Chemical composition of Ambrette Essential Oil

Chemical composition of Ambrette Essential Oil Chemical composition of the essential oil of ambrette (Abelmoschus moschatus Medik.) from subtropical region of north India Ram S. Verma*, Rajendra C. Padalia, Amit Chauhan ABSTRACT Abelmoschus moschatus (Family: Malvaceae), popularly known as ambrette or muskdana, is an important aromatic and medicinal plant of India. The plant is widely cultivated in tropical countries for their musk-scented seeds useful in perfumery and medicine. In this study, hydrodistilled ambrette seed essential oil produced in subtropical region of north India was investigated using gas chromatography-flame ionization detector (GC-FID) and GC-mass spectrometry (GC-MS). A total of 27 constituents, representing 90.4% of the total oil composition were identified. Major constituents of the oil were (2E,6E)-farnesyl acetate (58.0%), (Z)-oxacycloheptadec-8-en-2-one (12.1%), decyl acetate (4.8%), (2Z,6E)-farnesyl acetate (3.5%), (Z)-oxacyclopentadec-6-en-2-one (2.4%), dodecyl acetate (2.4%) and (2E,6Z)-farnesol (2.0%). Ambrettolide and its homologues, responsible for the characteristic musk-like odour, constitute 15.8% of oil composition. Keywords: Abelmoschus moschatus, Malvaceae, ambrette seed, essential oil, (2E,6E)-farnesyl acetate 1. Introduction Abelmoschus moschatus Medik. (syn. Hibiscus abelmoschus (L.), commonly known as ambrette, is native to India (1). It is cultivated in tropical regions of Asia, Africa and South America for their seeds. The seeds have been used as tonic, stimulant, carminative, diuretic, demulcent, and for stomachic property (2). The essential oil obtained by steam-distillation of ambrette seeds is a valuable material known for a rich, sweet, floral-musky, distinctly wine-like or brandy-like odor, which finds application in flavour and fragrance formulations (3). Moreover, the seed essential oil is used as anti-inflammatory, analgesic and antispasmodic drug. It is indicated against cramps and bowel disorders and also useful in the itching caused by insect bites. The leaves and the fruits of the plant are consumed in soups and the seeds are used as a spice (4). In addition to this, A. moschatus shows good antioxidant, antiproliferative and antimicrobial activities (5). The plant has been classified as â€Å"an herb of undefined safety† by the Food and Drug Administration (FDA), and the extracts are classified as generally recognized as safe (GRAS) for their use in baked foods, candies, and alcoholic beverages (6). The chemical composition of essential oil and extracts of ambrette seed have been reported from different countries (7-16). The ambrette seed oil has a much smoother odor than synthetic musk compounds, and the major compounds responsible for the characteristic musky odor include ambrettolide: (Z)-7-hexadecen-16-olide and (Z)-5-tetradecen-14-olide (17). Despite a long history of uses in traditional medicines and in perfumery, information on A. moschatus from subtropical region of India is meager. Therefore, in this study, volatile oil composition of the ambrette seed grown in north India (subtropical condition) has been investigated. 2. Experimental 2.1. Plant material and isolation of essential oil The ambrette seeds were collected from experimental field of CSIR-Central Institute of Medicinal and Aromatic Plants, Research Centre, Pantnagar (Uttarakhand) in the month of December (2009–2011). The experimental site is located between coordinates 29.02 °N, 79.31 °E and an altitude of 243 m in foothills of north India. Isolation of the essential oil from ambrette seeds was carried out by hydrodistillation in a Clevenger’s type apparatus for 5 hours. Isolated oil was dried over anhydrous Na2SO4 and stored at 4 °C until further analyses. 