NATURAL VOLATILES & ESSENTIAL OILS Volume 8, Issue 1, 2021

REVIEWS______

Pages 1-12 Review on Malaysian Goniothalamus essential oils and their comparative study using multivariate statistical analysis. Natasa Mohd Shakri, Wan Mohd Nuzul Hakimi Wan Salleh and Shazlyn Milleana Shaharudin

RESEARCH ARTICLES______

Pages 13-17 Essential oil composition of Strychnos axillaris Colebr. (Loganiaceae). Wan Mohd Nuzul Hakimi Wan Salleh, Shamsul Khamis, Hakimi Kassim and Alene Tawang

18-21 Volatile components and antimicrobial activity of the n-hexane extracts of Neomuretia pisidica (Kit Tan) Kljuykov, Degtjareva & Zakharova. Ayşe Esra Karadağ, Betül Demirci, Ömer Çeçen, Ayvaz Ünal and Fatma Tosun

22-28 Chemical composition of essential oil from the aerial parts of Santolina rosmarinifolia L. a wild Algerian medicinal plant. Djamel Sarri, Noui Hendel, Hadjer Fodil, Giuseppe Ruberto and Madani Sarri

29-38 Monarda essential oils as natural cosmetic preservative systems. Łukasz Gontar, Anna Herman, Ewa Osińska

39-48 The Essential Oil Profiles of Chaerophyllum crinitum and C. macrospermum Growing wild in Turkey. Hale Gamze Ağalar, Ayhan Altıntaş, and Betül Demirci

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 1-12 Shakri et al. DOI: 10.37929/nveo.786172

REVIEW Review on Malaysian Goniothalamus essential oils and their comparative study using multivariate statistical analysis

Natasa Mohd Shakri1, Wan Mohd Nuzul Hakimi Wan Salleh1,* and Shazlyn Milleana Shaharudin2

1Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris (UPSI), 35900 Tanjong Malim, Perak, MALAYSIA 2Department of Mathematics, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris (UPSI), 35900 Tanjong Malim, Perak, MALAYSIA

*Corresponding author. Email: [email protected] Submitted: 27.08.2020; Accepted: 24.12.2020

Abstract The genus Goniothalamus is belonging to the Annonaceae family, consists of ca. 2500 species and found in tropical Southeast Asia. The Goniothalamus essential oils were recognized to possess considerable biological activities with varied chemical composition. This article aims to overview the medicinal uses, chemical compositions, and biological activities of Malaysian Goniothalamus essential oils considered as a medicinal plants, widely used as traditional herbal medicines in the treatment of various diseases. The data were collected from the scientific electronic databases including SciFinder, Scopus, Elsevier, PubMed and Google Scholar. Ten Goniothalamus species have been reported for their essential oils and biological activities. It can be observed that the major components were α-cadinol, terpinen-4-ol, β-eudesmol, β-selinene, linalool, limonene, α-copaene, 1,8-cineole and β-cubebene. In addition, the selected chemical components from the bark, leaf and root oils were analysed using Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) and were able to cluster in four groups based on relationships and chemical patterns in essential oils. This multivariate data analysis may be used for the identification and characterization of essential oils from different Goniothalamus species that are to be used as raw materials of traditional herbal products.

Keywords: Annonaceae, Goniothalamus, essential oil, principal component analysis, hierarchical cluster analysis

Introduction Known as the most powerful therapeutic agents, essential oils are usually used as an alternative medicine known as aromatherapy to support human health and well-being. Additionally, essential oils have also been commonly used in the cosmetics, food, and agricultural industries. Essential oils are usually extracted from a natural source and have that particular plant's fragrance (Winska et al., 2019; Salleh et al., 2014a). The essential oil is normally stored within the plant's oil cells, glands, and vessels. It is released from the flowers as a fragrant fragrance, or retained in the plant's seeds, fruits, leaves, barks or roots until it gradually evaporates (Burger et al., 2019; Salleh et al., 2014b). Essential oils from aromatic and medicinal plants have been known since antiquity to possess biological activity, most notably antibacterial, antifungal and antioxidant properties (Salleh et al., 2015a, 2015b, 2015c, 2016a, 2016b, 2016c). Annonaceae is one of the plant families which numerous report on essential oils, due to its wide use in various traditional medicines and believed to have high medicinal values. Annonaceae family is the largest family of the order Magnoliales consisting of approximately 135 genera and more than 2,500 species. The family has a source of edible fruit that can be considered to have economic importance. Goniothalamus is a genus of the Annonaceae family, with approximately 160 species of trees and shrubs frequently found in tropical Southeast Asia throughout Indochina and Malaysia (Anary et al., 2016; Aslam et al., 2016). The botanical characteristics are simple, strongly aromatic bark, having few leaves that are simple, alternate and exstipulate. The secondary nerves are also oblique, straight and parallel to

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 1-12 Shakri et al. DOI: 10.37929/nveo.786172

scalariform reticulations. The axillary flowers are characteristically woody, fusiform and often dark green (Wiart, 2006, 2007; Nielsen, 1993). Goniothalamus species have been used as traditional medicines in Malaysia but information about the volatile composition quality of essential oils from these herbal materials is still limited. Previous studies have described the biological activities of various Goniothalamus species such as antibacterial (Funnimid et al., 2019), antimicrobial (Ghani et al., 2010), antifungal (Duc et al., 2016), antioxidant (Iqbal et al., 2015), and cytotoxicity activities (Kim et al., 2013). The available information on the essential oils of Goniothalamus species was collected via electronic searches such as Pubmed, SciFinder, Scopus, Google Scholar, and Web of Science. This work aims to give an overview of all published studies on the chemical composition and biological activities of Malaysian Goniothalamus essential oils. In addition, the multivariate statistical analysis was also determined for the leaf, bark and root oils of selected Goniothalamus species. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to characterize their essential oils components. Traditional uses Goniothalamus species are used to induce abortion, antiaging, body pain, rheumatism, skin irritation, typhoid fever, tympanites, stomach ache and fever in widespread medicinal commodities. Table 1 shows several Goniothalamus species and their medicinal uses.

Table 1. Medicinal uses of several Goniothalamus species

Species Local name Part Medicinal uses G. amuyon amúyon Seeds Used to treat scabies, rheumatism and tympanites (Ahmad et al., 1991) Fruits Used to treat stomachache (Quisumbing, 1951) G. cheliensis Stems Used for the treatment of liver cancer, lung carcinoma and chronic cough (Jiang et al., 2011) G. dolichocharpus bihidieng Roots It is boiled and taken orally by the Kelabit community to ease stomachache (Quisumbing, 1951) G. giganteus penawar hitam Roots Used in abortion and treatment of colds (Wiart, 2006) Leaves Heated leaves are applied to swellings (Wiart, 2006) G. lanceolatus selukai Leaves Used as a traditional remedy for fever, skin infection, postpartum, abortion, as well as a cancer treatment (Wiart, 2007) G. laoticus Khao Lam-dong Stem bark Used traditionally as a tonic and a febrifuge by the local people in the northeastern part of Thailand (Wu et al., 1991) G. macrophyllus selayak hitam Leaves Used to allay fever (Alkofahi et al., 1988) Roots Used as a postpartum remedy and to cause abortion, antiaging purposes, rheumatisms, skin complaints, eliminate excessive gas in the body and used as a lotion to treat body pains (Alkofahi et al., 1988) G. marcanii Leaves Used treating for infectious diseases in early childhood (Mahiwan et al., 2013) G. malayanus kenanga paya Roots Used for the treatment of rheumatism and fever (Ahmad et al., 1991) Stem bark Used to treat measles and as insect repellents (Ahmad et al., 1991) G. scortechinii gajah beranak Leaves Used as a postpartum protective remedy and were used to improve blood circulation (Burkill, 1966) G. sesquipedalis Leaves Used to treat fever, cough, colds, snakebite, pains, and infectious and inflammatory diseases (Akter et al., 2018)

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G. tapis kenarak Roots Used as an abortifacient during early months of pregnancy, an infusion of the roots is used to treat typhoid fever (Inayat-Hussain et al., 1999) G. uvarioides belindung Roots Used as postpartum protective remedies, abortifacients, and to treat typhoid fever, rheumatism, and headache (Moharam et al., 2012) G. velutinus kayu tas hitam Leaves Used as a traditional medicine for treating headache, food poisoning, as well as snakebite remedies, induce abortion and as a post-partum remedy (Inayat-Hussain et al., 1999)

Chemical composition of Malaysian Goniothalamus essential oils There are ten species of Goniothalamus originated from Malaysia that were successfully reported for their essential oil composition. The essential oils of ten local Goniothalamus which were G. andersonii, G. clemensii, G. macrophyllus, G. malayanus, G. ridleyi, G. tapis, G.tapisoides, G. velutinus, G. uvarioides, and G. woodii have been investigated (Jusoh et al., 2015; Moharam et al., 2010; Ghani et al., 2010; Ahmad et al., 2010; Jantan et al., 2005). The plant parts used for the extraction of essential oils includes flower, fruit, leaf, stem, bark, and root. However, most of the essential oils from Malaysian Goniothalamus were extracted from bark, leaf and root. Table 2 shows the major components identified in Goniothalamus essential oils, whereas Figure 1 so its chemical structures.

Table 2. Major components identified in Malaysian Goniothalamus essential oils

Species Locality Parts Yield Total Major components References (%) components (%) G. andersonii Sarawak Leaf 0.70 25 (85.30) Guaiol (28.60%) (1), elemol Jantan et al., (19.60%), β-caryophyllene (7.70%) 2005 G. clemensii Sarawak Bark 2.80 23 (98.10%) α-Cadinol (41.60%) (2), agarospirol Moharam et (19.00%), elemol (16.10%) al., 2010 G. macrophyllus N. Sembilan Root 0.05 14 (42.50%) Cyperene (9.80%) (3), geranyl Ghani et al., acetate (9.40%), camphene (7.50%) 2010 G. macrophyllus N. Sembilan Twig 0.14 21 (90.00%) Geranyl acetate (45.50%) (4), Ghani et al., geraniol (17.00%), linalool 2010 (12.70%), camphene (7.50%) G. macrophyllus 1 Pahang Bark 0.80 42 (97.80%) Terpinen-4-ol (38.80%) (5), 1,8- Jantan et al., cineole (18.10%), geranyl acetate 2005 (11.10%), geraniol (9.70%) G. macrophyllus 2 Sarawak Bark 0.70 41 (96.40%) Terpinen-4-ol (42.70%) (5), β- Jantan et al., ocimene (25.40%), α-terpineol 2005 (10.00%), 1,8-cineole (5.80%) G. malayanus Sarawak Bark 0.96 36 (93.90%) β-Eudesmol (32.20%) (6), γ- Jantan et al., eudesmol (21.80%), (E)-nerolidol 2005 (9.10%), elemol (6.70%), α- eudesmol (6.60%) G. malayanus 1 Sarawak Leaf 0.32 35 (86.30%) β-Selinene (33.60%) (7), viridiflorol Jantan et al., (13.10%), epiglobulol (7.70%) 2005 G. malayanus 1 Sarawak Root 0.18 36 (90.50%) β-Eudesmol (27.80%) (6), γ- Jantan et al., eudesmol (18.80%), (E)-nerolidol 2005 (6.50%), α-eudesmol (6.00%), elemol (5.10%) G. malayanus 2 Sarawak Leaf 0.32 35 (86.20%) β-Selinene (33.60%) (7), viridiflorol Jantan et al., (13.10%), epiglobulol (7.70%) 2005

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G. malayanus 2 Sarawak Root 0.18 35 (90.00%) β-Eudesmol (27.80%) (6), γ- Jantan et al., eudesmol (18.80%), (E)-nerolidol 2005 (6.50%), α-eudesmol (6.00%), elemol (5.10%) G. ridleyii 1 Kelantan Bark 0.11 50 (89.50%) Linalool (15.20%) (8), citronellal Jusoh et al., (Fresh) (10.90%), β-eudesmol (9.80%), 2015 limonene (7.50%), elemol (7.40%) G. ridleyii 1 Kelantan Stem 0.03 47 (90.10%) β-Eudesmol (27.10%) (6), γ- Jusoh et al., (Fresh) eudesmol (20.80%), (Z)-nerolidol 2015 (9.50%), elemol (6.60%) G. ridleyii 1 Kelantan Fruit 0.12 49 (89.80%) β-Cubebene (20.70%) (9), elemol Jusoh et al., (Fresh) (20.20%), hedycaryol (9.40%), β- 2015 eudesmol (9.10%), viridifloral (6.10%) G. ridleyii 2 (Dry) Kelantan Bark 0.38 39 (94.60%) Linalool (15.80%) (8), citronellal Jusoh et al., (12.90%), limonene (10.60%), β- 2015 eudesmol (7.60%), safrole (7.50%) G. ridleyii 2 (Dry) Kelantan Leaf 0.90 48 (95.10%) Linalool (23.40%) (8), α-copaene Jusoh et al., (19.80%), β-caryophyllene 2015 (11.40%), 1,8-cineole (7.00%), terpinen-4-ol (6.10%) G. ridleyii 2 (Dry) Kelantan Stem 0.03 35 (93.30%) β-Eudesmol (27.80%) (6), δ- Jusoh et al., eudesmol (19.90%), (Z)-nerolidol 2015 (7.50%), elemol (7.10%), linalool (5.50%) G. ridleyii 2 (Dry) Kelantan Branch 0.27 37 (93.10%) β-Selinene (35.90%) (7), viridifloral Jusoh et al., (16.30%), (Z)-nerolidol (5.90%) 2015 G. ridleyii 2 (Dry) Kelantan Root 0.15 47 (92.80%) Cyperene (22.80%) (3), α-copaene Jusoh et al., (10.90%), α-eudesmol (8.10%), 2015 hedicaryol (4.60%) G. ridleyii 2 (Dry) Kelantan Fruit 0.83 37 (93.20%) β-Cubebene (17.20%) (9), elemol Jusoh et al., (15.90%), β-eudesmol (9.50%), 2015 hedicaryol (8.50%), viridifloral (8.40%) G. tapis Sarawak Root 0.98 36 (88.00%) Cyperene (16.20%) (3), α-copaene Ahmad et al., (8.70%), α-eudesmol (5.70%), β- 2010 elemene (5.30%), γ-amorphene (5.20%) G. tapis 1 Sarawak Bark 2.85 23 (82.70%) Limonene (12.70%) (10), linalool Moharam et (13.00%), safrole (11.20%), al., 2010 citronellal (6.30%), α-eudesmol (5.80%) G. tapis 1 Sarawak Leaf 2.23 26 (92.40%) α-Copaene (23.80%) (11), linalool Moharam et (18.50%), β-caryophyllene al., 2010 (14.20%), 1,8-cineole (7.60%) G. tapis 2 Sarawak Bark 2.85 22 (83.00%) Limonene (12.70%) (10), linalool Ahmad et al., (13.00%), safrole (11.20%), 2010 citronellal (6.30%), α-eudesmol (5.80) G. tapis 2 Sarawak Leaf 2.23 29 (93.60%) α-Copaene (23.80%) (11), linalool Ahmad et al., (18.50%), β-caryophyllene 2010 (14.40%), 1,8-cineole (7.60%) G. tapisoides 1 Sarawak Bark 3.86 23 (99.00%) 1,8-Cineole (47.90%) (12), Moharam et terpinen-4-ol (22.50%), p- al., 2010 menthene (6.90%), α-pinene (6.60%), γ-terpinene (6.60%)

