Research Article |
Corresponding author: Milena Nikolova ( mtihomirova@gmail.com ) Academic editor: Kalina Danova
© 2022 Borislav Georgiev, Milena Nikolova, Ina Aneva, Anatoli Dzhurmanski, Boriana Sidjimova, Strahil Berkov.
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.
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
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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 IC50 value about 1 mg/ml and the last group with low activity, with IC50 value greater than 1 mg/ml. Essential oils of Origanum vulgare subsp. hirtum Ietswaart., 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.
Acetylcholinesterase, alkaloids, carvacrol, essential oils, galanthamine
The use of natural products as an alternative to synthetic insecticides is a priority for modern agriculture. At the root of the mechanism of action of organophosphorus insecticides is acetylcholinesterase inhibition (
In the present study, methanolic extracts and essential oils from species of Amaryllidaceae, Asteraceae, Brassicaceae and Lamiaceae were evaluated in vitro for AChE inhibitory activity.
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 & T.R. Dudley, Tanacetum parthenium L., Salvia forskaohlei L., Salvia sclarea L., Salvia aethiopis L., Thymus yankae L., Centaurea arenaria M.Bieb. ex Willd., Nepeta caria L. and Eupatorium perfoliatum L.) 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 Ietswaart., Monarda fistulosa L. and Satureja pilosa Vel.).
Methanolic extracts. Air-dried powdered plant material (1 g) was extracted with methanol for 24 hours at room temperature. After filtration, the organic solution was evaporated 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 inhibitory activity of all samples was determined using Ellman’s colorimetric method, as modified by
AChE (50 μl) at a concentration of 0.25 U/ml was dissolved in phosphate buffer (8 mM K2HPO4, 2.3 mM NaH2PO4, 0.15 M NaCl, pH 7.5) and 50 μl 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 μl of the substrate solution (0.04 M Na2HPO4, 0.2 mM DTNB, 0.24 mM ATCI, pH 7.5). The absorbance values 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 IC50 values were measured in triplicate and the results are presented as means.
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 × 0.25 mm inner diameter × 0.25 µm film thickness). The chromatographic conditions for methanolic extracts and essential oils are described by
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
Acetylcholinesterase inhibitory activity of essential oils and methanolic extracts.
Plant species | Extract/EO | AChE activity IC50 [mg/mL] |
---|---|---|
Amaryllidaceae | ||
Leucojum aestivum | MeOH | 0.20 |
Asteraceae | ||
Artemisia lerchiana | MeOH | 1.08 |
Artemisia santhonicum | MeOH | 0.94 |
Centaurea arenaria | MeOH | > 1 |
Eupatorium cannabinum | MeOH | 1.07 |
Tanacetum parthenium | MeOH | > 1 |
Brassicaceae | ||
Aurinia uechtritziana | MeOH | > 1 |
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 | > 1 |
Salvia sclarea | MeOH | > 1 |
Thymus yankae | MeOH | > 1 |
Monarda fistulosa | EO | 0.0042 |
Origanum vulgare ssp hirtum | EO | 0.30 |
Satureja pilosa | EO | 0.0069 |
Thymus longidentatus | EO | 0.72 |
Galanthamine | Positive control | 0.0011 |
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: IC50 4.03 μM = 0.0011 mg/ml), including the methanolic extract of L. aestivum and essential oils of M. fistulosa, S. pilosa, O. vulgare ssp hirtum and T. longedentatus; second group: plant products with moderate activity with IC50 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 IC50 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.
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
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 | ||
α-Thujene | 930 | 4.72 | 4.66 | – | – |
α-Pinene | 932 | 1.29 | 1.43 | – | – |
Sabinene | 971 | – | – | – | 0.43 |
ß-Myrcene | 988 | – | – | 4 | – |
p-Cymene | 1025 | 21.82 | 16.26 | 2.97 | – |
trans-ß-Ocimene | 1044 | – | – | – | 1.15 |
γ-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 | 1.73 | – |
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 | – |
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 detected, 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.
Four essential oils and 14 methanolic extracts were studied for AChE inhibitory activity. All studied essential oils showed significant activity and their profiles were determined by GC/MS. Isomers – carvacrol and thymol - were identified as the main 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 (
Essential oils of many plant species have been examined as an alternative to synthetic insecticides (
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 (
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. GC/MS analysis proved the presence of bioactive compounds in these plant products. Thus, we recommend them to be tested for insecticidal activity in the future.
This research was supported by the Bulgarian National Science Fund, Bulgarian Ministry of Education and Science (Grant DN 16/2, 11.12.2017).