PEER-REVIEW ARTICLE PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8797 Chemical Composition and Biological Activities of Essential Oils from Alyssoides utriculata (L.) Medik Osman Üçüncü * The chemical compositions, antioxidant activities, and antimicrobial activities of the essential oils acquired from the separated parts of air-dried flowers, leaves, and stems of Alyssoides utriculata L. plant growing in Turkiye were determined. Three volatile oil components were acquired via hydrodistillation using a Clevenger apparatus and analyzed by the Gas Chromatography-Mass spectrometry/Flame Ionization Detection (GC- MS/FID) analysis. A total of 75, 67, and 76 compounds in the volatile oils of flower, leaves, and stem of A. utriculata were identified, respectively. The highest percentage of chemical compounds in the essential oils of A. utriculata were determined to be monoterpenes in flowers and leaves, (72.4% and 66.5%) and hydrocarbons (29.2%) in stems. While α-pinene (62.5% and 46.7%) was defined as the major compound in the flowers and leaves, nonane (21.2%) was determined to be so in the stem essential oil. The antioxidant activity of the obtained essential oils was determined according to free radical scavenging and total phenolic content (TPC), and antimicrobial activity against 12 bacteria and 5 fungi, using the agar dilution method. The amount of TPC and scavenging activity of the flower oil were found to be 440.61 ± 6.26 mg GAE/L and 46.00 ± 1.28%, respectively. Based on the antimicrobial activity results, all the essential oils of A. utriculata were determined to have antimicrobial activity against Escherichia coli and Bacillus subtilis. DOI: 10.15376/biores.19.4.8797-8811 Keywords: Essential oil; Chemical composition; Antioxidant activity; Antimicrobial activity; α-Pinene; Alyssoides utriculata Contact information: Department of Pharmacy Services, Macka Vocational School, Karadeniz Technical University, Trabzon, 61750 Turkiye; *Corresponding author: osmanucuncu@yahoo.com INTRODUCTION Since the beginning of humanity, human beings have been using plants to meet their basic needs such as nutrition, treatment, and warmth. In complementary medicine for treatment purposes, whole plants, including leaves, roots, and flowers, are used, as well as various extracts and essential oils obtained from them (Al Abboud et al. 2024; Alghonaim et al. 2023). The Brassicaceae (also known as Cruciferae) family has economic, agricultural, nutritional, and medicinal qualities (Preedy 2015). Foods, such as cabbage, broccoli, Bok-choy, and mustard, which have an important place in daily nutrition, are from the Brassicaceae family and contain glucosinolates, minerals, carotenoids, soluble sugars, polyphenols, vitamins, and antioxidant compounds (Preedy 2015; Luo et al. 2022). Members of Brassicaceae are widely used in traditional medicine and as veterinary medicines for livestock (Salehi et al. 2021). The popularity and consumption of vegetable Brassicaceae family members are increasing due to their nutritional value and biological effects. Their phytochemical http://www.yourlinkhere.edu/ mailto:osmanucuncu@yahoo.com PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8798 composition has been studied, as they contain valuable secondary metabolites, such as glucosinolates, phenolic compounds (hydroxycinnamic acids, flavonoids, anthocyanins, tocopherols, and carotenoids), terpenes, and fatty acids, which are directly linked to different biological activities (Favela-González et al. 2020). Brassicaceae metabolites are used in the treatment of chronic diseases such as obesity, type-2 diabetes, stroke, hypertension, and cancer (Favela-González et al. 2020). Previous studies have reported that the essential oils and extracts of Brassicaceae species are rich in glucosinolates and that they have biological activities such as anticancer, anti-inflammatory, antimicrobial, anti-obesity, cardioprotective, gastroprotective, and antioxidant activities (Favela- González et al. 2020; Salehi et al. 2021). They are also known to contain high amounts of carotenoids, tocopherol, and ascorbic acid, which have antioxidant effects (Singh et al. 2017). The genus Alyssoides Mill., a member