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• Isoquinoline alkaloids were tested against cholinesterases in vitro and in silico. • N -norgalanthamine is the most active one inhibiting both cholinesterases. • 8-Hydroxy-9-methoxycrinine was a strong inhibitor of AChE. • (+)-9- O -demethylhomolycorine displayed a moderate BChE inhibition. • This is the first report on ChE inhibition by three off the tested alkaloids. All cholinesterase (ChE) inhibitory drugs in clinical use are nitrogenous compounds and among them, galanthamine as the latest anticholinesterase drug, contains a tetrahydroisoquinoline structure. For this reason, isoquinoline alkaloids seem to be attractive in search for novel drug candidates towards Alzheimer's disease (AD). Therefore, in the current work, we investigated inhibitory capacity of several alkaloids representing expanding group of biogenetically-related isoquinoline alkaloids from the Amaryllidaceae including lycorine, (+)-9- O -demethylhomolycorine, 6-hydroxybuphanisine, 9- O -demethoxymontanine, 3- epi- hydroxy-bulbispermine, tazettine, haemanthamine, (+)-haemanthidine, (-)-crinine, 8-hydroxy-9-methoxycrinine, and N -norgalanthamine against ChEs by microtiter enzyme inhibition assays. Our findings indicated that N -norgalanthamine is the most active one inhibiting both acetylcholinesterase (AChE, IC 50 = 2.42 ± 0.16 µg/mL) and butyrylcholinesterase (BChE, IC 50 = 20.87 ± 1.01 µg/mL) at 100 μg/mL, whereas IC 50 values of galanthamine used as the reference were 1.33 ± 0.11 µg/mL and 37.69 ± 2.93 µg/mL for AChE and BChE, respectively. Beside this, 8-hydroxy-9-methoxycrinine was a strong inhibitor of AChE (IC 50 = 6.92 ± 0.51 µg/mL), while (+)-9- O -demethylhomolycorine displayed a moderate BChE inhibition (IC 50 = 83.57 ± 1.36 µg/mL). Relevantly, 8-hydroxy-9-methoxycrinine and N -norgalanthamine were docked into active gorges of hAChE (PDB: 4EY6) and hBChE (PDB: 4TPK), which pointed out the detail that N -norgalanthamine displayed a very similar binding mode of to that of galanthamine. Moreover, both AChE-inhibiting alkaloids were found to stably bind to hAChE active site occupying the middle of the gorge between the catalytic site and the peripheral anionic site. 8-Hydroxy-9-methoxycrinine was also docked into the active site of BChE. We can conclude that among the tested alkaloids, N -norgalanthamine with dual ChE inhibitory effect seems to be the most promising anti-Alzheimer candidate molecule for future experiments. [ABSTRACT FROM AUTHOR]

Pteryxin is a dihydropyranocoumarin derivative found in Apiaceae family. In this study, pteryxin, which was previously isolated from the fruits of Mutellina purpurea , was investigated for its inhibitory potential against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which are the key enzymes in the pathology of Alzheimer's disease (AD). The compound was tested in vitro using ELISA microplate reader at 100 μg/ml and found to cause 9.30 ± 1.86% and 91.62 ± 1.53% inhibition against AChE and BChE, respectively. According to our results, pteryxin (IC 50 = 12.96 ± 0.70 μg/ml) was found to be a more active inhibitor of BChE than galanthamine (IC 50 = 22.16 ± 0.91 μg/ml; 81.93± 2.52% of inhibition at 100 μg/ml). Further study on pteryxin using molecular docking experiments revealed different possible binding modes with both polar and hydrophobic interactions inside the binding pocket of BChE. Top docking solution points out to the formation of two hydrogen bonds with the catalytic residues S198 and H438 of BChE as well as a strong π - π stacking with W231. Therefore, pteryxin as a natural coumarin seems to be a strong BChE inhibitor, which could be considered as a lead compound to develop novel BChE inhibitors for AD treatment. [ABSTRACT FROM AUTHOR]

