Stanojević et al. 2022, Biologica Nyssana 13(2) 13 (2) December 2022: 205-216 DOI: 10.5281/zenodo.7476279 Comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (Tagetes patula L.) flower petals Original Article Jelena Stanojević Faculty of Technology, University of Niš, Bulevar Oslobođenja 124, 16000 Leskovac, Serbia jstanojevic@tf.ni.ac.rs (corresponding author) Nataša Simonović Faculty of Technology, University of Niš, Bulevar Oslobođenja 124, 16000 Leskovac, Serbia Ljiljana Stanojević Faculty of Technology, University of Niš, Bulevar Oslobođenja 124, 16000 Leskovac, Serbia Zoran Ilić Faculty of Agriculture, University of Priština, Kopaonička bb, 38219 Lešak, Serbia Aleksandra Milenković Faculty of Technology, University of Niš, Bulevar Oslobođenja 124, 16000 Leskovac, Serbia Jelena Zvezdanović Faculty of Technology, University of Niš, Bulevar Oslobođenja 124, 16000 Leskovac, Serbia Dragan Cvetković Faculty of Technology, University of Niš, Bulevar Oslobođenja 124, 16000 Leskovac, Serbia Received: November 20, 2022 Revised: December 12, 2022 Accepted: December 15, 2022 Abstract: The present study aimed to determine and compare chemical composition and antioxidant activity of essential oils (EOs) isolated from marigolds (Tagetes patula L.) cultivated in the garden in southeast Serbia. The EOs were isolated from dry orange and red flower petals by Clevenger type hydrodistillation during 2 h by using 1:15 m/V hydromodule. Their qualitative composition was determined by GC/MS and quantitative by GC/FID method. The antioxidant activity was determined by using the DPPH assay. The most abundant components in the essential oil isolated from orange flower petals were geranyl acetate (36.7%) and (E)-caryophyllene (31.6%) while the one isolated from the red flower petals contained (E)-caryophyllene (69.4%) in the highest percentage. Since phototoxic thiophenes were identified in both EOs, they should not be used as components in cosmetic products for applications on areas of skin exposed to sunshine. Essential oils showed similar antioxidant activity, with orange flower EO being somewhat better. Key words: marigold, Tagetes patula L. flower petals, thiophenes, GC/MS, antioxidant activity Apstrakt: Uporedna analiza hemijskog sastava i antioksidativne aktivnosti etarskog ulja izolovanog iz narandžastih i crvenih cvetnih latica baštenske kadife (Tagetes patula L.) Cilj ovog rada bio je određivanje i poređenje hemijskog sastava i antioksidativne aktivnosti etarskih ulja izolovanih iz baštenske kadife (Tagetes patula L.) gajene u jugostočnoj Srbiji. Etarska ulja su izolovana iz suvih narandžastih i crvenih cvetnih latica Clevenger hidrodestilacijom u toku 2 h pri hidromodulu 1:15 m/V. Kvalitativni sastav izolovanih etarskih ulja određen je GC/MS metodom a kvantitivni sastav GC/FID metodom. Antioksidativna aktivnost određena je DPPH testom. Najzastupljeniji sastojci u etarskom ulju izolovanom iz narandžastih latica bili su geranil acetat (36.7%) i (E)-kariofilen (31.6%) dok je u etarskom ulju izolovanom iz crvenih latica (E)-kariofilen bio zastupljen u najvećem procentu. S obzirom na to da su fototoksični tiofeni identifikovani u oba etarska ulja, ova etarska ulja ne bi trebalo koristiti kao komponente u kozmetičkim proizvodima namenjenim nezi delova tela izloženih sunčevim zracima. Etarska ulja su pokazala slična antioksidativna svojstva, pri čemu je etarsko ulje izolovano iz narandžastih cvetnih latica nešto bolje. Ključne reči: baštenska kadifa, Tagetes patula L. cvetne latice, tiofeni, GC/MS, antioksi- dativna aktivnost Introduction The life of people and animals on the Earth, as mem- bers of delicate world ecosystem, is possible due to the most important link of the chain with which they live in symbiosis – plants. People use plants more than 10,000 years. They cultivate them, con- sume (as the main source of vitamin C), and use as animal feed, as well as for fuel production (Parisi et al., 2010). The “dark side” of ethnobotany is re- flected in phytodermatosis (dermatitis caused by plants) occurrence. It could be caused by the direct contact with plants or by association with