of the Brassicaceae family, is represented by two species (The Plant List 2013). Alyssoides is morphologically similar to the genus Physoptychis Boiss. according to Flora of Turkey (Cullen 1965) and distinguished from Physoptychis with less than 10 mm fruit diameter. According to phylogenetic-based studies, the genus Alyssoides is not monophyletic, and the members of the genus are grouped with members of Fibigia Medik. Alyssoides utriculata (L.) Medik is the only species of the genus found in Turkiye, and it is a yellow-flowered ornamental shrubby plant native to the country (Cullen 1965). This species is both an ornamental plant and used in some forms of treatment (rabies and hiccup) (Blazevic et al. 2013). Alyssoides utriculata var. utriculata is the only member of Alyssoides utriculata at the variety level in the Flora of Turkey (Cullen 1965; Mutlu 2012). Essential oils are complex mixtures of low concentrations derived from different parts of plants and evaporate easily at room temperature (Fidan et al. 2022). The essential oils exhibit refreshing, pain-relieving, stress-relieving, insecticidal, antimicrobial, antifungal, and antioxidant activities and are used in the food preservation and cosmetic industries (Polatoğlu et al. 2013; Yılar et al. 2016; Cüce and Basançelebi 2021; Saruhan and Oz 2023). It has been reported that that the essential oils of Brassicaceae family members contain interesting natural phytochemicals such as allyl isothiocyanate (B. juncea, B, nigra), 1-butene-4-isothiocyanate (B. juncea, B. napus), benzyl isothiocyanate and 2-phenylethyl-isothiocyanate (Sinapis alba) as sulfur-containing compounds, hexahydrofarnesyl acetone (Arabis alpina, Eruca vesicaria), pulegone, isomenthone (B. campestris), phytol (Capsella bursa-pastoris), and β-elemene as terpene derivatives, and 2,6,10-trimethyldecane, nonacosane (Arabis alpina, Capsella bursa-pastoris) as hydrocarbons (Singh et al. 2015; Hichri et al. 2016; Salehi et al. 2021; Ucuncu 2021; Gumusok et al. 2023). There is only one study in the literature on the essential oils and biological activities of A. utriculata. Blazevic et al. (2013) investigated the chemical composition of the essential oil obtained from A. utriculata and the acetyl cholinesterase activities of dichloromethane extracts. In the gas chromatography-mass spectrometry (GC/MS) analysis of essential oils of different parts of A. utriculata, chemical compounds belonging to the compound classes alcohols, carbonyls, alkanes, sulfur compounds, terpenes, fatty acids and esters, phenols, and phenylpropane derivatives were detected. According to this report, compounds such as but-3-enyl isothiocyanate, erucin, and sulforaphane, which are glucosinolates degradation products, are responsible for the acetylcholinesterase activity exhibited by the essential oil and extracts (Blazevic et al. 2013). https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/pulegone PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8799 The chemical compositions and biological activities of A. urticulata with respect to individual parts of the plant, which may be important towards potential use, have not been explored. The goal of the present research is to determine the chemical compositions of essential oils in the air-dried parts (flower, leaf, and stem) of A. utriculata, which can be considered as a member of the Brassicaceae family, and has been subject to limited studies, and to investigate their antimicrobial and antioxidant capacities. EXPERIMENTAL Plant Materials Alyssoides utriculata plant was collected from the roadsides between Torul and Kürtün, Gümüşhane: (40° 38' 30" N, 39° 11' 39" E at 800 m above sea level) in Turkiye (A7), a location with dry air and sandy soil, during June 2022. Flowers, leaves, and stems of A. utriculata were separated and air-dried at room temperature (20 to 22°C). The botanical identification of the plant was carried out by Prof. Kamil Coşkunçelebi in the Department of Biology, at Karadeniz Technical University (KTU), Trabzon, Turkiye. Voucher specimens were deposited with the number KTUB743 in the Herbarium of KTU. Separation and Analysis of the Essential Oils The volatile oils from air-dried plant parts (flower – 85 g, leaves – 54 g, and stems – 124 g) of A. utriculata were isolated using a modified Clevenger-type hydrodistillation apparatus (4 h, yields: 0.24%, 0.19%, and 0.08 % (w/w), respectively) (Ucuncu et al. 2019). Hydrodistillation for each sample was carried out three times, and the average value of the essential oil percentage (w/w) was used for the final evaluation and was detected on an air- dried weight basis. Essential oil yields were calculated with the following Eq. 1 (Fidan et al. 2022): 𝑌𝑖𝑒𝑙𝑑 (%) = (Amount of extracted essential oil (g)) (Amount of air−dried plant material (g)) × 100 (1) The essential oils obtained from the air-dried plants were taken by dissolving in 1.0 mL high-performance liquid chromatography (HPLC) grade n-hexane, dried over anhydrous sodium sulfate, and filtered (Ucuncu et al. 2019; Fidan et al. 2022). A HP-5MS capillary chromatographic apolar column (film thickness 0.2 μm 30 m × 0.25 mm ID) was used for GC-FID (Agilent-7890A) and GC-MS (Agilent 5975C) analyses. These analyses were employed as described previously (Ucuncu et al. 2019; Fidan et al. 2022; Oz 2022). The essential oils were analyzed twice. The GC peak areas of essential oil compounds were clarified by comparing the NIST and Willey libraries in the GC-MS device. The retention indices (RI) of components were determined through the retention times (RT) of homolog n-alkanes (C6-C32) and authentic compounds with linear interpolation. Identification of volatile compounds was determined by matching their RI values with NIST and Willey library data, comparing Kovats indices (KI) and literature value. Quantitative determination of components was performed with regard to peak area integration with GC-FID (Adams 2007; Ucuncu et al. 2019; Fidan et al. 2022; Oz 2022; Chemdata NIST 2023). PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8800 DPPH Assay and the Total Phenolic Content The antioxidant activities of essential oils were determined by free radical scavenging capacity (DPPH assay), the most frequently used antioxidant/antiradical test, and by total phenolic contents (TPC) analysis, which shows not only total phenolic content but also total reducing capacity of the sample and is widely accepted as an antioxidant test (Sanchez-Moreno et al. 1998; Kasangana et al. 2015). The free radical scavenging activities of volatile oils against stable 2,2-diphenyl-2- picrylhydrazyl hydrate (DPPH·) were spectrophotometrically determined. For this purpose, a DPPH solution prepared with 4 mL of 0.1 mM methanol was added to the volatile oils of A. utriculata. The change in color was measured at 517 nm on a UV-Vis spectrophotometer (Libra S60, Biochrom Ltd, Cambridge UK). The measurements were performed three times, and averaged. Trolox and ascorbic acid were used as standard antioxidants for comparison (Sağdıç et al. 2011; Ahmed et al. 2015). The TPC amounts of volatile oils were determined by the Folin-Ciocalteu method. For this purpose, the absorbances of the samples were measured at 765 nm. TPC in essential oils were expressed as gallic acid equivalents (GAE) according to the method described previously (Ucuncu et al. 2019). The measurements were performed three times, and averaged (Kasangana et al. 2015). Antimicrobial Activity Assessment The antimicrobial activities of essential oils were determined using the agar-well diffusion method against 12 bacteria and 5 yeast samples. The antimicrobial tests were made in Gümüşhane University Food Engineering Laboratories. For this purpose, the samples of volatile oils were dissolved in HPLC-grade n-hexane to prepare stock solutions. Measurements were made according to previously described methods (Sağdıç and Özcan 2003; Matuschek et al. 2014). The results were expressed as inhibition zones (mm) of test microorganisms. The results of antimicrobial activity are given in Table 3. Statistical Analysis Statistical analyses were performed using Microsoft Excel software with XLSTAT (Addinsoft, Version 2024 New York, NY, USA). The consistency of measurements across the analysis was assessed using the relative standard deviation of repeatability (RSDr%) and the predicted relative standard deviation (PRSDr%). RESULTS AND DISCUSSION Chemical Composition of Essential Oils The results of GC-MS and GC-FID analyses performed to determine the chemical compositions of essential oils of A. utriculata are given in Table 1, and their chemical class distributions are presented in Fig. 1. About 119 compounds were identified, constituting over 92.77%, 87.66%, and 86.26% total essential oil compositions of flowers, leaves, and stems of A. utriculata, respectively. The identified compounds are divided into 10 groups: alcohols, carbonyl compounds, fatty acids, hydrocarbons, monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, diterpene, and ‘other’. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8801 Table 1. Identified Components and Chemical Class Distribution in the Essential Oils of the Aerial Parts (Flower, Leaf, and Stem) of A. utriculata No. Compounds Concentration (%) Exp. RI b N. RI c Flower a Leaf a Stem a Alcohols 1 1-Octen-3-ol 0.20 977 976 2 (E)-2-Decen-1-ol 0.28 1270 1273 3 2-Methyl-Z,Z-3,13- octadecadienol 0.96 2145 MS Carbonyl Compounds 4 Hexanal 0.66 801 800 5 2-(E)-Hexenal 0.11 0.42 0.09 854 854 6 2-(E)-Heptenal 0.16 959 961 7 Benzaldehyde 0.08 962 966 8 2,3-Octadienone 0.09 982 986 9 6-Methyl-5-hepten-2-one 0.03 0.05 0.15 987 987 10 Octanal 0.11 1002 1001 11 3-Octen-2-one 0.16 1039 1037 12 Benzeneacetaldehyde 0.05 1044 1043 13 Acetophenoned 0.38 0.56 0.49 1066 1065 14 2-Nonanone 0.12 1093 1095 15 Nonanal 0.06 0.03 0.42 1105 1102 16 (E)-2-Nonenal 0.36 1160 1159 17 2-Methyl-3-phenyl-propanal 0.05 0.06 1241 1244 18 (E)-2-Decenal 0.23 1262 1261 19 2-Undecanone 0.06 1294 1294 20 (E,E)-2,4-Decadienal 0.01 1317 1317 21 (E)-2-Undecenal 0.44 1364 1364 22 (Z)-Jasmone 0.05 1400 1400 23 Z-3-Hexen-1-ol benzoate 0.31 1574 1573 24 Benzyl benzoate 0.03 1768 1770 25 Benzoic acid, octyl ester 0.15 1783 1779 26 Phthalic acid, isobutyl octyl ester 0.72 1871 MS Fatty Acids 27 Nonanoic acid 0.40 1275 1272 28 Dodecanoic acid 0.82 1569 1568 29 Tetradecanoic acid 1.01 1767 1767 30 Pentadecanoic acid 1.04 1867 1867 31 Hexadecanoic acid 0.06 18.37 1965 1968 32 Heptadecanoic acid 0.33 2078 2077 Hydrocarbons 33 Nonaned 0.36 0.46 21.23 901 900 34 Undecaned 0.31 0.25 4.26 1100 1100 35 Tricosaned 0.04 0.57 2299 2300 36 Tetracosaned 0.22 2400 2400 37 Pentacosaned 0.05 0.19 1.11 2500 2500 38 Hexacosaned 0.15 2601 2600 39 Heptacosaned 0.65 2700 2700 40 Nonacosaned 1.02 2900 2900 Monoterpene Hydrocarbons 41 α-Pinened 62.46 46.69 6.32 939 939 42 Camphene 1.18 3.54 0.26 949 947 43 Verbenene 0.21 0.17 953 951 PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8802 44 Sabinene 0.78 0.20 974 975 45 β-Pinene 1.75 0.72 0.13 978 979 46 α-Phellandrene 0.15 0.09 1005 1007 47 α-Terpinene 0.46 0.46 1017 1017 48 o-Cymene 3.35 6.42 0.89 1023 1021 49 Limononed 2.31 5.46 0.86 1027 1025 50 β-Ocimene 0.31 0.11 1038 1037 51 γ-Terpinene 0.95 1.47 0.26 1059 1060 52 α-Terpinolene 0.51 1.12 0.42 1089 1083 53 Mentha-1,4,8-triene 0.05 1112 1113 Oxygenated Monoterpenes 54 (E)-Linalool oxide 0.12 1072 1069 55 Fenchol 0.33 0.91 0.23 1114 1115 56 α-Thujone 0.05 1117 1114 57 1,3,8-p-Menthatriene 0.08 1119 1119 58 α-Campholenal 0.65 1.12 0.28 1127 1130 59 (E)-Pinocarveol 0.71 0.68 0.34 1140 1140 60 (Z)-Verbenol 0.12 0.08 1142 1141 61 Camphore 0.47 0.28 1146 1146 62 (E)-Pinocamphone 0.11 0.14 1161 1160 63 Pinocarvone 0.26 0.28 0.13 1164 1164 64 p-Mentha-1,5-dien-8-ol 0.25 0.29 1167 1167 65 Borneol 1.24 2.40 0.72 1168 1168 66 4-Terpineol 0.29 0.43 0.44 1179 1178 67 p-Cymen-8-ol 0.45 0.39 1187 1188 68 α-Terpineol 0.53 1.21 0.60 1193 1190 69 Mrytenol 0.37 0.41 0.33 1198 1198 70 Verbenone 0.76 0.41 1211 1212 71 trans-Carveol 0.31 0.42 0.10 1220 1220 72 α-Fenchyl acetate 0.03 1230 1228 73 D-Carvone 0.08 0.08 1243 1242 74 Carvotanacetone 0.04 0.15 1249 1250 75 3-Carvomenthenone 0.05 1256 1256 76 Thymol 0.03 0.11 1287 1286 77 Carvacrol 0.09 0.21 1321 1317 78 Geranyl acetone 0.18 1451 1452 Sesquiterpene Hydrocarbons 79 Bicycloelemene 0.05 1339 1338 80 α-Cubebene 0.06 1352 1351 81 Cyclosativene 0.54 1368 1368 82 α-Ylangene 0.13 0.03 0.30 1374 1374 83 α-Copaene 0.19 0.09 1.05 1378 1378 84 β-Bourbonene 0.05 0.10 1386 1386 85 β-Cubebene 0.18 1388 1388 86 β-Patchoulene 0.06 0.07 1390 1388 87 α-Gurjunene 0.12 1403 1408 88 Junipene 0.08 1405 