Modern green approaches for obtaining Satureja kitaibelii Wierzb. ex Heuff extracts with enhanced biological activity J. Serb. Chem. Soc. 87 (12) 1359–1365 (2022) Short communication JSCS–5599 Published 1 August 2022 1359 SHORT COMMUNICATION Modern green approaches for obtaining Satureja kitaibelii Wierzb. ex Heuff extracts with enhanced biological activity VANJA ŠEREGELJ1, OLJA ŠOVLJANSKI1*, JAROSLAVA ŠVARC-GAJIĆ1, TEODORA CVANIĆ1, ALEKSANDRA RANITOVIĆ1, JELENA VULIĆ1 and MILICA AĆIMOVIĆ2# 1Faculty of Technology Novi Sad, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia and 2Institute of Field and Vegetable Crops Novi Sad, Maksima Gorkog 30, 21000 Novi Sad, Serbia (Received 14 March, revised 14 May, accepted 16 May 2022) Abstract: Modern trends in phytochemical extraction from alternative crops support the use of alternative technologies, such as ultrasound- and microwave- -assisted extractions. Considering the reduction of toxic solvents, rapid and effective extraction process, the above-mentioned technologies have become the foundation of green chemistry approaches in a wide range of applications. These extractions have not been studied on Satureja kitaibelii Wierzb. ex Heuff, which is a highly potent plant when considering its aromatic and medi- cinal properties. This preliminary study presents an in vitro evaluation of bio- logical activities of ultrasound- and microwave-assisted extracts of S. kitaibelii, for the first time. Furthermore, it offers a totally green, modern, fast and repro- ducible method for extraction of phytochemicals from S. kitaibelii herba (Rtanj Mountain, Serbia). This short communication suggests that the applied micro- wave-assisted extraction, using only water as the solvent, can be a promising approach for obtaining green products with commercial potential. Keywords: ultrasound-assisted extraction; microwave-assisted extraction; in vitro antioxidant analysis; in vitro antimicrobial analysis; Lamiaceae. INTRODUCTION Satureja kitaibelii Wierzb. ex Heuff. or Rtanj tea is an endemic Lamiaceae species, mainly spread across the Balkan Peninsula.1 Satureja kitaibelii is well known for its aromatic and medicinal properties; hence it is used as a culinary herb in Mediterranean dishes, in aromatherapy, or in traditional medicine to treat various ailments.2 The extraction process is a crucial step in the valorisation of * Corresponding author. E-mail: vanjaseregelj@tf.uns.ac.rs # Serbian Chemical Society member. https://doi.org/10.2298/JSC220314043S ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ 1360 ŠEREGELJ et al. the plant sources; different extraction techniques and extracting solvents can inf- luence the final phytochemical composition and bioactive potential of the obtained extracts. After reviewing scientific-relevant literature it was found that the articles regarding S. kittaibelii usually include conventional extraction appro- aches, essential oil, and with two non-conventional method (subcritical water ext- raction and ultrasound-assisted extraction).1,3–5 Conventional extraction tech- niques have disadvantages like thermal degradation of bioactive compounds, or the use of a large quantity of organic solvents with toxic impact on the environ- ment or on human health. Recent trends in phytochemical extraction from plants recommend exploring the use of modern technologies; ultrasound-assisted (UA) and microwave-assisted extractions (MA) which become popular due to the fact that these techniques reduce the consumption of toxic solvents, increase the speed and extraction efficiency, thus being compliant to the principles of green chemistry.6 UA extracts (UAE) and MA extracts (MAE) of S. kittaibelii have not been studied until now. Thus, the aim of this study was to investigate the effects of UAE and MAE on the extraction of phytochemicals from this plant, in order to obtain valuable information regarding possible application in food and pharma- ceutical