Neuroprotective Effects of Chemical Constituents of Leaves of Euonymus hamiltonianus Wall.

Euonymus hamiltonianus Wall. is considered a medicinal plant and is used to treat pain, cough, dysuria, and cancer, but a clear phytochemical investigation of its biological activities has yet to be performed. Investigation of chemical constituents of the leaves of Euonymus hamiltonianus Wall. led to the isolation of three new compounds by chromatography techniques, euonymusins A–C (1, 10, and 11), and the acquisition of new spectroscopic data for euonymusin D (2), along with the identification of ten known compounds. The chemical structures of the compounds were established using extensive spectroscopic techniques, including NMR, MS, and hydrolysis, and compared with the published data. These compounds were tested in vitro for their inhibitory effects on beta amyloid production (Aβ42). Compounds 13 and 14 displayed weak inhibition, with IC50 values ranging from 53.15 to 65.43 µM. Moreover, these compounds were also assessed for their inhibitory effects on nitric oxide production. Of these compounds, 3, 4, and 14 displayed inhibitory effects on NO production, with IC50 values ranging from 14.38 to 17.44 µM. Compounds 3, 4, and 14 also suppressed LPS-induced expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein.


Introduction
Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS), affect the lives of millions of patients across the world and cause a variety of socio-economic problems [1].The main reasons for these symptoms are the formation and accumulation of extracellular senile plaques of amyloid beta (Aβ) [2,3].It was reported that Aβ is the main component and plays an important role in AD expression [4], causing astrocyte activation and neuronal apoptosis, leading to the deterioration of AD.Aβ is produced by proteolysis of amyloid precursor protein (APP), a type I transmembrane protein.APP can be cleaved in mediated stages through amyloidogenic pathways by β-secretase 1 (BACE1) and then γ-secretase [5].Therefore, inhibition of the aggregation of Aβ peptides is considered one of the potential pieces of evidence for the neuroprotective effect of natural products in the progression of neurodegenerative diseases [6].Additionally, during this progress, microglial cells are a type of CNS immune cell which play an essential role in immune defense and tissue repair with a self-limiting mechanism.Over-activation of microglia has been determined as a property of neurodegenerative diseases, leading to the production of various proinflammatory cytokines and neurotoxic substances, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α), and nitric oxide (NO) [1].NO is a product of the inducible isoforms of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) enzymes.It was demonstrated Plants 2024, 13, 1094 2 of 13 that iNOS is not normally expressed in the brain, but LPS upregulates iNOS expression in microglial cells, astrocytes, and possibly neurons [7].Two forms of COX, COX-1 and COX-2, are known to be induced in response to many stimulants and to be activated in sites of inflammation [8].Several studies have reported that COX-2 is associated with cytotoxicity in neurodegenerative diseases.Taken together, the inflammatory mediators, including iNOS and COX-2, are responsible for the symptoms of severe neuronal damage in neurodegenerative diseases [7].
Phytochemical compounds, originating from plant sources, have demonstrated promising capabilities in the treatment of neurodegenerative conditions [9].As more and more evidence suggests that chronic inflammation caused by microglia contributes to disorders in the central nervous system, it is becoming clear that anti-inflammatory natural products and their components can serve as powerful protectors against a range of CNS pathologies [10].Euonymus hamiltonianus Wall. is a medicinal plant belonging to the Celastraceae family, which is distributed in many Asian countries, including Korea, Japan, China, and India [9].This indigenous plant is a deciduous tree that has been used for traditional treatment of various conditions, such as diuretic, carminative, stimulant, gynecological, coughing, pain, and cancer-related diseases [10].This medicinal plant species, E. hamiltonianus, was used to treat pain, cough, dysuria, and cancer and exhibited effects of enhanced memory and cognitive abilities when applied as a functional food in the prevention and treatment of cognitive impairment-related diseases [11,12].The genus Euonymus of the family Celastraceae has been investigated and commonly compared to the species E. hamiltonianus.The genus Euonymus is reported to contain sesquiterpenes, flavonoids, and coumarins [13][14][15].As of this moment, there are only a limited number of reports on the phytochemistry and related activities performed by E. hamiltonianus.Previous studies have shown some of its phytochemicals by identifying novel coumarins in this indigenous plant, but these phytochemicals did not perform any related bioactivity [16].Furthermore, to date, only three triterpenoids, six sesquiterpenoids, two flavonoids, and two coumarins have been identified in this plant species, and further in-depth phytochemical investigation of E. hamiltonianus is still needed [17][18][19][20].