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G. tapisoides 1 Sarawak Leaf 3.05 26 (99.40%) 1,8-Cineole (79.00%) (12), α-pinene Moharam et (9.60%), α-terpineol (4.40%) al., 2010 G. tapisoides 1 Sarawak Root 1.45 20 (99.70%) 1,8-Cineole (56.10%) (12), Moharam et terpinen-4-ol (19.60%), γ-terpinene al., 2010 (5.70%) G. tapisoides 2 Sarawak Bark 3.86 24 (92.60%) 1,8-Cineole (47.90%) (12), Ahmad et al., terpinen-4-ol (22.50%), α-pinene 2010 (6.60%), γ-terpinene (6.60%) G. tapisoides 2 Sarawak Leaf 3.05 28 (99.50%) 1,8-Cineole (79.00%) (12), α-pinene Ahmad et al., (9.60%), α-terpineol (4.40%) 2010 G. tapisoides 2 Sarawak Root 1.45 19 (95.60%) 1,8-Cineole (56.10%) (12), Ahmad et al., terpinen-4-ol (19.6%), γ-terpinene 2010 (5.70%) G. uvarioides Sarawak Leaf 0.27 51 (92.10%) β-Cubebene (15.20%) (9), elemol Jantan et al., (9.70%), epi-α-cadinol (6.20%) α- 2005 muurolene (4.80%) G. uvarioides Sarawak Bark 0.98 28 (87.20%) β-Eudesmol (31.50%) (6), γ- Jantan et al., eudesmol (16.00%), hedycaryol 2005 (13.60%), α-eudesmol (5.60%), (Z)- nerolidol (5.20%) G. uvarioides Sarawak Root 0.35 28 (85.50%) Terpinen-4-ol (39.50%) (5), 1,8- Jantan et al., cineole (14.00%), α-terpineol 2005 (6.30%), p-cymene (5.10%) G. velutinus Sarawak Bark 1.40 45 (93.90%) α-Cadinol (14.00%) (2), α-eudesmol Moharam et (9.70%), t-muurolol (9.10%), β- al., 2010 selinene (6.10%), γ-muurolene (5.20%) G. woodii Sarawak Bark 1.80 36 (97.00%) α-Cadinol (21.90%) (2), elemol Moharam et (12.60%), agarospirol (8.00%) al., 2010

Figure 1. Chemical structures of several major components identified from Goniothalamus essential oils. H

O H O (1) (2) (3) (4) OH

OH H OH (5) (6) (7) (8)

O

(9) (10) (11) (12)

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The bark oil was extracted from nine species of Goniothalamus which are G. clemensii, G. macrophyllus, G. malayanus, G. ridleyi, G. tapis, G. tapisoides, G. uvarioides, G. velutinus, and G. woodii. Seven species were found for its richness in oxygenated sesquiterpenes. They were α-cadinol (41.6%), β-eudesmol (32.2%), γ- eudesmol (21.8%), agarospirol (19.0%), elemol (16.1%), hedycaryol (13.6%), α-eudesmol (9.7%), t-muurolol (9.1%), (E)-nerolidol (9.1%), cubenol (7.7%), and (Z)-nerolidol (5.2%). In addition, sesquiterpene hydrocarbons were also found in the bark oils of Malaysian Goniothalamus. They were revealed by the presence of β-selinene (6.1%) found in the bark oil of G. velutinus. Furthermore, oxygenated monoterpenes were found in four local Goniothalamus bark oils. They were found in the bark oil of G. macrophyllus, G. ridleyi, G. tapis, and G. tapisoides. They were characterised by 1,8-cineole (47.9%), terpinen-4-ol (42.7%), linalool (15.8%), α-terpineol (10.0%), and geraniol (9.7%). Moreover, monoterpene hydrocarbons were also found as the major group components in four bark oils of Goniothalamus collected from Malaysia. (Z)-β- Ocimene (25.4%) and limonene (7.5%-12.7%) were found in the bark oil of G. macrophyllus, G. ridleyi and G. tapis. Meanwhile, p-menthene (6.9%), α-pinene (6.6%), and γ-terpinene (6.6%) were found in the bark oil of G. tapisoides. Geranyl acetate (11.1%), an ester was the major component of G. macrophyllus bark oil. Aldehyde which is citronellal (6.3%-12.9%) and phenyl propanoid which is safrole (5.3%-11.2%) were found in the bark oil of G. ridleyi and G. tapis, respectively. Five essential oils of Malaysian Goniothalamus have been extracted by using leaf parts which are G. andersonii, G. malayanus, G. tapis, G. tapisoides, and G. uvarioides. The leaf oils were dominated by sesquiterpene hydrocarbons, oxygenated sesquiterpenes, monoterpene hydrocarbons, and oxygenated monoterpenes. Four out of five species have sesquiterpene hydrocarbons as their major components. They were identified as β-selinene (33.6%), α-copaene (23.8%), β-cubebene (15.2%), β-caryophyllene (14.4%), and α-muurolene (4.8%). In addition, oxygenated sesquiterpenes were also found in the leaf oil which was guaiol (28.6%), elemol (19.6%), viridiflorol (13.1%), and epi-globulol (7.7%). Moreover, oxygenated monoterpenes which were 1,8-cineole (7.6%-79.0%), linalool (18.5%), and α-terpineol (4.4%) were found in two leaf oils identified as G. tapis and G tapisoides. Besides, α-pinene (9.6%) was the only monoterpene hydrocarbon found from the leaf oil of G. tapisoides. The root oils of Malaysian Goniothalamus have been extracted from five species which were identified as G. macrophyllus, G. malayanus, G. tapis, G. tapisoides, and G. uvarioides. The components can be classified as oxygenated sesquiterpenes, sesquiterpene hydrocarbons, oxygenated monoterpenes, monoterpene hydrocarbons and ester. The root oil of G. malayanus was dominated by oxygenated sesquiterpenes which were characterized by elemol (5.1%), (E)-nerolidol (6.5%), and γ-eudesmol (18.8%). β-eudesmol (5.0%-27.8%) and α-eudesmol (5.7%-6.0%) was found in both G. malayanus and G. tapis root oils. Next, sesquiterpene hydrocarbons found in the root oils were cyperene (9.8%-16.2%), α-copaene (8.7%), β-elemene (5.3%), and γ-amorphene (5.2%). Furthermore, oxygenated monoterpenes were found in the root oils of G. tapisoides and G. uvarioides which are 1,8-cineole (14.0%-56.1%), terpinen-4-ol (19.6%-39.5%), and α-terpineol (6.3%). Monoterpene hydrocarbons which were camphene (7.5%), γ-terpinene (5.7%), and p-cymene (5.1%) were found in the root oils of G. macrophyllus, G. tapisoides, and G. uvarioides. Last but not least, geranyl acetate (9.4%) which is an ester was found in the root oil of G. macrophyllus. Meanwhile, the fruit oil only reported from G. ridleyii with β-cubebene, elemol, and β-eudesmol as the major components (Jusoh et al., 2015). Biological activities Although many members of the genus Goniothalamus are renowned for their valuable essential oils, the genus is still poorly explored as far as its biological activities of essential oil are concerned. A search of the literature revealed the occurrence of antimicrobial and antiplatelet aggregation and platelet-activating factor

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(PAF) receptor antagonistic activities that have been reported from the Malaysian Goniothalamus essential oils. The antimicrobial activity of root and twig oils of G. macrophyllus has been reported by Ghani et al. (2010). The oils were found to demonstrate the notable antimicrobial activity with the MIC values below the cut-off point of 1 mg/mL. The root oils were considerably more active than the twig oil in inhibiting all the bacterial and fungal strains except P. aeruginosa. The root oils had the strongest inhibitory effect against VISA24, S. epidermidis and C. albicans with the MIC values of 0.3 mg/mL. The twig oils demonstrated moderate to weak activity toward all bacterial strains tested with the MIC values ranging from 2.5 to 5 mg/mL. Both dermatophytes, T. rubrum and M. cannis however showed similar susceptibility to the twig and root oils with the MIC values of 2.5 mg/mL. Moharam et al. (2010) reported antiplatelet aggregation and platelet-activating factor (PAF) receptor antagonistic activity against nine Goniothalamus essential oils (G. velutinus, G. woodii, G. clemensii, G. tapis and G. tapisoides). The bark oil of G. velutinus was the most effective sample as it inhibited both arachidonic acid (AA) and ADP-induced platelet aggregation with IC50 values of 93.6 and 87.7 μg/mL, respectively. Among the studied oils, the bark oils of G. clemensii, G. woodii, G. velutinus and the root oil of G. tapis showed significant inhibitory effects on PAF receptor binding, with IC50 values ranging from 3.5 to 10.5 μg/mL. Multivariate statistical analysis Ten Malaysian Goniothalamus species (28 samples) have been selected for this study which are G. andersonii, G. clemensii, G. macrophyllus, G. malayanus, G. ridleyi, G. tapis, G. tapisoides, G. uvarioides, G. velutinus, and G. woodii. Information on plant materials used for multivariate statistical analysis as shown in Table 3.

Table 3. Information on plant materials used for multivariate statistical analysis. Species Collection Site Parts Lable References G. clemensii Sematan, Sarawak Bark GCB Moharam et al., 2010 G. macrophyllus 1 Fraser Hill, Pahang Bark GMB1 Jantan et al., 2005 G. macrophyllus 2 Lawas, Sarawak Bark GMB2 Jantan et al., 2005 G. malayanus Kota Samarahan, Sarawak Bark GLB Jantan et al., 2005 G. ridleyii 1 (Fresh) Gua Musang, Kelantan Bark GRB1 Jusoh et al., 2015 G. ridleyii 2 (Dry) Gua Musang, Kelantan Bark GRB2 Jusoh et al., 2015 G. tapis 1 Lawas, Sarawak Bark GTB1 Moharam et al., 2010 G. tapis 2 Lawas, Sarawak Bark GTB2 Ahmad et al., 2010 G. tapisoides 1 Sematan, Sarawak Bark GOB1 Moharam et al., 2010 G. tapisoides 2 Lawas, Sarawak Bark GOB2 Ahmad et al., 2010 G. uvaroides Merapok, Sarawak Bark GUB Jantan et al., 2005 G. velutinus Sematan, Sarawak Bark GVB Moharam et al., 2010 G. woodii Sematan, Sarawak Bark GWB Moharam et al., 2010 G. andersonii Kota Samarahan, Sarawak Leaf GAL Jantan et al., 2005 G. malayanus 1 Kota Samarahan, Sarawak Leaf GLL1 Jantan et al., 2005 G. malayanus 2 Kota Samarahan, Sarawak Leaf GLL2 Jantan et al., 2005 G. tapis 1 Lawas, Sarawak Leaf GTL1 Moharam et al., 2010 G. tapis 2 Lawas, Sarawak Leaf GTL2 Ahmad et al., 2010 G. tapisoides 1 Sematan, Sarawak Leaf GOL1 Moharam et al., 2010 G. tapisoides 2 Sematan, Sarawak Leaf GOL2 Ahmad et al., 2010 G. uvarioides Merapok, Sarawak Leaf GUL Jantan et al., 2005

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G. macrophyllus Pasoh Negeri Sembilan Root GMR Ghani et al., 2010 G. malayanus 1 Kota Samarahan, Sarawak Root GLR1 Jantan et al., 2005 G. malayanus 2 Kota Samarahan, Sarawak Root GLR2 Jantan et al., 2005 G. tapis Lawas, Sarawak Root GTR Ahmad et al., 2010 G. tapisoides 1 Sematan, Sarawak Root GOR1 Moharam et al., 2010 G. tapisoides 2 Sematan, Sarawak Root GOR2 Ahmad et al., 2010 G. uvaroides Merapok, Sarawak Root GUR Jantan et al., 2005

Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used to characterize their essential oils components. The components common to all essential oils were used to determine the similarity among species with a CA performed with the software Statistica 7.0. The Unweighted Pair Group Method with Arithmetic Mean (UPGMA) was used to cluster groups based on Euclidean distance. The PCA was carried out with the software Statistica 7.0. PCA was used to reveal interrelationships among the ten species of the genus Goniothalamus based on the essential oil common components of these species (Wickramagamage, 2010; Shaharudin et al., 2013, 2018). The HCA analysis revealed four distinct groups for each leaf bark, leaf, and root oils, based on the Euclidian distance as illustrated in Figure 2, Figure 3 and Figure 4, respectively. For Goniothalamus bark oils, the first group, Cluster I consisted of G. clemensii, G. velutinus and G. woodii. Cluster II included G. macrophyllus (1 and 2) and G. tapisoides (1 and 2). Meanwhile, Cluster III consisted of G. malayanus and G. uvaroides, whereas Cluster IV contained G. ridleyi (1 and 2) and G. tapis (1 and 2). For Goniothalamus leaf oil, the first group, Cluster I consisted of G. andersonii and G. uvarioides. Cluster II included G. malayanus (1 and 2), whereas Cluster III consisted of G. tapis (1 and 2). In addition, Cluster IV consisted of G. tapisoides (1 and 2). For Goniothalamus root oils, the first group, Cluster I consisted of G macrophyllus, while Cluster II comprised G. tapisoides (1 and 2) and G. uvaroides. Meanwhile, Cluster III consisted of G. malayanus (1 and 2), whereas Cluster IV contained G. tapis. Furthermore, to evaluate the accuracy of this classification, the cluster obtained was confirmed by PCA analysis. Similarly, the bark, leaf and roots oils of Goniothalamus were divided into four groups each, Cluster I-IV. The results were obtained by PCA based on forty-one (bark oil), thirty-four (leaf oil), and thirty-one (root oil) chemical components. Three factors explained 51.50% (bark oil), 59.76% (leaf oil), and 65.99% (root oil) of accumulated variation of the data analysed. The first three are considered the most important as they represent ≥50% of the accumulated variation. Furthermore, these results also may be correlated with other factors involving a genetic determination that could also be modulated by biotic pressures, volatile constituents during flowering influenced by pollinators, and during the vegetative phase by pathogens and herbivores, or differences in environmental conditions (Silva et al., 2007). Thus, the variation pattern in essential oil composition may reflect selective pressures in different ecological and geographical environments (ecotypes).

