Fadel et al. 2025, Biologica Nyssana 16(2) 16 (2) December 2025: DOI: 10.46793/BiolNyss.16.2.13F Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus Original Article Hamza Fadel Research Unit, Valorization of Natural Resources, Bioactive Molecules and Physicochemical and Bio- logical Analyzes (VARENBIOMOL), University of Mentouri Brothers Constantine 1, Ain El-Bey Road, 25000, Constantine, Algeria. Meriem Mermoune Research Unit, Valorization of Natural Resources, Bioactive Molecules and Physicochemical and Bio- logical Analyzes (VARENBIOMOL), University of Mentouri Brothers Constantine 1, Ain El-Bey Road, 25000, Constantine, Algeria. Sabrina Bicha Research Unit, Valorization of Natural Resources, Bioactive Molecules and Physicochemical and Bio- logical Analyzes (VARENBIOMOL), University of Mentouri Brothers Constantine 1, Ain El-Bey Road, 25000, Constantine, Algeria. Salah Akkal Research Unit, Valorization of Natural Resources, Bioactive Molecules and Physicochemical and Bio- logical Analyzes (VARENBIOMOL), University of Mentouri Brothers Constantine 1, Ain El-Bey Road, 25000, Constantine, Algeria. salah4dz@yahoo.fr (corresponding author) Nabila Slougui Research Unit, Valorization of Natural Resources, Bioactive Molecules and Physicochemical and Bio- logical Analyzes (VARENBIOMOL), University of Mentouri Brothers Constantine 1, Ain El-Bey Road, 25000, Constantine, Algeria. National Polytechnic School of Constantine Hichem Hazmoune Research Unit, Valorization of Natural Resources, Bioactive Molecules and Physicochemical and Bio- logical Analyzes (VARENBIOMOL), University of Mentouri Brothers Constantine 1, Ain El-Bey Road, 25000, Constantine, Algeria. Received: July 15, 2025 Revised: October 24, 2025 Accepted: October 27, 2025 Abstract: This study included the volatile composition and antioxidant potential of an Algerian Auresian Anacyclus clavatus. Anacyclus clavatus essential oil (ACEO) and crude methanol extract (ACME) were obtained by steam distillation and maceration, respectively. Phenols and flavonoids were found in moderate amounts in ACME. ACEO exhibited moderate antioxidant capacity using β-carotene bleaching assays (40.09±3.7% and 78.1±5.28%, respectively). The main volatile constituents of ACEO were octadecanol (33.08%), caryophyllene oxide (5.10%), cis-thujone (4.81%), tetracosane (4.36%), nonanal (4.05%), and phytone (3.66%), respectively, with nonanal and phytone as key compounds in ACEO for the first time. These results will certainly enrich our knowledge of the currently limited information on the chemical composition of ACEO and suggest that this species is a viable source of bioactive compounds with potential uses in medicines and nutraceuticals. Key words: Anacyclus clavatus, essential oil, GC-MS, phenolic content, flavonoid content, antioxidant activity Apstrakt: Hemijski sastav i antioksidativna aktivnost etarskog ulja Anacyclus clavatus iz Alžira Ovo istraživanje obuhvata ispitivanje isparljivih jedinjenja i antioksidativnog potencijala Anacyclus clavatus poreklom iz oblasti Aures u Alžiru. Etarsko ulje Anacyclus clavatus (ACEO) i sirovi metanolni ekstrakt (ACME) dobijeni su postupkom destilacije vodenom parom, odnosno maceracijom. Fenoli i flavonoidi su utvrđeni u umerenim količinama u ACME. ACEO je pokazalo umerenu antioksidativnu sposobnost primenom testa izbjeljivanja β-karotena (40,09±3,7% i 78,1±5,28%). Glavne isparljive komponente ACEO bile su oktadekanol (33,08%), kariofilen oksid (5,10%), cis-tujon (4,81%), tetrakozan (4,36%), nonanal (4,05%) i fiton (3,66%), pri čemu su nonanal i fiton po prvi put identifikovani kao ključna jedinjenja u ACEO. Ovi rezultati svakako doprinose proširenju postojećih, ograničenih podataka o hemijskom sastavu ACEO i ukazuju na to da ova vrsta može predstavljati značajan izvor bioaktivnih jedinjenja sa potencijalnom primenom u medicini i nutraceutici. Ključne reči: Anacyclus clavatus, etarsko ulje, GC–MS, sadržaj fenola, sadržaj flavonoida, antioksidativna aktivnost Introduction People have traditionally valued aromatic and therapeutic herbs because they recognize their importance in preserving health and averting illness (Mahato et al., 2025). Despite the development of synthetic medicines, these natural resources remain essential in both traditional and modern medicine, providing a wide range of medicinal compounds (Buragohain et al., 2025). Aromatic and therapeutic species contain a wide range of secondary metabolites, including polyphenols and flavonoids, which are widely used in the culinary, cosmetics, and pharmaceutical sectors (Dimitrijević et al., 2025; Šovljanski et al., 2025). The Asteraceae is one of the largest families, comprising 250,000 species and approximately 1,600 genera (Paksoy et al., 2016). It is known © 2025 Fadel 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. for its wide range of uses, not only in medicine but also in the cultivation of ornamental plants and the production of natural rubber, colorants, insecticides, and spices (Mechergui et