Synthetic Cooling Agents in Australian-Marketed E-cigarette Refill Liquids and Disposable E-cigarettes: Trends Follow the U.S. Market.

INTRODUCTION: E-cigarettes are becoming increasingly popular in Australia, especially amongst the younger population. The synthetic cooling molecules WS-3 and WS-23 have been identified in e-cigarette products from the United States and Europe. The extent of inclusion of these synthetic coolants in Australian e-liquids is unknown, particularly in newer disposable e-cigarettes. AIMS AND METHODS: E-cigarettes and e-liquids were purchased within Australia and anonymously donated by Australian users. Nicotine, WS-3, WS-23, and menthol were quantified in the e-liquids using gas chromatography-mass spectrometry (GC-MS). RESULTS: WS-23 and nicotine were detected in all of the disposable e-cigarettes with WS-23 often present in high concentrations. There was no correlation between cooling terms in the flavor name and the inclusion of cooling agents. Only three bottled e-liquids were found to contain WS-23 while none contained WS-3 above the limit of detection. CONCLUSIONS: Synthetic coolants were a common addition in disposable e-cigarettes while rarely added to e-liquid bottle refills. Their inclusion in these products is reflective of trends observed in United States and European e-cigarette products. IMPLICATIONS: The increase in synthetic cooling agents as components of e-liquids, particularly disposable e-cigarette devices, has been observed within Australian samples across a range of brands and flavors. WS-23 was present in every disposable e-cigarette analyzed in this study, often in relatively high concentrations. Its inhalational toxicology should be considered when evaluating the safety of these products.

A Wide Range of Flavoring-Carrier Fluid Adducts Form in E-Cigarette Liquids.

A range of flavoring molecules are used in electronic cigarette liquids (e-liquids), some of which have been shown to form cyclic acetal adducts with e-liquid solvent components propylene glycol (PG) and vegetable glycerine (VG). The objective of this study was to identify the range of flavoring molecules which form adducts in e-liquid products. Common e-liquid flavoring molecules (N = 36) from a range of chemical class groups were exposed to PG, VG, or methanol and analyzed by GC-MS over a time frame of 4 weeks to identify possible reaction products. Adduct formation was observed, with 14 of the flavoring molecules reacting with methanol, 10 reacting with PG, and 10 reacting with VG. Furfural PG and VG acetals, valeraldehyde PG and VG acetals, veretraldehyde PG and VG acetals, p-anisaldehyde PG and VG acetals, and piperonal VG acetal were confirmed for the first time. Adducts formed by reaction with ketone-containing flavoring molecules were also observed for the first time. The presence of these acetals was confirmed in 32% of commercial e-liquid products analyzed (N = 142). This study has established a range of flavoring molecules which are able to react with solvent components PG and VG in e-liquids under standard storage conditions. These newly identified adducts need to be further assessed to determine their toxicological safety.

Nicotine in electronic cigarette fluid: importation pathways to unequal harm.

The Australian Government recently walked away from changes to the importation of nicotine-containing electronic cigarette fluids, originally due to come into force on 1 January 2021. Additionally, the Therapeutic Goods Administration is in the process of rescheduling nicotine for use in e-fluids. We are concerned that the 270 000 daily vapers in Australia will purchase high concentrations of nicotine (≥100 mg/mL) for mixing with nicotine-free locally purchased e-fluids, which is a pathway of increased relative harm. We would like to see regulation of these products to limit the maximum concentration of nicotine, ensure appropriate child-resistant containers and compulsory labelling for all nicotine-containing e-fluid bottles.

Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt Bridge Formation.

Native mass spectrometry (MS) is a powerful means for studying macromolecular protein assemblies, including accessing activated states. However, much remains to be understood about what governs which regions of the protein (un)folding funnel, which can be explored by activation of protein ions in a vacuum. Here, we examine the trajectory that Cu/Zn superoxide dismutase (SOD1) dimers take over the unfolding and dissociation free energy landscape in a vacuum. We examined wild-type SOD1 and six disease-related point mutants by using tandem MS and ion-mobility MS as a function of collisional activation. For six of the seven SOD1 variants, increasing activation prompted dimers to transition through two unfolding events and dissociate symmetrically into monomers with (as near as possible) equal charges. The exception was G37R, which proceeded only through the first unfolding transition and displayed a much higher abundance of asymmetric products. Supported by the observation that ejected asymmetric G37R monomers were more compact than symmetric G37R ones, we localized this effect to the formation of a gas-phase salt bridge in the first activated conformation. To examine the data quantitatively, we applied Arrhenius-type analysis to estimate the barriers on the corresponding free energy landscape. This reveals a heightening of the barrier to unfolding in G37R by >5 kJ/mol-1 over the other variants, consistent with expectations for the strength of a salt bridge. Our work demonstrates weaknesses in the simple general framework for understanding protein complex dissociation in a vacuum and highlights the importance of individual residues, their local environment, and specific interactions in governing product formation.