Chemical Profiling of Phytochemicals 1-14
In this study, qualitative profile analysis was performed on the 30% EtOH crude extract of the leaves of E. hamiltonianus using HPLC.The isolated phytochemicals, 1-14, were used as standards and compared to the crude extract profile represented by a highperformance liquid chromatogram (Figure 3).While analyzing the chemical profile of the HPLC chromatogram, the isolated compound 2 showed signs of degradation.With comparative UV area normalization, compound 2 was confirmed as a pure compound initially when isolated (Figure S3.8).After compound 2 was stored for 27 days, the compound was re-evaluated through LC-MS analysis and showed signs of degradation (Figure S3.9).For reconfirmation, LC-MS analyses of both the dried powdered form of the compound when isolated and the compound in DMSO solution were performed (Figure S3.9), both sets of results showing signs of degradation.Compound 2 was degraded into three separate peaks, and the original compound 2 indicated by m/z 759.2 was verified by LC-MS analysis, and the peaks of 2a and 2b were indicated by m/z 465.1 and 627, respectively.This suggested that the original compound 2 was partially degraded into different quercetin glycosides, with possible hydrolysis of the sugar moieties.Due to the close resemblance of compound 2 to compound 1, it was investigated in the same manner after a significant length of time.Compound 1 did not show any sign of degradation (Figure S3.10), and the structural difference between 1 and 2 is the substitution of a methoxy group at C-3 ′ .Previous studies suggested that the presence of abundant hydroxyl groups in the chemical structures of flavonoids can facilitate degradation, whereas substituents of sugar moieties and methoxyl groups can possibly protect against degradation of flavonoids [35,36].

Inhibition of the Aβ Aggregation of Compound 13
Preventing the harmful aggregation of Aβ peptides has been indicated to be a promising strategy against neurodegenerative disease [37].Phytomedicines have been widely reported to be utilized in the treatment of neurodegenerative diseases and other conditions associated with Aβ [38].The total extraction and fractions (Figure S1) of E. hamiltonianus displayed inhibitory effects against Aβ production [19].Secretion levels of Aβ42 were significantly decreased by EA and BuOH fractions, with inhibitions of 50.6% and 50.4% at 25 µg/mL, respectively.All isolated compounds were tested for their inhibitory activity against Aβ42 production in APPsw-transfected HeLa cells.Among them, compound 13 showed weak inhibition of Aβ42 production with an IC50 value of 65.43 µM (Table 2).

Inhibition of the Aβ Aggregation of Compound 13
Preventing the harmful aggregation of Aβ peptides has been indicated to be a promising strategy against neurodegenerative disease [37].Phytomedicines have been widely reported to be utilized in the treatment of neurodegenerative diseases and other conditions associated with Aβ [38].The total extraction and fractions (Figure S1) of E. hamiltonianus displayed inhibitory effects against Aβ production [19].Secretion levels of Aβ42 were significantly decreased by EA and BuOH fractions, with inhibitions of 50.6% and 50.4% at 25 µg/mL, respectively.All isolated compounds were tested for their inhibitory activity against Aβ42 production in APPsw-transfected HeLa cells.Among them, compound 13 showed weak inhibition of Aβ42 production with an IC 50 value of 65.43 µM (Table 2).The accumulation of Aβ is a characteristic trait of both hereditary and random occurrences of AD.The intense focus of pharmaceutical developments for the treatment of AD is to decrease Aβ levels, a task that seems to be quite difficult [39].Inhibitors that have been directly proven to be effective in reducing Aβ production in the lab have encountered potential issues during clinical trials [40].This study shows that only compound 13 (Table 2) reduced Aβ levels in Hela-swAPP cells.This suggests that while compound 13 may not be highly potent or efficient, it does interact with Aβ in a way that results in inhibition.The inability to detect decreased Aβ in the supernatants of Hela cells could be due to the extremely low amounts of the peptide these cells secrete.Considering the relative content in the extract, the effect was similar between the subfraction Bu6 + 7 (Figure S1) and compound 13, and this indicates that 13 may possibly alleviate AD via targeting anti-β-amyloidosis during the pathogenesis of AD.