al., 2017). Asteraceae plants have been distinguished by their capitula, which include closely arranged flowers on a receptacle encircled by bracts. Asteraceae plants are distributed worldwide, including in subtropical, arid, and semi-arid climates, and most species have been used traditionally in conventional medicine due to their phytochemicals, including polyphenols, flavonoids, and volatile compounds (Chroho et al., 2022). The Asteraceae family includes a wide range of herbaceous plants, shrubs, and trees, many of which are economically and medicinally important (Heywood et al., 2007). Anacyclus, a species of Asteraceae, is found in the Mediterranean basin, including Algerian regions, and is known for its adaptability to challenging, dry environments. The genus Anacyclus (Quezel & Santa, 1963a) is distributed across the Mediterranean basin, including Algerian zones, and is known for its adaptability to challenging, dry environments (Vitales et al., 2018). Anacyclus species, encompassing both annual and perennial varieties, have historically been used to treat various ailments, including sore throat, toothache, and rhinitis (Baslam et al., 2023). They have various biological capacities, including antimicrobial, anticonvulsant, anxiolytic, anabolic, aphrodisiac, immunostimulating, and antioxidant properties (Sissi et al., 2024). Anacyclus clavatus (Desf.) Pers., also known as “white Anacyclus” (Quezel & Santa, 1963b), is an annual herb found in Mediterranean countries (Mifsud, 2002). Its flowering season starts in March and extends to May (Chroho et al., 2022). The plant grows in fields, on roads, and in wastelands. Its disc achenes are winged and have large capitula. The plant is used for skin, nervous system, digestive, reproductive, and renal issues. The aerial parts are used for fever, digestive problems, and stomach pain (Mechergui et al., 2017). Roots are used for rheumatism, respiratory problems, diabetes, toothaches, and paralysis (Manouze et al., 2017). Powdered roots are also used for kidney diseases, skin problems, reproductive issues, and allergies (Ouasti et al., 2024). In Algeria, limited research has examined the volatile content of A. clavatus, with the main volatile chemicals being germacrene D, δ-elemene, α- and β-thujone, and artemisia ketone (Aliboudhar et al., 2013; Aliboudhar & Tigrine-Kordjani, 2014). Thus, the current study aimed to enrich knowledge on the Algerian A. clavatus from the Aures area regarding its essential oil composition by using GC-MS, and to evaluate its antioxidant capacity. Materials and Methods Chemicals Methanol (95%) was purchased from Sigma-Aldrich (Deisenhofen, Germany); DPPH (Sigma, St. Louis, USA); H2O2 was obtained from Sigma-Aldrich, USA; and the analytical standards of ascorbic acid, gallic acid, Folin-Ciocalteu, Na2CO3, and α-tocopherol were obtained from Sigma-Aldrich, USA (purity ≥ 98%). Plant material The leaves, stems, and flowers of A. clavatus were collected in April 2023 from the Algerian region of Aures (Timgad, 35 km east of Batna, 35° 29′ 05′′ north, 6° 28′ 07′′ east, altitude: 1072 m). A voucher specimen (AC/118/VAR/04-23) was verified and placed in the VARENBIOMOL Research Unit’s Herbarium at the University of Constantine 1. Extraction and preparation of samples The powdered, air-dried aerial part of A. clavatus (100 g) was extracted with 80% aqueous methanol for 72 hours at room temperature. Following filtration through cotton, the crude methanolic extract ACME was concentrated to dryness under reduced pressure (20.2 g, yield = 20.2%, w/w). The steam distillation method (Yazıcı & Sevgili, 2024) was used to extract the volatile components from the fresh plant material (240 g), which was broken into small pieces and processed for three hours. The obtained ACEO was weighed (32.0 mg; yield = 0.01%, w/w) and stored at 4 °C until analysis. The ACEO and ACME yields were calculated in proportion to the plant weight using the following formula: Yield (%) = (W1 × 100) / W2; where W1: weight of the obtained sample; W2: initial weight of the plant materials. GC-MS and GC-FD analyses of ACEO The ACEO was analyzed using a SHIMADZU GCMS-QP2010 chromatograph with an RXI-5MS capillary column (30 m × 0.25 mm, film thickness 0.25 μm). The flow rate of the carrier gas, helium, was 1.44 mL/min. After 10 minutes at 45 °C, the oven was heated to 180 °C at a rate of 3 °C per minute and held there for 5 minutes. Following that, it was heated to 280 °C at a rate of 5 °C per minute and held for 5 minutes. Finally, it was heated to 330 °C at a rate of 10 °C/min for 2 minutes. The injector and detector (FID) temperatures were set at 330 °C. In split/splitless mode, 1 μL of diluted sample (in dichloromethane) was injected at a 30:1 split. For GC-MS detection, an electron ionization apparatus with an ionization energy of 70 eV was used. The BIOLOGICA NYSSANA ● 16 (2) December 2025: Fadel et al. ● Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus BIOLOGICA NYSSANA ● 16 (2) December 2025: Fadel et al. ● Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus capillary column components were identified by comparing their mass spectra fragmentation patterns and calculated retention indices (RI) with those available in commercial databases (Adams, 2017; Babushok et al., 2011) and literature. DPPH radical scavenging assay A methanolic solution of DPPH was prepared at a concentration of 0.04 mg/mL. The samples ranged in different concentrations. 