industries. The impact of these modern extraction technologies on the phytochemical composition and biological activity (antioxidant and antimicro- bial) was evaluated by varying different green extraction solvents. EXPERIMENTAL Plant material The Satureja kitaibelii herb was collected on the Rtanj Mountain, Serbia (43◦46′34′′ N; 21◦53′36′′ E) in July 2020. Voucher specimens (BUNS 2-1373) were used for the identific- ation of species. The collected aboveground flowering parts were dried naturally in shade at ambient temperature. Constant weight was gained after one week of drying. Dry plant mat- erial was placed in a soft paper bag until further analysis. Ultrasound-assisted extraction Ultrasound-assisted (UA) extraction was carried out in an ultrasonic bath (Iskra, Slo- venia) by placing samples in the proximity of the ultrasound source. Ground sample (5 g) was extracted at room temperature with 100 ml of solvent (70% methanol or distilled water) for 30 min. The extracts were filtered (Whatman paper No. 1) and stored at 4 °C until further analysis. Microwave-assisted extraction Microwave-assisted (MA) extraction was carried out in an adapted microwave oven des- cribed previously by Švarc-Gajić et al.7 Ground sample was extracted maintaining the same sample-to-solvent ratio, extraction time, and solvent type as in the case of UA extraction, for comparison reasons. The extraction was carried for 30 min applying magnetron power of 450 W. After completing the extraction process, the extracts were filtered (Whatman paper No. 1) and stored at 4 °C until further analysis. ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ ALTERNTIVE EXTRACTIONS OF S. kitaibelii 1361 Phytochemical analysis Phenolic quantification was performed using Shimadzu Prominence HPLC, connected to an SPD-20AV UV/Vis detector (Shimadzu, Kyoto, Japan). Separation was performed on a Luna C-18 RP column, 5 µm, 250 mm×4.6 mm (Phenomenex, Torrance, CA, USA) with a C18 guard column, 4 mm×9 mm×30 mm (Phenomenex, Torrance, CA, USA). The filtered extracts were examined by HPLC reverse phase analysis as described by Aćimović et al.4 In vitro antioxidant analysis Antioxidant activity of extracts was investigated using four in vitro assays, as outlined by Aćimović et al.:4 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzothiazo- line-6-sulphonic acid (ABTS), superoxide anion (SOA) and reducing power (RP). The anti- oxidant activities were expressed as μmol of Trolox equivalents per g of dry plant material. In vitro antimicrobial analysis Antimicrobial activity of S. kitaibelii extracts was determined against the following ATTC referent strains: Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Bacillus cereus, Staphylococcus aureus, Enterococcus faecalis and Listeria monocytogenes (bacteria), Saccharomyces cerevisiae, Candida albicans (yeasts), and Aspergillus brasiliensis (fungi). Disk diffusion and microdilution methods for in vitro evaluation of antimicrobial act- ivity as well as for testing minimal inhibitory concentration were performed by methods def- ined by Mićić et al.8 Statistical analysis Statistical analyses were carried out using Origin v. 8.0 SRO software. Significant dif- ferences were calculated by ANOVA (p < 0.05). Results are presented as mean value ± stan- dard deviation (n = 3). RESULTS AND DISCUSSION The phenolics profiles of the analyzed extracts are presented in Table I. They possess obviously different phenolics contents depending on the applied tech- nique and solvent, which could be related to different extraction mechanisms and polarity of the presented compounds. The highest concentration of phenolic com- pounds was found in the extracts prepared by MAE; in terms of used solvent, significantly higher phenolic content was noted in water extract. In extracts pre- pared by UAE, the concentration of phenolic compounds was lower, but not their number. In this case, 70 % methanol exhibited better efficiency for phenolic