Anti-Inflammation Effect of the Isolated Compounds 3, 4, and 14
The cytotoxic effects of the isolated compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) were evaluated by the MTT assay and did not affect the viability of BV-2 microglial cells following 12 h of treatment in the presence or absence of LPS, even at a concentration of 100 µM (Table 2).The anti-inflammatory effects of the fourteen compounds (1-14) isolated from E. hamiltonianus were evaluated in activated BV-2 microglial cells by the production of NO and proinflammatory cytokines.BV-2 microglial cells were stimulated with 0.1 µg/mL LPS for 12 h in the presence of the compounds at various concentrations, and the levels of NO in the culture supernatants were measured using Griess reactions.Dexamethasone was used as a positive control compound, with an IC 50 value of 1.24 µM.The results of the NO production assay, including IC 50 values, are summarized in Table 2.Among them, those for compounds 3, 4, and 14 are displayed, their IC 50 values ranging from 14.38 to 17.44 µM, but the other compounds were inactive.In a comparison of the activities of the pair of 4 (IC 50 = 14.38 µM) and 5 (IC 50 > 100 µM), the substituted hydroxy group was indicated to have a more potent activity against NO production than methoxy substitution at position C-3 ′ .In the same manner, the substituted sugar moiety at C-3 also showed a different inhibition of NO production compared with 3 (galactose, IC 50 = 14.38 µM) and 6 and 7 (sambubiose and glucose, respectively; IC 50 > 100 µM), showing greater potential inhibition for the substituted galactopyranoside unit.The compounds were continuously assessed to determine whether they suppressed the expression of iNOS and COX-2 in LPS-stimulated BV-2 microglial cells.The cells were stimulated with LPS in the presence of these compounds at various concentrations, and the expression levels of iNOS and COX-2 were determined by Western blotting.The results indicated that 3, 4, and 14 also downregulated expression levels of LPS-induced iNOS and COX-2 proteins in a concentration-dependent manner (Figure 4).Among them, compound 3 exhibited the most potent inhibitory activity against LPS-induced iNOS and COX-2 expression at treatment concentrations of 1.0, 2.0, and 4.0 µM.Although compound 3 inhibited NO production to a somewhat lesser extent than compound 4, 3 significantly suppressed iNOS and COX-2 protein expression compared to 4. Thus, compound 3 may be considered a potential anti-inflammatory agent.
the other compounds were inactive.In a comparison of the activities of the pair of 4 (IC50 = 14.38 µM) and 5 (IC50 > 100 µM), the substituted hydroxy group was indicated to have a more potent activity against NO production than methoxy substitution at position C-3′.In the same manner, the substituted sugar moiety at C-3 also showed a different inhibition of NO production compared with 3 (galactose, IC50 = 14.38 µM) and 6 and 7 (sambubiose and glucose, respectively; IC50 > 100 µM), showing greater potential inhibition for the substituted galactopyranoside unit.The compounds were continuously assessed to determine whether they suppressed the expression of iNOS and COX-2 in LPS-stimulated BV-2 microglial cells.The cells were stimulated with LPS in the presence of these compounds at various concentrations, and the expression levels of iNOS and COX-2 were determined by Western blotting.The results indicated that 3, 4, and 14 also downregulated expression levels of LPS-induced iNOS and COX-2 proteins in a concentration-dependent manner (Figure 4).Among them, compound 3 exhibited the most potent inhibitory activity against LPS-induced iNOS and COX-2 expression at treatment concentrations of 1.0, 2.0, and 4.0 µM.Although compound 3 inhibited NO production to a somewhat lesser extent than compound 4, 3 significantly suppressed iNOS and COX-2 protein expression compared to 4. Thus, compound 3 may be considered a potential anti-inflammatory agent.Lipopolysaccharide (LPS), a key constituent of the cell wall in Gram-negative bacteria, is instrumental in triggering inflammation, with the ability to activate immune cells and provoke an inflammatory reaction [41].And microglia residing in the CNS plays a crucial role in preserving the balance within its microenvironment.When microglia are activated by LPS, they release a large number of cytotoxic mediators, such as NO, causing neuronal damage [42].Phytochemicals, which are secondary metabolites produced by plants, serve as a defense mechanism against environmental pressures like LPS-induced inflammatory stress [43].The treatments with compounds 3, 4, and 14 effectively Lipopolysaccharide (LPS), a key constituent of the cell wall in Gram-negative bacteria, is instrumental in triggering inflammation, with the ability to activate immune cells and provoke an inflammatory reaction [41].And microglia residing in the CNS plays a crucial role in preserving the balance within its microenvironment.When microglia are activated by LPS, they release a large number of cytotoxic mediators, such as NO, causing neuronal damage [42].Phytochemicals, which are secondary metabolites produced by plants, serve as a defense mechanism against environmental pressures like LPS-induced inflammatory stress [43].The treatments with compounds 3, 4, and 14 effectively downregulated the production of inflammatory mediators, including NO and iNOS, against LPS.Moreover, some research reports have found that certain phytochemicals can inhibit the production of NO and iNOS without inhibiting COX-2 [44].In our study, a similar phenomenon was observed in the treatment of compound 14, which showed no significant effect in terms of COX-2 inhibition.It was indicated that induced COX-2 can mobilize excessive release of PGE2, which can be a great booster for the progress of inflammation [45].Therefore, inhibition of COX-2 will also be an essential effect when investigating the anti-inflammatory activity of natural products.Combined with iNOS and NO inhibition, compounds 3 and 4 showed significant anti-inflammatory activity, and compound 3 in particular can be a potential target for potential development as an anti-inflammation constituent of E. hamiltonianus.