50 µL of each sample was combined with 3 mL of the prepared DPPH solution under the same conditions. A spectrophotometer was used to measure absorbance at 517 nm after a 30-minute incubation at room temperature. Under the same circumstances, the ascorbic acid was used as a positive standard. All measurements were carried out in duplicate (n = 2), and the following formula was used to determine the anti-radical capacity: IP (%) = [(Abscontrol – Abssample) / Abscontrol] × 100, where IP (%) is the inhibition percentage, Abssample is the absorbance of the sample, and Abs control is the absorbance of the negative control. The obtained values were expressed as IC50 (inhibitory concentration required to inhibit 50% of DPPH) from linear regression in Microsoft Excel (Nadji et al., 2024). β-Carotene bleaching assay 0.5 mg of β-carotene was dissolved in 2 mL of chloroform with 25 μL of linoleic acid and 200 mg of Tween 40 to prepare a β-carotene/linoleic acid solution. After the chloroform evaporated, 100 mL of oxygenated water was added, and the mixture was vigorously stirred until an emulsion formed. Next, 0.5 mL of each aliquot was mixed with 3.5 mL of the previously prepared emulsion. The same conditions were used to test the α-tocopherol standard as a positive control. The decay of β-carotene can be rationalized in the presence of an excess of linoleic acid and O2 by assuming that a small amount of peroxyl radical generated readily reacts with both β-carotene and antioxidant (Takada et al., 2006). The bleaching of β-carotene was monitored at 490 nm at regular intervals for 120 minutes at 50 °C (Mahdavi et al., 2017). All determinations have been carried out in duplicate. The antioxidant activity was determined using the following formula: Inhibition = [Abssample t=2h / Abssample t=0h] x 100, where: Abssample t = 2h is the absorbance of the emulsion containing the sample after 2 hours. Abssample t = 0h is the initial absorbance of the emulsion containing the sample. Determination of total phenolic content The polyphenol content in ACME was evaluated using the colorimetric technique published by Singleton et al. (1999), with minor modifications. 250 μL of sample (1 mg/mL) was mixed with 500 μL of Folin-Ciocalteu (1 N) reagent. After 4 minutes of incubation at 25 °C, 20 μL of 20% sodium carbonate (Na2CO3) solution was added to the mixture, bringing the total volume to 770 μL. The resulting mixture was incubated in a dark environment for 120 minutes before being measured at 760 nm. Gallic acid at varied amounts was used as the standard. All measurements have been carried out in duplicate. The total phenol content was estimated in μg GAE/mg by extrapolating from a calibration curve of varied gallic acid concentrations. Determination of total flavonoid content Flavonoids were quantified using a procedure that involves the formation of a very stable combination between aluminum chloride and the oxygen atoms present on flavonoids’ carbon structures (Kim et al., 2003). ACME was prepared at a concentration of 1 mg/mL in methanol. 1 mL of the sample was then mixed with 1 mL of aluminum trichloride solution (AlCl3, 2%), to obtain a total volume of 2 mL. Following one hour of incubation, absorbance was measured at 420 nm. This experiment was performed in duplicate. The absorbance of a quercetin standard solution was measured under the same circumstances. The results were expressed as the calibration curve for quercetin in μg QE/mg. Statistical analysis For the antioxidant evaluation, results are represented as mean values ± standard deviation (SD). A one-way analysis of variance (ANOVA, p<0.05) was performed to compare group means. Statistical analyses were performed using Microsoft Excel 2007 (Microsoft, Redmond, WA, USA). Results and Discussion GC-MS analysis ACEO was isolated by steam distillation and then analyzed by GC-FID and GC-MS. Thirty-seven volatile chemicals were found in the acquired data (Tab. 1, Fig. 1), accounting for 95.78% of the total detected oil composition. Among these, 35.59% were fatty alcohols, 19.86% were sesquiterpenoids, 14.03% were monoterpenoids, and 9.43% were hydrocarbons. The main abundant compounds were octadecanol (33.08%), caryophyllene oxide (5.10%), cis-thujone (4.81%), tetracosane (4.36%), nonanal (4.05%), and phytone (3.66%), respectively. Nonanal and phytone, the two main ingredients, have not been described before in the ACEO, according to the literature. Some Anacyclus species, including A. maroccanus, A. radiatus, A. cyrtolepidioides, and A. pyrethrum, have been