Evaluation of Different Extraction Techniques for the Assay of Anti - Acetylcholinesterase Activity of Olive leaves (Olea europaea) Chimica Techno Acta ARTICLE published by Ural Federal University 2021, vol. 8(4), № 20218403 eISSN 2411-1414; chimicatechnoacta.ru DOI: 10.15826/chimtech.2021.8.4.03 1 of 5 Evaluation of Different Extraction Techniques for the Assay of Anti - Acetylcholinesterase Activity of Olive leaves (Olea europaea) S.S. Khizrieva* , S.N. Borisenko, E.V. Maksimenko , N.I. Borisenko Research Institute of Physical and Organic Chemistry of the Southern Federal University, 194/2 Stachki Ave., Rostov-on-Don 344090, Russia * Corresponding author: hizrieva@sfedu.ru This article belongs to the regular issue. © 2021, The Authors. This article is published in open access form under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Abstract The total phenol content and the anti-acetylcholinesterase activity were compared in olive leaf (OL) extracts obtained using both sub- critical water extraction (SbWE) and conventional solvent extraction (ethanol-water). The method proposed by Ellman (in vitro) was used to study the inhibitory activity of acetylcholinesterase (AChE). The total content of phenolic compounds and AChE activities of OL ex- tracts varied depending on the used extraction method. Thus, the ex- tract obtained using the subcritical water technique (220 °C) showed the highest amounts of total phenolic components, expressed as gal- lic acid equivalents, (70.4 mg/g raw material) and the highest inhibi- tory AChE-activity (IC50 = 0.35 mg/ml). The obtained values of the anti-AChE activity of the extracts of OL demonstrated that the inhibi- tory activity for SbW-extract 120 °C (IC50 = 2.92 mg/ml) and SbW- extract 180 °C (IC50 = 0.8 mg/ml) is higher than that of the tradi- tional extract (IC50 = 3.6 mg/ml), respectively. These results indicate a great potential of the subcritical water technique to develop the techniques to produce commercial extracts of OL, and these results could encourage improved utilization of the OL. The collected data on the anti - acetylcholinesterase activity of olive leaves clearly demon- strate the prospects for use of OL extracts in the development of novel pharmaceutical substances and nutraceuticals for the preven- tion and/or the treatment of Alzheimer's disease as well as some other neurodegenerative diseases. Keywords olive leaves subcritical water extracts polyphenols Alzheimer's disease anti-Acetylcholinesterase activity Ellman’s method Received: 19.05.2021 Revised: 21.09.2021 Accepted: 08.11.2021 Available online: 09.11.2021 1. Introduction Alzheimer's disease (AD) is a neurodegenerative dis- ease that usually affects the elderly. AD is charac ter- ized by memory loss, impaired behavior, decreased performance, and slowed-down thinking. At present according to the World Health Organization about 50 million people worldwide are struggle from AD de- mentia, and over 152 million people may be affected by 2050 globally [1, 2]. In recent years, a growing number of works aimed at the finding of new pharmaceutical substances based on secondary plant metabolites [3–6] for the treatment of various neurodegenerative diseases, including AD. One of these promising groups of plant metabolites are polyphe- nols, which are common components of plant raw materi- als and agricultural wastes. In the presented work, the medium of subcritical water (in the temperature range from 100 to 220 °C) [7] was used to obtain extracts from the leaves of olive (Olea eu- ropaea L.) [8] – the most common waste of the olive oil industry. The antioxidant and neuroprotective effect of plant ex- tracts depends on their qualitative and quantitative com- position. The phenolic group is found in the structure of many medicinal plants and substances and largely deter- mines their pharmacological, physicochemical, and chemi- cal properties [9]. Polyphenolic derivatives in OL (Fig. 1) are represented by five main groups of phenolic compounds [10]. http://chimicatechnoacta.ru/ https://doi.org/10.15826/chimtech.2021.8.4.03 