 200, LEU 188, GLN 185, LYS 183\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e336322\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRibalinidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVAL 135, ASN 186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTHR 138, TYR 134, ASP 133, LEU 132, ALA 83, LYS 85, VAL 110, ILE 62, GLY 63, GLY 65, CYS 199, ASP 200, VAL 70, GLY 183, GLN 185, LEU 188\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDonepezil hydrochloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLYS 85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eASN 186, LEU 188, LEU 132, TYR 134, VAL 135, PRO 136, VAL 70, GLU 137, THR 138, GLN 72, ARG 141, GLU 97, VAL 110, ALA 83, PHE 201, GLY 65, ASP 200, CYS 199, ILE 62, VAL 61, LYS 60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eInteractions of the top five compounds of \u003cem\u003eCaralluma dalzielii\u003c/em\u003e and standard compound against the target protein of human \u0026beta;-Secretase\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePubChem CID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eName of compound\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eInteractions\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH-bond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOther bonds\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCatechin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLYS 107, GLN 73, GLY 11, ASP 32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTRP 115, ILE 118, GLY 74, THR 72, TYR 71, LEU 30, GLY 34, SER 35, GLY 74, PHE 108, ILE 110, THR 221, GLY 219, ASP 217\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12306738\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAphyllidine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGLN 73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLYS 107, PHE 108, TRP 115, LEU 30, ILE 118, TYR 71, THR 72, GLY 74, ARG 224, THR 221, THR 220, GLY 219\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9294\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEphedrine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eASP 217, THR 220\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePHE 108, LEU 30, ASP 32, GLY 34, SER 35, TRP 115, ILE 118, ILE 215, GLY 219, ASP 217, GLY 219, VAL 321, TYR 71, THR 72, GLN 73, GLY 74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5280343\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eQuercertin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTHR 220, TYR 187, ARG 128, ILE 126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLEU 30, ASP 32, GLY 34, SER 35, SER 36, ILE 118, GLY 219, ASP 217, ILE 215, ARG 224, VAL 321, THR 72, TYR 71 PRO 70, TYR 187, ALA 127\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e439246\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNarigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eASP 217, PHE 108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGLN 73, THR 72, TYR 71, VAL 321, ARG 224, ILE 215, GLY 219, THR 220, GLY 34, ASP 32, LEU 30, ILE 118, TRP 115, ILE 110, PHE 109, LYS 107\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDonepezil hydrochloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eILE 110, TRP 115, PHE 108, GLN 73, THR 72, TYR 187, PRO 70, VAL 69, ARG 128, LEU 30, ASP 32, THR 220, GLY 219, GLY 34, SER 35, ASP 217, ILE 215, VAL 321, THR 318\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study aimed to explore the therapeutic potential of CDME in a transgenic \u003cem\u003eDrosophila melanogaster\u003c/em\u003e model of AD. This investigation not only corroborates the ethnopharmacological use of \u003cem\u003eC. dalzielii\u003c/em\u003e for treating neurological disorders but also extends our understanding of its mechanisms of action in the context of AD. Our results provide strong evidence that CDME can mitigate key AD pathologies, including oxidative stress, memory deficits, and neurodegeneration, highlighting its potential as a natural therapeutic agent for AD.\u003c/p\u003e \u003cp\u003eThe transgenic \u003cem\u003eDrosophila melanogaster\u003c/em\u003e model employed in this study has proven to be a reliable platform for studying AD, as it recapitulates critical features of human disease, including Aβ aggregation and synaptic dysfunction (Kasanin et al., 2022). In this model, the UAS/GAL4 system drives the expression of human Aβ42 and human tau, fused to a secretion signal to facilitate extracellular localization (Tsintzas and Niccoli, 2024). Nervous system-specific overexpression of these proteins induces progressive structural and behavioral phenotypes, including locomotor dysfunction, age-dependent neurodegeneration, and reduced lifespan, mirroring key pathological features of AD (Bongiorni et al., 2024; Kasanin et al., 2022). This model offers several advantages, such as rapid behavioral assessments and the ability to screen potential therapies in a genetically tractable system. In this context, CDME demonstrated significant improvements in both the survival rate and behavioral performance of AD flies, especially at the 10 mg/10 g dose. The increase in fly survival and improvements in locomotor function, as measured by the negative geotaxis assay, suggest that CDME may help preserve neuronal integrity, potentially by mitigating the neurotoxic effects of Aβ aggregates.