BIOLOGICA NYSSANA ● 16 (2) December 2025: Fadel et al. ● Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus Peak N° RT KICalc KI theo Component Area % 1 22.992 1017 1042 Artemisia ketone 1.98 2 25.476 1062 1062 cis-Thujone 4.81 3 25.559 1064 1069 Nonanal 4.05 4 26.137 1074 1112 trans-Thujone 0.54 5 29.575 1141 1177 4-Terpineol 0.97 6 30.554 1160 1194 Myrtenal 0.9 7 30.656 1162 1195 Myrtenol 0.66 8 34.01 1232 1233 Chrysanthenyl acetate 2.55 9 39.025 1345 1365 Neryl acetate 1.64 10 39.525 1357 1374 α-Copaene 0.89 11 39.952 1374 1388 β-Elemene 0.58 12 40.114 1404 1408 Caryophyllene Z 1.04 13 43.152 1444 1452 β-E-Farnesene 2.44 14 44.268 1472 1485 β-Copaene 2.7 15 46.068 1519 1523 δ-Cadinene 0.61 16 48.382 1580 1578 Spathulenol 3.39 17 48.499 1583 1582 Neryl isovalerate, butanoic acid 1.96 18 48.606 1585 1587 Caryophyllene oxide 5.1 19 49.036 1597 1595 Salvial-4(14)-en-1-one 1.28 20 49.654 1614 1616 1,3,12-Hexadecatriene 1.02 21 50.891 1648 1641 α-epi-Muurolol 1.47 22 51.294 1660 1649 β-Eudesmol 0.51 23 51.449 1664 1654 α-Cadinol 1.13 24 53.584 1724 1701 Pentadecanal 0.89 25 58.918 1864 1841 Phytone 3.66 26 60.667 1906 1884 Hexadecanol 0.87 27 64.266 2001 1977 Hexadecanoic acid 3.32 28 66.048 2062 1999 Octadecanal 0.75 29 67.989 2134 2081 Octadecanol 33.08 30 68.349 2149 2102 Heneicosane 0.53 31 68.763 2166 2106 Phytol 1.43 32 72.85 2360 2365 Heneicosanol 1.64 33 73.076 2371 2400 Tetracosane 4.36 34 73.695 2404 2412 Methyl-20-heneicosonoate 0.47 35 76.894 2596 2600 Hexacosane 2.27 36 80.22 2822 2800 Octacosane 1.35 37 83.882 3048 3000 Triacontane 0.92 Oil yield 0.01 Total identified 95.78 Fatty alcohols 35.59 Sesquiterpenoids 19.86 Monoterpenoids 14.03 Hydrocarbons 9.43 Others 16.87 Table 1. Chemical composition of ACEO using GC-MS analysis BIOLOGICA NYSSANA ● 16 (2) December 2025: Fadel et al. ● Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus Major compounds Percentage (%) Algeria Tunisia Batna Boumerdes Boumerdes Boumerdes Sousse (Aliboudhar et al., 2013) (Aliboudhar & Tigrine- Kordjani, 2014) (Aliboudhar et al., 2015) (Hammami et al., 2013) Octadecanol 33.08 0.06 - 0.1±0.0 to 0.2±0.1 0.8 Caryophyllene oxide 5.1 1.28 1.3±0.3 to 2.5±0.6 0.2±0.2 to 0.3±0.1 - cis-Thujone 4.81 - - 0.9±0.4 to 1.3±0.5 9.8 Tetracosane 4.36 tr - - 0.5 Nonanal 4.05 - - - - Phytone 3.66 - - - - Germacrene D - 16.84 - 12.3±1.6 to 16.1±1.4 2 β-Thujone - 11.16% - - - Artemisia ketone 1.98 0.53 6.5±0.5 to 10.0±0.8 0.4±0.3 0.4 α-Thujone - 0.3 10.6±1.0 to 11.9±1.1 - - δ-Elemene - 0.17 0.4±0.0 to 4.0±0.7 9.1±1.1 to 10.4±1.3 - trans-Chrysanthenyl acetate - - 0.5±0.1 to 3.6±0.8 - 12.3 Chrysanthenone - 0.1 - 0.1±0.1 8.2 Table 2. Major compounds of ACEO from different regions Fig 1. GC-FID Chromatogram of ACEO shown to contain tetracosane and caryophyllene oxide in their essential oils (El Baz et al., 2024; Sissi et al., 2024; Zardi-Bergaoui et al., 2008). Tab. 2 shows the presence of the main components of our sample in the ACEO collected from different regions of the world. We can note variability in the volatile chemical composition of our ACEO species compared to that studied in other regions. Only a few published studies have reported the chemical composition of ACEO in the Algerian and Tunisian regions (Aliboudhar et al., 2013; Aliboudhar & Tigrine-Kordjani, 2014; Aliboudhar et al., 2015; Hammami et al., 2013). By comparing the data (Tab. 2), a significant difference Aliboudhar & Tigrine-Kordjani; Aliboudhar et al., 2015; Hammami et al., 2013), were absent in our sample, except for artemisia ketone compounds. This chemical composition variation could be linked to several factors, including geographical and environmental factors (Lahmar et al., 2025; Sarmah & Bora, 2025; Burczyk et al., 2024), time of collection (Hazrati et al., 2024; Boaventura et al., 2025), extraction technique (Acosta-Vega et al., 2025; Suttiarporn et al., 2025; Ferraz & Silva, 2024), and genetic background (Jakovljević, 2025; BIOLOGICA NYSSANA ● 16 (2) December 2025: Fadel et al. ● Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus Samples Yield (%) Total phenolic content µg GAE/mg Total flavonoid content µg QE/mg DPPH radical scavenging IC50 (mg/mL) β-carotene (%) ACME 20.2 19.28±4.93 6.53±0.70 6.96±0.06 40.09±3.79 ACEO 0.01 78.1±5.28 Ascorbic acid 0.22±0.02 α-Tocopherol 61.42±3.64 Table 3. Antioxidant activity, total phenol and flavonoid contents of ACEO and ACME (Mean values ± SD, n = 2) Fig. 2. Gallic acid calibration curve for TPC Fig. 3. Quercetin calibration graph for TFC in chemical composition was observed, where the main compounds in our sample, octadecanol, caryophyllene oxide, cis-thujone, and tetracosane, were absent or present at low concentrations in the previously mentioned samples. As for the two compounds, nonanal and phytone, they were completely absent in the previously published samples. In contrast, germacrene D, β-thujone, α-thujone, δ-elemene, trans-chrysanthenyl acetate, and chrysanthenone, which were predominant in all published samples (Aliboudhar et al., 