http://creativecommons.org/licenses/by/4.0/ http://orcid.org/0000-0001-7064-2402 http://orcid.org/0000-0002-8715-4517 http://orcid.org/0000-0003-4733-1985 Chimica Techno Acta 2021, vol. 8(4), № 20218403 ARTICLE 2 of 5 1) Hydroxytyrosol, C8H10O3 Caffeic acid, C9H8O4 2) Oleuropein, C8H10O3 Verbascoside, C29H36O15 3) Luteolin 7-glucoside, C21H20O11 Apigenin 7-glucoside, C21H20O10 4) 5) Rutin, C27H30O16 Catechin (flavan-3-ol), C15H14O6 Fig. 1 Structural formulas of phenolic compounds in olive leaves (OL): 1 – substituted phenols: tyrosol (2-4-hydroxyphenylethanol), hydroxytyrosol (2-3,4-dihydroxyphenylethanol) and caffeic acid (3,4-dioxycinnamic acid); 2 – oleuroposides (oleuropein and verbasco- side); 3 – flavones (luteolin-7-glucoside, apigenin-7-glucoside, diosmethin-7-glucoside, luteolin and diosmetin); 4 – flavonoids (rutin); 5 – flavan-3-ols (catechin) Olive polyphenols are known to have anticholinester- ase activity [11]. Therefore, in this work, the assessment of the sum of polyphenols in OL extracts obtained both by the traditional method and in the medium of subcritical water will be carried out using the Folin-Ciocalteu method [12]. Also, the anti-acetylcholinesterase (anti-AChE) activi- ty of the obtained extracts in vitro will be estimated ac- cording to the Ellman method [13]. Therefore, the purpose of the present work is the eval- uation of different extraction techniques for the assay of anti-AChE activity, and the determination of total amount of phenolic components of OL extracts obtained using both subcritical water extraction (SbWE) and traditional sol- vent extraction (TSE). 2. Experimental 2.1. Materials and Method Materials. As an object of study, the olive leaves of the O. europaea obtained from the Oleaf Company («Okvel», Rus- sia) were used. Acetylcholinesterase (AChE) was obtained from Electrophorus electricus (electric eel) (type VI-S, 3.1.1.7, 200–1000 units/mg protein), acetylthiocholine io- dide (ATChI) (≥98%, USA), 5.5’-dithiobis (2-nitrobenzoic) Chimica Techno Acta 2021, vol. 8(4), № 20218403 ARTICLE 3 of 5 acid (DTNB) (99%, USA), Folin-Ciocalteu reagent (2 M) were supplied by Sigma-Aldrich Company. Gallic acid (not less than 98%, MW = 170.12) was pur- chased from DIA-M Company (Russia). Na2CO3 (anhy- drous, GOST 83–79) and glacial acetic acid (CH3COOH, GOST 61–75, chemically pure: puriss.) were supplied by JSC VEKTON Company (Russia). Hydrochloric acid (HCl, GOST 14261–77, ultra-high purity: puriss. spec.) was pur- chased from Sigma Tech Company. Instrumentation. a SPEKS SSP 705 spectrophotometer (UV-Vid, 190–1100 nm) (manufactured by ZAO Spectro- scopic Systems, RF) was used for the measurements. Methods. In this work the ways for preparation of ex- tracts from leaves of olive (Olea europaea L.) included two different extraction techniques: 1) the traditional solvent (ethanol-water) extraction techniques and 2) the tech- niques using the medium of subcritical water, as described earlier [8]. The traditional extraction. A 1.0 g sample of the dry ol- ive leaves of O. europaea (particle size 0.5–3.0 mm) was boiled under reflux with the addition of 15 mL of 70% aqueous solution of ethanol for 90 min (bath temperature 82 °C). The boiling procedure was repeated 3 times (ex- traction time 270 min). The obtained extracts were fil- tered, combined, and analyzed by HPLC. SBW-extraction. The treatment of olive leaves in SBW was carried out using a custom-made stainless-steel reac- tor (autoclave) [8]. The reactor has inner volume of 10 mL (Dinner = 12 mm). A portion of ground olive leaves (particle size 0.5–3.0 mm) weighing 0.5 g was placed in a reactor, to which 7 mL distilled water was then added. The reactor was hermetically sealed and placed in an oven, where it was kept at a certain temperature (accuracy ±1 °C) for 1 h. Then the reactor cooled to room temperature (15 min) in a tank filled with cold water. The contents of the reactor were filtered through a paper filter into a graduated cylin- der, washing the reaction mixture with 70% ethanol until the color was washed out (~V = 40 ml, 60 min). Aliquots of the resulting solutions were dried at room temperature under a fan. Then, the anti-acetylcholinesterase activity of the extracts, as well as the sum of polyphenols and flavo- noids in the dry product, were determined using direct and differential spectrophotometry methods. 