1405 89 β-Maaliene 0.21 1413 1414 90 (E)-β-Caryophyllene 0.47 0.11 0.23 1422 1419 91 γ-Elemene 0.06 1431 1434 92 α-Guaiene 0.21 1438 1439 93 Aromadendrene 0.38 0.17 0.88 1442 1443 94 α-Humulene 0.07 1457 1456 95 Alloaromadendrene 0.16 1465 1467 96 γ-Gurjunene 0.36 0.51 1478 1479 PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8803 97 α-Amorphene 1.63 0.63 3.25 1480 MS 98 Germacrene D 0.19 1486 1485 99 β-Selinene 0.64 0.64 0.80 1490 1489 100 Valencene 0.49 0.33 1496 1496 101 α-Selinene 0.73 0.38 0.84 1499 1498 102 α-Muurolene 0.15 0.07 0.78 1503 1505 103 Calamenene 0.71 1527 1531 104 δ-Cadinene 0.98 2.77 1528 1528 105 E-Cadina-1,4-diene 0.06 1536 1537 106 α-Calacorene 0.26 0.28 0.15 1541 1542 107 α-Cadinene 0.13 0.27 1544 1544 108 β-Calacorene 0.11 0.58 1566 1564 Oxygenated Sesquiterpenes 109 Spathulenol 1.10 2.15 0.55 1585 1582 110 Salvial-4(14)-en-1-one 0.18 0.29 1599 1599 111 Viridiflorol 0.19 0.31 1606 1605 112 Isospathulenol 0.15 1640 1640 113 α-Cadinol 0.32 1644 1645 114 τ-Muurolol 0.37 0.76 0.30 1646 1648 115 Cadalene 0.25 0.31 0.50 1684 1688 116 Hexahydrofarnesyl acetone 0.17 0.18 1.28 1846 1846 117 (E,E)-Farnesyl acetone 0.25 1921 1921 Diterpene 118 ent-Pimara-8,15-diene 0.13 1941 1942 Other 119 2-Pentyl furan 0.32 990 988 Total (%) 92.77 (NC: 75) 87.66 (NC: 67) 86.26 (NC: 76) a Percentages obtained by FID peak-area normalization; b Retention index calculated from retention times relative to n-alkanes (C6-C32) on the non-polar HP-5MS column. c Literature retention indices (RI) on HP-5MS column as seen in NIST, Willey, Kovats Index, and Adams (2007). d Included as authentic compound, NC: Numbers of compounds, and MS: Identification of mass spectrum. For flower, leaves, and stem essential oils (Table 1), 75, 67, and 76 compounds were determined by GC-MS and GC-FID analyses, respectively Among them, three alcohols, 23 carbonyl compounds, six fatty acids, eight hydrocarbons, 13 monoterpene hydrocarbons, 25 oxygenated monoterpenes, 30 sesquiterpene hydrocarbons, nine oxygenated sesquiterpenes, one diterpene, and one other compound were identified (Fig. 1). While monoterpene hydrocarbons were the main chemical class of flowers (74.4%), and leaf oils (66.5%), hydrocarbons were the abundant class of stem oils (29.21%). The main compounds were as follows: α-pinene (62.5%), o-cymene (3.4%), and limonene (2.3%) for flower oils; α-pinene (46.5%), o-cymene (6.4%), and limonene (5.5%) for leaf oils; nonane (21.2%), hexadecanoic acid (18.4%), and α-pinene (6.3%) for stem oils. Approximately 36 compounds were common to all three essential oils. Nonane, α- pinene, o-cymene, limonene, borneol, α-amorphene, β-selinene, α-selinene, spathulenol, and hexahydrofarnesyl acetone were common components with relatively high quantities in all parts of A. utriculata. A study conducted by Blazevic et al. (2013) showed that 31 compounds were detected in the essential oils of the whole plant (A. utriculata), of which PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8804 hexadecanoic acid (11.5%), nonacosane (10.0%), hexahydrofarnesyl acetone (5.9%), phytol (4.3%), and heptacosane (4.0%) made up the majority (Blazevic et al. 2013). When compared to the work of Blazevich et al. (2013) (E,E)-2,4-decadienal, tricosane, tetracosane, pentacosane, nonacosane, thymol, β-caryophyllene, α-cadinol, hexahydro- farnesyl acetone, tetradecanoic acid, pentadecanoic acid, and hexadecanoic acid were similarly detected in the current study. In another study, Saka et al. (2017) identified 34 and 39 compounds in the essential oils of Brassica rapa var. rapifera leaves and roots (Saka et al. 2017). In this study, methyl-5-hexenenitrile (52.6%), 2-phenylethanol (10.2%), menthol (5.3%), allyl isothiocyanate (4.6%), and hexahydrofarnesyl acetone (3.2%), were abundant compounds in leaf essential oil. When this study is compared with Saka et al. (2017), it is seen that terpene derivative compounds, such as geranyl acetone, hexahydrofarnesyl acetone, α-pinene, β-pinene, camphene, sabinene, α-terpinene, limonone, and α-terpineol, were similar. Fig. 1. Chemical class distributions of the components identified in the essential oils of the aerial parts (flower, leaf, and stem) of A. utriculata The