BioRisk | 7: 309-3 | 5 (2022) Ceasar ea aa ores doi: 10.3897/biorisk.17.77052 RESEARCH ARTICLE & B lO R IS k https://biorisk.pensoft.net Plant products with acetylcholinesterase inhibitory activity for insect control Borislav Georgiev', Milena Nikolova', Ina Aneva', Anatoli Dzhurmanski’, Boriana Sidjimova', Strahil Berkov' | Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria 2 Institute for Roses and Aromatic Plants, bul. Osvobozhdenie 49, 6100 Kazanluk, Bulgaria Corresponding author: Milena Nikolova (mtihomirova@gmail.com) Academic editor: Kalina Danova | Received 26 October 2021 | Accepted 13 January 2022 | Published 21 April 2022 Citation: Georgiev B, Nikolova M, Aneva I, Dzhurmanski A, Sidjimova B, Berkov S (2022) Plant products with acetylcholinesterase inhibitory activity for insect control. In: Chankova S, Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology. BioRisk 17: 309-315. https://doi.org/10.3897/biorisk.17.77052 Abstract Acetylcholinesterase (AChE) inhibitors are widely used in Alzheimer’s treatment, but they are also crucial for their action on organophosphorus insecticides. The latter exert their toxicity by inhibiting the AChE enzyme in insects, leading to their death. Amaryllidaceae alkaloids have been proven to be potent AChE inhibitors. In the present study methanolic extracts and essential oils being obtained from species of Asteraceae, Lamiaceae, Brassicaceae and Amaryllidaceae were evaluated in vitro for AChE inhibitory activity. Ellman’s colourimetric method, with modifications, was used for AChE activity evaluation. According to the activity level, the tested plant products were divided into three categories. First: plant products with strong activity comparable to that of galanthamine; second: plant products with medium activity, with IC,, value about 1 mg/ml and the last group with low activity, with IC,, value greater than 1 mg/ml. Essential oils of Origanum vulgare subsp. hirtum letswaart., Satureja pilosa Vel., Monarda fistulosa L., Thymus longedentatus (Degen & Urum.) Ronniger and the methanolic extract of Leucojum aestivum L. showed the most potent activity and were referred to as the first group. Carvacrol was identified as the main component of the most active essential oils. In L. aestivum extract, galanthamine was found as the main alkaloid. The obtained results indicate that essential oils and alkaloid-rich plant extracts possess the strongest AChE inhibitory activity. This gives us a reason to recommend these plant products to be tested for insecticidal activity in the future. Keywords Acetylcholinesterase, alkaloids, carvacrol, essential oils, galanthamine Copyright Borislav Georgiev et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 310 Borislav Georgiev et al. / BioRisk 17: 309-315 (2022) Introduction The use of natural products as an alternative to synthetic insecticides is a priority for mod- ern agriculture. At the root of the mechanism of action of organophosphotus insecticides is acetylcholinesterase inhibition (Lopez et al. 2002; Lopez and Pascual-Villalobos 2010; Pang 2014; Mladenovi¢ et al. 2018). Amaryllidaceae alkaloids and terpenoids are second- ary metabolites with proven high AChE inhibitory activity (Lopez et al. 2002; Elgorashi et al. 2004; Wojtunik-Kulesza et al. 2017). Plant species of Amaryllidaceae, Lamiaceae and Asteraceae are rich in compounds from these chemical groups. Mono-, sesqui- and diter- penes, together with phenols, esters, oxides, ketones, alcohols and aldehydes are the main components of essential oils. Extensive studies on essential oils reveal that they possess various biological activities of great importance, such as toxic and repellent (insecticidal) activity (Isman 2000; Pascual-Villalobos and Ballesta-Acosta 2003; Garcia et al. 2005). In the present study, methanolic extracts and essential oils from species of Ama- tyllidaceae, Asteraceae, Brassicaceae and Lamiaceae were evaluated in vitro for AChE inhibitory activity. Materials and methods Plant material Plant material was collected from natural localities of the studied species (Artemisia santhonicum L., Artemisia lerchiana L., Micromeria dalmatica Benth., Thymus longedentatus (Degen & Urum.) Ronniger, Aurinia uechtritziana (Bornm.) Cullen & TR. Dudley, Tanacetum parthenium L., Salvia forskaohlei L., Salvia sclarea ., Salvia aethiopis L., Thymus yankae L.., Centaurea arenaria M.Bieb. ex Willd., Nepeta caria L. and Eupatorium perfoliatum 1.) or ex situ collections from the Institute of Biodiversity