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 1-12 Shakri et al. DOI: 10.37929/nveo.786172

Figure 2. PCA and UPGMA analyses of the composition of Malaysian Goniothalamus bark oils

Variables (axes F1 and F2: 51.50 %) Dendrogram 1 Complete Linkage, Correlation Coefficient Distance GWBGVBGCB 0,75 33.75 0,5 GUB 0,25 GLB GRB2GRB1

0 55.83 y

GMB2 GTB1 t

GTB2 i

r

a l

-0,25 i F2 (21.55 %) (21.55 F2

GMB1 m i -0,5 S Cluster 1 Cluster 2 Cluster 3 Cluster 4 GOB1 77.92 -0,75 GOB2 -1 -1 -0,75 -0,5 -0,25 0 0,25 0,5 0,75 1 100.00 F1 (29.95 %) GCB GVB GWB GMB1 GMB2 GOB1 GOB2 GLB GUB GRB1 GRB2 GTB1 GTB2 Active variables Variables

Figure 3. PCA and UPGMA analyses of the composition of Malaysian Goniothalamus leaf oils

Variables (axes F1 and F2: 59.76 %) Dendrogram 1 Complete Linkage, Correlation Coefficient Distance GLL1 0,75 GLL2 GOL2 GOL1 27.26 0,5 0,25

0 51.51

y t

GTL1 i r

GTL2 a l

-0,25 i

F2 (21.73 %) (21.73 F2

m

i S -0,5 GUL GAL Cluster 1 Cluster 2 Cluster 3 Cluster 4 75.75 -0,75 -1 -1 -0,75 -0,5 -0,25 0 0,25 0,5 0,75 1 100.00 F1 (38.03 %) GAL GUL GLL1 GLL2 GTL1 GTL2 GOL1 GOL2 Active variables Variables

Figure 4. PCA and UPGMA analyses of the composition of Malaysian Goniothalamus root oils

Variables (axes F1 and F2: 65.99 %) Dendrogram 1 Complete Linkage, Correlation Coefficient Distance GLR2 0,75 GLR1 GOR2 33.05 0,5 GOR1 GUR 0,25

0 55.37

y

t

i

r

a l

-0,25 i F2 (24.68 %) (24.68 F2

GTR m Cluster 1 Cluster 2 Cluster 3 Cluster 4 i -0,5 S GMR 77.68 -0,75 -1 -1 -0,75 -0,5 -0,25 0 0,25 0,5 0,75 1 100.00 F1 (41.31 %) GMR GOR1 GOR2 GUR GLR1 GLR2 GTR Active variables Variables

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Conclusion In conclusion, our study reports a review of Malaysian Goniothalamus essential oils and their chemical variability. This information is critical when selecting species with economic potential for the pharmaceutical and cosmetics industry. In addition, the multivariate data analysis may be used as quality control tools for the identification and characterization of essential oils from different Goniothalamus species that are to be utilized as raw materials in traditional herbal products. Further studies need to be carried out to determine fingerprints and chemical compositions of other Goniothalamus species and those collected from different origins.

ACKNOWLEDGMENT

The authors would like to thank the Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris for research facilities.

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare. REFERENCES

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RESEARCH ARTICLE

Essential oil composition of Strychnos axillaris Colebr. (Loganiaceae)

Wan Mohd Nuzul Hakimi Wan Salleh1,*, Shamsul Khamis2, Hakimi Kassim3 and Alene Tawang3

1Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris (UPSI), 35900 Tanjung Malim, Perak, MALAYSIA 2School of Environmental and Natural Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, MALAYSIA 3Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris (UPSI), 35900 Tanjung Malim, Perak, MALAYSIA *Corresponding author. Email: [email protected] Submitted: 07.09.2020; Accepted: 27.10.2020

Abstract The essential oil composition from the leaves of Strychnos axillaris Colebr. (Loganiaceae) growing in Malaysia was examined for the first time. The essential oil was obtained by hydrodistillation and fully characterized by gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). In total, 15 components were identified in the essential oil, which made up 87.2% of the total oil. The essential is composed mainly of α-ionone (20.2%), β-ionone (19.5%), bicyclogermacrene (19.2%), and geranyl acetate (10.2%).

Keywords: Loganiaceae, Strychnos axillaris, essential oil, ionone, GC-MS

Introduction The Loganiaceae is a pantropical angiosperm family in the order Gentianales consisting of 16 genera and approximately 460 species, distributed in all tropical areas of the world, as well as reaching some subtropical areas (Francis and Suseem, 2016). The genus Strychnos, the largest genus of the family Loganiaceae, as tropical woody plants, consists of about 394 species. Meanwhile, 25 species occur in the Malaysian region. The flowers are small and usually white or creamy white in colour (Bisset, 1974). Several are important sources of drugs or poisons: strychnine, from the seeds of S. nux-vomica and other species; and curare, from the bark of S. toxifera and other species (Bisset and Phillipson, 1976). Strychnos was already being used medicinally in China in the 14th Century. It has been prescribed as a stomachic, febrifuge, vermifuge, anticholeric and tonic and to treat sores, wounds, eczema and snake bites in Indonesia and the Philippines. In India, the seeds have been used to obstinate vomiting, to treat cholera, diarrhoea, asthma, dropsy, rheumatism, paralytic and neuralgic affections. In Australia, the fruit pulp of S. lucida has been used to treat a variety of skin complaints. S. wallichiana is used in Vietnam to treat rabies, leprosy, and as an aphrodisiac (Guo et al., 2018; Patel et al., 2017). S. axillaris is commonly known as gajah tarik in Malaysia. It grows naturally in woodlands, mixed forests, deciduous woodlands and lowlands. It has been used in Peninsular Malaysia in the preparation of arrow poison; its leaves have been used in India as a suppurative and the seeds internally as a febrifuge (Valadeau, 2010). The chemical analyses of S. axillaris revealed the presence of phenolic and iridoid glucosides (Itoh et al., 2008). Meanwhile, S. axillaris has not been previously studied for its essential oil. As a continuation part of our systematic evaluation of the aromatic flora of Malaysia (Salleh et al., 2014a, 2014b, 2014c, 2015, 2016a, 2016b, 2016c), we here report the volatile components of S. axillaris leaves.

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Materials and Methods Plant material Sample of Strychnos axillaris was collected from Gambang, Pahang in September 2019, and identified by Dr. Shamsul Khamis from Universiti Kebangsaan Malaysia (UKM). The voucher specimen (SK22/18) was deposited at UKMB Herbarium, Faculty of Science and Technology UKM. Isolation and analysis of essential oil The fresh leaf (340 g) was subjected to hydrodistillation in Clevenger-type apparatus for 4 hours. The essential oil obtained was dried over anhydrous magnesium sulfate and stored at 4-6°C. Gas chromatography (GC-FID) analysis was performed on an Agilent Technologies 7890B equipped with HP- 5MS capillary column (30 m long, 0.25 μm thickness and 0.25 mm inner diameter). Helium was used as a carrier gas at a flow rate of 0.7 mL/min. Injector and detector temperatures were set at 250 and 280°C, respectively. The oven temperature was kept at 50°C, then gradually raised to 280°C at 5°C/min and finally held isothermally for 15 min. Diluted samples (1/100 in diethyl ether, v/v) of 1.0 μL were injected manually (split ratio 50:1). The injection was repeated three times and the peak area percent were reported as means ±SD of triplicates. Gas chromatography-mass spectrometry (GC-MS) analysis was recorded using a Hewlett Packard Model 5890A gas chromatography and a Hewlett Packard Model 5989A mass spectrometer. The GC was equipped with an HP-5 column. Helium was used as carrier gas at a flow rate of 1 mL/min. The injector temperature was 250°C. The oven temperature was programmed from 50°C (5 min hold) to 280°C at 10°C/min and finally held isothermally for 15 min. For GC-MS detection, an electron ionization system, with ionization energy of 70 eV was used. A scan rate of 0.5 s (cycle time: 0.2 s) was applied, covering a mass range from 50-400 amu. Identification of components For identification of essential oil components, co-injection with the standards (major components) were used, together with correspondence of retention indices and mass spectra with respect to those reported in Adams (2007). Semi-quantification of essential oil components was made by peak area normalization considering the same response factor for all volatile components. Percentage values were the mean of three chromatographic analyses. Results and Discussion The list of chemical components identified in the essential oil is shown in Table 1. The essential oil yielded 0.17% calculated from the fresh weight of the leaves. The GC-FID (Figure 1) and GC-MS analysis of the essential oil revealed the presence of 15 chemical components with the composition of 87.2%. The main fractions in the essential oil were sesquiterpene hydrocarbons (58.9%), followed by oxygenated monoterpenes (19.5%). The most abundant components of the essential oil were α-ionone (20.2%), β-ionone (19.5%), bicyclogermacrene (19.2%), and geranyl acetate (10.2%). The other minor components detected in the essential oil in more than 2% were nerol (2.5%), α-terpineol (2.4%), (E)-nerolidol (2.4%), linalool (2.2%), geraniol (2.2%) and eugenol (2.0%). On the other hand, monoterpene hydrocarbons and oxygenated sesquiterpenes made up a minor fraction in the oil which constituted 1.3% and 2.4% of the total oil, respectively. The genus Strychnos is still poorly explored as far as its essential oil composition is concerned. A review of the existing literature on essential oils of the genus Strychnos revealed the presence of few studies reporting from S. spinosa (Hoet et al., 2006) and S. cocculoides (Shoko et al., 2013). In comparison to

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both studies, the leaf oil of S. spinosa was reported to show high amounts of palmitic acid (34.3%), whereas the fruit oil of S. cocculoides gave isobutyl acetate (53.2%) as the major component. However, these two components were not detected in the essential oil studied. The differences of the essential oil composition could be due to the different environmental and genetic factors, chemotypes and nutritional status of the plants, which may influence the oil composition (Salleh et al., 2016a).

Table 1. Chemical composition of Strychnos axillaris essential oil No. RRIa RRIb Components Percentagec Identificationsd 1 1014 1015 α-Terpinene 0.5 ± 0.1 RI, MS 2 1025 1029 Limonene 0.8 ± 0.1 RI, MS 3 1095 1097 Linalool 2.2 ± 0.2 RI, MS 4 1185 1189 α-Terpineol 2.4 ± 0.2 RI, MS 5 1240 1245 Nerol 2.5 ± 0.1 RI, MS 6 1271 1270 Geraniol 2.2 ± 0.2 RI, MS 7 1374 1375 Eugenol 2.0 ± 0.1 RI, MS 8 1435 1435 α-Ionone 20.2 ± 0.1 RI, MS, Std 9 1497 1495 β-Ionone 19.5 ± 0.1 RI, MS, Std 10 1529 1530 Bicyclogermacrene 19.2 ± 0.2 RI, MS, Std 11 1563 1565 (E)-Nerolidol 2.4 ± 0.2 RI, MS 12 1700 1700 Heptadecane 0.5 ± 0.1 RI, MS 13 1720 1722 Dodecanal 1.1 ± 0.1 RI, MS 14 1758 1755 Geranyl acetate 10.2 ± 0.1 RI, MS 15 1945 1940 Phytol 1.5 ± 0.2 RI, MS Phenylpropanoid 2.0 ± 0.1 Monoterpene hydrocarbons 1.3 ± 0.1 Oxygenated monoterpenes 19.5 ± 0.2 Sesquiterpene hydrocarbons 58.9 ± 0.2 Oxygenated sesquiterpenes 2.4 ± 0.1 Others 3.1 ± 0.1 Total identified 87.2 ± 0.1 a b Linear retention index, experimentally determined using homologous series of C6-C30 alkanes. Linear retention index taken from Adams (2007). cRelative percentage values are means of three determinations ±SD. dIdentification methods: Std, based on comparison with authentic compounds; MS, based on comparison with Wiley, Adams, FFNSC2, and NIST08 MS databases; RI, based on comparison of calculated RI with those reported in Adams, FFNSC2 and NIST08.

Figure 1. GC chromatogram of Strychnos axillaris essential oil

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The presence of α- and β-ionone in the oil could be used as a chemotaxonomic marker for this species. These compounds are extensively used as ingredients and building blocks in flavors and fragrances, as well as in the pharmaceutical industry (Lalko et al., 2007). Ionones and their derivatives are also known to possess important pharmacological properties, such as antileishmanial, anti-inflammatory, and antimicrobial activities (dos Santos Costa et al., 2007). In addition, recent studies have demonstrated the great potential of ionone derivatives as anticancer agents (Ansari and Emami, 2016). In conclusion, this is the first report of the chemical composition of the essential oil from Strychnos axillaris and the results of this study could contribute to the valorisation of this Malaysian aromatic and medicinal plant.

ACKNOWLEDGMENT

The authors would like to thank the Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris for research facilities.

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare. REFERENCES

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 13-17 Salleh et al. DOI: 10.37929/nveo.791343

Salleh, W. M. N. H. W., Ahmad, F., & Khong, H. Y. (2014b). Chemical composition of Piper stylosum Miq. and Piper ribesioides Wall. essential oils and their antioxidant, antimicrobial and tyrosinase inhibition activities. Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromaticas, 13(5), 488-497.

Salleh, W. M. N. H. W., Ahmad, F., & Khong, H. Y. (2014c). Antioxidant and anti-tyrosinase activities from Piper officinarum C.DC (Piperaceae). Journal of Applied Pharmaceutical Sciences, 4(5), 87-91.

Salleh, W. M. N. H. W., Kamil, F., Ahmad, F., & Sirat, H. M. (2015). Antioxidant and anti-inflammatory activities of essential oil and extracts of Piper miniatum. Natural Product Communications, 10(11), 2005-2008.

Salleh, W. M. N. H. W., & Ahmad, F. (2016a). Antioxidant and anti-inflammatory activities of essential oils of Actinodaphne macrophylla and A. pruinosa (Lauraceae). Natural Product Communications, 11(6), 853-855.

Salleh, W. M. N. H. W., Ahmad, F., Khong, H. Y., & Zulkifli, R. M. (2016b). Essential oil composition of Malaysian Lauraceae: A mini review. Pharmaceutical Sciences, 22(1), 60-67.

Salleh, W. M. N. H. W., Ahmad, F., Khong, H. Y., Zulkifli, R. M., Sarker, S. D. (2016c). Madangones A and B: Two new neolignans from the stem bark of Beilschmiedia madang and their bioactivities. Phytochemistry Letters, 15, 168-173.

Shoko, T., Apostolides, Z., Monjerezi, M., Saka, J. D. K. (2013). Volatile constituents of fruit pulp of Strychnos cocculoides (Baker) growing in Malawi using solid phase microextraction. South African Journal of Botany, 84, 11-12.

Valadeau, C., Castillo, J. A., Sauvain, M., Lores, A. F., Bourdy, G. (2010). The rainbow hurts my skin: Medicinal concepts and plants uses among the Yanesha (Amuesha), an Amazonian Peruvian ethnic group. Journal of Ethnopharmacology, 127, 175-192.

