Abstract
In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60–90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.
Highlights
Plant species from the club moss genus Lycopodium and Huperzia (Lycopodiaceae) are known to be a rich source of compounds belonging to the alkaloid group, possessing unique heterocyclic skeletons usually containing C15N2, C16N, C16N2 and C27N3
Three plant species belonging to the Lycopodiaceae family: L. clavatum, L. annotinum, H. selago, were subjected to extraction using the pressurized liquid extraction (PLE) method
It was found that the highest percentage of Lycopodium alkaloids were isolated under high pressure and temperature (100 bar, 80 ◦C) from methanolic extracts, so these conditions were used in this approach
Summary
Plant species from the club moss genus Lycopodium and Huperzia (Lycopodiaceae) are known to be a rich source of compounds belonging to the alkaloid group, possessing unique heterocyclic skeletons usually containing C15N2, C16N, C16N2 and C27N3. The alkaloids are quinolizine, or pyridine and α-pyridone type, which have attracted the interest of researchers [1,2,3] These compounds possess numerous properties such as anti-inflammatory, antioxidant, anticancer and antimicrobial activities, among others [4,5]. They are known for important bioactivities such as the ability to inhibit acetylcholinesterase (AChE), an enzyme responsible for decreasing the concentration of a neurotransmitter acetylcholine (ACh) at the synaptic site in the brain [3,6,7]. HTuopedraztiae, sneurrmatear(oTuhsurnebse. aerxchMgurorruapys) aTrreevwiso,riktihnagstboeicsoomlateeathpirsocmomisipnogudnrdugasfowretlhl eastrneeawtm, epnotteonft Aallkzahleoiimdserwsitdhisneoavseel ssytrmucpttuormessa[n1d0]n. eTwo dmaetceh, annuimsmersooufsacretisoenasrcfrhogmrothuipssspareeciwesoarnkdinfgrotmo irseollaatteedtphliasnctosm[6p,1o1u–n1d3]a. s well as new, potent alkaloids with novel structures and new mechAanliasmrgseopfaarcttioofntshferopmiontheiesrsinpgecwieosraknodnfraolmkarloeliadtsedofptlhaentLsy[6co,1p1o–d13ia].ceae family was carriAedlaorugteinpaCrtanoafdthaeinpitohneemeriidndglewoofrtkhoen20atlhkacleonidtusroyf[t1h4e–1L6y]c. oTphoisdlieadcetaoetfhaemiisloylawtiaosn caanrdrieiddeonutitfiicnaCtioannaodfaminorthe ethmanid2d0le0 oaflkthaleo2id0sthfrcoemntu5r4yL[y1c4o–p1o6d]i.uTmhsispleecdietso, wthheicisho,liantiothne a1n9d90isd,ewnteirfiecactliaosnsiofifedmobrye Cthaannad2i0a0naslkciaelnotiidsstsfrAomyer54anLdycocpoo‐ldeiaudmersspe[1c7ie]s,inwtohifcohu, rinmtahien 1cccl9olaa9mss0ssspee,sos:w:ulnleyyrdcceoopcpphlooaleddsgsiinminfieeae,,rdlilynybcceyoo(ddFCiiinngaueen,,raefdfaa1iwwa)n[cc2ees,tt3ctt,iii1emm8n]iitnn.iTseethsaaennAcddyoemmmrpiiassonccueednlllldacaonsn-eetlhooeauautdssbeaaerllslkkoa[an1llgoo7i]itddoissnthtwwoeiiltftyhohcuottrhphemeodkakieinenyye pcctNtaArteichholschoelna-aeatmmtarmasziniesevnsrnypsnalee1admauotrta0thkn1snuAneB0ei0aydinddt,Cq0bcleadahotulhhhcony[mapEetcua2divtophv,vppshi1emonNleteeeo9erthari,ugu‐apes2rzmimsnnicob0isutinttdu]ieienunatn.eAtnsigrirhog[.TqidBel2CnnyleHuhes,,aleh1.benewonhn9E(reiws,FduiHhetl2ttiroifrperni0gotoocnuov]hehughwr.ggcereieertzbreThee,tun,finiovhaantmtr1haievecetea)othoretretao[nB,eoset2ndtmtfh,m,bwlodm3iwoyaesr,ioftc1tcleionhhonoto8t,snihhned]tvcttti.heeghithhfenrTeayioreeothseichyofirofigtardeclsettovtlasithhtcanfsewhttroeasrceuentasumtoedwlc,etyltcvotlfpwhLitocueosmuiyoeornorrncyruyadesltoeeiannoiftpxianoitradndoatertoferdehsmdwgtemicnheuttepelhtheemoanghLllawelrsesoeyatesia,klcrnt,blalhgoeaolkatfreypulnoeomragelcogpdorltteodooseohneesfidisrminuetgxdnttzim.aghsngiotgtmnToreoewrhroeh-eroAtpeatuisghiutyllslsphCeAkeeyppki,,aohcrlse,eahwyleofEwlornmaucetdeiihodltetidpilkaothpenanhesBrantoezrAkmel,.m‐zydoadwtaTCiomiiybnnodnhihtrlpetdroyeeesiEheess saelekmalsoipdrsomseiesminsgpfroormthiseintrgeaftomr ethnet otrfenaetmureondteogfenneeurarotidveegdeinseearasteisv.e diseases
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