Microsoft Word - 11377 NSB Sevindik 2023.03.22.docx Received: 09 Nov 2022. Received in revised form: 10 Feb 2023. Accepted: 07 Mar 2023. Published online: 22 Mar 2023. From Volume 13, Issue 1, 2021, Notulae Scientia Biologicae journal uses article numbers in place of the traditional method of continuous pagination through the volume. The journal will continue to appear quarterly, as before, with four annual numbers. SHSTSHSTSHSTSHST Horticulture and ForestryHorticulture and ForestryHorticulture and ForestryHorticulture and Forestry Society of TransylvaniaSociety of TransylvaniaSociety of TransylvaniaSociety of Transylvania Sevindik E et al. (2023) Notulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia BiologicaeNotulae Scientia Biologicae Volume 15, Issue 1, Article number 11377 DOI:10.15835/nsb15111377 Research ArticleResearch ArticleResearch ArticleResearch Article.... NSBNSBNSBNSB Notulae Scientia Notulae Scientia Notulae Scientia Notulae Scientia BiologicaeBiologicaeBiologicaeBiologicae Chemical composition of Chemical composition of Chemical composition of Chemical composition of Vitex agnusVitex agnusVitex agnusVitex agnus----castuscastuscastuscastus L. flowers collected from L. flowers collected from L. flowers collected from L. flowers collected from populations distributed in Aydpopulations distributed in Aydpopulations distributed in Aydpopulations distributed in Aydiiiinnnn, , , , TürkiyeTürkiyeTürkiyeTürkiye Emre SEVİNDİK1*, Deniz KARAKOYUN1, İbrahim M. GÜLDERE1, Fatih BORHAN1, Elif APAYDIN2, Emre KABİL3 1Aydın Adnan Menderes University, Faculty Agriculture, Department of Agricultural Biotechnology, South Campus, Cakmar, Aydin, Türkiye; ph.d-emre@hotmail.com (*corresponding author); denizkrkyn98@gmail.com; mguldere00@gmail.com; fatihborhan986@gmail.com 2Giresun University, Center Research Laboratory Application and Research Center, Giresun, Türkiye; eliferenapaydin@gmail.com 3Yalova University, Armutlu Vocational School, Food Processing Division, 77500 Yalova, Türkiye; emre.kabil@yalova.edu.tr AbstractAbstractAbstractAbstract Essential oils (EOs) are compounds that are biosynthesized autonomously by aromatic plants as their secondary metabolites. Vitex agnus-castus plant leaves, flowers and fruits are of pharmacological importance. In this study, the chemical composition determination of the essential oils of the Vitex agnus-castus flowers with different colors (white, pink, purple) growing in Aydin ecological conditions was carried out with the extraction using Clevenger hydro-distillation apparatus and then the essential oil composition was determined by Gas Chromatography-Mass Spectrometer (GC-MS). As a result of the study, ‘Çakmar’ pink-flowered population had the main component (-)-allospathulenol (14.59%), ‘Çakmar’ purple-flowered and ‘Koçarli’ purple-flowered populations had the main component pyrrolo (3,2,1-jk) carbazole (14.42% and 14.68%, respectively), while ‘Çakmar’ white flowered, ‘Koçarli’ white flowered and ‘Koçarli’ pink flowered populations had the main component caryophyllene (15.54%, 12.64%, and 19.05 respectively). Keywords:Keywords:Keywords:Keywords: GC-MS; essential oils; Vitex agnus-castus; Türkiye IntroductionIntroductionIntroductionIntroduction All over the world, various plants have been used for various reasons. Especially, medicinal and aromatic plants are grown for use in preventing diseases, as flavoring agents in incense, cosmetics, preserving food and improving the taste of many types of food (Toplan et al., 2022; Yilmaz et al., 2022). In addition, many studies reporting medicinal plants with important biological activities such as antioxidant, antimicrobial, antiviral, and antiproliferative activities have been revealed (Zhang et al., 2014; Suleman et al., 2021; Gecer, 2022). Vitex is one of the largest genera of the Lamiaceae family (formerly belonging to the Verbenaceae family), and consists of 217 species. Some of the known and studied Vitex species are Vitex negundo Vitex agnus-castus, Vitex trifolia, Vitex rotundifolia, Vitex cymosa and Vitex peduncularis (Souto et al., 2020; Kamal et al., 2022). The Vitex genus is represented by two species (V. agnus-castus and V. pseudo-negundo (Hausskn. Ex Bornm.) Hand.