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 18-21 Karadağ et al. DOI: 10.37929/nveo.825335

RESEARCH ARTICLE

Volatile components and antimicrobial activity of the n-hexane extracts of Neomuretia pisidica (Kit Tan) Kljuykov, Degtjareva & Zakharova

Ayşe Esra Karadağ1,2,*, Betül Demirci3, Ömer Çeçen4, Ayvaz Ünal5 and Fatma Tosun1

1Department of Pharmacognosy, School of Pharmacy, Istanbul Medipol University, 34810, Istanbul, TURKEY 2Depatment of Pharmacognosy, Graduate School of Health Sciences, Anadolu University, Eskişehir , TURKEY 3Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470, Eskişehir, TURKEY 4Department of Plant and Animal Production, Medical and Aromatic Plants Programme, Ermenek Vocational High School, Karamanoğlu Mehmetbey University, 70400, Ermenek, Karaman, TURKEY 5Department of Science Education, Faculty of Ahmet Keleşoğlu Education, Necmettin Erbakan University, 42090, Konya, TURKEY

*Corresponding author. Email: [email protected] Submitted: 13.11.2020; Accepted: 06.01.2021

Abstract

The fruits, aerial parts and roots of Neomuretia pisidica (Kit Tan) Kljuykov, Degtjareva & Zakharova were extracted with n-hexane. Total of 18 compounds were characterised by GC analyses of the n-hexane extracts. Main volatile components of the n-hexane extract of aerial parts were characterized as 1,8-cineole (23.4%), camphor (21.4%), 2-ethyl hexanol (14.6%), α-pinene (7.2%), and verbenone (6.4%). Methyl linoleate (19.3%), 1,8-cineole (16.5%), camphor (13.2 %), α-pinene (6.1 %) and 2-ethyl hexanol (4.9%) were found in the n-hexane extract of roots. Whereas, 1,8-cineole (23.3 %), camphor (20.3%), 2-ethyl hexanol (14.2 %), α-pinene (9.9%), and limonene (4.1%) were the major components of the n-hexane extract of fruits. Antimicrobial activity were identified using a microdilution assay against selected human pathogenic strains. The most potent inhibitor activities with 156 µg/mL concentrations were detected against S. aureus and E. faecalis.

Keywords: Neomuretia pisidica, Apiaceae, volatile compounds, antimicrobial activity

Introduction Neomuretia (Apiaceae) is a new genus of geophytic plants that represented by two species distributed in the Mediterranean region of Turkey and Northern Iraq. Neomuretia pisidica (Kit Tan) Kljuykov, Degtjareva & Zakharova (syn. Hellenocarum pisidicum) is an endemic species growing in the Karaman province of Turkey (Zakharova et al., 2016). Apiaceae species are among the richest in essential oils (Baser&Kirimer, 2014; Oroojelian et al., 2010; Sarebkhar&Iranshahi, 2010; Tabanca et al., 2006). Akalın et al., 2009 and Kljuykov et al., 2020 published recent botanical reviews on the family. According to our interviews with local people, basal leaves of this species are used as food and for the treatment of toothaches. The current study was aimed to investigate the volatile components and antimicrobial activity of the n- hexane extracts of N. pisidica. Materials and Methods Plant material The roots, aerial parts, and fruits of N. pisidica were collected from the northern slopes of Göksu river valley near Akçaalan village, Karaman Province in 2017, and identified by one of us (ÖÇ). Voucher specimens were deposited at the GAZI Herbarium (Herbarium No: 2986).

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 18-21 Karadağ et al. DOI: 10.37929/nveo.825335

Extraction of plant materials The air-dried plant materials (fruit, root, and aerial parts; each 200 g) were separately powdered and extracted with n-hexane (3x 200 mL) at room temperature and filtered. The n-hexane was removed in a rotary evaporator in vacuo. GC-MS analysis The GC-MS analysis was carried out using an Agilent 5975 GC-MSD system. The analysis conditions were as described in our previous publication (Karaca et.al., 2020). GC analysis The analyzes were carried out as described in previous publications (Karaca et.al., 2020). GC analysis results are given in Table 1. Computer matching against commercial (Wiley GC/MS Library, MassFinder Software 4.0) (1,2) and in-house “Başer Library of Essential Oil Constituents” built up by genuine compounds and components of known oils. Antibacterial activity The antibacterial activity was studied using broth microdilution assay following the methods described by the CLSI, Clinical and Laboratory Standards Institute Standards (CLSI, 2006). The potential minimum inhibitory concentrations (MIC) were calculated against the selected human pathogenic; Pseudomonas aeruginosa ATCC 10145, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 6538, and Escherichia coli NRLL B-3008. The activity was studied as described in previous publications (Karadağ et.al., 2019). The antibacterial evaluations were in triplicates and reported as mean in Table 2.

Results and Discussion The volatile constituents of the n-hexane extracts of N. pisidica fruits, roots, and aerial parts were analyzed using GC-FID and GC-MS which led to the identification of eighteen compounds. The main components of the n-hexane extract of the aerial parts were characterized as 1,8-cineole (23.4%), camphor (21.4%), 2-ethyl hexanol (14.6%), α-pinene (7.2%), and verbenone (6.4%). Methyl linoleate (19.3%), 1,8-cineole (16.5%), camphor (13.2%), α-pinene (6.1%) and 2-ethyl hexanol (4.9%) were identified in the n-hexane extract of roots. Whereas, 1,8-cineole (23.3%), camphor (20.3%), 2-ethyl hexanol (14.2%), α-pinene (9.9%), and limonene (4.1%) were the major components of the n-hexane extract of fruits.

Table 1.The Volatile Composition of Neomuretia pisidica n-hexane extracts

RRI Compounds Aerial part % Fruit % Root % 1032 -Pinene 7.2 9.9 6.1 1076 Camphene 1.8 3.2 1.6 1093 Hexanal - 1.3 - 1174 Myrcene 1.5 2.5 1.0 1194 Heptanal - 1.3 - 1203 Limonene 3.1 4.1 2.3 1213 1,8-Cineole 23.4 23.3 16.5 1280 p-Cymene 4.5 2.7 1.5 1496 2-Ethyl hexanol 14.6 14.2 4.9 1532 Camphor 21.4 20.3 13.2

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 18-21 Karadağ et al. DOI: 10.37929/nveo.825335

1536 Pinocamphone 3.2 3.0 1.6 1553 Linalool 3.5 2.5 1.1 1706 -Terpineol 1.3 0.8 0.5 1719 Borneol 5.0 2.8 2.2 1725 Verbenone 6.4 3.8 2.8 2242 Methyl hexadecanoate - - 4.3 2509 Methyl linoleate - - 19.3 2583 Methyl linolenate - - 4.1 Total 96.9 95.7 83.0 RRI: Relative retention indices calculated against n-alkanes. %: Calculated from FID data

Table 2. Antimicrobial activities of the n-hexane extracts of N. pisidica (MICs in mg/mL)

Sample P. aeruginosa S. aureus E. coli E. faecalis

Fruit extract 2.5 2.5 0.625 0.156

Root extract 2.5 0.156 0.625 0.156

Aerial part extract 1.25 0.156 1.25 0.312 Tetracycline 16 0.25 >16 0.025

Antimicrobial activities of the n-hexane extracts of N. pisidica against bacterial strains were listed, in Table 2. The results revealed that the tested extracts are effective on S. aureus and E. faecalis at between 312-156 µg/mL concentration. In previous studies, the antimicrobial activities of 1,8-cineole (Hendry et al., 2009; Kifer et al., 2016; Vuuren et al., 2007) and camphor (Jirovetz et al., 2005) are studied and demonstrated that camphor and 1,8-cineole have remarkable antimicrobial capacity. Based on this, it can be thought that the antimicrobial effect of N. pisidica essential oils is caused by camphor and 1,8-cineole. Furthermore, the antinociceptive and antiinflammatory activities of 1,8-cineole, camphor and essential oils that contain large proportional amounts of 1,8-cineole and camphor were proven (Lenardão et al., 2016; Chandrakanthan et al., 2020; Santos et al., 2000; Barkin, 2013). Thus, antinociceptive and antiinflammatory activities may explain the folkloric usage of N. piscidica for toothache. Essential oils are known for their antimicrobial effects, and the different volatile components they contain may be responsible for this effect. In studies conducted with n-hexane extracts rich in volatile components, it has the potential of antibacterial effect as much as essential oils.

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare. REFERENCES

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Barkin, R. L. (2013). The pharmacology of topical analgesics. Postgraduate Medicine, 125(sup1), 7-18.

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Jirovetz, L., Buchbauer, G., Denkova, Z., Stoyanova, A., Murgov, I., Schmidt, E., & Geissler, M. (2005). Antimicrobial testinas and gas chromatoaraphic analysis of pure oxyaenated monoterpenes 1.8-cineole, α-terpineol, terpinen-4-ol and camphor as well as target comoounds in essential oils of pine (Pinus pinaster), rosemary (Rosmarinus officinalis), tea tree (Melaleuca alternifolia). Scientia Pharmaceutica, 73(1), 27-39.

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Karadağ, A. E., Demirci, B., Çaşkurlu, A., Demirci, F., Okur, M. E., Orak, D., Başer, K. H. C. (2019). In vitro antibacterial, antioxidant, anti-inflammatory and analgesic evaluation of Rosmarinus officinalis L. flower extract fractions. South African Journal of Botany, 125, 214-220.

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Kljuykov, E. V., Petrova, S. E., Degtjareva, G. V., Zakharova, E. A., Samigullin, T. H., & Tilney, P. M. (2020). A taxonomic survey of monocotylar Apiaceae and the implications of their morphological diversity for their systematics and evolution. Botanical Journal of the Linnean Society, 192(3), 449-473.

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Oroojalian, F., Kasra-Kermanshahi, R., Azizi, M., & Bassami, M. R. (2010). Phytochemical composition of the essential oils from three Apiaceae species and their antibacterial effects on food-borne pathogens. Food chemistry, 120(3), 765- 770.

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 22-28 Sarri et al. DOI: 10.37929/nveo.827601

RESEARCH ARTICLE Chemical composition of essential oil from the aerial parts of Santolina rosmarinifolia L. a wild Algerian medicinal plant Djamel Sarri1,*, Noui Hendel2, Hadjer Fodil1,3, Giuseppe Ruberto4 and Madani Sarri1,5,*

1Department of Nature and Life Sciences, Faculty of Sciences, University of M’sila, 28000, M’sila, ALGERIA 2Department of Microbiology and Biochemistry, Faculty of Sciences, University of M’sila, 28000, M’sila, ALGERIA 3Laboratory of Biology, Water and Environment (LBWE), Faculty of Nature and Life Sciences and Earth and Universe Sciences, University of May 8, 1945, 24000, Guelma, ALGERIA 4Istituto di Chimica Biomolecolare del Consiglio Nazionale delle Ricerche (ICB-CNR), Via Paolo Gaifami, 18, 95126 Catania, ITALY 5Laboratory of Phytotherapy Applied to Chronic Diseases (LPACD), Faculty of Nature and Life Sciences, University of Setif1, 19000, Setif, ALGERIA

*Corresponding authors. Email: [email protected]; [email protected] Submitted: 21.11.2020; Accepted: 11.01.2021

Abstract The analysed essential oil in this study was obtained by hydrodistillation from the aerial parts of Santolina rosmarinifolia L. (Asteraceae) collected from Hodna area of Algeria. This species is a medicinal herb traditionally used in Algeria. Its essential oil has been analyzed by combining GC-FID and GC-MS. The analysis led to the identification of eighty-two components, representing 91.84% of the whole composition of the sample. The main components were capillene (32.8%), 1,8-cineole (15.1%) and β-myrcene (14.0%).

Keywords: Santolina rosmarinifolia L., Asteraceae, Essential oil, GC-MS Analysis, Algeria

Introduction The Santolina L. is a genus belonging to the Asteraceae family (formerly known as the family Compositae, tribe Anthemideae). It is characterized by over ten widely distributed species in the Mediterranean region (Derbesy et al., 1989). Rare in Algeria, the genus Santolina is represented by only one species, Santolina rosmarinifolia L., called rosemary leaves; a polymorphous plant colonizing forests, pastures and mountainous regions. It is a sub-bushy shrub with closely linear leaves, all tubular flowers, hermaphrodite (peripheral anther sometimes sterile). Inflorescence in dense corymbs, monocle branches and woody stems (Quezel & Santa, 1963). Known in Algeria as Qeissoum and Jaeda (Quezel & Santa, 1963; Baba Aissa, 1999). Some species of the genus Santolina such as S. chamaecyparissus L., S. etrusca (Lacaita) Marchi & D'Amato, S. insularis (Gennari ex Fiori) Arrigoni, S. neapolitana Jord. & Fourr. and S. oblongifoliaBoiss. have been reported in traditional medicine in Spain, Italy, Mediterranean area and India, (Tundis & Loizzo, 2018). In the Algerian traditional pharmacopoeia, S. rosmarinifoliaL. is used in the treatment of dermatoses in the form of a decoction (Beniston & Beniston, 1984). Also, recommended in the form of flower bouquets arranged in cabinets to protect the linen against moths (Baba Aissa, 1999). These aerial parts are indeed used as vermifuge, stomachic, antispasmodic (Beloued, 2005) and widely pre-registered traditionally as a healing plant (Boudjelal et al., 2013; Sarri et al., 2014). Several Santolina species have been studied for their antimicrobial, antifungal, antiviral, and anti-inflammatory activities (Tundis & Loizzo, 2018).The infusion from S. rosmarinifolia fresh or dried flower heads was reported to have antipyretic, antihypertensive, hepatoprotective and intestinal anti-inflammatory properties (Ioannou et al., 2007). This study reports the essential oil composition of S. rosmarinifolia from Hammam Dalaa (M’sila Provence) with a distinct chemical profile for the first time.