chemical differences of the essential oils obtained in the present study can be used as alternative additives in foods, medicines, and cosmetic preparations. Terpene and terpene-related compounds in the essential oils in the current study are known for their important biological activities in humans (Saka et al. 2017). For example, among the identified compounds, α-pinene was an abundant compound of flower, leaf, and stem essential oils in ratios 62.46%, 46.49%, and 6.32%, respectively. There are studies in the literature showing that α-pinene is used for its anti-inflammatory, antimicrobial, anticancer, antiulcerogenic, and gastroprotective properties, and its ability to aid memory retention (Salehi et al. 2021). Possessing high amounts of α-pinene in its volatile oils, A. utriculata appears to be a potentially good source of biological effects. According to a literature survey, attractive natural phytochemicals, such as isothiocyanates, thymol, limonene, 1,5-heptadiene, 3-methyl-3-butenenitrile, α-farnesene, and linalool, have been reported from essential oils of Brassicaceae with wide bioactivities 0 10 20 30 40 50 60 70 80 % Amount Number of compound % Amount Number of compound % Amount Number of compound Flower Leaf Stem 74.42 12 66.50 13 9.14 77.12 20 10.18 22 3.47 11 6.94 22 4.45 15 13.38 18 0.76 4 0.9 3 29.21 8 0.89 9 1.48 7 4.32 16 Chemical class distributions of the components identified in the essential oils of the aerial parts (flower, leaf, and stem) of A. utriculata Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Diterpene Hydrocarbones Carbonyl Compounds Fatty acids PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8805 (Salehi et al. 2021). The essential oils in this family generally contain characteristic sulfur and nitrogen compounds (Salehi et al. 2021; Ucuncu 2021). However, sulfur and nitrogen compounds were not found in any of the essential oils in the current study. Some differences within the chemical composition of volatile oils from Brassicaceae and in the present study were observed, and it is probably related to different species, agronomical factors, extraction techniques, and several physical and chemical environmental factors (Salehi et al. 2021). Biological Activities The antioxidant capacities of essential oils (flowers, leaves, and stems) were investigated using TPC and DPPH. The antioxidant analysis results of the essential oils of A. utriculata are given in Table 2. The TPC and DPPH analyses of essential oils demonstrated very good repeatability. The indices showed excellent consistency (RSDr% < PRSDr%) across the values obtained from three separate analytical trials. The repeatability values (RSDr% < RSDr%) of the flower, leaf, and stem EO samples for TPC and DPPH tests were determined to be 1.42 < 4.23, 1.99 < 4.59, 2.29 < 4.72, and 1.11 < 4.28, 2.86 < 4.08, 2.25 < 4.20, respectively. The values of TPC and DPPH scavenging of flower oil were higher than those of other oils. Brassicaceae family members are known to have antioxidant properties (Golkar and Moattar 2019). Table 2. Total Phenolic Content and Reducing Activity of the Essential Oils from Aerial Parts of A. utriculata Sample Total Phenolic Content (mg GAE/L) DPPH Scavenging Activity (%) DPPH IC50 (µg/mL) Flower EO 440.61 ± 6.24 % 46.00 ± 1.28 478.92 ± 5.82 Leaf EO 255.04 ± 5.11 % 24.10 ± 0.98 529.13 ± 15.34 Stem EO 210.20 ± 4.83 % 21.65 ± 0.82 485.45 ± 12.43 Ascorbic Acid - %98.66 ± 1.39 119.46 ± 11.93 Trolox - %98.88 ± 1.47 148.45 ± 15.44 EO: Essential oil, and GAE/L: Gallic acid equivalent per L, ±: Standart deviation In this study, the TPC of the flower, leaf, and stem essential oil samples were found to be 440.61 ± 6.24, 255.04 ± 5.11, and 210.20 ± 4.83 mg GAE/L, respectively. According to the results, the TPC of the sample oils were similar to those reported by Ucuncu (2021) in the essential oils of the flower (485.60 ± 7.28 mg GAE/L) and aerial parts (140.00 ± 3.24 mg GAE/L) of Arabis alpina. In another study, the TPC of Iberis amara essential oils were 32.9 ± 0.7 (mg/g GAE/g DW) and 28.3 ± 1.7 (mg/g GAE/g DW) in leaf and bud explants, respectively (Golkar and Moattar 2019). In the present study, DPPH values of the samples were defined as 46.00 ± 