A study of Pt(II)-phenanthroline complex interactions with double-stranded and G-quadruplex DNA by ESI-MS, circular dichroism, and computational docking.

The binding interactions of a series of square-planar platinum(II)-phenanthroline complexes of the type [Pt(PL)(AL)]2+ [where PL = variously methyl-substituted 1,10-phenanthroline (phen) and AL = ethane-1,2-diamine (en)] were assessed with a G-quadruplex DNA (5'-TTG GGG GT-3', G4DNA) and a double-stranded DNA (5'-CGC GAA TTC GCG-3', dsDNA) sequence by ESI-MS. The results indicate a strong correlation between G4DNA affinity and increasing phenanthroline methyl substitution. Circular dichroism (CD) spectroscopy and molecular docking studies also support the finding that increased substitution of the phenanthroline ligand increased selectivity for G4DNA. ESI-MS was used to probe the interaction of a range of square-planar Pt(II)-phenanthroline complexes with double-stranded and G-quadruplex DNA.

Formaldehyde-activated WEHI-150 induces DNA interstrand crosslinks with unique structural features.

Mitoxantrone is an anticancer anthracenedione that can be activated by formaldehyde to generate covalent drug-DNA adducts. Despite their covalent nature, these DNA lesions are relatively labile. It was recently established that analogues of mitoxantrone featuring extended side-chains terminating in primary amino groups typically yielded high levels of stable DNA adducts following their activation by formaldehyde. In this study we describe the DNA sequence-specific binding properties of the mitoxantrone analogue WEHI-150 which is the first anthracenedione to form apparent DNA crosslinks mediated by formaldehyde. The utility of this compound lies in the versatility of the covalent binding modes displayed. Unlike other anthracenediones described to date, WEHI-150 can mediate covalent adducts that are independent of interactions with the N-2 of guanine and is capable of adduct formation at novel DNA sequences. Moreover, these covalent adducts incorporate more than one formaldehyde-mediated bond with DNA, thus facilitating the formation of highly lethal DNA crosslinks. The versatility of binding observed is anticipated to allow the next generation of anthracenediones to interact with a broader spectrum of nucleic acid species than previously demonstrated by the parent compounds, thus allowing for more diverse biological activities.

BSD2 is a Rubisco-specific assembly chaperone, forms intermediary hetero-oligomeric complexes, and is nonlimiting to growth in tobacco.

The folding and assembly of Rubisco large and small subunits into L8 S8 holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco-BSD2 assembly complexes in the model C3 plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m2 ) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L8 S8 pool. RNAi-silencing BSD2 production in transplastomic tobacco producing bacterial L2 Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi-bsd2 alleles into wild-type tobacco however impaired L8 S8 Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2-silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.

Structure-activity relationships of pyrazole-4-carbodithioates as antibacterials against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-acquired infections and is responsible for significant morbidity and mortality in residential care facilities. New agents against MRSA are needed to combat rising resistance to current antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA structure-activity relationships evaluated and selectivity versus human HKC-8 cells determined. Minimum inhibitory concentrations (MIC) ranged from 0.5 to 64 µg/mL and up to 16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The study supports further work aimed at elucidating the molecular targets of this interesting new class of anti-MRSA agents.

Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone.

Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO2-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco's biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob(AtL) plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob(AtL-R1) plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob(AtL-R1) and Arabidopsis leaves at 10-15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L8(A)S8(t) Rubisco [comprising AtL and tobacco small (S) subunits] in tob(AtL-R1) leaves compared with tob(AtL), despite >threefold lower steady-state Rubisco mRNA levels in tob(AtL-R1). Accompanying twofold increases in photosynthetic CO2-assimilation rate and plant growth were measured for tob(AtL-R1) lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.

Reversible and formaldehyde-mediated covalent binding of a bis-amino mitoxantrone analogue to DNA.