and Ecosystem Research (Leucojum aestivum L.) and the Institute of Roses and Aromatic Plants (Origanum vulgare subsp. hirtum letswaart., Monarda fistulosa L. and Satureja pilosa Vel.). Extraction of plant material Methanolic extracts. Air-dried powdered plant material (1 g) was extracted with metha- nol for 24 hours at room temperature. After filtration, the organic solution was evapo- rated and the dry extract stored at 4 °C until analysis. Essential oils. The essential oil was extracted on a Clevenger apparatus by water distillation from 50 g of dry plant material in a flask with 500 ml water for 2 hours. Acetylcholinesterase (AChE) inhibition assay Acetylcholinesterase inhibitory activity of all samples was determined using Ellman’s colorimetric method, as modified by Lépez et al. (2002). The assay was performed in 96- Acetylcholinesterase inhibitory activity of plant extracts ola well microplates. Acetylthiocholine iodide (ATCT) in a solution with 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB) was used asa substrate for AChE from Electrophorus electricus (Sigma-Aldrich, Germany). Methanolic extracts and essential oils from the studied plants with concentrations between 0.001 and 1000 ug/ml were tested. AChE (50 ul) at a concentration of 0.25 U/ml was dissolved in phosphate buffer (8 mM K,HPO,, 2.3 mM NaH,PO,, 0.15 M NaCl, pH 7.5) and 50 ul of the sample dissolved in the same buffer were added to the wells. The plates were incubated for 30 minutes at room temperature before the addition of 100 ul of the substrate solution (0.04 M Na,HPO,, 0.2 mM DTNB, 0.24 mM ATCI, pH 7.5). The absorbance val- ues were read on a microplate reader (BIOBASE, ELISA-EL10A, China) at 405 nm after 3 minutes. Enzyme activity was calculated as an inhibition percentage compared to an assay using a buffer without any inhibitor. Galanthamine was used as a positive control. The AChE inhibitory data were analysed in Microsoft Excel and the software package Prism 9 (Graph Pad Inc., San Diego, USA). The il Gon values were measured in triplicate and the results are presented as means. GC/MS analysis The most active samples were analysed for their bioactive components by GC/MS. The spectra were recorded on a Thermo GC, equipped with a Focus DSQ II mass detector, coupled to a HP-5MS capillary column (30 m length x 0.25 mm inner di- ameter x 0.25 um film thickness). The chromatographic conditions for methanolic extracts and essential oils are described by Berkov et al. (2021) and by Traykova et al. (2019), respectively. The components were identified by comparing their mass spectra and retention indices (RI) with those of authentic standards and the National Institute of Standards and Technology (NIST) spectra library. Results Eighteen samples — 4 essential oils and 14 methanolic extracts of 17 plant species - were examined for AChE inhibitory activity by Ellman’s colorimetric method, with modifications by Lopez et al. (2002). The results are presented in Table 1. According to the level of activity, the tested plant samples were divided into three groups. First group: plant samples with strong activity comparable to that of galanthamine (positive control: IC,, 4.03 uM = 0.0011 mg/ml), including the methanolic extract of L. aestivum and essential oils of M. fistulosa, S. pilosa, O. vulgare ssp hirtum and I! longedentatus; second group: plant products with moderate activity with IC. value about 1 mg/ml, including the methanolic extracts of S. aethiopsis, A. lerchiana, A. santhonicum, E.cannabinum, N. caria, M. dalmatica and O. vulgare ssp hirtum and the last group with low activity with a IC,, value above 1 mg/ml; the third group comprises the rest of the samples which showed low activity. For O. vulgare ssp hirtum, it was shown that the essential oil is a stronger inhibitor of AChE than the methanolic extract. 