1.28, 24.10 ± 0.98, and 21.65 ± 0.82%, respectively. The percentage DPPH scavenging values of trolox and ascorbic acid were found to be 98.66 ± 1.39% and 98.88 ± 1.47%, respectively, at a 200 μg/mL concentration. Ucuncu (2021) also determined the DPPH scavenging activity in flower (as 49.85 ± 1.22) and in aerial parts (as 23.20 ± 0.76%) in the essential oils of A. alpina. In another study, Balpinar (2018) detected DPPH scavenging activity (as 76.3%) in the flower-fruit-seed ethanol extract of Arabis alpina L. subsp. brevifolia. In a study by Xiao et al. (2019), the DPPH radical scavenging capacity of different varieties of the Brassicaceae ranged from 157.3 to 806.3 μmol of Trolox equivalents (TE)/100 g of fresh vegetable. These values correspond to TPC ranging from 88.6 to 811.2 mg of gallic acid equivalents (GAE)/100 g of fresh vegetable (Xiao et al. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8806 2018). The DPPH and TPC values obtained in the present study are consistent with the literature. According to the results of antioxidant analysis, essential oils showed reduction in the stable violet DPPH radical to the yellow-colored diphenylpicryl hydrazine, reaching 50% of reduction with IC50 values ranging 478.92 ± 5.82, 529.13 ± 15.34, and 485.45 ± 12.43 µg/mL for flower, leaves and stem, respectively (Table 2). The DPPH-radical scavenging activities of essential oils were lower compared to standard antioxidants ascorbic acid and trolox (IC50 119.46 ± 11.93 and 148.45 ± 15.44, respectively). According to the IC50 values it can be suggested that components present in the studied essential oils that are capable to scavenge DPPH radicals. The antioxidant capacity values of the essential oils found in this study are moderate. Monoterpene hydrocarbons, which are the main group components of the flower and leaf essential oils of A. utriculata, are known to act as radical scavenging agents (Golkar and Moattar 2019). The terpenic compounds, such as α-pinene, limonene, and o- cymene, play a significant role in electron transfer/hydrogen donating ability, and these compounds were found in the essential oils of A. utriculata (Pandey and Rizvi 2009). The antioxidant capacity of α-pinene (62.46% and 46.69%), which is abundant in flower and leaf essential oils, is well known (Bouzenna et al. 2017; Wang et al. 2019). The volatile oils of A. utriculata exhibited different inhibition levels against selected four gram (+), eight gram (-) bacteria as well as five fungi, as shown in Table 3. Five different concentrations (50, 100, 200, 500, and 1000 ppm) of essential oils were tested in this study. No antimicrobial activity of essential oils was observed at 50, 100, and 200 ppm concentrations. The inhibition zone increased with an increased concentration of A. utriculata volatile oils. A 10-ppm streptomycin sulfate was used as a standard antimicrobial. A 30-ppm nistasine was used as a standard antifungal. All the essential oils showed antibacterial activity against gram (-) ESC and gram (+) BS at 1000 ppm. The flower, leaves, and stem essential oil showed good antimicrobial activity against ESC (inhibition zone (mm) > 5.20 mm, 5.39 mm, and 5.30 mm, respectively). In contrast, flower oil exhibited moderate inhibition activity against KP and LM. The leaf and stem essential oils were effective fungi SC, whereas they showed no antifungal activity for the fungi studied in the present work. The antimicrobial activities of the samples were lower than the standards. For the antimicrobial activity analysis of the essential oils (RSDr% < RSDr%), repeatability was very good. According to the literature, Brassicaceae plants have shown good antimicrobial activity against bacteria and fungi (Balpinar 2018; Favela-González et al. 2020; Salehi et al. 2021; Ucuncu 2021). In one of the studies, the ethanol extracts of Brassica oleracea showed the maximum zone of inhibition for Aspergillus fumigatus, Citrobacter divergens and Klebsiella pneumonia at a concentration of 200 μg/200 μL (Paul et al. 2012). In another study, the essential oils of black mustard (Brassica nigra) exhibited antifungal activity against Botrytis cinera, Aspergillus niger, Aspergillus ochraceus, and Penicillium