Context:Xeranthemum annuumL. (Asteraceae) (XA) is an ornamental and medicinal species with limited bioactivity and phytochemical data. Objective:Identification of anticholinesterase, antioxidant, anti-inflammatory and analgesic effects of the flower and root–stem (R-S) extracts of XA. Materials and methods:Anticholinesterase (at 100 μg mL−1) and antioxidant (at 1000 μg mL−1) effects of various extracts were evaluated via microtiter assays, while anti-inflammatory and analgesic effects of the R-S extracts were tested using carrageenan-induced hind paw oedema (100 and 200 mg kg−1) andp-benzoquinone (PBQ) writhing models (200 mg kg−1) in male Swiss albino mice. The R-S ethanol extract of XA was subjected to isolation studies using conventional chromatographic methods. Results:Most of the extracts showed inhibition over 85% against butyrylcholinesterase and no inhibition towards acetylcholinesterase. The flower chloroform and the R-S ethyl acetate extracts were most effective (97.85 ± 0.94% and 96.89 ± 1.09%, respectively). The R-S ethanol extract displayed a remarkable scavenging activity against DPPH (77.33 ± 1.99%) and in FRAP assay, while the hexane extract of the R-S parts possessed the highest metal-chelating capacity (72.79 ± 0.33%). The chloroform extract of the R-S caused a significant analgesic effect (24.4%) in PBQ writhing model. No anti-inflammatory effect was observed. Isolation of zierin and zierin xyloside, which were inactive in anticholinesterase assays, was achieved from the R-S ethanol extract. Discussion and conclusion:This is the first report of anticholinesterase, antioxidant, analgesic and anti-inflammatory activities and isolation of zierin and zierin xyloside from XA. Therefore, XA seems to contain antioxidant and BChE-inhibiting compounds. [ABSTRACT FROM AUTHOR]

Abstract: In the current study, neuroprotective effect of the essential oil and aromatic waters of rose was investigated by in vitro and in silico methods. The samples were tested for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Since oxidative damage is associated with neurodegeneration, antioxidant activity of the samples was determined by DPPH radical scavenging, metal-chelation, and ferric-reducing antioxidant power (FRAP) assays. Their chemical composition was elucidated by GC–MS. Anti-cholinesterase effects of the main components (citronellol, geraniol, nerol, and phenylethyl alcohol) were also examined. The rose oil showed a noteworthy inhibition against AChE (60.86±1.99%) and BChE (51.08±1.70%) at 1000μg/mL and low activity in DPPH radical scavenging and FRAP tests. Phenylethyl alcohol exerted higher cholinesterase inhibition than other components and applied further to molecular docking simulations. Docking and binding energies supporting experimental results show that the compound is more selective towards BChE than AChE. [Copyright &y& Elsevier]

Abstract: In this study, cholinesterase inhibitory potential relevant to Alzheimer''s disease and antioxidant activities of the dichloromethane, ethyl acetate, and ethanol extracts from the aerial parts and/or roots of fourteen Salvia (sage) species (S. argentea, S. bracteata, S. caespitosa, S. cryptantha, S. glutinosa, S. indica, S. microstegia, S. multicaulis, S. pinnata, S. quezelii, S. syriaca, S. tobeyi, S. verticillata subsp. amasiaca, and S. viscosa) were investigated. Cholinesterase inhibition was determined by Ellman method at 25, 50, and 100μgml−1. Their antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, metal-chelation capacity, and ferric-reducing antioxidant power (FRAP) assays. Total phenol and flavonoid contents of the extracts were also determined. The dichloromethane and ethanol extracts of the aerial parts of S. cryptantha were the most active ones against AChE (56.22±1.07%) and BChE (33.80±4.18%), respectively. The ethanol extracts exhibited better scavenging activity and FRAP. Anticholinesterase activity of 9 Salvia species (S. argentea, S. bracteata, S. caespitosa, S. indica, S. pinnata, S. quezelii, S. syriaca, S. tobeyi, and S. viscosa) is reported for the first time in the current study. [Copyright &y& Elsevier]

Abstract: In the current study, the n-hexane, chloroform, ethyl acetate, water, and n-butanol fractions obtained from the main ethanol extract of Cistus laurifolius L. were evaluated for their cholinesterase inhibitory effects against acetyl- (AChE) and butyrylcholinesterase (BChE), at 50, 100, and 200μg/ml, using an ELISA microplate reader. The antioxidative effect of the extract and fractions was also determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferrous ion-chelation capacity, and ferric-reducing antioxidant power (FRAP) test systems. Total phenol and flavonoid contents of the extract and fractions were calculated using Folin-Ciocalteau and aluminium chloride reagents. Three flavonoid derivatives; 3-O-methylquercetin (1), 3,7-O-dimethylquercetin (2), and 3,7-O-dimethylkaempferol (3) isolated from the CHCl3 fraction were also tested in the same manner. Our experimental findings indicated that the ethanol extract exerted the highest AChE inhibition (80.07±1.06% at 200μg/ml). The ethyl acetate and n-butanol fractions displayed the best activity against DPPH and FRAP assays. [Copyright &y& Elsevier]