ext- raction. The obtained results were in correlation with the literature data; Maš- ković et al.6 reported that MAE ethanol extract of summer savory (Satureja hort- ensis L.) was richer in phenolic compounds than UAE. In general, microwaves induce a sudden increase in temperature inside the cellular structure, which leads to rupturing of cell walls and fast release of phytochemicals into extracting medium. The efficiency of microwave-assisted extraction lies in the fact that the energy of microwaves is directly converted to heat, by instantaneous absorption, i.e., by rapid alignment of sample dipoles with the frequency of microwaves, thus generating heat inside the matrix.10 Consequently, an induced sudden increase in temperature inside cells causes rupture of cell walls and fast release of phyto- ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ 1362 ŠEREGELJ et al. chemicals into the extracting medium. Apart from solvent selectivity towards the analyte, the dielectric constant is a significant factor for obtaining high-quality extracts as well. According to Vladić et al.,9 the chosen solvent should possess a high dielectric constant and strongly absorb the microwave energy; water has the highest dielectric constant, followed by methanol and ethanol. Conversely, in the case of the UAE, the cavitation phenomenon and free-radical formation can cause degradation of phytochemicals.10 TABLE I. HPLC analysis of phenolic compounds in extracts (c / mg g-1) obtained by ultra- sound-assisted (UAE) and microwave-assisted extraction (MAE); values in rows with differ- ent superscripts are significantly different at p < 0.05 Compound UAE 70 % methanol UAE water MAE 70 % methanol MAE water Vanilic acid 3.15±0.01b – 1.13±0.00a – Epicatechin gallate 7.94±0.01d 0.15±0.01a 0.40±0.00b 7.31±0.05c Syringic acid 31.03±0.03b 5.17±0.04a 58.62±0.07c 59.76±0.02d Coumarin acid 1.65±0.00c 0.36±0.01a 0.81±0.00b 2.50±0.02d Caffeic acid 4.26±0.03d 0.55±0.00a 0.96±0.00b 2.45±0.01c Gentisic acid – 1.61±0.01a 2.95±0.03b – Sinapic acid 4.01±0.02c 1.14±0.02a 1.45±0.01b 4.65±0.02d Rosmarinic acid 4.44±0.03c 1.45±0.00a 2.23±0.06b 8.63±0.03d Ferulic acid 4.01±0.01b 3.12±0.02a 7.97±0.03c 17.96±0.1d Rutin 0.04±0.00a 0.45±0.00b 0.95±0.00c 4.25±0.02d Luteolin – – 0.05±0.00a – Total phenolic compounds 60.53±0.14b 14.00±0.11a 77.52±0.20c 107.51±0.28d HPLC analysis showed that the dominant compound in all extracts was syr- ingic acid, which ranged from 5.17 m/g (UAE water extract) to 59.76 mg/g (MAE water extract). The highest content of syringic acid was earlier confirmed in S. kitaibelii subcritical water extract.4 Ćetković et al.3 have also classified syr- ingic acid among the most main phenolic compounds in S. kitaibelii extracts obtained by a conventional extraction technique and different organic solvents. In general, a single assay method is not sufficient for in vitro assessment of antioxidant activity of endogenous phytochemicals. Antioxidant molecules differ in polarities, thus they can act by different mechanisms. Antioxidant activity of S. kitaibelii extracts was challenged by four methods (Table II); significant anti- oxidant potential was found in water and 70 % methanol extracts, obtained by MAE. More precisely, the antioxidant potential of tested samples decreased res- pectively: MAE water > MAE 70 %methanol > UAE 70 % methanol > UAE water. There were considerable differences noted in antimicrobial effects against tested microorganisms between UAE and MAE extracts (Table III). The UAE extracts did not show antimicrobial effect, with the exception of water extract which showed low inhibition potential against A. brasiliensis. Consequently, the ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ ALTERNTIVE EXTRACTIONS OF S. kitaibelii 1363 defined minimal inhibitory concentration is above the initial concentration of ext- ract, and further antimicrobial potential of the concentrated extracts is required. However, the