The ability of a bis-amino mitoxantrone anticancer drug (named WEHI-150) to form covalent adducts with DNA, after activation by formaldehyde, has been studied by electrospray ionisation mass spectrometry and HPLC. Mass spectrometry results showed that WEHI-150 could form covalent adducts with d(ACGCGCGT)2 that contained one, two or three covalent links to the octanucleotide, whereas the control drugs (daunorubicin and the anthracenediones mitoxantrone and pixantrone) only formed adducts with one covalent link to the octanucleotide. HPLC was used to examine the extent of covalent bond formation of WEHI-150 with d(CGCGCG)2 and d(CG(5Me)CGCG)2. Incubation of WEHI-150 with d(CG(5Me)CGCG)2 in the presence of formaldehyde resulted in the formation of significantly greater amounts of covalent adducts than was observed with d(CGCGCG)2. In order to understand the observed increase of covalent adducts with d(CG(5Me)CGCG)2, an NMR study of the reversible interaction of WEHI-150 at both CpG and (5Me)CpG sites was undertaken. Intermolecular NOEs were observed in the NOESY spectra of d(ACGGCCGT)2 with added WEHI-150 that indicated that the drug selectively intercalated at the CpG sites and from the major groove. In particular, NOEs were observed from the WEHI-150 H2,3 protons to the H1' protons of G3 and G7 and from the H6,7 protons to the H5 protons of C2 and C6. By contrast, intermolecular NOEs were observed between the WEHI-150 H2,3 protons to the H2'' proton of the (5Me)C3 in d(CG(5Me)CGCG)2, and between the drug aliphatic protons and the H1' proton of G4. This demonstrated that WEHI-150 preferentially intercalates at (5Me)CpG sites, compared to CpG sequences, and predominantly via the minor groove at the (5Me)CpG site. The results of this study demonstrate that WEHI-150 is likely to form interstrand DNA cross-links, upon activation by formaldehyde, and consequently exhibit greater cytotoxicity than other current anthracenedione drugs.

Isolation and structural analysis of the covalent adduct formed between a bis-amino mitoxantrone analogue and DNA: a pathway to major-minor groove cross-linked adducts.

The major covalent adduct formed between a 13C-labelled formaldehyde activated bis-amino mitoxantrone analogue (WEHI-150) and the hexanucleotide d(CG5MeCGCG)2 has been isolated by HPLC chromatography and the structure determined by NMR spectroscopy. The results indicate that WEHI-150 forms one covalent bond through a primary amine to the N-2 of the G2 residue, with the polycyclic ring structure intercalated at the 5MeC3pG4/G10p5MeC9 site. Furthermore, the WEHI-150 aromatic ring system is oriented approximately parallel to the long axis of the base pairs, with one aliphatic side-chain in the major groove and the other side-chain in the minor groove. This study indicates that mitoxantrone derivatives like WEHI-150 should be capable of forming major-minor groove cross-linked adducts that will likely produce considerably different intracellular biological properties compared to known anthracycline and anthracenedione anticancer drugs.

Narrative Review of the Pharmacodynamics, Pharmacokinetics, and Toxicities of Illicit Synthetic Cannabinoid Receptor Agonists.

BACKGROUND: Synthetic cannabinoid receptor agonists (SCRAs) are the most diverse class of new psychoactive substances worldwide, with approximately 300 unique SCRAs identified to date. While the use of this class of drug is not particularly prevalent, SCRAs are associated with several deaths every year due to their severe toxicity. METHODS: A thorough examination of the literature identified 15 new SCRAs with a significant clinical impact between 2015 and 2021. RESULTS: These 15 SCRAs have been implicated in 154 hospitalizations and 209 deaths across the US, Europe, Asia, and Australasia during this time period. CONCLUSION: This narrative review provides pharmacodynamic, pharmacokinetic, and toxicologic data for SCRAs as a drug class, including an in-depth review of known pharmacological properties of 15 recently identified and emerging SCRAs for the benefit of researchers, policy makers, and clinicians who wish to be informed of developments in this field.

Labelling and composition of contraband electronic cigarettes: Analysis of products from Australia.

BACKGROUND: The sale of nicotine-containing electronic cigarettes (e-cigarettes) is prescription only in Australia, regulated under the Standard for Nicotine Vaping Products (TGO110). Australian e-cigarette users, however, are purchasing e-cigarette products outside of the intended pathways. METHODS: The labelling of e-cigarette packaging (N = 388 boxes) and the chemical composition of disposable e-cigarettes and pods (N = 428) were analysed for adherence to the current Australian regulations. These samples were confiscated from over-the-counter retailers in NSW by the NSW Ministry of Health during 2022 for non-compliance with Australian regulations. RESULTS: Following the announcement of the prescription only model for nicotine-containing e-cigarettes in Australia in mid-2021 there was a clear shift in the labelling of products. Any mention of the word 'nicotine' was removed from e-cigarette packaging by early 2022 and nicotine warnings were replaced with generic underage sale warnings. Despite this labelling, the vast majority (98.8 %) of devices analysed contained nicotine, most (89 %) at high concentration (>30 mg/mL) and 4.2 % contained at least one chemical prohibited by the TGO110. CONCLUSIONS: It appears that manufacturers have removed any mention of nicotine from the original packaging of nicotine-containing disposable e-cigarettes to circumvent restrictions on nicotine-containing products and continue their sale. The packaging of e-cigarette products in Australia is generally not indicative of their contents, particularly nicotine, and most did not display required warnings. Ingredients with associated health risks, prohibited in legal vapes by the TGO110, were found in samples. Consequently, the risks of e-cigarette use cannot be appropriately identified from the information supplied on the packaging or device.