Plant Materials
The leaves of E. hamiltonianus Wall.were collected at Hantaek Botanical Garden (37.094376, 127.406739; entrance of the botanical garden) in Yongin-si, Gyeonggi-do, Republic of Korea, from August to October 2019.Botanical identification was performed by Prof. Hyun Ok Yang, and the voucher specimen KGC07EH was deposited at the Herbarium of the Korea Institute of Science and Technology (Gangneung, Gangwon-do, Republic of Korea).

Hydrolysis of 1
Compound 1 (1.0 mg) was hydrolyzed by 4.0 mL of 5% HCl/MeOH for 3 h at 95 • C.After the reaction, the mixture was dried and separated in one part CH 2 Cl 2 to three parts water (15 mL CH 2 Cl 2 ), after which the water residue was evaporated in vacuo to dryness with MeOH, then analyzed by HPLC, and the results were compared with authentic samples [20].

Aβ Assay
APPsw-transfected HeLa cells were cultured with the isolated compounds or DMSO in Dulbecco's modified Eagle medium (DMEM, Welgene, Gyeongsan-si, Republic of Korea) for 8 h, and then the cell culture supernatants were collected for the following analyses.For the detection of Aβ secretion, kits for Aβ42 (KHB3442) and Aβ40 (KHB3482) were purchased from the Invitrogen Company and used according to the supplier's instructions [46].

Cell Cultures and MTT Assay for Cell Viability
For the Aβ-Hela-APPsw model, Hela cells were stably transfected with an APP carrying the Swedish mutation (APPsw) using BioT (Bioland Scientific LLC, Paramount, CA, USA) according to the manufacturer's instructions, which was provided by Prof. Tae-Wan Kim (Department of Pathology, Columbia University of Medical Center, New York, NY 10032, USA).APPsw-transfected HeLa cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Lafayette, CA, USA), 1% penicillin/streptomycin (Thermo Fisher Scientific, Lafayette, CA, USA), 260 µg/mL Zeocin, and 400 µg/mL G418.The cells were maintained at 37 • C under a humidified atmosphere of 5% CO 2 .The stock sample solution was dissolved in DMSO and kept at −20 • C and diluted to the final concentration in fresh medium before each experiment.For the cell viability assay, Hela cells were cultured in 96-well plates for 24 h and then various concentrations of comp 27 (0-10 µM) were treated for 12 h.The EZ-Cytox kit (DAEILLAB, Cheongwon, Republic of Korea) was applied to detect the cell viability, following the manufacturer's instructions, and measurements were taken by a microplate reader (BIO-TEK ® Dower-Wave; BioTek, Winooski, VT, USA) under absorbance at 450 nm.The results were presented as the percentage of MTT reduction, compared with the control group as 100%.For the LPS-induced microglia model, the BV-2 mouse microglia cell line was provided by Prof. Lee Sung Jung's research team in the College of Medicine, Seoul National University, and was cultured in DMEM-F12 with 5% FBS and 1% penicillin/streptomycin at 37 • C under a humidified atmosphere containing 5% CO 2 .And the cell viability assay was performed using the Aβ-Hela-APPsw model.