-Mazz) in https://www.notulaebiologicae.ro/index.php/nsb/index Sevindik E et al. (2023). Not Sci Biol 15(1):11377 2 the flora of Türkiye (Eryiğit et al., 2015). Vitex agnus-castus is defined as vitex, agnos and lygos by ancient literature (Kolancı, 2017). In Turkish, is known as 'hayıt, ayd, hayed, beşparmak grass, and bitter ayit' (Kavaz et al., 2022). It spreads especially in the Eastern Black Sea, Marmara, Aegean, Mediterranean, and partially in the Southeastern Anatolia region of Türkiye (Fakir et al., 2014). When this plant is looked at from an aesthetic point of view; the length of its spike-shaped flowers, its plump flower structure, its rich colour range from white- light pink to purple, its sustained blooming, and the striking appearance of its large seeds on the spike formed after its flowers, increase the variety of usage areas in herbal landscaping (Girmen and Karagüzel, 2005). Vitex agnus-castus is rich in numerous bioactive substances it is a well-known herbal plant (Zhelev et al., 2022). V. agnus castus fruits, flowers and leaves contain phenolic acids and derivatives, flavonoids, tannins, iridoids and diterpenoids (Sarikurkcu et al., 2009; Săvulescu et al., 2019; Bakr et al., 2020). Since ancient times, local flowers and fruits have been applied in various cultures (Özderin, 2021). Traditionally, chasteberry extract has been used to treat many gynecological conditions, including menstrual disorders, premenstrual syndrome (PMS), corpus luteum insufficiency, infertility, acne, menopause, impaired lactation, and various gynecological problems (Stojković et al., 2011; Ogaly et al., 2021). In this study, essential oil content of different colored (white, pink, purple) V. agnus-castus flowers grown in Aydın ecological conditions was determined. Materials and MethodsMaterials and MethodsMaterials and MethodsMaterials and Methods Collection of plant samples and extraction of essential oils For the essential oil extraction process, approximately 500 grams of V. agnus-castus flower samples were collected from different regions of Aydın, Türkiye, six populations with white, purple, and pink flowers in three different colors. The collected flower samples were kept with their moisture evaporated in an oven at 35 °C for 24 hours, then distilled for about 4 hours by hydro-distillation method in the Clevenger apparatus to extract the essential oil, and kept at +4 °C for chromatographic analysis. Chromatographic analysis by GC-MS The qualitative analysis of the essential oil obtained from the plant sample was dedected by Gas Chromatography-Mass Spectrometry (GC-MS) analysis system. Helium was used as the carrier gas and separation was performed with an HP-5MS capillary column coated with 5% phenyl-methylpolysiloxane (30.0 m x 250µm x 0.25 µm). GC-MS temperature program was applied from 50 °C to 80 °C ramped with 5 °C/min waiting for 2 minutes, 80 °C to 100 °C ramped with 2 °C/min waiting for 1 minute, 100 °C to 150 °C ramped with 2 °C/min waiting for 1 minute, 150 °C to 240 °C ramped with 2 °C/min waiting for 1 minute and finally from 240 °C to 270 °C ramped with 2 °C/min waiting for 7 minutes (Rambla et al., 2015; Apaydın, 2018). The volatile compounds which dedected were identified with Wiley and NIST mass spectrometry libraries. Results Results Results Results and Discussionand Discussionand Discussionand Discussion Essential oils (EOs) which are biosynthesized by