2013; Ganić et al., 2025; Solgi et al., 2025; Mustafa et al., 2024). Moreover, the high concentration of octadecanol in ACEO, a fatty alcohol known as a vaginal drug-delivery vehicle (Yuan et al., 2010), and caryophyllene oxide, a potent antioxidant, anti- inflammatory (Kumar et al., 2025), and antiparasitic (López-López et al., 2025) sesquiterpenoid, suggests this particular chemotype may exhibit an enhanced transethosomal system for enhanced transdermal delivery and therapeutic biological effects. Antioxidant capacity The percentage yields (Tab. 3) of ACEO and ACME were calculated based on the initial mass of the plant material as follows: Yield (%) = m1×100 / m2, where m1 is the final obtained mass of the oil/extract, and m2 is the initial mass of the species. The equation y = 0.018x + 0.236 was used to determine phenol concentrations at 765 nm (Fig. 2), with a correlation coefficient (R2) of 0.997. Gallic acid was used as the standard for the linear curve. The obtained results (Tab. 3) showed that the ACME had a moderate amount of phenols with a TPC of 19.28±4.93 µg GAE/mg. The flavonoid amount was measured using the calibration curve equation y = 0.0337x + 0.054 at 420 nm (Fig. 3), with a correlation coefficient (R2) of 0.997. Quercetin was used as the standard for the linear curve. The results showed that the ACME had moderate flavonoid content (6.53±0.70 µg QE/mg). Regarding the antioxidant evaluation of ACME using the DPPH assay, ascorbic acid was used as a reference, and the results were expressed as IC50 values (Tab. 3). The linear curve equation y =7.208x–0.136 (R2 = 0.993) was used to determine the IC50 (Fig. 4), and the linear curve equation y=240.8x+0.145 (R2=0.999) was used to determine the IC50 of the ascorbic acid (Fig. 5 and Fig. 6). The values indicate a weak antioxidant capacity for ACME compared to ascorbic acid (IC50 = 6.96±0.06 and 0.22±0.02 mg/ml, respectively), whereas, the β-carotene assay (Fig. 7), revealed a moderate antioxidant capacity (40.09±3.7%) and a high antioxidant capacity of ACEO (78.1±5.28%) compared to the α-tocopherol standard with 61.42±3.64% (ACEO ˃ α-tocopherol ˃ ACME). This could be explained by the difference in antioxidant mechanisms (Famutimi et al., 2025). Several published works reported the antioxidant capacities of A. clavatus extracts and essential oils (Chroho et al., 2022; Bouriche et al., 2016; Aliboudhar et al., 2013). Furthermore, A. clavatus extracts and essential oils have been shown, according to many studies, to possess various biological activities, including anticancer, anti-inflammatory, antibacterial, antifungal, and antioxidant capacity (Adiba et al., 2019; Hammami et al., 2013; Hasan et al., 2025). A number of published works demonstrated the direct relationship between the phenols and biological potentials, such as antioxidant capacity (Vlocskó et al., 2025; Hazarika et al., 2025), including the primary constituents of ACEO, caryophyllene oxide (Shabana et al., 2023), phytone (Gao et al., 2025), and compounds that follow (Tab. 1), such as spathulenol (do Nascimento et al., 2018), hexadecanoic acid (Ganesan et al., 2024), and chrysanthenyl acetate (Di Napoli et al., 2020). BIOLOGICA NYSSANA ● 16 (2) December 2025: Fadel et al. ● Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus Fig. 4. Linear curve of ACME Fig. 5. Linear curve of ascorbic acid standard Fig. 6. Antioxidant activity by DPPH assay of ACME and ascorbic acid Conclusion This study’s aim was to enrich knowledge of the volatile composition of Anacyclus clavatus, a member of the Asteraceae family collected in BIOLOGICA NYSSANA ● 16 (2) December 2025: Fadel et al. ● Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus Fig. 7. Antioxidant activity by β-carotene test of ACME and ACEO Algeria’s Aures zone, and to assess its crude methanol extract for total phenol and flavonoid contents. Using GC-MS and GC-FID, the primary constituents of ACEO were identified as octadecanol, caryophyllene oxide, thujone, tetracosane, nonanal, and phytone. The notably high antioxidant activity of ACEO and moderate amounts of flavonoids and phenols found in ACME imply that this species could be a useful natural resource for medicinal and nutritional uses. Future phytochemical and in vivo biological studies should be focused on the isolation of major compounds, particularly octadecanol and caryophyllene oxide, to confirm their individual contributions to the observed strong antioxidant activity and to test for other potential bioactivities, such as anti-inflammatory, antiparasitic, and vaginal drug-delivery vehicle activities. Acknowledgement: Pr. Rebbas Khellaf of M’sila University and Pr. Bicha Sabrina of Constantine 1 University are acknowledged by the authors for their help in identifying the plant material. References Acosta-Vega, L., Cifuentes, A., Ibáñez, E., & Galeano Garcia, P. (2025). Exploring natural deep eutectic solvents (nades) for enhanced essential oil extraction: current insights and applications. Molecules, 30(2), 