sunlight. Skin reactions caused by contact with plants could © 2022 Stanojević et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 205 14th Symposium on the Flora of Southeastern Serbia and Neighboring Regions be divided as follows: dermatitis by physical trauma, dermatitis by pharmacological action, IgE-mediated dermatitis, dermatitis by irritation, sunlight-induced dermatitis and dermatitis by sensitization. The let- ter occurs when sensitizing substances are present. Among other plant families, these substances are present in ornamental plants such as chrysantemum, daisies, and marigolds from sunflower (Asteraceae) family (dos Reis, 2010). The genus Tagetes consists of annual and perennial herbaceous plants, commonly called marigolds. It is native to the area from south-western America to Argentina, with maximum variations occurring in Mexico, but also naturalized all over the globe (Singh et al., 2015). Phytochemical studies of different plant parts revealed the presence of phenylpropanoids, carotenoids (such as lutein, zeaxanthin, neoxanthin plus violaxanthin, β-carotene, lycopene, α-cryptoxanthin, phytoene and phytofluene), flavonoids, thiophenes, and triterpeniods (Priyanka et al., 2013; Salehi et al., 2018; Singh et al., 2020). There are at least fifty-six Tagetes species (Salehi et al., 2018) and the most widely known are T. erecta (also known as Mexican marigold), T. patula (French marigold, dwarf marigold) and T. minuta (black mint or stinking roger). They are mainly studied in the field of agriculture, because of fungicidal, bactericidal, and insecticidal activities (Singh et al., 2015; Salehi et al., 2018). T. minuta is mainly cultivated for its essential oil, while T. patula and T. erecta have floricultural use. The flowers of African marigolds are yellow to orange and do not include red coloured marigolds, while French marigolds have red, orange and yellow as well as red and orange bicolour patterns flowers (Fig. 1). The marigold species are traditionally used as analgesics, antiseptics, carminatives, diuretics, stimulants etc. in Mexico; in food seasoning and for insect repellence in USA; and in ceremonial purposes in India, Mexico and Guatemala. Recently, they have been recognised as commercial resources of essential oils and biologically active compounds for potential use as agrochemicals, colorants and nutritional supplements as well as in cosmetics (Singh et al., 2015). The aerial parts of marigolds are rich sources of strongly aromatic EO (Tagetes oil), mainly used for perfumes production (Singh et al., 2020). Generally, Tagetes essential oils are rich in monoterpene hydrocarbons (such as ocimenes, limonene, etc.) and in acyclic monoterpene ketones (such as tagetone, dihydrotagetone, and tagetenone) which are the primary odorants – Fig. 2. There are also sesquiterpene hydrocarbons and oxygenated compounds but in much lower amounts. However, the chemical diversity is quite high (Tomova et al., 2005; Singh et al., 2015). Marigolds are very popular as garden plants in southeastern Serbia. However, there is scarce information on the chemical composition and antioxidant properties of their essential oil. Therefore, the aim of the present study was to determine the chemical composition of essential oils isolated from red and orange Tagetes patula L. flower petals grown in southeastern Serbia in order to detect potentially present phototoxic compounds as well as to determine and compare their antioxidant activities. Materials and Methods Plant material The seeds of Tagetes patula L. (Tagetes Patula Mix 5436) were purchased in local agricultural pharmacy (producer: Semenarna Ljubljana, Slovenia). Plant material was cultivated in the garden in village Manastirište (municipality of Vlasotince, southeastern Serbia; 42.9612° N, 22.1590° E). The orange and red flowers in their full flowering state (July and August 2022) were collected and dried in a shadowy place, at room temperature. Before 206 BIOLOGICA NYSSANA ● 13 (2) December 2022: 205-216 Stanojević et al. ● Comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (Tagetes patula L.) flower petals Fig. 1. The aerial