2.2. Anti-acetylcholinesterase (anti-AChE) assay The activity of AChE (in vitro) was measured by the modi- fication [11] of Ellman's method [13]. This method measures the activity of AChE serving acetylcholine as the substrate. Stock solutions of olive leaf extracts in 50% ethanol and then diluted to working concentrations with phos- phate buffer (pH = 7.4) were prepared. The AChE enzyme inhibitory assay was carried out ac- cording to Ellman's method [13], as described elsewhere [11], with slight modifications. Olive extracts (0.6 ml) in various concentrations (0.05–4 mg/ml) were added to the 1.88 mM ATChI substrate (0.36 ml), followed by addition of 1.44 ml of Ellman's reagent (0.25 mM). Sodium phos- phate buffer (0.1 M, pH 7.4) was used for all the prepara- tions and reaction mixture; however, the AChE enzyme (2 Units/mL) was prepared in sodium phosphate buffer (0.02 M, pH 7.0). After incubation of the reaction mix for 5 min at room temperature, AChE enzyme (0.12 ml) was added to the reaction mixture. This mixture was stirred (5 s) and after that the optical density was measured at a wavelength of 412 nm within 6 minutes from the start of the reaction on a spectrophotometer (UV-Vid, 190–1100 nm). To exclude the influence of the absorption of extract solutions on the absorption in the reaction mixture control (blank) solutions of the extracts were prepared. That is, the values of its absorption at a wavelength of 412 nm were subtracted from the values of the absorption of the reaction mixture. Phosphate buffer (pH 7.4) was added to the control reaction mixture instead of the test substance solution. The assay was performed in triplicate (n = 3). The per- centage inhibition was calculated using the formula: % inhibition = 1 − [ Absorption of the test sample at 412 nm Absorption control at 412 nm ] × 100 (1) The results of AChE inhibition were expressed as IC50: concentration of extracts (mg/mL) that resulted in 50% inhibition of enzyme activity. The values of IC50 were ob- tained from the dose-response curves. Total phenolic content assay. Total phenolic content was evaluated spectrophotometrically by the modified Folin-Chocalteu method [12]. The total phenolic contents of the OL were expressed as gallic acid equivalents in mil- ligram per gram of dried leaves. Gallic acid as a polyphe- nol standard was used. The optical density of the solutions was measured after 30 minutes in a 1 cm quartz cuvette at a wavelength of 750 nm. Based on the obtained data, a calibration curve was plotted (y = 107.3x + 0.003, R² = 0.997). In addition, the total phenolic content was evaluated in the extracts of OL obtained in different ways. 3. Results and discussion As a first stage of this study, the total phenolic content (TPC) of the TSE-extract was determined. It was found that the TSE extract contained 42.6 mg/g of TPC (in terms of standard gallic acid). The value of the inhibitory activity of the AChE enzyme of the TSE extract, based on the dose-response curves, was IC50 = 3.6 mg/ml, respectively. As a next stage, the set of the SbW-extracts (at 120 °C, 180 °C, and 220 °C) were obtained. Also, the yields of the TPC of the SbW-extracts, ob- tained in the temperature range 120–220 °C, were deter- mined. The yields of the TPC of SbW-extracts at 120 °C, 180 °C, and 220 °C were 32.7 mg/g, 41.8 mg/g, and Chimica Techno Acta 2021, vol. 8(4), № 20218403 ARTICLE 4 of 5 70.4 mg/g, respectively (Table 1). After that, the values of the AChE-activity for the obtained SbW-extracts were de- termined. The calculated values of the AChE activity, ex- pressed as IC50, were: IC50 = 2.92 mg/ml (120 °C); IC50 = 0.8 mg/ml (180 °C); IC50 = 0.35 mg/ml (220 °C), respectively. The obtained results are presented in Table 1. As can be seen from Table 1, the total content of phenol- ic compounds and AChE