\u003c/p\u003e \u003cp\u003eIn terms of memory performance, the CDME treatment showed promising results in the learning phase (PC0) of the APS assay, although no significant effect on short-term memory (PC3) was observed. This finding suggests that CDME may facilitate learning but does not fully rescue memory retention, possibly due to the complex nature of memory processes, which involve various molecular pathways that may not be entirely modulated by CDME in this model. These results are consistent with previous studies that have highlighted the role of natural compounds in enhancing cognitive function in AD models (da Rosa et al., 2022), though further research is needed to better understand the time-dependent effects of CDME on memory consolidation and retention.\u003c/p\u003e \u003cp\u003eBiochemical analysis further supports the neuroprotective role of CDME. Dysregulation of GSH levels is increasingly recognized as a significant factor in the pathogenesis of AD. Studies, including both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e AD models, have shown that AD pathology is associated with reductions in GSH levels (Mandal et al., 2015; Hashim et al., 2024). Furthermore, postmortem examinations of AD brains have consistently revealed decreased GSH concentrations compared to controls (Chen et al., 2022; Foret et al., 2024). The significant elevation of glutathione (GSH) levels in treated AD flies suggests that CDME has antioxidant properties, which is a key mechanism given the role of oxidative stress in AD pathogenesis (Ekundayo et al., 2024).\u003c/p\u003e \u003cp\u003eMoreover, the dose-dependent increase in SOD activity provides additional evidence that CDME can mitigate oxidative damage, which is known to contribute to neurodegeneration in AD. SOD is a crucial antioxidant enzyme that catalyzes the dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen. This is a critical step in reducing oxidative stress, as superoxide is a highly reactive free radical produced during normal cellular metabolism and exacerbated by various pathological conditions, including AD (Olufunmilayo et al., 2023). An increase in SOD activity following treatment with CDME would suggest a protective effect against oxidative stress.\u003c/p\u003e \u003cp\u003eAgain, in AD, the extracellular accumulation of Aβ is associated with the generation of hydrogen peroxide, lipid peroxides, and degradation products, including MDA (Xia et al., 2017). Studies have indicated that increasing SOD expression can mitigate lipid peroxidation, as demonstrated in a rat model of ischemic brain damage (Rao et al., 2021). The relationship between oxidative stress and lipid peroxidation is well-documented in models of transient brain injury. MDA levels are commonly measured to assess the extent of lipid peroxidation, as it is a primary end product of this process. In contrast, MDA levels, an indicator of lipid peroxidation, were significantly reduced in the CDME-treated groups, further supporting the extract's ability to alleviate oxidative stress. These findings are in line with the known antioxidant properties of various phytochemicals identified in \u003cem\u003eC. dalzielii\u003c/em\u003e, including flavonoids and saponins, which have been linked to the modulation of oxidative stress pathways in the brain (Ugwah-Oguejiofor et al., 2024). AD in transgenic drosophila model caused a reduction in TAG level (Heier and K\u0026uuml;hnlein, 2018), a lipid metabolism alteration, but on treatment with donepezil and CDME the levels were improved. This suggests that these interventions may help restore lipid metabolism, potentially mitigating neurodegenerative processes associated with altered lipid profiles in AD.\u003c/p\u003e \u003cp\u003eAdditionally, the reduction in acetylcholinesterase (AChE) activity, a hallmark of AD-associated cholinergic dysfunction, observed in the CDME-treated flies points to its potential role in enhancing cholinergic signaling. This effect, although less pronounced compared to the standard drug donepezil, suggests that CDME may offer a dual mechanism of action in AD therapy, both by enhancing cholinergic transmission and reducing oxidative damage. This finding is particularly noteworthy given the limited efficacy and side effects associated with current pharmacological treatments targeting the cholinergic system (Chin et al., 2022).