Insignoic acids A - E, unusual α, ß-unsaturated keto fatty acids isolated from the exocarp of Australian rainforest tree Endiandra insignis (Lauraceae).

Anti-inflammatory bioassay-guided compound isolation from the exocarp of the Australian rainforest tree Endiandra insignis (family Lauraceae) has led to the discovery and structural elucidation of unusual α, ß-unsaturated twenty-four carbon fatty acids and their positional isomers, insignoic acids A - E (1a - 5c). The stereochemistry and position of the double bond within the aliphatic chain were independently determined via NMR spectroscopy and Ozone-Induced Dissociation (OzID) Mass Spectrometry, respectively. Compounds (1a - 5c) displayed good to moderate anti-inflammatory activity in the range of 8-84 µM. The low therapeutic index observed when assessing the cell viability in the RAW macrophage cell lines, prompted us to investigate the anticancer potential of these unusual fatty acids. The anti-cancer activity was assessed in A-431 carinoma cell lines and MM649 melanoma cell lines. Insignoic acid C (3a-f) exhibited the highest level of potency with an IC50 value of 5-7 µM against both the cell lines. The insignoic acids are the first of their kind known for incorporating an alpha-beta unsaturated system flanked next to a keto group with an additional level of oxygenation at C-6 in a 24­carbon fatty acid backbone.

Continuous Electrospray Ionization Mass Spectrometry Assay for Measuring Phospholipase Activity against Liposomes.

Phospholipases have diverse roles in lipid and cell membrane biology. In animal venoms, they can have roles as neurotoxins or myotoxins that disrupt the integrity of cell membranes. In this work, we describe a temperature-controlled, continuous electrospray ionization mass spectrometry (ESI-MS) assay for measuring phospholipase A2 activity against liposomes. The enzyme used in this assay was paradoxin, which is a neurotoxic trimeric phospholipase A2 from inland taipan snake venom. Previously developed ESI-MS-based phospholipase assays have been discontinuous and analyzed hydrolysis of single lipid molecules by liquid chromatography ESI-MS. In this work, a continuous assay was developed against liposomes, a more complex substrate that more closely reflects the natural substrate for paradoxin. The assay confirmed the requirement for Ca2+ and allowed measurement of Michaelis-Menten-type parameters. The use of ESI-MS for lipid detection enabled nuanced insights into the effect of changing assay conditions not only on the enzyme but also on the liposome substrate. Changing the metal ion concentrations did not significantly change the liposomes but did affect enzymatic activity. Increasing temperature did not substantially affect the secondary structure of paradoxin but affected liposome size, resulting in increased enzymatic activity consistent with the disruption of the phosphatidylcholine membrane, increasing accessibility of sn-2 ester bonds. The continuous ESI-MS method described herein can be applied to other enzyme reactions, particularly those which utilize complex lipid substrates.

Effects of sewer biofilms on the degradability of carbapenems in wastewater using laboratory scale bioreactors.

Carbapenems are last-resort antibiotics used to treat bacterial infections unsuccessfully treated by most common categories of antibiotics in humans. Most of their dosage is secreted unchanged as waste, thereby making its way into the urban water system. There are two major knowledge gaps addressed in this study to gain a better understanding of the effects of their residual concentrations on the environment and environmental microbiome: development of a UHPLC-MS/MS method of detection and quantification from raw domestic wastewater via direct injection and study of their stability in sewer environment during the transportation from domestic sewers to wastewater treatment plants. The UHPLC-MS/MS method was developed for four carbapenems: meropenem, doripenem, biapenem and ertapenem, and validation was performed in the range of 0.5-10 µg/L for all analytes, with limit of detection (LOD) and limit of quantification (LOQ) values ranging from 0.2-0.5 µg/L and 0.8-1.6 µg/L respectively. Laboratory scale rising main (RM) and gravity sewer (GS) bioreactors were employed to culture mature biofilms with real wastewater as the feed. Batch tests were conducted in RM and GS sewer bioreactors fed with carbapenem-spiked wastewater to evaluate the stability of carbapenems and compared against those in a control reactor (CTL) without sewer biofilms, over a duration of 12 h. Significantly higher degradation was observed for all carbapenems in RM and GS reactors (60 - 80%) as opposed to CTL reactor (5 - 15%), which indicates that sewer biofilms play a significant role in the degradation. First order kinetics model was applied to the concentration data along with Friedman's test and Dunn's multiple comparisons analysis to establish degradation patterns and differences in the degradation observed in sewer reactors. As per Friedman's test, there was a statistically significant difference in the degradation of carbapenems observed depending on the reactor type (p = 0.0017 - 0.0289). The results from Dunn's test indicate that the degradation in the CTL reactor was statistically different from that observed in either RM (p = 0.0033 - 0.1088) or GS (p = 0.0162 - 0.1088), with the latter two showing insignificant difference in the degradation rates observed (p = 0.2850 - 0.5930). The findings contribute to the understanding about the fate of carbapenems in urban wastewater and the potential application of wastewater-based epidemiology.