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Materials and Methods Plant material The aerial parts of Santolina rosmarinifolia L. (Asteraceae) were collected in Hammam Dalaa (M’sila Provence) in May 2017. Avoucher specimen (SD2832/17) was deposited at the Herbarium of the Department of Nature and Life Sciences, Mohamed Boudiaf University (M’sila, Algeria). Essential oil extraction Washed aerial parts of the S. rosmarinifolia were dried at room temperature in a shady place and then ground to a powder form. The essential oil of the aerial parts of the S. rosmarinifolia (300 g) was obtained by hydrodistillation in Clevenger-type apparatus. This operation was carried out for 3 hours. The oil yield was 0.15%, as estimated on the dry weight basis (v/w). Analysis of the essential oil On a Hewlett-Packard gas-chromatograph model 5890, fitted with a flame ionization detector, gas chromatographic (GC) analysis was performed (FID). Analyses of GC-FID were carried out under the following analytical conditions: ZB-5 capillary column (30 m x 0.25 mm i.d. x 0.25 m film thickness); helium as carrier gas; split mode injection (1:50); 250 and 280°C as injector and detector temperatures, respectively. A programmed oven temperature (40°C to 300 at 2°C/min) was applied. On the same gas chromatograph, the gas chromatography-mass spectrometry (GC-MS) was performed in connection with a Hewlett-Packard mass spectrometer (model 5971A), 70eV ionization energy, 180°C ion source temperature, mass spectra data were collected over the scan mode in m/z range 40-400. The individual volatile constituents were identified by comparison of their identical retention indices with those of the compounds known from literature data (Adams, 2007). Furthermore, the chemical identification was confirmed by computer matching of spectral MS data with those stored in the Wiley 275 library and the comparison of the fragmentation patterns with those reported in literature. Results and Discussion Chemical composition of the essential oil Table 1 summarizes the composition and percentage of the essential oil compounds. They have been grouped according to the following classes of components: monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpenes and others, the last class comprises only the non-terpenoid compounds characterized in the essential oil. On the whole, eighty-two compounds were found, accounting for 91.84% of the total oil. The most plentiful class in the essential oil of the Algerian Santolina was the ‘others’ amounting to ca. 35% with 17 components, oxygenated monoterpenes was the second class with ca. 22% and 24 components, the following class was monoterpene hydrocarbons with 13 components and an amount of ca. 21%, sesquiterpenes was the last class with the lowest amount (ca. 13%) but with the highest number of components (28). The main constituents were identified as capillene (32.79%), 1,8-cineole (15.08%) and β- myrcene (13.98%). Species of the genus Santolina, particularly S. rosmarinifolia and its subspecies, clearly exhibit remarkable variability in terms of essential oil chemical composition (Table 2). This variability is very clear while comparing the major compounds of S. rosmarinifolia harvested in a steppe biotope (region of Hodna) which is the subject of this study to that harvested in another forest biotope (Aures region). In this context,

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 22-28 Sarri et al. DOI: 10.37929/nveo.827601

Mehalaine & Chenchouni (2018) showed that certain climatic factors in regions with a semiarid climate had an effect on the quality and composition of the essential oil. This last sample, in fact, showed the presence of two components not found in our sample, namely tricosane (10.6%) and pentacosane (6.7%); a further significant difference was the consistent presence of germacrene-D and α-pinene (30.2 and 10.1%, respectively), together with the absence of capillene (Chibani et al., 2013). A greater similarity with compositional data of our sample has been reported for a sample of S. rosmarinifolia ssp. rosmanifolia (Palà- Paùl et al., 1991, 2001). The phytochemical analysis of S. rosmarinifolia collected in Portugal showed that almost all the main compounds found in our sample were present but in lower amount (Ioannou et al., 2007). The flowers oils of S. rosmarinifolia collected in Spain presented some similarities when compared with our sample, which in turn did not show the presence of ar-curcumene and β-eudesmol (Pérez-Alonso & Velasco- Negueruela, 1988). Finally, some other reports on different species of Santolina showed the presence of some components such as 1,8-cineole (Villar et al., 1986; Giner et al., 1933; Cherchi et al., 2001; Flamini & Cioni, 2007; Tirillini et al., 2007; Grosso et al., 2009), β-myrcene (Poli et al., 1997; Zaiter et al., 2015) and capillene (Malti et al., 2019).

Table 1: Chemical composition of essential oil from aerial parts of Santolina rosmarinifolia L.

#.a Class/Compounds RIb %c

Monoterpene hydrocarbons 20.9 4 α-Thujene 932 0.1 5 α-Pinene 939 1.3 6 Camphene 955 0.3 7 Thuja-2,4(10)-diene 960 t 9 Sabinene 979 0.7 10 β-Pinene 983 3.5 11 β-Myrcene 997 14.0 12 Phellandrene 1009 t 13 α-Terpinene 1021 0.4 14 p-Cymene 1029 0.3 16 β-Z-Ocimene 1049 t 19 γ-Terpinene 1072 0.3 22 Terpinolene 1093 0.1 Oxygenated monoterpenes 22.3 15 1,8-Cineole 1041 15.1 21 cis-Sabinene hydrate 1089 0.2 23 Perillene 1100 0.5 25 Dehydro sabina ketone 1126 0.1 26 α-Campholenal 1130 0.1 28 trans-Pinocarveol 1146 0.5 29 Camphor 1151 1.0 30 Karahanaenone 1164 t 31 Sabina ketone 1166 t 32 cis-Chrysanthenol 1172 2.0 33 Borneol 1175 0.1 34 cis-Pinocamphone 1182 t 35 Terpinen-4-ol 1186 1.5

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36 Thuj-3-en-10-al 1192 0.1 38 α-Terpineol 1199 0.9 39 Myrtenol 1205 0.4 40 trans-Carveol 1227 t 41 cis-Carveol 1237 t 44 Geranial 1278 t 45 p-Menth-1-en-7-al 1283 t 46 cis-Verbenyl acetate 1286 t 47 γ-Terpinen-7-al 1287 t 48 Perilla alcohol 1306 0.1 49 Myrtenyl acetate 1334 t Sesquiterpenes 13.0 51 α-Copaene 1388 t 52 Bourbonene 1390 t 53 Z-Jasmone 1395 0.1 55 α-Gurjunene 1408 t 56 β-Caryophyllene 1422 t 57 β-Copaene 1431 0.1 58 trans-α-Bergamotene 1445 0.1 59 β-Farnesene 1464 t 60 Sesquisabinene 1471 0.1 61 γ-Gurjunene 1484 0.1 62 γ-Muurolene 1488 0.2 63 Curcumene 1489 0.2 64 Germacrene D 1496 t 66 γ-Cadinene 1526 t 67 -Cadinene 1535 0.4 69 Sphatulenol 1595 3.2 70 Globulol 1598 0.2 71 Salvial-4(14)-en-1-one 1608 0.3 72 Carotol 1618 0.2 73 Oplopenone 1623 0.1 74 β-Atlantol 1626 0.3 75 β-Acorenol 1638 t 76 γ-Eudesmol 1641 t 78 3-Thujopsanone 1657 2.1 79 Eudesma-4(15)-7-dien-1-β-ol 1698 0.3 80 5-neo-Cedranol 1703 0.5 81 Eudesma-4,11-dien-2-ol 1706 3.4 82 β-Acorenone 1708 1.7 Others 35.29 1 3-Hepten-1-ol acetate 773 t 2 Hexanal 803 t 3 E-2-hexenal 859 t 8 Benzaldehyde 966 0.4

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 22-28 Sarri et al. DOI: 10.37929/nveo.827601

17 Benzene acetaldehyde 1058 t 18 2-Octen-2-methyl-6-methylened 1063 0.7 20 Acetophenone 1077 t 24 Isopentylisovalerate 1103 t 27 4- acetyl-1-methylcyclohexaned 1135 0.1 37 3,3,5-Trimethyl-1,4-hexadiened 1195 0.1 42 cis-3-Hexenyl isovalerated 1249 0.1 43 p-Ethylacetophenone 1262 t 50 Decanoic acidd 1377 t 54 Methyl eugenol 1402 0.1 65 Capillened 1519 32.8 68 Hexenylbenzoate 1580 0.1 a The numbering refers to elution order on ZB-5 capillary column; b Retention index relative to standard mixture of n-alkanes on ZB- 5 capillary column; c Values (area%) represent averages of three determinations (t = trace, <0.05%); d Tentatively identified by MS data only.

Table 2. Principal volatile constituents (%) in the essential oils of some specimens of S. rosmarinifolia L. and S. rosmarinifolia L. ssp. rosmarinifolia

Origin / Status / Nature Part used Principal components (%) References germacrene-D (30.2), β-myrcene (12.0), tricosane Batna-Aures (Algeria) - Santolina Flowering Chibani et al., (10.6), β-pinene (10.1), sabinene (7.0) and rosmarinifolia L. / Wild aerial parts 2013 pentacosane (6.7). β-eudesmol (13.5), 1,8-cineole (12.9), camphor (8.0), Flower heads borneol (5.1), ar-curcumene (4.8), terpinen-4-ol (4.5), Botanical Gardens of Iasi (Romania) spathulenol (4.4) Ioannou et al., / Santolina rosmarinifolia L. / 2007 Cultivated ar-curcumene (9.6), β-phellandrene (8.1), Leaves spathulenol (7.5), β-pinene (6.0), γ-muurolene (5.8), myrcene (5.2), camphor (5.2). Sabinene (0.3-12), β-pinene (17.0-26.5), myrcene Puente de Madrid (Spain) / (0.3-15.5), β-phellandrene (14.4-27.6), limonene (3.1- Palá-Paúl et al., Santolina rosmarinifolia L. ssp. Aerial parts(*) 5.0), 1,8-cineole (0.9-1.7), artemisia ketone (1.0-2.4), 2001 rosmarinifolia / Wild terpinen-4-ol (0.5-4.0), capillene (t-5.1), ar- curcumene (1.0-2.4), β-eudesmol (0.4-5.0). Puerto de Nava-cerrad a Madrid capillene (35.2), β-phellandrene (14.9), myrcene Palá-Paúl et al., (Spain) / Santolina rosmarinifolia L. Aerial parts (13.1), β-pinene (7.8), sabinene (5.5), ar-curcumène 1999 ssp. rosmarinifolia / Wild (4.3) Pérez-Alonso & El Escorial and Puerto de Galapagar 1,8-cineole (8.9), β-pinène (8.9), myrcene (7.8), Velasco- a Madrid (Spain) / Santolina Flowers sabinene (6.6), ar-curcumene (5.8), β-eudesmol Negueruela, rosmarinifolia L. / Wild (13.4). 1988 (*) Components of the EO (in %) of Santolina rosmarinifolia L. ssp. rosmarinifolia over one year (min-max); t=traces Conclusion This study forms the first report on the chemical composition of the essential oil from Santolina rosmarinifolia L. harvested in a steppe biotope. The obtained results could contribute to the valorisation of this Algerian medicinal plant.

ACKNOWLEDGMENT

The authors are grateful to CNEPRU (D01N01UN280120150001) and we wish to thank Consiglio Nazionale delle Ricerche (C.N.R., Rome) for financial support. The authors would also to thank Mr. A. MAIZA and N. BEZAİ for the technical support.

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CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare. REFERENCES

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Chibani, S., Labed, A., Kabouche, A., Semra, Z., Smati, F., Aburjai, T., Kabouche, Z. (2013). Antibacterial activity and chemical composition of essential oil of Santolina rosmarinifolia L. (Asteraceae) from Algeria. Der Pharmacia Letter, 5(2), 238-241.

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Pérez-Alonso, M.J., Velasco-Negueruela, A. (1988). The essential oils of four Santolina species. Flavour and Fragrance Journal, 3(1), 37-42.

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 29-38 Gontar et al. DOI: 10.37929/nveo.772848

RESEARCH ARTICLE Monarda essential oils as natural cosmetic preservative systems Łukasz Gontar1,2, Anna Herman1,3,*, Ewa Osińska2 1Faculty of Cosmetology, The Academy of Cosmetics and Health Care, Warsaw, POLAND 2Department of Vegetable and Medicinal Plants, Faculty of Horticulture, Biotechnology and Landscape Architecture, Warsaw University of Life Sciences, Warsaw, POLAND 3Faculty of Health Sciences, Warsaw School of Engineering and Health, Bitwy Warszawskiej 1920 18 street, 02-366 Warsaw, POLAND

*Corresponding author. Email: [email protected] Submitted: 30.07.2020; Accepted: 26.01.2021

Abstract

The aim of the study was to compare the antimicrobial activity of essential oils (EOs) obtained from leaves and inflorescences of Monarda media, M. didyma, and M. citriodora found in different phenological stages of development, to examine the inhibition of microbial growth by Monarda EOs in O/W emulsions. Thymol, p-cymene, and carvacrol were the main constituents characterized in Monarda EOs. The strongest antimicrobial activity was obtained from the leaves of Monarda EOs in the flowering phase of the development. M. media EO fulfilled the criterion of the preservative effectiveness test for all evaluated bacteria and fungi, while M. didyma and M. citriodora EOs were not sufficient for Gram-negative bacteria. As an overall result, M. media EO may be an effective candidate as natural cosmetic preservative, and finally create self-preserving system in O/W emulsion, while M. didyma and M. citriodora EOs may only reduce the amount of synthetic preservatives used in O/W emulsions.

Keywords: Monarda citriodora, Monarda didyma, Monarda media, essential oils, preservative system

Introduction The preservatives are added to cosmetic formulations to prevent microbial growth during production process, packing, storage and entire period of use by consumers to ensure their safety. The cosmetic industry recommends the use of combination of various preservatives in the smallest concentrations for the protection of cosmetic products from potential microorganism contaminations. Unfortunately, synthetic preservatives are one of the main factors causing allergies and irritant contact dermatitis to users (Andersen, 1993). Therefore, many cosmetic manufacturers, also increasingly aware consumers draw their attention to cosmetics marked as preservative-free, which are referred as formulations without the well known preservatives listed in Annex VI of the Commission Directive 76/768/EEC, but other cosmetics ingredients with antimicrobial activity. Among the cosmetic ingredients used in self-preserving preparations are essential oils (EOs) and plant extracts (Herman, 2019). Therefore, EOs with antimicrobial activity can reduce the amounts as well as the concentration of synthetic preservatives in cosmetic products, or even completely eliminate the use and finally create a preservative free or self-preserving system. The aim of the present study was to compare the antimicrobial activity of EOs obtained from leaves and inflorescences of M. media L., M. didyma L., and Monarda citriodora L. found in different phenological stages of development, to examine the inhibition of microbial growth by Monarda EOs in O/W emulsions. Materials and Methods Microorganisms Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC8739, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404 were used. The microorganisms were

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activated through double passaging: bacteria on TSA medium (Trypticase Soy Agar; BioMerieux, France) (37°C, 48h), yeast on SDA medium (Sabouraud Dextrose Agar; BioMerieux, France) (25°C, 72h) and mould on SDA medium (BioMerieux, France) (25°C, 7days). Plant collection The fresh herbs of M. citriodora, M. didyma and M. media were harvested from June to September 2016 from an experimental field at the Department of Vegetable and Medicinal Plants, Warsaw University of Life Sciences (Poland) at 3 different stages of development: I – budding stage (shoots with only leaves), II – flowering stage (full bloom stage), III - maturity stage (seed forming stage). The raw materials, leaves and inflorescences (if available), were air-dried at 35°C in the dark. Essential oils extraction The Monarda EOs were isolated according to the European Pharmacopeia 9th edition. 30 g of each air- dried raw materials were distilled with 1 L of distilled water in a 2-liter round bottom glass flask using a Clevenger-type apparatus. The distillation time took about 180 minutes. Obtained Monarda EOs were stored in amber vials at 4°C until chemical and microbiological investigations. Analysis of essential oils The EOs from leaves of Monarda in the flowering phase of the development were analysed by Agilent 7890A/5975C GC-MS system inert XL MSD with Triple-Axis Detector (Agilent-Technologies, Little Falls, CA, USA), equipped with a non-polar capillary column (HP-5MS 5% phenylmethylsiloxane; 30.00 m × 0.25 mm, 0.25 μm film thickness). Oven temperature was kept at 50°C for 5 min initially, and then raised at the rate of 3°C min−1 to 240°C, and then was kept for 1 min at 240°C. Injector temperature was set at 290 °C. Helium was used as carrier gas at a flow rate of 0.8 mL min−1, and 1 μL samples (5% of EO in hexane) were injected automatically by the (7683B Injector) in the split mode (1:20). For MS detection, an electron ionization mode was used with ionization energy of 70 eV and scan range of 35 to 350 m/z. Acquiring and analyses of data were carried out using a built-in data-handling program provided by the manufacturer of the GC/MS (Agilent ChemStation). Quantification by % peak area calculations was performed using the non-polar HP- 5MS column. The identification of the EOs constituents were based on a comparison of their MS spectra with Mass spectra library of NIST 08 and Wiley 8th ed. Mass spectra were confirmed by comparing linear retention indices (LRI) calculated relative to (C8-C20) n-alkanes with LRI database, included in NIST 08 mass spectra library. Determination of antimicrobial activity of Monarda essential oils Several colonies of overnight cultures of individual organisms were suspended in saline to obtain density equal to 0.5 McFarland turbidity standard (approximate cell density of 1.5 × 108 CFU/mL). The antibacterial and antifungal activity was evaluated by using the disc diffusion method. Suspensions of microorganisms were spread over the TSA and SDA agar plates (BioMerieux, France), respectively by using sterile cotton swabs. The sterile paper discs of 6 mm in diameter (BTL, Poland) were impregnated with 10 µL mixture of Monarda EO / 96 % ethanol (1:1) and placed on the agar surface. Tetracycline (30 μg), Erythromycin (15 μg), Miconazole (10 μg) and Nystatin (100 U) (BTL, Poland) were used as controls. All bacterial plates were incubated at 37°C for 24 h and fungal plates at 25°C for 72h. The diameter of the zone of inhibition was measured in mm. For each test, three replicates were performed.