\u003c/p\u003e \u003cp\u003eThe molecular docking analysis of compounds from CDME against GSK3 and BACE1 revealed significant insights into their potential as therapeutic agents for AD. The docking scores for these compounds ranged from \u0026minus;\u0026thinsp;7.856 to -3.040 kCal/mol for GSK3 and \u0026minus;\u0026thinsp;6.742 to -2.541 kCal/mol for BACE1, indicating a wide range of binding affinities. The top compounds, such as catechin, flavone, naringenin, flavanones, and ribalinidine, showed higher binding affinities against GSK3 compared to the reference compound donepezil hydrochloride. Similarly, against BACE1, compounds like catechin, aphyllidine, ephedrine, quercetin, and naringenin demonstrated strong binding affinities, often surpassing that of donepezil hydrochloride. These findings suggest that these compounds could be effective inhibitors of GSK3 and BACE1, enzymes implicated in AD pathology (Shri et al., 2023). The interaction analysis revealed that these compounds formed multiple hydrogen bonds and other interactions with key residues in both GSK3 and BACE1. For instance, catechin interacted with residues like ASP 133, VAL 135, and ASP 200 in GSK3, while forming bonds with LYS 107, GLN 73, and ASP 32 in BACE1. Hydrogen bonds were the primary contributors to the energetic interactions between the compounds from CDME and their protein targets, underscoring their pivotal role in molecular recognition (Ugwah-Oguejiofor et al., 2025). These bonds, often described as crucial for protein-ligand interactions, were complemented by other significant interactions such as pi-cation, salt bridges, and pi-pi stacking. These hydrophobic interactions are essential for stabilizing the ligand-protein complexes, highlighting the multifaceted nature of molecular binding (Johnson et al., 2022). The identification of compounds with strong binding affinities for both GSK3 and BACE1 is significant for AD treatment. GSK3 is involved in tau phosphorylation, a hallmark of Alzheimer's pathology, while BACE1 plays a critical role in amyloid-beta production. Compounds that can inhibit both enzymes may offer a more comprehensive therapeutic approach by targeting multiple aspects of the disease. Catechins the best-performing compound have been extensively studied. Extensive research in both human and animal models has documented catechins' multifaceted biological activities (Ide et al., 2018; Sheng et al., 2023). These polyphenolic compounds demonstrate anti-inflammatory properties through modulation of key inflammatory signaling pathways like NF-κB (Ravindranath and Ravindranath, 2011) and subsequent downregulation of cytokines including TNF-α and IL-6 (Coșarcă et al., 2019). Their antioxidant mechanisms involve dual action - neutralizing free radicals through electron donation (Bawono et al., 2023) and binding transition metals to prevent Fenton reactions (Cai et al., 2018). At the molecular level, catechins exhibit neuroprotective potential by interfering with pathological processes in neurodegenerative diseases. Experimental evidence suggests they inhibit abnormal tau protein hyperphosphorylation through PP2A activation while disrupting amyloid-beta fibril formation via direct molecular interactions (Basurto-Islas et al., 2025). The compounds also regulate apoptotic pathways by balancing Bcl-2 family proteins and caspases (\u0026Ouml;zduran et al., 2023). Current investigations focus on optimizing catechin bioavailability and synergies with other bioactive compounds to maximize therapeutic efficacy. Flavones (Wang et al., 2025), naringenin (Zhu et al., 2024), quercetin (Elreedy et al., 2023) and kaempferol (Dong et al., 2023) have been reported as potent against AD while others such as aphyllidine and ribalinidine are yet to be determined. Further studies are needed to validate some of these findings through experimental assays and to assess their pharmacokinetic properties. Additionally, molecular dynamics simulations could provide insights into the stability and dynamics of these protein-ligand interactions, further supporting their potential as therapeutic agents.