Several extracts of the leaves and fruits of Juglans regia L. were assessed for their neuroprotective effects through antioxidant and anti-cholinesterase methods. Anticholinesterase activity was determined against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the enzymes vital for Alzheimer's disease, at 50, 100 and 200 μg ml− 1. Antioxidant activity was tested using radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH), N, N-dimethyl- p-phenylenediamine (DMPD), superoxide (SO), nitric oxide (NO) and hydrogen peroxide (H2O2) radicals, as well as ferric ion-chelating capacity, ferric- and phosphomolybdenum-reducing antioxidant power at 500, 1000 and 2000 μg ml− 1. Total phenol and flavonoid quantification of the extracts was calculated. The extracts scavenged DPPH radical in varying degrees; however, they did not scavenge DPMD and H2O2. Only the dichloromethane and water extracts were able to quench SO (10.09 ± 1.38%) and NO (24.09 ± 2.19%) radicals, respectively, at low level. The extracts showed either low or no BChE inhibition and no AChE inhibition. [ABSTRACT FROM AUTHOR]

Abstract: In vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory and antioxidant activities of the aqueous and ethanol extracts of the leaves, ripe fruits, and unripe fruits of Juniperus communis ssp. nana, Juniperus oxycedrus ssp. oxycedrus, Juniperus sabina, Juniperus foetidissima, and Juniperus excelsa were investigated in the present study. Cholinesterase inhibition of the extracts was screened using ELISA microplate reader. Antioxidant activity of the extracts was tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical scavenging, ferrous ion-chelating, and ferric-reducing antioxidant power (FRAP) assays. Total phenol and flavonoid contents of the extracts were determined spectrophotometrically. The extracts had low or no inhibition towards AChE, whereas the leaf aqueous extract of J. foetidissima showed the highest BChE inhibition (93.94±0.01%). The leaf extracts usually exerted higher antioxidant activity. We herein describe the first study on anticholinesterase and antioxidant activity by the methods of ferrous ion-chelating, superoxide radical scavenging, and ferric-reducing antioxidant power (FRAP) assays of the mentioned Juniperus species. [Copyright &y& Elsevier]

Abstract: Dichloromethane, ethyl acetate, acetone, methanol, and water extracts prepared from the fruits and leaves of Laurocerasus officinalis Roem. (LO) (Rosaceae) were screened for their cholinesterase inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the key enzymes in pathogenesis of Alzheimer''s disease (AD), using ELISA microplate reader at 50, 100, and 200μgmL−1. As AD is associated with oxidative stress, the antioxidant activity of the extracts was also tested by radical-forming methods against 2,2-diphenyl-1-picrylhydrazyl (DPPH), N,N-dimethyl-p-phenylendiamine (DMPD), and superoxide radicals as well as iron-related methods; iron-chelating capacity and ferric-reducing antioxidant power (FRAP) assays. Total phenol and flavonoid quantification was achieved using Folin–Ciocalteau and AlCl3 reagents, respectively. The highest AChE (44.01±1.75%) and BChE (19.91±0.37%) inhibition was caused by the LO-leaf-methanol extract 200μgmL−1, while it showed the best radical-scavenging activity against DPPH at 2000μgmL−1. Only, the dichloromethane and water extracts of the fruits and the leaf water extract had an iron-chelating capacity, while the leaf methanol extract displayed the highest FRAP. The leaf methanol extract (113.45±0.71mgg−1 extract) was found to be the richest in total phenols, while the leaf acetone extract (139.90±4.64mgg−1 extract) had the most abundant amount of total flavonoids. [Copyright &y& Elsevier]