parts of Tagetes patula L. (a, c) with dry orange (b) and red (d) flower petals used in this study BIOLOGICA NYSSANA ● 13 (2) December 2022: 205-216 Stanojević et al. ● Comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (Tagetes patula L.) flower petals 207 the analysis, the dried flower petals were separated from the capitula and ground in an electric grinder (Moulinex Multi moulinette 3 in 1, 500 W). Chemicals and reagents The following chemicals and reagents were used in this study: ethanol, 96% (Centrochem, Zemun, Serbia); 1,1-diphenyl-2-picrylhydrazyl (DPPH radical), butylated hydroxytoluene (BHT); alkane standard solution C8-C20 (~40 mg/l each, in hexane), alkane standard solution C21-C40; (E)-caryophyllene (≥98.0%, sum of enantiomers); β-pinene (≥99%); γ-terpinene (97%); linalool (97%) (Sigma Chemical Company, St. Louis, MO, USA); terpinen-4-ol (97%, J&K Scientific Ltd., Beijing, China); HPLC grade hexane (≥95%, Fisher Scientific, UK), and redistilled water. Isolation of essential oil The essential oils (EOs) were isolated by Clevenger type hydrodistillation from dry flower petals during 2 h by using 1:15 m/V hydromodule. The volatile compounds were collected in hexane (1 ml) added Fig. 2. Chemical structures of the primary odorants present in Tagetes EOs in a graduated tube of the apparatus. The hexane solution was separated from the water phase and evaporated at room temperature. The residual essential oil was measured gravimetrically and stored at 4 °C until analysed. The yield of the essential oils was determined in g per 100 grams of the extracted plant material (g/100 g p.m.). Gas chromatography-mass spectrometry (GC/ MS) and gas chromatography-flame ionization detection (GC/FID) analyses GC/MS analysis was performed on Agilent Technologies 7890B gas chromatograph, equipped with nonpolar, silica capillary column, HP-5MS (5% diphenyl- and 95% dimethyl-polysiloxane, 30 m × 0.25 mm, 0.25 μm film thickness; Agilent Technologies, Santa Clara, CA, USA) and coupled with inert, selective 5977A mass detector of the same company. The essential oils were dissolved in diethyl ether in concentrations of ~19 mg/ml (OFEO) and ~33 mg/ml (RFEO). One μl of the solution prepared was injected to the GC column through a split/splitless inlet set at 220 °C in 20:1 208 BIOLOGICA NYSSANA ● 13 (2) December 2022: 205-216 Stanojević et al. ● Comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (Tagetes patula L.) flower petals split mode. Helium was used as the carrier gas, at a constant flow rate of 1 ml/min. The oven temperature increased from 60 °C to 246 °C at the rate of 3 °C/ min. Temperatures of the MSD transfer line, ion source and quadrupole mass analyzer were set at 300 °C, 230 °C and 150 °C, respectively. The ionization voltage was 70 eV and mass range m/z 41-415. GC/FID analysis was carried out under identical experimental conditions as GC/MS. The flows of the carrier gas (He), make up gas (N2), fuel gas (H2), and oxidizing gas (Air) were 1, 25, 30, and 400 ml/ min, respectively. The temperature of the flame- ionization detector (FID) was set at 300 °C. Data processing was performed using MSD ChemStation, MassHunter Qualitative Analysis and AMDIS_32 softwares (Agilent Technologies, USA). Retention indices of the components from the analyzed samples were experimentally determined using a homologous series of n-alkanes from C8-C20 and C21-C40 as standards. Essential oil constituents identification was based on the comparison of their retention indices (RIexp) with those available in literature (Adams, 2007) (RIlit); their mass spectra with those of authentic standard as well as with those from Willey 6, NIST2011 and RTLPEST3 libraries (MS) and wherever possible, by co-injection with an authentic standard (Co-I). Quantification was done by an external standard method according to the procedure described by Sparkman et al. (2011). The standards used were in the concentration ranges as follows: β-pinene (0.125- 2 mg/ml), linalool (1.67-15 mg/ml), γ-terpinene (0.75-5 mg/ml), terpinen-4-ol (0.0625-1 mg/ml) and (E)-caryophyllene (0.0625-1 mg/ml). The response factor (RF) for each