citrinum (Salehi et al. 2021). In a different study, B. rapa var. rapifera root and leaves essential oils showed great antimicrobial activity against Listeria monocytogenes and Candida albicans, moderate great activity against Staphylococcus aureus, Escherichia coli, and Aspergillus flavus, and moderate activity against Klebsiella pneumoniae and Pseudomonas aeruginosa (Saka et al. 2017). Especially, the results obtained for Klebsiella pneumoniae, Aspergillus niger, and Penicillium in the current study agree with the literature. PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8807 Table 3. Screening Results for Antimicrobial Activity of the Essential Oil from the Aerial Parts (Flower, Leaf, and Stem) of A. utriculata (expressed as inhibition zone diameter in mm) Gram (-) Bacteria Flower EO Leaf EO Stem EO 1000 ppm 500 ppm 1000 ppm 500 ppm 1000 ppm 500 ppm Streptomsin Sulfate 10 ppm Nistasine 30 ppm AH - - - - - - 17.15 ± 0.05 NT EC - - - - - - - NT ESC 5.20± 0.05 - 5.39 ± 0.05 - 5.30 ± 0.05 - 7.10 ± 0.05 NT ECO - - - - - - 15,10 ± 0,05 NT KP 6.39 ± 0.05 4.00 ± 0.05 - - - - 16.18 ± 0.05 NT PV - - - - - - 14.02 ± 0.05 NT PA - - - - - - 17.00 ± 0.05 NT ST - - - - - - 17.88 ± 0.05 NT Gram (+) Bacteria BC - - - - - - 16.00 ± 0.05 NT BS 6.29 ± 0.05 - 6.45 ± 0.05 4.30 ± 0.05 4.19 ± 0.05 - 19.20 ± 0.05 NT LM 4.14 ± 0.05 - - - - - 19.25 ± 0.05 NT SA - - - - - - 11.95 ± 0.05 NT Fungus SC - - 7.24 ± 0.05 5.27 ± 0.05 4.10 ± 0.05 - NT 18.10 ± 0.05 CA - - - - - - NT 12.20 ± 0.05 AN - - 4.00 ± 0.05 - - - NT 14.28 ± 0.05 AP - - - - - - NT 11.10 ± 0.05 P - - - - - - NT 12.08 ± 0.05 EO: Essential oil, －: No activity observed, NT: Not tested, AH: Aeromonas hydrophila ATCC 35654, EC: Enterobacter cloacae ATCC 13047, ESC: Escherichia coli ATCC 25922, ECO: Escherichia coli O157: H7 ATCC 35150, KP: Klebsiella pneumoniae ATCC 13883, PV: Proteus vulgaris FMC, PA: Pseudomonas aeruginosa ATCC 27853, ST: Salmonella typhimurium ATCC 23566, BC: Bacillus cereus ATCC 9634, BS: Bacillus subtilis ATCC 6633, LM: Listeria monocytogenes ATCC 7644, SA: Staphylococcus aureus ATCC 25923, SC: Saccharomyces cerevisiae S288C, CA: Candida albicans ATCC 10231, AN: Aspergillus niger, AP: Aspergillus flavus ATCC 46283, P: Penicillium The main component in the essential oils obtained in the current investigation is α- pinene. In one study, this compound has broad potential in antimicrobial therapy to inhibit the growth of bacteria as a synergist of antibiotics (Borges et al. 2022). The use of plant-derived antimicrobials and antioxidants is increasing continually, and plants are a great source of bioactive metabolites. The natural antimicrobials and antioxidants are a good option for the development of new alternative antimicrobials and antioxidants against resistance caused by the abuse of conventional synthetic drugs (Favela-González et al. 2020). The different antimicrobial and antioxidant effects of the PEER-REVIEWED ARTICLE bioresources.cnr.ncsu.edu Üçüncü (2024). “Essential oils activity and chemistry,” BioResources 19(4), 8797-8811. 8808 essential oils obtained in this study may be due to the different phytochemicals they contain. The reason for these differences can be attributed to agronomical, physical, chemical and environmental factors. Minor (α-pinene, o-cymene, limonene, hexadecanoic acid, etc.) or trace compounds in the present essential oils might also give rise to the exhibited biological activities. Possible synergistic effects of compounds in essential oils should also be considered. CONCLUSIONS This study characterized the identities, phytochemical content, antimicrobial and activities, and antioxidant activities of essential oils of Alyssoides utriculata L. plant. 1. The results indicated that essential oils had moderate antimicrobial and antioxidant activities. The essential oils obtained in this study were effective against 4 microorganisms and 2 fungi at different doses. The findings suggest that the essential oils of A. utriculata contain a valuable source of bioactive compounds such as α-pinene, o-cymene, and limonene. These essential oils can be used as effective tools to control foodborne pathogenic microorganisms. 2. 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