284. https://doi.org/10.3390/ molecules30020284 Adams, R. P. (2017). Identification of essential oil components by gas chromatography/ mass spectrometry (ed. 4.1). Biology Department Baylor University. Aliboudhar, H., & Tigrine-Kordjani, N. (2014). Effect of extraction technique on the content and antioxidant activity of crude extract of Anacyclus clavatus flowers and their essential oil composition. Natural Product Research, 28(23), 2140–2149. https://doi.org/10.1080/14786419.2014.927872 Aliboudhar, H., Tigrine-Kordjani, N., & Youcef Meklati, B. (2015). Competition of microwave- assisted hydro-distillation in highlighting volatile phytochemicals of Anacyclus clavatus species. Journal of Essential Oil Research, 27(4), 355–362. https://doi.org/10.1080/10412905.2015.1029083 Aliboudhar, H., Tigrine-Kordjani, N., Hanifi, N., & Meklati, B. Y. (2013). Volatiles profiling and antioxidant activity evaluation of different parts of a medicinal plant: Anacyclus clavatus. Journal of Herbs, Spices and Medicinal Plants, 19(1), 33–47. https://doi.org/10.1080/10496475.2012.735215 Babushok, V. I., Linstrom, P. J., & Zenkevich, I. G. (2011). Retention indices for frequently reported compounds of plant essential oils. Journal of Physical and Chemical Reference Data, 40(4), 043101–043147. https://doi.org/10.1063/1.3653552 Baslam, A., Aitbaba, A., Aboufatima, R., Agouram, F., Boussaa, S., Chait, A., & Baslam, M. (2023). Phytochemistry, antioxidant potential, and antibacterial activities of Anacyclus pyrethrum: promising bioactive compounds. Horticulturae, 9(11), 1196. https://doi.org/10.3390/ horticulturae9111196 Boaventura, T. P., dos Santos, F. A. C., de Oliveira, P. E. C. M., Braga, N. G., Assis, Y. P. A. S., de Oliveira, J. E., Luz, R. L., & Gisele Cristina Favero, G. C. (2025). Use of the essential oil of Thymus vulgaris (Thyme) and its nanoemulsion as sedatives during tambaqui (Colossoma macropomum) transport: water quality, survival and physiology. Aquaculture International, 33(2), 150. http://dx.doi.org/10.2139/ssrn.4846016 Buragohain, D., Kundu, P., Nath, R., Kityania, S., Giri, A., & Das Talukdar, A. (2024). Importance of traditional resources in pharmaceutical industries. In Das Talukdar, A., Patra, J. K., Das, G., & Nath, D. (Eds.), Traditional resources and tools for modern drug discovery. Interdisciplinary Biotechnological Advances. https://doi.org/10.1007/978-981-97- 4600-2_15 Burczyk, J., Wierzchowska-Renke, K., Głowniak, K., Głowniak, P., & Marek, D. (2024). Geographie and environmental influences on the variation of essential oil and coumarins in Crithmum maritimum BIOLOGICA NYSSANA ● 16 (2) December 2025: Fadel et al. ● Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus L. Breeding Research on Aromatic and Medicinal Plants, 17(2), 305–311. https://doi.org/10.1300/ J044v09n04_07 Chroho, M., Aazza, M., Bouymajane, A., Majdoub, Y. O. E., Cacciola, F., Mondello, L., Zair, T., & Bouissa, L. (2022). HPLC-PDA/ESI- MS analysis of phenolic compounds and bioactivities of the ethanolic extract from flowers of Moroccan Anacyclus clavatus. Plants, 11(24), 3423. https:// doi.org/10.3390/plants11243423 Di Napoli, M., Maresca, V., Varcamonti, M., Bruno, M., Badalamenti, N., Basile, A., & Zanfardino, A. (2020). (+)-(E)-Chrysanthenyl acetate: A molecule with interesting biological properties contained in the Anthemis secundiramea (Asteraceae) flowers. Applied Sciences, 10(19), 6808. https://doi.org/10.3390/app10196808 Dimitrijević, M., Stojanović-Radić, Z., Radulović, N., Nešić, M., & Pejčić Pejić, M. P. (2025). Chemical composition and antifungal effect of the essential oil of Helichrysum italicum (Roth) G. Don against clinical isolates of Candida spp. Biologica Nyssana, 16(1), 177–187. http://dx.doi. org/10.46793/BiolNyss.16.1.6D do Nascimento, K. F., Moreira, F. M. F., Santos, J. A., Kassuya, C. A. L., Croda, J. H. R., Cardoso, C. A. L., Carmo Vieira, M., Tasca Góis Ruiz, A. L., Foglio, M. A., de Carvalho, J. E., & Formagio, A. S. N. (2018). Antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of Psidium guineense Sw. and spathulenol. Journal of Ethnopharmacology, 210, 351–358. https://doi.org/10.1016/j.jep.2017.08.030 El Baz, A., Mrabti, H. N., Ashmawy, N. S., Khan, S. A., Abdallah, E. M., Al-Mijalli, S. H., Alenazy, R., Alshabrmi, F. M., Bouyahya, A., El Hachlafi, N., Ardianto, C., Ifadotunnikmah, F., & Hmimid, F. (2024). Phytochemical characterization, antimicrobial properties and in silico modeling perspectives of Anacyclus pyrethrum essential oil. Heliyon, 10(16), e35079. https://doi.org/10.1016/j. heliyon.2024.e35079 Famutimi, O. G., Masha, S., Maluleke, R., Ncapayi, V., Lebepe, T. C., Mgedle, N., Kungwa, C. M., Fanoro, O. T., Adewale, I. O., & Oluwafemi, O. S. (2025). Assessment of antioxidant potential of carbon-based nanomaterials from different sources. Antioxidants, 14(10), 1227. https://doi.org/10.3390/ antiox14101227 Ferraz, L. P., & Silva, E. K. (2024). Pulsed electric field-assisted extraction techniques for obtaining vegetable oils