The dichloromethane (DCM), acetone, ethyl acetate and methanol extracts of the leaves and berries of Myrtus communis L. were screened against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase (TYRO), the enzymes linked to neurodegenerative diseases, at 200 μg ml− 1. Antioxidant activity was tested using radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and N, N- dimethyl- p-phenylenediamine (DMPD) radicals, metal chelation capacity, ferric-reducing antioxidant power (FRAP) and phosphomolybdenum-reducing antioxidant power (PRAP) assays. Total phenol and flavonoid quantification of the extracts was calculated spectrophotometrically. The extracts showed a moderate AChE (17.49 ± 3.99% to 43.15 ± 1.55%) and TYRO inhibition (4.48 ± 1.50% to 40.53 ± 0.47%). The leaf extracts were ineffective against BChE, while the berry extracts displayed inhibition between 21.83 ± 3.82% and 36.80 ± 2.00%. The polar extracts exerted remarkable scavenging effect against DPPH and DMPD and also in the FRAP assay, where the DCM extract of the berries had the best metal chelation capacity (79.29 ± 1.14%). This is the first study that demonstrates in vitro neuroprotective effects of myrtle. [ABSTRACT FROM AUTHOR]

Abstract: Various herbal coffee varieties are consumed traditionally in Turkey. In the current study, the ethanol extracts of the coffee and seed samples obtained from Gundelia tournefortii (tumble thistle), Nigella sativa (black cumin), Phoenix dactylifera (date), and Ceratonia siliqua (carob) as well as a sample of instant coffee (Nescafe®, green blend) were tested against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and tyrosinase, the enzymes connected to neurodegeneration. Since oxidative stress is strongly associated with neurodegeneration, antioxidant activity of the extracts was also determined. Antioxidant activity of the extracts was measured using anti-radical and metal-related methods. Total phenol and flavonoid contents were calculated using Folin–Ciocalteau''s and AlCl3 reagents, respectively. Fatty acid compositions of the seed oils of tumble thistle and black seed were analyzed by GC–MS. Our results showed that the date sample exerted the highest AChE and BChE inhibition at 300μgmL−1 (52.96% and 83.22%, respectively). [Copyright &y& Elsevier]

Cholinesterase (ChE) inhibition is an important treatment strategy for Alzheimer's disease (AD) as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are involved in the pathology of AD. In the current work, ChE inhibitory potential of twenty-four natural products from different chemical classes (i.e., diosgenin, hecogenin, rockogenin, smilagenin, tigogenin, astrasieversianins II and X, astragalosides I, IV, and VI, cyclocanthosides E and G, macrophyllosaponins A-D, kokusaginin, lamiide, forsythoside B, verbascoside, alyssonoside, ipolamide, methyl rosmarinate, and luteolin-7-O-glucuronide) was examined using ELISA microtiter assay. Among them, only smilagenin and kokusaginine displayed inhibitory action against AChE (IC50 = 43.29 ± 1.38 and 70.24 ± 2.87 µg/mL, respectively). BChE was inhibited by only methyl rosmarinate and kokusaginine (IC50 = 41.46 ± 2.83 and 61.40 ± 3.67 µg/mL, respectively). IC50 values for galantamine as the reference drug were 1.33 ± 0.11 µg/mL for AChE and 52.31 ± 3.04 µg/mL for BChE. Molecular docking experiments showed that the orientation of smilagenin and kokusaginine was mainly driven by the interactions with the peripheral anionic site (PAS) comprising residues of hAChE, while kokusaginine and methyl rosmarinate were able to access deeper into the active gorge in hBChE. Our data indicate that similagenin, kokusaginine, and methyl rosmarinate could be hit compounds for designing novel anti-Alzheimer agents. [ABSTRACT FROM AUTHOR]

Cholinesterase inhibition is one of the most treatment strategies against Alzheimer's disease (AD) where metal accumulation is also strongly associated with pathology of the disease. In the current study, we assessed inhibitory effect against acetyl- (AChE) and butyrylcholinesterase (BChE) and metal-chelating capacity of twelve diterpenes: arucadiol, miltirone, tanshinone IIa, 1-oxomiltirone, cryptotanshinone, 1,2-didehydromiltirone, 1,2-didehydrotanshinone IIa, 1β-hydroxycryptotanshinone, 15,16- dihydrotanshinone, tanshinone I, isotanshinone II, 1(S)-hydroxytanshinone IIa, and rosmarinic acid, isolated from Perovskia atriplicifolia and Salvia glutinosa. The compounds were tested at 10 μg/mL using ELISA microtiter assays against AChE and BChE. QSAR and molecular docking studies have been also performed on the active compounds. All of the compounds showed higher [e.g., IC50 = 1.12 ± 0.07 mg/mL for 1,2-didehydromiltirone, IC50 = 1.15 ± 0.07 mg/mL for cryptotanshinone, IC50 = 1.20 ± 0.03 μg/mL for arucadiol, etc.)] or closer [1,2-didehydrotanshinone IIa (IC50 = 5.98 ± 0.49 μg/mL) and 1(S)-hydroxytanshinone IIa (IC50 = 5.71 ± 0.27 μg/mL)] inhibition against BChE as compared to that of galanthamine (IC50 = 12.56 ± 0.37 μg/mL), whereas only 15,16-dihydrotanshinone moderately inhibited AChE (65.17 ± 1.39%). 1,2-Didehydrotanshinone IIa (48.94 ± 0.26%) and 1(S)-hydroxytanshinone IIa (47.18 ± 5.10%) possessed the highest metal-chelation capacity. The present study affords an evidence for the fact that selective BChE inhibitors should be further investigated as promising candidate molecules for AD therapy. [ABSTRACT FROM AUTHOR]