standard used was calculated as follows: RF=Areastd/Ssd where Areastd is the peak area of the analyte standard and Cstd is the concentration of the standard used. The values of the mean response factors for the 5-points calibration curves of each standard used were: 8.39×106; 2.45×107; 3.02×107; 8.36×106; and 6.83×106 for β-pinene, γ-terpinene, linalool, terpinen-4-ol, and (E)-caryophyllene, respectively. Then, the mean value of response factors (RFmean=1.56×10 7 for RFEO and 1.51×107 for OFEO) was calculated and used for quantification according to the following formula: Cx=Areax/RFmean where Cx is the concentration of an analyte in the sample (in mg/ml of EO), Areax is the peak area of the analyte in the sample and RFmean is the mean response factor of the standards used (Sparkman et al., 2011). The calculated concentrations of the β-pinene, and γ-terpinene in RFEO were: 0.02 mg/ml and 0.03 mg/ ml respectively, while the concentrations of linalool, terpinen-4-ol, and (E)-caryophyllene were: 0.16 mg/ ml; 0.15 mg/ml; and 45.79 mg/ml in RFEO and 0.20 mg/ml; 0.11 mg/ml, and 12.12 mg/ml in OFEO, respectively. Finally, the content of each component in the sample expressed in % was normalized to get percents according to the formula: C (%)=(Cx/ΣCx)x100 where Cx is the concentration of each component in the sample and ƩCx is the total concentration of all components in the sample. DPPH assay The ability of the essential oils to scavenge free DPPH radicals was determined using the DPPH assay. Essential oils were dissolved in ethanol and a series of different concentrations (0.4-13.5 mg/ml and 0.3-10.3 mg/ml for EO isolated from red (RFEO) and orange flower petals (OFEO, respectively)) were prepared. Ethanol solution of DPPH radical (0.3 ml, 300 μmol solution (3×10-4 mol/l)) was added to 0.75 ml of the prepared essential oil solutions („sample”) and the absorbance was measured at 517 nm after 20 min, 40 min, 60 min, 90 min, and 120 minutes incubation with radical (AS). Absorbance at 517 nm was determined for ethanolic solution of DPPH radical („control” - AC), diluted in the aforementioned ratio (0.3 ml of the DPPH radical of the given concentration with 0.75 ml ethanol added) as well as for the ethanolic solution of the essential oil which is not treated with DPPH radical solution (“blank” - AB). Ethanol was used as a blank. Free radical scavenging activity was calculated according to the formula: DPPH radical scavenging capacity (%)=100-[(AS-AB)x(100/AC)] All absorbances were measured on UV-VIS VARIAN-Cary 100 Conc. Spectrophotometer. BHT (dissolved in ethanol; concentration range 0.04-1.3 mg/ml) was used as a positive control. Results and discussion Qualitative and quantitative composition of essential oils The yields of pale yellow coloured essential oils were 0.03 g/100 g p.m and 0.01 g/100 g p.m for RFEO and OFEO, respectively. Total Ion Chromatograms (TICs) of EOs studied are given in Fig. 3, while their qualitative and quantitative composition is given in Tab. 1. The chemical structures of the most abundant compounds are shown in Fig. 4. 209 BIOLOGICA NYSSANA ● 13 (2) December 2022: 205-216 Stanojević et al. ● Comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (Tagetes patula L.) flower petals Fig. 3. TIC chromatogram of (a) OFEO, and (b) RFEO Fig. 4. Chemical structures of the most abundant compounds present in isolated EOs 210 BIOLOGICA NYSSANA ● 13 (2) December 2022: 205-216 Stanojević et al. ● Comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (Tagetes patula L.) flower petals Table 1. Chemical composition of essential oils isolated from orange and red flower petals of T. patula L. No. tret, min Compound RIexp RIlit Method of identification Content (%) c (mg/ml of EO) OFEO RFEO OFEO RFEO 1 7.92 Sabinene 965 969a RI, MS - tr - tr 2 8.03 β-Pinene 968 974a RI, MS, Co-I - tr - tr 3 8.46 2-Pentyl furan 983 984a RI, MS - tr - tr 4 10.11 (Z)-β-Ocimene 1,030 1,032a RI, MS 0.4 2.7 0.1 0.9 5 10.49 (E)-β-Ocimene 1,040 1,044a RI, MS - 0.2 - 0.1 6 10.92 γ-Terpinene 1,051 1,054a RI, MS, Co-I - tr - tr 7 12.06 Terpinolene 1,081 1,086a RI, MS - tr - tr 8 12.25 p-Cymenene 1,086 