2.2. GC and GC-MS analyses GC analysis of the essential oil was carried out on a Nucon gas chromatograph model 5765 equipped with DB-5 capillary column (30 m Ãâ€" 0.25 mm internal diameter, film thickness 0.25  µm) and flame ionization detector (FID). The oven column temperature ranged from 60–230  °C, programmed at 3  °C/min, using H2 as carrier gas at 1.0 mL/min, a split ratio of 1:35, an injection size of 0.03  µL neat, and injector and detector temperatures were 220  °C and 230  °C, respectively for Nucon gas chromatograph model 5765. GC/MS analysis of the essential oil sample was carried out on a Clarus 680 GC interfaced with a Clarus SQ 8C mass spectrometer of PerkinElmer fitted with Elite-5 MS fused-silica capillary column (30 m Ãâ€" 0.25 mm i.d., film thickness 0.25  µm). The oven temperature program was from 60–240  °C, at 3  °C/min, and programmed to 270  °C at 5  °C /min; injector temperature was 250  °C; transfer line and source temperatures were 220  °C; i njection size 0.03  µL neat; split ratio 1:50; carrier gas He at 1.0 mL/min; ionization energy 70 eV; mass scan range 40-450 amu. Characterization was achieved on the basis of retention index (RI, determined using a homologous series of n-alkanes, C8-C30 hydrocarbons), mass spectra library search (NIST/EPA/NIH version 2.1 and Wiley registry of mass spectral data 7th edition) and by comparing the observed RI and mass spectral data with the literature (18,19). The relative amounts of individual components were calculated based on the relative % peak areas (FID response), without using a correction factor. 2.3. Statistical analysis To compare of the examined essential oil composition of ambrette seed from subtropics with the reported compositions from other regions, seven samples (1: present study and 2-7: other regions) (8,9,11,13,14) were treated as operational taxonomic units. The percentage of nine major components, representing composition up to 82.8-89.0% of ambrette essential oil (decyl acetate, dodecyl acetate, (E)-ÃŽ ²-farnesene, (Z)-oxacyclopentadec-6-en-2-one, (2Z,6E)-farnesyl acetate, (2E,6E)-farnesyl acetate, (2E,6E)-farnesol, (Z)-oxacycloheptadec-8-en-2-one, and (E)-2,3-dihydrofarnesyl acetate) were used to determine the chemical relationship among the different essential oil samples by hierarchical cluster analysis using the average method (20). This software computes the hierarchical clustering of a multivariate dataset based on dissimilarities. The derived dendrogram depicts the grouping of chemical compositions as per their chemical constituents. 3. Results and discussion The essential oil yield and chemical composition of ambrette seeds observed in subtropics, north India is presented in Table 1. The seeds gave 0.12  ± 0.01% (v/w) of essential oil on hydrodistillation. However, essential oil yield was 0.15–0.20% in ambrette seeds under eastern Indian conditions (12). The resulting essential oil was analysed using GC-FID and GC-MS techniques. Altogether, 27 constituents, representing 90.4% of the total oil composition were identified. Major constituents of the oil were (2E,6E)-farnesyl acetate (58.0%), (Z)-oxacycloheptadec-8-en-2-one (12.1%), decyl acetate (4.8%), (2Z,6E)-farnesyl acetate (3.5%), (Z)-oxacyclopentadec-6-en-2-one (2.4%), dodecyl acetate (2.4%), (2E,6Z)-farnesol (2.0%), (Z)-oxacyclononadec-10-en-2-one (1.3%) and (E)-nerolidol (0.7%). The essential oil composition of ambrette seed has been investigated earlier from different countries and mainly five types of compositions are described. Garnero and Buil (1978) identified (2E,6E)-farnesol (39.0%) and (E,E)-farnesyl acetate (35.4%) as the major constituents of ambrette seed oil (13). Dung et al (1999) reported two different compositions, viz. (E)-2,3-dihydrofarnesyl acetate (67.3%) type, and (E,E)-farnesyl acetate (35.5%) and (E)-2,3-dihydrofarnesyl acetate (32.9%) type for ambrette seed oil from Vietnamese (14). However, ambrette seed oils from Ecuador and China are reported to have (E,E)-farnesyl acetate (59.1% and 64.22%) and (Z)-oxacycloheptadec-8-en-2-one (7.8% and 14.9%) as major constituents (8,9). According to an earlier study from Odisha (eastern India), the main constituents of ambrette seed oil were (E,E)-farnesyl acetate (47.6%), (E)-ÃŽ ²-farnesene (9.6%) and (Z)-oxacycloheptadec-8-en-2-one (9.0%) (11). Moreover, to compare the examined essential oil com position with earlier reported compositions, the contents (%) of nine major components of different oils were subjected to the hierarchical cluster analysis. The derived dendrogram clearly demonstrate dissimilarity based on the percentages of the constituents present among the different compositions (Figure 1). Thus, composition of the examined oil from subtropical northern India was closer to the oil composition reported from Ecuador (8). However, it was rather different from China (9) and eastern Indian (11) ambrette seed oils due to the content (%) of other constituents, viz. (E)-ÃŽ ²-farnesene and decyl acetate. 