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Preparation of O/W emulsions The composition of formulations is showed in Table 1. Emulsifier Ceteareth-20 (Eumulgin ® B2,CognisPolska Sp. z.o.o.), Isopropyl Myristate (Cognis Polska Sp. z.o.o.), Ethylhexyl stearate (Cetiol® 868, Cognis Polska Sp. z.o.o.), Octyldecanol (Eutanol G®, Cognis Polska Sp. z.o.o.), Cyclopentasiloxane and Dimethicone (Dow Corning®1411 Fluid, Dow Corning Europe S.A.), water, glycerine (POCH S.A, Poland) and preservative - Germaben II (Propylene Glycol (and) Diazolidinyl Urea (and) Methylparaben (and) Propylparaben; Ashland, USA) /essential oil were homogenized for 3 min using Heidolph SilentCrusher M homogenizer (Heidolph Instruments GmbH & Co. KG, Germany) at approximately 15 000 rpm. Then emulsion was gently stirred using blender RW 16 (IKA® Werke GmbH & Co. KG, Germany) and essential oils and the Sodium Polyacrylate (Cosmedia SP, Cognis Polska Sp. z.o.o.) was added stepwise. Stirring continued for an additional 30 min. Monarda EOs from leaves in flowering phase of the development were added at 1.5% concentration. Preservative was added at 1.0 % concentration as recommended use level for emulsion specified by producer (Ashland, USA). Emulsion without preservative and EOs was a references sample.

Table 1. Composition of the emulsions: E1 - emulsion without preservative / essential oils; E2 - emulsion with preservative; E3 – emulsion with essential oils

Percentage by weight Ingredients E1 E2 E3 SodiumPolyacrylate 0.8 0.8 0.8 Ceteareth-20 3 3 3 IsopropylcMyristate 4 4 4 EthylhexylSearate 3 3 3 CetearylIsononanoate 3 3 3 CyclopentasiloxaneandDimethicone 3 3 3 Glycerine 3 3 3 PropyleneGlycol (and) DiazolidinylUrea (and) Methylparaben (and) Propylparaben 0 1 0 Essential oil 0 0 1.5 Aqua 80.2 79.2 78.7 aINCI Name, bpreservative system (Germaben II)

Determination of the preservation efficacy of the O/W emulsions Evaluation of the antimicrobial protection of a cosmetic product was performed according to the EN ISO 11930:2012 (Cosmetics - Microbiology - Evaluation of the antimicrobial protection of a cosmetic product). Inoculation of test microorganism suspension was prepared by addition of 0.02 mL calibrated inoculum (each strain separately) to 20 g emulsion sample to obtained final concentration of bacteria between 1 x 105 - 1 x 106 CFU/ml and fungi between 1 x 104 - 1 x 105 CFU/ml in the formulation. The inoculated containers were mixed thoroughly and incubated in the dark at 20°C to 25°C. The number of viable microorganism in formulations was determined by the plate count method at the proper times 0, 7, 14 and 28 days after inoculation. A sample of 1 mL emulsion was transferred to 9 mL Eugon LT 100 broth (Graso Biotech, Poland) and pre-incubated for 30 minutes at room temperature, then 10-fold dilutions method was done. Triplicate plating of each dilution was performed with TSA agar for bacteria, SDA agar for yeast and PDA agar for mould. The plates were incubated at 37°C for 72h (bacteria and C. albicans) and 25°C for 7 days (A. brasiliensis), respectively. The number of surviving microorganisms per gram of tested cosmetic product was determined by count the CFU per plate (30-300 colonies for bacteria and C. albicans, 15-150

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colonies for A. brasiliensis). The results were expressed as log reduction value (log CFU/g). For each time and each strain, the log reduction value is calculated and compared to the minimum values required for evaluation criteria A or B of preservation efficacy test (Annex B in EN ISO 11930:2012). Formulation meets Criterion A when the cosmetic product is protected against microbial proliferation (recommended efficacy of antimicrobial preservation for topical preparations). Formulation meets Criterion B when the microbiological risk analysis shall demonstrate the existence of control factors not related to the formulation (e.g. a protective package such as pomp provides a higher level of protection than a jar) indicating that the microbiological risk for topical preparations is tolerable. All tests were conducted in triplicate and data from experiments were calculated as mean ±SD. Standard deviation for the test of microorganism population viability not exceeding 0.3 logarithmic unit. Statistical analysis All tests were conducted in triplicate and data from experiments were calculated as mean ±SD. Standard deviation for the test of microorganism population viability not exceeding 0.3 logarithmic unit. Results and Discussion Chemical composition and antimicrobial activity of Monarda sp. EOs In the recent years, many publications have notified that antimicrobial activity of EOs is closely related to their chemical compositions, which depend on growth stage and the part from which the raw material is obtained (Mohammadi and Saharkhiz, 2011; Saharkhiz et al., 2009). The main constituents of Monarda sp. EOs determined by the GC-MS method were: M. citriodora EO: thymol (58.0%), p-cymene (13.4 %) and carvacrol (11.7 %); M. media EO: thymol (41.9 %) and p-cymene (18.0 %); M. didyma EO: carvacrol (26.2 %) and p-cymene (11.6 %) (Table 2), what corresponds with the results obtained by other researchers (Collins et al., 1994; Fraternale et al., 2006; Mattarelli et al., 2017). Moreover, it is well known that thymol and carvacrol are bioactive compounds with strong antimicrobial activity against bacteria and fungi, even resistant isolates (Memar et al., 2017; Naghdi Badi et al., 2017).

Table 2. Chemical composition of Monarda sp. essential oils obtained from leaves in flowering phase of the development

Essential oil Retention LRI LRI database Relative content [%]

constituents time calculated (Nist 08) M. citriodora M. didyma M. media α-Thujene 8.421 925 928 1.37 1.31 1.96 α-Pinene 8.675 930 932 0.57 0.91 0.79 Camphene 9.309 944 946 0.10 0.69 0.13 Sabinene 10.610 970 975 0.02 7.77 0.17 1-Octen-3-ol 10.951 980 980 1.54 2.58 3.66 3-Octanone 11.300 987 988 0.05 0.23 0.06 Myrcene 11.461 991 993 0.34 1.09 0.40 3-Octanol 11.746 997 997 0.06 1.38 0.14 α-Phellandrene 11.978 1002 1006 0.04 0.17 0.18 α-Terpinene 12.600 1015 1018 1.19 2.00 3.14 ρ-Cymene 13.091 1026 1027 13.43 11.55 17.99 Limonene 13.211 1028 1030 0.72 1.18 4.09 1,8-Cineole 13.335 1031 1036 0.10 2.82 0

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y-Terpinene 14.703 1057 1058 0.76 5.79 0.94 cis-Sabinene hydrate 15.108 1065 1069 0.70 0.58 0.87 α-Terpinolene 16.147 1087 1090 0.11 0.33 0.12 trans-Sabinene hydrate 16.660 1095 1098 0.04 0.24 0.12 Linalool 16.833 1101 1101 0.16 6.46 0.16 Nonanal 17.057 1104 1106 0.03 0.22 0.14 Borneol 19.880 1164 1169 0.20 0.88 0.15 Terpinen-4-ol 20.472 1176 1180 0.94 1.07 0.72 α-Terpineol 21.155 1188 1193 0.08 2.55 0.12 Thymol methyl ether 23.269 1236 1234 0.02 0.67 0.15 Carvacrol methyl ether 23.693 1245 1243 2.14 10.99 6.69 Thymoquinone 23.929 1254 1252 0.66 0.21 1.81 Thymol 26.399 1295 1293 57.96 0.99 41.92 Carvacrol 26.654 1306 1303 11.70 26.15 4.44 β-Bourbonene 29.848 1382 1382 0.08 0.39 0.10 Caryophyllene 31.280 1417 1423 0.42 1.03 0.92 Germacrene D 33.821 1478 1479 0.06 2.67 0.90 δ-Cadinene 35.564 1511 1514 0.14 0.15 0.24 Thymohydroquinone 36.888 1556 1558 0.82 0.50 1.04 Caryophyllene oxide 37.806 1580 1580 0.34 0.14 0.22 Total identified (%) 96.9 95.68 94.48

The antimicrobial activity of Monarda sp. EOs were determined using disc-diffusion methods and presented in Table 3. The EOs from the leaves of M. media collected in the budding phase, and the EOs from inflorescences of M. media and M. didyma in the maturity phase showed the strongest antimicrobial activity against S. aureus growth. EOs from the leaves of M. media and M. citriodora inflorescences, both collected during the full flowering phase have the strongest antibacterial activity against P. aeruginosa and E. coli growth, respectively. The EOs from the leaves of M. citriodora harvested in the budding phase and full flowering phase as well EO from M. citriodora inflorescences collected in the maturity phase showed the stronger antifungal activity against C. albicans growth. The EOs from the inflorescences of M. citriodora harvested in the full flowering phase showed the strongest antifungal activity against A. brasiliensis, while EOs from the inflorescences of M. didyma collected in the maturity phase showed no activity against mould. Summarizing, the results showed that Monarda EOs obtained from the leaves in the flowering phase strongly inhibited the growth of Gram-positive bacteria and fungi but was less effective against Gram-negative bacteria. Literature data showed that M. citriodora EO has a strong antibacterial activity against E. coli, B. subtilis and S. albus (Lu et al., 2011) as well antifungal activity against 15 fungal species (Bishop and Thornton, 1997). M. didyma EO strongly inhibited the growth of S. aureus, E. coli, B. subtilis, B. cereus, P. fluorescens, S. typhimurium and C. albicans, while weak antibacterial activity was observed against L. monocytogenes and P. aeruginosa (Ghabraie et al., 2016; Wróblewska et al., 2019). Antifungal activity of M. didyma EO was completely inhibited mycelial growth and spore germination of Botrytis cinerea (Adebayo et al., 2013). The EOs from leaves and flower M. didyma collected during the vegetative phase inhibited the growth of fungi Rhizoctonia solani and B. cinerea (Fraternale et al., 2006). Moreover, it was shown that the main active ingredient of M. didyma EO - thymol and carvacrol showed inhibitory activity against R. solani (Gwinn et al., 2010). Unfortunately, the strong antimicrobial activity of Monarda EOs confirmed by disc-diffusion test are not enough to confirm their potential use as preservatives in cosmetics. Only a challenge test is able to confirm whether EOs can replace synthetic preservatives in

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cosmetics / reduce the concentration of preservatives added to cosmetics / or do not have sufficiently strong antimicrobial activities that would protect the cosmetics against microbial contamination. However, some literature data showed that the use of EOs in formulation significantly reduce or even eliminate the use of synthetic preservatives (Herman, 2019).

Table 3. Antibacterial and antifungal activity of Monarda essential oils (10 µl) in agar disc diffusion method

Plant Raw Microorganisms Species growth material stage S. aureus P. aeruginosa E. coli C. albicans A. brasiliensis Budding Leaf 57±2.0 11±0.5 41±6.0 66±0.0 62±2.0 Leaf 65±1.5 16±1.0 44±1.0 66±1.0 67±2.0 Flowering M. citrodora Inflorenscence 56±1.5 15±4.0 46±0.5 65±0.0 68±2.5 Leaf 74±3.5 14±0.0 43±2.5 61±0.5 63±1.0 Maturity Inflorenscence 53±0.5 17±2.0 43±3.0 66±2.0 60±5.0 Budding Leaf 73±6.5 17±0.5 36±0.5 52±1.0 52±1.5 Leaf 24±0.5 11±0.5 24±1.5 47±3.0 26±6.0 Flowering M. didyma Inflorenscence 84±0.0 15±0.5 30±2.0 49±4.0 33±4.0 Leaf 53±3.0 20±5.5 29±0.0 23±1.0 12±0.0 Maturity Inflorenscence 89±0.0 9±0.5 14±0.5 17±0.0 0 ± 0.0 Budding Leaf 89±0.0 19±2.0 34±1.5 59±2.0 55±1.5 Leaf 87±1.0 22±2.0 34±0.5 56±2.0 58±2.0 Flowering M. media Inflorenscence 80±2.0 21±1.0 31±0.5 58±2.0 55±5.0 Leaf 82±2.0 21±0.5 37±1.5 60±0.5 54±1.0 Maturity Inflorenscence 89±0.0 12±0.5 19±1.0 24±1.0 9±0.5 Ethanol 0±0.0 0±0.0 0±0.0 10±0.5 0±0.0 Tetracycline (30 μg) 40±0.0 16±1.5 25±2.5 - - Erythromycin (15 μg) 38±0.5 26±6.0 13±0.5 - - Miconazole (10 μg) - - - 14±0.5 14±0.5 Nystatin (100 U) - - - 20±0.5 12±0.5 Diameter of inhibition zones (mm) including the diameter of disc (6 mm), values are given as mean ± SD of triplicate experiment. Antimicrobial effectiveness EOs from leaves of M. citriodora, M. didyma and M. media added to the O/W emulsion at a concentration of 1.5% effectively reduced the growth of all tested references microorganisms (Figure 1). Among all tested essential oils, M. media EO completely inhibited growth of S. aureus, E. coli and C. albicans in emulsion after 28 days of incubation and significant inhibited the growth of P. aeruginosa and A. brasiliensis compared to emulsions with M. didyma, M. citriodora EOs and synthetic preservative. Antimicrobial activity of M. didyma and M. citriodora EOs in emulsions were comparable with activity of synthetic preservative against all tested references strains. The weakest antimicrobial activity against P. aeruginosa was observed in all tested emulsion with Monarda EOs and preservative. According to the PN-EN ISO 11930: 2012 standard, cosmetic formulations must meet the criterion A or B of the preservative effectiveness test for all references microorganisms, what is equivalent that the cosmetic product is protected against microbial proliferation (Table 4). Therefore, the EO from the leaves of M. media (1.5%) can acts a substitutes for synthetic preservatives in O/W emulsions and create self-preserving system. In turn, EOs from the leaves of

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M. citriodora and M. didyma can be valuable raw materials supporting the preservatives system, which can significantly reduce the concentration of preservatives used in cosmetic formulation.