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study demonstrates that CDME holds considerable promise as a potential therapy for AD. The extract\u0026rsquo;s ability to reduce oxidative stress, enhance learning, and improve locomotor function in the transgenic Drosophila melanogaster model of AD suggests that it may serve as a valuable source for developing novel therapeutic strategies for AD. However, future studies are needed to isolate and identify the specific bioactive compounds responsible for these effects, as well as to explore the long-term therapeutic potential of CDME in more complex vertebrate models. Additionally, clinical trials will be crucial to evaluate the safety and efficacy of CDME in human populations affected by AD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the technical staff of CAMRET, Usmanu Danfodiyo University for their technical support and Nigerian Tetfund for the financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Chinenye J. Ugwah-Oguejiofor, Funding acquisition: Chinenye J. Ugwah-Oguejiofor, Mustapha U. Imam, Aliyu H. Ahmed, Zainab Almustapha, Formal analysis, and Investigation: Evelyn Hassan and Dhaakirah Yakubu, Supervision: Chinenye J. Ugwah-Oguejiofor and Mustapha U. Imam, Writing: Chinenye J. Ugwah-Oguejiofor, Evelyn Hassan and Dhaakirah Yakubu, \u0026nbsp;Review and editing: Mustapha U. Imam and Chinenye J. Ugwah-Oguejiofor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was funded from the grant from the Institutional Based Research (IBR) funds TETF/DR\u0026amp;D/CE/UNI/SOKOTO/IBR/2024/VOL.II\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are included in the study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not require ethics approval\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlves SD, Lisboa-Filho PN, Zilli Vieira CL, Piacenti-Silva M (2025) Alzheimer\u0026apos;s Disease and Gut-Brain Axis: Drosophila melanogaster as a Model. 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Commun Biol 7(1):912.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Caralluma dalzielii, Neuroprotection, Alzheimer’s disease, Drosophila melanogaster, Phytochemicals, Molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-6128472/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6128472/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the neuroprotective potential of the methanol extract of \u003cem\u003eCaralluma dalzielii\u003c/em\u003e (CDME) against Alzheimer’s disease (AD) using transgenic \u003cem\u003eDrosophila melanogaster\u003c/em\u003e as a model organism. Fresh plant materials were collected, authenticated, shade-dried, and subjected to maceration extraction with 70% methanol. Phytochemical screening and High-Performance Liquid Chromatography (HPLC) analysis identified secondary metabolites in the extract. Transgenic AD model flies expressing human Aβ-42 peptide were generated and exposed to dietary supplementation with CDME at 1 mg, 10 mg, and 20 mg/10 g diet for 14 days as the case may be. Behavioral assays, including negative geotaxis, grooming, and aversive phototaxis suppression (APS), were conducted to assess locomotion, anxiety-like behavior, and memory performance. Neurobiochemical assays evaluated glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), acetylcholinesterase (AChE), and triacylglycerol (TAG) levels in fly heads. Results showed significant improvements in survival rate, locomotor activity, and learning performance at both CDME doses compared to the control (p\u0026lt;0.05). CDME administration led to dose-dependent increases in GSH and SOD levels, reductions in MDA and AChE activity, and enhanced TAG levels. Molecular docking analysis revealed strong binding affinities of CDME-derived compounds to glycogen synthase kinase 3 (GSK3) and β-secretase (BACE 1), key enzymes in AD pathogenesis, with several compounds outperforming the reference drug donepezil. These findings suggest that CDME possesses neuroprotective properties, potentially mitigating AD symptoms through antioxidative and cholinergic mechanisms. Further studies are recommended to isolate active compounds and explore their therapeutic potential. This study highlights the promise of \u003cem\u003eCaralluma dalzielii \u003c/em\u003eas a natural candidate for AD management.\u003c/p\u003e","manuscriptTitle":"Caralluma dalzielii as a Potential Therapy for Alzheimer’s Disease: Insights from a Transgenic Drosophila melanogaster Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-11 07:09:39","doi":"10.21203/rs.3.rs-6128472/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"63849b42-314d-4198-ace2-4dcec9e0f404","owner":[],"postedDate":"March 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-23T22:23:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-11 07:09:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6128472","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6128472","identity":"rs-6128472","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}