1,089a RI, MS - tr - tr 9 12.72 Linalool 1,098 1,095a RI, MS, Co-I 0.4 tr 0.1 tr 10 14.44 (E)-Tagetone 1,132 1,139a RI, MS - tr - tr 11 15.98 Terpinen-4-ol 1,169 1,174a RI, MS, Co-I tr tr tr tr 12 16.58 p-Cymen-8-ol 1,184 1,179a RI, MS - tr - tr 13 17.95 Coahuilensol, methyl ether 1,216 1,219a RI, MS - tr - tr 14 18.19 (Z)-Ocimenone 1,222 1,226a RI, MS tr 0.3 tr 0.1 15 18.60 (E)-Ocimenone 1,232 1,235a RI, MS - tr - tr 16 19.15 Piperitone 1,245 1,249a RI, MS - 0.3 - 0.1 17 20.30 Isobornyl acetate 1,276 1,283a RI, MS tr - tr - 18 21.97 Silphiperfol-5-ene 1,324 1,326a RI, MS - tr - tr 19 22.25 Presilphiperfol-7-ene 1,330 1,334a RI, MS - tr - tr 20 22.89 Piperitenone 1,337 1,340a RI, MS 0.5 1.5 0.1 0.5 21 23.04 α-Terpinyl acetate 1,340 1,346a RI, MS tr - tr - 22 23.73 Eugenol 1,357 1,356a RI, MS, Co-I tr tr tr tr 23 23.93 Piperitenone oxide 1,361 1,366a RI, MS - tr - tr 24 24.09 α-Copaene 1,365 1,374a RI, MS tr tr tr tr 25 24.59 Geranyl acetate 1,377 1,379a RI, MS 37.6 - 7.2 - 26 26.21 (E)-Caryophyllene 1,416 1,417a RI, MS, Co-I 31.6 69.4 6.0 22.8 27 26.51 α-trans-Bergamotene 1,423 1,432a RI, MS tr - tr - 28 27.30 Geranyl acetone 1,443 1,453a RI, MS tr tr tr tr 29 27.38 α-Humulene 1,444 1,452a RI, MS 1.0 1.7 0.2 0.6 30 28.53 Germacrene D 1,488 1,484a RI, MS 2.8 8.2 0.5 2.7 31 29.08 Bicyclogermacrene 1,490 1,500a RI, MS 1.1 3.6 0.2 1.2 32 29.39 (E)-Methyl isoeugenol 1,493 1,491a RI, MS tr - tr - 33 29.45 (E,E)-α-Farnesene 1,495 1,505a RI, MS - 0.6 - 0.2 34 29.56 β-Bisabolene 1,498 1,505a RI, MS 12.5 - 2.4 - 35 30.10 γ-Cadinene 1,511 1,513a RI, MS tr tr tr tr 36 30.78 (E)-γ-Bisabolene 1,529 1,529a RI, MS 0.9 0.4 0.2 0.1 37 32.51 Caryophyllene oxide 1,574 1,582a RI, MS 5.7 4.5 1.1 1.5 38 34.65 Caryophylla- 4(12),8(13)-dien-5α-ol 1,631 1,639a RI, MS tr tr tr tr 211 BIOLOGICA NYSSANA ● 13 (2) December 2022: 205-216 Stanojević et al. ● Comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (Tagetes patula L.) flower petals 39 35.32 α-Cadinol 1,648 1,652a RI, MS tr tr tr tr 40 36.63 Shyobunol 1,684 1,688a RI, MS tr tr tr tr 41 36.81 Eudesm-7(1l)-en-4-ol 1,688 1,698a RI, MS 0.9 tr 0.2 tr 42 37.18 (2E)-Tridecenol acetate 1,698 1,703a RI, MS 0.8 tr 0.2 tr 43 41.65 Hexahydrofarnesyl acetone 1,850 1,846b RI, MS 0.6 0.3 0.1 0.1 44 44.05 (5Z,9E)-Farnesyl acetone 1,897 1,889a RI, MS - tr - tr 45 44.27 5-(3-buten-1-ynyl)- 2,2'-bithiophene (BBT) 1,902 1,892c RI, MS - 0.4 - 0.1 46 46.12 Palmitic acid 1,960 1,959b RI, MS 1.2 1.9 0.2 0.6 47 49.29 5-(3-penten-1-ynyl)- 2,2-bithiophene (PBT) 2,053 2,043c RI, MS 0.7 1.5 0.1 0.5 48 53.28 2,2':5',2''-Terthiophene (α-Terthiophene) 2,181 2,171c RI, MS - 0.9 - 0.3 49 55.35 Tricosane 2,296 2,300a RI, MS, Co-I 0.5 0.7 0.1 0.2 Total identified 99.2 99.1 19.0 32.6 Grouped components (%) (mg/ml of EO) Monoterpene hydrocarbons (1, 2, 4-8) 0.4 2.9 0.1 1.0 Oxygenated monoterpenes (9-21, 23, 25, 28) 38.5 2.1 7.4 0.7 Sesquiterpene hydrocarbons (24, 26, 27, 29-31, 33-36) 49.9 83.9 9.5 27.6 Oxygenated sesquiterpenes (37-41, 43, 44) 7.2 4.8 1.4 1.6 Phenylpropanoids (22, 32) tr tr tr tr Thiophenes (45, 47, 48) 0.7 2.8 0.1 0.9 Others (3, 42, 46, 49) 2.5 2.6 0.5 0.8 tret.: Retention time; RI lit - Retention indices from literature (aAdams (2007); bBalogun et al. (2017); cSzarka et al. (2007)); RIexp: Experimentally determined retention indices using a homologous series of n-alkanes (C8-C20 and C21-C40) on the HP-5MS column. MS: constituent identified by mass-spectra comparison; RI: constituent identified by retention index matching; Co-I: constituent identity confirmed by GC co-injection of an authentic sample; tr = trace amount (<0.05%; <0.1 mg/ml); OFEO - essential oil isolated from orange flower petals, RFEO - essential oil isolated from red flower petals According to the results of GC/MS analysis, 30 and 42 compounds were identified in OFEO and RFEO, comprising 99.2%, and 99.1% of total EO composition, respectively. The most abundant groups of compounds in OFEO were sesquiterpene hydrocarbons (49.9%) and oxygenated monoterpenes (38.5%). On the other side, sesquiterpene hydrocarbons with 83.9% in the total essential oil composition were the dominant group in RFEO (Tab. 