aromatic plants as their secondary metabolites and represented by a strong odor are natural, volatile and complex mixtures of lipophilic compounds, often including terpenes, phenol-derived aromatic compounds and aliphatic compounds (Bakkali et al., 2008; Santos et al., 2016; Marić et al., 2021). There have been numerous studies to determine the chemical composition of essential oils obtained from chasteberry (Moudachirou et al., 1998; Senatore et al., 2003; Sarikurkcu et al., 2009; Stojković et al., 2011; Duymuş et al., 2014; Fakir et al., 2014; Gulsoy Toplan et al., 2015; Ulukanlı et al., 2015; Eryiğit et al., 2015; Tin et al., 2017; Zhelev et al., 2022 etc.). In our study, essential oil composition of V. Sevindik E et al. (2023). Not Sci Biol 15(1):11377 3 agnus-castus flowers analyzed (components with a total ratio of ≥2%) is shown in Table 1, while GC-MS chromatogram images are shown in Figures 1, 2, 3, 4, 5 and 6. As a result of the study, ‘Çakmar’ pink-flowered population had the main component (-)-allospathulenol (14.59%), ‘Çakmar’ purple-flowered and ‘Koçarlı’ purple-flowered populations had pyrrolo (3,2,1-jk)carbazole (14.42% and 14.68% respectively), while ‘Çakmar’ white flowered, ‘Koçarlı’ white flowered and ‘Koçarlı’ pink flowered populations had the main component of caryophyllene (15.54%, 12.64%, and 19.05 respectively). PCA (Principal Component Analysis) (simca 14.1) was used to determine the effect of flower varieties on volatile compounds. Table 1. Table 1. Table 1. Table 1. Composition of the essential oil from Vitex agnus-castus flowers of different populations Çakmar pink flowersÇakmar pink flowersÇakmar pink flowersÇakmar pink flowers Çakmar purple flowersÇakmar purple flowersÇakmar purple flowersÇakmar purple flowers Çakmar white flowersÇakmar white flowersÇakmar white flowersÇakmar white flowers RTRTRTRT ComponentComponentComponentComponent %%%% RTRTRTRT ComponentComponentComponentComponent %%%% RTRTRTRT ComponentComponentComponentComponent %%%% 7.423 α-pinene 2.45 21.516 cyclohexene 3.24 10.530 1.8-cineole 2.22 8.539 sabinene 2.99 24.446 trans β-farnesene 4.63 21.499 α-terpinyl propionate 4.90 15.108 cyclopentasiloxane 2.05 25.556 bicyclogermacrene 6.08 23.490 caryophyllenecaryophyllenecaryophyllenecaryophyllene 15.54 21.591 1-p-menthen-8-yl acetate 6.19 27.839 spathulenol 6.11 24.423 trans-β-farnesene 4.66 23.554 caryophyllene 5.53 27.965 caryophyllene oxide 4.28 27.782 spathulenol 6.36 24.480 trans β-farnesene 4.35 28.480 ledol 2.37 27.925 caryophyllene oxide 5.85 28.051 ((((----))))----allospathulenolallospathulenolallospathulenolallospathulenol 14.59 29.418 delta.-cadinene 4.08 29.390 delta.-cadinene 5.57 28.120 caryophyllene oxide 3.71 34.866 guaia-3,9-diene 2.07 36.187 n-(m-fluorophenyl)- maleımıde 4.68 28.675 ledol 2.56 36.250 n-(m- fluorophenyl)- maleımıde 13.70 36.977 pyrrolo(3,2,1-jk) carbazole 11.46 29.447 . α-cadinol 2.57 36.685 1-(3-hexyl)-2,5- dimethylbenzene 3.57 38.688 α-selinene 4.29 36.279 4(1h)-pteridinone 6.54 37.040 pyrrolo(3,2,1pyrrolo(3,2,1pyrrolo(3,2,1pyrrolo(3,2,1----jk)jk)jk)jk) carbazolecarbazolecarbazolecarbazole 14.42 39.346 sigmosceptrellin-b 2.34 37.103 pyrrolo(3,2,1-jk) carbazole 10.77 38.728 bakkenolıde-a-dıol 6.28 38.763 cycloheptane 4.67 39.386 kolavelool 2.29 Koçarlı purple flowersKoçarlı purple flowersKoçarlı purple flowersKoçarlı purple flowers Koçarlı white flowersKoçarlı white flowersKoçarlı white flowersKoçarlı white flowers Koçarlı pink flowersKoçarlı pink flowersKoçarlı pink flowersKoçarlı pink flowers RTRTRTRT ComponentComponentComponentComponent %%%% RTRTRTRT ComponentComponentComponentComponent %%%% RTRTRTRT componentcomponentcomponentcomponent %%%% 8.493 sabinene 3.49 9.987 β-phellandrene 2.12 6.811 α-pinene 3.00 10.513 1.8-cineole 3.79 25.179 (+)-2-carene 6.10 8.058 sabinene 4.24 21.505 4-terpinenyl ester of isobutanoicacid 5.63 28.915 caryophyllenecaryophyllenecaryophyllenecaryophyllene 19.05 9.998 β-phellandrene 2.19 23.485 caryophyllene 7.47 32.468 bicyclogermacrene 6.19 15.360 cyclopentasiloxane 5.32 24.423 trans-.