antimicrobial effect was observed in the case of MAE extract, especially in the one prepared by using water as a solvent. TABLE II. In vitro antioxidant activity (μmol TE/g) of Satureja kitaibelii extracts obtained by ultrasound (UAE)- and microwave-assisted extraction (MAE); values in rows with different superscripts are significantly different at p < 0.05 Antioxidant UAE 70 % methanol UAE water MAE 70 % methanol MAE water DPPH 78.99±14.33b 33.64±2.07a 225.63±11.45c 385.38±16.56d ABTS 744.66±9.74b 685.30±17.73a 1757.86±82.45c 2571.12±76.58d RP 153.20±2.99b 133.50±1.64a 336.71±5.68c 414.93±20.07d SOA 3730.81±20.57b 1993.66±45.94a 4417.17±15.30c 4506.69±0.53d TABLE III. In vitro antimicrobial activity of Satureja kitaibelii extracts obtained by ultra- sound (UAE)- and microwave-assisted extraction (MAE) Test organism UAE 70 % methanol UAE water MAE 70 % methanol MAE water Inhibition zone, mma E. coli ATCC 25922 nd nd 13.33± 0.57 40.00±0.00 P. aeruginosa ATCC 27853 nd nd nd 29.00±0.00 S. Typhimurium ATCC 13311 nd nd nd nd B. cereus ATCC 11778 nd nd nd 28.00±0.00 S. aureus ATCC 25923 nd nd 27.33±0.57 27.00±1.00 E. faecalis ATCC 19433 nd nd nd nd L. monocytogenes ATCC 35152 nd nd 24.00±1.00 17.33±0.57 S. cerevisiae ATCC 9763 nd nd nd nd C. albicans ATCC 10231 nd nd nd 18.33±0.57 A. brasiliensis ATCC 16404 nd 11.00±0.0 nd nd Minimal inhibitory concentration, mg/mLb E. coli ATCC 25922 > 50 > 50 > 50 0.78 P. aeruginosa ATCC 27853 > 50 > 50 > 50 1.56 S. Typhimurium ATCC 13311 > 50 > 50 > 50 > 50 B. cereus ATCC 11778 > 50 > 50 > 50 0.78 S. aureus ATCC 25923 > 50 > 50 12.5 0.78 E. faecalis ATCC 19433 > 50 > 50 > 50 > 50 L. monocytogenes ATCC 35152 > 50 > 50 25 > 50 S. cerevisiae ATCC 9763 > 50 > 50 > 50 > 50 C. albicans ATCC 10231 > 50 > 50 > 50 > 50 A. brasiliensis ATCC 16404 > 50 > 50 > 50 > 50 a< 22 mm – low; 22–26 mm – intramedier; >26 mm – high antimicrobial activity, nd – not detected; baccording to the initial concentration of the extracts Both MAE extracts showed the antimicrobial effect against E. coli, S. aureus and L. monocytogenes, while water extract expressed a similar effect against P. ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ 1364 ŠEREGELJ et al. aeruginosa, B. cereus and C. albicans. These differences can be explained by the phenolic compounds profile of the tested samples. Unlike the UAE samples, the methanolic MAE sample contains syringic acid, while the water MAE sample contains syringic acid, ferulic acid and rutin. All three mentioned phenolic com- pounds have previously been reported as antimicrobial agents; ferulic acid is an inhibition factor for P. aeruginosa, S. aureus, E. coli and L. monocytogenes growth,11 syringic acid inhibits the growth of S. aureus, while E. coli, P. aeru- ginosa, B. cereus and C. albicans are sensitive to the presence of rutin.12 The minimal inhibitory concentrations of the S. kitaibelii extracts varied according to strain level in the range of 0.78 to 25 mg/mL (Table III). The lower MIC values were obtained for the water extract compared with methanolic, which also lead to differences in chemical compositions. CONCLUSION In summary, this preliminary study indicates that the extract prepared with MAE and water as solvent exhibited the highest biological activity. Special significance of the presented approach is reflected in a totally green, modern, fast and reproducible process technology. According to the bioactivity screening, this research suggests that the MAE water extract of S. kitaibelii could be used as a natural source of antioxidants for developing a wide range of safe and functional products which will be investigated further. Acknowledgement. This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Contract No. 451-03-68/2022-14/ /200134 and 451-03-68/2022-14/200032. И З В О Д МОДЕРНИ „ЗЕЛЕНИ“ НАЧИНИ ЗА ДОБИЈАЊЕ Satureja kitaibelii WIERZB. EX HEUFF ЕКСТРАКТА СА ИЗРАЖЕНОМ БИОЛОШКОМ АКТИВНОШЋУ ВАЊА ШЕРЕГЕЉ1, ОЉА ШОВЉАНСКИ1, ЈАРОСЛАВА