Multi-Targeting Neuroprotective Effects of Syzygium aromaticum Bud Extracts and Their Key Phytocompounds against Neurodegenerative Diseases

Abstract

Alzheimer's disease (AD) is a neurodegenerative disease that causes a gradual loss of normal motor and cognitive function. The complex AD pathophysiology involves various factors such as oxidative stress, neuroinflammation, amyloid-beta (Aβ) aggregation, disturbed neurotransmission, and apoptosis. The available drugs suffer from a range of side effects and are not able to cover different aspects of the disease. Therefore, finding a safer therapeutic approach that can affect multiple targets at a time is highly desirable. In the present study, the underlying neuroprotective mechanism of an important culinary spice, Syzygium aromaticum (Clove) extract, and major bioactive compounds were studied in hydrogen peroxide-induced oxidative stress in human neuroblastoma SH-SY5Y cell lines as a model. The extracts were subjected to GC-MS to identify important bioactive components. The extracts and key bio-actives reduced reactive oxygen species (ROS), restored mitochondrial membrane potential (MMP), and provided neuroprotection from H₂O₂-induced oxidative stress in cell-based assays due to the antioxidant action. They also reduced lipid peroxidation significantly and restored GSH content. Clove extracts have also displayed anti-acetylcholinesterase (AChE) activity, anti-glycation potential, and Aβ aggregation/fibrilization inhibition. The multitarget neuroprotective approach displayed by Clove makes it a potential candidate for AD drug development.

Keywords: Alzheimer’s disease; H2O2 induced stress; MDS; Syzygium aromaticum; anti-acetylcholine esterase activity; anti-glycation; oxidative stress.

Figure 1

The anti-glycation activity exhibited by Clove extracts and eugenol. IC₅₀ values were estimated using the Glucose-BSA model. Data are means of triplicate. Results were calculated using GraphPad Prism 9.5. Abbreviations: AG—Aminoguanidine; CL–H—Clove–Hexane; CL-EA—Clove–Ethyl Acetate.

Figure 2

IC₅₀ curves of Clove extracts, eugenol, and β-Caryophyllene with inhibitor control (Galantamine) against AChE. The IC₅₀ values were calculated using GraphPad Prism 9.5. Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate.

Figure 3

Lineweaver-Burk plot of AChE in the presence of (A) CL-EA, (B) CL-H, (C) eugenol, and (D) β-Caryophyllene. Experiments were performed in triplicate. The graph was plotted using GraphPad Prism 9.5. Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate.

Figure 4

Inhibition effect of Aβ oligomerization by Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA. The relative amount of Aβ oligomers at 0 (A), 2 (B), and 4 h (C) and the value of Aβ oligomerization area under the curve (D). All data are presented as mean ± SEM (n = 3). A significant difference * (p < 0.05) and ** (p < 0.01) using one-way ANOVA followed by Dunnett’s post-hoc was observed in the reduction in oligomerization vs. the negative control (no treatment). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.

Figure 5

Fibrilization inhibition in the presence of Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA. The values are expressed as the mean ± SD (n = 3). Phenol Red (50 μM) was used as a positive control. A significant difference * (p < 0.05), ** (p < 0.01), and (*** p < 0.001) using one-way ANOVA followed by Dunnett’s post hoc was observed in the reduction in oligomerization vs. the negative control (buffer + Aβ). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.

Figure 6

Cytotoxicity assay of Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA on the SH-SY5Y cells. The cells were treated for 24 h with varying extract concentrations (1, 10, 25, and 50 μg/mL). The cell viability was reported as the percentage of the control group (100%). All data are presented as mean ± SEM (n = 3). A significant difference * (p < 0.05) and ** (p < 0.01) using one-way ANOVA followed by Dunnett’s post hoc was observed in the % of cell viability vs. the control group (no treatment). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.

Figure 7

Neuroprotective effects of Clove extracts (CL–H and CL–EA), Eugenol, β-Caryophyllene, and TMA in H₂O₂-induced neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H₂O₂ (100 μM) treatment. The results indicate % cell viability vs. the control cells mean ± SEM (n = 3). A significant difference */^# (p < 0.05), **/^## (p < 0.01), and ***/^### (p < 0.001), using one-way ANOVA followed by Dunnett’s test, was observed in the % of cell viability vs. untreated cells (^*) and H₂O₂ treated cells (^#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.

Figure 8

Effect of Clove extracts, eugenol, β-Caryophyllene, and TMA on H₂O₂-induced ROS production in SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H₂O₂ (100 μM) treatment. The results indicate the % of the ROS level vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A significant difference */^# (p < 0.05), **/^## (p < 0.01), ***/^### (p < 0.001), and ****/^#### (p < 0.0001) was observed in the % ROS vs. untreated cells (^*) and H₂O₂ treated cells (^#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; ROS: Reactive oxygen species.

Figure 9

Mitochondrial membrane potential in SH-SY5Y cells exposed to 200 μM H₂O₂ for 2 h after 12 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate % ∆Ψm vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A significant difference */^# (p < 0.05), **/^## (p < 0.01), ***/^### (p < 0.001), and ^#### (p < 0.0001) was observed in the % cell viability vs. untreated cells (^*) and H₂O₂ treated cells (^#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.; ∆Ψm: Mitochondrial membrane potential.

Figure 10

Reduced glutathione content in SH-SY5Y cell lysate exposed to 100 μM H₂O₂ for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSH (μM/μg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A significant difference */^# (p < 0.05), **/^## (p < 0.01), and ***/^### (p < 0.001) was observed in comparison to untreated cells (^*) and H₂O₂ treated cells (^#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; GSH: reduced glutathione.

Figure 11

Oxidized glutathione content in the SH-SY5Y cell lysate exposed to 100 μM H₂O₂ for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSSG (μM/μg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A significant difference of */^# (p < 0.05), and **/^## (p < 0.01) was observed in comparison to untreated cells (*) and H₂O_2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.; GSSG: oxidized glutathione.

Figure 12

GSH/GSSG ratio in SH-SY5Y cell lysate exposed to 100 μM H₂O₂ for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSH/GSSG ratio in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A significant difference of */^# (p < 0.05), **/^## (p < 0.01), and ***/^### (p < 0.001) was observed in comparison to untreated cells (*) and H₂O₂ treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; GSH: reduced glutathione; GSSG: oxidized glutathione.

Figure 13

Malondialdehyde (MDA) content in SH-SY5Y cells lysate exposed to 100 μM H₂O₂ for 6 h after 24 h pre-treatment with Clove extracts, Eugenol, β-Caryophyllene, and TMA. The results indicate MDA (μM) in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by One-way ANOVA followed by Dunnett’s test. A significant difference */^# (p < 0.05 was observed in comparison to untreated cells (^*) and H₂O_2-treated cells (^#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; MDA: Malondialdehyde.