and essential oils: Recent progress and opportunities for the food industry. Separation and Purification Technology, 354(2), 128833. https:// doi.org/10.1016/j.seppur.2024.128833 Ganesan, T., Subban, M., Christopher Leslee, D. B., Kuppannan, S. B., & Seedevi, P. (2024). Structural characterization of n-hexadecanoic acid from the leaves of Ipomoea eriocarpa and its antioxidant and antibacterial activities. Biomass Conversion and Biorefinery, 14(13), 14547–14558. https://doi.org/10.1007/s13399-022-03576-w Ganić, T., Pećinar, I., Nikolić, B., Kekić, D., Tomić, N., Cvetković, S., Vuletić, S., & Mitić- Ćulafić, D. (2025). Evaluation of Cinnamon essential oil and its emulsion on biofilm-associated components of Acinetobacter baumannii clinical strains. Antibiotics, 14(1), 106. https://doi. org/10.3390/antibiotics14010106 Gao, J. Y., Zou, Y. Y., Liang, Y., Zhang, Y. Y., & Zhang, Y. (2025). The essential oil composition of Lycopodium japonicum and its antioxidant activities. Chemistry of Natural Compounds, 1–3. https://doi. org/10.1007/s10600-025-04813-0 Hammami, R., Fernandez, B., Lacroix, C., & Fliss, I. (2013). Anti-infective properties of bacteriocins: an update. Cellular and Molecular Life Sciences, 70(16), 2947–2967. https://doi.org/10.1007/s00018- 012-1202-3 Hazarika, T. K., Debbarma, P., Malsawmkima, G., Barman, K., & Nath, P. C. (2025). Biochemical profiling and antioxidant potential of fruit tissues: a comparative study of Citrus cultivars indigenous to Northeast India. Journal of Food Processing and Preservation, 2025(1), 8833248. https://doi. org/10.1155/jfpp/8833248 Hazrati, S., Mousavi, Z., & Nicola, S. (2024). Harvest time optimization for medicinal and aromatic plant secondary metabolites. Plant Physiology and Biochemistry, 212, 108735. https:// doi.org/10.1016/j.plaphy.2024.108735 Heywood, V. H., Brummitt, R. K., Culham, A., & Seberg, O. (2007). Flowering plant families of the world. Ontario: Firefly Books. Jakovljević, D. (2025). Genetic diversity, cultivation, and utilization of aromatic plants. In Industrial Crops Improvement: Biotechnological Approaches for Sustainable Agricultural Development, 19, 171– 182. https://doi.org/10.1007/978-3-031-75937-6_10 Kumar, G. S., Sholapuri, P., K, D., Shaily Enugonda, M., & BP, G. (2025). In vivo and in silico anti-inflammatory activity of Artemisia vulgaris and β-caryophyllene oxide in carrageenan- induced paw edema in Wistar rats. Drug and Chemical Toxicology, 48(2), 426–439. https://doi.or BIOLOGICA NYSSANA ● 16 (2) December 2025: Fadel et al. ● Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus g/10.1080/01480545.2024.2415349 Lahmar, I., Yotova, L., & Belghith, K. (2025). Environmental impact on phonological stages in Lavandula officinalis: Chemical profiling of essential oil and extract, antioxidant activity, and acetylcholinesterase inhibition potential. Euro-Mediterranean Journal for Environmental Integration, 7, 1–12. https://doi.org/10.1007/ s41207-024-00714-w López-López, L. P., Hernández-Cuevas, N. A., Acosta-Viana, K. Y., Arana-Argáez, V. E., Torres-Romero, J. C., & Polanco-Hernández, G. M. (2025). Activity of β-Caryophyllene oxide and benznidazole mixture against Trypanosoma cruzi and in silico prediction of anti-trypanocidal interaction. Scientia Pharmaceutica, 93(3), 40. https://doi.org/10.3390/scipharm93030040 Mahato, D., Mahto, H., & Kumari, S. (2025). Medicinal and aromatic plant cultivation and sustainable development. In Industrial Crops Improvement. Sustainable Landscape Planning and Natural Resources Management. https://doi. org/10.1007/978-3-031-75937-6_8 Mahdavi, B., Yaacob, W. A., & Din, L. B. (2017). Chemical composition, antioxidant, and antibacterial activity of essential oils from Etlingera sayapensis AD Poulsen & Ibrahim. Asian Pacific Journal of Tropical Medicine, 10(8), 819–826. https://doi. org/10.1016/j.apjtm.2017.08.006 Manouze, H., Bouchatta, O., Gadhi, A. C., Bennis, M., Sokar, Z., & Ba-M’hamed, S. (2017). Anti-inflammatory, antinociceptive, and antioxidant activities of methanol and aqueous extracts of Anacyclus pyrethrum roots. Frontiers in Pharmacology, 8, 598. https://doi.org/10.3389/ fphar.2017.00598 Mechergui, K., Khaldi, S., & Jaouadi, W. (2017). Assessment of phenology and morphological diversity of 3 species of Asteraceae: Anacyclus clavatus, Chamaemelum fuscatum and Tanacetum parthenium. Asian Journal of Biology, 3(2), 1–12. https://doi.org/10.9734/AJOB/2017/34533 Mifsud, S. (2002). Anacyclus clavatus (White Anacyclus). MaltaWildPlants.com—the online Flora of the Maltese Islands. Mustafa, K. H., Khorshidi, J., Vafaee, Y., Rastegar, A., Morshedloo, M. R., & Hossaini, S. (2024). Phytochemical profile and antifungal activity of essential oils obtained from different Mentha longifolia L. accessions growing wild in Iran and Iraq. BMC Plant Biology, 24(1), 461. https://doi. org/10.1186/s12870-024-05135-z Nadji, W., Yousfi, B., Barhouchi, B., Salem, R., Yassad, S., & Djekoun, A. (2024). Formulation and development of an antiacne patch containing black seed and clove extracts by using chitosan-gelatin as polymers matrix. Journal of Molecular and Pharmaceutical Sciences, 3(1), 26–37. Ouasti, I., Ouasti, M., Panda, S., Mahanty, D. S., Bussmann, R. W., & Elachouri, M. (2024). Anacyclus clavatus (Desf.), Anacyclus monanthos (L.) Thell., Anacyclus monanthos (L.) Thell. subsp. cyrtolepidioides (Pomel) Humphries, Anacyclus pyrethrum (L.) Lag., Anacyclus radiatus Loisel., Anacyclus × valentinus L. – Asteraceae. In Ethnobotany of Northern Africa and Levant (pp. 1–7). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-031-43105-0_31 Paksoy, M. Y., Selvi, S., Sevindik, E., & Uysal, H. (2016). Soil content in the Anacyclus L. (Asteraceae) genus growing in Turkey. Turkish Journal of Life Sciences, 1(1), 8–12. Quezel, P., & Santa, S. (1963a). Nouvelle flore de l’Algérie et des régions désertiques méridionales. Tome II. Paris: Editions du Centre National de la Recherche Scientifique, 15, quai Anatole-France. Quezel, P., & Santa, S. (1963b). Nouvelle flore de l’Algérie et des régions désertiques méridionales. Tome II. Paris: Editions du Centre National de la Recherche Scientifique, 15, quai Anatole-France. Sarmah, A., & Bora, D. J. (2025). Carbon farming and the green economy: a science-based framework for sustainable agricultural transformation. In Advances in Modern Agriculture: Research and Innovation, 25(2), 209–225. Bhumi Publishing, India. Shabana, S. M., Gad, N. S., Othman, A. I., Mohamed, A. F., & El-Missiry, M. A. (2023). β-caryophyllene oxide induces apoptosis and inhibits proliferation of A549 lung cancer cells. Medical Oncology, 40(7), 189. https://doi.org/10.1007/ s12032-023-02022-9 Singleton, V. L., Orthofer, R., & Lamuela- Raventós, R. M. (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods in Enzymology, 299, 152–178. https://doi.org/10.1016/ S0076-6879(99)99017-1 Sissi, S., Ouhaddou, S., Dilagui, I., Soraa, N., Bellioua, S., Larhsini, M., & Markouk, M. (2024). Chemical composition, antioxidant and antimicrobial activities of Anacyclus maroccanus Ball. and Anacyclus radiatus Loisel. essential oils against multidrug resistant microbial BIOLOGICA NYSSANA ● 16 (2) December 2025: Fadel et al. ● Chemical composition and antioxidant activity of essential oil from Algerian Anacyclus clavatus pathogens. Natural Product Communications, 19(10), 1934578X241289844. https://doi. org/10.1177/1934578X241289844 Solgi, M., Bagnazari, M., Mohammadi, M., & Azizi, A. (2025). Thymbra spicata extract and arbuscular mycorrhizae improved the morphophysiological traits, biochemical properties, and essential oil content and composition of Rosemary (Rosmarinus officinalis L.) under salinity stress. BMC Plant Biology, 25(1), 220. https://doi. org/10.1186/s12870-025-06221-6 Šovljanski, O., Kljakić, A. C., & Tomić, A. (2025). Antibacterial and antifungal potential of plant secondary metabolites. In Plant Specialized Metabolites: Phytochemistry, Ecology and Biotechnology. https://doi.org/10.1007/978-3-031- 51158-5_6 Suttiarporn, P., Taithaisong, T., Namkhot, S., & Luangkamin, S. (2025). Enhanced eugenol composition in clove essential oil by deep eutectic solvent-based ultrasonic extraction and microwave- assisted hydrodistillation. Molecules, 30(3), 504. https://doi.org/10.3390/molecules30030504 Takada, H., Kokubo, K., Matsubayashi, K., & Oshima, T. (2006). Antioxidant activity of supramolecular water-soluble fullerenes evaluated by β-carotene bleaching assay. Bioscience, Biotechnology, and Biochemistry, 70(12), 3088– 3093. https://doi.org/10.1271/bbb.60491 Vitales, D., Feliner, G. N., Valles, J., Garnatje, T., Firat, M., & Alvarez, I. (2018). A new circumscription of the Mediterranean genus Anacyclus (Anthemideae, Asteraceae) based on plastid and nuclear DNA markers. Phytotaxa, 349(1), 1–17. https://doi.org/10.11646/phytotaxa.349.1.1 Vlocskó, R. B., Mastyugin, M., Török, B., & Török, M. (2025). Correlation of physicochemical properties with antioxidant activity in phenol and thiophenol analogues. Scientific Reports, 15(1), 73. https://doi.org/10.1038/s41598-024-83982-4 Yazıcı, Ö., & Sevgili, L. M. (2024). Obtaining cumin (Cuminum cyminum) essential oils by steam distillation. Part I: Investigation of operation parameters on essential oil yield and distribution of oil composition. Journal of Chemical Technology and Biotechnology, 99(2), 481–498. https://doi. org/10.1002/jctb.7550 Yuan, D., Ju, C., Ding, S., Jing, X., & Zhang, C. (2010). Synthesis of 1-octadecanol-modified water- swelling polyurethane hydrogels as vaginal drug- delivery vehicle. Journal of Biomaterials Science, Polymer Edition, 21(4), 493–505. https://doi. org/10.1163/156856209X427032 Zardi-Bergaoui, A., Harzallah-Skhiri, F., Hammami, S., Chreaif, I., Cheriaa-Issa, J., Jannet, H. B., & Mighri, Z. (2008). Composition and antibacterial activity of the essential oil from flowerheads of Anacyclus cyrtolepidioïdes Pomel from Tunisia. Journal of Essential Oil Bearing Plants, 11(6), 577–585. https://doi.org/10.1080/097 2060X.2008.10643669