312 Borislav Georgiev et al. / BioRisk 17: 309-315 (2022) Table |. Acetylcholinesterase inhibitory activity of essential oils and methanolic extracts. Plant species Extract/EO AChE activity IC,, [mg/mL] Amaryllidaceae Leucojum aestivum MeOH 0.20 Asteraceae Artemisia lerchiana MeOH 1.08 Artemisia santhonicum MeOH 0.94 Centaurea arenaria MeOH = al Eupatorium cannabinum MeOH 1.07 Tanacetum parthenium MeOH tal Brassicaceae Aurinia uechtritziana MeOH = al Lamiaceae Micromeria dalmatica MeOH 1.16 Nepeta caria MeOH 1.12 Origanum vulgare ssp hirtum MeOH 0.73 Salvia aethiopis MeOH 1.23 Salvia forskaohlei MeOH x al Salvia sclarea MeOH eel Thymus yankae MeOH aa Monarda fistulosa EO 0.0042 Origanum vulgare ssp hirtum EO 0.30 Satureja pilosa EO 0.0069 Thymus longidentatus EO O72 Galanthamine Positive control 0.0011 The most active samples were analysed for their bioactive components by GC/MS. The essential oil profiles of the studied samples are presented in Table 2. Monoterpe- noid phenols — isomers carvacrol and thymol - were identified as main components of the essential oils of S. pilosa, O. vulgare ssp. hirtum and M. fistulosa. p-Cymene (16.26%) and y-terpinene (16.07%) were found as the next most abundant constitu- ents of O. vulgare ssp. hirtum. In the essential oil profile of M. fistulosa, thymoquinone (25.41%) and p-cymene (21.82%) were present in significant amounts. Neral and ge- ranial were identified as major components of the essential oil of 7’ longedentatus with 24.9% and 27.95%, respectively. In L. aestivum methanolic extract, galanthamine was found as the main alkaloid. In the methanolic extract of O. vulgare ssp., hirtum carvacrol (15.67%) was also de- tected, but in a much smaller amount compared to the essential oil. Rosmarinic acid (6.06%), flavonoid glycosides (1.49%), malic acid (1.09%) and catechin (0.23%) were also identified as bioactive compounds. Discussion Four essential oils and 14 methanolic extracts were studied for AChE inhibitory ac- tivity. All studied essential oils showed significant activity and their profiles were de- termined by GC/MS. Isomers — carvacrol and thymol - were identified as the main Acetylcholinesterase inhibitory activity of plant extracts 313 Table 2. Main compounds identified in the essential oils of studied species (Mf M. fistulosa; Ovh: O. vulgare ssp hirtum; Sp: S. pilosa; Tl: T. longedentatus); Area (%). Compounds RI Studied essential oils Mf Ovh Sp Tl a-Thujene 930 4.72 4.66 - - oa-Pinene 932 1.29 1.43 = Sabinene 971 — — - 0.43 -Myrcene 988 — - 4 — p-Cymene 1025 2Ae82 16.26 Wi Hs = trans-B-Ocimene 1044 - — - Lo kS y-Terpinene 1059 — 16.07 1.04 — Camphor 1141 — — - 1.34 Terpinen-4-ol 1175 0.98 - - 0.74 Neral 1227 — - ~ 24.9 Carvacrol methyl ether 1245 1.98 2.54 ier) — Thymoquinone 1250 25.41 - - 0.22 Geranial 1264 ~ - — 27.96 Thymol 1290 19.75 — 30.58 — Carvacrol 1299 12.24 51.18 50.54 0.19 Neryl acetate 1359 - — — 12.79 Caryophyllene 1466 0.69 1.43 = = Caryophyllene oxide 1590 ~ — 1.18 — components of the essential oils of S. pilosa, M. fistulosa and O. vulgare ssp. hirtum. Carvacrol is a compound with a previously demonstrated strong AChE inhibitory activity (Jukic et al. 2007) and probably determines the activity of the essential oils. The established profile of O. vulgare ssp. hirtum is in accordance with data reported in literature for the natural populations of the species in Bulgaria from East Rhodopes, Strumska Valley and cultivated areas (Konakchiev et al. 2004; Alekseeva et al. 2021; Baycheva and Dobreva 2021). The composition of S. pilosa and M. fistulosa corre- sponds to that reported by Semerdjieva et al. (2020) and Ghosh et al. (2020), respec- tively. Citral isomers neral and geranial were determined as main components of the essential oil profile of 7’ longedentatus (Aneva et al. 2019). Essential oils of many plant species have been examined as an alternative to syn- thetic insecticides (Isman 2000; Pascual-Villalobos and Ballesta-Acosta 2003; Garcia et al. 2005). To the best of our knowledge, we report for the first time AChE activity of the essential oils of O. vulgare ssp. hirtum, T: longedentatus S. pilosa and M. fistulosa. The established strong inhibitory activity of the tested essential oils is a prerequisite for the presence of insecticidal activity. For O. vulgare ssp hirtum, it was shown that the essential oil is a stronger inhibitor of AChE than the methanolic extract. Assuming the activity is dependent on the presence of carvacrol, the difference in its content between the essential oil and the methanolic extract may also determine the difference in AChE activity. As galanthamine is a classic example for a substance with potent AChE inhibitory activity (Sramek et al. 2000), it undoubtedly determines the activity of the methanolic extract of L. aestivum. 314 Borislav Georgiev et al. / BioRisk 17: 309-315 (2022) Conclusion The obtained results indicate that the essential oils of Monarda fistulosa, Satureja pilosa, Origanum vulgare subsp. hirtum and Thymus longedentatus and the methanolic extract of Leucojum aestivum possess the strongest AChE inhibitory activity. 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