1). Among sesquiterpene hydrocarbons (E)-caryophyllene was the dominant compound in both EOs, with 31.6% and 69.4%, in total OFEO and RFEO composition, respectively while β-bisabolene, with 12.5% was second most dominant compound in OFEO. β-bisabolene was not detected in RFEO. Among oxygenated monoterpenes, the dominant one was geranyl acetate with 37.6% in total OFEO composition (structures given in Fig. 3). It was not detected in RFEO. The results obtained are in agreement with the study by Szarka et al. (2007). The authors hydrodistilled EO from T. patula flowers that had been air-dried, and the most abundant compound found there was (E)-caryophyllene, which accounted for 50.2% (compared to 69.4% in herein study). Geographical origin plays an important role in the chemical diversity not just among species but even in the same species. For example, Krishna et al. (2002) analysed EOs of the capitula, leaves and shoots of Tagetes patula L. raised in the CIMAP Experimental BIOLOGICA NYSSANA ● 13 (2) December 2022: 205-216 Stanojević et al. ● Comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (Tagetes patula L.) flower petals 212 Farm at Lucknow (India). The main constituents of EO isolated from capitula were (Z)-β-ocimene (19.9%), (Z)-tagetenone (12.4%), (E)-tagetenone (10.4%), piperitenone (5.8%) and (E)-caryophyllene (15.1%) (Krishna et al., 2002). On the other hand, the EO isolated from cultivated T. patula flowers grown in Mandal (Uttarakhand, India) contained β-ocimene (22.11%), α-terpinolene (14.59%), and (E)-caryophyllene (12.69%) as the most abundant compounds (Negi et al., 2013). The most abundant components of the EO isolated from fresh flowers of T. patula grown in New Delhi (India) were (E)- caryophyllene (3.92-42.76%), germacrene-D (1.48- 6.72%), (Z)-tagetone (1.29-4.38), caryophyllene oxide (0.68-24.3%), and piperitone oxide (0.11- 1.23) (Tamut et al., 2019). In the EO isolated from T. patula flowers sampled during August in areas of Erbil Province (Iraq), (E)-caryophyllene (20.59%), and (E)-ocimenone (12.08%) were the most abundant components while geranyl acetate was not identified (Safar et al., 2020). Zarate-Escobedo et al. (2018) reported the presence of geranyl acetate for genus Tagetes for the first time. They determined the chemical composition of the essential oil hydrodistilled from floral stems of 14 T. lucida populations from North and South of the State of Mexico, where six types of soils and six climatic conditions were detected. The plant material was collected from September to October 2014 and in September 2015. In Southern populations with a warm climate, the major compounds were monoterpenes: geranyl acetate (ranging from 12% to 40%) and β-ocimene (14% to 24%) depending on the location (Zarate-Escobedo et al., 2018). It is postulated that terpenoid compounds play a role in an ecological interaction of plants with biotic and abiotic factors of their environment by defending plants from herbivores and pathogens (toxins or repellents) or being the signals and rewards to pollinators. The constant evolution of new terpenoids structures is enabled by the evolution of new genes encoding new enzymes capable of making such new metabolites (Pichersky & Raguso, 2016). What is more, according to the available literature data single compounds such as bisabolene, (E)- caryophyllene, camphor, (E)-β-farnesene, pinene, and linalol have been recognized as good repellents towards aphids and various pests (Hori, 1998; Isman, 2000; Halbert et al., 2009; Pascual-Villalobos et al., 2017; Dardouri et al., 2019). Taking into account that (E)-caryophyllene and β-bisabolene were present in considerable amounts in the EOs isolated in this study (Tab. 1), they could be considered as a potential source of natural pesticides. It is well known that acyclic monoterpenes (ocimenones) including (Z)-β- and (E)-β-ocimene, (Z)- and (E)-tagetone and (Z)- and (E)-tagetenone are formed by chemical modification (such as hydrolysis, dehydration, oxidation and reduction which are catalyzed by specific enzymes) of either GPP or neryl pyrophosphate (NPP). On the other hand, the biosynthesis