Vaping preferences of individuals who vaporise dry herb cannabis, cannabis liquids and cannabis concentrates.

In 2019 an estimated 200 million people aged 15-64 used cannabis, making cannabis the most prevalent illicit substance worldwide. The last decade has seen a significant expansion in the cannabis vaporiser market, introducing cannabis vaporisation as a common administration method alongside smoking and ingestion. Despite reports of increased prevalence of cannabis vaporisation there has been little research into the use of these devices. To remedy the current dearth of data in this area this study utilised an anonymous online survey of individuals who self-reported past cannabis vaporisation. The respondents (N = 557) were predominantly young (<35 years) and male. Most (91.4 %) stated they had ever vaped dry herb cannabis, 59.1 % reported vaporisation of cannabis oil or liquids, and 34.0 % reported vaporisation of cannabis concentrates. This study identifies the types of vaporisation devices (including brands and models) employed by cannabis vapers, as well as the vaporisation temperatures and puff durations commonly used for dry herb, cannabis liquids and cannabis concentrates. To the best of our knowledge, this is the first time the usual operating temperatures of these vaporisation devices and user specific consumption patterns such as puff duration have been reported for cannabis vaping. This information will allow for a more realistic understanding of patterns of recreational use and improve experimental conditions in research settings to reflect the user's context.

Impact of molar absorbance on anthocyanin content of the foods.

Anthocyanins are present in bright colored fruit and vegetables with growing evidence for their health benefits. Several methods exist in the literature to measure the total monomeric anthocyanin content in foods. Although the simplest method uses UV-Vis spectrophotometry, it requires the use of anthocyanin molar absorption coefficients (Ɛ). While commonly reported for some compounds, these values vary substantially between studies. This study collated and compared existing Ɛ values for a range of anthocyanin-3-glucosides, measured new Ɛ values for these compounds and underwent an inter-laboratory validation of spectrometry methods. The Ɛ values used for the determination of anthocyanin content in Australian blueberries, were shown to greatly affect the estimated total anthocyanin. Significant differences in the Ɛ values were observed when measured at 520 nm, or their absorbance maximum and substantial difference in the estimated total anthocyanins were observed when expressed as equivalent of cya-3-glu or mal-3-glu.

Molecular networking based dereplication of AChE inhibitory compounds from the medicinal plant Vincetoxicum funebre (Boiss. & Kotschy).

Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting 47 million people worldwide. While acetylcholinesterase (AChE) inhibitors such as donepezil and galantamine are leading drugs in the symptomatic treatment of AD, new AChE inhibitors continue to be explored for improved potency and selectivity. Herein, a molecular networking approach using high resolution (HR-MS) and tandem mass spectrometry (MS2) has been used for rapid chemical profiling of an extract of the medicinal plant Vincetoxicum funebre Boiss. & Kotschy (Apocynaceae family) that was active against AChE. A total of 44 compounds were identified by combining the MN with traditional natural product methods, including the isolation and identification of five known compounds (13, 41-44) and a novel C13-norisoprenoid (40). In addition, the potential inhibitory activity of all 44 compounds was evaluated against the AChE enzyme via molecular docking to provide further support to the proposed structures. The glycosylated flavonoid querciturone (31) exhibited the highest affinity with a docking score value of -13.43 kJ/mol. Another five compounds showed stronger docking scores against AChE than the clinically used donepezil including the most active isolated compound daucosterol (44), with a binding affinity of -10.11 kJ/mol towards AChE. These findings broaden our understanding of Vincetoxicum metabolites and highlight the potential of glycosylated flavonoids as AChE inhibitors.Communicated by Ramaswamy H. Sarma.