Figure 1. Inhibition of growth of the microorganisms

7 7 CF CF CF + preservative 6 CF + preservative CF + M. citriodora A 6 CF + M. citriodora B CF + M. didyma CF + M. didyma 5 CF + M. media 5 CF + M. media 4 4

3 3

2 2

Viable cells in log [CFU/ml] log in cells Viable 1 1 Viable cells in log [CFU/ml] login cells Viable 0 0 0 7 14 21 28 0 7 14 21 28 Time [day] Time [day]

7 CF 7 CF CF + preservative CF + preservative 6 CF + M. citriodora C 6 CF + M. citriodora CF + M. didyma CF + M. didyma CF + M. media D 5 CF + M. media 5

4 4

3 3

2 2 Viable cells in log [CFU/ml] log in cells Viable

1 1 Viable cells in log [CFU/ml] login cells Viable

0 0 0 7 14 21 28 0 7 14 21 28 Time [day] Time [day]

7 CF CF + preservative 6 CF + M. citriodora E CF + M. didyma 5 CF + M. media

4

3

2

1 Viable cells in log [CFU/ml] login cells Viable 0 0 7 14 21 28 Time [day]

Staphylococcus aureus ATCC 6538(A), Pseudomonas aeruginosa ATCC 9027(B), Escherichia coli ATCC 8739(C), Candida albicansATCC 10231(D), Aspergillus brasiliensis ATCC 16404(E) in o/w formulation(CF) with Monarda citriodora, Monarda didyma, Monarda media essential oils and preservative Germaben II (Propylene Glycol (and) Diazolidinyl Urea (and) Methylparaben (and) Propylparabe).

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Table 4. Criteria of preservation efficacy test for o/w emulsion with preservatives and Monarda EOs according to PN- EN ISO 11930: 2012 standard o/w S. aureus P. aeruginosa E. coli C. albicans A. brasiliensis emulsion Base -a - - - - Preservative Bc - - - Ab M. citriodora B - - A A M. didyma A - - A A M. media A B B A A aBeyond the criterion. bRecommended efficacy of antimicrobial preservation for topical preparations. cMicrobiological risk for topical preparations is tolerable. Literature data showed that EOs from Calaminta officinalis(2%) in O/W emulsion and shampoo (Nostro et al., 2004), Cinnamomum zeylanicum (2.5%) in O/W emulsion (Herman et al., 2013; Herman, 2014), Artemisia afra and Pteronia incana in aqueous cream formulation (Muyima et al., 2002), Lavandula angustifolia (Turgut et al., 2017),Lavandulla officinallis (1.5%), Rosmarinus officinalis (1.5%) or their mixture (0.5% L. officinallis oil and 0.5% R. officinalis oil) in an aqueous cream (Muyima et al., 2002), and C. officinalis (0.5% and 1% v/v) in combination with 2.0 mM EDTA in cetomacrogol cream (Nostro et al., 2002) can create a self-preservative system. In turn, Thymus vulgaris EO (3%) in O/W and W/O emulsions (Manou et al., 1998), L. officinalis and Melaleuca alternifolia essential oils (2,5%) in O/W emulsion (Herman et al., 2013), C. officinalis essential oil (1%) in O/W emulsion (Nostro et al., 2004), lavender, lemon and tea tree oils alone (0.5%) or as mixtures (1%) of EOs in O/W body milks formulation (Kunicka‐Styczynska et al., 2009), lemon oil (1%) / lavender oil (1%) / tea tree oil (1%) as well their mixtures (0.5% each oil) in washing liquids (Kunicka-Styczyńska et al., 2011) can reduce the addition of preservatives used to cosmetic formulations. Moreover, using the lowest possible concentration of EOs with preservative activities will exclude from cosmetics too intense fragrance and formation allergies and skin irritation to users. Additionally, EOs as multifunctional ingredients can not only effect on cosmetics preservation system but also offers cosmetic industries many valuable properties, i.e. EOs can independently penetrate through the skin or increase the penetration of other active compounds from topically applied formulation into the lower layers of the skin (Herman and Herman, 2015), EOs with antimicrobial, antioxidant and anti- inflammatory properties are used in topical formulation for the treatment of many skin diseases as acnes, alopecia, psoriasis and wound healing (Reuter et al., 2010). Conclusions According to the experimental results observed it can be concluded that, M. media EO may be suggested as an effective candidate as natural cosmetic preservative in O/W emulsion, and create an artificial preservative free or self-preserving system. In addition, the M. didyma and M. citriodora EOs can reduce concentration of preservatives in O/W emulsions, at the same time contributing for the microbiological safety of the cosmetic formulation for its use and storage.

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare. REFERENCES

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 39-48 Ağalar et al. DOI: 10.37929/nveo.871951

RESEARCH ARTICLE The Essential Oil Profiles of Chaerophyllum crinitum and C. macrospermum Growing wild in Turkey Hale Gamze Ağalar1,*, Ayhan Altıntaş1,2 and Betül Demirci1

1Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470, Eskisehir, TURKEY 2Yunus Emre Vocational School of Health Services, Anadolu University, 26470, Eskisehir, TURKEY

*Corresponding author. Email: [email protected] Submitted: 31.01.2021; Accepted: 18.02.2021

Abstract

The current study reports the essential oil compositions of the aerial parts of Chaerophyllum crinitum Boiss. and Chaerophyllum macrospermum (Willd. ex Spreng.) Fisch. & C.A. Mey. ex Hohen. collected from Bitlis and Hakkari in Turkey, respectively. The essential oils, which were obtained with water distillation were analyzed by GC and GC-MS, simultaneously. The essential oil of C. crinitum were characterized with (E)-β-ocimene (38.1%) and terpinolene (12.7%), while terpinolene (21.4%), myristicin (18.9%), p-cymen-8-ol (11.9%) were identified as major components for C. macrospermum essential oil.

Keywords: Chaerophyllum crinitum, Chaerophyllum macrospermum, Apiaceae, essential oil, (E)-β-ocimene

Introduction Apiaceae is a large family consisting of 3780 species in 434 genera. Members of Apiaceae are mainly distributed in northern hemisphere. Species belonging to the Apiaceae family are of economic importance and are used in industrial processes such as beverage, perfumery, pharmaceutical, and cosmetic industries (Zengin et al., 2020). The genus Chaerophyllum L. has economic value-added species. In the Flora of Turkey, the genus Chaerophyllum L. is represented by 15 species of which four are endemic (Davis, 1972). “Chairo” (to please) and “phylum” (a leaf) words from Greek consist of Chaerophyllum and Chaerophyllum means the fragrant character of the foliage (Ebrahimabadi et al., 2010). Chaerophyllum species are used as aromatizer in food industry. In Turkey, C. macropodum Boiss. is used in cheese production for its strong aroma (Demirci et al., 2007, Tarakci et al., 2006). C. macropodum, (local name: mendi, mendo), is wildly grown in Van and is used in production of very famous cheese known as “Van Otlu Peynir” (Çelik et al., 2008; Tarakci & Temiz, 2009). Chaerophyllum species were characterized with lignans, polyacetylenes, essential oils, phenolic acids, and flavone derivatives (Rollinger et al., 2003; Gonnet, 1985; Dall’Acqua et al., 2004; Ayala Garay et al., 2001). Essential oils of Chaerophyllum species have been determined due to their economic values. Previous studies reported that C. crinitum was characterized with (E)-β-ocimene (50.5%) by Nematollahi et al. (2005); - terpinolene (20.3%) by Hayta and Celikezen (2016) while C. macrospermum seed essential oil was characterized with (E)-β-farnesene (27.1%), (Z)-β-ocimene (18.8%), p-cymene (14.3%), α-fenchyl acetate (12.7%) (Razavi & Nejad-Ebrahimi (2010)). The crushed fruits of C. aksekiense an endemic to Turkey were characterized with heptacosane (10.1%) by Başer et al. (2010). β-Phellandrene (17.6%) and limonene (15.9%) were found as major compounds in the essential of C. libanoticum growing wild in Turkey (Demirci et al., 2007).

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 39-48 Ağalar et al. DOI: 10.37929/nveo.871951

In Turkey, due to the use of Chaerophyllum species as food ingredients, these species were evaluated for their potential biological effects, mostly antimicrobial and antioxidant activities (Ağaoğlu et al., 2005; Durmaz et al., 2006; Sagun et al., 2006; Hayta and Celikesen, 2016; Kurkcuoglu et al., 2018). In the literature, a few studies on Chaerophyllum species growing wildly in Turkey were reported until now. Furthermore, the volatile composition of these species is very important because of the uses as food and as foodstuff. The present work reported that the essential oil compositions from aerial parts from both Chaerophyllum crinitum and C. macrospermum obtained by water distillation. Materials and Methods Plant materials Chaerophyllum crinitum was collected from Bitlis; Adilcevaz, Armutlu Village, Yeşilce Area at an altitude of 2140 m in Turkey. C. macrospermum was gathered from Hakkari; Cilo Mountain, between Kırıkdağ Village- and Cehennem Dere Plateau at an altitude of 2500 m in Turkey. Both of the samples are kept in Anadolu University, Pharmacy Faculty, Pharmacognosy Laboratory. Isolation of essential oils of the samples The essential oils from aerial parts of Chaerophyllum crinitum and C. macrospermum were isolated by water distillation for 3 h using a Clevenger-type apparatus. GC and GC-MS analysis The methods for GC (Agilent 6890N GC system) and GC-MS analysis (Agilent 5975 GC-MSD system) were given by our previous study (Duymuş et al., 2014). Identification of components Individual components were identified by computer matching with commercial libraries (Wiley GC-MS Library, MassFinder 3 Library) and in-house “Baser Library of Essential Oil Constituents”, which includes over 3200 authentic compounds with Mass Spectra and retention data from pure standard compounds. In addition, components of known oils as well as MS literature data, were also used for the identification (Duymuş et al., 2014). Result and Discussion The aerial parts of Chaerophyllum crinitum and C. macrospermum were subjected to water distillation using Clevenger apparatus for 3 h. Each essential oil was analyzed by GC and GC-MS systems, simultaneously. The essential oil of C. crinitum obtained by lower than 0.1% yielding was characterized with sixty compounds comprising 95.8% of the oil. C. macrospermum essential oil were obtained by 0.1% yielding from the aerial parts. Thirty-eight components were identified comprising 94.1% of total oil. The essential oil compositions of both samples were given at Table 1. The main constituents of the essential oil of C. crinitum were found as (E)-β-ocimene (38.1%), terpinolene (12.7%), α-pinene (5.5%), p-cymene (5.3%), limonene (5.3%) and p-cymen-8-ol (2.2%). Terpinolene (21.4%), myristicin (18.9%), p-cymen-8-ol (11.9%), limonene (6.1%), α-pinene (4.6%), spathulenol (4.4%), and α-p- dimethyl-styrene (3.6%) were the major compounds for C. macrospermum essential oil.

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 39-48 Ağalar et al. DOI: 10.37929/nveo.871951

Table 1. The essential oil compositions of Chaerophyllum crinitum and C. macrospermum

C. crinitum C. macrospermum RRI Compounds IM (%) (%)

1032 α-Pinene 5.5 4.6 tR, MS

1035 α-Thujene 0.1 - tR, MS

1076 Camphene 0.4 - tR, MS

1093 Hexanal 0.1 - tR, MS

1118 β-Pinene 1.7 0.4 tR, MS

1132 Sabinene 1.8 - tR, MS

1174 Myrcene 0.5 0.5 tR, MS

1176 α-Phellandrene 0.1 0.2 tR, MS

1188 α-Terpinene 0.4 - tR, MS

1194 Heptanal tr - tR, MS

1203 Limonene 5.3 6.1 tR, MS

1218 β-Phellandrene 1.2 1.7 tR, MS 1246 (Z)-β-Ocimene 0.4 0.7 MS

1255 γ-Terpinene 1.1 0.2 tR, MS 1266 (E)-β-Ocimene 38.1 2.4 MS

1280 p-Cymene 5.3 1.4 tR, MS

1290 Terpinolene 12.7 21.4 tR, MS

1296 Octanal 1.7 0.2 tR, MS 1398 2-Nonanone 0.3 - MS

1400 Nonanal 0.2 - tR, MS 1413 Rose furane 0.3 - MS 1452 α-p-Dimethylstyrene 0.9 3.6 MS 1477 4,8-Epoxyterpinolene 0.8 1.6 MS

1482 Fencylacetate 0.3 - tR, MS 1497 α-Copaene 0.2 - MS 1498 (E)-β-Ocimene epoxide 0.1 - MS 1548 (E)-2-Nonenal 0.2 - MS

1586 Pinocarvone 0.2 - tR, MS

1590 Bornyl acetate 1.3 - tR, MS

1611 Terpinen-4-ol - tR, MS 0.2 1612 β-Caryophyllene 0.8 tR, MS 1648 Myrtenal 0.2 - MS 1655 (E)-2-decenal 0.8 - MS 1668 (Z)-β-Farnesene 0.3 - MS

1670 trans-Pinocarveol 0.2 - tR, MS

1684 trans-Verbenol 0.2 - tR, MS 1700 Limonen-4-ol - 1.7 MS

1706 α-Terpineol - 0.2 tR, MS

1726 Germacrene D 0.3 1.3 tR, MS

1751 Carvone 0.2 - tR, MS 1773 δ-Cadinene 0.2 - MS 1755 Bicyclogermacrene - 0.8 MS

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Nat. Volatiles & Essent. Oils, 2021; 8(1): 39-48 Ağalar et al. DOI: 10.37929/nveo.871951

1797 p-Menthylacetophenone 0.3 0.9 MS

1827 Cuminaldehyde tr tr tR, MS 1802 (E, E)-2,4-decadienal 0.2 - MS

1845 trans-Carveol 0.4 - tR, MS 1864 p-Cymen-8-ol 2.2 11.9 MS 1868 (E)-Geranyl acetone tr - MS

2008 Caryophyllene oxide 1.6 2.7 tR, MS 2037 Salvial-4(14)-en-1-one - tr MS

2050 (E)-Nerolidol - tr tR, MS 2071 Humulene epoxide 2 0.3 - MS 2131 Hexahydrofarnesyl acetone 0.1 - MS 2144 Spathulenol 0.7 4.4 MS 2245 Elemicine - 0.4 MS 2243 Torilenol - 0.4 MS 2247 trans-α-Bergamotol - 0.3 MS

2255 α-Cadinol - 0.4 tR, MS

2296 Myristicin 0.3 18.9 tR, MS

2298 Decanoic acid 0.2 - tR, MS 2369 Eudesma-4(15)-7-dien-1-betaol 0.2 0.6 MS 2392 Caryophyllenol-II 0.3 0.4 MS 2404 trans-Isoelemicine - 0.5 MS

2500 Pentacosane tr 0.3 tR, MS

2503 Dodecanoic acid 0.2 - tR, MS

2655 Benzyl benzoate 0.3 - tR, MS

2670 Tetradecanoic acid 1.8 - tR, MS

2700 Heptacosane tr 0.4 tR, MS

2900 Nonacosane 0.9 0.7 tR, MS

2931 Hexadecanoic acid 2.0 1.1 tR, MS TOTAL 95.8 94.1

RRI, Relative retention indices calculated against n-alkanes % calculated from FID data. IM, Identification methods. tR, identification based on the retention times of genuine compounds on the HP Innowax column. MS, identified on the basis of computer matching of the mass spectra with those of the Wiley and MassFinder libraries and comparison with literature data; tr, trace amount <0.1%.