activities of OL extracts varied de- pending on the used extraction method. The extract ob- tained using the subcritical water technique (220 °C) showed the highest amounts of total phenolics (70.4 mg/g raw material) and the highest AChE inhibitory activity (IC50 = 0.35 mg/ml). Also, the obtained values of the anti- AChE activity of the extracts OL demonstrated that the in- hibitory activity for SbW-extract 120 °C (IC50 = 2.92 mg/ml) and SbW-extract 180 °C (IC50 = 0.8 mg/ml) are higher than those observed for the traditional extract (IC50 = 3.6 mg/ml), respectively. The obtained results indi- cated that a percentage (%) of inhibition of the AChE en- zyme activity by OL extracts is determined by the total con- tent of polyphenols in the investigated extracts. At the same time, it should be noted that an increase of the temperature of subcritical water from 120 to 220 °C causes a change in the physicochemical characteristics of water [7, 14]. Therefore, one can expect an increase in the solubility of plant metabolites, on the one hand, and the chemical transformation of polyphenols from the native OL, on the other hand. Recently, the authors have shown that in a subcritical water medium in a temperature range 180–220 °C a hydrolysis of rutin to quercetin took place (Fig. 2) [14–16]. Table 1 The values IC50 and total phenolic content of OL extracts, obtained by different extraction techniques: the TSE and the SbWE The methods of extraction TPC in terms of stand- ard gallic acid, mg/g of raw material Anti-AChE activity (IC50), mg/ml TSE 42.6 3.6 SbWE – 120 °C 32.7 2.92 SbWE – 180 °C 41.8 0.8 SbWE – 220 °C 70.4 0.35 Fig. 2 Hydrolysis of rutin in a medium of subcritical water According to the literature [17], quercetin is more ac- tive against the AChE enzyme than its glycoside rutin. The latter circumstance suggests that in SBW-extracts (180 and 220 °C) the aglycons, such as quercetin, are the main components to determinate the anti-AChE activity of the corresponding extracts. Thus, it was shown that the solution of the extract ob- tained in SBW at 220 °C has the best effect of inhibition of AChE, in contrast to the extract obtained by the traditional method. 4. Conclusions For the first time, the total phenol content and the anti- acetylcholinesterase activity were studied in the extracts of olive leaves obtained using both subcritical water and conventional (ethanol-water) extraction. It was shown that the total content of phenolic com- pounds and the anti-AChE activity of OL extracts varies depending on the extraction method used. The obtained SBW-extract (220 °C) showed the highest amounts of total phenolics, expressed as gallic acid equivalents, (70.4 mg/g raw material), and the highest inhibitory AChE-activity (IC50 = 0.35 mg/ml). The obtained values of the anti-AChE activity of the ex- tracts of olive’s leaves demonstrated that the inhibitory activities for SbWE 120 °C (IC50 = 2.92 mg/ml) and SbWE 180 °C (IC50 = 0.8 mg/ml) are higher than that of the tra- ditional extract (IC50 = 3.6 mg/ml), respectively. The obtained research results demonstrate the pro- spects for widespread use of the extracts of olive leaves in the development of pharmaceutical substances and nutraceuticals for the prevention or the treatment of Alz- heimer's disease, as well as other neurodegenerative dis- eases. Acknowledgements This work was supported by the Ministry of Science and Higher Education of the Russian Federation (State assign- ment in the field of scientific activity, project No 0852- 2020-0031) and the Russian Foundation for Basic Re- search (RFBR, grant no. 19-33-90211-Aspiranty (S.S. Khizrieva)). References 1. Mahadik VJ, Chavare MN, Patil SS, Wadkar KA. Cognition Enhancing potential of Sesbania grandiflora fruit extract in Scopolamine induced Amnesia in mice. Res J Pharm Technol. 2020;13(11):5057–62. doi:10.5958/0974-360X.2020.00886.0 2. Sharma VK. Current Therapeutic Strategies for Alzheimer’s disease: A Lost Direction or A Hope Remains? Res J Pharm Pharmacodynamics. 2010;2(3):215–20. 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