β.-farnesene 4.08 39.129 (+)-epi- bicyclosesquiphelland rene 2.54 25.110 (+)-2-carene 3.34 25.522 bicyclogermacrene 5.23 45.486 2-methyl-6- propylpyridine-3- carboxylic acid 3.02 28.829 caryophyllenecaryophyllenecaryophyllenecaryophyllene 12.64 27.811 spathulenol 7.25 48.810 (z)-β-caryophyllene 3.32 30.563 trans-. β-farnesene 5.38 27.942 caryophyllene oxide 4.23 50.407 1,2,4-trıazolo(3,4- c)(1,2,4)- benzotrıazın-1(5h)- one 2.98 32.463 bicyclogermacrene 4.50 29.396 α-cadinol 3.53 51.437 β-isomethylıonone 4.96 45.520 2h-1,4-benzothiazine 3.04 36.222 n-(m-fluorophenyl)- maleımıde 13.15 53.685 levopimaradiene 2.38 48.822 (z)-β-caryophyllene 3.08 36.674 1-(3-hexyl)-2,5- dimethylbenzene 3.54 55.682 cycloheptane 2.60 50.464 1,2,4-trıazolo(3,4- c)(1,2,4)-benzotrıazın- 1(5h)-one 3.49 37.006 pyrrolo(3,2,1pyrrolo(3,2,1pyrrolo(3,2,1pyrrolo(3,2,1----jk)jk)jk)jk) carbazolecarbazolecarbazolecarbazole 14.68 51.431 β-ısomethylıonone 3.74 38.711 bakkenolıde-a-diol 6.72 52.879 3-fluoro-5,7- dimethylquinol-2(1h) one 3.67 Sevindik E et al. (2023). Not Sci Biol 15(1):11377 4 39.358 sigmosceptrellin-b 2.62 55.654 methylpropyleyne 7.73 56.604 kolavelool 3.57 68.706 4,4'-dimethyl-2,2',5,5'- tetramethoxy-1,1'- biphenyl 2.02 Figure 1. Figure 1. Figure 1. Figure 1. Gas chromatogram of the essential oil of ‘Çakmar’ pink flowers Figure 2. Figure 2. Figure 2. Figure 2. Gas chromatogram of the essential oil of ‘Koçarlı’ purple flowers Sevindik E et al. (2023). Not Sci Biol 15(1):11377 5 Figure 3. Figure 3. Figure 3. Figure 3. Gas chromatogram of the essential oil of ‘Çakmar’ purple flowers Figure 4. Figure 4. Figure 4. Figure 4. Gas chromatogram of the essential oil of ‘Çakmar’ white flowers Sevindik E et al. (2023). Not Sci Biol 15(1):11377 6 Figure 5. Figure 5. Figure 5. Figure 5. Gas chromatogram of the essential oil of ‘Koçarlı’ white flowers Figure 6. Figure 6. Figure 6. Figure 6. Gas chromatogram of the essential oil of ‘Koçarlı’ pink flowers The biplot diagram for the first and second components shows the correlation of the studied flowers based on the volatile compounds detected. According to the data obtained, the first and second components (PC1 and PC2) explained 59.7% of the total variance. Except for ‘Çakmar’ purple flower, ‘Çakmar’ and ‘Koçarlı’ flower populations were collected in two different groups according to PC1 (Figure 7). Sevindik E et al. (2023). Not Sci Biol 15(1):11377 7 Figure 7.Figure 7.Figure 7.Figure 7. Biplot of the first two principal components In chasteberry fruits collected from Aydın,Türkiye, Tin et al. (2017) determined 1.8 cineole (8.24%) as the main component. Gulsoy Toplan et al. (2015) examined the essential oils of V. agnus-castus fruits collected from Zonguldak, Edirne, Balıkesir, Muğla, Antalya and the main components were determined as β- caryophyllene (11.7%), bicyclogermacrene (22.1%), 1.8-cineole (17.3%), 1.8-cineole (13.2%), sabinene and bicyclogermacrene (12.1%), respectively. In the essential oils of the fruits of the chasteberry plant collected from Izmir/Turkey, Eryiğit et al. (2015) determined trans-caryophyllene (19.17%) as the main component. Duymuş et al. (2014) determined sabinene (22.7%) and 1.8-cineole (22.4%) as the main components of fruits and the fruitless herbs, respectively, of V. agnus-castus collected from İzmir, Türkiye. Fakir et al. (2014) determined essential oil content of V. agnus castus collected from Isparta, Türkiye during the flowering period and the main component was α-pinene (26.99), while the main component was 1.8 cineole (28.34%) during the fruit ripening period. Ulukan et al. (2015) determined