ШВАРЦ-ГАЈИЋ1, ТЕОДОРА ЦВАНИЋ1, АЛЕКСАНДРА РАНИТОВИЋ1, ЈЕЛЕНА ВУЛИЋ1 и МИЛИЦА АЋИМОВИЋ2 1Технолошки факултет Нови Сад, Универзитет у Новом Саду, Бул. цара Лазара 1, 21000 Нови Сад и 2Институт за ратарство и повртарство Нови Сад, Максима Горког 20, 21 000 Нови Сад Савремени трендови у фитохемијској екстракцији из алтернативних усева подржа- вају истраживање употребе алтернативних технологија као што су екстракције уз помоћ ултразвука и микроталаса. С обзиром на редукцију токсичних растварача и брз и ефи- касан процес екстракције, поменуте технологије су постале темељ приступа “зеленој” хемији у широком спектру примена. С друге стране, ове екстракције нису проучаване на Satureja kitaibelii Wierzb. ex Heuff, која је веома значајна ендемична биљка с обзиром на ароматична и лековита својства. Ова прелиминарна студија је по први пут представила in vitro процену биолошке активности ултразвучних и микроталасних екстраката S. kit- aibelii. Нудећи апсолутно “зелену”, модерну, брзу и поновљиву методу за екстракцију фитокемикалија из S. kitaibelii (планина Ртањ), овo истраживање сугерише да приме- њена екстракција уз помоћ микроталаса, користећи само воду као растварач, може бити ________________________________________________________________________________________________________________________ (CC) 2022 SCS. Available on line at www.shd.org.rs/JSCS/ ALTERNTIVE EXTRACTIONS OF S. kitaibelii 1365 перспективан приступ за добијање “зелених” производа са комерцијалним потенци- јалом. (Примљено 14. марта, ревидирано 14. маја, прихваћено 16. маја 2022) REFERENCES 1. M. Aćimović, L. Pezo, V. Tešević, I. Čabarkapa, M. Todosijević, Ind. Crops Prod. 154 (2020) 112752 (https://dx.doi.org/10.1016/j.indcrop.2020.112752) 2. A. Đorđević, I. Palić, G. Stojanović, N. Ristić, R. Palić, Int. J. 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Available on line at www.shd.org.rs/JSCS/ @Article{Seregelj2022, author = {{\v{S}}eregelj, Vanja and {\v{S}}ovljanski, Olja and {\v{S}}varc-Gaji{\'{c}}, Jaroslava and Cvani{\'{c}}, Teodora and Ranitovi{\'{c}}, Aleksandra and Vuli{\'{c}}, Jelena and A{\'{c}}imovi{\'{c}}, Milica}, journal = {Journal of the Serbian Chemical Society}, title = {{Modern green approaches for obtaining Satureja kitaibelii Wierzb. ex Heuff extracts with enhanced biological activity}}, year = {2022}, issn = {1820-7421}, month = {aug}, number = {12}, pages = {1359--1365}, volume = {87}, abstract = {Modern trends in phytochemical extraction from alternative crops support the use of alternative technologies, such as ultrasound- and micro­wave-assisted extractions. Considering the reduction of toxic solvents, rapid and effective extraction process, the above-mentioned technologies have become the foundation of green chemistry approaches in a wide range of applications. These extractions have not been studied on Satureja kitaibelii Wierzb. ex Heuff, which is a highly potent plant when considering its aromatic and medi­cinal properties. This preliminary study presents an in vitro evaluation of bio­logical activities of ultrasound- and microwave-assisted extracts of S. kitaibelii, for the first time. Furthermore, it offers a totally green, modern, fast and repro­ducible method for extraction of phytochemicals from S. kitaibelii herba (Rtanj Mountain, Serbia). This short communication suggests that the applied micro­wave-assisted extraction, using only water as the solvent, can be a promising approach for obtaining green products with commercial potential.}, doi = {10.2298/JSC220314043S}, file = {:D\:/OneDrive/Mendeley Desktop/{v{S}}eregelj et al. - 2022 - Modern green approaches for obtaining Satureja kitaibelii Wierzb. ex Heuff extracts with enhanced biological ac.pdf:pdf;:02_11688_5599.pdf:PDF}, keywords = {Lamiaceae, assisted extraction, in vitro antimicrobial analysis, in vitro antioxidant analysis, microwave}, publisher = {Serbian Chemical Society}, url = {https://www.shd-pub.org.rs/index.php/JSCS/article/view/11688}, }