4. Conclusions In conclusions, the chemical composition of ambrette seed oil produced in subtropics was rich in (E,E)-farnesyl acetate (58.0%), and ambrettolide and its homologues (15.8%). The ambrette seed oil has a promising value for fragrance and fixative purposes. Based on the results of this study, it can be said that ambrette can also produced good quality essential oil in the subtropical conditions of north India. Acknowledgements Council of Scientific and Industrial Research (CSIR), New Delhi is thankfully acknowledged for the financial support to carrying out the work (Project: BSC0203). Authors are also thankful to the Director, CSIR-Central Institute of Medicinal and Aromatic Plants for encouragement and the Central Chemical Facility (CSIR-CIMAP) for providing facility for GC and GC/MS analyses. 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Grieder, (Z)-5-tetradecen-14-olide, a new macrocyclic lactone, and two unsaturated straight chain acetates from ambrette seed absolute. Helv. Chim. Acta, 60, 1155-1160 (1977). R.P. Adams, Identification of essential oil components by gas chromatography /mass spectrometry. Allured Publishing Corp., Carol Stream, Illinois, USA (2007). http://www.pherobase.com/database/kovats/kovats-detail-Z5-12Ac.php Wessa, P. (2013). Hierarchical clustering (v1.0.3) in free statistics software (v1.1.23-r7), office for research development and education. URL: http://www.wessa.net/ Table 1: Chemical composition of ambrette (Abelmoschus moschatus Medik.) seed essential oil from north India S. no. Compounda RIb RIc Content (%)d S. no. Compounda RIb RIc Content (%)d 1 ÃŽ ±-Pinene 933 932 0.1  ± 0.09 15 Decyl propanoate 1502 1501 0.2  ± 0.06 2 ÃŽ ²-Pinene 972 974 t 16 (E)-Nerolidol 1560 1562 0.7  ± 0.06 3 6-Methyl-5-hepten-2-one 978 981 0.1  ± 0.04 17 (Z)-5-Dodecenyl acetate 1588 1592* 0.5  ± 0.06 4 ÃŽ ±-Terpinene 1014 1014 t 18 Dodecyl acetate 1609 1607 2.4  ± 0.25 5 p-Cymene 1022 1020 0.2  ± 0.25 19 (2Z,6Z)-Farnesol 1696 1698 0.1  ± 0.00 6 Limonene 1026 1024 0.2  ± 0.16 20 (2E,6Z)-Farnesol 1713 1714 2.0  ± 0.93 7 1,8-Cineole 1028 1026 0.2  ± 0.21 21 (Z)-Oxacyclopentadec-6-en-2-one†  1719 2.4  ± 2.43 8 Linalool 1100 1095 0.4  ± 0.46 22 (2Z,6E)-Farnesyl acetate 1822 1821 3.5  ± 1.15 9 Camphor 1146 1141 t 23 (2E,6E)-Farnesyl acetate 1850 1845 58.0  ± 3.13 10 n-Decanol 1270 1266 0.3  ± 0.35 24 (2E,6E)-Farnesyl propanoate 1919 0.4  ± 0.17 11 Undecanal 1304 1305 t 25 (Z)-Oxacycloheptadec-8-en-2-one † Ã¢â‚¬   1928 1929 12.1  ± 4.88 12 Decyl acetate 1407 1407 4.8  ± 0.90 26 (Z)-Oxacyclononadec-10-en-2-one 2128 1.3  ± 0.79 13 (E)-ÃŽ ²-Farnesene 1458 1454 0.2  ± 0.33 27 Linoleic acid 2129 2132 t 14 10-Undecenol acetate 1499 1498 0.1  ± 0.10 Total identified (%) 90.4  ± 6.25 aMode of identification: retention index (RI), mass spectral data (GC–MS); RIb: Experimental Retention Index (relative to n-alkane); RIc: Retention Index from literature (18); dMean ( ± standard deviation) of three samples; † also known as (Z)-5-tetradecen-14-olide; † Ã¢â‚¬  also known as (Z)-7-hexadecen-16-olide (= musk ambrette); *KI: Kovat Index (19). Figure 1: Hierarchical cluster analysis of the essential oil compositions of ambrette (Abelmoschus moschatus Medik.) seed. 1: present study [(2E,6E)-farnesyl acetate (58.0%), (Z)-oxacycloheptadec-8-en-2-one (12.1%)]; 2: [(2E,6E)-farnesol (39.0%), (2E,6E)-farnesyl acetate (35.4%)] (Garnero and Buil, 1978); 3: China [(2E,6E)-farnesyl acetate (64.22%), (Z)-oxacycloheptadec-8-en-2-one (14.96%)] (Tang et al., 1990); 4: Ecuador [(2E,6E)-farnesyl acetate (59.1%), (Z)-oxacycloheptadec-8-en-2-one (7.8%)] (Cravo et al., 1992); 5: Vietnam [(E)-2,3-dihydrofarnesyl acetate (67.3%), (2E,6E)-farnesyl acetate (14.9%)] (Dung et al., 1999); 6: Vietnam [(2E,6E)-farnesyl acetate (35.5%), (E)-2,3-dihydrofarnesyl acetate (32.9%)] (Dung et al., 1999); 7: Eastern India [(2E,6E)-farnesyl acetate (47.6%), (E)-ÃŽ ²-farnesene (9.6%)] (Rout et al., 2004). 1