Determination of NO Production and the Cell Viability Assay
Cells were grown in 6-well plates (5 × 10 5 cells per well) for 24 h, and after incubation for 24 h, the cells were pre-administrated 5, 10, and 20 µM concentrations of the sample for 1 h, then treated with 0.1 µg/mL LPS for 12 h.The media were collected and centrifuged at 13,000 rpm for the removal of dead cells.The supernatants were collected and mixed with an equal volume (50 µL) of Griess reagents (1% sulfanilamide and N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% H 3 PO 4 ) for 5 min at room temperature, and the Nitric Oxide concentration was measured at 540 nm using a microplate reader and compared with the sodium nitrite standard curve.

Western Blot Analysis
The total protein samples (10 µg) were loaded in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels with 10% acrylamide/bis and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA).Then, the membranes were blocked with 5% silky milk dissolved in TBST (Tris Buffered saline Tween) for 1 h under room temperature and incubated overnight at 4 • C with specific primary antibodies (1:1000).After being washed with TBST, the membranes were incubated with secondary HRP-conjugated IgG (1:2000) at room temperature for 1 h and visualized using enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific, Lafayette, CA, USA).Densitometry analysis of the bands was performed with the fusion solo system (Vilber, Paris, France).The band densities were determined as the intensities of protein by TotalLab TL120 software v2009 (TotalLab, Newcastle, UK).

Statistical Analysis
Data were expressed as the mean ± SD of each independent repeat.For comparison of three or more repeats, data were analyzed by one-way analysis of variance (ANOVA) with Dunnett's post hoc test.A value of p < 0.05 was considered statistically significant.Statistical tests were carried out using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).

Conclusions
The chemical investigation of the leaves of E. hamiltonianus was carried out and brought a general phytochemical view with a structural characterization of eight flavonols (1-8), one lignan (9), and five phenols (10-14, including one isocoumarin and four phenolic derivatives).Among them, one novel flavonoid glycoside (1), one phenolic acid (10), and one coumarin (11) were identified as new compounds, and this is the first study to present the purified spectroscopic data of euonymusin D (2).The neuroprotective effects of the isolated compounds were elucidated via assessment of their inhibition of Aβ and NO production.Their inhibitory effects were weak with respect to Aβ production, whereas compounds 3, 4, and 14 displayed good inhibitory effects against NO production.Furthermore, these compounds suppressed the expression levels of iNOS and COX-2 in LPS-stimulated BV-2 microglial cells.This also suggests that the isolated compounds 3, 4, and 14 may be a possible target for future neuroprotective agents.Among published studies, this is the first to report an investigation into the relationship between secondary metabolites obtained from E. hamiltonianus species and their bioactivity.

Figure 1 .
Figure 1.The chemical structures of compounds (1-14) isolated from the leaves of E. hamiltonianus Wall.

Figure 1 .
Figure 1.The chemical structures of compounds (1-14) isolated from the leaves of E. hamiltonianus Wall.Compound 2 was obtained as a yellowish, amorphous powder.HR-ESIMS data showed a molecular ion peak at m/z 759.2010 [M + H] + (calcd.for C 32 H 39 O 21 , 759.1978), indicating a molecular formula of C 32 H 38 O 21 .Its IR spectrum showed the presence of hydroxyl (3309 cm −1 ), carbonyl (1731 cm −1 ), and aromatic ring (1454 cm −1 ) absorption bands.The UV spectrum suggested the appearance of the highest absorption band at 250 nm.The spectroscopic data of 2 closely resembled those of 1 (Table1), except for the fact the latter had one less methoxy group. 2 was previously identified as quercetin-3-O-(2 ′′ -O-(β-D-xylopyranosyl)-β-D-glucopyranoside)-7-O-β-D-glucopyranoside by the personal empirical experience of Fiasson, K. G. and co-workers without significant spectroscopic data to support the identification (Figure1)[23].This study reports the first full spectroscopic data for a purified compound, 2 (FigureS3.1-S3.6).Therefore, the structure of 2 was completely determined, and it was named euonymusin D.

Figure 4 .
Figure 4. Inhibition of LPS-induced iNOS and COX-2 expression in BV-2 microglial cells by 3, 4, and 14.Respective Western blots presenting the expression level of iNOS and COX-2.Each group of experiments was repeated three times (n = 3).

Figure 4 .
Figure 4. Inhibition of LPS-induced iNOS and COX-2 expression in BV-2 microglial cells by 3, 4, and 14.Respective Western blots presenting the expression level of iNOS and COX-2.Each group of experiments was repeated three times (n = 3).
a Cells were pretreated with isolated compounds (50 µM) for 1 h and incubated with 100 ng/mL LPS for 24 h.The results are presented as means ± SDs (n = 3).b Positive control-not detected.