The ethanol (EtOH) extracts of thirty-four taxa from the Amelanchier , Cotoneaster , Pyrus , and Sorbus genera (Rosaceae) have been screened for their inhibitory potential against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at 200 μg/mL as well as their antioxidant activity using metal-chelating capacity, ferric-reducing antioxidant power (FRAP), and N , N -dimethyl- p -phenylenediamine (DMPD) radical scavenging activity at 2000 μg/mL using ELISA microtiter assays. Our results indicated that two extracts of Sorbus umbelleta from different localities had 56.20 ± 5.05% (IC 50 = 182.1 μg/mL) and 58.18 ± 3.77% (IC 50 = 177.8 μg/mL) of inhibition against AChE, while 50.67 ± 0.32% of BChE inhibition (IC 50 = 206.5 μg/mL) was caused by the extract of Cotoneaster meyeri . Among the tested extracts, S. subfusca displayed the highest FRAP value (1.618 ± 0.09) and DMPD radical scavenging activity (31.05 ± 0.01%), while the best metal-chelating capacity (27.13 ± 2.01%) was found to belong to the extract of Sorbus kusnetzovii . The EtOH extracts of the active species ( e.g. Cotoneaster meyeri , two samples of Sorbus umbelleta , and Sorbus subfusca ) in enzyme inhibition assays have also been subjected to HPLC analysis to quantify some selected phenolic acids and flavonoid derivatives. Chlorogenic acid (28.83 ± 0.67–149.10 ± 0.46 mg/100 g) and rutin (46.08 ± 0.63–109.08 ± 0.07 mg/100 g) were quantified in all four extracts. Caffeic acid (0.79 ± 0.18 mg/100 g) was only present in C. meyeri , whereas ferulic acid and apigenin were detected in three extracts in minor amounts. Our findings also pointed out to a marked variation in bioactivity and phytochemistry among the same plant species growing in different places. This is the first anticholinesterase and antioxidant study on most the Rosaceae species studied herein. Among them, Sorbus umbellata appears to be a promising herb due to its noticeable anti-AChE and antioxidant activities. [ABSTRACT FROM AUTHOR]

The hydroalcoholic extracts of the Turkish traditional coffee samples from 18 commercial brands were tested for their neurobiological effects through enzyme inhibition based on enzyme-linked immunosorbance microtiter assays against acetylcholinesterase, butyrylcholinesterase, and tyrosinase, linked to Alzheimer's and Parkinson's diseases. The extracts were also subjected to several antioxidant test systems to define their antiradical, metal-chelation capacity, and reducing power. Total phenol and flavonoid contents in the extracts were delineated by spectrophotometric methods, while chlorogenic acid in the coffee samples was quantified by high-pressure liquid chromatography. The extracts displayed low to moderate inhibition (from 2.13 ± 0.01% to 36.12 ± 1.07% at 200 mg/mL) against the tested enzymes, whereas they had notable 2,20-diphenyl-1-picrylhydrazyl radical scavenging activity up to 56.15 ± 2.03% at 200 mg/mL. The extracts exerted a remarkable ferric-reducing antioxidant power values, while chlorogenic acid was found to range between 0.288 ± 0.005% and 2.335 ± 0.010%. [ABSTRACT FROM AUTHOR]