of sesquiterpenes proceeds through the precursor farnesyl pyrophosphate (FPP) which is formed by condensation of GPP with one molecule of IPP (Singh et al., 2015). Given that terpenoids’ biosynthesis is genetically determined, finding the reason for the difference in chemical composition between OFEO and RFEO, regarding the most abundant components, goes beyond the scope of this paper. Geranyl acetate is an acyclic monoterpenes ester with great economic value. It is widely used in cosmetic industry due to its „rose-like” odor. In Cymbopogon spp. geranyl acetate is biosynthesed by acetylation of geraniol catalyzed by geraniol acetyl transferase (GAT) (Ganjewala & Luthra, 2010). Phosphatase (GPPase) mediates the formation of geraniol while geranyl acetate esterase (GAE) catalizes deacetylation of geranyl acetate into geraniol. Thus, the content of geraniol depends on the relative activities of these three (GPPase, GAT, and GAE) enzymes. Geraniol was not identified in herein studied EO. The possible reason could be that the plant material was collected in the phase when GAE (catalyzing transformation of geranyl acetate to geraniol) had little or no activity. Having in mind that essential oils isolated from „geranyl- acetate rich” plants like palmarosa (Cymbopogon martini (Roxb.) Wats. var. motia Burk.) contained 4.3-14.8% of geranyl acetate (Rajeswara Rao et al., 2009); wild carrot (Daucus carota L. ssp. carota) mature umbels 16.5% (Staniszewska et al., 2005); lemongrass (Cymbopogon flexuosus (Steud) Wats.) 25.9% (Kulkarni et al., 1997); pastinocello fruits (Daucus carota ssp. major) 34.2% (Flamini et al., 2014); coriander (Coriandrum sativum L.) fruits 46.27% (Msaada et al., 2007); and the OFEO isolated in this paper (containing 37.6%) could be considered as a potential source of geranyl acetate. The thiophenes, with BBT, PBT, and α-terthienyl as representatives, were also identified, with 0.7% and 2.8% in OFEO and RFEO, respectively (Tab. 1). Although the thiophenes are the main secondary metabolites of Tagetes roots in this paper they were identified in flower petals, which is in agreement with the study of Szarka et al. (2007). Namely, Szarka et al. (2007) studied the composition of EOs isolated from hairy roots, normal roots and flowers and the major thiophene of flower EO was PBT with 6.0% in the total EO composition (Szarka et al., 2007). The identity of thiophenes identified in this paper was confirmed by comparing mass spectrum from the hit 213 BIOLOGICA NYSSANA ● 13 (2) December 2022: 205-216 Stanojević et al. ● Comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (Tagetes patula L.) flower petals list in mass spectra libraries but also with the mass spectra given in the study of Szarka et al. (2006) as well as by comparing experimentally obtained retention indices with the retention indices given in the study of Szarka et al. (2007). On the other side, in the study of Arciniegas et al. (2020), the authors determined the antioxidant and photosensitizer activities of extracts and isolates of genus Dyssodia (Asteraceae, Tageteae). The antioxidant activity was determined by the interactions with copper ion (Cu2+) observed in EPR, as well as by the DPPH and the thiobarbituric reactive substances (TBARS) methods. Their photosensitizer activities were observed as the the abilities to produce 1O 2 by electron paramagnetic resonance (EPR). They isolated seven thiophene derivatives, among others 2,2′:5′,2′′‐terthiophene, and 5‐(3‐buten‐1‐ynyl)‐2,2′‐bithiophene, which were identified in this study as well. Both thiophenes had no antioxidant activity determined by the DPPH assay, so they surely not contribute to the antioxidant activity of the EOs studied in this paper. On the other side, α‐terthiophene and related compounds are known as photosensitizers by their activity to generate singlet oxygen (1O2) under UV irradiation regime (Arciniegas et al., 2020). What is more, these compounds, and especially α-terthienyl show enhanced nematocidal activity in the presence of sunlight (UV-A), but also antibiotic, ovicidal, algicidal, larvicidal, and antifeedant activities (D’Auria