the main component as 1.8-cineole (24.38%) from essential oils of chasteberry leaves collected from Osmaniye, Türkiye. Sarikurkcu et al. (2009) identified 1.8- cineole (24.98%) as the main component of chasteberry fruits from Manisa, Türkiye. Senatore et al. (2003) determined 1.8-cineol (21.6%) as the most abundant in the essential oil obtained from the white flowered fruit of Vitex agnus-castus collected from İçel, Türkiye, and caryophyllene (30.9%) as the most in essential oil obtained from the fruits of the purple flowered ones. In Montenegro, Stojkovic et al. (2011) found the main component as sabinene (17.8%) in immature chasteberry fruits, 1.8-cineole (16.3%) in mature fruits, and 1.8- cineol (22.0%) in leaves. Bakr et al. (2020) have determined 1.8 cineole (44.98 %) as the most abundant component in leaves of V. agnus-castus collected from the city of Giza, Egypt. Zhelev et al. (2022) identified 1.8-cineole (16.9–18.8%) as the main component in chasteberry fruits collected from South-Central Bulgaria and North-East Bulgaria. Moudachirou et al. (1998) determined 1.8-cineole (22.6%) as the main component in the essential oil content of the leaves of Vitex agnus-castus collected from Benin. In the past, in some studies of essential oils on the fruits, leaves and flowers of the Vitex agnus-castus, 1.8 cineole, α-pinene, caryophyllene, trans-caryophyllene, β-caryophyllene, sabinene and bicyclogermacrene were determined as main components. Sevindik E et al. (2023). Not Sci Biol 15(1):11377 8 When we compare our study with previous ones, the results are not consistent. The reason for this could be that the geographical and environmental conditions in which the samples distributed were different, confirming that the essential oil composition and content of the plants could be different (Sevindik et al., 2016). In our study, for ‘Çakmar’ white flower, ‘Koçar’ white flower and ‘Koçarlı’ pink flower populations, the main component was determined as caryophyllene. Sesquiterpenoids are an extremely large group of secondary metabolites found in plants. Among them, the caryophyllene or β-caryophyllene group is the most common in nature. In the last few years, β-caryophyllene has represented an important topic of study (Francomano et al., 2019; Gyrdymova and Rubtsova, 2021). In the past, the antimicrobial effect of β-caryophyllene has been tested against some human pathogens (Dahham et al., 2015). ConclusionsConclusionsConclusionsConclusions In this study, we evaluated the chemical composition of essential oils obtained from white, pink and purple chasteberry flowers. As a result, we found (-)-allospathulenol, pyrrolo(3,2,1-jk)carbazole and caryophyllene as main components. These findings may be a source for further antimicrobial, anti- inflammatory, anti-cancer, and antioxidant studies. Authors’ ContributionsAuthors’ ContributionsAuthors’ ContributionsAuthors’ Contributions E.S, D.K İ.M.G and F.B collected flowers samples. The experiments were performed and analyzed by E.S. E.A and E.K. E.S, E.A and E.K wrote the paper. All authors read and approved the final manuscript. Ethical approvalEthical approvalEthical approvalEthical approval (for researches involving animals or humans) Not applicable. AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements This research was supported by the TUBITAK 2019-2/2209-A (Project no: 1919B011904175) Conflict of InterestsConflict of InterestsConflict of InterestsConflict of Interests The authors declare that there are no conflicts of interest related to this article. Sevindik E et al. (2023). Not Sci Biol 15(1):11377 9 ReferencesReferencesReferencesReferences Apaydın E (2018). Investigation of antioxidant and antimicrobial activity of essential oil from Salvadora persica (miswak). 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