The dichloromethane (DCM), ethyl acetate (EtOAc), and methanol extracts from the leaves, roots, and flowers of the five species of Gentiana ( Gentiana asclepiadea, Gentiana cruciata, Gentiana olivieri, Gentiana septemfida, and Gentiana verna) and Gentianella caucasea were investigated for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and antioxidant effect using 2,2-diphenyl-1-picrylhydrazyl radical scavenging, metal-chelation capacity, and ferric-reducing antioxidant power assays. Total phenol and flavonoid contents of the extracts were determined spectrophotometrically. The presence of some characteristic compounds found in Gentiana species (gentiopicroside, swertiamarin, isoorientin, isovitexin and vitexin) was analyzed in the extracts by thin layer chromatography. The flower DCM extract of G. verna exerted the highest inhibition against AChE (53.65 ± 1.03%), whereas the root EtOAc extract of G. cruciata was the most effective in BChE inhibition assay (50.72 ± 0.75%) at 100 μg ml− 1. The extracts of G. verna were also found to be more active in the antioxidant tests. [ABSTRACT FROM AUTHOR]

Abstract: In the current work, the acetone, ethyl acetate, and ethanol extracts and essential oils of the twigs and needles of five Turkish Pinus species; P. brutia, P. halepensis, P. nigra, P. pinea, and P. sylvestris along with pycnogenol (the bark extract of P. pinaster) were examined for their inhibitory effects against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at 200μgmL−1. Their antioxidant activity was tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and N,N-dimethyl-p-phenylendiamine (DMPD) radical scavenging, metal-chelation, and ferric-reducing antioxidant power (FRAP) assays. Total phenol and flavonoid contents of the extracts were determined by Folin–Ciocalteau and AlCl3 reagents, respectively. GC–MS technique was applied for compositional profile of the essential oils of the tested species. Accordingly, the best AChE and BChE inhibition was caused by the twig essential oil (83.91±3.95%) and the needle ethanol extract (82.47±5.57%) of P. halepensis, respectively. Pycnogenol also caused a remarkable inhibition against AChE (63.33±0.22%) and BChE (83.67±0.22%). The extracts and essential oils usually showed a strong activity in the antioxidant assays. Pycnogenol exhibited a potent antioxidant effect and had the richest total phenol content. In the essential oils, α- and β-pinene were identified the major components. These results reveal that the Pinus species and pycnogenol are the potential sources of active metabolites with cholinesterase and antioxidant properties. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Galium species have been reported to be used against epilepsy in traditional Turkish folk medicine. Aim of study: The present work was undertaken to evaluate the in vivo anticonvulsant and in vitro neuroprotective effects of Galium spurium L. and to determine the major constituents by UPLC–TOF-MS. Materials and methods: Anticonvulsant activity of the aerial parts of Galium spurium was investigated using pentylenetetrazole, picrotoxin, and maximal electroshock-induced seizure animal models. In order to evaluate the safety, neurotoxicity (Rota rod test) of the ethanol extract was also determined. In vitro neuroprotective effect of the ethanol extract of Galium spurium was assessed by acetylcholinesterase and butrylcholinesterase inhibitions. Ultra Performance Liquid Chromatography–Time of Flight Mass Spectrometer (UPLC–TOF-MS) was used to identify the major compounds in the extract. Results: In pentylenetetrazole-induced seizure, the ethanol extract at doses of 250 and 1000mg/kg prolonged the onset of seizures. Similarly, Galium spurium (250 and 500mg/kg) significantly delayed the onset of picrotoxin-induced seizures in mice and these doses also exhibited 12.5% and 17% protection, respectively, against picrotoxin-induced seizures. Furthermore, Galium spurium extract showed a significant protective effect against maximal electroshock-induced seizures at doses of 250 and 1000mg/kg (50% and 37.5%, respectively) and also all tested doses prolonged the onset of seizures. No motor co-ordination was observed with intraperitoneal injection of Galium spurium extract at doses of 500 and 1000mg/kg. The extract exhibited 16.2% inhibition against butrylcholinesterase at 200μg/mL concentration, whereas it did not inhibit acetylcholinesterase. Phytochemical analysis of the extract based on the MS data by UPLC–TOF-MS, ten major compounds (phenolic and triterpenic acids, flavonoids and iridoids) were determined. Conclusions: The results indicate that Galium spurium may have anticonvulsant activity against picrotoxin and maximal electroshock-induced seizures in mice. Phenolic acids, flavonoids and iridoids might be responsible for anticonvulsant activity. The results offer possible beneficial effects by the plant''s aerial parts and may suggest a realistic explanation for its traditonal usage in epilepsy. [Copyright &y& Elsevier]