et al., 1987). Antioxidant activity The antioxidant activity of isolated EOs was determined by the DPPH assay and compared both mutually, and with BHT as a positive control. Their DPPH radical scavenging activity determined after 20 min, 40 min, 60 min, 90 min, and 120 min incubation with the DPPH radical is shown in Fig. 5. The EC50 values obtained from the graphs given in Fig. 5 are presented in the Tab. 2. According to the results obtained, antioxidant activity of both EOs and BHT was dependant on both concentration and incubation time with the DPPH radical, as already stated (Stanojević et al., 2016). Generally, the duration of incubation time of DPPH radical with samples depends on the type of sample, taking longer time for interaction with the weak antioxidants (Bal et al., 2021). The effect of incubation time studied in this paper indicated that DPPH radical scavenging activity of both EOs and BHT reached the maximum after 120 minutes of incubation (Tab. 2). However BHT, as a phenolic representative of synthetic antioxidants used in this paper, reduced the 50% of the initial DPPH radical concentration (the EC50 value) after 20 minutes of incubation and showed ~12 and ~14 times stronger Fig. 5. Antioxidant activity of (a) OFEO, (b) RFEO, and (c) BHT antioxidant activity in comparison to OFEO and RFEO after 120 minutes of incubation, respectively. Therefore, the isolated EOs had similar antioxidant BIOLOGICA NYSSANA ● 13 (2) December 2022: 205-216 Stanojević et al. ● Comparative analysis of chemical composition and antioxidant activity of essential oil isolated from orange and red marigold (Tagetes patula L.) flower petals 214 activity and both are weaker antioxidants than BHT. Comparing the chemical composition of isolated EOs, the main groups were sesquiterpenes hydrocarbons (83.9% vs. 55.6%) and oxygenated monoterpenes (2.1% vs. 38.5%) in RFEO and OFEO, respectively. According to the study by Ruberto & Barrata (2000), oxygenated monoterpenes have better antioxidant activity in comparison to monoterpenes hydrocarbons, sesquiterpenes hydrocarbons, and oxygenated sesquiterpenes. The best antioxidant activity in the mentioned study showed phenolic compounds (such as thymol and carvacrol) due to their redox properties, and the ability to neutralize free radicals (Ruberto & Barrata, 2000). Considering that OFEO contained considerable amount of oxygenated monoterpenes (38.5%) in comparison to RFEO (2.1%), its slightly better antioxidant activity could be ascribed to their presence, especially to the presence of geranyl acetate due to its capacity to reduce free radical stability via electron or hydrogen donating mechanisms (Seema Farhath et al., 2013). On the other side, phenolic compounds (particularly eugenol) were identified in traces in both isolated EOs, so their weak antioxidant activity is not surprising. Conclusions The essential oils hydrodistilled from dry red and orange Tagetes patula L. flower petals cultivated in south-eastern Serbia are rich sources of sesquiterpene hydrocarbons ((E)-caryophyllene in RFEO comprising 69.4% of total EO composition) and oxygenated monoterpenes (geranyl acetate in OFEO comprising 37.6% of total EO composition). They have also contained sulphurated tiophenes (BBT, PBT and TTP) with 2.8% and 0.7% in total RFEO and OFEO composition, respectively. Having in mind that α-terthienyl, also called terthiophene (TTP) and 5-(3-penten-1-ynyl)-2,2-bithienyl (PBT) are phototoxic compounds, they should not be used as components in cosmetic products for applications on areas of skin exposed to sunshine. Being weaker antioxidants in comparison to the synthetic antioxidant BHT, they could not be used as its natural alternative. On the other side, the overuse of synthetic pesticides causes pest resistance and makes them one of the major pollutants in soil and water, as well as toxic substances for humans and animals. Taking into account that (E)-caryophyllene and β-bisabolene present in considerable amounts in the EOs isolated in this study are recognized as good repellents (besides thiophenes). 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