Essential oil compositions of Chaerophyllum species were determined by several studies. Table 2 shows previous reports on different Chaerophyllum species. According to Table 2, C. aksekiense (Turkey), C. aromaticum (Turkey), C. aureum (Serbia), C. azoricum (Portugal), C. bulbosum (Iran), C. bulbosum ssp. Bulbosum (Greece), C. byzantinum (Turkey), C. coloratum (Montenegro), C. crinitum (Iran, Turkey), C. hirsutum (Serbia), C. libanoticum (Turkey), C. macropodum (Iran, Turkey), C. macrospermum (Iran, Nakhichevan), C. prescotti (Siberia), C. temulum (Serbia), C. villosum (India) were analyzed for their essential oil compositions by different researchers. Different techniques such as water distillation, steam distillation, microwave assisted hydrodistillation, microdistillation, simultaneous distillation-extraction were used to isolate volatile compounds. Different parts of the plants (fruits, seeds, umbels, inflorescences, roots, shoots, stems, and aerial parts) were subjected to distillation procedures. In general, monoterpenes and diterpenes were the main groups in essential oils. Recent data have shown that factors such as seasonal variation, edaphic and genetic factors, harvest time, vegetative stage affected the composition of essential oil (Evergetis and Haroutounian, 2020).

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When our findings compared with previous data, C. crinitum essential oils were characterized with (E)-β- ocimene (50.5%) in Iran (Nematollahi et al., 2005), and characterized with -terpinolene (20.3%), -cubebene (9.3%), -terpineol (7.2%) and limonene (5.8%) in Turkey (Hayta and Celikezen, 2016). Our results indicated (E)-β-ocimene (38.1%), terpinolene (12.7%), α-pinene (5.5%), p-cymene (5.3%), limonene (5.3%) and p- cymen-8-ol (2.2%) as main constituents. C. macrospermum essential oils were obtained from seeds (Iran), aerial parts (Iran) and inflorescences (Nakhichevan). The seed essential oil consisted of (E)-β-farnesene (27.1 %), (Z)-β-ocimene (18.8%), p-cymene (14.3%), α-fenchyl acetate (12.7 %) and spathulenol (8.8%) (Razavi and Nejad-Ebrahimi, 2010), the essential oil from aerial parts contained as main constituents (E)-β-ocimene (55.9%), terpinolene (9.8%), α-pinene (7.5%), β-phellandrene (4.3%) and β-pinene (4.2%) (Sefidkon and Abdoli, 2005), while the inflorescence essential oil was characterized with 1,8-cineole (7.2%), linalool (6.7%), δ-3-carene (4.4%), α-terpineol (4.7%), farnesol (4.0%) (Mamedova, 1995). Our study reported that terpinolene (21.4%), myristicin (18.9%), p-cymen-8-ol (11.9%), limonene (6.1%), α-pinene (4.6%), spathulenol (4.4%), and α-p-dimethyl-styrene (3.6%) were major compounds in the essential oil.

Table 2. Previous studies on essential oils of Chaerophyllum species

Plant / Origin Method/ Results Reference C. aksekiense A. Duran et Water distillation, GC-MS analysis Başer et al., 2000 Duman 77 components, representing 82.0% of the oil; Heptacosane (10.1%), Crushed fruits (Turkey) humulene epoxide II (7.8%), (E)-β-farnesene (6.2%), caryophyllene oxide (6.0%), α-humulene (5.5%), terpinolene (5.5%), nonacosane (5.3%) and terpinen-4-ol (4.6%)

C. aromaticum L. Water distillation, GC and GC-MS analysis Kurkcuoglu et al., Aerial parts (Turkey) 18 compounds, 99.2% of the oil; Sabinene (28.1%), terpinolene (16.7%) and 2018 γ-terpinene (16.1%)

C. aureum L. Water distillation, GC and GC-MS analysis Lakusic et al., 2009 Aerial parts and fruits 50 compounds, representing 97.7% of aerial parts (Suva mountains), 97.0% (Serbia) of aerial parts (Kopaonik mountains) and 98.5% of fruits (Kopaonik mountains); Sabinene (18.5-31.6%), p-cymene (7.9-25.4%) and limonene (1.9-10.9%)

C. azoricum Trel. Simultaneous Distillation-Extraction, GC and GC-MS analysis Pedro et al., 1999 Leaves+stems (Portugal) 34-37 compounds, 98-99% of the oil; Terpinolene (44-62%) and γ-terpinene (9-31%) of the oils

C. bulbosum L. Water distillation, GC and GC-MS analysis Masoudi et al., 2011 Aerial parts (Iran) 29 compounds, 92.2% of the oil; (E)-β-Farnesene (22.3%), (Z)-β-ocimene (18.8%), and myristicin (17.1%), caryophyllene oxide (6.6%), allo-ocimene (5.1%), and (E)-β-ocimene (4.0%)

C. bulbosum L. ssp. GC and GC-MS analysis Kokkalou and bulbosum (Greece) 29 compounds, 95% of the oil; Apiol (37%), trimethyl-3,7,11-dodecatrien- Stefanou, 1989 1,6,10-ol-3 (8.5%), linalool (7.7%), myristicin (6.9%) and eugenol (5.8%)

C. byzantinum Boiss. Water distillation, GC and GC-MS analysis Kürkçüoğlu et al., Aerial parts (Turkey) 65 compounds, 94.6% of the oil; Sabinene (30.0%), p-cymen-8-ol (16.0%) 2006

C. coloratum L. Water distillation, GC and GC-MS analysis Stesevic et al., 2016 Root and stem Root: 56 components, 99.2% of the oil; Myrcene (72.2%), β-phellandrene (Montenegro) (5.5%) Stem: 18 compounds, 94.4% of the oil; (E)-β-ocimene (33.6%), (Z)-β- ocimene (20.4%) and terpinolene (10.8%)

C. coloratum Water distillation, GC and GC-MS analysis Vajs et al., 1995

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Ripe fruits, umbels Ripe fruit: 8 compounds, 95.1% of the oil; (E)-β-Farnesene (79.2%), β- (Montenegro) pinene (7.0%), (Z)-β-ocimene (4.3%) Umbel: 8 compounds, 80.2% of the oil; (E)-β-Farnesene (68.5%), (Z)-β- ocimene (5.5%)

C. crinitum Boiss. Water distillation, GC and GC-MS analysis Nematollahi et al., Aerial parts (Iran) 11 components, 84.3% of the oil; (E)-β-ocimene (50.5%) 2005

C. crinitum Water distillation, GC and GC-MS analysis Hayta and Celikezen, Aerial parts (Turkey) 64 compounds, 85.5% of the oil; -Terpinolene (20.3%), -cubebene 2016 (9.3%), -terpineol (7.2%) and limonene (5.8%)

C. hirsutum L. Water distillation, GC and GC-MS analysis Petrovic et al., 2017 Roots, shoots, Root: 70 compounds, 97.3% of the oil; γ-Terpinene (15.8%), acorenone B inflorescences (Serbia) (14.2%), (Z)-Falcarinol (7.9%), isothymol methyl ether (6.5%) Shoot: 55 compounds, 95.2% of the oil; Acorenone B (57.0%), endo-Fenchyl acetate (9.8%), 5-neo-cedranol (6.0%) Inflorescence: 69 compounds, 98.8% of the oil; Acorenone B (44.6%), endo- Fenchyl acetate (19.1%), γ-terpinene (6.4%)

C. libanoticum Boiss. et Water distillation, GC and GC-MS analysis Demirci et al., 2007 Kotschy 73 components, 98.3% of the total oil; β-Phellandrene (17.6%), limonene Crushed fruits (Turkey) (15.9%), β-pinene (8.8%), and sabinene (8.5%)

C. macropodum Boiss. Water distillation (WD) and Microwave assisted hydrodistillation (MAHD), Khajehie et al., 2017 Aerial parts (Iran) GC-MS analysis (E,Z)-β-Ocimene (29.3 and 29.3%), myrcene (14.5 and 14.6%), and terpinolene (14.4 and 14.3%) in WD and MAHD, resp.

C. macropodum Water distillation, GC and GC-MS analysis Ebrahimabadi et al., Leaves and flowers (Iran) Leaf: 37 compounds, 98.3% of the oil; Spathulenol (10.4%), myrcene 2010 (10.0%), germacrene D (8.7%), limonene (6.7%), β-caryophyllene (6.2%) Flower: 31 compounds, 99.4% of the oil; trans-β-Farnesene (27.5%), trans- β-ocimene (20.9%), limonene (12.0%), cis-β-ocimene (4.3%)

C. macropodum Water distillation, GC and GC-MS analysis Shafaghat, 2009 Flowers, leaf, stem (Iran) Flower: 10 compounds, 98.5% of the oil; Myristicin (42.5%) and trans-β- ocimene (41.0%) Leaf: 18 compounds, 99.3% of the oil; trans-β-Ocimene (24.9%), myristicin (15.7%), terpinolene (14.5%), fenchyl acetate (13.9%), cis-β-ocimene (6.3%) and sabinene (6.1%) Stem: 11 compounds, 97.1% of the oil; trans-β-Ocimene (54.2%), myristicin (22.4%) and sabinene (8.9%)

C. macropodum Microdistillation, GC and GC-MS analysis Başer et al., 2006 Fruits (Turkey) 41 compounds, 92% of the sample; p-Cymene (39.3%), spathulenol (7.3%), p-cymen-8-ol (5.9%), octanal (5.2%), (E)-β-ocimene (4.5%)

C. macropodum Water distillation, GC and GC-MS analysis Nematollahi et al., Aerial parts (Iran) 28 compounds, 98.5% of the oil; α-Pinene (23.0%), β-pinene (17.3%) and 2005 fenchyl acetate (13.8%)

C. macrospermum Water distillation, GC-MS analysis Razavi and Nejad- (Spreng.) Fisch et C.A. 15 compounds, 94.6% of the oil; (E)-β-Farnesene (27.1%), (Z)-β-ocimene Ebrahimi, 2010 Mey. (18.8%), p-cymene (14.3 %), α-fenchyl acetate (12.7%) and spathulenol Seeds (Iran) (8.8%)

C. macrospermum Water and Steam distillation, GC and GC-MS analysis Sefidkon and Abdoli, Aerial parts (Iran) 27 compounds; (E)-β-Ocimene (55.9%), terpinolene (9.8%), α-pinene 2005 (7.5%), β-phellandrene (4.3%) and β-pinene (4.2%)

C. macrospermum Water distillation, GC and GC-MS analysis Rustaiyan et al., 2002

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Aerial parts (Iran) 16 compounds, 99.6% of the oil; (E)-β-Ocimene (40.0%), tricyclene (19.4%), δ-3-carene (18.3%) and mycrene (10.1%)

C. macrospermum Steam distillation, GC analysis Mamedova, 1994 Inflorescences 33 compounds; Cineole (7.2%), linalool (6.7%), δ-3-carene (4.4%), a- (Nakhichevan) terpineol (4.7%), farnesol (4.0%)

C. prescotti DC Steam distillation, GC and GC-MS analysis Letchamo et al., 2005 Flowering tops (Siberia) 18 compounds, 99% of the oil; (E)-β-Ocimene (35.6%), (Z)-β-ocimene (19.4%), γ-terpinene (18.8%), myrcene (10.6%) and terpinolene (4.6%)

C. temulum L. Water distillation, GC and GC-MS analysis Stamenkovic et al., Root, stem, inflorescence, Root at the flowering stage: 35 compounds, 90.4% of the oil; (Z)-Falcarinol 2015 fruit (Serbia) (61.7%) Root at the fruiting stage: 31 compounds, 91.2% of the oil; (Z)-Falcarinol 62.3% Stem at the flowering stage: 43 compounds, 94.8% of the oil; Germacrene D (38.4), phytol (12.4%), α-humulene (10.1%) Stem at the fruiting stage: 45 compounds, 92.3% of the oil; Germacrene D (32.5%), phytol (11.6%), α-humulene (8.1%) Inflorescence: 74 compounds, 96.8% of the oil; (Z, E)-α-Farnesene (23.4%), germacren-D-4-ol (9.0%), (E)-β-farnesene (9.0%), (E)-β-ocimene (7.4%), germacrene D (6.6%), β-phellandrene (6.1%), Fruit: 41 compounds, 95.6% of the oil; Germacren-D-4-ol (27.6%), (Z, E)-α- farnesene (13.4%), α-zingiberene (8.6%), (E)-β-farnesene (7.8%)

C. villosum Wall. ex DC. Water distillation (for root), Steam distillation (for leaf), GC and GC-MS Joshi, 2013a; Joshi, Root, leaf (India) analysis 2013b Root: 31 compounds, 91.5% of the oil; Carvacrol methyl ether (31.1%), myristicin (19.1%), thymol methyl ether (18.6 %), γ-terpinene (11.7%) Leaf: γ-Terpinene (74.9%), p-cymene (10.0%), terpinolene (2.9%) and β- pinene (2.5%)

As conclusion, significant differences between the essential oils obtained by our study and the previous studies which analyzed Chaerophyllum species gathered from different countries (geographic regions), originated from different parts of the plants (fruit, stem, flower, seed, etc) and plant parts at different phases of growth were observed. It may be concluded that large differences in the environmental conditions have contributed to the large variability in the composition and major compounds of Chaerophyllum essential oils. Due to the rather heterogeneous data on the chemistry of the different Chaerophyllum species and the insufficient number of taxa studies, it may not be sufficient to make detailed comparison within the genus and obtain possible chemical diagnostic properties. Hence, detailed further studies on evaluation of essential oils of these species are needed.

ACKNOWLEDGEMENT

This study presented as a poster presentation at International Symposium of Essential Oils held on September 11-14, 2011 in Turkey was published as an abstract in JEOR’s 23th volume (sup 1).

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare.

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